Plant hormones play a crucial role in acclimation to abiotic stress and regulate the growth and development and often alter gene expression.Our study revealed that the expression of several genes involved with hormones were changed due to the impact of OsCam1–1 over expression under salt stress.Lipoxygenaseen coded by three HT salt-responsive DEGs including homologs of AtLOX2 and AtLOX5, is the enzyme in the early step of the jasmonate biosynthesis pathway.JA plays a role in the physiological response in plants under biotic and abiotic stress.An earlier report has shown that the absence of AtLOX2 expression results in no change under normal conditions, but JA accumulation induced by wounding is absent and the expression of vsp, a wound-JA-induced gene, is also suppressed.In addition, three HT salt-responsive 12-oxo-PDA-reductasegenes identified encode JA precursor-catalyzed enzyme that catalyzes the cis- 12-oxophytodienoic acid reduction reaction.These findings suggest that the JA content might be enhanced by the over expression of OsCam1–1 under salt stress by enhancing the production of enzymes in the JA biosynthesis pathway.In addition, the expression of NCED and AAO genes participating in ABA biosynthesis was altered by the influence of OsCam1–1 over expression under salt stress.Our previous report has shown that the expression levels of NCED and AAO, and ABA content are enhanced in transgenic rice over-expressing OsCam1–1 under salt stress in comparison to wild type.Collectively,vertical hydroponic nft system the transcriptome indicates that OsCam1– 1 over expression likely has effects across biotic and abiotic stresses via plant hormonal regulation through JA and ABA.It has been suggested that biotic-abiotic stress crosstalk may occur via the MAPK/MPK cascade to regulate the plant hormone response to stress.
Transcription factors play roles as master regulators controlling clusters of genes in the plant regulation of the stress response.AP2/EREBP encoded by several HT salt-responsive DEGs, is in a large gene family of TFs that function in plant growth, primary and secondary metabolism, and response to hormones and environmental stimuli.Two AP2/EREBP DEGs were identified as OsDREB1A and OsDREB1B, and a previous report has shown that OsDREB1A-overexpressing transgenic Arabidopsis exhibit induced expression of target stress-inducible genes of Arabidopsis DREB1A and increased tolerance to drought, high salt and freezing stress, as compared with wild type.MYB, which was found encoded by several HT salt-responsive DEGs, is an important gene family of TFs, and several Arabidopsis MYB genes respond to hormone or stress.A previous report has shown that over expression of OsMYB48–1, which is a member of those DEGs, resulted in enhanced salt and drought tolerance in rice.Furthermore, OsMYB48–1 also controlled ABA biosynthesis by regulating the expression of OsNCED4 and OsNCED5 in response to drought stress.WRKY is a large TF family that responds to plant stress by regulating the plant hormone signal transduction pathway and is also involved in the biosynthesis of carbohydrate and secondary metabolites, senescence, and development.According to several reports, WRKY genes identified here as HT salt-responsive DEGs are involved in the biotic stress response.The evidence shows that OsWRKY53 can bind to mitogen-activated protein kinases, OsMPK3 and OsMPK6, and inhibit their activity, resulting in a reduction of JA, jasmonoylisoleucine and ethylene production and causing a suppression of herbivore defense ability.The expression of OsWRKY71 was induced by salicylic acid , JA, and 1-aminocyclo-propane-1-carboxylic acid.Over expression of OsWRKY71 affected the induction of OsNPR1 and OsPR1b expression, which are defense signaling genes, resulting in an enhancement of bacterial plant pathogen resistance.WRKY13 has been shown to regulate crosstalk between abiotic and biotic stress by suppressing the SNAC1 and WRKY45–1genes, which are involved in drought and bacterial infection, by binding to W-like-type cis-elements on their gene promoters.
OsWRKY62, which was down-regulated by effect of OsCam1–1 over expression and salt stress, was found in two splicing forms, short and full-length forms.Over expression of the full-length form of OsWRKY62 resulted in the suppression of blast fungus resistance.In contrast, the knockout Oswrky62 line showed an enhanced defense-related gene expression level and accumulation of phytoalexins.Based on the transcriptome profiles, OsCam1–1 over expression clearly affected the expression of transcription factors that are well-known to regulate both biotic and abiotic stress responses.Therefore, OsCam1–1 likely functions through the activity of these transcription factors in mediating biotic-abiotic crosstalk regulation via diverse mechanisms.According to our transcriptomics data analysis, plant hormones might mediate the regulation of these TFs, leading to the downstream acclimated phenotypes in response to diverse stresses.Secondary metabolites play important roles in acclimating the plant to the environment and stress conditions.A hydroxyphenylpyruvate dioxygenase , which participates in the first committed reaction in the vitamin E biosynthesis pathway, was highly expressed and enhanced by the effect of either OsCam1–1 over expression or salt stress.Previous evidence has shown that the expression of HPPD responded to oxidative stress in barley leaf because it was induced by senescence, methyl jasmonate, ethylene, hydrogen peroxide and herbicide; paraquat and 3–1,1-dimethylurea.Furthermore, a report on the expression of two rice laccasegenes , which were identified as HT salt-responsive DEG here, in yeast cells suggested that the laccases played roles in atrazine and isoproturon herbicide detoxification.In Arabidopsis, atlac1 and atlac2 mutants exhibited deficient root elongation under polyethylene glycoltreatment, while the atlac8 mutant showed early flowering and the atlac15 mutant showed abnormal seed color.In addition, the evidence revealed that the expression level of AtLAC2 was enhanced by salt and PEG treatment.
Previous evidence has shown that 3-ketoacyl-CoA synthase encoded by three HT salt-responsive DEGs, plays a role in wax biosynthesis.The kcs1–1 mutant exhibited reduced wax content, a thin seedling stem and low moisture sensitivity.Another report has shown that the expression of KCS20 and KCS2/DAISY, two other Arabidopsis 3-ketoacyl-CoA synthase genes, is induced by salt, ABA and drought conditions and that these genes play roles in cuticular wax and suberin biosynthesis in root.In addition, previous evidence has shown that Arabidopsis genes encoding class III triacylglycerol lipase encoded by four HT salt-responsive DEGs, are involved in many processes.At4g16070, which is orthologous to one of HT salt-responsive DEGs, was predicted to be a gene involved in stress or the Ca2+ signaling pathway.At4g16820 and At4g18550, which are orthologous to other rice DEGs are involved in seed germination, senescence or the stress response.At1g02660, which is orthologous to another rice DEG, is involved in the plant defense response signaling pathway.Finally, an early report showed that acyl-CoA dehydrogenase, which was identified encoded by another salt-responsive DEG, functions in mitochondrial β-oxidation in maze root tip under glucose starvation conditions.Based on these results, the activity of OsCam1–1 might affect lipid metabolism and possibly be linked to energy metabolism during salt stress.genes.A previous study has demonstrated that the transcript level of glucose-6-phosphate/phosphate translocatorin Arabidopsis correlates with the sugar level in leaf, and another study has suggested that GPT2 functions as a plastid anti-porter transporting glucose-6-phosphate into the plastid to support starch biosynthesis.Recently, a proteomic study has found that cucumber seed germination is enhanced by melatonin under high salt conditions via regulated energy metabolism and the up-regulation of proteins involved in glycolysis, the TCA cycle and the glyoxylate cycle.The transcriptome results herein illustrated possible changes in the cellular respiratory pathway in transgenic rice under salt stress.These lines of evidence suggest that OsCam1–1 may confer salt tolerance by regulating central energy metabolism.Salt stress inhibited the activity of granule-bound starch synthaseand suppressed the expression of GSSBI and GSSBII, resulting in a decrease in starch content in rice leaf.An earlier report has shown that Pokkali, the standard salt-tolerant rice, shows significantly higher starch concentration under salt stress than KDML 105, which was identified as a salt-sensitive cultivar.The transgenic KDML105 rice examined herein exhibited significant decrease in starch levels, but to a lesser extent than the wild type, while it showed improved maintenance of sucrose levels under salt stress, which probably reflected the higher salt tolerance ability.The transcriptome results revealed that several sucrose and starch degradation genes were up-regulated.An early report revealed that sucrose synthase, which its gene expression level was up-regulated to a higher level in transgenic rice, plays a role in starch synthesis by generating ADP-glucose or UDP-glucose through the cleavage of sucrose,nft hydroponic system which can be used for starch polymerization.Moreover, the findings showed that sucrose synthase activity correlated with starch and ADP-glucose accumulation in developing barley seed and that transgenic potato plants with a disrupted sucrose synthase gene were defective in starch accumulation.However, the transcriptome results herein showed that lower expression levels of several genes in the starch biosynthetic pathways in transgenic compared with wild type rice.These findings suggested that starch metabolism in higher plant might be regulated by several mechanisms: post-translational modifications such as redox modulation and protein phosphorylation, or allosteric modulation by metabolites, which is related to the metabolic flux.Additionally, three invertase genes, encoding a sucrose-digesting enzyme, were identified as HT salt-responsive DEGs.A double mutant of two isoforms of Arabidopsis neutral invertase genes, inv1/ inv2, has been shown to exhibit severe growth defects, and therefore the authors suggested that cytosolic invertase may play role in supplying sucrose to Arabidopsis non-photosynthetic cells.
Together with the altered expression levels of these genes, our results for increased starch and sucrose levels in transgenic rice suggest that starch and sucrose metabolism are likely downstream components that are regulated by OsCam1–1 under salt stress.The down-regulation of photosynthetic genes due to the impact of OsCam1–1 and up-regulation of genes involved in lipid metabolism suggests that transgenic rice may balance carbon and energy metabolism under salt stress by obtaining monosaccharide units through the mobilization of lipids, which might be converted to sugar via the glyoxylate cycle and gluconeogenesis, as in previous discussions and/or the cell wall, which is a large carbon reservoir in the cell.Four known CaM-interacting proteins previously identified in other plants were obtained from the rice cDNA expression library screening indicating specific CaM target identification.Cyclic nucleotide-gated channels , one of the four known CIPs, are activated by binding cyclic nucleotide monophosphates, which play roles in ion-homeostasis control, development, and biotic or abiotic stress defense.In Arabidopsis, AtCNGC10 functions in cation uptake in root, and AtCNGC10 anThisense Arabidopsis lines are more salt-sensitive than wild type.Another well-known CIP identified herein, glutamate decarboxylase , is the enzyme that converts L-glutamate to γ-amino butyric acid , which is involved in amino acid metabolism.CaM binds to GAD and regulates its activity, resulting in a balance of glutamate-GABA metabolism.Transgenic tobacco expressing petunia GAD lacking the CaM-binding domain exhibits a severely abnormal morphology associated with a high level of GABA but low level of glutamate.Calmodulin-binding transcription activators are found in several species of multicellular organisms.Herein, an OsCAMTA was confirmed to be a CaM target, even though the transcriptome showed that over expression of OsCam1–1 and salt stress did not significantly affect the expression levels of its gene.In Arabidopsis, CAMTA3 regulates a set of biotic stress-responsive genes, and the camta3 Arabidopsis mutant showed enhanced biotic stress tolerance.A rice kinesin motor domain-containing protein was also confirmed as a CaM target in this study.Some evidence has revealed that kinesin motordomain-containing protein plays a role in cell developmental processes.In late anaphase, the amino-terminal motor kinesinis accumulated along the microtubule toward the spindle mid-zone and then localized to microtubules near the future cell plate area, suggesting that AtPAKRP1 may play a role in the maintenance or establishment of the phragmoplast microtubule array.Another report has revealed that AtPAKRP2 first exhibits a punctate pattern in late anaphase, and then is concentrated at the division site following the appearance of the phragmoplast microtubule array in the mirror pair.Treatment with brefeldin A, which inhibits protein transportation from the endoplasmic reticulum to the Golgi apparatus, resulted in the alteration of AtPAKRP2 localization, so the authors suggested that AtPAKRP2 functions in Golgi-derived vesicles transportation in the phragmoplast.Here, six CaM-interacting proteins that have not been found in other plant species were identified in rice.In Arabidopsis, hydroxycinnamoyl-coenzyme A shikimate/quinate hydroxycinnamoyltransferase, a homolog of LOC_Os02g39850, which was identified as CIP herein, contains acyltransferase activity capable of catalyzing the conversion p-coumaroyl-CoA to caffeoylCoA, which plays a role in the lignin biosynthesis pathway.Silencing of this acyltransferase gene in Arabidopsis results in a dwarf phenotype and change in lignin composition.LOC_Os09g36220 was identified as OsPRR95, a pseudo response regulator, which takes part in circadian systems by binding a core oscillator to define rhythm to adapt to the daily changing environment.