constitutive knox1 gene expression in dandelion (taraxacum officinale, web.) changes leaf morphology...

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Planta (2006) 224: 1023–1027 DOI 10.1007/s00425-006-0288-y ORIGINAL ARTICLE Kai J. Müller · Xinqiang He · Rainer Fischer · Dirk Prüfer Constitutive knox1 gene expression in dandelion (Taraxacum officinale, Web.) changes leaf morphology from simple to compound Received: 10 January 2006 / Accepted: 31 March 2006 / Published online: 9 May 2006 © Springer-Verlag 2006 Abstract Seed plants with compound leaves constitute a polyphyletic group, but studies of diverse taxa show that genes of the class 1 KNOTTED-LIKE HOMEO- BOX (KNOX1) family are often involved in compound leaf development. This suggests that knox1 genes have been recruited on multiple occasions during angiosperm evolution (Bharathan et al. in Science 296:1858–1860, 2002). In agreement with this, we demonstrate that the simple leaf of dandelion (Taraxacum oYcinale Web.) can be converted into a compound leaf by the constitutive expression of heterologous knox1 genes. Dandelion is a rosette plant of the family Asteraceae, characterised by simple leaves with deeply lobed margins and endogenous knox1 gene expression. Transgenic dandelion plants con- stitutively expressing the barley (Hordeum vulgare L.) hooded gene (bkn3, barley knox3) or the related bkn1 gene, developed compound leaves featuring epiphyllous rosettes. We discuss these results in the context of two current models of compound leaf formation. Keywords Compound leaf · Dandelion · Epiphylly · Leaf shape complexity · Knox1 family Abbreviations KNOX: Knotted1 related homeobox · SAM: Shoot apical meristem · SEM: Scanning electron microscopy · RT-PCR: Reverse-transcription polymerase chain reaction Introduction The enormous morphological variation observed among the leaves of diVerent plants predominantly reXects adaptations for speci Wc functions, such as opti- mised photosynthesis (Fleming 2003). In nearly all plant species, the basic leaf architecture is either simple or compound. Simple leaves have a single blade, com- prising lamina tissue supported by a midrib and subsid- iary lateral veins. Morphological variations of the lamina margins are common (Fig. 1a, b). In contrast, compound leaves have multiple blade structures (termed leaXets or pinnae), each supported by a lateral vein joined to a midrib devoid of lamina tissue, here known as the rachis (Fig. 1c). Leaves originate repeat- edly from groups of founder cells in the Xanks of the shoot apical meristem (SAM). These develop into a leaf primordium, which matures gradually through the expansion of the lamina. In one model of compound leaf development, the compound leaf is considered a modiWed simple leaf, developing though incomplete expansion of the lamina. An alternative interpretation correlates compound leaf with shoot formation, i.e. the compound leaf is considered a collection of simple leaves on a common petiole. The latter reXects the exis- tence of compound leaf primordia, i.e. young leaves whose organisation resembles that of the shoot, with the leaf proper being the shoot and the leaXets its lat- eral organs (Kaplan 2001; Fleming 2003; Lacroix et al. 2003; Champagne and Sinha 2004). Electronic Supplementary Material Supplementary material is available for this article at http://dx.doi.org/10.1007/s00425-006- 0288-y and is accessible for authorized users. K. J. Müller · R. Fischer · D. Prüfer Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Forckenbeckstrasse 6, 52074 Aachen, Germany K. J. Müller (&) · D. Prüfer Westfälische Wilhelms-Universität Münster, Institute for Biochemistry and Biotechnology of Plants, Hindenburgplatz 55, 48143 Munster, Germany E-mail: [email protected] Tel.: +49-241-8028121 Fax: +49-241-8020145 X. He Department of Plant Molecular and Developmental Biology, Peking University, 5 Yi He Yuan Lu, Beijing, 100871, People's Republic of China

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Page 1: Constitutive knox1 gene expression in dandelion (Taraxacum officinale, Web.) changes leaf morphology from simple to compound

Planta (2006) 224: 1023–1027 DOI 10.1007/s00425-006-0288-y

ORIGINAL ARTICLE

Kai J. Müller · Xinqiang He · Rainer Fischer · Dirk Prüfer

Constitutive knox1 gene expression in dandelion (Taraxacum officinale, Web.) changes leaf morphology from simpleto compound

Received: 10 January 2006 / Accepted: 31 March 2006 / Published online: 9 May 2006© Springer-Verlag 2006

Abstract Seed plants with compound leaves constitutea polyphyletic group, but studies of diverse taxa showthat genes of the class 1 KNOTTED-LIKE HOMEO-BOX (KNOX1) family are often involved in compoundleaf development. This suggests that knox1 genes havebeen recruited on multiple occasions during angiospermevolution (Bharathan et al. in Science 296:1858–1860,2002). In agreement with this, we demonstrate that thesimple leaf of dandelion (Taraxacum oYcinale Web.) canbe converted into a compound leaf by the constitutiveexpression of heterologous knox1 genes. Dandelion is arosette plant of the family Asteraceae, characterised bysimple leaves with deeply lobed margins and endogenousknox1 gene expression. Transgenic dandelion plants con-stitutively expressing the barley (Hordeum vulgare L.)hooded gene (bkn3, barley knox3) or the related bkn1gene, developed compound leaves featuring epiphyllousrosettes. We discuss these results in the context of twocurrent models of compound leaf formation.

Keywords Compound leaf · Dandelion · Epiphylly · Leaf shape complexity · Knox1 family

Abbreviations KNOX: Knotted1 related homeobox · SAM: Shoot apical meristem · SEM: Scanning electron microscopy · RT-PCR: Reverse-transcription polymerase chain reaction

Introduction

The enormous morphological variation observedamong the leaves of diVerent plants predominantlyreXects adaptations for speciWc functions, such as opti-mised photosynthesis (Fleming 2003). In nearly allplant species, the basic leaf architecture is either simpleor compound. Simple leaves have a single blade, com-prising lamina tissue supported by a midrib and subsid-iary lateral veins. Morphological variations of thelamina margins are common (Fig. 1a, b). In contrast,compound leaves have multiple blade structures(termed leaXets or pinnae), each supported by a lateralvein joined to a midrib devoid of lamina tissue, hereknown as the rachis (Fig. 1c). Leaves originate repeat-edly from groups of founder cells in the Xanks of theshoot apical meristem (SAM). These develop into a leafprimordium, which matures gradually through theexpansion of the lamina. In one model of compoundleaf development, the compound leaf is considered amodiWed simple leaf, developing though incompleteexpansion of the lamina. An alternative interpretationcorrelates compound leaf with shoot formation, i.e. thecompound leaf is considered a collection of simpleleaves on a common petiole. The latter reXects the exis-tence of compound leaf primordia, i.e. young leaveswhose organisation resembles that of the shoot, withthe leaf proper being the shoot and the leaXets its lat-eral organs (Kaplan 2001; Fleming 2003; Lacroix et al.2003; Champagne and Sinha 2004).

Electronic Supplementary Material Supplementary material isavailable for this article at http://dx.doi.org/10.1007/s00425-006-0288-y and is accessible for authorized users.

K. J. Müller · R. Fischer · D. PrüferFraunhofer Institute for Molecular Biology and Applied Ecology (IME), Forckenbeckstrasse 6, 52074 Aachen, Germany

K. J. Müller (&) · D. PrüferWestfälische Wilhelms-Universität Münster, Institute for Biochemistry and Biotechnology of Plants, Hindenburgplatz 55, 48143 Munster, GermanyE-mail: [email protected].: +49-241-8028121Fax: +49-241-8020145

X. HeDepartment of Plant Molecular and Developmental Biology, Peking University, 5 Yi He Yuan Lu, Beijing, 100871, People's Republic of China

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Genes of the class 1 KNOTTED-LIKE HOMEOBOX(KNOX1, Kerstetter et al. 1994) family are involved incompound leaf development in many plants (Cham-pagne and Sinha 2004). One major function of thesegenes is to maintain the meristematic state within theSAM. In simple-leaved plants such as maize, Arabidopsis,tobacco and snapdragon (and in pea, which has com-pound leaves), the absence of knox1 expression withinthe SAM marks the position of the leaf founder cells,indicating a substantial contribution of knox1 genes toregulating the initiation of both simple and compoundleaf development (Jackson et al. 1994; Lincoln et al.1994; Nishimura et al. 1999; Hofer et al. 2001; Golz et al.2002). However, there are also species which expressknox1 genes throughout the SAM and in young leaves –these include simple-leaved barley and compound-leavedtomato and potato (Hareven et al. 1996; Müller et al.2001; Rosin et al. 2003).

During leaf development, knox1 gene expressionhelps to retard the expansion of the lamina and appearsto promote the growth of leaXet primordia in com-pound-leaved species. For example, inducible over-expression of knox1 in Arabidopsis leaves shows that leafprimordia are competent to respond to knox1 for a lim-ited developmental time window, resulting in the appear-ance of lobes where serrations normally occur in wildtype Arabidopsis leaves (Chuck et al. 1996; Hay et al.2003). Additionally, knox1 expression at the site of

reduced lateral lamina outgrowth is mirrored by reportergene expression under the control of the knox1 promoterin the sinuses of the lobes of Arabidopsis loss-of-functionmutants with lobed phenotypes (Ori et al. 2000). More-over, dramatic leaf shape modiWcations result from con-stitutive knox1 expression in Arabidopsis and tobacco,e.g. lobing, reduced leaf size and ectopic (or epiphyllous)shoot development (Sinha et al. 1993; Chuck et al. 1996;Parnis et al. 1997; Nishimura et al. 1999, 2000; seeFig. 1e). In several compound-leaved species, knox1expression in leaf primordia correlates with the forma-tion of leaXet primordia (Bharathan et al. 2002). Constit-utive knox1 expression in compound-leaved tomato andpotato causes the repeated generation of higher-orderleaXets, and occasionally epiphyllous meristem or shootformation occurs on these “super-compound” leaves(Hareven et al. 1996; Parnis et al. 1997; Kim et al 2003;Rosin et al. 2003; see Fig. 1f, g). However, until nowthere has been no demonstration that a simple leaf canbe converted into a compound leaf by constitutive orleaf-speciWc expression of a knox1 gene (Fleming 2003).Initially, we anticipated it would be possible to generatea compound leaf in a simple-leaved plant by constitu-tively expressing knox1 and hence interfering with thenormally complete growth of the lamina. For this pur-pose, we considered dandelion, with its deeply lobed leafmargins, as a perfect model system (Figs. 1b, 2a, b). Wechose the barley bkn3 and bkn1 cDNAs as representative

Fig. 1 Leaf architecture in wild type and knox1-transgenic plants, showing morphological changes induced by knox1. Sketches are depicted of simple leaves emphasising entire, ser-rated (a) and lobed (b) lamina margins (blue arrows). In c com-pound leaf architectures are sorted schematically by increas-ing numbers and orders of leaX-ets (red arrows). A wild type, simple tobacco leaf (d) upon bkn3 and bkn1 co-over-expres-sion develops lobes (e) and epiphyllous shoots (insert). A wild type, unipinnate potato leaf (f) upon bkn3 over-expression develops up to four orders of leaXets (g). The degree of bkn1 or bkn3-induced leaf modiWca-tion increases with leaf number in such transgenic potato material

lamina

a Simple leaf

midrib (midvein)

entire margin

serrated margin

b Simple leaf

lobed „deeply lobed“

lateralvein rachis

f g e d

bipinnate unipinnate(2nd order leaflets)

(1st order leaflets)

c Compound leaf

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knox1 genes since they have been shown to induce epi-phylly in tobacco (Müller et al. 1995; Lin and Müller2002) and super-compound leaves in potato (Fig. 1g). In

addition, we studied the expression of resident knox1genes in dandelion leaves and monitored the expressionof the knox1 gene Dandeknox1.

Fig. 2 35S::knox1-induced alterations of leaf characters in dande-lion. a A wild type dandelion plant is sketched and b displays thesimple character of the Wrst leaf following the cotyledons in the wildtype; Lp leaf primordium; Co cotyledon. Note that already at thisearly stage lobing (arrow) can occur. c A wild type leaf primordiumof approximately stage 4 or 5 of a plant that developed roughly 30leaves is shown: yellow arrows point to the lobes of the primordiumand the insert emphasises the involute appearance of the same pri-mordium. d The heteroblastic leaf series of a wild type dandelion ro-sette is depicted. e PCR experiments are shown that detect theendogenous knox1 gene Dandeknox1. Lanes 1 and 2, Wrst strand cD-NAs of RNAs prepared from wild type leaves of 1–3 mm in lengthwere used as templates for the PCRs (controls without reverselytranscribed RNAs are shown in lanes 6 and 7); lanes 4 and 5, samplessubjected to RT-PCR were prepared from leaves that reached

10 mm in length (controls without RT in lanes 8 and 9); lane 10,genomic DNA of wild type dandelion was used as a template. Thesize of the ampliWed DNA fragment correlates with two intronspresent within DANDEKNOX1. Lane 3, Invitrogen DNA 1 kb sizemarker. f The growth habit of the 35S::bkn1-a dandelion plant isshown and a leaf series of a rosette from this plant in g. At the baseof a leaXet (h) epiphyllous shoots emerge as hairy bulges (red ar-rows). A cross section through a lamina-borne epiphyllous meristemof 35S::bkn3-b dandelion is shown in i and thereof derived epiphyl-lous rosettes in j and k. The expression of the indexed transgenes (l)was assured by RT-PCR using gene speciWc pairs of primers thatampliWed 541 and 556 bp for bkn1 and bkn3, respectively, fromcDNA but not from cDNA after DNAse treatment and RNA with-out reverse transcription (green, red and yellow asterisks, respec-tively). Bars 500 �m (b, c), and 20 �m (i, j)

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Materials and methods

Dandelion seeds were obtained as certiWed material fromBlauetikett-Bornträger GmbH, 67591 OVstein, Ger-many. To establish tissue culture, seeds were surface ster-ilized in 0.12% sodium hypochlorite. Construction of the35S::bkn1 and 35S::bkn3 cDNA vectors, in which thebarley bkn1 and bkn3 genes, respectively, are driven bythe cauliXower mosaic virus 35S promoter, has beendescribed previously and Agrobacterium-mediated trans-formation and plant selection procedures were carriedout according to standard methods (Müller et al. 1995).

Microscopy and RT-PCR analysis were carried outaccording to standard procedures (see Lin and Müller2002). SEM samples were young shoots with cotyledonsjust spreading and older plants that had developedapproximately 30 leaves. Fresh material was processedon a cooling stage and examined directly at an accelerat-ing voltage of 5–10 kV in a Weld emission environmentalscanning electron microscope (Quanta 200F, FEI, Hills-boro, OR, USA). RNA isolates were derived from wildtype or transgenic leaf material and the primer combina-tions used in these experiments were speciWc for bkn3 orbkn1 cDNAs. Based on PileUP multiple sequence align-ments of Asteraceae knox1 gene sequences, the geneDandeknox1 was isolated by PCR using the primer com-bination 5�-KX1 and 3�-KX2 (GATCAGTTCATGGAAGCTTAC and GCATCCATTACCACAAATTGC,respectively) and as template both, dandelion genomicDNAs and cDNAs. The sequence is deposited at Gen-Bank (Accession number AY958202). A multiplesequence Wle comparing ten KNOX1 and six KNOX2proteins demonstrates that Dandeknox1 belongs to theknox1 gene family (supplementary Fig. 0).

Results

Visual analysis of wild type dandelion plants

Dandelion (Fig. 2a) belongs to the family Asteraceae andforms leaf rosettes rather than undergoing vegetativeshoot elongation. Consequently, the leaves that originatefrom the SAM are located more or less on a platformand the youngest leaf primordia are protected by hairsthat probably evolved to provide a humid environmentat the shoot apex (data not shown). SigniWcant shootgrowth in dandelion is usually associated with the for-mation of compound inXorescences that reside abovehollow inXorescence stems and appear as main or subse-quent side shoots over the period of vegetative growth.Dandelion is a perennial plant and lateral shoots candevelop into elaborate neighbouring rosettes. Dandelionhas lobed, simple leaves, and subtle lobing can already beobserved on the Wrst true leaf emerging after the cotyle-dons (Fig. 2b), becoming more prominent in older leaves(Fig. 2c). The leaf architecture in each rosette always

remains simple, but the leaves comprise a heteroblasticseries, starting with entire margins that later become ser-rated and in mature leaves deeply lobed as well as ser-rated (Fig. 2d). Endogenous knox1 gene expression indandelion leaves at diVerent developmental stages can bedetected in RT-PCR experiments using a primer combi-nation speciWc for the gene Dandeknox1 (Fig. 2e).

Endogenous and heterologous expression of knox1 genes in dandelion

We generated knox1-neomorphic transgenic dandelionplants, two expressing 35S::bkn1 and two expressing35S::bkn3. In addition to antibiotic selection of dandelioncallus after leaf disc transformation, knox1-transgenicplant material was identiWed visually during sterile tissueculture, as such callus pieces showed palmate venation pat-terns on leaves emerging from the Wrst shoots. Later, suchplants showed more dramatic knox1-speciWc phenotypicalterations in comparison to wild type dandelion (Fig. 2f).The knox-transgenic dandelion leaves displayed a pro-nounced heteroblastic series of leaf architectures, andwithin a single rosette the earlier simple leaves tended tobecome more compound-shaped and complex. Earlierleaves were simple with serrated and irregularly-lobed orlocally frayed margins, and the typical knox1 venation pat-tern changed from palmate to nearly parallel over largerareas. Later in development, the leaves became compound-shaped and the number of leaXets increased with leaf suc-cession. No lamina expansion was visible around the mid-rib or the major parts of departing lateral veins,emphasising the compound architecture of these leaves.The leaXets in turn displayed similar margin alterations asseen in earlier simple leaves (Fig. 2g). Epiphyllous meriste-matic areas were Wrst recognised macroscopically as hairywhite bulges that typically developed above the veins.Most epiphylls arose in the sinuses of the irregular lobes ofsimple leaves or leaXets, and to the left and right at thebase of individual leaXets on the rachis (Fig. 2h). Such epi-phylls were derived from individual meristems, layered andorganized similarly to vegetative shoot meristems (Fig. 2i)and in structure identical with epiphylls emerging on juve-nile leaves of bkn1- or bkn3-transgenic tobacco (Lin andMüller 2002). Later in development, such epiphyllous mer-istems occasionally developed into rosettes on leaves(Fig. 2j) that did not display substantial vegetative shootelongation (Fig. 2k). Transgene expression was conWrmedby RT-PCR (Fig. 2l) and no substantial phenotypic diVer-ence between the bkn1- and bkn3-transgenic dandelionplants was observed. Although cultivated for 2.5 yearsknox1-dandelion transformants showed no inXorescencedevelopment, even after vernalisation (2 months, 4°C).

Discussion

The constitutive expression of the barley knox1 genesbkn1 or bkn3 in dandelion, in addition to the normal

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expression of endogenous Dandeknox1, results in a mor-phological shift from simple to compound leaf architec-ture. Furthermore, the presence of lobe primordia ondandelion leaf primordia suggests that the dandelion leafis a result of secondary morphogenesis, i.e. a compoundprimordial structure originally precedes the Wnally sim-ple, deeply lobed leaf. In the light of this, the addition ofbkn1 or bkn3 to the expression of Dandeknox1 in dande-lion leaves promotes the disclosure of a subtly com-pound nature of the dandelion leaf.

Together with barley, dandelion forms a minority ofsimple-leaved plant species that expresses knox1 geneswithin leaves. While barley does not respond to endoge-nous bkn1, bkn3 or transgenically expressed knox1 withspeciWc modiWcations of foliar leaves (Williams-Carrieret al. 1997; Müller et al. 2001) dandelion does. Therefore,in this sense, the competence of the tissue to respond toleaf-speciWc knox1 expression is diVerent in the two spe-cies. Moreover, the transgenic expression of knox1 genesin addition to endogenous knox1 expression supports theconclusion that a certain threshold of knox1 gene prod-ucts is necessary to induce the phenotypic alterationsobserved in dandelion.

There are two models of knox1 activity in leaf develop-ment which could be involved in the morphological shiftwe observed: Wrst, the retardation of lamina outgrowth,and second the promotion of leaXet outgrowth. At pres-ent, we can only speculate that both eVects could havecontributed to the development of compound leaves in thebkn1- or bkn3-transgenic dandelion plants, since ourobservations Wt with both models. More importantly, ourexperiments support the hypothesis that an evolutionarystep from simple to compound leaves could have occurreddirectly through increased knox1 gene expression. ThisreXects the fact that other developmental pathways havebeen shown to control compound leaf formation, e.g. inpea and tomato (Hareven et al. 1997; Sinha 1997; Gourlayet al. 2000) and that compound leaves evolved indepen-dently on multiple occasions (Bharathan et al. 2002).

Acknowledgements We gratefully acknowledge the assistance ofJulia Boike, Barbara Kampmann, (Fraunhofer Institute for Molec-ular Biology and Applied Ecology, Schmallenbeg-Grafschaft, Ger-many), Catheriné Schöpper and Dr. Klaus Tenberge (WestfälischeWilhelms-Universität Münster, Germany).

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