Abstract

Plants have evolved different abilities to adapt to the ever-fluctuating environments for sessility. Calcium-dependent protein kinase (CDPK) is believed to play a pivotal role in abiotic stress signaling. So far, study on the specific substrates that CDPK recognized in response to adversity is limited. In the present study, we revealed a potential interaction between CDPK and a bHLH transcription factor under salt stress in Chenopodium glaucum. First, we identified a CgCDPK, which was up-regulated under salt and drought stress; then by Y2H screening, CgCDPK was detected to be involved in interaction with a bHLH TF (named as CgbHLH001), which also positively respond to salt and drought stress. Further computational prediction and experiments including GST-pulldown and BiFC assays revealed that potential interaction existed between CgCDPK and CgbHLH001, and they might interact on the plasma membrane. In addition, CgCDPK-overexpressed transgenic tobacco line could significantly accumulate transcripts of NtbHLH (a homolog of CgbHLH001 in N. tabacum), which provided another evidence of correlation between CgCDPK and CgbHLH001. Our results suggest that CgbHLH001 can interact with CgCDPK in signal transduction pathway in response to abiotic stress, which should provide new evidence for further understanding of the substrate specificity of plant CDPK signaling pathway.

Highlights

  • Plants have evolved different abilities to adapt to the ever-fluctuating environments for sessility

  • We observed that red [DiI staining for plasma membrane (PM) marker] and green (CgCDPK-green fluorescent protein (GFP) overexpression) fluorescent signals were completely merged into yellow color on the PM of the epidermal cells of tobacco leaf (Fig. 1b), it revealed that CgCDPK located on the PM

  • We identified an interaction component of Calcium-dependent protein kinase (CDPK) - a basic helix-loop-helix transcription factor (TF) from C. glaucum by yeast two hybrid (Y2H) screening, further in vitro pulldown and in vivo bi-molecular fluorescence complementation (BiFC) assay as well as transgenic plant verification all provided evidence that CgCDPK and CgbHLH001 could positively respond to abiotic stress and potentially interact in transduction of signal to enhance stress tolerance

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Summary

Introduction

Plants have evolved different abilities to adapt to the ever-fluctuating environments for sessility. Our results suggest that CgbHLH001 can interact with CgCDPK in signal transduction pathway in response to abiotic stress, which should provide new evidence for further understanding of the substrate specificity of plant CDPK signaling pathway. An increasing evidence in CDPK signaling pathways reveals the interaction between CDPK and its downstream transcription factors (TFs) or proteins in response to stresses[14,15,16]. Interactions between AtSLAH3 (S-type anion channel) and AtCPK21, or between AtSLAC1 (another S-type anion channel) and AtCPK23 have been verified by GST-pulldown assay in vitro and confirmed by BiFC in vivo[28, 29]. These data suggest that Y2H, GST-pulldown, and BiFC are effective ways in PPI analysis

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