Final Report Summary - RSL4 (RSL4 is a master regulator of cell growth and size)
Root hairs are projections of the root epidermal cells that help in nutrient and water uptake, anchorage and biotic interactions. Root hairs grow by tip growth and provide a good model to study cell growth mechanisms. The Dolan lab identified a transcription factor RSL4 that positively regulates the growth of root hairs. One of the aims of this project was to find how RSL4 controls the cell growth mechanism. To answer this question we looked for direct downstream targets of RSL4. The method adopted was to introduce GR-RSL4 (RSL4 fused to Glucocorticoid receptor) into mutants that lack functional RSL4. GR traps the fusion protein in the cytoplasm and only upon addition of Dexamethasone (DEX) GR-RSL4 can translocate to the nucleus and control the expression of downstream genes. Addition of cycloheximide together with dexamethasone prevents any new protein being synthesized. This inhibits the expression of genes further downstream or the indirect targets of RSL4. We performed a microarray to compare the transcriptomes of plants harboring GR-RSL4, before and after treatment with DEX and DEX+Cycloheximide. Our analysis identified 33 genes that showed greater than two-fold induction upon Dex treatment and were insensitive to addition of cycloheximide, indicating that they might be direct targets of RSL4. Since RSL4 is involved in root hair development the direct targets of RSL4 might have similar function. To find this we ordered T-DNA insertion mutants of the direct targets and looked at their root hair development. We identified five homozygous mutants that show defective root hair development. One of them codes for a protein involved in exocytosis has been previously reported to have a root hair defect. The other four genes code for a kinase, phosphoinositide-phosphatase, peroxidase and a bZIP transcription factor. All the mutants have less root hairs and the length of the root hairs are also shorter than in the wild type, similar to rsl4. We have made overexpression and GFP-fusion constructs for these genes and are currently awaiting seeds from these lines to decipher their precise role in the cell growth mechanism.
Previous results from our lab show that root hairs continue to grow as long as RSL4 is present in those cells. Since the cessation of growth coincides with the disappearance of RSL4, the turnover of the RSL4 protein can provide clues about the fundamental question ‘How does a cell stop growing’. We identified a peptide motif called D-box in RSL4 that acts as a recognition sequence for targeting proteins for degradation via the ubiquitin-mediated 26S proteosomal pathway. Our hypothesis was that if the D-box is indeed required for the destruction of RSL4 then we could predict that mutant forms of RSL4 in which the D-box is mutated will be more stable than WT protein producing longer root hairs. To confirm this we made a genomic mch-RSL4 fusion in which the D-box is mutated and introduced it into the mutants lacking RSL4 function. At the same time we introduced the mch-RSL4 fusion with a wild type D-box into rsl4 and rsl2 rsl4 as a control. rsl4 mutants when transformed with the mutated or non-mutated forms of RSL4 complemented the rsl4 phenotype producing root hairs similar to the wildtype. Interestingly, we found that mutations in the D-box of RSL4 resulted in the production of long hairs in rsl2 rsl4. Since the transgenic lines with the mutated and non-mutated forms of RSL4 have similar root lengths, we decided to measure the stability of the mch-RSL4 protein by measuring the distance to which mch-RSL4 protein persisted in root hair cells from the root tip. Our results indicate that the D-box mutated RSL4 protein was more stable than the non-mutated RSL4 as the mch-RSL4 protein persisted in root hair cells at a greater distance from the root tip. We are currently characterizing the stability of the RSL4 protein in more detail. Understanding of this mechanism can provide clues about the growth cessation process.
Previous results from our lab show that root hairs continue to grow as long as RSL4 is present in those cells. Since the cessation of growth coincides with the disappearance of RSL4, the turnover of the RSL4 protein can provide clues about the fundamental question ‘How does a cell stop growing’. We identified a peptide motif called D-box in RSL4 that acts as a recognition sequence for targeting proteins for degradation via the ubiquitin-mediated 26S proteosomal pathway. Our hypothesis was that if the D-box is indeed required for the destruction of RSL4 then we could predict that mutant forms of RSL4 in which the D-box is mutated will be more stable than WT protein producing longer root hairs. To confirm this we made a genomic mch-RSL4 fusion in which the D-box is mutated and introduced it into the mutants lacking RSL4 function. At the same time we introduced the mch-RSL4 fusion with a wild type D-box into rsl4 and rsl2 rsl4 as a control. rsl4 mutants when transformed with the mutated or non-mutated forms of RSL4 complemented the rsl4 phenotype producing root hairs similar to the wildtype. Interestingly, we found that mutations in the D-box of RSL4 resulted in the production of long hairs in rsl2 rsl4. Since the transgenic lines with the mutated and non-mutated forms of RSL4 have similar root lengths, we decided to measure the stability of the mch-RSL4 protein by measuring the distance to which mch-RSL4 protein persisted in root hair cells from the root tip. Our results indicate that the D-box mutated RSL4 protein was more stable than the non-mutated RSL4 as the mch-RSL4 protein persisted in root hair cells at a greater distance from the root tip. We are currently characterizing the stability of the RSL4 protein in more detail. Understanding of this mechanism can provide clues about the growth cessation process.