FUNCTIONAL CHARACTERIZATION OF THREE ARABIDOPSIS GENES AT5G17960, AT5G51190, AT5G58680 INVOLVED IN PLANT STRESS RESPONSE

Ph.DDOCTOR OF PHILOSOPH

A Hormone-Responsive C1-Domain-Containing Protein At5g17960 Mediates Stress Response in <i>Arabidopsis thaliana</i>

<div><p>Phytohormones play a critical role in mediating plant stress response. They employ a variety of proteins for coordinating such processes. In Arabidopsis thaliana, some members of a Cys-rich protein family known as C1-clan proteins were involved in stress response, but the actual function of the protein family is largely unknown. We studied At5g17960, a C1-clan protein member that possesses three unique C1 signature domains viz. C1_2, C1_3 and ZZ/PHD type. Additionally, we identified 72 other proteins in A. thaliana that contain all three unique signature domains. Subsequently, the 73 proteins were phylogenetically classified into IX subgroups. Promoter motif analysis of the 73 genes identified the presence of hormone-responsive and stress-responsive putative cis-regulatory elements. Furthermore, we observed that transcript levels of At5g17960 were induced in response to different hormones and stress treatments. At1g35610 and At3g13760, two other members of subgroup IV, also showed upregulation upon GA3, biotic and abiotic stress treatments. Moreover, seedlings of independent transgenic A. thaliana lines ectopically expressing or suppressing At5g17960 also showed differential regulation of several abiotic stress-responsive marker genes. Thus, our data suggest that C1-domain-containing proteins have a role to play in plant hormone-mediated stress responses, thereby assigning a putative function for the C1-clan protein family.</p></div

Response of <i>At5g17960</i> to different phytohormone treatments.

<p>The transcript levels of <i>At5g17960</i> were detected in 12-day-old WT seedlings at the indicated time points upon treatments with (A) 10 µM GA³, (B) 10 µM ACC, (C) 10 µM SA and (D) 10 µM MeJA. The mock treatments for GA3, SA and MeJA were done using ethanol, whereas, the mock treatment for ACC was done using sterile water. The expression levels were normalized against the expression of <i>TUB</i>. Data from three independent biological replicates were averaged and presented here with error bars representing SD. Bars with asterisks are significantly different from the mock treatments at the corresponding time points, as per Student’s <i>t</i>-test (P ≤ 0.05).</p

Response of <i>At1g35610</i> and <i>At3g13760</i> to GA³ treatment.

<p>The transcript levels of (A) <i>At1g35610</i> and (B) <i>At3g13760</i> were detected in 12-day-old WT seedlings at the indicated time points upon treatments with 10 µM GA³. The mock treatment was done using ethanol. The expression levels were normalized against <i>TUB</i> expression. Data presented here depict mean from three independent biological replicates with error bars representing SD. Bars with asterisks are significantly different from the mock treatments at the corresponding time points, as per Student’s <i>t</i>-test (P ≤ 0.05).</p

Detection of putative <i>cis</i>-elements and associated TFs in 73 C1-clan genes.

<p>**Total motif enrichment score = sum of the % occurrences of all motif species belonging to the same TF family.</p><p>Detection of putative <i>cis</i>-elements and associated TFs in 73 C1-clan genes.</p

Response of stress-inducible marker genes in transgenic <i>At5g17960</i> seedlings upon abiotic stress treatments.

<p>(A) Expression levels of <i>DREB2A</i> in 12-day-old seedlings of the WT, amiR::<i>At5g17960</i> #1–1 and 35S::<i>At5g17960</i> #9–1 upon treatment with 200 mM NaCl were examined at different time points. (B) Expression levels of <i>RD29A</i> were checked upon exposure of the seedlings to 20 min drought treatment. (C) <i>COR15A</i> expression levels were examined upon exposure of the WT, amiR::<i>At5g17960</i> #1–1 and 35S::<i>At5g17960</i> #9–1 seedlings to 10°C for indicated time points. (D) Expression levels of <i>ELIP2</i> in the WT and transgenic seedlings were examined after exposing the seedlings to 10 min UV irradiation. The expression levels were normalized against the expression of <i>TUB</i>. Data from three independent biological replicates were averaged and presented here with error bars representing SD. Bars with asterisks are significantly different from the WT at the corresponding time points, as per Student’s <i>t</i>-test (P ≤ 0.05).</p