vavilovВавиловский журнал генетики и селекцииVavilov Journal of Genetics and Breeding2500-3259Institute of Cytology and Genetics of Siberian Branch of the RAS10.18699/VJ21.057vavilov-3108Research ArticleГЕНЕТИЧЕСКИЕ РЕСУРСЫ И БИОКОЛЛЕКЦИИPLANT GENETICSФилогенетический и структурный анализ аннексинов у гороха (Pisum sativum L.) и их роль в развитии бобово-ризобиального симбиозаPhylogenetic and structural analysis of annexins in pea (Pisum sativum L.) and their role in legume-rhizobial symbiosis developmenthttps://orcid.org/0000-0003-0528-5618ПавловаО. А.PavlovaO. A.

Пушкин, Санкт-Петербург

Pushkin, St. Petersburg

https://orcid.org/0000-0002-2158-0855ЛеппяненИ. В.LeppyanenI. V.

Пушкин, Санкт-Петербург

Pushkin, St. Petersburg

КустоваД. В.KustovaD. V.

Пушкин, Санкт-Петербург

Pushkin, St. Petersburg

https://orcid.org/0000-0003-4061-435XБовинА. Д.BovinA. D.

Пушкин, Санкт-Петербург

Pushkin, St. Petersburg

https://orcid.org/0000-0002-5375-0943ДолгихЕ. А.DolgikhE. A.

Пушкин, Санкт-Петербург

Pushkin, St. Petersburg

dol2helen@yahoo.comВсероссийский научно-исследовательский институт сельскохозяйственной микробиологииРоссияAll-Russia Research Institute for Agricultural MicrobiologyRussian Federation202110092021255502513Copyright © Павлова О.А., Леппянен И.В., Кустова Д.В., Бовин А.Д., Долгих Е.А., 20212021Павлова О.А., Леппянен И.В., Кустова Д.В., Бовин А.Д., Долгих Е.А.Pavlova O.A., Leppyanen I.V., Kustova D.V., Bovin A.D., Dolgikh E.A.This work is licensed under a Creative Commons Attribution 4.0 License.https://vavilov.elpub.ru/jour/article/view/3108

Аннексины являются Ca2+/фосфолипид-связывающими белками, которые вовлечены в контроль многих биологических процессов, необходимых для роста и развития растений. Ранее выполненный протеомный анализ позволил нам выявить два аннексина, синтез которых усиливается в ответ на ризобиальную инокуляцию. В этой работе с помощью филогенетического анализа два аннексина были классифицированы как PsAnn4 и PsAnn8 на основании их гомологии с аннексинами других бобовых растений. С помощью молекулярного моделирования мы изучили структурные особенности этих аннексинов, которые могут влиять на их функциональную активность. Для анализа функции PsAnn4 и PsAnn8 были проведены сравнительный протеомный анализ, эксперименты с ингибиторами поступления кальция в клетку и локализация в тканях растений. Отсутствие активации синтеза PsAnn4 у мутанта гороха P56 (sym10), не способного формировать клубеньки, предполагает участие этого аннексина в бобово-ризобиальном симбиозе. Количественная ПЦР, совмещенная с обратной транскрипцией, показала, что экспрессия гена PsAnn4 увеличивается на ранних стадиях развития симбиоза начиная с 1–3-го дня после инокуляции до 5-го дня, тогда как блокатор Ca2+ канала LaCl3 подавляет эту экспрессию. Для изучения локализации PsAnn4 в клетках растений были получены конструкции для синтеза этого белка, слитого с такими флуорофорами, как красный флуоресцентный белок (RFP) и желтый флуоресцентный белок (YFP) при транскрипционной регуляции под промотором 35S в листьях Nicotiana benthamiana при инфильтрации Agrobacterium tumefaciens. Локализация PsAnn4 в клеточной стенке или плазматической мембране клеток растений указывает на возможность участия этого аннексина в ионном транспорте или модификации мембраны. Обсуждается возможная роль аннексина PsAnn4 в регуляции ранних стадий развития симбиоза у гороха.

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 identified 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 influence their functional activity. To verify the functions of these annexins, we performed comparative proteomic analysis, experiments with calcium influx 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 influence on this expression. To follow the PsAnn4 protein localization in plant cells, it was fused to the fluorophores such as red fluorescent protein (RFP) and yellow fluorescent protein (YFP) and expressed under the transcriptional regulation of the 35S promoter in Nicotiana benthamiana leaves by infiltration with Agrobacterium tumefaciens. The localization of PsAnn4 in the cell wall or plasma membrane of plant cells may indicate its participation in membrane modification or ion transport. Our results suggest that PsAnn4 may play an important role during the early stages of pea-rhizobial symbiosis development.

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The authors declare that there are no conflicts of interest present.