Impregnace - lignifikace a suberinizace
Model diferenciace xylemu in vitro u r. Zinnia
Barvení kyselým fuchsinem(A) a phloroglucinolem(B,C) na lignin.
Lignin
Phenylpropanoidní metabolismus vedoucí ke vzniku monolignolů – pkumaryl 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, Fentonovou reakcí (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 obraně
Colletotrichum atakuje b. kukuřice obranná papila akum. kalosu a lignin.
Důležitá úloha aktinu!
Wall Apposition
Biologicky aktivní oligosacharidy spouštějí obrannou eakci.
LIGNINy a DOBÝVÁNÍ SOUŠE
Evoluční perspektiva - G- a Slignin 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
Populus trichocarpa
Suberinizace Caspariho "prstýnky" v endodermis brambora… Lipofilní polyalifatické a polyaromatické domény - polyestery
hlavní monomer hydroxy-alifatické kyseliny (C16-C30)
Pod=PEROXIDÁZY
• KUTIKULA
SUDAN III
značení radioakt. radioakt. mastnými kyselinami!! Kutikula – lipoidní polyester - Lipofilní polyalifatický.
Kutikulární mutanti
Jak kutikulární mutanty poznáme?
Origin of Leaf Hydrophobicity Cause: Waxy Outer Layer and Surface Roughness •Wax Crystals on Epidermal Cells :Crystal Density determines actual hydrophobicity. •Long hair like structures (trichomes) and bumpy protrusions induce surface roughness. 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.
C. graminicola
C
“Biological nano-indenter”
Pathogenic Attack On Plant Surfaces Issues: 1) Adsorption of pathogens 2) Mechanical resistance Adapted from Bechinger et al. Science (1999) 1896.
A closer look at the membrane . . . Epicuticular surface (coated with thin layer of lipids for waterproofing) CH3-(CH2)n-CH3; CH3-(CH2)n-CH2-OH; n ~ 30 ~ 6 µm thick
Biopolyester support (cutin) OH HO-CH2(CH2)5CH(CH2)8-COOH
(Side view, SEM of tomato fruit cuticle)
Removed cells (Pectin degraded)
(also some embedded lipids pre-dominantly fatty acids)
We examine a model system
tomato fruit cuticle (chemically simple)
1) Enymatically isolated to remove outer underlying plant cell 2) Investigate investigate influence of water on the surface and intact membrane bulk rheology isolated biopolymer support (lipids extracted)
Agrochemical Delivery Importance • Agrochemical spray on leaves
Ways to enhance spreading • Temperature • Coating the surface • Addition of a surfactant ----> Surfactant enhanced spreading
Alex Couzis, CCNY
Prekurzory kutikuly jsou na povrch epidermis „pumpovány“ ABC transportery.
Podobně jako při ukládání 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.
WT
cer mutanti Arabidopsis
Zinnia elegans „růže“ z Mexika
MODEL XYLOGENEZE
Xylogenese – AGP-LTP XYLOGEN, polárně 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 Xyloglucan Endotransglycosylase 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, USA 2Toyobo Research Institute1-1 Katata 2-Chome, Ohtsu, Shiga, 520-02, JAPAN Abstract Cotton is the mostly widely used textile fiber and production of cotton fibers produces over $100 billion in farm income worldwide. Cotton fibers are singlecelled 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 xyloglucan endotransglycosylase (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.
Introduction Xyloglucan 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 acaulis mutants 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 overexpress 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.
10
EXT Activity
8 Coker 312 KC22-5 KC22-3 KC22-10
6 4 2
A BCDE F
A BCD
KC22-10
KC22-5
PCR Genotype
0
KC22-3
Genotype
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.
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.
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 greenhouseand 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 KC22expressing or non-expressing lines. *Indicates significant differences between expressing and non-expressing plants (P<0.005). These results indicate that the KC22 transgene specifically affects fiber length.
Table II. Comparison of cotton fiber quality parameters measured by AFIS and stelometer (strength). Fiber samples were from fieldgrown 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.
Figure 3. Co-segregation of increased fiber length with the 35SKC22 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.
Generation: Plant ID: Genotype: Length:
17:6
Generation: T1 Plant ID: 10-49 Genotype: -/Length: 1.09 0:13
Generation T0, Greenhouse KC22 Transgene
_ (35SGUS)
+
Greenhouse -
T1,
+
T1, Field -
UQL Finenes Strengt Length SFC Maturity Genotype (inches s h (inches) (%<0.5) Ratio ) (mTex) (g/Tex)
+
HVI Length (inches)
1.07 ±0.06
1.27 ±0.01*
1.07 ±0.05
1.30 ±0.02*
1.06 ±0.07
1.30 ±0.03*
HVI Strength (g/tex)
22. 0 ±2.0
23.0 ±1.8
25.0 ±1.3
27.5 ±0.5
26.1 ±2.5
28.0 ±0.5
HVI Micronaire
n.d.
n.d.
4.4 ±0.01
4.6 ±0.6
4.6 ±0.5
4.3 ±0.7
35S-GUS (Control)
1.04 ±0.04
1.21 ±0.07
5.8 ±1.1
189 ±11
22.0 ±2.1
0.95 ±0.02
35S-KC22 Expresse r
1.25 ±0.02*
1.47 ±0.03*
4.6 ±0.6*
190 ±7.0
20.2 ±1.8
0.98 ±0.03
T0 10 +/1.33
Generation: T2 Genotype: -/Length (Avg): 1.07
Generation: T1 Plant ID: 10-173 Genotype: +/Length: 1.30 15:5
Generation: T2 Plant ID: 10-173-71 Genotype: +/Length 1.25
Generation: Plant ID: Genotype: Length:
T2 10-173-389 +/+ 1.24 21:0
Generation: T3 Genotype: +/+ Length (Avg): 1.25
Figure 4. Example pedigree for transgenic plant line derived from T0 plant 35S-KC22 #10. Offspring plants that inherit the transgene produced fibers between 1.25 and 1.30 inches in length while offspring that did not inherit the transgene produced fibers similar to nontransformed control plants (<1.10 inches). The 35S-KC22 transgene acts as a fully dominant fiber quality allele.
