Functional Study Of StGRIK1 And Deciphering Mechanism For Cold Induced Expression Of StBAM1 And StBAM9 In Potato

Potato(Solanum tuberosum L.)is the third most important food crop for direct human consumption,providing world food security.With properties of wide adaptation,high yield,and rich and balanced nutrition,potato has important effect in a steady growth of food in China.Starch served as the main photosynthetic product in potato leaves and the main component in tuber dry matter,the regulation of its metabolic process predominates tuber yield and quality formation.Meanwhile,starch is also the source of reducing sugar accumulation during cold-induced sweetening.Several potato starch metabolism genes have been functionally characterized;however,the expression of these starch metabolism genes is synergistic or not and their transcriptional regulation mechanisms are poorly understood.Previous studies have shown that Sn RK1 plays an important role in starch-sugar metabolism in potato,but its upstream kinase GRIKs have not been identified.Our previous work found that ?-amylases St BAM1 and St BAM9 play vital role in cold-induced sweetening,the mechanism of their cold-induced expression in tubers is still unclear.Therefore,the object of this study was to characterize the function of potato Sn RK1 upstream kinase St GRIK1,and to decipher mechanism for cold-induced expression of ?-amylases St BAM1 and St BAM9.The main results are as follows:1.Identification and Functional Characterization of Potato Sn RK1 Upstream Kinase St GRIK1By identifying and analyzing the Sn RK1 upstream kinase GRIKs,it was found that there are two GRIKs homologous genes in the potato genome,St GRIK1 and St GRIK2.St GRIK2 has multiple alternative splicing forms,and none of them have the potential to encode active kinases;while St GRIK1 encodes a conserved GRIK protein with kinase activity and can directly phosphorylate St Sn RK1? in vitro,indicating that St GRIK1 might be the only upstream kinase of Sn RK1 in potato.Co-localization analysis showed that St GRIK1 is a tonoplast protein.Sequence analysis revealed that St GRIK1 does not contain a transmembrane domain,while there are three potential S-acylated cysteineresidues at its N-terminus.Subsequently,the truncation and mutant localization analysis of St GRIK1 demonstrated that the three acylated cysteine residues at the N-terminus of St GRIK1 are essential its tonoplast localization,while the N-terminal domain of St GRIK1 containing all the three S-acylated cysteine residues is insufficient to mediate tonoplast targeting;and further screening and verification of St GRIK1 binding protein found that St GRIK1 can interact with vesicle traffic-related protein St VAP1 in the vicinity of the tonoplast,suggesting that St VAP1 may assist St GRIK1 tonoplast targeting through vesicle traffic pathway.Suppressing St GRIK1 resulted in decreased starch and reducing sugar content in potato leaves and reduced endogenous phosphorylation of Sn RK1,while Sn RK1 plays a pivotal role in potato starch-sugar metabolism.Transcriptome analysis indicated that the expression of multiple starch metabolism genes was affected by suppressing St GRIK1,suggesting that St GRIK1 may affect the expression of starch metabolism genes in potato leaves depending on phosphorylating Sn RK1,thereby regulating starch-sugar metabolism in potato leaves and ultimately influencing potato yield.Suppressing St GRIK1 led to increasing potato salt sensitivity and accelerating the dark-induced leaf senescence process.Previous studies have shown that there is crosstalk between salt stress and senescence,salt stress can stimulate the expression of many senescence-associated genes,subsequently,initiate leaf senescence,the ROS signaling pathway was downstream of both salt stress and senescence.Bi FC analysis indicated that St GRIK1-St ABI2-St SOS2 form a protein complex in tonoplast,while SOS2 is a key factor in the salt stress response pathway.ROS-associated CAT2 and NDPK2 has been identified as interactors of SOS2 in arabidopsis,we also found that their homologous in potato are potential interacting proteins of St GRIK1,suggesting that there might be a more complicated protein complex involved in mediating potato response to salt stress and senescence.In addition,St GRIK1 is highly expressed in flower buds and flowers.Suppressing St GRIK1 resulted in inhibition of flowering in long days,indicating that St GRIK1 might play essential roles in potato flowering and flower development.2.Deciphering Mechanism for Cold Induced Expression of ?-Amylases St BAM1 and St BAM9 in Potato TubersBy cloning and sequence analysis of the promoters of St BAM1 and St BAM9,and determining their expression patterns,it was found that their promoters contained ABA-response elements(ABRE),and their expression was also induced by ABA,indicating that St BAM1 and St BAM9 may be directly regulated by AREB/ABF/ABI5 transcription factors(TFs),which have been shown to recognize ABRE and regulate the expression of ABA-induced genes in the ABA signaling pathway.Through genome-wide identification and analysis,we found that there are 7 members of the AREB/ABF/ABI5 subfamily in the potato genome.Subcellular localization,yeast transcriptional activity and expression pattern analysis showed that potato AREB/ABF/ABI5 s were nuclear proteins,and St AREB1,St AREB2 and St AREB4 were both transcriptionally active and ABA-inducible,suggesting that these genes may be the core TFs in ABA signaling pathway.In vitro ABRE binding activity assay and in vivo promoter of St BAM1 and St BAM9 activation analysis showed that all the potato AREB/ABF/ABI5 s can recognize and bind ABRE,and only St AREB2,St AREB3,St AREB4 and St ABL2 can simultaneously activate the promoter of St BAM1 and St BAM9,suggesting that these members may directly regulate the expression of St BAM1 and St BAM9.The determination of ABA content and the expression of NCED involved in ABA synthesis showed that cold induced expression of St NCED1 led to accumulation of ABA in tubers during cold storage.Finally,expression patterns of potato ARB/ABF/ABI5 subfamily members and St BAM1 and St BAM9 during cold storage were analyzed,the results showed that St AREB2 had a similar expression pattern with St BAM1 and St BAM9 during tuber cold storage.To summarize,our results revealed that,cold could induce the accumulation of ABA by stimulating the expression of St NCED1,followed by activation of thedownstream TF St AREB2,which can activate the expression of St BAM1 and St BAM9 by recognizing and binding ABRE in their promoter region,ultimately promoted starch degradation and accumulation of reducing sugars in tubers during cold storage.