Screening And Identification Of The Proteins Interacting With LCU In Arabidopsis

Transcription factors play important roles in the regulation of plant growth and development, as well as their response to environment. The interaction between TFs and genomic DNA defines many functional features and biochemical processes through control target genes expression. Except for conservative transcription factors, for example NF-Y family, there are plant-specific transcription factors, such as TCP family proteins. In Arabidopsis, there are 24 putative members in TCP family. Studies had shown that classⅡmembers regulate growth in different organs, through the inhibition of cell proliferation or growth. While much less is known about calss I members, many are remain to be detected.LCU in this paper is a putative member of TCP transcription factor gene family found through forward genetic method, which is also termed TCP7. The semi-dominant mutant lcu-D had many obvious phenotypes, such as leaf upwardly curling. To investigate the mechanism of LCU function, we constructed LCU/lcu fused to Ga14 DNA binding domain, BD-LCU, BD-lcu. And then the bait plasmind and seedling cDNA libriary were cotransformated into yeast strain AH 109. After screening, sequencing and retested in yeast, we screened 11 proteins interesting interacted with LCU and lcu, such as transcription factor TCP 14, TCP21, NF-YC1, NF-YC3. Meawhile, we found proteins interacted with lcu only. But we did not find proteins specifically interacted with LCU. These results may display mutant protein lcu has other novel functions besides LCU.Yeast two-hybrid analysis indicated LCU and lcu interacted with most members of TCP Class I subfamily to form homodomer or heterodimer, except TCP 19 and TCP20, and these proteins prefered to lcu. NF-YC1, NF-YC3, NF-YC4 and NF-YC9 also interacted with LCU or lcu.TCP21, which is the most homolog of LCU, interacted with NF-YC proteins.We detected LCU and lcu co-localized in nucleus with NF-YC1 and NF-YC3, so it provided space for their interaction. To detect whether LCU and NF-YC expression are regulated by each other, we analysed the gene expression level of LCU or NF-YC in corresponding mutants by Real-time PCR. The results demonstrated that NF-YC were upregulated in lcu-D mutant, especially NF-YC4;while in nf-yc mutants the expression level of TCP7 and TCP21 had no obvious change.To illustrate the target genes regulated by LCU and NF-Y together, we detected expression level of CYCD3;1 and CYCD4;1. CYCD3;1 was up-regulated while CYCD4; 1 was down-regulated in lcu-D mutant. And the two cyclin gene had slight down-regulated in LCU microRNA lines, LCU over-expressed lines, che-1 mutants and nf-yc349 triple mutants.In brief, LCU interacted with NF-YC protein to regulate plant morphogenesis directly or indirectly through some unkown way to regulate cell cycle genes.