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

Boron (B) is an essential nutrient for N₂‐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.     

Relationship between boron and calcium in the N2-fixing legume–rhizobia symbiosis

Because boron (B) and calcium (Ca²⁺) seem to have a strong effect on legume nodulation and nitrogen fixation, rhizobial symbiosis with leguminous plants, grown under varying concentrations of both nutrients, was investigated. The study of early pre‐infection events included the capacity of root exudates to induce nod genes, and the degree of adsorption of bacteria to the root surface. Both phenomena were inhibited by B deficiency, and increased by addition of Ca²⁺, resulting in an increase of the number of nodules. The infection and invasion steps were investigated by fluorescence microscopy in pea nodules harbouring a Rhizobium leguminosarum strain that constitutively expresses green fluorescent protein. High Ca²⁺ enhanced cell and tissue invasion by Rhizobium, which was highly inhibited after B deficiency. This was combined with an increased B concentration in nodules of plants grown on B‐free medium and supplemented with high Ca²⁺ concentrations, and that can be attributed to an increased B import to the nodules. Histological examination of indeterminate (pea) and determinate (bean) nodules showed an altered nodule anatomy at low B content of the tissue. The moderate increase in nodular B due to additional Ca²⁺ was not sufficient to prevent the abnormal cell wall structure and the aberrant distribution of pectin polysaccharides in B‐deficient treatments. Overall results indicate that the development of the symbiosis depends of the concentration of B and Ca²⁺, and that both nutrients are essential for nodule structure and function.     

Diffusion and reaction of oxygen in the central tissue of ureide-producing legume nodules

Previous simulation models for the diffusion and reaction of oxygen in legume nodules were based on infected cells and neglected adjacent uninfected cells. This study uses a three-dimensional model of the central zone of legume nodules made up of the two cell types represented by a geometrically defined, space-filling, binary combination of polyhedra, each with bevelled edges to allow for a network of intercellular gas spaces. The model predicted a distinctively compartmentalized distribution of [O₂] between uninfected and infected cells; with high O₂ concentrations for an uninfected cell being consistent with, and necessary for, efficient operation of uricase and ureide synthesis and low O₂ concentrations across most of the infected cell providing a suitable environment for N₂-fixation. Compartmentalization of O₂ also predicted significant O₂ fluxes between cell types, compromising maintenance of low [O₂] in infected cells, as well as high [O₂] in uninfected cells. The results predict that there might be significant resistance to O₂ diffusion across the cell : cell interface due to the plasmalemma and cell walls.     

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.     

Genetic dissection of the initiation of the infection process and nodule tissue development in the Rhizobium-pea (Pisum sativum L.) symbiosis

Twelve non-nodulating pea (Pisum sativum L.) mutants were studied to identify the blocks in nodule tissue development. In nine, the reason for the lack of infection thread (IT) development was studied; this had been characterized previously in the other three mutants. With respect to IT development, mutants in gene sym7 are interrupted at the stage of colonization of the pocket in the curled root hair (Crh- phenotype), mutants in genes sym37 and sym38 are blocked at the stage of IT growth in the root hair cell (Ith- phenotype) and mutants in gene sym34 at the stage of IT growth inside root cortex cells (Itr- phenotype). With respect to nodule tissue development, mutants in genes sym7, sym14 and sym35 were shown to be blocked at the stage of cortical cell divisions (Ccd- phenotype), mutants in gene sym34 are halted at the stage of nodule primordium (NP) development (Npd- phenotype) and mutants in genes sym37 and sym38 are arrested at the stage of nodule meristem development (Nmd- phenotype). Thus, the sequential functioning of the genes Sym37, Sym38 and the gene Sym34 apparently differs in the infection process and during nodule tissue development. Based on these data, a scheme is suggested for the sequential functioning of early pea symbiotic genes in the two developmental processes: infection and nodule tissue formation.     

Effects of boron and calcium nutrition on the establishment of the Rhizobium leguminosarum–pea (Pisum sativum) symbiosis and nodule development under salt stress

The effects of modifying boron (B) and calcium (Ca²⁺) concentrations on the establishment and development of rhizobial symbiosis in Pisum sativum plants grown under salt stress were investigated. Salinity almost completely inhibited the nodulation of pea plants by Rhizobium leguminosarum bv. viciae 3841. This effect was prevented by addition of Ca²⁺ during plant growth. The capacity of root exudates derived from salt‐treated plants to induce Rhizobium nod genes was not significantly decreased. However, bacterial adsorption to roots was highly inhibited in plants grown with 75 mM NaCl. Moreover, R. leguminosarum 3841 did not grow in minimal media containing such salt concentration. High Ca²⁺ levels enhanced both rhizobial growth and adsorption to roots, and increased nodule number in the presence of high salt. Nevertheless, the nodules developed were not functional unless the B concentration was also increased. Because B has a strong effect on infection and cell invasion, these processes were investigated by fluorescence microscopy in pea nodules harbouring a R. leguminosarum strain that expresses green fluorescent protein. Salt‐stressed plants had empty nodules and only those treated with high B and high Ca²⁺ developed infection threads and exhibited enhanced cell and tissue invasion by Rhizobium. Overall, the results indicate that Ca²⁺ promotes nodulation and B nodule development leading to an increase of salt tolerance of nodulated legumes.     

CERBERUS, a novel U-box protein containing WD-40 repeats, is required for formation of the infection thread and nodule development in the legume–Rhizobium symbiosis

Endosymbiotic infection of legume plants by Rhizobium bacteria is initiated through infection threads (ITs) which are initiated within and penetrate from root hairs and deliver the endosymbionts into nodule cells. Despite recent progress in understanding the mutual recognition and early symbiotic signaling cascades in host legumes, the molecular mechanisms underlying bacterial infection processes and successive nodule organogenesis are still poorly understood. We isolated a novel symbiotic mutant of Lotus japonicus , cerberus , which shows defects in IT formation and nodule organogenesis. Map-based cloning of the causal gene allowed us to identify the CERBERUS gene, which encodes a novel protein containing a U-box domain and WD-40 repeats. CERBERUS expression was detected in the roots and nodules, and was enhanced after inoculation of Mesorhizobium loti . Strong expression was detected in developing nodule primordia and the infected zone of mature nodules. In cerberus mutants, Rhizobium colonized curled root hair tips, but hardly penetrated into root hair cells. The occasional ITs that were formed inside the root hair cells were mostly arrested within the epidermal cell layer. Nodule organogenesis was aborted prematurely, resulting in the formation of a large number of small bumps which contained no endosymbiotic bacteria. These phenotypic and genetic analyses, together with comparisons with other legume mutants with defects in IT formation, indicate that CERBERUS plays a critical role in the very early steps of IT formation as well as in growth and differentiation of nodules.     

Rhizobium leguminosarum bv. viciae genotypes interact with pea plants in developmental responses of nodules, roots and shoots

The variability of the developmental responses of two contrasting cultivars of pea (Pisum sativum) was studied in relation to the genetic diversity of their nitrogen-fixing symbiont Rhizobium leguminosarum bv. viciae. A sample of 42 strains of pea rhizobia was chosen to represent 17 genotypes predominating in indigenous rhizobial populations, the genotypes being defined by the combination of haplotypes characterized with rDNA intergenic spacer and nodD gene regions as markers. We found contrasting effects of the bacterial genotype, especially the nod gene type, on the development of nodules, roots and shoots. A bacterial nod gene type was identified that induced very large, branched nodules, smaller nodule numbers, high nodule biomass, but reduced root and aerial part development. The plants associated with this genotype accumulated less N in shoots, but N concentration in leaves was not affected. The results suggest that the plant could not control nodule development sustaining the energy demand for nodule functioning and its optimal growth. The molecular and physiological mechanisms that may be involved are discussed.     

钙、硼对花生生长、产量和品质的影响

在土壤代换性钙和有效硼含量分别为0.84me/100g和0.163mg/kg的条件下,研究了施钙、施硼及钙、硼配施对花生生长、产量和品质的影响。结果表明,施用钙肥能提高花生的产量和品质;施用硼肥能改善品质,但在产量上却产生负效应;钙、硼配施也能提高产量,但主效应仍然是钙。施用钙硼肥料不但使花生成熟期植株中钙、硼含量提高,而且种植花生后土壤中的钙、硼残留量也明显增加。     

Influence of boron and calcium on the tolerance to salinity of nitrogen-fixing pea plants

The effects of different levels of B (from 9.3 to 93 M B) and Ca (from 0.68 to 5.44 mM Ca) on plant development, nitrogen fixation, and mineral composition of pea (Pisum sativum L. cv. Argona) grown in symbiosis with Rhizobium leguminosarum bv. viciae 3841 and under salt stress, were analysed. The addition of extra B and extra Ca to the nutrient solution prevented the reduction caused by 75 mM NaCl of plant growth and the inhibition of nodulation and nitrogen fixation. The number of nodules recovered by the increase of Ca concentration at any B level, but only nodules developed at high B and high Ca concentrations could fix nitrogen. Addition of extra B and Ca during plant growth restored nodule organogenesis and structure, which was absolutely damaged by high salt. The increase in salt tolerance of symbiotic plants mediated by B and Ca can be co-related with the recovery of the contents of some nutrients. Salinity produced a decrease of B and Ca contents both in shoots and in nodulated roots, being increased by the supplement of both elements in the nutrient solution. Salinity also reduced the content in plants of other nutrients important for plant development and particularly for symbiotic nitrogen fixation, as K and Fe. A balanced nutrition of B and Ca (55.8 M B, 2.72 mM Ca) was able to counter-act the deficiency of these nutrients in salt-stressed plants, leading to a huge increase in salinity tolerance of symbiotic pea plants. The necessity of nutritional studies to successfully cultivate legumes in saline soils is discussed and proposed.     

Structural changes in the innercortex cells of soybean root nodules are induced by short-term exposure to high salt or oxygen concentrations

After a 2 h exposure of intact soybean nodules to high concentrations of NaCl (100mol m_?3) or oxygen (8OkPa O₂), morphometric computations carried out using an image analysis technique on semi-thin sections showed that both treatments induced a decrease in the area of the inner-cortex cells, which were then characterized by a tangential elongation. In contrast, no significant change in area occurred in the middle-cortex cells although their elongation decreased. Electron microscopic observations showed that in the inner-cortex cells changes included the presence of wall infoldings, an enlarged periplasmic space and a lobate nucleus whose chromatin distribution differed from that of the control. Structural changes also occurred in the endoplasmic reticulum, microbodies, mitochondria and plastids. From several of these changes, which are similar to those noted in osmocontractil cells in response to external stimuli, it can be hypothesized that the inner cortex may provide a potential mechanism for the control of oxygen diffusion through the nodules.     

缺硼对大豆根瘤结构和功能的影响

在营养液培养条件下以普通结结瘤大豆Ｂｒａｇｇｃｖ．「Ｇｌｙｃｉｎｅｍａｘ（Ｌ．）Ｍｅｒｒ」及其超结瘤突变体ｎｔｓ３８２为实验材料,运用光学显微方法研究了硼对大豆根瘤结构的影响,并测定了根瘤固氮酶活性结果表明,缺硼使根瘤结构受到严重破坏,并使固氮酶活性显著下降,缺硼使根瘤结构受到破坏是导致固氮酶活性下降的可能原因。     

Analysis of calcium spiking using a cameleon calcium sensor reveals that nodulation gene expression is regulated by calcium spike number and the developmental status of the cell

Rhizobium-made Nod factors induce rapid changes in both Ca(2+) and gene expression. Mutations and inhibitors that abolish Nod-factor-induced Ca(2+) spiking block gene induction, indicating a specific role for Ca(2+) spiking in signal transduction. We used transgenic Medicago truncatula expressing a "cameleon" Ca(2+) sensor to assess the relationship between Nod-factor-induced Ca(2+) spiking and the activation of downstream gene expression. In contrast to ENOD11 induction, Ca(2+) spiking is activated in all root-hair cells and in epidermal or pre-emergent root hairs cells in the root tip region. Furthermore, cortical cells immediately below the epidermal layer also show slow Ca(2+) spiking and these cells lack Nod-factor-induced ENOD11 expression. This indicates a specialization in nodulation gene induction downstream of Nod-factor perception and signal transduction. There was a gradient in the frequency of Ca(2+) spiking along the root, with younger root-hair cells having a longer period between spikes than older root hairs. Using a Ca(2+)-pump inhibitor to block Ca(2+) spiking at various times after addition of Nod factor, we conclude that about 36 consecutive Ca(2+) spikes are sufficient to induce ENOD11-GUS expression in root hairs. To determine if the length of time of Ca(2+) spiking or the number of Ca(2+) spikes is more critical for Nod-factor-induced ENOD11 expression, jasmonic acid (JA) was added to reduce the rate of Nod-factor-induced Ca(2+) spiking. This revealed that even when the period between Ca(2+) spikes was extended, an equivalent number of Ca(2+) spikes were required for the induction of ENOD11. However, this JA treatment did not affect the spatial patterning of ENOD11-GUS expression suggesting that although a minimal number of Ca(2+) spikes are required for Nod-factor-induced gene expression, other factors restrict the expression of ENOD11 to a subset of responding cells.     

CGR2 and CGR3 have critical overlapping roles in pectin methylesterification and plant growth in Arabidopsis thaliana

Pectins are critical polysaccharides of the cell wall that are involved in key aspects of a plant's life, including cell‐wall stiffness, cell‐to‐cell adhesion, and mechanical strength. Pectins undergo methylesterification, which affects their cellular roles. Pectin methyltransferases are believed to methylesterify pectins in the Golgi, but little is known about their identity. To date, there is only circumstantial evidence to support a role for QUASIMODO2 (QUA2)‐like proteins and an unrelated plant‐specific protein, cotton Golgi‐related 3 (CGR3), in pectin methylesterification. To add to the knowledge of pectin biosynthesis, here we characterized a close homolog of CGR3, named CGR2, and evaluated the effect of loss‐of‐function mutants and over‐expression lines of CGR2 and CGR3 in planta. Our results show that, similar to CGR3, CGR2 is a Golgi protein whose enzyme active site is located in the Golgi lumen where pectin methylesterification occurs. Through phenotypical analyses, we also established that simultaneous loss of CGR2 and CGR3 causes severe defects in plant growth and development, supporting critical but overlapping functional roles of these proteins. Qualitative and quantitative cell‐wall analytical assays of the double knockout mutant demonstrated reduced levels of pectin methylesterification, coupled with decreased microsomal pectin methyltransferase activity. Conversely, CGR2 and CGR3 over‐expression lines have markedly opposite phenotypes to the double knockout mutant, with increased cell‐wall methylesterification levels and microsomal pectin methyltransferase activity. Based on these findings, we propose that CGR2 and CGR3 are critical proteins in plant growth and development that act redundantly in pectin methylesterification in the Golgi apparatus.     

Effects of boron deficiency and calcium supply on the calcium metabolism in tomato plant

Summary Tomato plants were grown in water-culture with a different supply of Ca (10, 100 ppm) and B (0, 0.2 ppm), and the effects of B deficiency on the translocation and subcellular distribution of Ca in tomato plants were studied by using⁴⁵CaCl₂ as a carrier of Ca. Boron deficiency slight increased the total Ca uptake by the plant and inhibited the Ca translocation to the upper leaves. The incorporation of⁴⁵Ca into the cell wall in the upper leaves was increased by B deficiency at both Ca levels. As Ca supply decreased, the distribution of⁴⁵Ca in the 1N NaCl fraction of the cell wall increased only at 0.2 ppm B. As B supply decreased, the distribution of⁴⁵Ca in the 0.6N HCl fraction increased at both Ca levels. These results suggest that B deficiency inhibit the translocation of Ca, and induce the abnormal changes of the Ca metabolism in the cell wall.     

The physiology and biochemistry of cultivar-strain interactions in the white clover-Rhizobium symbiosis

Two white clover cultivars were inoculated with two Rhizobium leguminosarum bv. trifolii strains in a factorial series of experiments. Plants were grown in axenic conditions in nitrogen free nutrient solution in a controlled environment room. Variations in nitrogen fixation were dependent partly upon general strain effects, partly upon general cultivar effects but there were also substantial differences attributable to precise interactions between specific combinations. The physiological and biochemical basis of these differences was examined. There were variations in the onset of nodulation and nitrogenase (acetylene reduction) activity. The rate at which nitrogenase activity developed also differed between associations as did the average size and number of nodules but none of these effects correlated well with differences in plant dry matter accumulation. Studies on nodule biochemistry revealed that the major nitrogen fixation enzymes were present in all four associations. Nodule protein content and enzyme activity (on a g nodule fresh weight basis) were substantially greater in associations formed by the more effective strain but cannot explain the interactive effect on dry matter accumulation. The relevance of these data to our understanding of factors regulating variations in nitrogen fixation is discussed.     

硼添加对铝胁迫栝楼生长和生理的影响

为揭示硼对缓解栝楼(Trichosanthes kirilowii Maxim.)铝(Aluminum)毒害的生理机制,以耐铝性强的安国栝楼和耐铝性弱的浦江栝楼为材料,研究了50μmol/L硼酸(H₃BO₃)对300μmol/L Al³⁺胁迫下栝楼幼苗生长、铝积累、抗氧化能力和细胞壁组分的影响。结果表明：铝胁迫下,植株的根长、株高、鲜重和干重降低,超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性受到显著抑制,安国栝楼、浦江栝楼根尖丙二醛(MDA)含量分别增加256.1%、278.2%,细胞壁多糖含量、果胶甲酯酶(PME)活性、根尖铝积累量显著提高,果胶甲基酯化度(DM)和3-脱氧-D-甘露-辛酮糖酸(KDO)含量降低。外源硼可以缓解铝胁迫对栝楼幼苗生长的抑制作用,安国与浦江栝楼的抗氧化酶活性显著提升,MDA含量、细胞壁多糖含量、PME酶活性均降低,安国栝楼果胶DM值恢复至正常水平的91.5%,浦江栝楼KDO含量较单铝处理组上升52.0%,活性铝结合位点减少,有效降低了根尖铝含量并维持其形态结构。因...     

Cell wall integrity: Targeted post-synthetic modifications to reveal its role in plant growth and defense against pathogens

The plant cell wall, a dynamic network of polysaccharides and glycoproteins of significant compositional and structural complexity, functions in plant growth, development and stress responses. In recent years, the existence of plant cell wall integrity (CWI) maintenance mechanisms has been demonstrated, but little is known about the signaling pathways involved, or their components. Examination of key mutants has shed light on the relationships between cell wall remodeling and plant cell responses, indicating a central role for the regulatory network that monitors and controls cell wall performance and integrity. In this review, we present a short overview of cell wall composition and discuss post-synthetic cell wall modification as a valuable approach for studying CWI perception and signaling pathways.     

Role of pectin methylesterases in cellular calcium distribution and blossom-end rot development in tomato fruit

Blossom-end rot (BER) in tomato fruit (Solanum lycopersicum) is believed to be a calcium (Ca(2+) ) deficiency disorder, but the mechanisms involved in its development are poorly understood. Our hypothesis is that high expression of pectin methylesterases (PMEs) increases Ca(2+) bound to the cell wall, subsequently decreasing Ca(2+) available for other cellular functions and thereby increasing fruit susceptibility to BER. The objectives of this study were to evaluate the effect of PME expression, and amount of esterified pectins and Ca(2+) bound to the cell wall on BER development in tomato fruit. Wild-type and PME-silenced tomato plants were grown in a greenhouse. At full bloom, flowers were pollinated and Ca(2+) was no longer provided to the plants to induce BER. Our results show that suppressing expression of PMEs in tomato fruit reduced the amount of Ca(2+) bound to the cell wall, and also reduced fruit susceptibility to BER. Both the wild-type and PME-silenced fruit had similar total tissue, cytosolic and vacuolar Ca(2+) concentrations, but wild-type fruit had lower water-soluble apoplastic Ca(2+) content and higher membrane leakage, one of the first symptoms of BER. Our results suggest that apoplastic water-soluble Ca(2+) concentration influences fruit susceptibility to Ca(2+) deficiency disorders.