The Mechanism Of NPH3Regulating Hypocotyls Curve In Arabidopsis

Light is an important environmental factor regulating plant growth and development. Plants can getmore light through phototropism. Phototropism is an adaptive response for plants to complex andchangeable external environment. The plant phototropism adjustment involves a number of light receptorsand phototropins is the main blue light receptors. Phototropins mainly mediate phototropism, stomatalopening, chloroplast relocation movements, rapid inhibition of hypocotyl growth, as well as leaf expansion.Phototropins have a serine/threonine kinase domain located within the C terminus and LOV domain in theN terminus, phototropins can phosphorylate itself in response to blue light, and a FMN molecule bound tothe LOV domain through the cysteine.In phototropic responses, PHOT1and PHOT2can occur autophosphorylation and bind to NPH3,PKS1or other proteins to regulate the polarized auxin transport. This will be lead to a asymmetricdistribution of auxin in the hypocotyls to promote bending toward the light. In addition, NPH3can directlyinteract with PHOT1and PHOT2based on yeast two-hybrid. nph3mutant shows no hypocotylphototropism in response to both weak and strong unilateral blue light, which suggested NPH3mayfunction as a molecular switch to determine the downstream transmission of PHOT1and PHOT2.Moreover, yeast two-hybrid showed direct interaction between NPH3and two auxin carriers, AUX1andPIN1, which indicated that NPH3may regulate auxin transport, and then cause a differential distribution onboth sides of hypocotyl. Our previous studies have confirmed that Ca2+involves in regulating hypocotylphototropism under strong blue light, but yeast two-hybrid showed that NPH3had no interaction with Ca2+binding proteins CaM, which suggesting that there may be other mechanisms involved in the regulation ofhypocotyl phototropism. What’s more, our previous studies show that PKS1interact with PHOT1, PHOT2,NPH3and calmodulin (CAM4/CAM5/CAM7). However, phenotypic analysis show that PKSs(including PKS1, PKS2and PKS4) single gene mutation, have a similar to the wild type performancecurved toward the light, double mutant was significantly lower in the light of its bending, whichcorresponds to three mutations in Arabidopsis hypocotyl bending hardly occurs to the light. These resultsimply that PKSs protein in the regulation of Arabidopsis hypocotyl reaction to light bending functionalredundancy. Because of the nph3mutant showed nonphototropic hypocotyl under almost all experimentalconditions, taken together with the above analysis results, we speculate that the hypocotyls bend toward thelight is mainly mediated by NPH3, and PKSs family proteins play an important role in signal transductionpathways.Phototropism is due to the asymmetrical distribution of auxin and plant asymmetric growth. Inaddition, the nph3mutant showed nonphototropic hypocotyl, which whether is depended on its regulationof auxin transport have not been reported. So we constructed some kinds of genetic materials such as nph3DR5:GFP, nph3PIN1:GFP, nph3AUX1:GFP, looking forward to observe the differential distribution ofauxin. Unfortunately, blue light treatment on those materials showed no difference with wild type byscanning in accordance with time course. Considering that the mutant is screened by EMS mutagenesis, Compared with wild type, NPH3had a point mutation (G-to-A) at1517bp in the gene DNA which lead tothe codon encoding the tryptophan becomed the termination codon. So there is a expression about the frontpart of NPH3. We have know that the protein structure of NPH3is divided into three parts：an N-terminalBTB (Broad-Complex/Tramtrack/Bric-a-brac) domain, an NPH3domain in the middle of each protein, aC-terminal coiled coil. The coiled-coil region of NPH3interaction with PHOT, the BTB domain interactswith CULLIN3(CUL3), which regulates plant growth and development in Arabidopsis thaliana. Theexperiments show that the asymmetric distribution of auxin is not light-induced and extending downwardfrom the hook of the hypocotyl even toward the light source. Therefore we suppose that the point mutationof NPH3lead to optical signal not transmitted to the downstream and the part of NPH3protein willinteracts with CUL3, PIN1, AUX1to regulate auxin transport, furthermore because there is no lightinduced and auxin only unilateral downward transport in hypocotyls. We suppose that it can also makesome other gene mutated, such as EXPA1or EXPA8, which can encode a-relaxin and a-relaxin effectivelypromote the extension of the cell wall, it may be a key for hypocotyl curve to light. Of course, it is just aspeculation now, we need to further research. So we prepare to use T-DNA insertion mutant of nph3andhybridization with WT DR5:GFP, WT AUX1:GFP, WT PIN1:GFP for the next step research.