The Regulation of Pea (Pisum sativum L.) Symbiotic Nodule Infection and Defense Responses by Glutathione, Homoglutathione, and Their Ratio

Abstract

In this study, the roles of glutathione (GSH), homoglutathione (hGSH), and their ratio in symbiotic nodule development and functioning, as well as in defense responses accompanying ineffective nodulation in pea (Pisum sativum) were investigated. The expression of genes involved in (h)GSH biosynthesis, thiol content, and localization of the reduced form of GSH were analyzed in nodules of wild-type pea plants and mutants sym33-3 (weak allele, “locked” infection threads, occasional bacterial release, and defense reactions) and sym33-2 (strong allele, “locked” infection threads, defense reactions), and sym40-1 (abnormal bacteroids, oxidative stress, early senescence, and defense reactions). The effects of (h)GSH depletion and GSH treatment on nodule number and development were also examined. The GSH:hGSH ratio was found to be higher in nodules than in uninoculated roots in all genotypes analyzed, with the highest value being detected in wild-type nodules. Moreover, it was demonstrated, that a hGSHS-to-GSHS switch in gene expression in nodule tissue occurs only after bacterial release and leads to an increase in the GSH:hGSH ratio. Ineffective nodules showed variable GSH:hGSH ratios that correlated with the stage of nodule development. Changes in the levels of both thiols led to the activation of defense responses in nodules. The application of a (h)GSH biosynthesis inhibitor disrupted the nitrogen fixation zone in wild-type nodules, affected symbiosome formation in sym40-1 mutant nodules, and meristem functioning and infection thread growth in sym33-3 mutant nodules. An increase in the levels of both thiols following GSH treatment promoted both infection and extension of defense responses in sym33-3 nodules, whereas a similar increase in sym40-1 nodules led to the formation of infected cells resembling wild-type nitrogen-fixing cells and the disappearance of an early senescence zone in the base of the nodule. Meanwhile, an increase in hGSH levels in sym40-1 nodules resulting from GSH treatment manifested as a restriction of infection similar to that seen in untreated sym33-3 nodules. These findings indicated that a certain level of thiols is required for proper symbiotic nitrogen fixation and that changes in thiol content or the GSH:hGSH ratio are associated with different abnormalities and defense responses.

Effects of Elevated Temperature on Pisum sativum Nodule Development: I—Detailed Characteristic of Unusual Apical Senescence

“…The upregulation of genes encoding the GSH biosynthesis enzymes (PsGSH1, PsGSHS) in heat-stressed nodules observed in this study may also indicate the link to oxidative stress. Indeed, in early senescent nodules of the mutant sym40-1, the genes of glutathione biosynthesis were upregulated, while the content of GSH was low compared to the wild type [37].…”

“…viciae 3841 strain on the pea line SGE was investigated. This symbiotic pair is actively used as a model system for various studies of nodule development in peas [32,[37][38][39][40]. Heat-stressed nodules changed their color from pink to green at their In nodules exposed to 21 • C for 12 days, a green color was seen at the base of the nodule, which indicated the formation of the senescence zone (Figure 7G,H).…”

“…viciae 3841 strain on the pea line SGE was investigated. This symbiotic pair is actively used as a model system for various studies of nodule development in peas [32,[37][38][39][40]. Heat-stressed nodules changed their color from pink to green at their apical parts (Figure 1), which can be considered as the induction of degradation associated with leghemoglobin oxidation [41,42].…”

“…To characterize heat stress-induced changes in gene expression, we analyzed marker genes for which increased expression was previously shown to be enhanced during symbiotic nodule senescence [34,35] and the development of defense responses [36,37]. In this study, this set of genes was supplemented with heat-shock protein genes.…”

Signaling in Legume–Rhizobia Symbiosis

“…On the other hand, the important factor of the senescence onset is the age-related shift in the nodule redox status. Thus, Ivanova and co-workers showed that a functional metabolically active nodule requires high amounts of reduced glutathione (GSH) in tissues, whereas accumulation of its oxidized form (GSSG) correlated with nodule senescence [192]. Fukudome et al also found that the nodule content of nitric oxide (NO) is one of the key regulators of nodule senescence.…”