Populus trichocarpa clade A PP2C protein phosphatases: their stress-induced expression patterns, interactions in core abscisic acid signaling, and potential for regulation of growth and development
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Overexpression of the poplar PP2C protein phosphatase gene PtrHAB2 resulted in increased tree height and altered leaf morphology and phyllotaxy, implicating PP2C phosphatases as growth regulators functioning under favorable conditions.
We identified and studied Populus trichocarpa genes, PtrHAB1 through PtrHAB15, belonging to the clade A PP2C family of protein phosphatases known to regulate abscisic acid (ABA) signaling. PtrHAB1 through PtrHAB3 and PtrHAB12 through PtrHAB15 were the most highly expressed genes under non-stress conditions. The poplar PP2C genes were differentially regulated by drought treatments. Expression of PtrHAB1 through PtrHAB3 was unchanged or downregulated in response to drought, while all other PtrHAB genes were weakly to strongly upregulated in response to drought stress treatments. Yeast two-hybrid assays involving seven ABA receptor proteins (PtrRCAR) against 12 PtrHAB proteins detected 51 interactions involving eight PP2Cs and all PtrRCAR proteins with 22 interactions requiring the addition of ABA. PtrHAB2, PtrHAB12, PtrHAB13 and PtrHAB14 also interacted with the sucrose non-fermenting related kinase 2 proteins PtrSnRK2.10 and PtrSnRK2.11, supporting conservation of a SnRK2 signaling cascade regulated by PP2C in poplar. Additionally, PtrHAB2, PtrHAB12, PtrHAB13 and PtrHAB14 interacted with the mitogen-activated protein kinase protein PtrMPK7. Due to its interactions with PtrSnRK2 and PtrMPK7 proteins, and its reduced expression during drought stress, PtrHAB2 was overexpressed in poplar to test its potential as a growth regulator under non-stress conditions. 35S::PtrHAB2 transgenics exhibited increased growth rate for a majority of transgenic events and alterations in leaf phyllotaxy and morphology. These results indicate that PP2Cs have additional roles which extend beyond canonical ABA signaling, possibly coordinating plant growth and development in response to environmental conditions.
KeywordsAbscisic acid PP2C Poplar Drought stress Leaf morphology SnRK2
We thank Drs. Xiaoyan Sheng and Kristi DeCourcy for expert technical assistance. This work was supported by the United States Department of Energy (DOE) Office of Science (Biological and Environmental Research), Grant Nos. DE-SC0008570 to AMB and DE-FG02-07ER64449 to EPB and AMB and the United States Department of Agriculture-National Institute of Food and Agriculture (USDA-NIFA) Grant No. 2014-67013-21580 to EPB and AMB. Support was also provided by the Virginia Agricultural Experiment Station and the Hatch Program of USDA-NIFA, Project No. VA-135994.
SBR, HEP, AMB, and EPB designed experiments and wrote the manuscript. SBR and HEP produced vectors for Y2H and for transient and stable gene expression in plants. SBR conducted Y2H and BiFC assays, prepared and characterized transgenic poplar plants, and measured gene expression. SBR analyzed RNAseq data. SBR and SPW analyzed and documented BiFC results using confocal microscopy.
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