Analysis of the interaction between OsRZFP34, a rice RING zinc-finger protein, with its up-regulated proteins

• Main Authors: Pei-Jyun Lian, 連珮君
• Other Authors: Ching-Hui Yeh
• Format: Others
• Language: zh-TW
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/4992nb

碩士 === 國立中央大學 === 生命科學系 === 103 === RING zinc-finger proteins (RZFPs) are known to be essential in the regulation of plant processes, including responses to biotic and abiotic stress. By oligo microarray expression profiling, we identified a rice RZFP gene, OsRZFP34, whose gene expression increased with high temperature or abscisic acid (ABA) treatment. Our previous study has shown that OsRZFP34 is involved in transpiration cooling and may modulate the genes implicated in Ca2+ sensing, K+ regulator, and ABA response to control stomata opening in rice (Hsu et al., 2014). Here we further investigate the interaction between OsRZFP34 and a calmodulin binding protein OsCaMBP (RAP-DB accession no. Os12g0556200), a potassium transporter OsHAK5 (RAP-DB accession no. Os01g0930400), or an ABA response transcriptional factor OsWRKY80 (RAP-DB accession no. Os09g0481700) and OsRZFP34-regulated mechanism of stomatal opening. Firstly, we determined subcellular localization of these proteins and used bimolecular fluorescence complementation (BiFC) to analyze the interaction between OsRZFP34 and each of the 3 proteins. The results of green fluorescent protein 6 (GFP6) localization experiment showed that OsCaMBP-GFP6 and OsWRKY80-GFP6 fusion proteins were mainly associated with nucleus, while OsHAK5-GFP6 fusion protein was localized in the plant plasma membrane of onion epidermal cells that corresponded with Horie et al., 2011. By BiFC analysis, we found that our OsRZFP34 physically interacted with OsCaMBP and OsWRKY80 only in the nucleus, and with OsHAK5 in the nucleus and plasma membrane. In contrast, the fluorescent signals for co-expression of OsRZFP34 and OsHAK5 were found on the plasma membrane under high temperature. Then we used the Ca2+-sensitive fluorescent dye Fluo-3/AM to detect the [Ca2+]cyt of guard cells treated with heat, ABA, H2O2, Ca2+, or K+. The results indicated that the guard cells of OsRZFP34-overexpressing plants showed lower [Ca2+]cyt than that of the WT after treatment. Taken together, these results demonstrate that OsRZFP34 can interact with OsCaMBP, OsHAK5, and OsWRKY80 and modulate [Ca2+]cyt of guard cells to regulate stomatal opening in rice.