arabis alpina pajares와 - seoul national...
TRANSCRIPT
1
이학박사학위논문
다년생 식물 Arabis alpina Pajares와
일년생 식물 Arabidopsis thaliana Sy-0의
분자·생리학적 비교를 통한 다년생 생활사 연구
Comparative analysis of molecular and
physiological traits between
perennial Arabis alpina Pajares
and annual Arabidopsis thaliana Sy-0
2018년 2월
서울대학교 대학원
생명과학부
박 종 윤
2
CONTENTS
CONTENTS ------------------------------------------------------------ 2
LIST OF FIGURES --------------------------------------------------- 7
LIST OF TABLES -------------------------------------------------- 10
ABBREVIATIONS ------------------------------------------------- 11
CHAPTER I. BACKGROUND ---------------------------------- 13
Life strategies in plants: Monocarpy and Polycarpy
1.1.1. Life strategies of monocarpic annuals and polycarpic
perennials ....................................................................... 14
1.1.2. Polycarpic perennial Arabis alpina Pajares ............... 16
1.1.3. Monocarpic annual Arabidopsis thaliana Sy-0 .......... 17
1.2. Aims of the thesis ............................................................. 19
1.3. REFERENCES ................................................................ 21
CHAPTER II. -------------------------------------------------------- 23
Comparative analysis of molecular traits between perennial
Arabis alpina Pajares and annual Arabidopsis thaliana Sy-0
2.1. ABSTRACT ..................................................................... 24
2.2. INTRODUCTION ........................................................... 26
3
2.2.1. Life strategies of perennial and annual plants ......... 26
2.2.2. Vernalization and flowering ...................................... 27
2.2.3. Vernalization requiring polycarpic perennial Arabis
alpina Pajares ................................................................ 28
2.2.4. Features of juvenile plants ......................................... 29
2.2.5. Regulation of juvenile to adult transition ................. 30
2.2.6. miR156-SPL regulatory module ................................ 31
2.2.7. Purpose of this study ................................................... 32
2.3. MATERIALS AND METHODS ................................... 34
2.3.1. Plant materials and growth conditions ..................... 34
2.3.2. Characterization of miR156 precursors in Arabis
alpina Pajares ................................................................ 34
2.3.3. Sampling of axillary shoot apices in Arabis alpina
Pajares and Arabidopsis thaliana Sy-0 ........................ 37
2.3.4. Microscopic analysis ................................................... 37
2.3.5. Transcript expression analysis .................................. 37
4
2.3.6. Oligonucleotide primers ............................................. 38
2.4. RESULTS ......................................................................... 40
2.4.1. Distinctive developmental features of Arabis alpina
Pajares during vegetative growth ................................ 40
2.4.2. In Arabis alpina Pajares, axillary shoots undergoing
different developmental phases coexist in the same
plant ................................................................................ 46
2.4.3. Homologs of miR156 precursors in Arabis alpina ... 48
2.4.4. MicroRNA156 expression levels in axillary shoot
apices are variable depending on the developmental
stages in Arabis alpina Pajares ..................................... 52
2.4.5. After vernalization, some axillary shoot apices
expressing high levels of pre-miR156s in Arabis alpina
maintain vegetative phase ............................................ 56
2.4.6. Developmental features of winter annual
Arabidopsis thaliana Sy-0 .............................................. 60
5
2.4.7. MicroRNA156 levels in all the axillary shoot apices
of Arabidopsis thaliana Sy-0 are similar independent
of developmental stages ................................................ 63
2.4.8. Differential responses to vernalization in Arabis
alpina Pajares and Arabidopsis thaliana Sy-0 ............. 64
2.5. DISCUSSION .................................................................. 71
2.6. REFERENCES ................................................................ 76
CHAPTER III. ------------------------------------------------------- 85
Transcriptome analysis of Arabis alpina Pajares to find
regulators for initiating vegetative axillary meristems
3.1. ABSTRACT ..................................................................... 86
3.2. INTRODUCTION ........................................................... 88
3.2.1. Key regulators in initiation and activation of axillary
meristems in Arabidopsis ............................................. 89
3.2.2. Hormonal regulation in initiation and activation of
axillary meristems in Arabidopsis ............................... 90
3.3. MATERIALS AND METHODS ................................... 93
3.3.1. Plant materials and growth condition ....................... 93
6
3.3.2. Transcriptome analysis .............................................. 93
3.4. RESULTS ......................................................................... 95
3.4.1. Distinctive morphological traits of Arabis alpina
Pajares during and after vernalization ....................... 95
3.4.2. Transcriptome analysis of primary stems in both
vegetative and reproductive phases ........................... 106
3.5. DISCUSSION ................................................................ 120
3.6. REFERENCES .............................................................. 122
CHAPTER IV. CONCLUSION --------------------------------- 126
A study to elucidate the molecular factors responsible for the
perennial traits in Arabis alpina Pajares
ABSTRACT IN KOREAN --------------------------------------- 129
감사의 글 ------------------------------------------------------------ 132
7
LIST OF FIGURES
Figure 1. A life cycle of Arabis alpina Pajares --------------------------------------- 42
Figure 2. Leaf developments of both primary and axillary shoots of Arabis
alpina Pajares show similar transition from juvenile to adult phase ----- 43
Figure 3. Developmental patterns of primary and axillary shoots in Arabis
alpina Pajares and categorization of developmental stages from S1 to S5
------------------------------------------------------------------------------------------ 44
Figure 4. Proportion of axillary shoots undergoing particular developmental
stages at each node ------------------------------------------------------------------ 47
Figure 5. Six homologs of miR156 precursors in Arabis alpina Pajares ------- 49
Figure 6. Secondary structure of six homologs of miR156 precursors in Arabis
alpina Pajares ------------------------------------------------------------------------ 50
Figure 7. Relative expression levels of miR156 precursors in the primary shoot
apices of Arabis alpina Pajares during 2 to 6-week vegetative growth --- 51
Figure 8. Expression levels of miR156 precursors in the primary and axillary
shoot apices during vegetative growth of Arabis alpina Pajares ----------- 53
8
Figure 9. Expression levels of miR156 precursors in the primary and S1 axillary
shoot apices during vegetative growth of Arabis alpina Pajares ----------- 55
Figure 10. Expression levels of miR156 precursors in the primary and axillary
shoot apices of Arabis alpina Pajares after vernalization ------------------- 57
Figure 11. Flowering competence of axillary shoot apices after vernalization in
Arabis alpina Pajares depends on developmental stages -------------------- 59
Figure 12. Expressions of several genes in the axillary shoots of winter annual
Arabidopsis thaliana, Sy-0 were synchronized --------------------------------- 61
Figure 13. Vernalization responsive gene expression of Arabidopsis thaliana Sy-
0 according to age ------------------------------------------------------------------- 67
Figure 14. Vernalization-mediated floral transition in the primary shoot
apices of Pajares at different developmental ages ---------------------------- 69
Figure 15. Vernalization-mediated flowering response according to age after
germination in Arabidopsis thaliana Sy-0 -------------------------------------- 70
Figure 16. Developmental patterns of primary stems of Pajares under
continuous vegetative and cold temperature ---------------------------------- 97
Figure 17. Establish adventitious roots at nodes especially during vernalization
9
treatment ------------------------------------------------------------------------------ 99
Figure 18. After 3 weeks growth of excised stem segments with or without
adventitious roots ------------------------------------------------------------------ 100
Figure 19. Outgrowth of axillary buds when they returned to warm
temperature following vernalization ------------------------------------------- 102
Figure 20. A schematic diagram of an experiment for searching energy and
nutrient resources in Pajares ---------------------------------------------------- 104
Figure 21. Survivability and development of shoots from type I to type IV - 105
Figure 22. Overview of transcriptome analysis of reproductive stems in Pajares
----------------------------------------------------------------------------------------- 108
Figure 23. Comparison of amino-acids sequence similarity between AaRAXs
and AtRAXs ------------------------------------------------------------------------- 115
Figure 24. Expression analysis of AaRAXs at various stages of leaf axils
according to size of axillary buds and developmental phases ------------- 117
10
LIST OF TABLES
Table 1. Nucleotides sequences of miR156 precursors in Pajares ................. 36
Table 2. Sequences of oligonucleotide primers used in qRT-PCR analysis .. 39
Table 3. A categorization of developmental stages of axillary shoots, S1 to S5,
according to the numbers of elongated internodes and leaves generated
from axillary shoots ................................................................................... 45
Table 4. Classification of developmental stages of axillary shoots in
Arabidopsis thaliana Sy-0 .......................................................................... 62
Table 5. Flowering efficiency in Arabis alpina Pajares after prolonged cold
treatment .................................................................................................... 65
Table 6. Genes Ontology data related to hormones ...................................... 110
Table 7. Up-regulated transcription factors that related phytohormones .. 112
Table 8. Relative expression of AtRAXs homologs in Pajares ...................... 114
Table 9. Up-regulated transcription factors in reproductive stems of Pajares
that related with auxin or cytokinin, and expressed in meristematic
region ........................................................................................................ 119
11
ABBREVIATIONS
Aa
At
miR156
pre-miR156
SPL
Arabis alpina
Arabidopsis thaliana
microRNA 156
precursor of microRNA 156
SQUAMOSA PROMOTER BINDING PROTEIN-
LIKE
PEP1-PEP2
FLC
AP1-AP2
LFY
PP2A
RT-PCR
qRT-PCR
PERPETUAL FLOWERING 1-2
FLOWERING LOCUS C
APETALA 1-2
LEAFY
PROTEIN PHOSPHATASE 2A
Reverse Transcription-Polymerase Chain Reaction
quantitative RT-PCR
ART1 AERIAL ROSETTE 1
REV
CUC1-CUC3
REVOLUTA
CUP-SHAPED COTYLEDON1-3
12
RAX1-RAX3
ROX
LAS
SAM
AM
AVT
eFP
REGULATOR OF AXILLARY MERISTEMS1-3
REGULATOR OF AXILLARY MERISTEM
FORMATION
LATERAL SUPPRESSOR
Shoot Apical Meristem
Axillary Meristem
AtGenExpress Visualization Tool
Electronic Fluorescent Pictograph
13
CHAPTER I. BACKGROUND
Life strategies in plants: Monocarpy and Polycarpy
14
1.1.1. Life strategies of monocarpic annuals and polycarpic
perennials
The life strategies in plants and animal are largely divided into semelparity and
iteroparity. Semelparous species invest most of their energy and resources in
reproduction to maximize the number of offsprings produced in a life cycle. They
reproduce once in their lifetime and die soon after, while iteroparous species survive
through multiple events of reproduction in their lifetime (Amasino, 2009).
Arabidopsis thaliana, one of the most popular model organisms in the plant
kingdom, is a well known example of the semelparous species. They complete the
life cycle within two months producing a number of progenies. After reproduction,
Arabidopsis senesces and dies even under favorable growth condition. Iteroparous
species in plants includes most of trees, which can live for several years with multiple
cycles of reproduction. The semelparity and iteroparity in plants are generally
referred as monocarpy and polycarpy, respectively. The monocarpic plants are
represented by all annuals and a few perennial species such as bamboo, agave and
silversword, which live for many years without flowering and die after flowering
once. The polycarpic plants are usually perennial that survive for many years during
which they undergo multiple reproducing events (Albani and Coupland, 2010;
Amasino, 2009; Keifer et al., 2017).
The status of being monocarpic annual or polycarpic perennial is decided by the
function of meristem determinacy together with the processes of senescence. To
15
preserve perenniality in plants, at least one of the apical meristems from their shoot
axes should remain indeterminate after reproductive phase (Thomas et al., 2000). In
most perennial trees, meristems show dormant state that is insensitive to growth
promoting signals until it became sensitized and resume growth (Munne´-Bosch,
2008). In tree researches, dormancy is defined as the absence of visible growth in
any plant structure containing a meristem region. It has pivotal roles for survival of
indeterminacy meristems under unfavorable environment (Arora et al., 2003; Rohde
and Bhalerao, 2007).
Although meristem determinacy is important for life style of annual or perennial
plants, programmed senescence and cell death processes being implemented
progressively also make significant contributions. In perennial shrubs and trees,
organs developed during the growing season of the year are green and fresh, while
those generated in previous years are suberized and lignified (Thomas et al., 2000).
There are two types of senescence in monocarpic plants. During vegetative
development, senescence occurs mostly in leaves and progresses along the shoot axis
resulting in remobilization of nutrients from older to younger leaves. During
reproductive phase, holistic senescence occurs to allow remobilization of nutrients
to the seeds. Therefore, significant differences in lifestyles between monocarpic and
polycarpic plants are made at the levels of indeterminacy and totipotency of
meristems, and partial senescence (Munne´-Bosch, 2008).
Evolutionary transitions between perenniality and annuality appear to have
occurred in higher plants. In most cases of phylogenetic lineages, annuals have been
16
derived from perennial ancestors. However, in some genera, the reversal to
perenniality has also been documented. In theory, annuality evolves as an adaptive
response to unpredictable circumstances. In practice, annuals are usually found in
open, dry and hot habitats, but perennials are more commonly found in mild habitats
with high seedling mortality. Generally, a life history evolves to optimize the number
of offspring produced with minimal cost of reproduction (Friedman and Rubin, 2015;
Keifer et al., 2017).
1.1.2. Polycarpic perennial Arabis alpina Pajares
The Brassicaceae is comprised of approximately 340 genera and 3,350 species
including Brassica crops and the model plant Arabidopsis thaliana. The divergence
of Brassica-Arabidopsis had occurred at nearly 14-20 million years ago (Koch et al.,
2000; Wang, 2007). Arabis alpina is widely distributed in southern mountain regions,
from the northern amphi-Atlantic area and the European mountains including the
Mediterranean to the Caucasus, Iran and Iraq, and reaches to the Arabian Peninsula
in the southeast; in addition, remote populations are known from East African
mountain regions. In central Europe, it is found mostly in mountain and subalpine to
alpine habitats (Koch et al., 2006).
Recently, there are several molecular studies done in Arabis alpina. For example,
PERPETUAL FLOWEING 1 (PEP1), an ortholog of FLOWERING LOCUS C (FLC),
has been found to contribute to the perennial traits in Arabis alpina. It limits the
17
duration of flowering, and prevents formation of some branches of floral transition,
and confers flowering response to the winter temperature. In contrast to Arabidopsis
thaliana FLC, PEP1 is only transiently repressed by vernalization. The pep1 mutants
that were generated by EMS mutagenesis continuously flower in the absence of
vernalization without affecting perennial characteristics (Wang et al., 2009b). On the
other hand, Arabis alpina TFL1 inhibits flowering and prevents AaLFY expression
in young plants exposed to long-term cold. In older plants, AaTFL1 increases the
duration of vernalization required for activating expression of AaLFY and flowering
(Wang et al., 2011b). Arabis alpina PEP2, an ortholog of Arabidopsis thaliana
flowering repressor AP2, prevents flowering before vernalization. In addition,
miR156 abundance in young plants explains regulation of age-dependent flowering
response to vernalization (Bergonzi et al., 2013).
1.1.3. Monocarpic annual Arabidopsis thaliana Sy-0
Arabidopsis thaliana has been the most popular model for molecular studies of
plants, especially in flowering-time regulation. Diverse Arabidopsis accessions can
be grouped into two categories based on the need for a prolonged period of cold
exposure-vernalization, to facilitate flowering. A rapid-cycling type, known as
summer-annual accessions, including most laboratory strains, flower rapidly without
vernalization. In contrast, flowering is strongly delayed in the absence of
vernalization in winter-annual accessions. Arabidopsis thaliana Sy-0 is a natural
18
strain that requires long period of cold exposure to induce early flowering. The
timing of anthesis is more than two months after seed germination, at which time the
basal rosettes contain up to 70-80 leaves. With the transition to reproductive stage,
bolting produces an elongated primary stem bearing 10-12 cauline leaves. At the
axils of these cauline leaves, additional leaves emerge which resemble the basal
rosette leaves. These subtending cauline leaves are similar in structure to the basal
rosettes, but they are seen in the aerial portion of the plant. Aerial rosettes develop
up to 18 leaves in the axils of cauline leaves. The establishment of axillary meristems
are initiated only after the primary shoot apical meristem (SAM) undergoes the
transition to flowering in most Arabidopsis. However, in Sy-0 and other late-
flowering varieties, axillary meristems are initiated at the axils of the oldest mature
leaves even before the transition to reproductive phase (Grbic´ and Bleecker, 1996).
In early flowering Arabidopspis thaliana, the development of axillary meristem
occurs basipetally, following axils from younger leaves to older leaves during
conversion from vegetative phase to reproductive phase. By contrast, prolonged
vegetative phase of the late flowering Arabidopsis thaliana including Sy-0 develops
axillary buds acropetal direction such that axillary meristems are initiated from older
leaves to young leaves even under vegetative phase (Grbic' and Bleecker, 2000;
Hempel and Feldman, 1994).
Previous reports revealed the mechanisms leading to novel plant architecture in
Sy-0 ecotype of Arabidopsis thaliana that generates aerial rosettes in the subtending
cauline leaves, and exhibits inflorescence and floral reversion. This heterochronic
19
shift in reproductive development of shoot meristems results from interaction among
dominant alleles at AERIAL ROSETTE 1 (ART1), FRIGIDA (FRI) and FLOWERING
LOCUS C (FLC) loci. Subsequently, HUA2 was identified as a candidate for ART1,
which is a putative pre-mRNA processing factor and acts as a repressor of floral
transition by reducing the expression of several MADS box genes including
FLOWERING LOCUS C (FLC) and FLOWERING LOCUS M (FLM). Interestingly,
an allelic form of HUA2 in Landsberg erecta (Ler) was found to have a premature
stop codon (Doyle et al., 2005; Poduska et al., 2003; Wang et al., 2007).
1.2. Aims of the thesis
In sessile plants, adopting a specific life strategy of annuality or perenniality is a
means to adapt and protect the species under unexpected environments. Due to
additional challenge in studying perennials, such as the long life cycle, a scarcity of
genetic resources, and difficulties in molecular handling, the detailed mechanisms
by which perenniality is maintained are not well understood yet.
Perennial plants can largely maintain their life styles in two ways. One way is by
retaining the vegetative state even after flowering in several shoot apical meristems
in the same plant for the following year. Alternatively, they establish new vegetative
meristems that are not affected by senescence after reproduction.
As the polycarpic perennial Arabis alpina is a close relative to the popular model
Arabidopsis thaliana, it is imperative to study the molecular regulation of
20
perenniality by comparative analysis of the two plants. In addition, both Arabis
alpina Pajares and Arabidopsis thaliana Sy-0, the two natural accessions that were
used in this research, show the requirement of vernalization to accelerate flowering
in common. The developmental patterning of acropetal axillary branches is also
comparable between these two accessions, making them ideal materials to
understand the mechanisms maintaining annuals and perennials.
Therefore, this thesis will focus on understanding the differences in behavior of
axillary shoots by comparative analysis of axillary meristems at a molecular level,
including transcriptome analysis to find molecular candidates for initiating
vegetative axillary buds to preserve perenniality.
21
1.3. REFERENCES
Albani, M.C., and Coupland, G. (2010). Comparative analysis of flowering in
annual and perennial plants. Curr Top Dev Biol 91, 323-341.
Amasino, R. (2009). Floral induction and monocarpic versus polycarpic life
histories. Genome Biology 10, 1-3.
Arora, R., Rowland, L.J., and Tanino, K. (2003). Induction and release of bud
dormancy in woody perennials: a science comes of age. HortScience 38, 911-921.
Doyle, M.R., Bizzell, C.M., Keller, M.R., Michaels, S.D., Song, J., Noh, Y.-S.,
and Amasino, R.M. (2005). HUA2 is required for the expression of floral
repressors in Arabidopsis thaliana. Plant Journal 41, 376-385.
Friedman, J., and Rubin, M.J. (2015). All in good time: understanding annual and
perennial strategies in plants. American Journal of Botany 102, 497-499.
Grbic', V., and Bleecker, A.B. (2000). Axillary meristem development in
Arabidopsis thaliana. Plant Journal 21, 215-223.
Grbic´, V., and Bleecker, A.B. (1996). An altered body plan is conferred on
Arabidopsis plants carrying dominant alleles of two genes. Development 122,
2395-2403.
Hempel, F.D., and Feldman, L.J. (1994). Bi-directional inflorescence development
in Arabidopsis thaliana: Acropetal initiation of flowers and basipetal initiation of
paraclades. Planta 192, 276-286.
Keifer, C., Severing, E., Karl, R., Bergonzi, S., Koch, M., Tresch, A., and
Coupland, G. (2017). Divergence of annual and perennial species in the
22
Brassicaceae and the contribution of cis-acting variation at FLC orthologues.
Molecular Ecology 26, 3437-3457.
Koch, M.A., Haubold, B., and Mitchell-Olds, T. (2000). Comparative evolutionary
analysis of chalcone synthase and alcohol dehydrogenase loci in Arabidopsis,
Arabis, and related genera (Brassicaceae). Mol Biol Evol 17, 1483-1498.
Munne´-Bosch, S. (2008). Do perennials really senesce? TRENDS in Plant Science
13, 216-220.
Poduska, B., Humphrey, T., Redweik, A., and Grbic´, V. (2003). The synergistic
activation of FLOWERING LOCUS C by FRIGIDA and a new flowering gene
AERIAL ROSETTE 1 underlies a novel morphology in Arabidopsis. Genetics 163,
1457-1465.
Rohde, A., and Bhalerao, R.P. (2007). Plant dormancy in the perennial context.
TRENDS in Plant Science 12, 217-223.
Thomas, H., Thomas, H.M., and Ougham, H. (2000). Annuality, perenniality and
cell death. Journal of Experimental Botany 51, 1781-1788.
Wang, Q., Sajja, U., Rosloski, S., Humphrey, T., Kim, M.C., Bomblies, K.,
Weigel, D., and Grbic´, V. (2007). HUA2 caused natural variation in shoot
morphology of A. thaliana. Current Biology 17, 1513-1519.
23
CHAPTER II.
Comparative analysis of molecular traits between
perennial Arabis alpina Pajares and
annual Arabidopsis thaliana Sy-0
24
2.1. ABSTRACT
Comparative analysis of molecular traits between perennial
Pajares and annual Arabidopsis thaliana Sy-0
Jong-Yoon Park
School of Biological Sciences
The Graduate School
Seoul National University
Annual plants complete their life cycle within a year while perennial plants
maintain growth for several years. Arabis alpina, a polycarpic perennial, is a close
relative of monocarpic annual Arabidopsis. Pajares is an accession of Arabis alpina
requiring vernalization, a long-term cold treatment, for flowering. Arabidopsis
shows holistic flowering whereas Pajares shows idiographic flowering, producing
axillary branches under variable developmental phases from juvenile, adult
vegetative to reproductive phases. To understand the molecular mechanism behind
25
diverse phases of axillary branches, the levels of primary miR156 expressions were
analyzed as miR156-SPL module is a key regulator for developmental phase
transition. It was found that in Pajares, miR156 levels were highly variable among
the axillary branches, which causes differential sensitivity to vernalization. Thus, the
axillary branches expressing high levels of miR156 remain in juvenile phase even
after vernalization, whereas the axillary branches expressing low levels of miR156
produce flowers after vernalization. In contrast, every axillary branches of
Arabidopsis winter annual Sy-0 expressed similar levels of miR156 and
synchronously responded to vernalization, which causes holistic flowering.
Therefore, it suggests that variable miR156 expression levels and the resulting
differential response to vernalization among axillary branches are distinctive
features determining polycarpic perenniality of Arabis alpina Pajares.
Keywords : annual plants, perennial plants, life cycle, juvenility, floral
competency, vernalization, longevity
Student Number : 2010 - 30933
26
2.2. INTRODUCTION
2.2.1. Life strategies of perennial and annual plants
Plant kingdom is largely divided into semelparous monocarpic and iteroparous
polycarpic plants depending on the life cycle strategies. The monocarpic species
include all annuals and some perennials such as bamboo. They show holistic
senescence following massive flowering at once to maximize the number of
offspring. Arabidopsis is a representative annual model plant, which lives a single
growing season and complete their life cycle within a year. In Arabidopsis, rapid
cycling accessions such as Columbia and Landsberg erecta execute early-flowering
whereas winter annual accessions require prolonged cold for 4~8 weeks to accelerate
flowering. Some perennial species, such as bamboo, maintain vegetative phase for
many years before flowering and then undergo holistic senescence to die (Albani and
Coupland, 2010). On the other hand, most polycarpic species repeat several cycles
of reproduction (iteroparous reproduction) and ensure long-term survival. Polycarpic
perennials prolong their lives by maintaining vegetative shoots and / or producing
new vegetative shoots even after transition to reproductive phase (Albani and
Coupland, 2010; Amasino, 2009; Bergonzi and Albani, 2011).
27
2.2.2. Vernalization and flowering
During plant development, a series of phase transitions occur to their life cycle in
response to environmental and endogenous signals. Especially, the phase transition
from vegetative to reproductive state is elaborate regulation through complicated
genetic mechanisms. The five regulatory pathways, photoperiod (day length and
light intensity), vernalization (long-term cold treatment), gibberellic acid (GA),
autonomous and aging pathways for reproductive transition are known in
Arabidopsis. Vernalization is one of the environmental pathways to accelerate
flowering. It requires two dominant genes, FRIGIDA (FRI) and FLOWERING
LOCUS C (FLC). FLC is a gene encoding MADS domain transcription factor and
acts as a floral repressor. In winter annual Arabidopsis, FLC is highly expressed and
prevents transition to reproductive development before winter season. FRI is
required for the expressional activation of FLC (Choi et al., 2011; Michaels and
Amasino, 1999). When plants exposed to long-term cold environment, FLC converts
from active state to silenced state. A rapid reduction of FLC transcription occurs as
early as two weeks after exposure to cold through antisense transcript, COOLAIR
(Swiezewski et al., 2009). Another cold-induced a sense long non-coding RNA
(COLDAIR) interacts with the histone methyltransferase subunit of the Polycomb
repressive complex 2 (PRC2) (Heo and Sung, 2011). Prolonged cold induces
accumulation of polycomb-mediated histone modification, H3K27me3, at a specific
site in FLC. Once FLC expression has been down-regulated, it is epigenetically
silenced by a PHD-PRC2 complex (Bastow et al., 2004; Sung and Amasino, 2004).
28
The epigenetic repression of FLC is maintained when plants return to warm
condition for reproductive development.
2.2.3. Vernalization requiring polycarpic perennial Arabis
alpina Pajares
Polycarpic perennial Arabis alpina (Alpine rock-cress) is a close relative to
Arabidopsis which belongs to Brassicaceae family. Arabis alpina is widely spread
in mountainous areas of Europe, North and East Africa, Central and East Asia, and
North America (Ansell et al., 2011). Genome size of Arabis alpina is 392 Mb and it
is self-compatible (Eaux et al., 2014). Pajares is a vernalization requiring accession
among more than 140 accessions of Arabis alpina (Bergonzi, 2012; Koch et al., 2000;
Koch et al., 2006; Wang, 2007; Wang et al., 2009b). Recently, several studies
demonstrated flowering mechanism in vernalization requiring Pajares. PERPETUAL
FLOWERING 1 (PEP1), genetic ortholog of Arabidopsis FLOWERING LOCUS C
(FLC), sustained vegetative phase until its expression repressed by exposure to
vernalizaion. In contrast to Arabidospis, in which FLC stably repressed through
vernalization, prolonged cold exposure transiently represses FLC in Pajares. In these
results, some shoots of a plant expressing resumption of FLC are remained
vegetative phase in warm temperature returning (Wang et al., 2009b). TERMINAL
FLOWER 1 (TFL1) of Arabis alpina inhibits flowering when young plants exposed
to long-term cold treatment to prevent precocious flowering. AaPEP2, an ortholog
29
of Arabidopsis floral repressor APETALA2 (AP2), is involved vernalization
responsive flowering together with PEP1 (Bergonzi et al., 2013; Wang et al., 2011a;
Wang et al., 2009b). Moreover, Pajares undergo more than 5 weeks of vegetative
growth for floral competence. It should be exposed cold temperature for at least 10
weeks for vernalization-mediated flowering (Wang, 2007; Wang et al., 2011b).
2.2.4. Features of juvenile plants
A life cycle of plants usually consists of discrete developmental phases. During
germination, seedlings sprout from seeds through embryonic to post-embryonic
developmental transition. The seedlings increase their mass during vegetative phase.
The vegetative phase is further divided into juvenile and adult phases, and plants
acquire competence to flower through such changes. Before transition to
reproductive phase, plants become competent to flower and eventually produce
reproductive organs and seeds (Bäurle and Dean, 2006; Huijser and Schmid, 2011).
Plants show heteroblasty because the same plants have leaves with different
morphological traits developed from juvenile to adult vegetative phases. The early
rosettes, juvenile leaves of Arabiodpsis are small and almost round in shape with
smooth margins and long petioles, while adult rosette leaves are enlarged and
elongated with visible leaf serrations. Leaf trichomes are detected on both abaxial
and adaxial surfaces in adult vegetative leaves, but juvenile leaves have leaf
trichomes only on adaxial side (Wu and Poethig, 2006).
30
2.2.5. Regulation of juvenile to adult transition
During plant development, many environmental and endogenous cues affect phase
transition. The environmental signals such as light intensity, photoperiod, and
ambient temperature as well as endogenous hormones and aging influence the timing
of phase transition. A post-transcriptional regulatory module, microRNA156 -
SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL), is highly conserved
in various plant species and acts as an age-dependent timer regulating phase
transitions (Bergonzi et al., 2013; Chuck et al., 2007; Hyun et al., 2016; Morea et
al., 2016; Wang et al., 2011a; Wu and Poethig, 2006; Xie et al., 2006; Xu et al.,
2016; Zhou et al., 2013). The expression level of miR156 is higher at seedling stage
and is gradually decreased according to age. A wide range of transgenic plant species
including Arabidopsis, rice, maize, poplar hybrid tree overexpressing miR156
produce excessive number of juvenile leaves and flower extremely late (Bergonzi et
al., 2013; Chuck et al., 2007; Schwab et al., 2005; Wang et al., 2011a; Wu and
Poethig, 2006; Xie et al., 2006; Zhou et al., 2013). Such reports suggest that miR156
has functions promoting juvenile phase and delaying developmental phase transition.
In Arabidopsis, 11 of 17 genes encoding SPL transcription factors are revealed as
targets of miR156, while direct upstream factors of miR156 remain to be discovered
(Huijser and Schmid, 2011). The SPL proteins redundantly act in developmental
phase transitions from embryogenesis to reproductive phase (Fornara and Coupland,
2009; Huijser and Schmid, 2011; Hyun et al., 2016; Wang et al., 2009a; Wang et al.,
2008; Wu et al., 2009; Wu and Poethig, 2006; Xu et al., 2016).
31
2.2.6. miR156-SPL regulatory module
In Arabidopsis, microRNAs play pivotal roles of developmental processes and
responses such as leaf polarity, leaf morphogenesis, flowering, root cap formation,
auxin signaling and stress response. Plant miRNA156 is an important factor in aging
and phase transition-related development. The overexpression of MIR156B in
Columbia background showed delayed flowering with significantly increased
number of juvenile leaves and weakened apical dominance. Constitutively
expressing the a, b, c, d, e and f isoforms of miR156 also presented similar
phenotypes to MIR156B overexpresser (Wu and Poethig, 2006). SQUAMOSA
PROMOTER BINDING PROTEIN-LIKE (SPL) proteins are highly conserved
plant-specific transcription factors, containing a DNA binding SPB-domain.
Transcriptional expression of ten genes out of 17 SPL family genes in Arabidopsis
was declined in the plants overexpressing MIR156B (Schwab et al., 2005). The
translational repression of SPL genes is through cleavage of target SPL mRNAs by
nearly perfect base pairing with mature miRNA (Reinhart et al., 2002). MiRNA156
mediated post-transcriptional regulatory mechanism is a conserved feature in various
species of plants. In previous researches, AaSPL showed post-transcriptional
repression by miR156, and their inverse proportional expression is dependent on age
(Wang et al., 2009a; Wang et al., 2008). In this report, Pajares overexpressing
MIR156B could not transit to reproductive phase even though PEP1 repressed by the
exposure to long-term cold. In addition, transcriptional expression pattern of miR156
in pep1-1 was indistinguishable from the wild type Pajares. Even though flowering
32
mechanisms repressed by miR156 and PEP1 were parallel, miR156 might play an
important role in vernalization response. Since 3-week-old transgenic plants, which
constitutively expressing a miR156 mimicry construct (MIM156), presented
vernalization-responsive floral competence while wild-type Pajares could fully
respond to vernalization only in the case of older than 5-week-old plants (Bergonzi
et al., 2013).
2.2.7. Purpose of this study
In recent decades, several genetic factors to regulate developmental phase
transition have been studied in Arabidopsis. However, molecular studies in
perennials are limited because of long life cycle, a scarcity of genomic resources,
and difficulties in handling. As regulation of flowering is particularly important in
biomass and yield, researchers studying perennial plants have focused on the
orthologs of Arabidopsis flowering genes (Foster et al., 2003; Hsu et al., 2011; Hsu
et al., 2006; Jensen et al., 2001; Lin et al., 2005; Mimida et al., 2009). To figure out
a life strategy of perennial plants, comparative analysis of the molecular and
physiological features between a perennial plant, Pajares, and a close relative annual,
Arabidopsis thaliana Sy-0 was performed. These results indicated that
asynchronized expression of pre-miR156s in axillary shoots of Arabis alpina results
in the variable responsiveness to vernalization, thus the axillary shoots incompetent
to flower remained as vegetative branches after winter cold. In contrast, winter
33
annuals of Arabidopsis thaliana showed synchronized expression of pre-miR156 in
all the axillary shoots. Therefore, it suggests that variable expression of miR156 in
axillary shoots confers polycarpic perenniality to Arabis alpina.
34
2.3. MATERIALS AND METHODS
2.3.1. Plant materials and growth conditions
Arabis alpina Pajares and A. thaliana ecotype Sy-0 seeds were surface sterilized
in 75% ethanol and 0.05% tween-20 solution. After sterilization, seeds were sown
on one-half-strength MS medium supplemented with 1% (w/v) sucrose and 1% (w/v)
plant agar. The Pajares and Sy-0 seeds were stratified under dark at 4℃ for 10 days
and 3 days, respectively. After stratification, the seeds were germinated on MS
medium, then seedlings were transplanted to soil under controlled condition of 16-
hour light and 8-hour dark at 22℃. For long-term cold treatment, vernalization,
plants were transferred to vernalization chamber (8-hour light and 16-hour dark at
4℃).
2.3.2. Characterization of miR156 precursors in Arabis alpina
Pajares
To obtain the information of nucleotide sequences of miR156 precursors in
Arabis alpina Pajares, BLAST tool of NCBI
(ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/) was used with Genome
database of Pajares (Research, 2014)
(https://www.ncbi.nlm.nih.gov/bioproject/PRJNA241291). Based on the genomic
sequences, I cloned six homologs of miR156 precursors which are highly similar
35
with miR156 precursors in Arabidopsis. The sequences covering target-binding site
were used for quantitative RT-PCR with the specific primers presented in Table 2.
Nucleotide sequences of miR156 precursors in Arabis alpina Pajares are annotated
(Table 1).
36
Table 1. Nucleotides sequences of miR156 precursors in Pajares. Red colored
nucleotides indicate a target binding site of miR156s
37
2.3.3. Sampling of axillary shoot apices in Arabis alpina
Pajares and Arabidopsis thaliana Sy-0
For expression analysis of miR156 precursors in various stages of axillary shoots
of Arabis alpina Pajares, S1 to S5 stages of axillary shoot apices were harvested
from 8-week-old Pajares before and after vernalization (Figure 8, 9 and Figure 10).
The axillary shoot apices undergoing the same developmental stages were
collectively harvested regardless of the node positions.
2.3.4. Microscopic analysis
To determine meristem identity, microscopic analysis was performed in various
stages of axillary shoots of Pajares after long-term cold treatment. Vegetative 8-
week-old Pajares, which have S1 ~ S5 axillary shoots, were exposed to vernalization
for 12 weeks. The shoot apices of primary and axillary shoots were observed by
digital light-microscope (DIMIS-M® , CMOS sensor) before returning to warm
temperature.
2.3.5. Transcript expression analysis
For gene expression analysis, total RNA was extracted from apical and axillary
shoot apices in certain developmental stages and chronological ages using the
38
RNeasy® plant mini kit (QIAGEN 74904). Contaminated genomic DNA was
eliminated with recombinant DNaseI (Takara 2270A). cDNA was synthesized using
total RNA with reverse transcriptase (Fermentas EP0441) and oligo (dT).
Quantitative PCR was performed using the iQTM SYBR® Green Supermix (Bio-Rad
170-8882) and analyzed by the CFX96 real-time PCR detection system.
2.3.6. Oligonucleotide primers
The sequences of oligonucleotide primers used in this work were listed in Table
2.
39
Table 2. Sequences of oligonucleotide primers used in qRT-PCR analysis
40
2.4. RESULTS
2.4.1. Distinctive developmental features of Arabis alpina
Pajares during vegetative growth
Arabis alpina Pajares has distinctive features compared with a close relative
annual plant, Arabidopsis thaliana. For example, Pajares shows internode elongation
and outgrowth of axillary shoots from each node during vegetative phase whereas A.
thaliana develops rosette leaves due to lack of internode elongation during
vegetative growth (Figure 1B). In addition, Pajares has to pass through at least 5
weeks of juvenile phase to respond to vernalization, a long-term winter cold for
flowering, and absolutely requires more than 10 weeks of vernalization for flowering
(Wang et al., 2011b). The most interesting feature of Pajares is that each axillary
shoot undergoes whole life cycle, from juvenile to adult, and reproductive phases,
like an individual plant (Figure 1C and D). In a primary shoot, the four basalmost
true leaves show juvenile features such as unexpanded leaves with smooth margins
(Figure 2A and C). These four juvenile leaves are produced within about 3 weeks
after germination (Figure 3A). Similarly, juvenile leaves are produced at the 3 basal
nodes of axillary shoots (Figure 2B and C). To compare the growth patterns of
axillary shoots with those of primary shoots, I measured shoot-length, number of
true leaves every week and they showed similar growth rate (Figure 3A and B). For
the analysis of growth patterns in axillary shoots, I categorized each axillary shoot
depending on the developmental stage from S1 to S5 (description of developmental
41
stage in Figure 3C ~ G and Table 3). The S1 axillary shoot is the latest and S5 axillary
shoot is the oldest one that used for the analysis.
42
Figure 1. A life cycle of Arabis alpina Pajares
(A) A juvenile vegetative Arabis alpina Pajares, grown on soil for 3 weeks after
germination. Scale bar = 1cm
(B) A 5-week-old, vegetative adult Pajares generates axillary shoots at almost nodes
of a primary stem (in box). Arrows indicate axillary shoots. Scale bar = 1cm
(C) A 8-week-old Pajares before long-term cold, vernalization.
(D) Axillary shoots generated from 8-week-old Pajares. Arrows indicate axillary
shoots in diverse size. Scale bar = 1cm
(E) A flowering Pajares which grown for 3 weeks under long-day, warm condition
after 12 weeks of vernalziation. Scale bar = 1cm
43
Figure 2. Leaf developments of both primary and axillary shoots of Arabis
alpina Pajares show similar transition from juvenile to adult phase
(A) Morphology of the leaves from a primary shoot in 9-week-old Arabis alpina
Pajares showing both juvenile and adult leaves. Scale bar=1cm
(B) Morphology of the leaves from an axillary shoot generated at the 2nd
node
showing both juvenile and adult leaves. Scale bar = 1cm
(C) Average numbers of leaf serration from the 1st
to the 8th
leaves developed
after cotyledon (n=15). The number of leaf serration increases according to
developmental phase, which is observed in both primary and axillary shoots.
44
Figure 3. Developmental patterns of primary and axillary shoots in Arabis
alpina Pajares and categorization of developmental stages from S1 to S5
(A) The numbers of true leaves and the shoot length from primary shoots were
measured weekly from 3 weeks to 8 weeks. (n=24).
(B) The numbers of true leaves and the shoot length from axillary shoots were
measured, weekly. The axillary shoots produced at the 2nd node from cotyledons
were used for this measurement. (n=24)
(C~G) Various developmental stages of axillary shoots were categorized as S1~S5
stages of which characterizations were described in Table 3. (C) to (G) are
representative photos for stage S1 to S5. (Scale bar=1cm)
45
Table 3. A categorization of developmental stages of axillary shoots, S1 to S5,
according to the numbers of elongated internodes and leaves generated from
axillary shoots
46
2.4.2. In Arabis alpina Pajares, axillary shoots undergoing
different developmental phases coexist in the same plant
An 8-week-old Pajares, which is in vegetative phase before vernalization, has
several axillary shoots undergoing various developmental stages from S1 to S5 and
I labelled them as 8WS1 to 8WS5. The 8WS5 shoots have been developed at the 4
basalmost nodes of primary shoot. To monitor branching order, proportion of
axillary shoots in each developmental stage from all node-positions from cotyledons
to shoot apices was calculated using 24 plants of 8-week-old Pajares (Figure 4). In
general, axillary shoots in Pajares developed acropetal direction, but there were some
exceptions at the two basalmost nodes. Most axillary shoots in 8WS5 were found at
the 1st and 2nd nodes from cotyledons. In contrast to this, the axillary shoots in
8WS1 were mostly located near the shoot apices, at the 7th to 10th nodes from
cotyledons. Therefore, the observation of Pajares growth strongly indicates that
axillary shoots generated from the same plant show asynchronous development with
variable developmental stages from S1 to S5.
47
Figure 4. Proportion of axillary shoots undergoing particular developmental
stages at each node
Composition of axillary shoots undergoing from S1 to S5 stages at each node of 8-
week-old plants, Arabis alpina Pajares. Y axis indicates position of each node
counted from cotyledons (Ct). X axis indicates proportion of axillary shoots at
particular developmental stages. (n=24)
48
2.4.3. Homologs of miR156 precursors in Arabis alpina
Age-dependent decrease of miR156 level is conserved in diverse plant species
(Morea et al., 2016). To test whether miR156 level is also decreased according to
developmental progress in axillary shoots of Arabis alpina Pajares, I cloned six
homologs of miR156 precursor genes using Arabis alpina database (Research, 2014).
These six homologs were designated as pre-miR156a, b, c, d, f and g depending on
the phylogenic analysis and prediction of secondary structure (Figure 5 and 6)
(Reinhart et al., 2002).
Since expression levels of miRNA precursors reflect mature miRNA levels in
several plant species (Wang et al., 2009a; Wu et al., 2009; Zhou et al., 2013), I
analyzed quantities of six miR156 precursors by qRT-PCR, which amplifies the
precursor sequences covering the target binding site (131 to 276 bp) (Table 1). In
primary shoot apices, the relative expression levels of pre-miR156a, b, c, and d were
gradually decreased upon aging, especially the expression levels of pre-miR156a and
c were dramatically reduced from 2 to 3-week (Figure 7). Transcript levels of pre-
miR156f and g were too low to compare even in young shoot apices.
49
Figure 5. Six homologs of miR156 precursors in Arabis alpina Pajares
(A) Nucleotides-sequences of miR156 precursors were analyzed by multi-
alignment program (CLC Main Workbench 7.7.1. QIAGEN®
https://www.qiagenbioinformatics.com/products/clc-main-workbench/). A target
binding site of miR156 was highly conserved in both Arabis alpina and
Arabidopsis (asterisks).
(B) According to alignment analysis, a phylogenetic tree was generated by CLC
Main Workbench to compare similarity of nucleotides sequences between
Arabidopsis and Arabis alpina Pajares.
50
Figure 6. Secondary structure of six homologs of miR156 precursors in Arabis
alpina Pajares
A target binding site containing transcripts of six miR156 precursors were cloned.
Based on these nucleotides-sequences, secondary structures of miRNAs were
predicted by RNA structure tool of CLC Main Benchwork 7.7.1. QIAGEN® program.
Target binding conserved sequences are highlighted.
51
Figure 7. Relative expression levels of miR156 precursors in the primary shoot
apices of Arabis alpina Pajares during 2 to 6-week vegetative growth
Quantitative RT-PCR results show that the transcript levels of miR156a, b, c and d
precursors are declined age-dependently in the primary shoot apices during
vegetative growth.
52
2.4.4. MicroRNA156 expression levels in axillary shoot apices
are variable depending on the developmental stages in Arabis
alpina Pajares
Relative expression levels of miR156 precursors was examined in the axillary
shoot apices, which are in various developmental stages, developed from 8-week-
old Pajares. For this analysis, I collectively harvested the axillary shoot apices in the
same developmental stage (based on the size as shown in Figure 3C ~ G) from all
positions of nodes. The expression levels of pre-miR156a and b were gradually
decreased according to progressive development from 8WS1 to 8WS5 stages. Levels
of pre-miR156c and d in axillary shoots were low and not strictly dependent on the
development (Figure 8). Thus, Pajares shows asynchronous development of axillary
shoots undergoing variable stages from S1 to S5, which is manifested in the levels
of pre-miR156s expression.
53
Figure 8. Expression levels of miR156 precursors in the primary and axillary
shoot apices during vegetative growth of Arabis alpina Pajares
The primary shoot apices of 3-week (3W), 5-week (5W), and 8-week (8W) old
Arabis alpina Pajares were collected for the comparison of expression levels. The
axillary shoot apices in each developmental stage from S1 to S5, labelled as 8WS1
to 8WS5, were collected from 8-week-old plants and the expression levels were
compared. Relative expression levels of primary miR156a, b, c and d were
normalized to PP2A expression.
54
To elucidate whether developmental stage-dependent expressions of miR156 are
completely independent from the physical age of their primary shoot apices, levels
of miR156 precursors were compared among S1 shoot apices harvested from 4, 5, 6,
7, and 8 weeks old plants. The transcript levels of pre-miR156a among S1 shoot
apices were declined according to the age of plants. For instance, pre-miR156a level
in 4WS1, S1 axillary shoot apices from 4-week-old Pajares, was 1.9-fold and 4.6-
fold higher than 5WS1 and 6WS1 respectively (Figure 9). Although the transcript
levels of pre-miR156a were dramatically reduced from 4WS1 to 6WS1, the levels
from 6WS1 to 8WS1 were not reduced further and maintained that quantity, which
was similar level observed in 3 weeks old primary shoot apices. Such quantitative
results of pre-miR156a levels in axillary shoot apices from different ages, the time
from germination, and different developmental stages indicate that the molecular
behavior of axillary shoots is influenced by both their ages when branching started
and their developmental stages when harvested. Recent studies concerning floral
competency of Arabis alpina Pajares reported that all the 5-week-old plants but none
of 3-week-old plants can flower after more than a year of vernalization (Bergonzi et
al., 2013; Wang et al., 2011b). Thus, it is noteworthy that all the S1 axillary shoot
apices from plants with diverse ages show higher levels of pre-miR156a than 5-
week-old primary shoot apices and would not reduce the level below than 3-week-
old primary shoot apices. It indicates that all the S1 axillary shoots are incompetent
to flower regardless of their physical ages after germination.
55
Figure 9. Expression levels of miR156 precursors in the primary and S1 axillary
shoot apices during vegetative growth of Arabis alpina Pajares
Expression levels of pre-miR156a, b, c and d in S1 shoot apices developed from
4-week to 8-week-old plants were compared. Gray bars, miR156 precursor levels in
the primary shoot apices from 3-week to 8-week-old (3W~8W) plants. Black bars,
miR156 precursor levels in the S1 axillary shoot apices from 4-week to 8-week-old
(4WS1~8WS1) plants.
56
2.4.5. After vernalization, some axillary shoot apices
expressing high levels of pre-miR156s in Arabis alpina
maintain vegetative phase
To make it clear whether miR156 expression levels of primary and axillary shoot
apices are related to floral transition, I investigated the transcript levels of pre-
miR156a, b and c before and after vernalization. For vernalization treatment, 8-
week-old vegetative Pajares plants were exposed to 12 weeks of cold (8WV). After
vernalization, each axillary shoot from these 8WV plants was categorized into VS1
to VS5 according to developmental stages (described in Table 3 and Figure 3C ~ G).
To compare the transcript levels of pre-miR156s, I collectively harvested the axillary
shoot apices undergoing the same developmental stage from all node positions. The
VS1 and VS2 shoots, which were mostly generated during vernalization period,
expressed prominently high levels of pre-miR156a and b. Such levels of pre-
miR156a and b were higher than the levels detected from the primary shoot apices
in juvenile phase, i.e, younger than 3 weeks old primary shoots (Figure 10). As
developmental stages were progressed from VS1 to VS5, the transcript levels of pre-
miR156a and b were steadily reduced similar to the pattern observed in the primary
shoots. However, the pre-miR156c level was very low in the axillary shoot apices
after vernalization (Figure 10).
57
Figure 10. Expression levels of miR156 precursors in the primary and axillary
shoot apices of Arabis alpina Pajares after vernalization
Transcript levels of pre-miR156a, b and c were checked in the plants treated with
12 weeks of vernalizartion after 8-week growth in long days. 8WV, plants
vernalized with 12 weeks of cold after 8 weeks of growth in room temperature. VS1
to VS5 indicate S1 to S5 stages of axillary shoot apices from 8WV plants. Primary
shoot apices of 3W, 5W, 7W-old vegetative plants were compared with axillary
shoots from 8-week vernalized plants for comparison of miR156 levels.
58
I also quantified the transcript levels of floral marker LEAFY (LFY) to determine
the meristem identity of these axillary shoots (Figure 11A). In contrast to the pre-
miR156s expressions, LFY was eminently expressed in the VS3 to VS5 axillary shoot
apices but was low in the VS1 and VS2. Consistent with this, the VS3, VS4 and VS5
shoots developed floral meristems together with primary shoots (Figure 11D and E).
Meanwhile, most of the VS2 and all the VS1 apices were in the vegetative phase
even after vernalization (Figure 11B and C). These results indicate that some axillary
shoots, expressing high levels of pre-miR156s, maintain vegetative phase even after
vernalization.
59
Figure 11. Flowering competence of axillary shoot apices after vernalization in
Arabis alpina Pajares depends on developmental stages
(A) Expression levels of AaLFY in the primary and axillary shoot apices undergoing
various developmental stages after vernalization (8WV, vernalization-treated plants
after 8 weeks growth in long days; VS1 to VS5, axillary shoots of S1 to S5 stages
from 8WV plants).
(B) ~ (E) Morphologies of shoot apices in different developmental stages. (B, C)
VS2 axillary shoots show either vegetative (B) or reproductive (C) development. All
of VS3 axillary shoot apices (D) and all the primary shoot apices (E) of 8WV plants
show inflorescence development.
60
2.4.6. Developmental features of winter annual
Arabidopsis thaliana Sy-0
To see if asynchronous expression of miR156 precursors in the axillary shoots of
Arabis alpina Pajares is a unique feature of perennial plant, I compared the
expression with that in a close annual relative, A. thaliana. In Pajares, pre-miR156b,
c, and d showed relatively weak expression compared with pre-miR156a, thus I
focused on pre-miR156a level in the axillary shoot apices of winter annual
Arabidopsis ecotype, Sy-0. In contrast to rapid cycling accessions of Arabidopsis
such as Columbia (Col) and Landsberg erecta (Ler), Sy-0 shows acropetal
development of axillary shoots subtended by cauline leaves. This is quite dissimilar
with Col, Ler, and late-flowering mutants obtained from these genetic background
such that they show basipetal development of axillary shoots after flowering (Grbic'
and Bleecker, 2000; Hempel and Feldman, 1994; Hempel et al., 1998; Shi et al.,
2016). Because Sy-0 shows acropetal development of axillary shoots and produces
aerial rosette leaves after bolting, the axillary shoots from Sy-0 seem to be equivalent
to the axillary shoots developed in Pajares after vernalization.
The axillary shoots of Sy-0 can transit to reproductive phase after producing more
than ~12 aerial rosette leaves. Those shoots containing aerial rosettes of 14-week-
old Sy-0 were classified into 3 categories based on the number of aerial rosette leaves
and the position of axillary branching (the description of each categories are in
Figure 12A and Table 4). The S1 axillary shoots have fewer than 4 aerial rosette
leaves and developed more lately than the other axillary shoots.
61
Figure 12. Expressions of several genes in the axillary shoots of winter annual
Arabidopsis thaliana, Sy-0 were synchronized
(A) A 14-week-old Arabidopsis thaliana Sy-0 grown under long-day condition
developed aerial rosettes in diverse developmental stages in axillary shoots.
Magnified aerial rosettes are shown in box.
(B) Relative expression levels of floral marker genes in the primary shoot apices (FS)
and axillary shoot apices (S1, S2 and S3) from 14-week-old Arabidopsis thaliana
Sy-0.
(C) Relative expression levels of pre-miR156a, SPL3 and SPL9 in the primary shoot
apices (FS) and axillary shoot apices (S1, S2, and S3) from 14-week-old Arabidopsis
thaliana Sy-0.
62
Table 4. Classification of developmental stages of axillary shoots in Arabidopsis
thaliana Sy-0
Developmental stages of axillary shoots from 14 weeks old winter annual, Sy-0
were categorized according to the number of aerial rosette leaves and the position of
branching.
63
2.4.7. MicroRNA156 levels in all the axillary shoot apices of
Arabidopsis thaliana Sy-0 are similar independent of
developmental stages
To check whether all the axillary shoots are at the vegetative phase, expressions
of APETALA1 (AP1) and LFY, two floral meristem identity genes, were analyzed in
S1 ~ S3 shoot apices. Floral organ specific AP1 was expressed only in the flowering
primary shoot apices, but not detected in their axillary shoot apices. On the other
hand, S1 ~ S3 axillary shoot apices expressed LFY higher than the primary shoot
apices. As axillary shoots developed from S1 to S3, LFY expression was decreased.
Since LFY is expressed not only in floral organs but also in leaf primordia (Blázquez
et al., 1997), it is likely that S1 containing more primordia than S2 or S3 exhibits
higher level of LFY (Figure 12B). The transcript level of pre-miR156a was examined
in these vegetative S1 ~ S3 and primary shoot apices from 14-week-old Sy-0. All
stages of axillary shoot apices showed similar levels of pre-miR156a. The other
miR156 precursors were expressed too low to compare their values. The transcript
levels of SPL3 and SPL9, direct target genes of miR156, were also expressed in
similar level in all the axillary shoot apices (Figure 12C). These results indicate that
the level of miR156 expression in axillary shoot apices is irrelevant to the
developmental stages of axillary shoots in the annual Arabidopsis thaliana Sy-0.
Therefore, these results demonstrate that the developmental fate of all the axillary
shoots from the same Sy-0 plant is synchronized at a molecular level.
64
2.4.8. Differential responses to vernalization in Arabis alpina
Pajares and Arabidopsis thaliana Sy-0
In Pajares, 5-week-old plants can transit from vegetative to reproductive phase if
they are sufficiently exposed to vernalization treatment, but 3-week-old plants fail
to progress reproductive phase even though they are exposed to prolonged cold
environment (Wang et al., 2011b). Expression levels of miR156 in juvenile Pajares
plants, younger than 3-week-old, are almost unchanged during vernalization,
whereas the levels drop rapidly after returning to warm environment (Bergonzi et al.,
2013). Consistently, my experiments also showed that all the 5-week-old Pajares
plants perfectly respond to vernalization but all the 3-week-old plants failed to flower
even after 12 weeks vernalization (n=64, respectively). However, in the case of 4-
week-old Pajares, 31.75% of vernalized plants among 63 flowered (Table 5).
65
Table 5. Flowering efficiency in Arabis alpina Pajares after prolonged cold
treatment
Flowering efficiency depending on aging was measured in 3-week, 4-week and 5-
week-old Pajares plants. In the case of 5-week-old Pajares, all 64 plants progressed
into reproductive phase. The 20 plants out of 63 Pajares could make flowers when
4-week-old Pajares exposed to 12 weeks of vernalization. 3-week-old, juvenile
Pajares maintain vegetative phase even after long-term cold treatment.
66
To decide whether juvenility is also an important factor for vernalization response
in winter annuals of Arabidopsis, I checked vernalization effect according to the
expression levels of pre-miR156a and the ages of Sy-0. Transcript levels of pre-
miR156a from the 7-day-old Sy-0 was dramatically reduced to 35.0% after 6 weeks
of vernalization, as opposed to juvenile Pajares which maintains high levels of pre-
miR156a during vernalization (Bergonzi et al., 2013). The expression of pre-
miR156a was increased 7-fold higher after vernalization than before long-term cold.
In the case of 30 days old Sy-0, the transcript levels of pre-miR156a increased to
4.3-fold after vernalization compared with plants before vernalization, instead of
decreasing. Therefore, the transcript levels of pre-miR156a at the end of 6-week
vernalization were considerably different in each age (Figure 13A). I also checked
the transcript levels of LFY for these Sy-0 plants vernalized at different ages. Before
vernalization, the transcript levels of LFY were very low since Sy-0 is a very late-
flowering winter annual. However, after vernalization, LFY expression in Sy-0 was
highly activated and the activation of LFY was stronger in juvenile plants than older
plants (Figure 13B).
67
Figure 13. Vernalization responsive gene expression of Arabidopsis thaliana Sy-
0 according to age
(A) Expression levels of pre-miR156a in the primary shoot apices of 7-, 10-, 20-, and
30-day-old Sy-0 before vernalization (black bars) and after 6 weeks vernalization
(white bars) treatment were examined.
(B) Expression levels of LFY were analyzed before (black bars) and after (white bars)
vernalization in Sy-0. Plants in variable ages from 7 days to 30 days old were
exposed to 6 weeks vernalization.
68
In contrast to Sy-0, AaLFY in juvenile Pajares was not activated by vernalization.
AaLFY expression was highly activated by vernalization only in floral-competent 5-
week-old Pajares; AaLFY expression was increased to about 7-fold at the end of
vernalization comparing to non-vernalized condition (Figure 14). Then,
vernalization response of Sy-0 according to physical ages after germination was
analyzed. Sy-0 showed earlier flowering because of stronger vernalization response,
if exposed to vernalization in younger stage before floral inductive long-day
condition (Figure 15). These results show that Arabidopsis winter annuals do not
have juvenile insensitivity to vernalization, instead juvenile Sy-0 is more sensitive
to vernalization than adult plants. Therefore, developmental maturity is a critical
factor for vernalization-mediated flowering in perennial Pajares whereas winter
annual Sy-0 can respond to vernalization regardless of the ages when vernalized.
69
Figure 14. Vernalization-mediated floral transition in the primary shoot
apices of Pajares at different developmental ages
Comparison of the transcript levels of AaLFY before (white bars) and after (black
bars) 12 weeks of vernalization was performed in Arabis alpina Pajares at different
ages.
70
Figure 15. Vernalization-mediated flowering response according to age after
germination in Arabidopsis thaliana Sy-0
Flowering time was measured by counting the number of rosette leaves produced
when flowering. Plants in variable ages from 2 days to 30 days old were exposed to
6 weeks vernalziation.
71
2.5. DISCUSSION
In terms of longevity, plants can be largely divided into perennial and annual
plants (Amasino, 2009). Most of perennial plants are polycarpic, that is, repetitively
producing flowers every year whereas annual plants are monocarpic, producing
flowers once in a life time. Recent studies using perennial plants Arabis alpina and
Cardamine flexuosa provided important insights of a molecular nature of
perenniality but still we are devoid of complete understanding (Bergonzi et al., 2013;
Zhou et al., 2013). To provide molecular basis of perenniality, I directly compared
the molecular nature of Arabis alpina Pajares and Arabidopsis thaliana Sy-0. In both
Arabidopsis winter annual Sy-0 and perennial Arabis alpina Pajares, axillary shoots
in various developmental stages are produced in the same plant (Figure 4, Figure
12A, Table 3 and 4). However, in Pajares, each axillary shoot expresses differential
levels of pre-miR156s according to its developmental stage and age of a primary
shoot when branching initiated (Figure 8 and 9). Such differential expression of
miR156 precursors depending on the developmental stages of axillary shoot apices
was observed even after primary shoots were in flowering phase (Figure 10). In
contrast to this, pre-miR156a levels in the axillary shoots from the same Sy-0 were
similar irrespective of developmental stages, thus synchronized (Figure 12C). Since
miR156 is a general key player in phase transitions of plants and Pajares
overexpressing MIR156B fails to flower even after long-term cold exposure
(Bergonzi et al., 2013), such asynchronous expression of miR156 in the axillary
shoots of Pajares seems to be the basis of maintenance of vegetative shoots after
72
winter. For example, some axillary shoots of Pajares expressing high pre-miR156s
levels maintain vegetative phase even though main shoots are under flowering phase
(Figure 10 and 11). On the contrary, Sy-0 shows holistic flowering, because it cannot
maintain vegetative growth due to low levels of pre-miR156a (Figure 12B and C).
Therefore, these results clearly demonstrate that variation of miR156 levels in
axillary shoots of Pajares confers polycarpic perenniality but synchronous reduction
of miR156 levels in the axillary shoots of Sy-0 confers monocarpic traits.
The axillary shoots subtended by cauline leaves in Arabidopsis, which is
produced after floral transition, may not be comparable to the axillary shoots
produced in Arabis alpina Pajares. Because the axillary shoots produced in Arabis
alpina Pajares are developed acropetally on average, asynchronous though, whereas
axillary shoots produced after floral transition in rapid cycling accessions of
Arabidopsis are developed basipetally (Grbic' and Bleecker, 2000; Hempel and
Feldman, 1994; Hempel et al., 1998; Shi et al., 2016). In addition, the molecular
basis of axillary shoots developed during vegetative phase, which is produced in the
axils of rosette leaves, are dissimilar with the axillary shoots developed during
reproductive phase, which is produced in the axils of cauline leaves in Arabidopsis
(Wang et al., 2014b). However, there is a contradictory report showing that axillary
shoots produced during reproductive phase in a winter annual Arabidopsis are
developed acropetally (Suh et al., 2003), which is similar to the axillary shoot
development in Arabis alpina Pajares. Sy-0 is another winter annual accession of
Arabidopsis thaliana (Poduska et al., 2003; Wang et al., 2007) and has a unique
73
feature developing aerial rosettes, which are unusual vegetative leaves at the nodes
of elongated stem produced after bolting (Grbic' and Bleecker, 2000; Grbic ́ and
Bleecker, 1996; Schultz and Haughn, 1991; Wang et al., 2007). Furthermore, Sy-0
shows acropetal development of axillary shoots in contrast to rapid cycling
accessions of Arabidopsis or late-flowering mutants derived from such accessions
(Figure 12A). Therefore, the axillary shoots developed in Sy-0 during reproductive
phase are more likely to the axillary shoots produced in Pajares. To confirm such
hypothesis, further molecular analyses will be required using molecular markers
specific for axillary meristem development.
The heteroblasty caused by differential vegetative phase transitions is more
common in perennial plants (Poethig, 1990, 2010). For instance, morphological and
physiological characters, such as plastochron, phyllotaxis, internode length,
thorniness, photosynthetic efficiency, adventitious rooting, disease and insect
resistance, are distinguishable between juvenile and adult phases (Poethig, 1990).
Likewise, Arabis alpina Pajares shows typical heteroblastic characteristics such that
leaves produced at a juvenile phase are relatively small and simple compared to
leaves produced at an adult phase. The basalmost 4 leaves of primary shoots show
such juvenile morphology in addition, the shoot apices developed within about 3
weeks after germination expressed pre-miR156s relatively high levels, thus are
insensitive to vernalization (Bergonzi et al., 2013). Similar with primary shoots,
basalmost 3 leaves of axillary shoots of Pajares are also small and having smooth
margins (Figure 2 and 3). The axillary shoots produced only 3 leaves are categorized
74
as developmental stage 1 (S1). The S1 axillary shoot apices express high levels of
pre-miR156s, thus are unable to respond to vernalization. In contrast, Arabidopsis
Sy-0 does not undergo such juvenile phase incompetent to vernalization response.
Instead, Sy-0 shows higher sensitivity to vernalization at younger stage. Such
difference seems to be due to the differential maintenance of pre-miR156s levels
after vernalization in juvenile stage. In Pajares, the expression levels of pre-miR156s
are maintained during vernalization (Bergonzi et al., 2013), whereas in Arabidopsis
Sy-0, miR156 levels are decreased if vernalized at younger ages but increased if
vernalized at old ages (Figure 13A). Therefore, Arabidopsis winter annuals show
higher sensitivity to vernalization at younger ages (Figure 15). The aim of life in
monocarpic annuals is maximizing the number of offspring by exhausting most of
their resources. On the other hand, a life strategy of polycarpic perennials is
extension of life span as long as possible through multiple times of flowering
(Amasino, 2009). In Arabis alpina Pajares, vernalization-mediated flowering shoots
senesce after reproduction like annual plants (Astrid Wingler, 2011). Therefore,
polycarpic perennials including Pajares require shoots in juvenile phase, insensitive
to floral inductive signals such as vernalization, to maintain vegetative growth,
which allows sustaining perennial traits.
A molecular study in perennial plants is still rare since it has many obstacles, for
instance, long generation time, and difficulties in generating transgenic plants. Thus,
this study to compare the molecular differences in miR156 expressions and
vernalization responses between the perennial Arabis alpina and a close relative
75
annual Arabidopsis thaliana Sy-0 will be useful for future study. The most urgent
question is the molecular mechanism behind the synchronous and asynchronous
expression of miR156 in the axillary shoots in annuals and perennials.
76
2.6. REFERENCES
Albani, M.C., and Coupland, G. (2010). Comparative analysis of flowering in
annual and perennial plants. Curr Top Dev Biol 91, 323-341.
Amasino, R. (2009). Floral induction and monocarpic versus polycarpic life
histories. Genome Biology 10, 1-3.
Ansell, S.W., Stenøien, H.K., Grundmann, M., Russell, S.J., Koch, M.A.,
Schneider, H., and Vogel, J.C. (2011). The importance of Anatolian mountains
as the cradle of global diversity in , a key artic-alpine species. Annals of Botany
108, 241-252.
Astrid Wingler (2011). Interactions between flowering and senescence regulation
and the influence of low temperature in Arabidopsis and crop plants. Ann Appl
Biol 159, 320-338.
Bäurle, I., and Dean, C. (2006). The timing of developmental transitions in plants.
Cell 125, 655-664.
Bastow, R., Mylne, J.S., Lister, C., Lippman, Z., Martienssen, R.A., and Dean,
C. (2004). Vernalization requires epigenetic silencing of FLC by histone
methylation. Nature 427, 164-167.
Bergonzi, S. (2012). The regulation of reproductive competence in the perennial . In
Mathematisch-Naturwissenschaftlichen (Universität zu Köln).
77
Bergonzi, S., and Albani, M.C. (2011). Reproductive competence from an annual
and a perennial perspective. J Exp Bot 62, 4415-4422.
Bergonzi, S., Albani, M.C., Themaat, E.V.L.v., Nordström, K.J.V., Wang, R.,
Schneeberger, K., Moerland, P.D., and Coupland, G. (2013). Mechanisms of
age-dependent response to winter temperature in perennial flowering of . Science
340, 1094-1097.
Blázquez, M.A., Soowal, L.N., Lee, I., and Weigel, D. (1997). LEAFY expression
and flower initiation in Arabidopsis. Development 124, 3835-3844.
Choi, K., Kim, J., Hwang, H.-J., Kim, S., Park, C., Kim, S.Y., and Lee, I. (2011).
The FRIGIDA complex activates transcription of FLC, a strong flowering
repressor in arabidopsis, by recruiting chromatin modification factors. The Plant
Cell 23, 289-303.
Chuck, G., Cigan, A.M., Saeteurn, K., and Hake, S. (2007). The heterochronic
maize mutant Corngrass1 results from overexpression of a tandem microRNA.
Nature Genetics 39, 544-549.
Eaux, S.L., Manel, S., and Melodelima, C. (2014). Development of an Arabis
alpina genomic contig sequence data set and application to single nucleotide
polymorphisms discovery. Mol Ecol Resour 14, 411-418.
Fornara, F., and Coupland, G. (2009). Plant phase transitions make a SPLash. Cell
78
138, 625.
Foster, T., Johnston, R., and Seleznyova, A. (2003). A morphological and
quantitative characterization of early floral development in apple (Malus x
domestica Borkh.) Annals of Botany 92, 199-206.
Grbic', V., and Bleecker, A.B. (2000). Axillary meristem development in
Arabidopsis thaliana. Plant Journal 21, 215-223.
Grbic´, V., and Bleecker, A.B. (1996). An altered body plan is conferred on
Arabidopsis plants carrying dominant alleles of two genes. Development 122,
2395-2403.
Hempel, F.D., and Feldman, L.J. (1994). Bi-directional inflorescence development
in Arabidopsis thaliana: Acropetal initiation of flowers and basipetal initiation of
paraclades. Planta 192, 276-286.
Hempel, F.D., Zambryski, P.C., and Feldman, L.J. (1998). Photoinduction of
flower identity in vegetatively biased primordia. Plant Cell 10, 1663-1675.
Heo, J.B., and Sung, S. (2011). Vernalization-mediated epigenetic silencing by a
long intronic noncoding RNA. Science 331, 76-79.
Hsu, C.-Y., Adams, J.P., Kima, H., No, K., Ma, C., Strauss, S.H., Drnevich, J.,
Vandervelde, L., Ellis, J.D., Rice, B.M., (2011). FLOWERING LOCUS T
duplication coordinates reproductive and vegetative growth in perennial poplar.
79
Proc Natl Acad Sci USA 108, 10756-10761.
Hsu, C.-Y., Liu, Y., Luthe, D.S., and Yuceer, C. (2006). Poplar FT2 Shortens the
Juvenile Phase and Promotes Seasonal Flowering. Plant Cell 18, 1846-1861.
Huijser, P., and Schmid, M. (2011). The control of developmental phase transitions
in plants. Development 138, 4117-4129.
Hyun, Y., Richter, R., Vincent, C., Martinez-Gallegos, R., Porri, A., and
Coupland, G. (2016). Multi-layered regulation of SPL15 and coperation with
SOC1 itegrate edogenous flowering pathways at the Arabidopsis shoot meristem.
Developmental Cell 37, 254-266.
Jensen, C.S., Salchert, K., and Nielsen, K.K. (2001). A TERMINAL FLOWER1-
Like gene from perennial ryegrass involved in floral transition and axillary
meristem identity. Plant Physiol 125, 1517-1528.
Koch, M.A., Haubold, B., and Mitchell-Olds, T. (2000). Comparative evolutionary
analysis of chalcone synthase and alcohol dehydrogenase loci in Arabidopsis,
Arabis, and related genera (Brassicaceae). Mol Biol Evol 17, 1483-1498.
Koch, M.A., Kiefer, C., Ehrich, D., Vogel, J., Brochmann, C., and Mummenhoff,
K. (2006). Three times out of asia minor: the phylogeography of Arabis alpina L.
(Brassicaceae). Molecular Ecology 15, 825-839.
Lin, S.-I., Wang, J.-G., Poon, S.-Y., Su, C.-l., Wang, S.-S., and Chiou, T.-J. (2005).
80
Differential regulation of FLOWERING LOCUS C expression by vernalization in
Cabbage and Arabidopsis. Plant Physiol 137, 1037-1048.
Michaels, S.D., and Amasino, R.M. (1999). FLOWERING LOCUS C Encodes a
Novel MADS Domain Protein That Acts as a Repressor of Flowering. The Plant
Cell 11, 949-956.
Mimida, N., Kotoda, N., Ueda, T., Igarashi, M., Hatsuyama, Y., Iwanami, H.,
Moriya, S., and Abe, K. (2009). Four TFL1 / CEN-Like genes on distinct linkage
groups show different expression patterns to regulate vegetative and reproductive
development in Apple ( Malus×domestica Borkh.). Plant Cell Physiol 50, 394-412.
Morea, E.G.O., Silva, E.M.d., Silva, G.F.F.e., Valente, G.T., Rojas, C.H.B.,
Vincentz, M., and Nogueira, F.T.S. (2016). Functional and evolutionary analyses
of the miR156 and miR529 families in land plants. BMC Plant Biology 16, 153.
Poduska, B., Humphrey, T., Redweik, A., and Grbic´, V. (2003). The synergistic
activation of FLOWERING LOCUS C by FRIGIDA and a new flowering gene
AERIAL ROSETTE 1 underlies a novel morphology in Arabidopsis. Genetics 163,
1457-1465.
Poethig, R.S. (1990). Phase change and the regulation of shoot morphogenesis in
plants. Science 250, 923-930.
Poethig, R.S. (2010). The past, present, and future of vegetative phase change. Plant
81
Physiol 154, 541-544.
Reinhart, B.J., Weinstein, E.G., Rhoades, M.W., Bartel, B., and Bartel, D.P.
(2002). MicroRNAs in plants. Genes & Dev 16, 1616-1626.
Research, Max Plank Istitute for Plant Breeding Research. (2014). Arabis alpina
cultivar:Pajares (gray rockcress). Transcriptional networks and their evolution in
Brassicaciae.
Schultz, E.A., and Haughn, G.W. (1991). LEAFY, a homeotic gene that regulates
inflorescence development in Arabidopsis. Plant Cell 3, 771-781.
Schwab, R., Palatnik, J.F., Riester, M., Schommer, C., Schmid, M., and Weigel,
D. (2005). Specific effects of microRNAs on the plant transcriptome.
Developmental Cell 8, 517-527.
Shi, B., Zhang, C., Tian, C., Wang, J., Wang, Q., Xu, T., Xu, Y., Ohno, C.,
Sablowski, R., Heisler, M.G., (2016). Two-step regulation of a meristematic cell
population acting in shoot branching in Arabidopsis. PLoS Genetics 12, e1006168.
Suh, S.-S., Choi, K.-R., and Lee, I. (2003). Revisiting phase transition during
flowering in Arabidopsis. Plant Cell Physiol 44, 836-843.
Sung, S., and Amasino, R.M. (2004). Vernalization in Arabidopsis thaliana is
mediated by the PHD finger protein VIN3. Nature 427, 159-164.
82
Swiezewski, S., Liu, F., Magusin, A., and Dean, C. (2009). Cold-induced silencing
by long antisense transcripts of an Arabidopsis Polycomb target. Nature 462, 799-
803.
Wang, J.-W., Czech, B., and Weigel, D. (2009a). miR156-regulated SPL
transcription factors define an endogenous flowering pathway in Arabidopsis
thaliana. Cell 138, 738-749.
Wang, J.-W., Park, M.Y., Wang, L.-J., Koo, Y., Chen, X.-Y., Weigel, D., and
Poethig, R.S. (2011a). MiRNA control of vegetative phase change in trees. PLoS
Genetics 7, e1002012.
Wang, J.-W., Schwab, R., Czech, B., Mica, E., and Weigel, D. (2008). Dual effects
of miR156-Targeted SPL Genes and CYP78A5/KLUH on plastochron length and
organ size in Arabidopsis thaliana. Plant Cell 20, 1231.
Wang, Q., Sajja, U., Rosloski, S., Humphrey, T., Kim, M.C., Bomblies, K.,
Weigel, D., and Grbic´, V. (2007). HUA2 caused natural variation in shoot
morphology of A. thaliana. Current Biology 17, 1513-1519.
Wang, R. (2007). Flowering-time control and perennialism in , a perennial relative
of Arabidopsis thaliana. In Mathematisch-Naturwissenschaftlichen (Universität
zu Köln).
Wang, R., Albani, M.C., Vincent, C., Bergonzi, S., Luan, M., Bai, Y., Kiefer, C.,
83
RosaCastillo, and Coupland, G. (2011b). Aa TFL1 confers an age-dependent
response to vernalization in perennial . Plant Cell 23, 1307-1321.
Wang, R., Farrona, S., Vincent, C., Joecker, A., Schoof, H., Turck, F., Alonso-
Blanco, C., Coupland, G., and Albani, M.C. (2009b). PEP1 regulates perennial
flowering in . Nature 459, 423-427.
Wang, Y., Wang, J., Shi, B., Yu, T., Qi, J., Meyerowitz, E.M., and Jiao, Y. (2014).
The stem cell niche in leaf axils is established by auxin and cytokinin in
Arabidopsis. Plant Cell 26, 2055-2067.
Wu, G., Park, M.Y., Conway, S.R., Wang, J.-W., Weigel, D., and Poethig, R.S.
(2009). The sequential action of miR156 and miR172 regulates developmental
timing in Arabidopsis. Cell 138, 750-759.
Wu, G., and Poethig, R.S. (2006). Temporal regulation of shoot development in
Arabidopsis thaliana by miR156 and its target SPL3. Development 133, 3539-
3547.
Xie, K., Wu, C., and Xiong, L. (2006). Genomic organization, differential
expression, and interaction of SQUAMOSA promoter-binding-like transcription
factors and microRNA156 in rice. Plant Physiol 142, 280-293.
Xu, M., Hu, T., Zhao, J., Park, M.-Y., Earley, K.W., Wu, G., Yang, L., and
Poethig, R.S. (2016). Developmental functions of miR156 regulated SQUAMOSA
84
PROMOTER BINDING PROTEIN-LIKE (SPL) Genes in Arabidopsis thaliana.
PLoS Genetics 12, 1-29.
Zhou, C.-M., Zhang, T.-Q., Wang, X., Yu, S., Lian, H., Tang, H., Feng, Z.-Y.,
Zozomova-Lihová, J., and Wang, J.-W. (2013). Molecular basis of age-
dependent vernalization Cardamine flexuosa. Science 340, 1097-1100.
85
CHAPTER III.
Transcriptome analysis of Arabis alpina Pajares
to find regulators for
initiating vegetative axillary meristems
86
3.1. ABSTRACT
Transcriptome analysis of Arabis alpina Pajares to find
regulators for initiating vegetative axillary meristems
Jong-Yoon Park
School of Biological Sciences
The Graduate School
Seoul National University
Generally, polycarpic perenniality can be characterized in two ways. First,
polycarpic perennials maintain at least one SAM in vegetative state after flowering.
Second, they establish new vegetative lateral shoots during reproductive phase.
Branching is one of the distinctive features of plants that allows to initiate new organs
during postembryonic development. In addition, it contributes to the yield and
quality of crop species. By monitoring the developmental patterns during and after
vernalization, it was noted in this study that activation of axillary buds was rapid
when Pajares returned to warm temperature after vernalization. During last decades,
several transcription factors have been identified as pivotal players for initiation and
activation of axillary meristems in Arabidopsis thaliana, such as SHOOT
MERISTEMLESS (STM), LATERAL SUPPRESSOR (LAS), REGULATOR OF
87
AXILLARY MERISTEMS1-3 (RAX1-RAX3), and REGULATOR OF AXILLARY
MERISTEM FORMATION (ROX). In addition, three classes of plant hormones,
auxin, cytokinin and strigolactones are known to act as key regulators to control
activation of axillary meristems. Auxin and strigolactones inhibit outgrowth of
axillary meristems, whereas cytokinin promotes activation of buds. To search for
regulators of vegetative branching during reproductive phase in Arabis alpina
Pajares, transcriptome analysis was performed using primary stem segments under
vegetative and reproductive phases. The result showed that 105 transcription factors
were elevated during reproductive phase. RAX2, a member of R2R3 MYB
transcription factor, was identified as one of these up-regulated transcription factors.
The expression of other RAX family genes, RAX1 and RAX3, was also slightly
increased in reproductive stems. Fifty-eight of these transcription factors turned out
to be the ones related with various phytohormone metabolisms. Finally, the study
also sorted out seven transcription factors whose expression was influenced by auxin
or cytokinin, and specifically expressed in shoot apical meristems.
Keywords : annuality, perenniality, vegetative phase, reproductive phase,
axillary meristem, branching, transcription factors,
phytohormone
Student Number : 2010 - 30933
88
3.2. INTRODUCTION
One of the developmental features distinguishing plants from animals is that
animal body plans are established during embryogenesis, while plants are capable of
initiating new organs during postembryonic development (Grbic´ and Bleecker, 1996;
Wang et al., 2014b). The architecture of plant body is basically established during
embryogenesis too. During embryogenesis, an apical-basal axis develops forming a
root apical meristem at one end and a shoot apical meristem (SAM) at the other.
During postembryonic development after this bipolar organization, each phytomer,
a functional unit of a plant produced by meristems, consists of a node containing
more than an axillary meristem at each leaf axil in the shoot system. One of the
critical events during postembryonic development is the formation of secondary axes
of growth, such as vegetative shoots, inflorescence shoots, or flowers (McSteen and
Leyser, 2005; Schmitz and Theres, 2005). Branching, a secondary growth axes,
initiates from buds subtending leaf axils. Formation of these buds requires the
establishment of axillary meristems (Wang et al., 2014b).
There are two hypotheses explaining the ontogeny of the axillary meristems
(AMs). One is that AMs initiates de novo in leaf axils. It suggests that the original
cells of AMs in the boundary zone acquire meristematic activity. The other is
‘detached meristem’ hypothesis. It explains that the cells of AMs are derived and
detached from the cells of SAM during the initiation of leaf primordium (Guo et al.,
2015; McSteen and Leyser, 2005; Wang and Li, 2008).
89
3.2.1. Key regulators in initiation and activation of axillary
meristems in Arabidopsis
The expression of SHOOT MERISTEMLESS (STM) is required for SAM initiation
and maintenance. STM encodes a class I KNOTTED-like homeodomain protein. The
transcriptional expression of STM is detected in all types of SAMs during whole life
span. Therefore, STM is used as popular marker of SAM fate (Long and Barton, 2000;
Shi et al., 2016).
Maintenance of meristem formation competence and the subsequent initiation of
AMs are regulated by various transcription factors. For instance, REVOLUTA (REV)
is a member of the Arabidopsis HD-Zip III, REGULATOR OF AXILLARY
MERISTEMS1-3 (RAX1-RAX3) and LATERAL SUPPRESSOR (LAS) belongs to the
R2R3-type MYB transcription factors, REGULATOR OF AXILLARY MERISTEM
FORMATION (ROX) is a family member of bHLH transcription factors, CUP-
SHAPED COTYLEDON1-3 (CUC1-CUC3) encodes NAC domain transcription
factors. Many of these genes have conserved function in the regulation of AM
initiation in dicots and monocots, such as tomato (Solanum lycopersicum), maize
(Zea mays), and rice (Oryza sativa) (Keller et al., 2006; ller et al., 2006; Raman et
al., 2008; Schmitz and Theres, 2005; Shi et al., 2016; Wang and Li, 2008; Yang et
al., 2012).
The RAX genes were defined as orthologs of tomato (Solanum lycopersicum)
Blind gene. Results from several gain/loss-of-function mutant analyses of RAXs
90
show that they have redundant roles in initiation and activation of axillary meristems
together with LAS during early step of AMs establishment (Keller et al., 2006; ller
et al., 2006). ROX is also functionally redundant with RAX1 and LAS for regulating
AMs formation, and its transcription is positively regulated by RAX1 and LAS (Yang
et al., 2012). More recently, EXCESSIVE BRANCHES1 (EXB1/WRKY71) encoding
a WRKY transcription factor has been found to control AM initiation by positively
regulating the RAXs’ expression and auxin homeostasis (Guo et al., 2015).
3.2.2. Hormonal regulation in initiation and activation of
axillary meristems in Arabidopsis
Three different plant hormones, auxin, cytokinin and strigolactones or their
derivatives play key roles in the control of bud activation. Auxin has a broad effect
on plant development, particularly on AM initiation and development including
leaves, branches, and flowers (Domagalska and Leyser, 2011; Wang and Li, 2008) .
It is required for AM formation, and reduced auxin response in the axils delays
terminal differentiation of those axils (McSteen and Leyser, 2005). Polar auxin
transport (PAT) involves influx and efflux carriers. PIN1, an auxin efflux carrier and
one of the eight members of the PIN protein family, is well characterized for its role
in mediating auxin distribution and AM initiation (Wang and Li, 2008). In many
plant species, growth of axillary buds is inhibited by primary shoot apex and remains
dormant until later time to resume growth according to their developmental program
91
or in response to environmental cues (Shimizu-Sato and Mori, 2001). In this process,
known as apical dominance, auxin derived from the primary shoot apex is a main
repressor while cytokinin derived from the roots is considered as a main activator of
lateral bud development (Schmitz and Theres, 2005; Wang and Li, 2008).
There are two hypothetical models explaining the regulation of auxin-mediated
axillary branching; auxin transport canalization-based model, and the second
messenger hypothesis, respectively. According to the canalization-based hypothesis,
the PAT stream following the primary shoot can inhibit bud outgrowth by regulating
the ability of axillary buds. The second messenger model states that auxin acts by
influencing the levels of mobile signals entering the bud and directly regulate its
meristematic activity. Therefore, both hypotheses provide explanations for inhibited
bud outgrowth by apical dominance (Borghi et al., 2016; Domagalska and Leyser,
2011).
Cytokinin is mostly synthesized in the root and transported acropetally. It acts as
an activator of buds outgrowth. Auxin suppresses cytokinin synthesis by regulating
the expression of isopentenyl transferase (IPT) gene encoding a rate limiting enzyme
for cytokinin biosynthesis (Chatfield et al., 2000; Domagalska and Leyser, 2011).
Strigolactones, a group of newly identified phoytohormones, act together with auxin
to inhibit AM outgrowth. Strigolactones are synthesized in both the roots and the
shoots and are transported acropetally to repress bud activity. Strigolactones reduce
auxin transport while auxin promotes the expression of strigolactone biosynthetic
genes, thus production of strigolactones is controlled by an indirect feedback
92
inhibition through modulation of auxin flow (Borghi et al., 2016; Wang et al., 2014b).
Besides auxin, cytokinin, and strigolactones, ABA was also suggested to act as an
inhibitor for axillary bud growth, although no concrete evidence supporting this
proposal has been reported (Chatfield et al., 2000; Wang and Li, 2008).
Recent studies revealed that PIN1 proteins lead to the accumulation of auxin to
the maximum level where a new leaf primordium to be formed. After the leaf
primordium bulges out, PIN1s are translocated toward the tip of the leaf primordium.
As a consequence, auxin is transported out of the axil region resulting in an auxin
minimum with a subsequent cytokinin signaling to follow in the leaf axil after leaf
elongation. Finally, an AM is established in the site of low auxin level (Wang et al.,
2014a; Wang et al., 2014b). In addition, low levels of STM sustain meristematic
competence while high levels of STM establish meristem initiation. The maintenance
of STM expression depends on the auxin minimum at the leaf axils. Low level of
STM expression is required but not sufficient for AM initiation, and subsequent
elevated expression of STM induces axillary bud formation. The initial expression
STM requires the auxin minimum in the leaf axil and the transcription factor REV
directly up-regulates STM expression (Shi et al., 2016).
93
3.3. MATERIALS AND METHODS
3.3.1. Plant materials and growth condition
Arabis alpina Pajares seeds were surface sterilized in 75% ethanol and 0.05%
tween-20 solution. After sterilization, the seeds were sown on one-half-strength MS
medium supplemented with 1% (w/v) sucrose and 1% (w/v) plant agar. The Pajares
seeds were stratified under dark at 4℃ for 10 days to two weeks. After stratification,
the seeds were germinated on MS medium, then 2 weeks old seedlings were
transplanted to soil under controlled condition of 16-hour light and 8-hour dark at
22℃. For long-term cold treatment, vernalization, plants were transferred to
vernalization chamber (8-hour light and 16-hour dark at 4℃) for 12 weeks. To
analyze growth pattern in reproductive phase, vernalization treated Pajares returns
to normal condition (16-hour light and 8-hour dark at 22℃) for flowering.
3.3.2. Transcriptome analysis
Vegetative and reproductive primary stem segments were used in this experiment.
For a control sample, leaves, lateral branches, roots and shoot apices were eliminated
from vegetative primary stem which grown under normal condition (16-hour light
and 8-hour dark at 22℃) for 8 weeks. For an experimental sample, 8 weeks old
Pajares plants exposed to vernalization (8-hour light and 16-hour dark at 4℃) for 12
94
weeks. At the end of additional 10 days growth under normal condition, primary
stem segments were harvested as same way as the control sample. Total RNA was
extracted from vegetative and reproductive stem segments by using RNeasy® plant
mini kit (QIAGEN 74904). Contaminated genomic DNA was eliminated with
recombinant DNaseI (Takara 2270A). Transcriptome analysis was performed at
Beijing Genomics Institute (BGI)-HONG KONG CO., LIMITED by using Illuimina
HiSeqTM 2000 platform.
95
3.4. RESULTS
3.4.1. Distinctive morphological traits of Arabis alpina Pajares
during and after vernalization
Pajres seeds were sown to MS-agar medium, or they were germinated and
cultivated directly in soil. To increase germination efficiency, Pajares seeds require
cold treatment for about 2 weeks for stratification. Pajares seedlings are grown on
soil more than 5 weeks to subject to long-term cold treatment, vernalization. Notable
features observed during the vegetative phase were elongation of internodes and
outgrowing of axillary buds. In contrast to a close relative, Arabidopsis thaliana,
which develops only basal rosettes before the onset of reproductive phase, primary
stems of Pajares were elongated starting from early developmental stage (Figure 1A
and B). In Arabidopsis thaliana, most axillary buds are dormant during vegetative
state (Shimizu-Sato and Mori, 2001). On the other hand, axillary buds became visible
in a little after 3 weeks old Pajares and steadily protruded. Pajares needs exposure to
vernalization environment at least for 10 weeks. During vernalization, Pajares show
growth retardation. During vegetative growth under warm temperature, average
length of the primary shoots of Pajares increased dramatically from 0.63±0.29
centimeters to 12.68±0.98 centimeters during 5 weeks of growth. However, the
increase in the primary shoots length was less than 2 centimeters when 3 weeks old
Pajares were exposed to long-term cold condition for 12 weeks. In the case of rate
of true leaves development, more than 13 leaves were newly produced from 3 weeks
96
to 8 weeks old vegetative Pajares while about 5 leaves were newly developed under
12 weeks of cold condition. Interestingly, new leaves grew up explosively only for
10 days under warm circumstance after cold (Figure 16).
97
Figure 16. Developmental patterns of primary stems of Pajares under
continuous vegetative and cold temperature
The elongation of primary shoot (black/blue triangle) was measured weekly
starting from 3 weeks after germination under continuous warm condition with long-
day (black triangle) or cold temperature with short-day for 12 weeks (blue triangle).
Leaf production was also analyzed under continuous warm, long-day (black circle)
and 12 weeks of cold, short-day (blue circle).
98
Another distinct morphological feature of Pajares was development of adventitious
roots during vernalization. When Pajares were exposed to long-term cold condition,
short roots occasionally developed at stems above the ground (Figure 17). Intrigued
to know whether these short adventitious roots can uptake water and nutrients, the
survivability of stem segments with or without these adventitious roots was
examined. The two types of stem segments were excised then re-planted on soil.
About 3 weeks after transplanting, floral organs were only detected at a shoot apex
containing the adventitious roots (Figure 18A). In addition, short adventitious roots
were actively growing in these shoots (Figure 18B).
99
Figure 17. Establish adventitious roots at nodes especially during vernalization
treatment
(A) During prolonged cold treatment, new vegetative shoots were formed at nodes
of a primary shoot (arrows).
(B) Adventitious small and short roots were detected the other side of the shoot in
panel (A).
(C) Enlarged adventitious roots (arrows in (A)).
100
Figure 18. After 3 weeks growth of excised stem segments with or without
adventitious roots
(A) Stem segments with/without adventitious roots were excised then transplanted
to soil to test their survivability. After 3 weeks of growth, the shoots with
adventitious roots made floral organs.
(B) During 3 weeks of growth, the shoot with adventitious roots had actively
developed root system. However, the other shoot without adventitious roots had
withered.
101
At the end of long-term cold treatment, newly produced axillary buds were visible
at most of the lignified stem nodes in Pajares. These axillary buds actively developed
leaves when they returned to warm ambient temperature. Distinctive feature of their
development was reduction in shoot length in comparison with that of vegetative
axillary shoots developed before vernalization (Figure 19).
102
Figure 19. Outgrowth of axillary buds when they returned to warm
temperature following vernalization
Growing pattern of axillary buds were observed for 10 weeks after returning to
warm condition. A bulging, subtending leaf actively developed for subsequent life
cycle.
103
It was of interest to know the energy and nutrient resources for perennial life
strategy. Therefore, to define requisite organs for surviving through the next life
cycles, various parts of shoots were eliminated after flowering. The first group of
plants had only basal primary stems without any axillary shoots and leaves. In the
second type of plants, enlarged mature leaves and elongated shoots as well as
inflorescences were removed except young vegetative axillary shoots. The third type
had primary stem without inflorescences but contained vegetative axillary buds and
shoots. The fourth group of plants contained inflorescence with only young
vegetative shoots (Figure 20). Axillary shoots development was active in type III and
IV which contained enlarged leaves developed in inflorescence or developing shoots.
These results indicate that active photosynthetic organs might be important to
generate new vegetative shoots for next year (Figure 21).
104
Figure 20. A schematic diagram of an experiment for searching energy and
nutrient resources in Pajares
Several types of shoot systems were designed as type I to type IV. Type I plants
did not have any lateral organs such as inflorescences, leaves, and even visible
axillary buds. Type II plants contained visible axillary buds but no elongated axillary
shoots and any subtending leaves. Type III plants had compromised vegetative
axillary shoots except subtending leaves. Type IV had inflorescences that would
senesce soon leaving only small axillary buds like type II.
105
Figure 21. Survivability and development of shoots from type I to type IV
A shoot system in type I (A) did not produce any lateral organs after 4 weeks of
cultivation (B). The axillary buds formed nodes of a primary stem in type II (C)
withered after 4 weeks (D). Type III shoot system before (E) and after (F) 4 weeks of
growth. Axillary shoots in type III actively developed (F). Axillary buds in type IV
enlarged and elongated after 4 weeks of growth (H) compared with previous stage (G).
106
3.4.2. Transcriptome analysis of primary stems in both
vegetative and reproductive phases
In most perennial strategies including Pajares, new vegetative axillary meristems
are initiated during or after reproductive phase. The mechanisms for initiation and
elongation of axillary shoots have been studied lately. There are several transcription
factors, such as GRAS-, MYB-, bHLH-type, that have pivotal roles in early steps of
this process (Keller et al., 2006; ller et al., 2006; Raman et al., 2008; Schmitz and
Theres, 2005; Yang et al., 2012). On the other hand, phytohoromone-mediated
regulation also plays key roles in axillary bud initiation and activation. Auxin
minimum at leaf axil is required to initiate axillary bud formation. Both perception
and signaling of cytokinin are also required for establishment of axillary meristems.
Auxin inhibits axillary meristem outgrowth, but cytokinin promotes bud activation.
In addition, strigolactones or their derivatives act together with auxin to suppress
outgrowing of axillary meristems (Borghi et al., 2016; Chatfield et al., 2000;
Domagalska and Leyser, 2011; Grbic' and Bleecker, 2000; Long and Barton, 2000;
McSteen and Leyser, 2005; Schmitz and Theres, 2005; Shi et al., 2016; Wang et al.,
2014a; Wang et al., 2014b).
Transcriptome analysis was performed by using Illumina HiSeq2000 to identify
central players for initiation and outgrowth of axillary buds during reproductive
phase in Arabis alpina Pajares. To this end, vegetative and reproductive primary
stems containing leaf axils without leaf blades were collected. For vegetative
primary stems, primary stems of 8-week-old Pajares grown under long-day, warm
107
condition were prepared and were eliminated of shoot apices, lateral organs
including leaves and axillary branches leaving only leaf axils from which axillary
buds to be generated. For reproductive primary stems, 8-week-old Pajares were
exposed to 12 weeks of vernalization followed by additional 10 days of growth under
long-day, warm condition. To monitor inductive signals for initiation of axillary
meristems and minimize other factors affected by cold exposure, Pajares were
returned to normal growth condition after vernalization. During 10 days of recovery
condition, the expression of PEP1, an ortholog of floral repressor of Arabidopsis
FLC, was found to be de-repressed to the level of pre-vernalization. Therefore, it
could be assumed that these reproductive primary stems were harvested and treated
the same way as that of vegetative primary stems.
According to transcriptome analysis results, sequence information of whole
transcriptome of Pajares showed 47.9% and 43.2% similarity with Arabidopsis
lyrata and Arabidopsis thaliana, respectively. Overall, 1018 genes (2.3% of total
number of obtained unigenes) were up-regulated and 697 genes (1.57% of total
number of obtained unigenes) were down-regulated (Figure 22).
108
Figure 22. Overview of transcriptome analysis of reproductive stems in Pajares
(A) Comparative analysis of sequence similarities with other plant species.
(B) The number of unigenes fluctuated in reproductive stems in comparison with
vegetative stems.
109
As several transcription factors act as key regulators of the initiation of axillary
meristems, special attention was made out to the transcription factors whose
expression fluctuated more than two folds in reproductive stems. Among those, 105
transcription factors showed more than two-fold increase in expression, whereas 27
transcription factors showed reduction in expression to less than one-half level in
reproductive stems. Phytohormones have central roles for lateral organ development.
Especially, three classes of hormones auxin, cytokinin and strigolactones have been
defined as central players for axillary bud initiation and activation (Domagalska and
Leyser, 2011; Wang and Li, 2008). In effect, the Genes Ontology data of the
transcriptome analysis revealed that many of these genes are likely to be involved in
a number of different processes of phytohormone-related metabolisms (Table 6A).
Phytohormone abscisic acid (ABA), auxin, ethylene, jasmonic acid (JA), and
salicylic acid (SA) - related genes showed differential expression patterns before and
after vernalization. Although cytokinin perception and signaling are both required
for axillary meristem initiation, differential expression of cytokinin-related genes
was not significant in this expression (Table 6B).
110
Table 6. Genes Ontology data related to hormones
(A) Overview of gene ontology that are related to hormone metabolisms and
functions. Overall, 1269 genes are categorized as process ontology.
(B) GO data show that the frequency of unigene clusters which are related with
specific hormones.
111
Since there is no microarray data of Arabis alpina available, expression profile for
the transcription factor genes of Arabis alpina corresponding to those selected from
transcriptome analysis of Pajares was tested by using AtGenExpress Visualization
Tool (Kilian et al., 2007) (http://jsp.weigelworld.org/expviz/expviz.jsp) to search for
plant hormone-related transcription factors in this species. Among 105 up-regulated
genes for the transcription factors in vernalized primary stems of Pajares, orthologs
of 58 genes were confirmed to be influenced by exogenous treatment of several
hormones in Arabidopsis thaliana (Table 7).
112
Table 7. Up-regulated transcription factors that related phytohormones
In transcriptome ananlysis results, 105 transcription factors were increased more
than two folds in reproductive stems (REP) in comparison with vegetative stems
(VEG) of Pajares. Among them, the expression of 58 transcription factors was
influenced by phytohormones (http://jsp.weigelworld.org/expviz/expviz.jsp). The
expression was caculated by FPLK (Fragments Per Killobase Million fragments).
113
Of these hormone-related transcription factors, REGULATOR OF AXILLARY
MERISTEMS 2 (RAX2/MYB38) showed about two folds of elevated expression in
reproductive stems (Table 7). Expressions of RAX1 and RAX3, other members of the
RAX family, were also slightly increased in vernalization treated stems of Pajares
(Table 8). However, other well-defined regulators for axillary meristem initiation,
such as LAS, CUCs, ROX and meristematic marker STM, were not detected in this
result.
Upon this finding, the sequence similarity for RAX genes was first compared
between Arabidopsis thaliana and Arabis alpina Pajares. The genomic DNA and
mRNA sequences of RAX2 gene were 82% and 88% identical, respectively. The
sequence of amino-acids encoded by AaRAX2 was 83% identical to AtRAX2 protein
(Figure 23B). Sequence similarities of the other AaRAXs to AtRAX were also more
than 80% at the amino-acids level (Figure 23A and C).
114
Table 8. Relative expression of AtRAXs homologs in Pajares
AaRAX1, AaRAX2, and AaRAX3 were slightly increased in reproductive primary
stems relative to vegetative primary stems of Pajares in the transcriptome analysis
result.
115
Figure 23. Comparison of amino-acids sequence similarity between AaRAXs
and AtRAXs
Putative amino-acids sequences of AtRAXs homologs of Pajares were 85%, 83%,
and 83% similar with AtRAX1, AtRAX2, and AtRAX3 respectively.
116
To examine whether these genes were differentially expressed in Pajares at
different developmental stage of axillary buds, primary stem-nodes with leaf axils
were harvested and collected according to the size of axillary buds and by the timing
of the harvest. The leaf axils in the first group were barren axils that do not have any
initiation of axillary meristems. The second group with axillary buds contained
outward bulges on the meristems, and the third group had outgrowing lateral
branches with more than 6 leaves developed (Figure 24A, B and C). In addition, the
node samples from the three groups were harvested at specific developmental phases.
The plants at mature vegetative (8W, vegetative 8 weeks growth ), vernalization
(7WV, 12 weeks of vernalization following 7 weeks of vegetative growth), and
reproductive phase that were recovered into warm temperature after vernalization
(7WVRT, 10 days growth under warm temperature after 7WV) were used to verify
the activity of these genes in axillary meristem development. AaRAX1 was expressed
in actively growing axillary buds (group II) especially after vernalization. The
expression of AaRAX2 was elevated at leaf axils in group II and group III during
vegetative growth. AaRAX3 was not significantly influenced by activity of axillary
meristems (Figure 24D). These results suggest that AaRAXs might have roles in
initiation and activation of axillary meristems in Arabis alpina Pajares, but specific
functions could be different from those in Arabidopsis thaliana. For examples,
AaRAX2 was mainly expressed in leaf axils with developing buds under vegetative
phase but not in reproductive phase, while AtRAX2 is active in both late vegetative
and reproductive phase (Keller et al., 2006; ller et al., 2006).
117
Figure 24. Expression analysis of AaRAXs at various stages of leaf axils
according to size of axillary buds and developmental phases
(A ~ C) The leaf axils were harvested based on the size of axillary buds such as
barren axils (A, group I), axils with outward bulges (B, group II) and axils with
elongated axillary shoots (C, group III). White boxes indicate regions that were
excised for the expression analysis.
(D) Relative expression level of AaRAXs in group I ~ III axils harvested at different
developmental phases (8W, vegetative 8 weeks; 7WV, vernalized following 7 weeks
growth; 7WVRT, 10 days after 7WV).
118
Of particular interest in this study was to find transcription factors controlling the
initiation or activation of axillary meristems in response to phytohormones since
auxin, cytokinin, and strigolactones have been implicated as important regulators for
lateral organ formation. Among the transcription factor genes up-regulated in the
reproductive stems of Pajares, candidates that are related with the auxin, cytokinin
or strigolactones signaling or metabolism were searched for. Several genes that are
associated with auxin and/or cytokinin related processes were found, but no such
genes with strigolactones-related functions were identified. In addition, genes
encoding the transcription factors that are actively expressed in meristematic tissues
were also examined using the developmental map available on the electronic
fluorescent pictograph (eFP) browser (Winter1 et al., 2007)
(http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi). Based upon these results, seven
transcription factors that exhibit SAM-specific expression and auxin or cytokinin-
related functions were selected as candidates for probable regulators of axillary
meristem formation. In particular, the expression of ARABIDOPSIS THALIANA
HOMEOBOX2 (ATHB2), one of the Homeodomain-Leucine zipper (HD-Zip) class
II members, was found to be influenced by both auxin and cytokinin. Moreover,
ATHB2 was previously reported as functionally redundant with HAT3 and ATHB4 in
establishing bilateral symmetry and in controlling SAM activity (Turchi et al., 2013).
Thus, ATHB2 is likely to have a function in modulating development of new axillary
meristems in perennial Pajares (Table 9).
119
Table 9. Up-regulated transcription factors in reproductive stems of Pajares
that related with auxin or cytokinin, and expressed in meristematic region
Among the 58 up-regulated transcription factor genes in Table 7, seven
transcription factors showed their gene expression were affected by auxin or
cytokinin. In addition, their expressions were meristem-specific according to the
electronic fluorescent pictograph (eFP) microarray data
(http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi). (REP, primary stems in reproductive
phase; VEG, primary stems in vegetative phase; FPKM, Fragments Per Killobase
Million fragments)
120
3.5. DISCUSSION
In perennial plants, at least one of the shoot apical meristem should remain
indeterminate even under environment for floral induction (Thomas et al., 2000). In
addition, both initiation and activation of axillary buds are also critical for ensuring
growth in the following year (Munne´-Bosch, 2008). Several transcription factors
and plant hormones are involved in these processes (Borghi et al., 2016; Domagalska
and Leyser, 2011; Keller et al., 2006; ller et al., 2006; McSteen and Leyser, 2005;
Raman et al., 2008; Schmitz and Theres, 2005; Shi et al., 2016; Yang et al., 2012).
To obtain a better insight into the mechanisms by which molecular regulators
mediate the initiation and activation of axillary buds, transcriptome analyses were
performed on tissue samples derived from vegetative and reproductive stems of
Arabis alpina Pajares. At the time of the tissue sampling, outward bulges were
clearly noticeable at stem-nodes, axils of subtending leaves, especially under
condition for floral induction following vernalization (Figure 19). For the
reproductive stem segments, Pajares plants made inductive to flowering by
cultivating under warm temperature for 10 days after 12 weeks of vernalization were
used with with vegetative Pajares as a control sample. The result revealed that 105
transcription factor genes were up-regulated in reproductive stems compared with
vegetative stems. The expression of an ortholog for the Arabidopsis AtRAX2, a
member of R2R3 MYB family and a positive regulator for initiation of axillary buds,
showed almost a 2-fold increase in reproductive Pajares’ stems (Table 7). In fact, in
support of their proposed roles, AaRAX1 and AaRAX2 were found to be actively
121
expressed in the axils with axillary buds (Figure 24). Three different phytohormone,
auxin, cytokinin and strigolactones have been implicated as important players in the
initiation and activation of axillary buds (Borghi et al., 2016; Chatfield et al., 2000;
McSteen and Leyser, 2005; Wang et al., 2014a; Wang and Li, 2008; Wang et al.,
2014b). Reflecting this feature, seven transcription factor genes, IDD14, CRF6, AIL6,
CRF11, ARF18, ATHB2, and RD26 were found to be up-regulated under floral
inductive condition in this study (Figure 9). Microarray data indicate that all of these
transcription factors are commonly expressed in the SAM and their expressions are
influenced by auxin or cytokinin (http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi;
http://jsp.weigelworld.org/expviz/expviz.jsp). Especially, ATHB2 is a well-known
factor required for meristem maintenance and its expression is regulated by both
auxin and cytokinin (Turchi et al., 2013). Therefore, these seven transcription factors
might be the molecular switches involved in the regulation of branching patterning
in perennial Arabis alpina Pajares.
122
3.6. REFERENCES
Borghi, L., Martinoia, E., Liu, G.-W., Emonet, A.l., and Kretzschmar, T. (2016).
The importance of strigolactone transport regulation for symbiotic signaling and
shoot branching. Planta 243, 1351-1360.
Chatfield, S.P., Stirnberg, P., Forde, B.G., and Leyser, O. (2000). The hormonal
regulation of axillary bud growth in Arabidopsis. Plant Journal 24, 159-169.
Domagalska, M.A., and Leyser, O. (2011). Signal integration in the control of shoot
branching. Nat Rev Mol Cell Biol 12, 211-221.
Grbic', V., and Bleecker, A.B. (2000). Axillary meristem development in
Arabidopsis thaliana. Plant Journal 21, 215-223.
Grbic´, V., and Bleecker, A.B. (1996). An altered body plan is conferred on
Arabidopsis plants carrying dominant alleles of two genes. Development 122,
2395-2403.
Guo, D., Zhang, J., Wang, X., Han, X., Wei, B., Wang, J., Li, B., Yu, H., Qingpei
Huang, a.H.G., Qu, L.-J., et al. (2015). The WRKY transcription factor
WRKY71/EXB1 controls shoot branching by transcriptionally regulating RAX
genes in Arabidopsis. Plant Cell 27, 3112-3127.
Keller, T., Abbott, J., Moritz, T., and Doerner, P. (2006). Arabidopsis
REGULATOR OF AXILLARY MERISTEMS1 controls a leaf axil stem cell niche
123
and modulates vegetative development. Plant Cell 18, 598-611.
Kilian, J., Whitehead, D., Horak, J., Wanke, D., Weinl, S., Batistic, O., D’Angelo,
C., Bornberg-Bauer, E., Kudla, J.r., and Harter, K. (2007). The AtGenExpress
global stress expression data set: protocols, evaluation and model data analysis
of UV-B light, drought and cold stress responses. Plant Journal 50, 347-363.
ller, D.r.M., Schmitz, G., and Theres, K. (2006). Blind homologous R2R3 Myb
genes control the pattern of lateral meristem Initiation in Arabidopsis. Plant Cell
18, 586-597.
Long, J., and Barton, M.K. (2000). Initiation of axillary and floral meristems in
Arabidopsis. Developmental Biology 218, 341-353.
McSteen, P., and Leyser, O. (2005). Shoot branching. Annu Rev Plant Biol 56, 353-
374.
Munne´-Bosch, S. (2008). Do perennials really senesce? TRENDS in Plant Science
13, 216-220.
Raman, S., Greb, T., Peaucelle, A., Blein, T., Laufs, P., and Theres, K. (2008).
Interplay of miR164, CUP-SHAPED COTYLEDON genes and LATERAL
SUPPRESSOR controls axillary meristem formation in Arabidopsis thaliana.
Plant Journal 55, 65-76.
Schmitz, G., and Theres, K. (2005). Shoot and inflorescence branching. Curr Opin
124
Plant Biol 8, 506-511.
Shi, B., Zhang, C., Tian, C., Wang, J., Wang, Q., Xu, T., Xu, Y., Ohno, C.,
Sablowski, R., Heisler, M.G., et al. (2016). Two-step regulation of a
meristematic cell population acting in shoot branching in Arabidopsis. PLoS
Genetics 12, e1006168.
Shimizu-Sato, S., and Mori, H. (2001). Control of outgrowth and dormancy in
axillary buds. Plant Physiol 127, 1405-1413.
Thomas, H., Thomas, H.M., and Ougham, H. (2000). Annuality, perenniality and
cell death. Journal of Experimental Botany 51, 1781-1788.
Turchi, L., Carabelli, M., Ruzza, V., Possenti, M., Sassi, M., Peñalosa, A., Sessa,
G., Salvi, S., Forte, V., Morelli, G., et al. (2013). Arabidopsis HD-Zip II
transcription factors control apical embryo development and meristem function.
Development 140, 2118-2129.
Wang, Q., Kohlen, W., Rossmann, S., Vernoux, T., and Theres, K. (2014a). Auxin
depletion from the leaf axil conditions competence for axillary meristem
formation in Arabidopsis and Tomato. Plant Cell 26, 2068-2079.
Wang, Y., and Li, J. (2008). Molecular basis of plant architecture. Annu Rev Plant
Biol 59, 253-279.
Wang, Y., Wang, J., Shi, B., Yu, T., Qi, J., Meyerowitz, E.M., and Jiao, Y. (2014b).
125
The stem cell niche in leaf axils is established by auxin and cytokinin in
Arabidopsis. Plant Cell 26, 2055-2067.
Winter1, D., Vinegar, B., Nahal, H., Ammar, R., Wilson, G.V., and Provart, N.J.
(2007). An ‘‘Electronic Fluorescent Pictograph’’ browser for exploring and
analyzing large-scale biological data sets. PLoS One 2, e718-e718.
Yang, F., Wang, Q., Schmitz, G., ller, D.r.M., and Theres, K. (2012). The bHLH
protein ROX acts in concert with RAX1 and LAS to modulate axillary meristem
formation in Arabidopsis. Plant Journal 71, 61-70.
126
CHAPTER IV. CONCLUSION
A study to elucidate the molecular factors responsible
for the perennial traits in Arabis alpina Pajares
127
Polycarpic perenniality is a remarkable and interesting phenomenon that can
extend the plant longevity to an extreme. However, the molecular foundation for
perenniality is still poorly understood because there are many unique challenges for
studying perenniality, such as difficulties of sample manipulation and shortage of
genetic resources. This study attempted to obtain insights into the molecular factors
involved in maintaining perenniality by employing comparative analyses between
two close relatives, annual Arabidopsis thaliana Sy-0 and Arabis alpina Pajares. In
general, perennial plants preserve their lives by sustaining vegetative state in some
branching lateral shoots during reproductive phase. In Arabis alpina Pajares, age-
dependent expression of miR156 precursors was found to be asynchronously
controlled in different parts of axillary shoots on the same plant. The lateral shoots
with strong expression of miR156 precursors sustained vegetative state while those
with weak expression of miR156 precursors turned on the transition into
reproductive phase after vernalization. This asynchronous expression of pre-
miR156s was specific in the perennial Arabis alpina Pajares, which was not observed
in the annual Arabidopsis thaliana Sy-0. On the other hand, new vegetative shoots
were actively produced even during the process of inflorescence senescence.
Through the transcriptome analysis of reproductive stems of Pajares, putative
candidates for regulating axillary branching under floral inductive condition were
searched for. The homologs of Arabidopsis thaliana RAX1 and RAX2 turned out to
be elevated in expressions at stem-nodes with axillary buds. In addition, seven genes
encoding transcription factors were up-regulated in reproductive stems of Pajares.
All of these are involved with functions related with auxin and/or cytokinin and are
128
expressed in the meristematic region, which make a strong case for the products of
these genes to be the candidates for the factors that regulate the initiation and/or
activation of axillary buds in reproductive phase of Arabis alpina Pajares.
129
ABSTRACT IN KOREAN
식물은 생의 지속 기간에 따라 일년생, 이년생, 다년생 식물로 구분한
다. 다년생 식물이 여러 해 동안 삶을 지속할 수 있는 이유는 크게 두
가지 측면에서 살펴 볼 수 있다. 첫째, 적어도 한 개 이상의 정단 분열
조직이 개화기 이후에도 영양 생장 상태를 유지한다는 점이다. 둘째, 새
로운 영양 생장 줄기를 새롭게 생성한다는 것이다. 본 연구에 사용한
Arabis alpina는 다년생 식물이면서 식물 과학의 연구 모델인
Arabidopsis thaliana의 근연 종 이다. Arabidopsis thaliana는 일년생
식물로 한 해 동안 단 한 차례 개화를 거쳐 식물 전체적인 노화를 통해
생을 마감하는 형태의 대표적인 식물이다. 반면에, Arabis alpina는 영양
생장과 생식 생장을 반복하며 여러 해 동안 생명을 지속한다. Pajares는
Arabis alpina 중에서 장기간의 저온 처리인 춘화 과정을 거쳐야만 개화
가 가능한 식물로 생식 생장을 이룬 조직에 한하여 노화를 보이기 때문
에 여러 해 살이가 가능하다. 이는 Pajares가 하나의 개체 내에서 발달
한 여러 곁가지들이 유년기부터 생식 생장기에 이르는 다양한 발달 단계
를 개별적으로 나타내기 때문이다. Pajares에서 다양한 발달 단계의 곁
가지들이 혼재할 수 있는 분자적인 원인을 알아보고자 본 연구를 진행했
다. 한편, 식물에서 발견된 여러 마이크로 RNA중 microRNA156와 이
의 작용체인 SPL 유전자들 간의 전사 후 조절 작용에 의해 식물의 발달
단계 전환이 조절됨이 Arabidopsis를 비롯한 여러 식물에서 알려져 있
130
다. 이 때문에 하나의 Pajares 개체 내에서 나타나는 다양한 발달 단계
의 곁가지 정단 조직을 이용해 miR156의 발현량을 확인해 보고자 했다.
그 결과, 하나의 개체 내에 함께 존재하는 곁가지라 하더라도 발달 단계
가 진행된 정도에 따라 miR156의 발현량이 차등적으로 감소하는 현상
을 보였고, 발현량의 차이에 따라 춘화를 통한 개화 능력도 다르게 나타
남을 알 수 있었다. 즉, Pajares가 장기간의 저온에 노출되어 개체의 정
단부 및 일부의 곁가지가 개화기에 진입하더라도 miR156의 발현량이
유년기 식물의 수준으로 높게 나타나는 곁가지의 경우에는 여전히 영양
생장기에 머무는 것을 확인했다. 본 결과를 일년생 식물과 비교하기 위
해 일년생 겨울 종으로 Pajares와 유사하게 생식 생장기에도 다양한 발
달 단계의 곁가지가 발달하는 Arabidopsis thaliana Sy-0를 연구했다.
Sy-0는 Pajares와는 다르게 발달 단계가 다른 곁가지임에도 하나의 개
체 내에 존재하는 경우에 유사한 정도의 miR156의 발현량을 나타내었
고, 따라서 개화 현상이 개체 단위에서 일괄적으로 나타났다. 뿐만 아니
라, Pajares에서 유년기 식물은 춘화 처리에 대한 개화 반응이 없었던
것과 다르게 Sy-0에서는 어린 식물체도 춘화 처리를 통한 개화 촉진이
유발되며 오히려 어린 시기의 식물일수록 춘화를 통한 개화 촉진에 더욱
민감함을 알 수 있었다.
이를 통해, Arabis alpina Pajares의 다년생 생활사의 분자적인 한 원인
으로 식물의 개체 내에서 개별적으로 발달한 곁가지가 발달 정도에 따라
131
miR156 발현량의 차이를 보임으로써 곁가지 별로 춘화 반응이 서로 다
르게 나타나는 것을 생각 할 수 있다. 이로써 생식 생장기 이후에도 영
양 생장 상태를 유지하는 곁가지가 존재할 수 있다.
다음으로, 영양 생장 줄기와 생식 생장 줄기에서 전사체 양을 비교 분
석한 실험을 통해 생식 생장기의 Pajares에서 영양 생장 줄기의 발달을
유도하는 분자적 원인을 규명하고자 했다. 그 결과, 105개의 전사 인자
의 발현이 2배 이상 증가함을 확인했고, 이 중에 REGULATOR OF
AXILLARY MERISTEMS2 (RAX2)라 하는 곁가지 발달 촉진 유전자가
존재함을 확인했다. 105개의 전사 인자 중에서 식물 호르몬에 의해 발현
이 변화하는 58개의 유전자를 우선 선별했다. 이어서, 이들 중에서 특히
곁가지 발달에 주요한 기능을 담당하는 옥신, 사이토키닌에 의한 발현
변화를 보이면서 식물의 정단 분열 조직에서 발현이 나타나는 전사 조절
자 7개를 선별할 수 있었다. 이들 유전자는 다년생Arabis alpina
Pajares가 생식 생장기 이후에 새로운 영양 생장 줄기를 형성하여 다음
해에 생명을 연장할 수 있도록 기능할 가능성이 높을 것이라 추측한다.