Stably TransformedLotus japonicusPlants Overexpressing Phytoglobin LjGlb1-1 Show Decreased Nitric Oxide Levels in Roots and Nodules as Well as Delayed Nodule Senescence

Abstract

The class 1 phytoglobin, LjGlb1-1, is expressed in various tissues of the model legume Lotus japonicus, where it may play multiple functions by interacting with nitric oxide (NO). One of such functions is the onset of a proper symbiosis with Mesorhizobium loti resulting in the formation of actively N 2 -fixing nodules. Stable overexpression lines (Ox1 and Ox2) of LjGlb1-1 were generated and phenotyped. Both Ox lines showed reduced NO levels in roots and enhanced nitrogenase activity in mature and senescent nodules relative to the wild-type (WT). Physiological and cytological observations indicated that overexpression of LjGlb1-1 delayed nodule senescence. The application to WT nodules of the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) or the phytohormones abscisic acid (ABA) and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) repressed nitrogenase activity, induced the expression of three senescence-associated genes, and caused cytological changes evidencing nodule senescence. These effects were almost completely reverted by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. Our results reveal that overexpression of LjGlb1-1 improves the activity of mature nodules and delays nodule senescence in the L. japonicus-M. loti symbiosis. These beneficial effects are probably mediated by the participation of LjGlb1-1 in controlling the concentration of NO that may be produced downstream in the phytohormone signaling pathway in nodules.

Plant hemoglobins: a journey from unicellular green algae to vascular plants

“…Ljap1‐1 is the only Glb of L. japonicus that is induced by NO or its immediate precursor NO 2 − (Shimoda et al , ). Plants overexpressing Ljap1‐1 or A. firma Glb1 exhibit better symbiotic performance and produce nodules with lower NO concentrations than the wild‐type plants (Shimoda et al , ; Fukudome et al , ). Similar beneficial effects of overexpressing Mtru1‐1 have been very recently reported in the Sinorhizobium meliloti–Medicago truncatula symbiosis (Berger et al , ).…”

Physiological significance of pedospheric nitric oxide for root growth, development and organismic interactions

“…NO production also has been reported in mature N 2 -fixing nodules, especially in infected rhizobia-harbouring cells (Calvo-Begueria et al, 2018;Horchani et al, 2011) and during nodule senescence (Cam et al, 2012;Fukudome et al, 2019). Accumulating evidence also supports a multifaceted role for NO during N 2 -fixation.…”

“…Recent studies in both major model legumes indicated a role of PHYTOGB1.1 in the modulation of NO content throughout the symbiosis. In L. japonicus, the overexpression of LjGlb1-1 positively correlated with reduced NO content and enhanced nitrogenase activity, while delaying nodule senescence (Fukudome et al, 2019). Similarly, in M. truncatula production of indeterminate nodules, MtPHYTOGB1.1 silencing, and overexpression studies suggested that the formation of the PHYTOGB 1.1-NO complex contributes to the regulation of NO levels and limits its toxic effects, with high NO levels promoting the defence responses, while low NO levels promote the infection process and nodule development (Berger, Guinand, Boscari, Puppo, & Brouquisse, 2020).…”

Reactive Sulfur Species Interact with Other Signal Molecules in Root Nodule Symbiosis in Lotus japonicus

“…In contrast, Lotus japonicus forms determinate nodules, and NO levels that are too high can inhibit infection and nodulation [26]. Regardless of nodule type, NO inhibits nitrogenase activity, such that decreased levels of NO in the nodules contribute to higher nitrogenase activity and delayed nodule senescence [26][27][28][29][30][31]. Generally, infection with symbiotic microbes suppresses the expression of pathogenesis-related genes; in M. truncatula, ROS are necessary for symbiosis because of their function as negative regulators of pathogenesis-related genes [32].…”