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Further Characterization of Calcineurin B-Like Protein and Its Interacting Partner CBL-Interacting Protein Kinase from Pisum sativum.

ABSTRACT: The recently discovered calcium sensor calcineurin B-like proteins (CBLs), and their interacting partners CBL-interacting protein kinases (CIPKs), have emerged as a key network in response to stress and calcium signaling in plants. The studies on CBL and CIPK are so far mainly restricted to Arabidopsis and work on dissecting this pathway in higher plants is inadequate. Our recent studies revealed that calcium sensor CBL from pea gets phosphorylated by pea CIPK. Furthermore, the transcript levels of both the CBL and CIPK from pea were coordinately upregulated in response to various stresses including high salinity, cold, wounding, salicyclic acid and calcium but not to abscisic acid and dehydration. Here we report the results on the computational analysis of EF hands of PsCBL protein, which indicate that it contains all the functional domains required for calcium binding activity. We have also focused on homology based computational modeling of PsCBL and PsCIPK proteins using AtCBL2 and Chk1 as templates respectively, which suggested the high degree of conservation between AtCBL2, PsCBL and OsCBL and between Chk1, PsCIPK and AtCIPK18. Furthermore, the direct interaction of PsCBL and PsCIPK mutant proteins was studied by the yeast 2-hybrid system, which confirmed that FISL domain is an important interaction module required for PsCBL and PsCIPK interaction.

SUBMITTER: Tuteja N

PROVIDER: S-EPMC2634211 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundProanthocyanidins (PAs) accumulate in the seeds, fruits and leaves of various plant species including the seed coats of pea (Pisum sativum), an important food crop. PAs have been implicated in human health, but molecular and biochemical characterization of pea PA biosynthesis has not been established to date, and detailed pea PA chemical composition has not been extensively studied.ResultsPAs were localized to the ground parenchyma and epidermal cells of pea seed coats. Chemical analyses of PAs from seeds of three pea cultivars demonstrated cultivar variation in PA composition. 'Courier' and 'Solido' PAs were primarily prodelphinidin-types, whereas the PAs from 'LAN3017' were mainly the procyanidin-type. The mean degree of polymerization of 'LAN3017' PAs was also higher than those from 'Courier' and 'Solido'. Next-generation sequencing of 'Courier' seed coat cDNA produced a seed coat-specific transcriptome. Three cDNAs encoding anthocyanidin reductase (PsANR), leucoanthocyanidin reductase (PsLAR), and dihydroflavonol reductase (PsDFR) were isolated. PsANR and PsLAR transcripts were most abundant earlier in seed coat development. This was followed by maximum PA accumulation in the seed coat. Recombinant PsANR enzyme efficiently synthesized all three cis-flavan-3-ols (gallocatechin, catechin, and afzalechin) with satisfactory kinetic properties. The synthesis rate of trans-flavan-3-ol by co-incubation of PsLAR and PsDFR was comparable to cis-flavan-3-ol synthesis rate by PsANR. Despite the competent PsLAR activity in vitro, expression of PsLAR driven by the Arabidopsis ANR promoter in wild-type and anr knock-out Arabidopsis backgrounds did not result in PA synthesis.ConclusionSignificant variation in seed coat PA composition was found within the pea cultivars, making pea an ideal system to explore PA biosynthesis. PsANR and PsLAR transcript profiles, PA localization, and PA accumulation patterns suggest that a pool of PA subunits are produced in specific seed coat cells early in development to be used as substrates for polymerization into PAs. Biochemically competent recombinant PsANR and PsLAR activities were consistent with the pea seed coat PA profile composed of both cis- and trans-flavan-3-ols. Since the expression of PsLAR in Arabidopsis did not alter the PA subunit profile (which is only comprised of cis-flavan-3-ols), it necessitates further investigation of in planta metabolic flux through PsLAR.

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Further Characterization of Calcineurin B-Like Protein and Its Interacting Partner CBL-Interacting Protein Kinase from Pisum sativum.

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