What Can Boron Deficiency Symptoms Tell Us about Its Function and Regulation?

On the eve of the 100th anniversary of Dr. Warington's discovery of boron (B) as a nutrient essential for higher plants, "boronists" have struggled to demonstrate a role beyond its structural function in cell walls dimerizing pectin molecules of rhamnogalacturonan II (RGII). In this regard, B deficiency has been associated with a plethora of symptoms in plants that include macroscopic symptoms like growth arrest and cell death and biochemical or molecular symptoms that include changes in cell wall pore size, apoplast acidification, or a steep ROS production that leads to an oxidative burst. Aiming to shed light on B functions in plant biology, we proposed here a unifying model integrating the current knowledge about B function(s) in plants to explain why B deficiency can cause such remarkable effects on plant growth and development, impacting crop productivity. In addition, based on recent experimental evidence that suggests the existence of different B ligands other than RGII in plant cells, namely glycolipids, and glycoproteins, we proposed an experimental pipeline to identify putative missing ligands and to determine how they would integrate into the above-mentioned model.

Altered plant organogenesis under boron deficiency is associated with changes in high-mannose N-glycan profile that also occur in animals.

Boron (B) deficiency affects the development of Pisum sativum nodules and Arabidopsis thaliana root meristems. Both organs show an alteration of cell differentiation that result in the development of tumor-like structures. The fact that B in plants is not only able to interact with components of the cell wall but also with membrane-associated glycoconjugates, led us to analyze changes in high mannose type N-glycans (HMNG). The affinoblots with concanavalin A revealed alterations in the N-glycosylation pattern during early development of nodules and roots under B deprivation. Besides, there is increasing evidence of a B role in animal physiology that brought us to investigate the impact of B deficiency on Danio rerio (zebrafish) development. When B deficiency was induced prior to early cleavage stages, embryos developed as an abnormal undifferentiated mass of cells. Additionally, when B was removed at post-hatching, larvae undergo aberrant organogenesis. Resembling the phenomenon described in plants, alteration of the N-glycosylation pattern occurred in B-deficient zebrafish larvae prior to organogenesis. Overall, these results support a common function of B in plants and animals associated with glycosylation that might be important for cell signaling and cell fate determination during development.

A partner-switching system controls activation of mixed-linkage ß-glucan synthesis by c-di-GMP in Sinorhizobium meliloti.

Sinorhizobium meliloti synthesizes a linear mixed-linkage (1 → 3)(1 → 4)-ß-d-glucan (ML ß-glucan, MLG) in response to high levels of cyclic diguanylate (c-di-GMP). Two proteins BgsA and BgsB are required for MLG synthesis, BgsA being the glucan synthase which is activated upon c-di-GMP binding to its C-terminal domain. Here we report that the product of bgrR (SMb20447) is a diguanylate cyclase (DGC) that provides c-di-GMP for the synthesis of MLG by BgsA. bgrR is the first gene of a hexacistronic bgrRSTUWV operon, likely encoding a partner-switching regulatory network where BgrR is the final target. Using different approaches, we have determined that the products of genes bgrU (containing a putative PP2C serine phosphatase domain) and bgrW (with predicted kinase effector domain), modulate the phosphorylation status and the activity of the STAS domain protein BgrV. We propose that unphosphorylated BgrV inhibits BgrR DGC activity, perhaps through direct protein-protein interactions as established for other partner switchers. A bgrRSTUWV operon coexists with MLG structural bgsBA genes in many rhizobial genomes but is also present in some MLG non-producers, suggesting a role of this partner-switching system in other processes besides MLG biosynthesis.

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.

The Sinorhizobium fredii HH103 Genome: A Comparative Analysis With S. fredii Strains Differing in Their Symbiotic Behavior With Soybean.

Sinorhizobium fredii HH103 is a fast-growing rhizobial strain infecting a broad range of legumes including both American and Asiatic soybeans. In this work, we present the sequencing and annotation of the HH103 genome (7.25 Mb), consisting of one chromosome and six plasmids and representing the structurally most complex sinorhizobial genome sequenced so far. Comparative genomic analyses of S. fredii HH103 with strains USDA257 and NGR234 showed that the core genome of these three strains contains 4,212 genes (61.7% of the HH103 genes). Synteny plot analysis revealed that the much larger chromosome of USDA257 (6.48 Mb) is colinear to the HH103 (4.3 Mb) and NGR324 chromosomes (3.9 Mb). An additional region of the USDA257 chromosome of about 2 Mb displays similarity to plasmid pSfHH103e. Remarkable differences exist between HH103 and NGR234 concerning nod genes, flavonoid effect on surface polysaccharide production, and quorum-sensing systems. Furthermore a number of protein secretion systems have been found. Two genes coding for putative type III-secreted effectors not previously described in S. fredii, nopI and gunA, have been located on the HH103 genome. These differences could be important to understand the different symbiotic behavior of S. fredii strains HH103, USDA257, and NGR234 with soybean.

The interaction of boron with glycolipids is required to increase tolerance to stresses in Anabaena PCC 7120.

Boron (B) is an essential nutrient for heterocystous cyanobacteria growing under diazotrophic conditions. Under B-deficient conditions, the heterocyst envelope is highly disorganized, and the glycolipid layer is predominantly lost. Therefore, we examined whether B is implicated in the regulation of synthesis or processing and/or stability of glycolipids in Anabaena PCC 7120. RT-PCR analysis indicated that the expression of hglE was not significantly changed under B deficiency, suggesting that the synthesis of glycolipids during heterocyst formation was not compromised. In contrast, the overexpression of devB and hepA, encoding a glycolipid and a carbohydrate transporter, respectively, results in the instability of the envelope under B-deficient conditions. The capacity of borate to bind and stabilize molecules is considered the basis of any B biological function. Using a borate-binding-specific resin and thin layer chromatography, we detected the glycolipids that interact with B. Several heterocyst-specific glycolipids were detected as putative B ligands, suggesting a role for B in stabilizing the heterocyst envelope. Moreover, the glycolipids of Anabaena growing in non-diazotrophic conditions were also detected as putative B ligands. Although B is not essential for Anabaena under non-N2-fixing conditions, the presence of this micronutrient increased the tolerance of Anabaena to detergent treatment, salinity and hyperosmotic conditions. Taken together, the results of the present experiment suggest a beneficial role for B in environmental adaptation. Furthermore, we discuss the nutrient requirement for living organisms growing in nature and not under laboratory conditions.

Boron deficiency results in early repression of a cytokinin receptor gene and abnormal cell differentiation in the apical root meristem of Arabidopsis thaliana.

The development of Arabidopsis thaliana was dramatically altered within few hours following boron (B) deprivation. This effect was particularly evident in the apical root meristem. The essentiality of boron in plants has been clearly linked to its structural role in the cell wall, however the diversity and rapidity alterations of plant organogenesis when the micronutrient is absent suggest that B deficiency could also affect gene regulation during plant development. Therefore, the effect of B deficiency on cell elongation, apical root meristem cell division, and early differentiation of root tissues was investigated in A. thaliana seedlings. Dark-growth experiments indicated that hypocotyl elongation was inhibited 2 days after removing B, but apical root growth ceased almost immediately following B deprivation. Detection of cycline B1 by GUS staining of a promoter-reporter construct revealed that low B led to a reduced zone of cell division. The expression of CRE1/WOL/AHK4, encoding an integral membrane protein with histidine kinase domain that mediates cytokinin signaling and root xylem differentiation, was inhibited under B deficiency resulting in arrested xylem development at the protoxylem stage. Because the transition from cell division to cell differentiation in apical root meristems is controlled by cytokinins, this result support the hypothesis that signaling mechanisms during cell differentiation and organogenesis are highly sensitive to B deficiency, and together with previous reports that link the micronutrient with auxin or ethylene control of root architecture, suggests that B could play a role in regulation of hormone mediated early plant development signaling.

Genome sequence of the soybean symbiont Sinorhizobium fredii HH103.

Sinorhizobium fredii HH103 is a fast-growing rhizobial strain that is able to nodulate legumes that develop determinate nodules, e.g., soybean, and legumes that form nodules of the indeterminate type. Here we present the genome of HH103, which consists of one chromosome and five plasmids with a total size of 7.22 Mb.

Symbiotic properties and first analyses of the genomic sequence of the fast growing model strain Sinorhizobium fredii HH103 nodulating soybean.

Glycine max (soybean) plants can be nodulated by fast-growing rhizobial strains of the genus Sinorhizobium as well as by slow-growing strains clustered in the genus Bradyrhizobium. Fast-growing rhizobia strains with different soybean cultivar specificities have been isolated from Chinese soils and from other geographical regions. Most of these strains have been clustered into the species Sinorhizobium fredii. The S. fredii strain HH103 was isolated from soils of Hubei province, Central China and was first described in 1985. This strain is capable to nodulate American and Asiatic soybean cultivars and many other different legumes and is so far the best studied fast-growing soybean-nodulating strain. Additionally to the chromosome S. fredii HH103 carries five indigenous plasmids. The largest plasmid (pSfrHH103e) harbours genes for the production of diverse surface polysaccharides, such as exopolysaccharides (EPS), lipopolysaccharides (LPS), and capsular polysaccharides (KPS). The second largest plasmid (pSfrHH103d) is a typical symbiotic plasmid (pSym), carrying nodulation and nitrogen fixation genes. The present mini review focuses on symbiotic properties of S. fredii HH103, in particular on nodulation and surface polysaccharides aspects. The model strain S. fredii HH103 was chosen for genomic sequencing, which is currently in progress. First analyses of the draft genome sequence revealed an extensive synteny between the chromosomes of S. fredii HH103 and Rhizobium sp. NGR234.

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.

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 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.

Interactions between salinity and boron toxicity in tomato plants involve apoplastic calcium.

The lack of consensus about the mutual relations between salinity and boron (B) toxicity with respect to the physiological response of plants necessitates investigation of the interactions of soluble B with salinity. In this investigation, the effect of B was compared with Ca in order to elucidate whether the two nutrients have similar effects and/or to elucidate a relationship under salinity. Following addition of B or Ca, salinity was applied to tomato plants and the cell wall and plasma membrane permeability, measured as water permeability and electrolyte leakage, in relation to amino acid and ion cell wall composition, were determined. As the relationship between B and salinity was complex, several hypotheses are established. The increase of aquaporin functionality due to the presence of B and Ca compared with NaCl-treated plants could be the most feasible, whereas there is currently no satisfactory explanation for the results for the cell wall amino acid composition. In addition, the elemental composition results revealed that, in addition the known interactions between B and Ca with respect to cell wall stability, Mg and Mn were also increased in NaCl+B and NaCl+Ca treatments, suggesting their possible involvement in the cell wall function necessary for plant growth.

Gene SMb21071 of plasmid pSymB is required for osmoadaptation of Sinorhizobium meliloti 1021 and is implicated in modifications of cell surface polysaccharides structure in response to hyperosmotic stress.

Megaplasmid pSymB of the nitrogen-fixing symbiont Sinorhizobium meliloti, implicated in adaptation to hyperosmotic stress, contains 11 gene clusters that apparently encode surface polysaccharides. However, only 2 of these clusters, containing the exo and exp genes, have been associated with the synthesis of the acidic exopolysaccharides succinoglycan and galactoglucan, respectively. The functions of the other 9 clusters remain unsolved. The involvement of one of those regions, pSymB cluster 3, on surface polysaccharide synthesis and its possible implication in osmoadaptation were investigated. In silico analysis of cluster 3 showed that it putatively encodes for the synthesis and transport of a methylated surface polysaccharide. Mutants affected in this cluster were symbiotically effective but showed defects in growth under saline and nonsaline osmotic stress. The gene SMb21071, encoding a putative initiating glycosyltransferase, is transcriptionally induced under hyperosmotic conditions. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis and silver staining showed that osmotic stresses changed the profiles of surface polysaccharides of wild-type and mutants strains in different ways. The overall results suggest that cluster 3 is important for growth under saline stress and essential for growth under nonsaline hyperosmotic stress, and it appears to be implicated in maintaining and (or) modifying surface polysaccharides in response to osmotic stress.

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.

Developmentally regulated membrane glycoproteins sharing antigenicity with rhamnogalacturonan II are not detected in nodulated boron deficient Pisum sativum.

The peribacteroid membrane (PBM) of symbiosomes from pea root nodules developed in the presence of boron (+B) was labelled by anti-rhamnogalacturonan II (RGII) (anti-rhamnogalacturonan II pectin polysaccharide) antiserum. However, in nodules from plants grown at low boron (-B), anti-RGII pectin polysaccharide did not stain PBMs. Given that RGII pectin binds to borate, and that symbiosomes differentiate aberrantly in -B nodules because of abnormal vesicle traffic, anti-RGII pectin polysaccharide antigens were further analysed. Following electrophoresis and electroblotting, anti-RGII pectin polysaccharide immunostained three bands in +B but not in -B nodule-derived PBMs. A similar banding pattern was observed after the immunostaining of membrane fractions from uninfected roots, indicating that anti-RGII pectin polysaccharide antigens are common to both peribacteroid and plasma membranes. Protease treatment of samples led to disappearance of anti-RGII pectin polysaccharide labelling, indicating that the three immunostained bands correspond to proteins or glycoproteins. The immunochemical study of RGII antigen distribution during nodule development showed that it is strongly present on the PBM of dividing (undifferentiated) symbiosomes but progressively disappeared during symbiosome maturation. In B-deficient nodules, PBMs were never decorated with RGII antigens, and there was an abnormal targeting of vesicles containing pectic polysaccharide (homogalacturanan) to cell membranes. Overall, these results indicate that RGII, boron and certain membrane (glyco)-proteins may interact closely and function cooperatively in membrane processes associated with symbiosome division and general cell growth.

Cell surface interactions of Rhizobium bacteroids and other bacterial strains with symbiosomal and peribacteroid membrane components from pea nodules.

Samples of Rhizobium bacteroids isolated from pea nodule symbiosomes reacted positively with a monoclonal antibody recognizing N-linked glycan epitopes on plant glycoproteins associated with the peribacteroid membrane and peribacteroid fluid. An antiserum recognizing the symbiosomal lectin-like glycoprotein PsNLEC-1 also reacted positively. Samples of isolated bacteroids also reacted with an antibody recognizing a glycolipid component of the peribacteroid membrane and plasma membrane. Bacterial cells derived from free-living cultures then were immobilized on nitrocellulose sheets and tested for their ability to associate with components of plant extracts derived from nodule fractionation. A positive antibody-staining reaction indicated that both PsNLEC-1 and membrane glycolipid had become associated with the bacterial surface. A range of rhizobial strains with mutants affecting cell surface polysaccharides all showed similar interactions with PsNLEC-1 and associated plant membranes, with the exception of strain B659 (a deep-rough lipopolysaccharide mutant of Rhizobium leguminosarum). However, the presence of a capsule of extracellular polysaccharide apparently prevented interactions between rhizobial cells and these plant components. The importance of a close association between peribacteroid membranes, PsNLEC-1, and the bacterial surface is discussed in the context of symbiosome development.