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Phylogenetic and structural analysis of annexins in pea (Pisum sativum L.) and their role in legume-rhizobial symbiosis development.

ABSTRACT: Annexins as Ca2+/phospholipid-binding proteins are involved in the control of many biological processes essential for plant growth and development. In a previous study, we had shown, using a proteomic approach, that the synthesis of two annexins is induced in pea roots in response to rhizobial inoculation. In this study, phylogenetic analysis identif ied these annexins as PsAnn4 and PsAnn8 based on their homology with annexins from other legumes. The modeling approach allowed us to estimate the structural features of these annexins that might inf luence their functional activity. To verify the functions of these annexins, we performed comparative proteomic analysis, experiments with calcium inf lux inhibitors, and localization of labeled proteins. Essential down-regulation of PsAnn4 synthesis in a non-nodulating pea mutant P56 (sym10) suggests an involvement of this annexin in the rhizobial symbiosis. Quantitative RT-PCR analysis showed that PsAnn4 was upregulated at the early stages of symbiosis development, starting from 1-3 days after inoculation to up to 5 days after inoculation, while experiments with the Ca2+ channel blocker LaCl3 revealed its negative inf luence on this expression. To follow the PsAnn4 protein localization in plant cells, it was fused to the f luorophores such as red f luorescent protein (RFP) and yellow f luorescent protein (YFP) and expressed under the transcriptional regulation of the 35S promoter in Nicotiana benthamiana leaves by inf iltration with Agrobacterium tumefaciens. The localization of PsAnn4 in the cell wall or plasma membrane of plant cells may indicate its participation in membrane modif ication or ion transport. Our results suggest that PsAnn4 may play an important role during the early stages of pea-rhizobial symbiosis development.

SUBMITTER: Pavlova OA

PROVIDER: S-EPMC8453364 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The genome sequences of several legume species are now available allowing the comparison of the nitrogen (N) transporter inventories with non-legume species. A survey of the genes encoding inorganic N transporters and the sensing and assimilatory families in pea, revealed similar numbers of genes encoding the primary N assimilatory enzymes to those in other types of plants. Interestingly, we find that pea and Medicago truncatula have fewer members of the NRT2 nitrate transporter family. We suggest that this difference may result from a decreased dependency on soil nitrate acquisition, as legumes have the capacity to derive N from a symbiotic relationship with diazotrophs. Comparison with M. truncatula, indicates that only one of three NRT2s in pea is likely to be functional, possibly indicating less N uptake before nodule formation and N-fixation starts. Pea seeds are large, containing generous amounts of N-rich storage proteins providing a reserve that helps seedling establishment and this may also explain why fewer high affinity nitrate transporters are required. The capacity for nitrate accumulation in the vacuole is another component of assimilation, as it can provide a storage reservoir that supplies the plant when soil N is depleted. Comparing published pea tissue nitrate concentrations with other plants, we find that there is less accumulation of nitrate, even in non-nodulated plants, and that suggests a lower capacity for vacuolar storage. The long-distance transported form of organic N in the phloem is known to be specialized in legumes, with increased amounts of organic N molecules transported, like ureides, allantoin, asparagine and amides in pea. We suggest that, in general, the lower tissue and phloem nitrate levels compared with non-legumes may also result in less requirement for high affinity nitrate transporters. The pattern of N transporter and assimilatory enzyme distribution in pea is discussed and compared with non-legumes with the aim of identifying future breeding targets.

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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Phylogenetic and structural analysis of annexins in pea (Pisum sativum L.) and their role in legume-rhizobial symbiosis development.

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