Functional Studies Of OSCA1 Interacting Proteins And OSCA Family Members

Water is essential for plant growth and developement.Under water deficiency,the plant development is significantly reduced.OSCA1 is a hyperosmolality-gated non-selective cation channel,which plays an important role to deal with drought in plants,but it is not clear how OSCA1 is regulated during the response to hyperosmotic stress,and the biological functions of other members of OSCA family.Exploring the functional domains of the OSCA1 protein structure helps us understand the mechanism OSCA1 perceiving stimuli.In this study,we verified that OSIP-CM was an interaction factor of OSCA1 by using yeast two-hybrid and bimolecular fluorescence complementation?Bi FC?.By heterologous expression of OSCA1 and OSIP-CM together in HEK 293T cells,we found that OSIP-CM could feedback inhibit the OSCA1-mediated calcium signal induced by hypertonic stimulation.In order to artificially modify the channel characterization of OSCA1 to improve the sensitivity of OSCA1 to stress and genetically modify the drought-specific resistance in plant,we designed experiments to study the functional regions and key regulatory sites of OSCA1.By analyzing OSIP-CM binding site and OSCA1 protein structure,we made three site-directed amino acid mutations in OSCA1.The experiments showed that osca1 still interacted with OSIP-CM by yeast two-hybrid.It indicates that there are multiple binding sites between OSCA1 and OSIP-CM.Next,we will design multiple site mutations to find the key functional region?s?of OSCA1,therefore,the OSCA1 gene could be artificially modified to increase the channel activity and increase the sensitivity to permeation perception.Ca²⁺is a crucial second messenger that regulates pollen germination and tube growth,Ca²⁺is very important for the growth of pollen tubes.It has been found that the calcium ions on the top of pollen tubes were destroyed by calcium chelators,which inhibited the growth of pollen tubes.And the cytoplasmic osmolality of pollen tubes changes led to the ionic and growth kinetics changes.Therefore,both Ca²⁺and osmotic stress correlated with effects of the growth of pollen tube.During the growth of the pollen tubes,the periodic fluctuation of calcium concentration is also called calcium oscillation,and its oscillation period is the same as the period of the pollen tubes growth.Hypertonicity can change calcium oscillations in pollen tubes,but the mechanism is unclear.This project aims to discover the calcium ion channel?s?responding to pollen calcium oscillations by phenotypic screening pollen tip growth in OSCA family T-DNA lines.Based on OSCA1 as a mechanical channel probably perceiving the tension of the pollen tube cell membrane,we determined that there are two genes,OSCA D and OSCA F,played an important role in pollen tubes through analysis of transcriptome expression and phenotype.By studying the physiological phenotype of osca d-f,we found that the mutant pollen grain viability was as normal as Wild-type,but the pollen germination and tube growth were inhibited under high osmotic medium,and seeds development was also defective.Based on those phenotypes,we will characterize calcium channels responding to calcium oscillations of the pollen tips.