Identification and partial characterization of touch-induced genes from an ethylene-insensitivie Arabidopsis mutant
Open Access
- Author:
- Chotikacharoensuk, Thitinun
- Graduate Program:
- Horticulture
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- December 10, 2002
- Committee Members:
- Richard N Arteca, Committee Chair/Co-Chair
Majid R Foolad, Committee Member
Hong Ma, Committee Member
Ramesh Raina, Committee Member
Dennis R Decoteau, Committee Member - Keywords:
- mechanical stimulation
touch-induced genes
etr1-3
opr3
calcium dependent protein kinase
CDPK
jasmonic acid - Abstract:
- <html> <head> <title>Abstract of Chotikacharoensuk's PhD thesis</title> </head> <body> <p class="MsoNormal"> Touch has been shown to affect plant growth and development. Daily touch treatments often result in a reduction in plant height, an increase in stem diameter, a delay in flowering, and an acceleration of senescence in a variety of plant species. Ethylene has been shown to have similar effects and thought to cause touch responses. However, mechanisms in which touch is perceived, transduced, and used to activate other responses in plant systems remain unclear. Therefore, this study was initiated to better understand touch-induced responses in plants. Differential display PCR was used to identify touch-regulated genes from 3-week-light-grown ethylene insensitive <i>etr1-3</i> Arabidopsis (Columbia ecotype) mutant plants. The ethylene insensitive <i>etr1-3</i> Arabidopsis mutant was used to reduce ethylene effects and increase the chance of identifying touch-induced genes, which possibly were not regulated by ethylene. From the 24 combinations of primer sets used, 32 cDNA fragments were selected of which only 3 cDNA fragments (A8A, G5A, and G7F) were touch-inducible. The 3 cDNA fragments were activated 10 min following a 30 sec touch treatment. The fragments were then used to screen a cDNA library for a full-length or near full-length cDNA. </p> <p class="MsoNormal">A 1.2 kb fragment for <i>OPR3</i> was obtained from A8A screenings. This cDNA fragment encodes for 12-oxophytodienoate-10, 11-reductase (OPR), an enzyme in the jasmonic acid biosynthetic pathway. <i> OPR3</i> was induced 10 min following a touch treatment, reaching a maximum level at 60 min, and declining by120 min. Wounding also activated <i>OPR3</i> with similar kinetics to touch treatment. In addition to touch and wounding, treatments with methyl jamonate clearly activated <i>OPR3</i> expression, while ethylene had no effect. Treatment with NaCl as well as CaCl<sub>2</sub> strongly increased <i>OPR3</i> expression, whereas a slight induction was also observed from treatment with MgCl<sub>2</sub>. Darkness treatment, however, did not activate <i>OPR3</i>.</p> <p class="MsoNormal"> Effects of daily touch treatments on growth and development of jasmonic acid deficient <i>opr3</i> Arabidopsis mutant plants were also observed. Preliminary results showed that daily touch treatments for 4 weeks had no significant effect on leaf area and inflorescence length of the <i>opr3</i> plants, whereas smaller leaves and shorter inflorescences were observed from touch-treated wild-type Wassilewskija (WS) plants. However, leaves of touch-stimulated <i>opr3</i> plants showed signs of stress, which were increased necrotic regions on the leaves. Plant fresh and dry weights of control and touch-stimulated groups were not significantly different in both wild-type and <i>opr3 </i>plants. It was unclear what caused the reduced touch responses in the <i>opr3</i> mutant; therefore, more research is necessary in this area to better understand the involvement of jasmonic acid in plant mechanical responses.</p> <p class="MsoNormal"> The cDNA library screenings with G5A fragment resulted in a 2 kb cDNA encoding for a calcium-dependent protein kinase (CDPK32). Northern analysis showed that <i>CDPK32</i> expression was activated within 10 minutes, reaching a maximum level between 20-30 min, and declining to almost undetectable levels 60 min following a touch treatment. Wounding also induced <i>CDPK32</i> with a maximum expression at 30 min and large reduction at 120 min after the treatment initiation. Salt stress and darkness treatments increased <i>CDPK32</i> expression, whereas treatments with methyl jasmonate and ethylene had little or no effect on <i>CDPK32</i> expression. Exogenous application of 50 mM CaCl<sub>2 </sub>slightly increased <i>CDPK32</i> expression; however, the induction was lower than treatment with MgCl<sub>2</sub> indicating that the expression was possibly due to osmotic stress, not extracellular Ca<sup>2+</sup>. <i>CDPK32</i> expression, however, was increased by a 1 h darkness treatment and disappeared when subsequently transferred to light conditions for 1 h.</p> <p class="MsoNormal"> A 1.4 kb cDNA encoding for a novel protein was recovered from the cDNA library screenings with G7F fragment and designated <i>GDL </i>for GDA1-like cDNA. <i>GDL</i> was activated within 10 min, reaching a maximum level 20 min, and began to decline 40 min following touch treatment in the <i>etr1-3</i> plants. Similar kinetics of touch-induced <i>GDL</i> expression were observed in wild-type Arabidopsis plants. In addition to touch, wounding induced <i>GDL</i> with a maximum expression at 30 min and a large reduction at 120 min after treatment initiation. Treatment with 50 mM CaCl<sub>2</sub> strongly increased <i>GDL</i> expression, whereas slight expression of <i>GDL</i> was also observed from treatment with MgCl<sub>2</sub>. Treatments with 100 mM NaCl for 3 h and 100 mM MeJA for 6 hr clearly activated <i>GDL</i> expression, whereas there was no induction with ethylene and darkness treatments.</p> </body> </html>