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

O A Pavlova,I V Leppyanen,D V Kustova,E A Dolgikh,A D Bovin

doi:10.18699/vj21.057

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.

Highlights

Annexins are of particular research interest due to their abili ty to regulate various aspects of plant growth and develop ment
Phylogenetic analysis of annexins in pea and other legumes The search of the sequences presumably coding for annexins in legumes was performed using BlastX with 8 prev ious ly revealed M. truncatula and 13 P. vulgaris nucleotide sequences encoding these proteins (Kodavali et al, 2013; Carrasco-Castilla et al, 2018) as queries against different plant sequence databases: https://phytozome.jgi.doe.gov/pz/ portal.html for M. truncatula and P. vulgaris, http://www. kazusa.or.jp/lotus/ for L. japonicus, and the URGI data base v. 1 https://urgi.versailles.inra.fr/blast for P. sativum L. (Clark et al, 2001; Carrasco-Castilla et al, 2018; Kreplak et al, 2019)
The coding sequences for annexins from P. sativum were named based on their phylogenetic relationships with the corresponding homologous sequences from M. truncatula and P. vulgaris (Clark et al, 2012; Kodavali et al, 2013; Carrasco-Castilla et al, 2018)

Summary

Introduction

Annexins are of particular research interest due to their abili ty to regulate various aspects of plant growth and develop ment. Annexins as phospholipid-binding pro teins are being implicated in the fusion of membrane vesicles, as was shown for annexins from bell pepper and cotton (Clark et al, 2012; Lizarbe et al, 2013). They are involved in the regulation of exocytosis, e. The Arabidopsis thaliana annexin AtAnn, which is expressed in root cells, exhibits pH-dependent cation-channel activity, while Z. mays annexins cause active conductivity of Ca2+ in lipid bilayers at slightly acidic pH (Gorecka et al, 2005; Laohavisit et al, 2009). Since annexins can be Ca2+ sensors, these proteins are likely to be involved in signal transduction; for example, the annexin from Triticum aestivum was suggested to be engaged in low-temperature signaling (Breton et al, 2000)

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