Functional And Evolutionary Analysis Of Trehalose-6-phosphate Synthase In Moringa

Moringa oleifera Lam.is a perennial woody plant with high economic value.Because it is rich in dietary fiber,plant protein,vitamins,minerals,and flavonoids,Moringa has been used in food,health,and cosmetic products,and as a woody feed crop among other applications.Moringa is light-loving,heat-and drought-resistant,fast growing,flowers year round,and has a relatively small genome;this makes it an excellent model for studying the molecular mechanisms in flowering woody plants.Based on analysis of the Moringa leaf transcriptome under a long-day photoperiod,we identified candidate genes involved in photoperiod regulation.We analyzed the phylogeny,expression patterns,subcellular localization,and biological functions of M.oleifera trehalose-6-phosphate(Mo TPS).We characterized the growth phenotypes of a tps1 tps2 tps3/+ tps4 quadruple mutant in Arabidopsis.The effects of multiple TPS mutations on plant development,flowering time,phytohormone levels,and gene expression were also analyzed.The main results are as follows.1.In total,7,153 differentially expressed genes(DEGs)were identified based on comparison of two groups(ZT＿20h vs.ZT＿16h and ZT＿4h vs.ZT＿20h).From ZT=16 h to ZT=20h and ZT=4 h,cluster analysis showed that the number of downregulated DEGs was much greater than that of upregulated DEGs.Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis showed that DEGs in the starch and sucrose metabolism pathway,and the circadian rhythm pathway,were significantly enriched.In the starch and sucrose metabolism pathway,Mo TPS expression varied greatly between light and dark conditions.q RT-PCR analysis showed that most genes in the Mo TPS gene family expression levels that varied based on the light level under long-day conditions;this suggested that Mo TPS gene transcription is regulated by light and dark,and may also be involved in photoperiod regulation in Moringa leaves.2.The Mo TPS gene family has eight members,which are divided into subfamily I(Mo TPS1 and Mo TPS2)and subfamily II(Mo TPS3 to Mo TPS8).A large-scale replication event occurred in the Mo TPS gene family 150–167 million years ago.Mo TPS genes are undergoing strong purification selection;there are two positive selection sites,252-E and 45-E,which may accelerate new functions of Mo TPS proteins.3.The eight Mo TPS genes are expressed in different tissues,and the expression patterns differ markedly among subfamilies.Subfamily I expression levels are higher in stem tips and leaves than in other tissues,while the six genes of subfamily II are expressed at higher levels in petioles,stems,and roots.Relative to the control,subfamily II gene expression levels were increased in different tissues by high temperature stress(42°C),and Mo TPS2 and Mo TPS3 expression levels were increased by drought stress in most tissues.The expression of most Mo TPS genes was induced by salt stress(1 M Na Cl)in petioles,roots,and leaves,and by low temperature stress in stems.These results indicate that Mo TPS genes may be involved in abiotic stress responses in different tissues.4.The tps1Δtps2Δ double mutant YSH567 yeast functional complementation experiment demonstrated that Mo TPS1 has trehalose-6-phosphate synthase(Tre6P)activity and can complement the growth defect of YSH567 on glucose medium,while Mo TPS2 cannot.Under short-day conditions,the flowering of wild-type Col-0 Arabidopsis plants overexpressing Mo TPS1 or Mo TPS2 was significantly accelerated;however,it did not differ much from that of the wild type under long-day conditions.These results indicate that sufficient nutrients are accumulated during the day(for consumption at night)under long-day conditions,and that the Mo TPS1 or Mo TPS2 genes are functionally redundant with the At TPS genes.Under short-day conditions,however,the longer night time requires additional TPS/Tre6 P to regulate the degradation rate of starch,and the rate of sucrose synthesis.5.The amount of Tre6 P in tps1 tps2 tps3/+ tps4 quadruple mutant plants was significantly reduced.Compared with wild-type plants,the tps1 tps2 tps3/+ tps4 plants exhibited a lateflowering phenotype under both long-and short-day conditions,indicating that the At TPS gene is a positive regulator of flowering in Arabidopsis,and that mutation of the At TPS gene may affect the photoperiod pathway in Arabidopsis.The expression patterns of SOC1,FT,and TSF genes in tps1 tps2 tps3/+ tps4 plants were similar to those of wild-type Col-0,but the expression levels were significantly downregulated.These results indicate that the At TPS gene can participate in flowering regulation in Arabidopsis through the photoperiod pathway.6.The flowering time of tps1 tps2 and tps3/+ tps4 mutant plants was not only delayed,but also very irregular,with flowering occurring between days 42 and 79.Apical dominance was inhibited in the late flowering tps1 tps2 tps3/+ tps4 plants and multiple apical growth points appeared.In 25-day-old tps1 tps2 tps3/+ tps4 mutant plants,the auxin content was significantly reduced and the cytokinin content was significantly increased.Expression of the AO4 gene in the indole acetic acid(IAA)synthesis pathway was reduced 72-fold,and the expression of the cytokinin signal inhibitor AHP6 gene was decreased 42-fold.During plant organ initiation,AHP6 can determine the key period for flower organ primordium activation when auxin and cytokinin signals overlap.This indicates that At TPS mutation affects the levels of auxin and cytokinin in plants,leading to the loss of apical dominance.7.Under both long-and short-day conditions,spraying with the GA3 can still cause tps1tps2 tps3/+ tps4 plants to flower earlier,indicating that gibberellin metabolism and signal transduction may not be affected by an At TPS gene mutation.The expression of key genes involved in GA synthesis,metabolism,and signal transduction in tps1 tps2 tps3/+ tps4 mutants did not change significantly during the various developmental stages.These results indicate that the At TPS gene may not participate in a gibberellin-dependent flowering pathway.8.The FLOWERING LOCUS C(FLC)gene is a strong inhibitor of flowering,and a core component of the autonomic and vernalization pathways.As seedlings transition from vegetative growth to reproductive growth,FLC expression is gradually reduced.The pattern of FLC expression in the tps1 tps2 tps3/+ tps4 mutant during development was similar to that of wild-type Col-0,with the highest expression level seen on day 5 and the lowest on day 25.The level of FLC expression in the mutant was significantly higher than that in the wild-type Col-0,with expression on day 25 in the mutant higher being even than that on day 5 in the wild type.In the tps1 tps2 tps3/+ tps4 mutant,MAF4 and MAF5 expression levels were also significantly upregulated.MAF4,MAF5,and FLC belong to the MADS-box transcription factor family,which regulates plant flowering through vernalization/autonomous pathways.These results suggested that the At TPS gene may regulate flowering by participating in an autonomous/vernalization pathway.