Table_1_Gibberellins Inhibit Nodule Senescence and Stimulate Nodule Meristem Bifurcation in Pea (Pisum sativum L.).docx

The development of nitrogen-fixing nodules formed during Rhizobium–legume symbiosis is strongly controlled by phytohormones. In this study, we investigated the effect of gibberellins (GAs) on senescence of pea (Pisum sativum) symbiotic nodules. Pea wild-type line SGE, as well as corresponding mutant lines SGEFix--1 (sym40), SGEFix--2 (sym33), SGEFix--3 (sym26), and SGEFix--7 (sym27), blocked at different stages of nodule development, were used in the study. An increase in expression of the GA2ox1 gene, encoding an enzyme involved in GA deactivation (GA 2-oxidase), and a decrease in the transcript abundance of the GA20ox1 gene, encoding one of the enzymes involved in GA biosynthesis (GA 20-oxidase), were observed in analyzed genotypes during nodule aging. A reduction in the amount of bioactive GA3 was demonstrated by immunolocalization in the early senescent mutant and wild-type lines during aging of symbiotic nodules. Down-regulated expression of senescence-associated genes encoding cysteine proteases 1 and 15a, thiol protease, bZIP transcription factor, 1-aminocyclopropane-1-carboxylate (ACC) synthase, ACC oxidase, and aldehyde oxidase was observed in the nodules of wild-type plants treated with exogenous GA3 relative to the untreated plants. GA3-treated plants also showed increases in nodule size and the nitrogen fixation zone, and decreases in the number of nodules and the senescence zone. Immunogold localization revealed higher levels of GA3 in the peribacteroid spaces in symbiosomes than in the matrix of infection threads. Furthermore, a decrease in GA3 label in mature and senescent symbiosomes in comparison with juvenile symbiosomes was observed. These results suggest a negative effect of GAs on the senescence of the pea symbiotic nodule and possible involvement of GAs in functioning of the mature nodule. Simultaneously, GA3 treatment led to nodule meristem bifurcation, indicating a possible role of GAs in nodule meristem functioning.

氮固定根瘤的形成过程在根瘤菌-豆科植物共生过程中受到植物激素的强烈调控。本研究旨在探讨赤霉素（GAs）对豌豆（Pisum sativum）共生根瘤衰老的影响。研究采用了豌豆野生型系SGE及其对应的突变体系SGEFix--1（sym40）、SGEFix--2（sym33）、SGEFix--3（sym26）和SGEFix--7（sym27），这些突变体系在根瘤发育的不同阶段被阻断。在分析根瘤衰老过程中的基因型中，观察到GA2ox1基因（编码参与GA失活的酶，GA 2-氧化酶）表达量增加，以及GA20ox1基因（编码参与GA生物合成的酶之一，GA 20-氧化酶）转录丰度降低。通过免疫定位技术在根瘤衰老过程中，早期衰老的突变体和野生型系中证明了生物活性GA3含量减少。与未处理植株相比，在野生型植株的根瘤中，经外源GA3处理的植株表现出衰老相关基因（编码半胱氨酸蛋白酶1和15a、硫醇蛋白酶、bZIP转录因子、1-氨基环丙烷-1-羧酸（ACC）合酶、ACC氧化酶和醛氧化酶）下调的表达。GA3处理的植株也显示出根瘤大小和固氮区增加，以及根瘤数量和衰老区减少。免疫金定位显示，在共生体的周细菌空间中GA3的水平高于感染丝的基质。此外，与幼年共生体相比，在成熟和衰老的共生体中观察到GA3标记减少。这些结果提示赤霉素对豌豆共生根瘤衰老具有负面影响，并可能涉及成熟根瘤的功能。同时，GA3处理导致根瘤原基分叉，这表明赤霉素可能在根瘤原基功能中发挥可能的作用。