Dataset Information

New Insight into HPts as Hubs in Poplar Cytokinin and Osmosensing Multistep Phosphorelays: Cytokinin Pathway Uses Specific HPts.

ABSTRACT: We have previously identified proteins in poplar which belong to an osmosensing (OS) signaling pathway, called a multistep phosphorelay (MSP). The MSP comprises histidine-aspartate kinases (HK), which act as membrane receptors; histidine phosphotransfer (HPt) proteins, which act as phosphorelay proteins; and response regulators (RR), some of which act as transcription factors. In this study, we identified the HK proteins homologous to the Arabidopsis cytokinin (CK) receptors, which are first partners in the poplar cytokinin MSP, and focused on specificity of these two MSPs (CK and OS), which seem to share the same pool of HPt proteins. Firstly, we isolated five CK HKs from poplar which are homologous to Arabidopsis AHK2, AHK3, and AHK4, namely, HK2, HK3a, HK3b, HK4a, HK4b. These HKs were shown to be functional kinases, as observed in a functional complementation of a yeast HK deleted strain. Moreover, one of these HKs, HK4a, was shown to have kinase activity dependent on the presence of CK. Exhaustive interaction tests between these five CK HKs and the 10 HPts characterized in poplar were performed using two-hybrid and BiFC experiments. The resulting partnership was compared to that previously identified between putative osmosensors HK1a/1b and HPt proteins. Finally, in planta coexpression analysis of genes encoding these potential partners revealed that almost all HPts are coexpressed with CK HKs in four different poplar organs. Overall, these results allowed us to unravel the common and specific partnerships existing between OS and CK MSP in Populus.

SUBMITTER: Hericourt F

PROVIDER: S-EPMC6963366 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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New Insight into HPts as Hubs in Poplar Cytokinin and Osmosensing Multistep Phosphorelays: Cytokinin Pathway Uses Specific HPts.

Héricourt François F Larcher Mélanie M Chefdor Françoise F Koudounas Konstantinos K Carqueijeiro Inês I Lemos Cruz Pamela P Courdavault Vincent V Tanigawa Mirai M Maeda Tatsuya T Depierreux Christiane C Lamblin Frédéric F Glévarec Gaëlle G Carpin Sabine S

Plants (Basel, Switzerland) 20191211 12

We have previously identified proteins in poplar which belong to an osmosensing (OS) signaling pathway, called a multistep phosphorelay (MSP). The MSP comprises histidine-aspartate kinases (HK), which act as membrane receptors; histidine phosphotransfer (HPt) proteins, which act as phosphorelay proteins; and response regulators (RR), some of which act as transcription factors. In this study, we identified the HK proteins homologous to the Arabidopsis cytokinin (CK) receptors, which are first par ...[more]

PMID: 31835814

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Project description:Climate change with increasing periods of drought is expected to reduce the yield of biomass crops such as poplars. To combat yield loss, it is important to better understand the molecular mechanisms that control growth under drought. Here, the goal was to resolve the drought-induced changes of active cytokinins, a main growth hormone in plants, at the tissue level in different cell types and organs of poplars (Populus × canescens) in comparison with growth, biomass, leaf shedding, photosynthesis and water potential. Since cytokinin response is mediated by type-A response regulators, ARR5::GUS reporter lines were used to map cytokinin activity histochemically. The expression of PtaRR3 and PtaRR10 was examined in different stem sections. Young leaves showed strong cytokinin activity in the veins and low staining under drought stress, accompanied by diminished leaf expansion. Leaf scars, at positions where drought-shedding occurred, showed strong reduction of cytokinin activity. The pith in the differentiation zone of stem showed high cytokinin activity with distinct, very active parenchymatic cells and enhanced activity close to primary xylem. This pattern was maintained under drought but the cytokinin activity was reduced. Mature phloem parenchymatic cells showed high cytokinin activity and mature wood showed no detectable cytokinin activity. Cytokinin activity in the cambium was apparent as a clear ring, which faded under drought. Xylem-localized cytokinin activities were also mirrored by the relative expression of PtaRR3, whereas PtaRR10 showed developmental but no drought-induced changes. Primary meristems exhibited high cytokinin activity regardless of drought stress, supporting a function of this phytohormone in meristem maintenance, whereas declining cytokinin activities in apical pith tissues and cambium of drought-stressed poplars linked cytokinin in these cell types with the control of primary and secondary growth processes. Changes in cytokinin activity further imply a role in drought avoidance mechanisms of poplars, especially in the reduction of leaf area.

Project description:Plant hormones cytokinins (CKs) are one of the major mediators of physiological responses throughout plant life span. Therefore, a proper homeostasis is maintained by regulation of their active levels. Besides degradation, CKs are deactivated by uridine diphosphate glycosyltransferases (UGTs). Physiologically, CKs active levels decline in senescing organs, providing a signal to nutrients that a shift to reproductive tissues has begun. In this work, we show CK glucosides distribution in Arabidopsis leaves during major developmental transition phases. Besides continuous accumulation of N-glucosides we detected sharp maximum of the glucosides in senescence. This is caused prevalently by N7-glucosides followed by N9-glucosides and specifically also by trans-zeatin-O-glucoside (tZOG). Interestingly, we observed a similar trend in response to exogenously applied CK. In Arabidopsis, only three UGTs deactivate CKs in vivo: UGT76C1, UGT76C2 and UGT85A1. We thereby show that UGT85A1 is specifically expressed in senescent leaves whereas UGT76C2 is activated rapidly in response to exogenously applied CK. To shed more light on the UGTs physiological roles, we performed a comparative study on UGTs loss-of-function mutants, characterizing a true ugt85a1-1 loss-of-function mutant for the first time. Although no altered phenotype was detected under standard condition we observed reduced chlorophyll degradation with increased anthocyanin accumulation in our experiment on detached leaves accompanied by senescence and stress related genes modulated expression. Among the mutants, ugt76c2 possessed extremely diminished CK N-glucosides levels whereas ugt76c1 showed some specificity toward cis-zeatin (cZ). Besides tZOG, a broader range of CK glucosides was decreased in ugt85a1-1. Performing CK metabolism gene expression profiling, we revealed that activation of CK degradation pathway serves as a general regulatory mechanism of disturbed CK homeostasis followed by decreased CK signaling in all UGT mutants. In contrast, a specific regulation of CKX7, CKX1 and CKX2 was observed for each individual UGT mutant isoform after exogenous CK uptake. Employing an in silico prediction we proposed cytosolic localization of UGT76C1 and UGT76C2, that we further confirmed by GFP tagging of UGT76C2. Integrating all the results, we therefore hypothesize that UGTs possess different physiological roles in Arabidopsis and serve as a fine-tuning mechanism of active CK levels in cytosol.

Dataset Information

New Insight into HPts as Hubs in Poplar Cytokinin and Osmosensing Multistep Phosphorelays: Cytokinin Pathway Uses Specific HPts.

Publications

New Insight into HPts as Hubs in Poplar Cytokinin and Osmosensing Multistep Phosphorelays: Cytokinin Pathway Uses Specific HPts.

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