Solanum tuberosum StCDPK1 is regulated by miR390 at the posttranscriptional level and phosphorylates the auxin efflux carrier StPIN4 in vitro, a potential downstream target in potato development

Abstract

Among many factors that regulate potato tuberization, calcium and calcium-dependent protein kinases (CDPKs) play an important role. CDPK activity increases at the onset of tuber formation with StCDPK1 expression being strongly induced in swollen stolons. However, not much is known about the transcriptional and posttranscriptional regulation of StCDPK1 or its downstream targets in potato development. To elucidate further, we analyzed its expression in different tissues and stages of the life cycle. Histochemical analysis of StCDPK1::GUS (β-glucuronidase) plants demonstrated that StCDPK1 is strongly associated with the vascular system in stems, roots, during stolon to tuber transition, and in tuber sprouts. In agreement with the observed GUS profile, we found specific cis-acting elements in StCDPK1 promoter. In silico analysis predicted miR390 to be a putative posttranscriptional regulator of StCDPK1. Quantitative real time-polymerase chain reaction (qRT-PCR) analysis showed ubiquitous expression of StCDPK1 in different tissues which correlated well with Western blot data except in leaves. On the contrary, miR390 expression exhibited an inverse pattern in leaves and tuber eyes suggesting a possible regulation of StCDPK1 by miR390. This was further confirmed by Agrobacterium co-infiltration assays. In addition, in vitro assays showed that recombinant StCDPK1-6xHis was able to phosphorylate the hydrophilic loop of the auxin efflux carrier StPIN4. Altogether, these results indicate that StCDPK1 expression is varied in a tissue-specific manner having significant expression in vasculature and in tuber eyes; is regulated by miR390 at posttranscriptional level and suggest that StPIN4 could be one of its downstream targets revealing the overall role of this kinase in potato development.

StCDPK3 Phosphorylates In Vitro Two Transcription Factors Involved in GA and ABA Signaling in Potato: StRSG1 and StABF1

“…StCDPK3 transcripts were detected in early elongating stolons prior to tuber swelling while StCDPK1 expression was stronger in swollen stolons [38,49]. According to our qRT-PCR data (Fig 3), both StCDPK3 and StRSG1 are present in stolons, thus it is possible to speculate that StCDPK3 could control GA endogenous levels at the onset of tuberization through StRSG1 phosphorylation.…”

“…A similar phosphorylation assay was performed using 6xHisStCDPK1 [41] as the enzyme source. StCDPK1 is able to phosphorylate the hydrophilic loop of the auxin efflux carrier StPIN4 in vitro [49]. However, as observed in Fig 2E, StCDPK1 was unable to phosphorylate StRSG1; the band observed corresponds to the Ca 2+ -dependent autophosphorylation of StCDPK1.…”

“…StCDPK1 cannot phosphorylate StRSG1 in vitro suggesting that both kinases are involved in different signaling cascades. In fact, we recently showed that StCDPK1 is strongly associated with the vascular system and is able to phosphorylate the hydrophilic loop of the auxin transporter StPIN4 in vitro [49]. It remains to be confirmed whether StCDPK3 and StRSG1 interact in vivo .…”

TAS3 miR390-dependent loci in non-vascular land plants: Towards a comprehensive reconstruction of the gene evolutionary history

“…Biogenesis of ta-siRNAs includes initial AGO-dependent miRNA binding at single Axtell, 2013;Fei et al, 2013;Zheng et al, 2015;Xia et al, 2013; de 82 Felippes et al, 2017;Komiya, 2017;Deng et al, 2018). Moreover, miR390 may additionally 83 target and inhibit protein-coding gene transcripts, such as StCDPK1 related to auxin-responsive 84 pathway (Santin et al, 2017). 85 Previously, we described a new method for identification of plant TAS3 loci based on 86 PCR with a pair of oligodeoxyribonucleotide primers mimicking miR390.…”

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“…Axtell, 2013;Fei et al, 2013;Zheng et al, 2015;Xia et al, 2013; de 82 Felippes et al, 2017;Komiya, 2017;Deng et al, 2018). Moreover, miR390 may additionally 83 target and inhibit protein-coding gene transcripts, such as StCDPK1 related to auxin-responsive 84 pathway (Santin et al, 2017). 85 Previously, we described a new method for identification of plant TAS3 loci based on 86 PCR with a pair of oligodeoxyribonucleotide primers mimicking miR390.…”