Roles And Mechanisms Of Carbonic Anhydrase βCA3 In Elevated CO₂-induced Defense Against Pseudomonas Syringae In Tomato

Tomato（Solanum lycopersicum）is one of the most important vegetable crops in protected cultivation.However,it is often threatened by various pathogens,such as Pseudomonas syringae pv.tomato（Pst DC3000）often leads to the decline of yield and quality and the excessive use of pesticides,which is the bottleneck problem restricting the safe,efficient and high-quality production of protected crops.Many studies have found that CO₂ enrichment can not only improve crop photosynthesis,but also regulate plant disease resistance.However,the molecular mechanism of how plants sense the change of CO₂concentration to regulate their immunity remains unknown.Carbonic anhydrases（CAs）,which are CO₂-binding proteins that catalyze the interconversion between CO₂ and HCO₃^-,are essential components of CO₂ sensing and concentrating systems.In this study,tomato was used to study the role and mechanisms of carbonic anhydraseβCA3 in elevated CO₂-induced defense against Pst DC3000,through the gene expression,transcriptional regulation,protein phosphorylation,stomatal&hormone,and transcriptome analysis.The main results are as follows:1.The role of tomatoβCAs in elevated CO₂-induced defense against Pst DC3000was identified.q RT-PCR analysis showed thatβCA3 was highly expressed in response to elevated CO₂ and Pst DC3000 inoculation.At the same time,the response pattern ofβCAs to elevated CO₂ and Pst DC3000 inoculation in Arabidopsis and two other Solanaceae crops pepper and tobacco were analyzed,we found thatβCAs,which were up-regulated by elevated CO₂ and Pst DC3000 inoculation were located in the same branch in the phylogenetic tree.The green fluorescent protein（GFP）fluorescence signal showed that tomatoβCA3 is localized in cytosol,nucleus and cytomembrane,but it is absent from chloroplast.βCA3 knock-out and over-expression plants were generated to study the role of tomatoβCA3.Compared with WT,βca3 mutants were more sensitive to Pst DC3000,while OE-βCA3 was more resistant,and elevated CO₂-induced resistance was largely compromised inβca3 mutants.These results indicate that tomatoβCA3 is a key factor in the defense against Pst DC3000 induced by elevated CO₂.2.The transcriptional regulation mechanism ofβCA3 in response to elevated CO₂and Pst DC3000 inoculation was investigated.The expression ofβCA3 was significantly upregulated by elevated CO₂ and Pst DC3000 inoculation,but its transcriptional regulation mechanism is not clear.By analyzing the promoter region ofβCA3 gene,we uncovered four conserved NAC recognition sites（NACRSs）.Analyzing the transcriptome data of tomato inoculated with Pst DC3000,combined with the results of q RT-PCR validation,it showed that the expression of NAC43 was most significantly up-regulated after tomato inoculation with Pst DC3000.Meanwhile,NAC43 expression was significantly up-regulated by elevated CO₂.EMSA,Dual-Luciferase and Ch IP assay showed that NAC43 could be combined withβCA3 promoter and enhancesβCA3 promoter activity.Moreover,the upregulation ofβCA3 expression induced by Pst DC3000 infection and elevated CO₂ was significantly inhibited in the nac43 mutant compared with WT,but the fold ofβCA3 expression upregulation was significantly increased in the OE-NAC43.These results demonstrate that NAC43transcriptionally activatesβCA3 in response to elevated CO₂ and Pst DC3000inoculation.3.The protein modification mechanism of tomatoβCA3 in response to elevated CO₂ and Pst DC3000 inoculation was investigated.In this paper,a novel tomato LRR-RLK（Leucine rich-repeat receptor like kinase）gene GRACE1,was identified to interact withβCA3,which was induced by elevated CO₂and treatment with flagellin.Silencing GRACE1 not only suppressed the disease resistance induced by elevated CO₂,but also largely suppressed the disease resistance enhanced by overexpression ofβCA3,especially under elevated CO₂.Using in vitro phosphorylation and liquid chromatography tandem mass spectrometry,it was found that GRACE1 can phosphorylate the Ser-207 and Thr-225 sites ofβCA3 protein.The function of phosphorylation of these two sites in plant immunity was further tested in the transient overexpression in tobacco and tomatoβca3 mutant aplastic system by point mutation simulation dephosphorylation experiments.The results showed that the dephosphorylation of Ser-207 site reduced plant disease resistance,but not Thr-225.Together,these results argue that S207 phosphorylation onβCA3 by GRACE1 is a central feature ofβCA3-mediated immunity.4.The relationship betweenβCA3-induced disease resistance and stomatal and SA disease resistance pathways in tomato was investigated.The responses ofβca3 mutant and OE-βCA3 plants to elevated CO₂ and Pst DC3000 infection-induced stomatal closure were not significantly different from those of WT.The binding ability ofβCA3to SA was detected by thermal migration assay,and it was found thatβCA3 was not a binding protein of SA.βca3 mutant and OE-βCA3 plants showed the same level of SA content as WT after inoculation with Pst DC3000,as well as the expression levels of SA synthesis genes ICS,PALs and downstream response genes PR1 and PR4.In addition,exogenous SA can still improve tomato disease resistance inβca3 mutants.When the key factor NIM1（the Arabidopsis NPR1 homologous gene）of the SA defense pathway was silenced,overexpression ofβCA3 could still enhance the disease resistance.In conclusion,the mechanism ofβCA3-induced defense against Pst DC3000in tomato is independent of stomata and SA resistance pathways.5.Transcriptome analysis of WT andβca3 mutants inoculated with Pst DC3000under different CO₂ concentrations was performed by RNA-Seq technology.The results showed that compared with WT under normal conditions,knocking outβCA3did not cause significant changes in tomato gene transcription,only of 4.1%transcripts expression were changed.There were 927 genes were significantly up-regulated in WT after inoculation with Pst DC3000.Enrichment analysis of Gene Ontology（GO）categories revealed that 10 out of 21 GO items in the biological process category were associated with carbohydrate metabolism and signaling,and a large number of genes were involved in cell wall metabolism.The upregulation of cell wall-related genes Cellulose synthase（CESA）,Xyloglucan endotransglucosylase/hydrolase（XTH）and Polygalacturonase（PG）,as well as the contents of lignin and cellulose induced by Pst DC3000 inoculation,were significantly inhibited inβca3 mutant,under both CO₂conditions.These indicated that knocking outβCA3 led to changes in the metabolism of cell wall-related substances in plant immune responses.In conclusion,tomatoβCA3 was highly induced by elevated CO₂ and Pst DC3000inoculation,and played an important role in elevated CO₂-induced defense against Pst DC3000.βCA3 is transcriptionally activated by the transcription factor NAC43 and is also post-translationally regulated by the receptor-like kinase GRACE1 at S207 site.TheβCA3-mediated disease resistance pathway was independent of the stomatal and SA disease resistance pathways,but knocking outβCA3 affected the accumulation of cell wall substances in plant immune responses.This study provides theoretical basis for improving crop disease resistance by CO₂ enrichment in facility cultivation industry.