Molecular Mechanism Analysis Of Synergistic Participation Of KCS5 And KCS6/CER6 In Epidermal Wax Synthesis In Arabidopsis Thaliana

Plant epidermal wax is a layer of hydrophobic lipids covering the surface of the aboveground part of land plants.As the first barrier for plants to deal with the changes of the external environment,it plays an important role in plants to deal with biological and abiotic stress factors such as diseases and pests,ultraviolet radiation and drought.It is a potential universal target for crop stress resistance genetic improvement.Therefore,the study on the synthesis mechanism of plant epidermal wax not only has important theoretical significance,but also has high application value in crop genetic engineering improvement.Plant epidermal wax is synthesized in epidermal cells.Its components mainly include very long chain fatty acids(VLCFA)and their derivatives,aldehydes,alkanes,secondary alcohols,ketones,primary alcohols and esters.Fatty acid extension complex enzyme(FAE)is responsible for the extension process of wax synthesis precursor VLCFA-Co A and plays a key role in wax synthesis.As a key enzyme in FAE,KCS plays a key role in determining the chain length extension of VLCFA-Co A.Up to now,several KCS genes have been identified in Arabidopsis to participate in wax synthesis,including KCS1,KCS2,KCS6/CER6,KCS20 and KCS16.Among them,KCS6/CER6 is mainly involved in the extension process from C26-Co A to C28-Co A and plays the most key role in epidermal wax synthesis.KCS5 is highly homologous with KCS6/CER6 and is specifically differentiated from cruciferous plants.But is this gene involved in wax synthesis? Is there functional redundancy with KCS6/CER6? Both deserve in-depth discussion.In order to further clarify the function of KCS6/CER6 gene in wax synthesis,we first analyzed the epidermal wax components of stems,flowers and leaves of KCS6/CER6 mutants cer6-2 and cer6-3,and found that different mutations of KCS6/CER6 gene have different effects on wax synthesis.cer6-3 is a KCS6/CER6 function complete deletion mutant,and the synthesis of C26 and longer chain waxy components in its waxy is inhibited,while the synthesis of C28 and longer chain waxy components in the function partial deletion mutant cer6-2 is inhibited,indicating that KCS6/CER6 is mainly responsible for the extension process from C24-Co A to C26-Co A.In addition,the synthesis of C32 and longer chain waxy components was significantly affected in cer6-2 and cer6-3 leaves,but the waxy components related to C28 and C30 were not affected.Therefore,we speculate that KCS5 may also be involved in the synthesis of C28 and C30 waxes in the leaves of cer6 mutant.In order to verify the above speculation and explore the function of KCS5 in wax synthesis,we knocked out KCS5 gene by CRISPR-Cas9 method to obtain kcs5-1 and kcs5-2mutants.Through wax phenotype analysis,we found that KCS5 has a similar function to KCS6/CER6 in the wax synthesis of Arabidopsis flowers and rosette leaves,that is,it is responsible for the synthesis of C26 and longer carbon chain wax components,However,its effect on wax synthesis is significantly weaker than KCS6/CER6.We speculate that this may be related to the low expression of KCS5 gene.It is worth noting that,unlike KCS6/CER6,KCS5 mutation significantly inhibited the synthesis of C30 chain long waxy components,especially C29 alkanes,indicating that there are differences in their functions in leaf waxy synthesis.Then,through the heterologous expression analysis of yeast and tobacco,we found that KCS5 showed higher catalytic activity in the synthesis of C30 acid than KCS6/CER6.In addition,we also confirmed that KCS5 and KCS6 are similar and can also interact with CER2 and CER26 to catalyze the synthesis of C32-C34 fatty acids.In order to further explore the function and relationship of KCS5 and KCS6/CER6 in the wax synthesis of Arabidopsis,we introduced KCS6/CER6 mutation in the context of kcs5-1by CRISPR-Cas9 method to obtain kcs5-1 cer6-4 double mutant.Wax phenotype analysis showed that the wax content in kcs5-1 cer6-4 double mutant decreased significantly compared with kcs5-1 and cer6-4 single mutant,indicating that there was functional redundancy in KCS5 and KCS6/CER6 in the biosynthesis of epidermal wax.Our further study found that the non-stomatal water loss rate of kcs5-1 cer6-4 double mutant was significantly higher than that of kcs5-1,cer6-4 single mutant and wild type,and the accumulation of drought-induced wax was completely blocked in kcs5-1 cer6-4 double mutant,suggesting that KCS5 and KCS6/CER6 play a key role in responding to drought-induced wax synthesis and maintaining non-stomatal water loss.In conclusion,we analyzed the function of KCS5 through mutant analysis,yeast heterologous expression,Bi LC(Bimolecular Luciferase Complementation)technology and stress-related physiological experiments,clarified the molecular mechanism of KCS5 and KCS6/CER6 participating in Arabidopsis epidermal wax synthesis and further improved the fine network of plant wax synthesis,which has certain theoretical significance and practical value.