Role of the miR319-JAW-TCP module in the growth and development of the female reproductive organ in Arabidopsis thaliana

Abstract

Named after their founding members TEOSINTE BRANCHED1, CYCLOIDEA, and PROLIFERATING CELL FACTORs, the TCP transcription factors have been investigated for their diverse role in plant growth and development. The five miR319-regulated JAW-TCPs in Arabidopsis - TCP2, 3, 4, 10 & 24 - belong to the CINCINNATA-like class II TCP proteins. These five TCPs, along with three more non-JAW-TCPs (TCP5, 13 & 17), are specifically required for the growth & developmental processes of vegetative organs such as leaf morphogenesis & senescence, hypocotyl elongation, epidermal cell patterning and timing to flowering transition. Recent reports have implicated the role of the miR319 and JAW-TCPs module petal growth, patterning ovules and the apical part of the pistil. However, our knowledge of the role of these proteins in floral organ development is far from complete due to the inherent difficulty in studying the TCP function due to extensive functional redundancy among their members. In this thesis, we have addressed this lacuna by examining the role of miR319 and JAW-TCPs in pistil growth and development.

First, we show that the five JAW-TCPs are dispensable for pistil patterning but necessary for fruit morphogenesis. Through genetic interaction and biochemical studies, we demonstrate that JAW-TCPs directly activate the FRUITFULL gene to promote fruit morphogenesis. To analyze the pistil growth at the cellular level, we generated several tcp and fruitful loss-of-function lines where the plasma membrane was marked with a fluorescent protein to make the cell outline visible. We studied the cellular growth parameters during fruit growth by analyzing these marked mutant pistils using confocal microscopy. These experiments showed that the JAW-TCPs are required for anisotropic cell growth, specifically along the length axis, leading to fruit elongation. Further, RT-qPCR, reporter gene analysis, and genetic interaction studies showed that the genes that are restricted to the valve margin by the FRUITFULL protein are ectopically expressed in the TCP-FUL domain of the pistil, thus ascribing a gene patterning function to the JAW-TCPs in growing fruits.

Next, we analyzed the publicly available transcriptome datasets in JAW-TCP loss and gain-of-function mutants and made a preliminary list of transcriptional targets. Using two in silico filtering strategies, we arrived at a short list of high-confidence targets. Further, these targets were biochemically validated by RT-qPCR, ChIP-qPCR and EMSA studies. Subsequently, one target, ACS4, was chosen for further investigation. Previous studies have implicated ACS4 and its homologues in regulating the fertilization of ovules by the male gametes in the pollen tubes. By generating a pollen reporter line, pACA9::G3GFP-GUS, we identified a function for JAW-TCPs during pollen growth inside the female organ. Finally, we reported the role miR319 in several aspects of carpel patterning by using miR319-knockdown lines and the gain of TCP4 function in transgenic plants. Congenital carpel fusion was primarily affected in both types of transgenic lines. We observed additional patterning defects in the TCP4 gain of function lines, such as increased carpels, ovule patterning, and accessory tissue patterning defects. We conclude this chapter with a small-scale yeast two-hybrid screen to investigate the purported mechanism of TCP action in carpel development. We report the physical interaction between TCP4 and AINTEGUMENTA & SPATULA, the two proteins involved in carpel patterning, suggesting that miR319 repression of JAW-TCP may prevent the formation of an inhibitory heteromeric protein complex involving JAW-TCPs to promote carpel development.

In conclusion, we have uncovered previously unreported aspects of JAW-TCP functions in pistil patterning and fruit morphogenesis essential for plant reproduction.