Roles Of 13-Lipoxygenase In Stomatal Movement Regulation In Arabidopsis

Lipoxygenase is a kind of nonheme iron containing fatty acid dioxygenases,which are ubiquitous in plants and animals.LOXs catalyze the oxygenation of polyunsaturated fatty acid and generate a series of compounds called oxylipins.These substances are considered to be important signaling molecules involved in many physiological processes.Studies have been reported that LOX may regulate stomatal movement in response to various stimuli,but many of the details in this process are unclear.Based on previous research in our lab,this study focus on the function and possible mechanism of 13-LOXs?LOX2 and LOX6?,which are highly expressed in Arabidopsis guard cells,in regulating stomatal movement.In order to better understand the information of 13-LOXs,we determined the subcellular localization of LOX2 and LOX6 through the protoplasts transient expression and permanent expression of transgenic plants assays.It was found that LOX2 and LOX6 are both localized in chloroplasts.Quantitative PCR results revealed that the gene expression of LOX2 and LOX6 were upregulated when Arabidopsis seedlings were treated with NO donor SNP for 1 hour.Leaf detachment assay showed that the lox6 mutants lose water faster than those of wild-type plants though they have similar numbers of stomata.Therefore,the difference of water loss may be associated with stomatal aperture.Stomatal bioassay indicated that exogenous SNP treatment can induce stomatal closure in wild-type,but not for lox6 mutant.Collectively,these results showed that LOX6 was involved in SNP-induced stomatal closure.To further determine the relationship between LOX2 and LOX6,we obtained the lox2lox6 double mutant.Because previous report has revealed that LOX play a key role in plant roots,we focus our test on the phenotype of roots between wild-type and double mutant,and found that there was no obvious difference on primary root elongation and lateral root density,in addition they did not show any abnormal phenotype under SNP treatment.In terms of stomatal movement,lox2lox6 exhibited the similar water loss rate and stomatal density relative to wild-type.However,the lox2lox6 mutant,to some extent,impaired SNP-induced stomatal closure.On the other hand,we also detected changes of Ca²⁺ concentration in guard cells by confocal microscope after incubation of epidermal peels in the buffer containing fluorescence probe.It was found that SNP,ABA can increase Ca²⁺ concentration in cytoplasm,while in lox6 mutant,SNP-mediated improvement of Ca²⁺ was inhibited,but was not affected in ABA treatment;Absence of LOX2 did not affect the regulation of SNP but blocked ABA-induced Ca²⁺ increase;The same changes trend was occurred in lox2lox6 as well as lox6,it was also suppressed in SNP-induced Ca²⁺ increase.In conclusion,the stomatal movement induced by NO is mediated by the cytoplasmic Ca²⁺ change,which is affected by LOX6.LOX2 and LOX6 in guard cells may be functioning through different signaling pathways.