Nodulation Phenotypes of Gibberellin and Brassinosteroid Mutants of Pea

Abstract

The initiation and development of legume nodules induced by compatible Rhizobium species requires a complex signal exchange involving both plant and bacterial compounds. Phytohormones have been implicated in this process, although in many cases direct evidence is lacking. Here, we characterize the root and nodulation phenotypes of various mutant lines of pea (Pisum sativum) that display alterations in their phytohormone levels and/or perception. Mutants possessing root systems deficient in gibberellins (GAs) or brassinosteroids (BRs) exhibited a reduction in nodule organogenesis. The question of whether these reductions represent direct or indirect effects of the hormone deficiency is addressed. For example, the application of GA to the roots of a GA-deficient mutant completely restored its number of nodules to that of the wild type. Grafting studies revealed that a wild-type shoot or root also restored the nodule number of a GA-deficient mutant. These findings suggest that GAs are required for nodulation. In contrast, the shoot controlled the number of nodules that formed in graft combinations of a BR-deficient mutant and its wild type. The root levels of auxin and GA were similar among these latter graft combinations. These results suggest that BRs influence a shoot mechanism that controls nodulation and that the root levels of auxin and GA are not part of this process. Interestingly, a strong correlation between nodule and lateral root numbers was observed in all lines assessed, consistent with a possible overlap in the early developmental pathways of the two organs.Nodulation is a symbiotic process whereby bacteria of the genus Rhizobium invade compatible leguminous host plants (Mylona et al., 1995;Mathesius, 2003). The invasion ultimately leads to the formation of structures called nodules, in which the bacteria fix atmospheric nitrogen to be used by the plant. As with any developmental process, nodulation is multifaceted, requiring specific signaling events regulated temporally and spatially (Ferguson and Mathesius, 2003).Beginning in the 1980s, mutagenesis experiments using pea (Pisum sativum) produced abnormal nodulation phenotypes including nonnodulating (nod2), poorly nodulating (nod6), and hypernodulating (nod11) mutants, as well as those that fix nitrogen poorly or not at all (fix-; see refs. in Borisov et al., 2000). At present, over 200 nodulation mutants exist in pea (Borisov et al., 2000). Nodulation mutants have also been selected for in the model legume species Medicago truncatula and Lotus japonicus, which have smaller genomes than pea, making them more desirable tools for molecular studies. Mutants in these species have since been used to identify genes and gene products involved in nodule formation and functioning. This approach has been successful, and the orthologs of many nodulation genes discovered in M. truncatula or L. japonicus have subsequently been identified in important crop species such as pea (see refs. in Oldroyd and Downie, 2004).Here, we take the reverse approach to investig...

The Root Hair “Infectome” of Medicago truncatula Uncovers Changes in Cell Cycle Genes and Reveals a Requirement for Auxin Signaling in Rhizobial Infection

“…Pea (Pisum sativum) mutants deficient in BRs or GA had reduced nodulation (Ferguson et al, 2005), and SL levels were positively correlated with nodulation in pea and L. japonicus (Soto et al, 2010;Foo and Davies, 2011;Foo and Reid, 2013;Liu et al, 2013), but infection was not characterized in these studies. Our observation that CCD8 is induced in root hairs by S. meliloti taken together with the identification of SLs in M. truncatula root exudates (Liu et al, 2011) raises the possibility that SLs are secreted during infection.…”

Lignin Modification Leads to Increased Nodule Numbers in Alfalfa

“…A significant body of evidence also points to elevated GA levels, as recorded in the HCT down-regulated roots, as a potential cause of hypernodulation. For example, mutations in pea (Pisum sativum) that reduce GA levels strongly inhibit nodule development, and exogenous application of GA rescues the mutant phenotype (Ferguson et al, 2005). Moreover, GA response genes and GA biosynthetic pathway genes are up-regulated after rhizobial infection in Sesbania rostrata and soybean (Lievens et al, 2005;Hayashi et al, 2012), and constitutive GA Figure 7.…”

“…signaling mutants can also exhibit reduced nodule formation (Ferguson et al, 2011). Interestingly, GA 4 is the main accumulated bioactive GA in HCT downregulated alfalfa roots, compared with GA 1 or GA 3 in pea and Lotus japonicus (Ferguson et al, 2005(Ferguson et al, , 2011Maekawa et al, 2009), although knowledge of the role of this bioactive form in nodulation is limited.…”

Plant Hormonal Regulation of Nitrogen-Fixing Nodule Organogenesis

“…Thus, negative regulation of nodulation by BR might be mediated through the action of spermidine in shoots. In spite of the immobility of BR in the pea (Pisum sativum) (Symons and Reid, 2004), defective nodulation in a BRdeficient pea mutant was recovered in wild-type shoot grafting experiments, implying that BRs might regulate shoot-derived AON signaling via unknown pathways (Ferguson et al, 2005). It is clear that BR play a critical role in AON; however, the discrepancy between the negative effects of exogenous BR on nodulation and impaired nodule formation in BR-biosynthetic or BR-insensitive pea mutants suggests that BR might differentially control nodulation with respect to local and systemic regulation.…”

“…Application of GA biosynthesis inhibitors causes severe nodulation defects in Sesbania (Sesbania rostrata) (Lievens et al, 2005). A GA-deficient pea mutant also showed decreased nodule numbers (Ferguson et al, 2005;Ross et al, 2005). However, recent genetic and pharmacological studies of GA during nodulation have revealed a negative effect of GA on nodule development by L. japonicus.…”