PII Overexpression in Lotus japonicus Affects Nodule Activity in Permissive Low-Nitrogen Conditions and Increases Nodule Numbers in High Nitrogen Treated Plants

Abstract

We report here the first characterization of a GLNB1 gene coding for the PII protein in leguminous plants. The main purpose of this work was the investigation of the possible roles played by this multifunctional protein in nodulation pathways. The Lotus japonicus LjGLB1 gene shows a significant transcriptional regulation during the light-dark cycle and different nitrogen availability, conditions that strongly affect nodule formation, development, and functioning. We also report analysis of the spatial profile of expression of LjGLB1 in root and nodule tissues and of the protein's subcellular localization. Transgenic L. japonicus lines overexpressing the PII protein were obtained and tested for the analysis of the symbiotic responses in different conditions. The uncoupling of PII from its native regulation affects nitrogenase activity and nodule polyamine content. Furthermore, our results suggest the involvement of PII in the signaling of the nitrogen nutritional status affecting the legumes' predisposition for nodule formation.

Ectopic Expression of PII Induces Stomatal Closure in Lotus japonicus

“…These three transgenic lines have been propagated to isolate homozigous T2 lines with consistent patterns of GUS activity. The analysis of the pLjGLB1-gus A confirms the expression previously described in vascular bundle of transgenic roots (D'Apuzzo et al, 2015), giving the opportunity to identify an additional striking spatial profile of expression in leaf tissues where GUS activity is localized in main and secondary veins but also in mesophyll and guard cells (Figures 1A,B). The GUS staining in guard cells is even better visualized in leaves after epidermis peeling (Figures 1C,D).…”

“…We have recently described the GUS activity driven by the pr LjGLB1-gus A promoter-fusion, localized in vascular bundle of Lotus japonicus transgenic hairy roots and nodules with a slight activity observed in root tips (D'Apuzzo et al, 2015). In order to gain further inside the profile of LjGLB1 expression in different organs, we have used the same T-DNA construct to obtain L. japonicus transgenic plants via A. tumefaciens -mediated transformation.…”

“…Oligonucleotides and strategies for pr LjGLB1 - gus A, LjGLB1 -GFP fusions and PII over-expressing T-DNA constructs were described in D'Apuzzo et al (2015). …”

“…We recently reported the characterization of L. japonicus transgenic PII overexpressing plants that display alterations of the nodular N-fixation activity as well as the N dependent signaling pathway controlling the competence of Lotus plants for nodule formation (D'Apuzzo et al, 2015). In this article, we now investigate whether the un-coupling of PII from its native regulation may affect another process where N assimilation pathways play a crucial role such as the stomatal movement.…”

An In Vitro Procedure for Phenotypic Screening of Growth Parameters and Symbiotic Performances in Lotus corniculatus Cultivars Maintained in Different Nutritional Conditions

“…Interestingly, this is associated to an increased shoot growth capacity in symbiotic conditions, which does not represent an obvious combination of phenotypic traits. In fact, nitrogen-fixing root nodules are the organs where atmospheric N reduction and release take place, but at the same time these are optional C sink organs that need to assimilate large amount of energy source in the form of photosynthate products either for the formation of nodule primordium [50], and to provide energy for the N fixation performed by the Rhizobium and assimilation of the produced ammonium and starch biosynthesis [51,52]. Therefore a very fine regulatory circuit determines the number of nodules sufficient to satisfy N demand but still convenient in terms of energy expenses.…”