Molecular Genetic Analysis of Trichome Development in Arabidopsis thaliana

Abstract

Several mutants with aberrant trichome patterning and differentiation are known in Arabidopsis. The genes involved in these processes code for proteins with diverse functions such as transcription, protein degradation, microtubule arrangement, cell wall remodeling etc. Even though the role of these genes in trichome development have been studied by genetic and biochemical methods, the factors that in turn regulate them at the transcriptional, translational and post-translational levels have been less studied. Here, using mutational analysis, we have studied two such factors that control the trichome development genes and dissected the molecular mechanisms adopted by them. In the first part of the work, we have addressed the role of class II TCP genes in general, and TCP4 in specific, in trichome differentiation, and in the second part, we have addressed the role of TNI/UBP14 in controlling the proteins involved in trichome development. TCPs negatively regulate trichome development The TCP genes encode DNA-binding transcription factors and regulate multiple aspects of plant development including leaf shape and size. The Arabidopsis genome encodes 24 TCP genes, classified into two major groups; class I and class II, based on their sequence similarity. Among the eleven class II members, five (TCP2, 3, 4, 10, 24) are post-transcriptionally regulated by miR319. Role of miR319-targeted TCP genes are well established in leaf morphogenesis at the cellular level as a negative regulators of cell proliferation and as a positive regulators of cellular differentiation. It has been shown that in TCP loss-of-function, there is prolonged cell division and in their gain-of-function, there is a precocious differentiation at both organ and cellular levels. However, these studies focused mainly on the role of the TCP genes in the pavement cell differentiation without much attention to other epidermal cells with specialized structure and function, namely trichomes and stomata. Detailed phenotypic analysis of the class II TCP loss-of-function as well as and gain-of-function lines suggested that these TCP genes negatively regulate both trichome initiation and trichome differentiation. To learn more on the molecular mechanism of TCP4-mediated trichome development, we analyzed the results of the DNA microarray analysis where the transcriptome of the 9-day old seedlings of jaw-D;pTCP4:mTCP4:GR genotype was compared before and after TCP4 induction and identified four genes involved in trichome development, that was up regulated by TCP4. These genes are GLABROUS INFLORESCENCE STEMS (GIS), TRICHOMELESS1 & 2 (TCL1, 2) and ZINC FINGER PROTEIN8 (ZFP8) that have been previously shown to regulate both trichome initiation and differentiation. Of the four genes, GIS is reported to regulate the trichome branching. Analysis of GIS transcript levels in the TCP loss of- function mutants like jaw-D and tcp2;4;10 showed a down-regulation while it was upregulated in the gain-of-function lines TCP4:VP16, pBLS:rTCP4:GFP and upon TCP4 induction in the pTCP4:mTCP4:GR line. GIS transcript was also up-regulated when TCP4 was induced in the absence of additional protein synthesis, suggesting that the TCP4 protein is directly responsible for GIS activation. Further, TCP4 is capable of binding to its cognate sites present on the GIS locus. While GIS is massively activated in the TCP4:VP16 line leading to reduced trichome branching, The TCP4:VP16 protein failed to suppress trichome branching in the absence of GIS as seen in the gis;TCP4:VP16 line. Taken together, these results provide evidence that GIS is required for the TCP4-mediated inhibition of trichome differentiation. Previous analysis of several trichome mutants showed that there is a strong correlation between the endoreduplication status of a trichome and its extent of branching. However, there are several other genes including STI, BLT, NOK and GIS that regulate trichome branching independent of endoreduplication. However TCPs, regulate GIS transcription, independent of endoreduplication pathway. In addition to branching, analysis of trichome development in the TCP loss and gain-of-function mutants has shown that TCPs negatively regulate trichome initiation as well. From the analysis of the microarray results mentioned above, all the four identified genes - TCL1, 2, ZFP8, GIS - are reported to be involved in regulating trichome density. Among these, TCL1, and 2 suppress trichome initiation whereas ZFP8 & GIS promote it. It is possible that TCPs are involved in maintaining the homeostatic regulation of trichome density by activating both positive and negative regulators of trichome initiation. Gene expression analysis has shown that TCP4 directly up-regulate the expression of all these genes (Results in Chapter IV; Krishna Reddy Challa, PhD thesis, 2014). While TCP4:VP16 suppressed trichome density in Col-0, tcl1 mutation did not show increased trichome density, possibly because of a functional redundancy between TCL1 and TCL2. However, the tcl1;TCP4:VP16 plants failed to reduce trichome density, suggesting that TCP4 required TCL1 for inhibition of trichome initiation and TCL2 is not capable of compensating for the loss of TCL1. Analysis of the tcp2;4;10;zfp8 mutant showed that ZFP8 acts downstream to the TCP genes. Taken together, we conclude that TCPs act upstream to GIS, ZFP8, TCL1/2 to maintain a balanced distribution and differentiation of trichome cells. TARANI (TNI) negatively regulates the trichome differentiation Analysis of trichome development in the tni mutant showed that the TNI protein negatively regulates trichome differentiation alone, without affecting trichome density. TARANI encodes the deubiquitinase enzyme UBP14 in Arabidopsis (Premananda, K., PhD Thesis, 2014). Because of the G→A point mutation at the junction of third intron and fourth exon, two different transcripts are formed in the tni mutant; the wild type TNI transcript and an aberrant TNIintron transcript where the 3rd intron is incorporated in frame. This result in a two-fold down-regulation of the TNI transcript in homozygous tni plants compared to Col-0. To determine whether the tni trichome phenotype is caused by this decrease in TNI level or by the presence of the aberrant transcript TNIintron, we generated a transgenic line where TNI, TNIintron and an artificial micro RNA that targets TNI specifically in the trichome cells under the control of GL2 promoter. Analysis of the trichome-branching phenotype in these transgenic lines revealed that, interestingly, both under and over-expression of TNI leads to increased trichome branching, emphasizing the requirement for a balanced amount of TNI protein in the cell for proper trichome development. Genetic analysis of tni with known trichome branching mutants has shown that BLT is epistatic to TNI. Based on the genetic and molecular data, we hypothesize that in tni mutant, there is an increased amount of BLT protein that leads to increased trichome branching. To test this, we have raised a 35S::HA-BLT transgenic line, which shows hyper branched trichome phenotype. Analysis of the tni;35S::HA-BLT phenotype, and comparison of BLT protein level between Col-0 and tni using anti-HA antibody, would test our hypothesis that BLT is degraded by TNI to suppress trichome branching. We are currently in the process of generating the tni;35S::HA-BLT line and hope to obtain the data by the time of thesis defense examination. Thus, we have added one more transcription factor, TCP4 to the existing trichome developmental pathway (Fig. 6). We have investigated the detailed molecular mechanism for TCP-mediated regulation of trichome differentiation and have laid the foundation for further studies on role of TCP genes in trichome initiation. In the mutants of TCP genes, it is known that the pavement cell differentiation is reduced and the studies reported here have shown that the loss of TCP function has an opposite effect on trichome differentiation; the branching is increased in the TCP mutants. It is possible that the TCP proteins collaborate with different partners in these two cell types to bring about opposite effect on differentiation. From the second part of this study, we have added a protein degradation factor, TNI/UBP14, to the existing trichome differentiation pathway (Fig. 6). We have shown that, the balanced amount of TNI protein is required for normal trichome development. Unpublished data from our laboratory has shown that TNI is involved in conversion of free polyubiquitin chains into monoubiquitin, which is an essential step in proteasome-mediated degradation of all target proteins (Parinita Majumdar and Utpal Nath). BLT could be one such target protein that is upregulated in the tni mutant leading to hyper-branched trichomes. Increased trichome branching in the 35S::HA-BLT supports this hypothesis. Comparative estimation of the BLT levels between Col-0 and tni plants would enable us to demonstrate this.