| Long-chain acyl-Co A synthetase(LACS)is one of the important enzymes that play role in the metabolism of fatty acids such as phospholipids biosynthesis,and triacylglycerol(TAG)metabolism,wax,cutin,suberin synthesis,and fatty acid β-oxidation in plants.These molecules play a vital role in the demarcation of cells and organelles,as the source of stored energy,and as a barrier against biotic and abiotic stresses.LACS has been extensively studied in Arabidopsis,however,its evolutionary mechanism and functional role in green plants are still not fully explored.LACS genes possess diverse functions and expression patterns in different species.It is important to understand the classification and evolutionary relationship of LACS genes to better understand the functional roles of specific lineages.Here we performed genome-wide identification of the LACS gene family including phylogenetic analysis,gene structure identification,subcellular localization,tissue-specific expression,determination of conserved motifs,and role in suberin synthesis.In total 122 LACS genes were identified in green plant species including green algae,mosses,lycophytes,monocots,and eudicots.We identified 697 sequences but629 sequences were selected due to alignment and duplication errors.The whole data set consisted of Rhodophyta(2),Chlorophyta(13),Embryophyta(3),Tracheophyte(1),and Angiosperms(105).The retrieved amino acid sequences were in the range of 271 to 1056 residues.The intron/exon pattern revealed the diversity and the maximum of the LACS from land plants is up to 22 introns,indicating a potential increase of introns in land plant LACS.Phylogenetic analysis revealed six major clades based on different LACS genes relationship and distribution in different green plants.The classification pattern was well supported by the subsequent analysis such as gene structure and conserved motif analysis.LACS subcellular localization analysis revealed that genes are specifically distributed in various subcellular compartments such as endoplasmic reticulum(ER),plastids,plasma membrane,and peroxisomes.We obtained tissue specificity and expression pattern of LACS genes using the Gene Vestigator website.Among them we found that At LACS2 was specifically highly expressed in the suberinproducing endothelial layer,suggesting that this gene may be involved in the synthesis of suberin.In addition to the reported involvement in cutin synthesis,lacs2-3 mutant also showed expression in suberin synthesis.Suberin analysis also confirmed that At LACS2 plays an important role in suberin synthesis.Furthermore,the role of LACS genes was examined specifically in Glycine max which revealed six major clades based on the obtained 19 sequences from Arabidopsis and G.max.The q RT-PCR analysis revealed a differential expression pattern of Gm LACS2 in different organs.Gm LACS2-1 was predominantly expressed in mature and young leaves.Gm LACS2-2 was highly expressed in sprouts and young leaves whereas Gm LACS2-3 was significantly higher in mature leaves.The expression pattern analysis detected that Gm LACS2-4 was extremely expressed in the root,sprout,young pod,and mature pod.To find out which Gm LACS2 gene might be involved in cuticle synthesis,we checked the responses of these genes to several chemicals triggering drought stress including sodium chloride(Na Cl),polyethylene glycol(PEG),and abscisic acid(ABA).Overall results showed that Gm LACS2-2 and Gm LACS2-3 were strongly induced by all treatments,Gm LACS2-1 specifically responded to ABA treatment,and the expression of Gm LACS2-4 was not induced by the three stress factors.It indicates that these genes may play different roles under different abiotic stress conditions.To check the cuticle permeability,we stained Col-0,atlacs2,and atlacs2/Gm LACS2-3 C1 respectively with TB staining solution.The atlacs2 mutant showed dark staining due to cuticle defect,while the staining results of the atlacs2/Gm LACS2-3 C1 leaves were similar to that of wild type,indicating that the soybean Gm LACS2-3 gene can complement the phenotype of the Arabidopsis atlacs2 mutant leaf permeability defect,and also it is further shown that the function of LACS2 is highly conserved in both species.The overexpression of Gm LACS2-3 in wild-type Arabidopsis Col-0 significantly increased the amounts of cutin and suberin but had little effect on wax amounts.Drought tolerance analysis of transgenic lines found that overexpression of Gm LACS2-3 can now increase the tolerance of transgenic lines to drought.In conclusion,this study first revealed the evolutionary pattern and functional diversity of LACS genes,which not only enriched our understanding of the functions of LACS genes in different plants but also laid a foundation for further understanding and evaluation of LACS family genes.In addition,by verifying the function of the soybean Gm LACS2 s gene,we believe that Gm LACS2-3 plays an important role in preventing water loss,providing a potential target gene for future genetic improvement of soybean drought resistance... |
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