María Reguera

Sinorhizobium fredii HH103 cgs mutants are unable to nodulate determinate- and indeterminate nodule-forming legumes and overproduce an altered EPS.

Sinorhizobium fredii HH103 produces cyclic beta glucans (CG) composed of 18 to 24 glucose residues without or with 1-phosphoglycerol as the only substituent. The S. fredii HH103-Rifr cgs gene (formerly known as ndvB) was sequenced and mutated with the lacZ-gentamicin resistance cassette. Mutant SVQ562 did not produce CG, was immobile, and grew more slowly in the hypoosmotic GYM medium, but its survival in distilled water was equal to that of HH103-Rifr. Lipopolysaccharides and K-antigen polysaccharides produced by SVQ562 were not apparently altered. SVQ562 overproduced exopolysaccharides (EPS) and its exoA gene was transcribed at higher levels than in HH103-Rifr. In GYM medium, the EPS produced by SVQ562 was of higher molecular weight and carried higher levels of substituents than that produced by HH103-Rifr. The expression of the SVQ562 cgsColon, two colonslacZ fusion was influenced by the pH and the osmolarity of the growth medium. The S. fredii cgs mutants SVQ561 (carrying cgs::Omega) and SVQ562 only formed pseudonodules on Glycine max (determinate nodules) and on Glycyrrhiza uralensis (indeterminate nodules). Although nodulation factors were detected in SVQ561 cultures, none of the cgs mutants induced any macroscopic response in Vigna unguiculata roots. Thus, the nodulation process induced by S. fredii cgs mutants is aborted at earlier stages in V. unguiculata than in Glycine max.

Boron deficiency results in induction of pathogenesis-related proteins from the PR-10 family during the legume-rhizobia interaction.

Boron (B) deficiency has a strong effect on molecular and cellular plant-bacteria interactions during the development of the legume-rhizobia symbiosis, leading to reduced infection and early necrosis of nodules, resembling a pathogenic-like rather than a symbiotic interaction. Therefore, induction of pathogenesis-related (PRs) proteins was investigated here in legume root nodules. Following two-dimensional electrophoresis and MALDI-TOF spectrometry analysis of proteins extracted from Pisum sativum B-sufficient (+B) or B-deficient (-B) root nodules, two proteins from the family PR10, ABR17 and PR10.1, were identified as highly induced in -B nodules. Analysis of gene expression and the use of anti-ABR17 confirmed that induction occurred in B-deficient young nodules and increased during nodule development. ABR17 was also induced in -B nodules of Phaseolus vulgaris. Boron deficiency did not significantly increase the expression of these PR10 in uninfected plant tissues. Moreover, independent of B, induction was detected in senescent tissues, although at a level weaker than in -B nodules. The immunochemical study of ABR17 antigen distribution showed that it was localized in all tissues of poorly invaded B-deficient nodules and accumulated around bacteria, which showed advanced degradation. These results suggest that, under B deficiency, the rhizobia-legume dialogue fails and the bacterium is recognized as a pathogen by the plant, which reacts to prevent infection by inducing at least these two identified PR10 proteins.

Ligands of boron in Pisum sativum nodules are involved in regulation of oxygen concentration and rhizobial infection

Boron (B) is an essential nutrient for N(2)-fixing legume-rhizobia symbioses, and the capacity of borate ions to bind and stabilize biomolecules is the basis of any B function. We used a borate-binding-specific resin and immunostaining techniques to identify B ligands important for the development of Pisum sativum-Rhizobium leguminosarum 3841 symbiotic nodules. arabinogalactan-extensin (AGPE), recognized by MAC 265 antibody, appeared heavily bound to the resin in extracts derived from B-sufficient, but not from B-deficient nodules. MAC 265 stained the infection threads and the extracellular matrix of cortical cells involved in the oxygen diffusion barrier. In B-deprived nodules, immunolocalization of MAC 265 antigens was significantly reduced. Leghaemoglobin (Lb) concentration largely decreased in B-deficient nodules. The absence of MAC 203 antigens in B-deficient nodules suggests a high internal oxygen concentration, as this antibody detects an epitope on the lipopolysaccharide (LPS) of bacteroids typically expressed in micro-aerobically grown R. leguminosarum 3841. However, B-deprived nodules did not accumulate oxidized lipids and proteins, and revealed a decrease in the activity of the major antioxidant enzyme ascorbate peroxidase (APX). Therefore, B deficiency reduced the stability of nodule macromolecules important for rhizobial infection, and for regulation of oxygen concentration, resulting in non-functional nodules, but did not appear to induce oxidative damage in low-B nodules.

Boron dependent membrane glycoproteins in symbiosome development and nodule organogenesis A model for a common role of boron in organogenesis

During the last two decades, we have analyzed the roles of boron (B) in the development of the legume-rhizobia symbiosis and nodule organogenesis. As in other plant tissues, B is needed for the maintenance of nodule cell wall structure. Moreover, several symbiotic events including rhizobial infection, nodule cell invasion and symbiosome development that involve membrane related functions (i.e. vesicle targeting, secretion, or cell surface interactions) are affected by B deficiency. Using anti-rhamnogalacturonan II (anti-RGII) antiserum and immunological techniques, we recently described membrane glycoproteins (RGII-glycoproteins) developmentally regulated in Pisum sativum nodules, which are not detected by the antibody in B-deficient nodules. RGII-glycoproteins appeared related with development processes involving extensive membrane synthesis, like symbiosome maturation or cell growth, both of them negatively affected by B deficiency. Here, we suggest that, besides maintaining cell wall structure, B is both stabilizing components of the membrane glycocalyx and promoting interactions between cell surfaces glycoconjugates that are important during the establishment of the symbiosis and during nodule development. Moreover, we hypothesize that B is playing a similar role during plant or animal embryogenesis and development.

Borate promotes the formation of a complex between legume AGP-extensin and Rhamnogalacturonan II and enhances production of Rhizobium capsular polysaccharide during infection thread development in Pisum sativum symbiotic root nodules.

The capacity to bind to biomolecules is considered to be the basis for any physiological role of boron (B). Legume arabinogalactan protein-extensin (AGPE), a major component of the infection thread matrix of legume nodules is a potential B-ligand. Therefore, its role in infection threads development was investigated in Pisum sativum grown under B deficiency. Using the AGPE-specific antibody MAC265, immunochemical analysis revealed that a 175 kDa MAC265 antigen was abundant in +B but much weaker in -B nodule extracts. A B-dependent complex involving AGPE and rhamnogalacturonan II (RGII) could be co-purified using anti-RGII antiserum. Following fractionation of -B nodules, MAC265 antigens were mostly associated with the bacterial pellet. Immunogold staining confirmed that AGPE was closely associated with the surface of rhizobia in the lumen of threads in -B nodules whereas in +B nodules, AGPE was separated from the bacterial surface by a sheath of capsular polysaccharide. Interestingly, colonies of rhizobia grown in free-living culture without B developed low capsule production. Therefore, we propose that B could be important for apical growth of infection threads by strengthening thread wall through a B-dependent AGPE-RGII interaction and by promoting bacterial advance through a B-dependent production of a stable rhizobial capsule that prevents AGPE attachment.

Calcium mediates the cellular response of Chlamydomonas reinhardtii to the emerging aquatic pollutant Triclosan

The present study was aimed at investigating the role of intracellular free calcium, [Ca2+]c, in the early cellular response of the green alga Chlamydomonas reinhardtii to the emergent pollutant Triclosan (13.8μM; 24h of exposure). There is a growing concern about the persistence and toxicity of this antimicrobial in aquatic environments, where non-target organisms such as C. reinhardtii, a primary producer of ecological relevance, might be severely impacted. A mechanistic study was undertaken which combined flow cytometry protocols, physiological as well as gene expression analysis. As an early response, Triclosan strongly altered [Ca2+]c homeostasis which could be prevented by prechelation with the intracellular calcium chelator BAPTA-AM. Triclosan induced ROS overproduction which ultimately leads to oxidative stress with loss of membrane integrity, membrane depolarization, photosynthesis inhibition and mitochondrial membrane depolarization; within this context, Triclosan also induced an increase in caspase 3/7 activity and altered the expression of metacaspase genes which are indicative of apoptosis. All these adverse outcomes were dependent on [Ca2+]c. Interestingly, an interconnection between [Ca2+]c alterations and increased ROS formation by Triclosan was found. Taken altogether these results shed light on the mechanisms behind Triclosan toxicity in the green alga Chlamydomonas reinhardtii and demonstrate the role of [Ca2+]c in mediating the observed toxicity.

Boron deficiency inhibits root growth by controlling meristem activity under cytokinin regulation

Significant advances have been made in the last years trying to identify regulatory pathways that control plant responses to boron (B) deficiency. Still, there is a lack of a deep understanding of how they act regulating growth and development under B limiting conditions. Here, we analyzed the impact of B deficit on cell division leading to root apical meristem (RAM) disorganization. Our results reveal that inhibition of cell proliferation under the regulatory control of cytokinins (CKs) is an early event contributing to root growth arrest under B deficiency. An early recovery of QC46:GUS expression after transferring B-deficient seedlings to control conditions revealed a role of B in the maintenance of QC identity whose loss under deficiency occurred at later stages of the stress. Additionally, the D-type cyclin CYCD3 overexpressor and triple mutant cycd3;1-3 were used to evaluate the effect on mitosis inhibition at the G1-S boundary. Overall, this study supports the hypothesis that meristem activity is inhibited by B deficiency at early stages of the stress as it does cell elongation. Likewise, distinct regulatory mechanisms seem to take place depending on the severity of the stress. The results presented here are key to better understand early signaling responses under B deficiency.

The impact of different agroecological conditions on the nutritional composition of quinoa seeds

Quinoa cultivation has been expanded around the world in the last decade and is considered an exceptional crop with the potential of contributing to food security worldwide. The exceptional nutritional value of quinoa seeds relies on their high protein content, their amino acid profile that includes a good balance of essential amino acids, the mineral composition and the presence of antioxidants and other important nutrients such as fiber or vitamins. Although several studies have pointed to the influence of different environmental stresses in certain nutritional components little attention has been paid to the effect of the agroecological context on the nutritional properties of the seeds what may strongly impact on the consumer food’s quality. Thus, aiming to evaluate the effect of the agroecological conditions on the nutritional profile of quinoa seeds we analyzed three quinoa cultivars (Salcedo-INIA, Titicaca and Regalona) at different locations (Spain, Peru and Chile). The results revealed that several nutritional parameters such as the amino acid profile, the protein content, the mineral composition and the phytate amount in the seeds depend on the location and cultivar while other parameters such as saponin or fiber were more stable across locations. Our results support the notion that nutritional characteristics of seeds may be determined by seed’s origin and further analysis are needed to define the exact mechanisms that control the changes in the seeds nutritional properties.