Calcium-dependent protein kinases (CDPKs) are essential calcium sensors. In this work, we have studied StCDPK2 isoform from potato both at gene and protein level. StCdpk2 genomic sequence contains eight exons and seven introns, as was observed for StCdpk1. There is one copy of the gene per genome located in chromosome 7. StCDPK2 encodes an active CDPK of 515 aminoacids, with an apparent MW of 57 kDa, which presents myristoylation and palmitoylation consensus in its N-terminus. StCDPK2 is highly expressed in leaves and green sprouts; enhanced expression was detected under light treatment, which corresponds well with light responsive cis-acting elements found in its promoter sequence. Antibodies against the recombinant StCDPK2::6xHis protein detected this isoform in soluble and particulate fractions from leaves. StCDPK2 autophosphorylation and kinase activity are both calcium dependent reaching half maximal activation at 0.6 μM calcium. The active kinase is autophosphorylated on serine and tyrosine residues and its activity is negatively modulated by phosphatidic acid (PA). Our results reveal StCDPK2 as a signalling element involved in plant growth and development and show that its activity is tightly regulated.
Potato (Solanum tuberosum L.) tubers are an excellent staple food due to its high nutritional value. When the tuber reaches physiological competence, sprouting proceeds accompanied by changes at mRNA and protein levels. Potato tubers become a source of carbon and energy until sprouts are capable of independent growth. Transcript profiling of sprouts grown under continuous light or dark conditions was performed using the TIGR 10K EST Solanaceae microarray. The profiles analyzed show a core of highly expressed transcripts that are associated to the reactivation of growth. Under light conditions, the photosynthetic machinery was fully activated; the highest up-regulation was observed for the Rubisco activase (RCA), the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the Photosystem II 22 kDa protein (CP22) genes, among others. On the other hand, sprouts exposed to continuous darkness elongate longer, and after extended darkness, synthesis of chloroplast components was repressed, the expression of proteases was reduced while genes encoding cysteine protease inhibitors (CPIs) and metallocarboxypeptidase inhibitors (MPIs) were strongly induced. Northern blot and RT-PCR analysis confirmed that MPI levels correlated with the length of the dark period; however, CPI expression was strong only after longer periods of darkness, suggesting a feedback loop (regulation mechanism) in response to dark-induced senescence. Prevention of cysteine protease activity in etiolated sprouts exposed to extended darkness could delay senescence until they emerge to light.
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