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Functional characterization of tomato Sl-IAA3 and Sl-hls genes. Role ...

Functional characterization of tomato Sl-IAA3 and Sl-hls genes. Role ...

Chapitre IV:

Chapitre IV: Functional Characterization of Tomato Hookless Genes mutants with tomato Sl-HLS1 and Sl-HLS2 resulted in complete recovery of the greening phenotype and rescued the complemented lines from death (Figure 3). These indicated that beside their role in hook formation, and like At-HLS1 gene, both tomato hookless genes are required in the greening upon white light treatment. Sl-HLS1 and Sl-HLS2 Expression Restores Glucose Sensitivity to the hls1 Mutant Wild-type seedlings of Arabidopsis undergo growth arrest when cultivated in light in the presence of 6% (w/v) glucose whereas hls1 mutant is capable to grow in the presence of up to 6.5 % (w/v) glucose (Figure 4 A). Complementation of hls1 with Sl-HLS1 and Sl-HLS2 resulted in recovery of glucose-induced growth A -C2H4 +C2H4 WT hls1 hls1/ Sl-HLS2 numbers of leaves B 0% glucose 6.5% glucose 6.5% glucose - C2H4 + C2H4 WT hls1 hls1/Sl-HLS2 Figure 4. Glucose sensitivity of hls1 Mutant Complemented with Sl-HLS1 and Sl-HLS2. A. Wild type, hls1 mutant and complemented lines were lightgrown on MS medium containing 6.5% (w/v) glucose during 12 days in the presence of air or 10 µM of ethylene. For each line, 50 seedlings were grown and the data presented are representative of more than tree replicates. B. Leaf differenciation was assessed by counting the number of leaves emerging in each seedling lines. inhibition similar to that shown by the wild type (Figure 4 A). It is known that exogenous ethylene allows seedlings to overcome glucose-induced inhibition of growth. Accordingly, when seedlings were supplemented with 10 µL.L -1 ethylene, 6 5 4 3 2 1 0 106

Chapitre IV: Functional Characterization of Tomato Hookless Genes all lines including wild-type control, escape glucose-induced growth inhibition (Figure 4A). Upon ethylene treatment, the complemented seedlings show long hypocotyls, small and curved dark green cotyledons similar to wild type seedlings. This is in contrast with hookless mutant that show short hypocothyls, expanded and light green cotyledons and longer petioles (Figure 4A). Assseesing leaf differenciation in 12-day-old plantlets indicated that in absence of glucose, hls1 mutant bears higher number of developing leaves than wild type whereas in complemented lines there were twice less leaves than wild type (Figure 4B). Glucose treatment completely prevents leaf developemnt in wild type and complemented lines while it significantly reduced the number of leaves in hls1 mutant (Figure 4B). In the presence of glucose, ethylene treatment enhanced leaf development in all three types of lines. These data indicate that hookless genes are required for glucose-induced developmental arrest but their overexpression does not seem to enable overcaming the antagonistic effect of ethylene on seedling growth development. WT hls1 hls1/ Sl-HLS2 % of germinated seeds 100 80 60 40 20 0 WT hls1 hls1/Sl-HLS2 Figure 5. Effect of ABA on seed germination of Sl-HLS1 and Sl-HLS2 Complemented Lines. A. Etiolated seedlings of WT, hls1, and tomatocomplemented hls1 mutant were grown in presence of 100mM of ABA. B. The result in panel A were quantitatively expressed by examinating a large number of seedlings (n>50). 107

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