Phytosulfokine-δ: A Small Peptide, but a Big Player in Symbiosis Gene Regulation

Abstract

Nitrogen availability is one of the critical determinants of agricultural yield. Biological nitrogen fixation, such as legume–rhizobia symbiotic association, might function as a solution to fix nitrogen. Using phytosulfokine (PSK)-α sequences as a query, Yu et al., 2022 performed a comprehensive genome-wide search of legume species to identify PSK-δ, a divergent pentapeptide differing in single amino acid. Furthermore, PSK-δ exhibited nodule-specific expression with lower expression in the root, substantiating the nodule-specific temporal expression and suggesting its role in nodule development and nitrogen fixation. Additionally, in planta functional characterization in Medicago truncatula using overexpression and Tnt1-insertion mutant analysis indicated the role of PSK-δ in symbiotic nodulation. Interestingly, a similar phenotype of MtPSKδ mutant (mtpskδ) with that of wild-type control led to the hypothesis of its functional redundancy with PSK-α in nodule organogenesis. Further investigation regarding its position in the Nod-factor signaling pathway revealed the downstream function of PSK-δ in association with MtENOD11 in regulating nodule formation.

Nitrogen is involved in building DNA and proteins; however, plants can procure minimal soil inorganic nitrogen [1]. Therefore, nitrogen availability is a key factor determining agricultural yield. Biological nitrogen fixation (BNF), such as the legume–rhizobia symbiotic association, appeared as a solution to fix nitrogen [2,3]. Legume–rhizobia interaction starts with the secretion of flavonoids by the legumes, which acts as a signal for rhizobia; in turn, rhizobia secrete lipo-chitooligosaccharides (LCOs) or Nod-factors in response to plant flavonoids [4]. Upon LCOs perception by Nod-factor receptors, a phosphorylation signaling cascade is activated, also known as the common symbiotic pathway (CSP), leading to calcium spiking [5]. Several essential proteins, such as DMI1, DMI2 and DMI3, participate in the CSP to induce calcium spiking. The calcium spiking is interpreted by calcium calmodulin-dependent protein kinase to activate the transcription of important genes such as NIN, NSP2 and ENOD11, etc. [6]. The activation of these genes is accompanied by rhizobial entry into the host. Simultaneously the cortical cells divide to facilitate the formation of a niche for bacteria known as root nodules. Phytohormones auxin and cytokinin have been well characterized in inducing nodule organogenesis [7].

Small-signaling peptides recently appeared as signaling peptide species regulating different plant developmental processes, including arbuscular mycorrhizae association and root nodulation [8]. Phytosulfokine-α (PSK-α) is a signaling peptide (disulfated pentapeptide) perceived by leucine-rich repeat receptor kinases that induce cell expansion or elongation [9]. Genetic study and exogenous peptide application revealed that PSK-α mediated cell expansion or elongation in the root, hypocotyl, and leaf promotes their growth. Additionally, PSK-α regulates in vitro regeneration of recalcitrant legumes, somatic embryogenesis, immune responses, and drought-induced flower drop [10]. PSK-α is a positive regulator of nodule number and promotes nodule maturation from the primordium [11].

Yu et al., 2022 searched the genomes of legume species using reported PSK-α sequences as a query [11,12,13,14]. During their search, they identified legume-specific genes encoding pentapeptides differing in one amino acid, i.e., YIYTN instead of YIYTQ, and named as PSK-δ. Only one gene encoding PSK-δ was identified in Medicago truncatula and Lotus japonicus. In contrast, two genes were identified in Glycine max. The PSK-δ genes were identified from the three legumes; however, the experiments were performed in M. truncatula and L. japonicus. PSK-δ precursor proteins were ~80 amino acids long with an N-terminal secretory peptide and the pentapeptide motif at C-terminus. The expression pattern analysis revealed that the PSK-δ gene showed nodule-specific expression in all three species. Simultaneously, the nodule/root-specific peptide nature of PSK-δ was also established. Temporal expression pattern analysis in nodule tissue revealed that the PSK-δ gene was majorly involved in nodule development. Further, a promoter: GUS assay using pMtPSKδ:GUS transformation into hairy root displayed a GUS signal from the developing primordia, mainly in meristematic and infection zones.

Since the PSK-δ expression was mainly localized in nodules, the authors decided to perform in planta functional characterization using overexpression and Tnt1-insertion mutant analysis. Constitutive overexpression of PSK-δ resulted in an increased pink and white nodule number and a slight increase in primary root length. Moreover, the number of nodule primordia increased. However, infection thread formation remained unaffected, suggesting that MtPSKδ enhances nodule organogenesis, thereby promoting nodulation. To rule out the role of shoot signaling in the regulation of nodule numbers because of constitutive expression using 35S promoter, authors expressed the gene in the root by hairy root transformation. The expression in root tissue resulted in an increase in nodule number by 54% compared to the wild type, indicating a direct role of MtPSKδ in promoting nodulation. In addition to overexpression, the exogenous application of pentapeptide to M. truncatula and L. japonicus seedlings increased the nodule numbers. Thus, MtPSKδ has a conserved promotive effect across different legume species irrespective of determinate or indeterminate nodulation types.

It was expected that the mutant of MtPSKδ should exhibit a phenotype opposite to one observed upon overexpression or exogenous application of MtPSKδ. Interestingly, the homozygous Tnt1-insertion mutant of MtPSKδ (mtpskδ) showed similar parameters in terms of shoot height, root length, nodule number, shape, size, and color compared to wild type control. In a previous investigation by the same authors, they found that the MtPSKα family has three members, MtPSKα1, MtPSKα2, and MtPSKα3, with MtPSKα1 showing the highest expression [13]. The authors performed RNA interference of the MtPSKα1 gene to check the functional relevance of MtPSKα1 during nodulation. In the RNAi lines, the expression of MtPSKα1 was decreased; however, no change was observed in the expression of MtPSKα2, MtPSKα3, and MtPSKδ. The nodule number in MtPSKα1-RNAi lines remained the same as in the control. Therefore, the authors hypothesized that the unexpected phenotype of MtPSKδ knockout might be due to MtPSKα expression, and there is a functional redundancy between these two. To check this hypothesis, MtPSKα1-RNAi was performed in the mtpskδ Tnt1 mutant line. The MtPSKα1-RNAi in mtpskδ Tnt1 mutant background resulted in decreased shoot height, fresh weight, and several pink and white nodules. Additionally, the number of infection threads remained unaffected; however, a ~41% decrease in nodule primordia was observed. Therefore, these results prove that nodule organogenesis in M. truncatula is redundantly regulated by PSK-α and PSK-δ (Figure 1).

Finally, the authors tried to place the gene in the Nod-factor signaling pathway. Treatment of the seedlings with PSK-δ peptide for 12 and 24 h, followed by qRT-PCR expression analysis of three early nodulation genes, MtNIN, MtERN1, and MtENOD40, revealed no change in expression, indicating that PSK-δ does not function upstream of Nod-factor. However, the expression of MtPSKδ increased only in wild-type plants but not in Nod-factor signaling mutants nfp, dmi3, nsp1, and nin, suggesting that Nod-factor signaling induces MtPSKδ expression. The authors checked the expression of Nod-factor-inducible genes after PSK-δ treatment and found that only MtENOD11 expression was elevated. Hence, it was proposed that PSK-δ functions downstream of Nod-factor signaling and regulates nodule formation through MtENOD11 expression. The researchers concluded that the PSK-δ is a novel kind of PSK that is widespread in different legume species and is essential for many functions related to development, growth, and legume plants’ immune response. Overall, the study highlights the importance of PSKs in plant growth and the potential for these small peptide hormones to be utilized for practical applications in agriculture.