impregnace - lignifikace a suberinizace -...

Impregnace - lignifikace a suberinizace

Model diferenciace xylemu in vitro u r. Zinnia

Barvení kyselým fuchsinem(A) a phloroglucinolem(B,C) na lignin.

Lignin

Phenylpropanoidnímetabolismus

vedoucí ke vzniku monolignolů – p-kumaryl alkoholu,

koniferyl alkoholu, a sinapyl alkoholu.

Začíná se kyselinou skořicovou.

Stěnový cross-linking

Hydroxylové radikály (´OH) a H2O2 vznikající ve stěně činností PRX a O2 a NADH, Fentonovoureakcí (Fe2+ a H2O2 =ROS) či

NOX v plasmalemě; H2O2 pak je využito k oxidativní tvorbě

křížových vazeb peroxidasami.

U tabáku byla pomocí exprese spec. anti-mRNA blokována exprese kationtové isoformy

stěnové PRX.Transformanti obsahovali až o 50% méně ligninu, aniž by byl

narušen normální vývoj transgenních rostlin.

Lignifikace v obran ě

Colletotrichum atakuje b. kukuřice -obranná papila akum. kalosu a lignin.

Důležitá úlohaaktinu!

Wall Apposition

Biologicky aktivní oligosacharidyspouštějí obrannou eakci.

LIGNINyLIGNINyLIGNINyLIGNINy a DOBÝVÁNÍ SOUŠEa DOBÝVÁNÍ SOUŠEa DOBÝVÁNÍ SOUŠEa DOBÝVÁNÍ SOUŠE

Evoluční perspektiva - G- a S-lignin

nahosemenné - jen tracheidy (cévice) bohaté na guaiacyl lignin (G- lignin )

krytosemenné - vedle trachejí (céva) bohatých také na G-lignin , maji také vláknité sklerenchymatické podpůrné b. (libriformní,

"fiber") se syringyl ligninem (S - lignin ).

U krytosemenných došlo k vytvoření druhého b. typu: specializovaných typů elementů - vláknité sklerenchymatické

podpůrné b. s mechanickou fcí. užívají novou lignifikační dráhu přes syntézu sinapyl alkoholu.

Transkriptom tvorby dřeva topolu

Populustrichocarpa

SuberinizaceSuberinizaceSuberinizaceSuberinizace

Caspariho "prstýnky" v endodermisbrambora…

Lipofilní polyalifatické a polyaromatické domény - polyestery

hlavní monomerhydroxy-alifatickékyseliny (C16-C30)

Pod=PEROXIDÁZY

• KUTIKULA

SUDAN III

značení značení značení značení radioaktradioaktradioaktradioakt....mastnými kyselinami!!mastnými kyselinami!!mastnými kyselinami!!mastnými kyselinami!!

Kutikula – lipoidní polyester - Lipofilní polyalifatický.

Kutikulární mutanti

Jak kutikulární mutanty

poznáme?

Effect: Nature’s Self Cleaning Mechanism

•Dirt particles (spores, disease fungi) adhere more strongly to water than the leaf and are consequently washed away.

•Lack of water on surface prevents disease organisms from germinating and growing as they cannot survive.

Cause: Waxy Outer Layer and Surface Roughness

•Wax Crystals on Epidermal Cells :Crystal Density determinesactual hydrophobicity.

•Long hair like structures (trichomes) and bumpy protrusionsinduce surface roughness.

Origin of Leaf Hydrophobicity

C

Adapted from Bechinger et al. Science (1999) 1896.

Pathogenic Attack On Plant Surfaces

Issues: 1) Adsorption of pathogens2) Mechanical resistance

“Biological nano-indenter”C. graminicola

A closer look at the membrane . . .

Removed cells (Pectin degraded)

Epicuticular surface (coated with thin layer of lipids for waterproofing)

Biopolyester support (cutin) (Side view, SEM of tomatofruit cuticle)OH

HO-CH2(CH2)5CH(CH2)8-COOH

CH3-(CH2)n-CH3; CH3-(CH2)n-CH2-OH; n ~ 30

~ 6 µµµµm thick

We examine a model system tomato fruit cuticle (chemically simple)

1) Enymatically isolated to remove outer underlying plant cell2) Investigate investigate influence of water on the surface andbulk rheology intact membrane

isolated biopolymer support (lipids extracted)

(also some embedded lipidspre-dominantly fatty acids)

Importance• Agrochemical spray on leaves

Ways to enhance spreading

• Temperature• Coating the surface• Addition of a surfactant

----> Surfactant enhanced spreading

Alex Couzis, CCNY

Agrochemical Delivery

Prekurzory kutikuly jsou na povrch epidermis „pumpovány“

ABC transportery.

Podobně jako přiukládání suberinu.

Delší čas lezení mšic u

cer3 vedl k

odhalení C30

alkoholu (triaconta

nolu), jako

repelentu

Někteří cer mutanti jsou sterilní, mají narušenu

hydrataci pylu.

cer mutanti Arabidopsis

WTWTWTWT

Zinniaelegans„r ůže“ z Mexika

MODELXYLOGENEZE

Xylogenese – AGP-LTP XYLOGEN, polárn ě sekretovaný induktor

xylogeneze

Purifikace na ConA koloně a N-terminální sekvenování po deglykosylaci.

2 – deglykosylovaný xylogen

Ke stimulaci mRNA stačí auxin, ale bílkovina žádá také cytokinin!

mRNA

bílkovina

ZeXYP1 mRNA se hromadí v prokambiu a nezralém xylému.

(pc – prokambium; ix – nez. xylem; xp – xylem. parenchym; te – cévy; se – floem)

Xylogen je lokalizován apikálně v mladých vznikajících cévách (n).

Dvojitý mutant Arabidopsis atxyp1/2 (homolog xylogenu!) má narušený rozvoj venace listu a konektivity vodivých pletiv.

1. U Arabidopsis je znám mutant cotyledon vascular pattern1(cvp1), který má narušenou

funkci sterol metyltransferázy –enzymu účastnícího se tvorby

mj. také stigmasterolu. 2. Xylogen obsahuje v LTP dom. 8x cystein, který tvoří 4xdisulf. můstek. Vzpomeňme na redox

regulaci askorbátem!

• BIOTECHNOLOGIE BUNĚČNÉ STĚNY

• má dalekosáhlé implikace pro praktické využití.

Improved Fiber Quality in Transgenic Cotton Plants that Over-Express XyloglucanEndotransglycosylase

Teresa H. Burns1, Yoshihisa Kasukabe2, Koichi Fujisawa2, Susumu Nishiguchi2, Yoshihiko Maekawa2, Jeanie L. Heinen1, Mohamed Fokar1, Ginger G. Light1, Sundus A. Lodhi1, and Randy D. Allen1*

1Departments of Biological Sciences and Plant & Soil Sciences, Texas Tech University, Lubbock, TX 79409, USA2Toyobo Research Institute1-1 Katata 2-Chome, Ohtsu, Shiga, 520-02, JAPAN

AbstractCotton is the mostly widely used textile fiber and production of cotton fibers produces over $100 billion in farm income worldwide. Cotton fibers are single-celled trichomes that grow from the epidermis of the seed coat. Length is one of the most crucial factors in determining fiber quality and elongation during early phases of development results in mature fiber cells typically more than one inch (2.5 cm) in length. As in all plant cells, elongation of cotton fibers is driven by turgor pressure and limited by the resistance of the cell wall to expansion. To investigate the role of putative cell wall loosening enzymes in fiber elongation, transgenic plants were developed that over-express xyloglucanendotransglycosylase (XET). These plants have XET activity levels that are approximately twice those of wild-type plants. Fibers from primary transgenic plants (T0) grown in the greenhouse averaged 1.27 inches in length, compared to 1.07 inches for wild-type plants. After self-pollination, these plants produced seed that were grown in the field. Segregation of the XET transgene in these field-grown plants correlated with increased fiber length. Analysis of homozygous expressing and non-expressing segregant lines indicated that over-expression of XET led to the produced fibers that were consistently 15 to 20% longer than non-expressing plants without affecting other quality traits. This research demonstrates that XET is involved in regulating cell expansion and is a limiting factor in the elongation of cotton fibers.

IntroductionXyloglucan endotransglycosylase (XET) is able to transfer a high-molecular weight portion from a donor xyloglucan to a suitable acceptor such as a xyloglucan-derived nonasaccharide. Thus, XETs can cleave and rejoin intermicrofibrillar xyloglucan chains, causing reversible wall-loosening leading to cell expansion. XET activity has been detected in growing regions of virtually all plants tested and the levels of XET activity correlate well with the growth rate of the tissue. Further, XET expression responds to phytohormones that increase cell elongation and reduced expression of specific members of the XET gene family have been reported in acaulismutants of Arabidopsis which have defects in internode elongation. Expression of XETs in elongating tissues, including cotton fibers, suggests that they could still play an important role in promoting cell wall extensibility. To test this hypothesis, a cDNA (designated KC22) that had strong sequence similarity to other plant XETs was isolated from a cotton ovule cDNA library. The KC22 cDNA was used to develop a gene construct designed to over-express XET in cotton tissues. Analysis of the fiber from these plants indicated significant increases in fiber length, indicating that XET activity is one factor that limits fiber length.

4.3 ±0.7

4.6 ±0.5

4.6 ±0.6

4.4 ±0.01n.d.n.d.

HVI Micronaire

28.0 ±0.5

26.1 ±2.5

27.5 ±0.5

25.0 ±1.3

23.0 ±1.8

22. 0 ±2.0

HVI Strength (g/tex)

1.30 ±0.03*

1.06 ±0.07

1.30 ±0.02*

1.07 ±0.05

1.27 ±0.01*

1.07 ±0.06

HVI Length (inches)

+-+-+_

(35S-GUS)

KC22 Transgene

T1, Field

T1,

GreenhouseT

0, GreenhouseGeneration

Table I. Comparison of HVI-derived quality characteristics of fiber samples from greenhouse-grown T0 control (35S-GUS) and 35S-KC22-containing plants. Fiber quality data from greenhouse-and field-grown T1 sibling lines from three independent transgenic cotton plants segregating for the 35S-KC22 gene construct are also included. HVI analysis of fiber from T0 plants was performed at the Toyobo Research Institute, Otsu, Japan. HVI analysis of fiber from T1 plants was performed at the International Textile Research Center, Lubbock, TX. Each value represents the mean of two separate assays for three independent KC22-expressing or non-expressing lines. *Indicates significant differences between expressing and non-expressing plants (P<0.005). These results indicate that the KC22 transgenespecifically affects fiber length.

Figure 2. Comparison of XET specific activity in extracts from seedlings of control and three independent transgenic cotton plants that express the 35S-KC22 gene cassette. XET activity in transgenic plant was approximately double that than in control plants.

0

2

4

6

8

10

Genotype

EX

T A

ctiv

ity Coker 312KC22-5

KC22-3KC22-10

Figure 3. Co-segregation of increased fiber length with the 35S-KC22 transgene cassette T1 transgenic lines. Fiber length was determined by HVI was determined for individual field-grown T2 plants from three independently transformed lines segregating for the 35S-KC22 gene cassette. Inheritance of the 35S-KC22 cassette was assayed by PCR amplification. Plants that carry the 35S-KC22 cassette had fibers approximately 15 to 20% longer than those that did not inherit the cassette. These results indicate a direct relationship between XET over-expression and fiber length.

Genotype

PCR

A B C D

KC22-10 KC22-5 KC22-3

A B C D E F

17:6

15:5

21:0

0:13

Generation: TPlant ID: 10Genotype: +/-Length: 1.33

0

Generation: TPlant ID: 10-173Genotype: +/-Length: 1.30

1

Generation: TPlant ID: 10-173-71Genotype: +/-Length 1.25

2 Generation: TPlant ID: 10-173-389Genotype: +/+Length: 1.24

2Generation: TGenotype: -/-Length (Avg): 1.07

2

Generation: TPlant ID: 10-49Genotype: -/-Length: 1.09

1

Generation: TGenotype: +/+Length (Avg): 1.25

3

Figure 4. Example pedigree for transgenic plant line derived from T0 plant 35S-KC22 #10. Offspring plants that inherit the transgeneproduced fibers between 1.25 and 1.30 inches in length while offspring that did not inherit the transgene produced fibers similar to non-transformed control plants (<1.10 inches). The 35S-KC22 transgeneacts as a fully dominant fiber quality allele.

Figure 1. Comparison of derived amino acid sequences for plant XETs. The a cDNA for the Gossyipium hirsutum XET was used to develop the 35S-KC22 transgene for expression in cotton.

0.98 ±0.03

20.2 ±1.8

190 ±7.0

4.6 ±0.6*

1.47 ±0.03*

1.25 ±0.02*

35S-KC22Expresser

0.95 ±0.02

22.0 ±2.1

189 ±11

5.8 ±1.11.21 ±0.07

1.04 ±0.04

35S-GUS(Control)

MaturityRatio

Strength

(g/Tex)

Fineness

(mTex)

SFC(%<0.5)

UQL(inches

)

Length(inches)

Genotype

Table II. Comparison of cotton fiber quality parameters measured by AFIS and stelometer (strength). Fiber samples were from field-grown T1 transgenic cotton plants transformed with the 35S-KC22 gene construct or a reporter gene construct (35-GUS). AFIS analysis was performed at the International Textile Research Center, Lubbock, TX. Each value represents the mean of two separate assays for three independent KC22-expressing or control lines. *Indicates significant differences between expressing and control plants (P<0.005). Mean fiber length and upper quartile length are significantly increased in 35S-KC22 expressing plants while short fiber content (SFC) in decreased. These results indicate that XET over-expression increases overall fiber length rather than affecting just the longest or shortest fibers.