The requirement of trace amounts of boron (B) for plant growth was reported as early as 1923 (Warington, 1923) and over several decades this micronutrient has been related to most biochemical, physiological or anatomical plant processes. Cell wall stability through borate cross-linking of apiosyl residues of the pectin polysaccharide rhamnogalacturonan II (RGII) is the most convincingly demonstrated role of B (O’Neill et al., 2004). Moreover, there is increasing evidence that B is involved in the control of expression of some genes that are important for plant metabolism (Beato et al., 2010) or cell differentiation and organogenesis (Lowatt, 1985; Reguera et al., 2009). Furthermore, the ability of boric acid to inhibit the growth of cell lines of prostate and breast cancer (Meacham et al., 2007) or abnormal embryonic development in animals under low-B conditions (Rowe & Eckhert, 1999) points to a function of the micronutrient that is related to signalling mechanisms and to the control of gene expression.
Further progress on this research topic will depend on new methodologies with greater analytical capability and on the use of biological models that are highly dependent on the micronutrient. Such a model is available in relation to the symbiotic interaction between legumes and nitrogen (N2)-fixing rhizobia, which triggers the organogenesis of a new root organ, the nodule, following a highly regulated process (Foucher & Kondorosi, 2000; Oldroyd & Downie, 2008). The high sensitivity of nodule development to B deficiency is in line with there being a higher B content in nodules than in other plant tissues (Redondo-Nieto et al., 2003). Symptoms of B deficiency appear in nodules earlier than in other plant organs that can develop using B storage in seed (Bolaños et al., 1994). This supports the utility of nodule organogenesis as a model for B-nutrition studies. Indeed, 3 wk after inoculation with rhizobia, B-deficient low functional nodules led to plants suffering from N deficiency. The reduction of the shoot and root FW of those plants (which was c. 40% lower than B-sufficient controls) was attributed mainly to a lack of N rather than to an effect of B deficiency on root and shoot growth (Bolaños et al., 1994).
Typical symptoms of B deficiency appear in the structure of symbiotic nodules with irregular walls as a result of an abnormal assembly of components, such as pectins and proline- and hydroxyproline-rich glycoproteins (Bonilla et al., 1997a,b). In addition, B deficiency affects each stage of the symbiosis (summarized in Table 1): early plant bacteria recognition (Redondo-Nieto et al., 2001), the infection process (Bolaños et al., 1996; Reguera et al., 2010a), symbiosome development (Bolaños et al., 2001; Redondo-Nieto et al., 2007) and cell differentiation during nodule organogenesis (Reguera et al., 2009). Interestingly, several of those B deficiency symptoms, mainly early preinfection and infection events or nodule organogenesis, but not symbiosome development, could be partially prevented by addition of Ca2+ (Redondo-Nieto et al., 2003).
|Symbiotic event||−B||−B + 2Ca2+ (Redondo-Nieto et al., 2003)|
|nod gene activity (Redondo-Nieto et al., 2001)||Inhibited||Restored|
|Root colonization (Redondo-Nieto et al., 2001)||Decreased||Restored|
|Infection thread development (Redondo-Nieto et al., 2001; Reguera et al., 2010b,c)||Prematurely aborted||Partially restored|
|Rhizobial endocytosis (Bolaños et al., 1996)||Decreased||Partially restored|
|Bacteroid proliferation (Bolaños et al., 1996)||Decreased||Restored|
|Symbiosomal PsNLEC 1 glycoprotein (Bolaños et al., 2001)||Abnormally glycosylated||Not restored|
|Peribacteroid membrane RGII-glycoproteins (Redondo-Nieto et al., 2007)||Absent||Not studied|
|Anaphase-promoting complex (ccs52 gene) (Reguera et al., 2009)||Down-regulated||This study|
|Hydroxyproline/proline-rich proteins (Bonilla et al., 1997a)||Not covalently bound||Not studied|
|Cell wall pectins (Bonilla et al., 1997b)||Disorganized||Partially restored|
To test the interesting hypothesis that B (and the B–Ca2+ relationship) is affecting the overall control of gene expression during nodule development, we grew the legume model Medicago truncatula inoculated with Sinorhizobium meliloti 1021 in B-sufficient or B-deficient media supplemented with different Ca2+ concentrations (see details in Redondo-Nieto et al., 2003).
Total RNA was isolated from nodules derived from four replicate experiments with three different B and Ca2+ treatments, and following the procedure described in Mergaert et al. (2003), and retrotranscribed in the presence of [α-33P]-deoxycytidine triphosphate to obtain 33P-labelled cDNA. Transcriptome analysis was performed by hybridization of the cDNA on a nylon membrane printed with probes from a cDNA library prepared from M. truncatula nodule mRNA (Györgyey et al., 2000; Mergaert et al., 2003). The probe set on the arrays included genes coding for nodulins (nodule-specific genes), cell cycle proteins, signal transduction proteins, transcription factors, components and regulators of the cytoskeleton, proteins involved in cell wall synthesis, a group representing diverse proteins and a set of clones with no homology to previously characterized proteins. In total, 192 unique genes, repeated three times, were represented on the arrays (see more details and the full data in the Supporting Information, Table S1).
Overall, a general shift was detected in the gene expression of B-deficient nodules, affecting > 70% of genes analysed. The majority were up-regulated genes, although some genes crucial for nodule development and function (discussed later) were down-regulated under B deficiency (Fig. 1). Among the affected gene categories (Table 2), it is noticeable that transcripts coding enzymes important for cell wall synthesis (i.e. cellulose synthase, chitinases) or for structural proline-rich and glycine-rich wall proteins were accumulated. This was likely a secondary response to the unstable cell wall structure under B deficiency, resulting in a demand of cell wall components that increases the expression of genes encoding the wall-building machinery. Short-term alteration of other cell wall-related genes in B-deficient Arabidopsis roots has also been reported (Camacho-Cristóbal et al., 2008), and therefore a primary effect of B deficiency on the expression of wall-modifying and wall-synthesis enzyme genes is also possible.
|Category/gene||−B||−B + 2Ca2+|
|MRP-like ABC transporter||Up-regulated||Restored|
|14-3-3 like protein||Up-regulated||Partially restored|
|14-3-3 like protein||Up-regulated||Partially restored|
|GDP dissociation inhibitor||Up-regulated||Restored|
|Small GTP-binding protein||Up-regulated||Restored|
|Small GTP-binding protein||Up-regulated||Restored|
|Small GTP-binding protein (RAB1)||Up-regulated||Restored|
|AMP activated protein kinase beta||Up-regulated||Restored|
|Transcription facto TFIII||Up-regulated||Restored|
|Myb-like DNA binding protein||Up-regulated||Restored|
|WRKY3 DNA binding protein||Up-regulated||Up-regulated|
|Putative ripening-related protein||Up-regulated||Restored|
|Glycine-rich protein 2||Up-regulated||Up-regulated|
|Prolin-rich protein (PRP1)||Up-regulated||Up-regulated|
|Glycine-rich protein 1||Up-regulated||Up-regulated|
|Glycine-rich protein 3||Up-regulated||Up-regulated|
|MtN3 nodulin-like protein||Down-regulated||Restored|
|Early N-75 nodulin||Up-regulated||Up-regulated|
|Nodulin 25 precursor||Up-regulated||Up-regulated|
|Histone H1||Up-regulated||Partially restored|
|Phospholipid glutathione peroxidase||Up-regulated||Restored|
|Putative disease resistance protein||Up-regulated||Restored|
|Hypersensitive-induced response protein||Up-regulated||Restored|
|Putative myosin heavy chain||Up-regulated||Restored|
|cdc2Ms E (ADN7605)||Down-regulated||Restored|
|cdc2Ms D (ADN7604)||Down-regulated||Restored|
|cdc2Ms F (ADN7606)||Down-regulated||Restored|
Genes typically involved in nodulation were either up-regulated (including several early and late nodulins) or down-regulated, for example, leg-haemoglobin genes previously reported in B-deficient Pisum sativum nodules (Reguera et al., 2010c). One important effect of B deficiency is an aberrant nodule organogenesis in which abnormal cell proliferation, accompanied by a lack of differentiation, was attributed to the down-regulation of the ccs52a gene (Reguera et al., 2009), which is involved in cell cycle arrest and transition of mitotic to endoreduplication cycles (Cebolla et al., 1999; Vinardell et al., 2003). In our study, not only ccs52a but also other genes involved in cell cycle regulation, including some cdc genes, were down-regulated, although expression of cyclin genes was not significantly affected by B deficiency (Table S1). Moreover, other transcripts involved in cell cycle regulation were accumulated. This indicates a perturbation in the cell cycle regulation during low-B nodule organogenesis. Other important categories of genes up-regulated by B deficiency included most of those coding for proteins involved in signal transduction and genes expressed in response to several types of stresses, which may be expected under B deficiency, as has been reported in low-B cultures of Nicotiana tabacum BY-2 cells (Kobayashi et al., 2004).
Interestingly, the effect of the lack of the micronutrient on the expression of most genes (> 75% of the up-regulated or down-regulated ones) was, at least partially, prevented by adding Ca2+ during plant growth and nodulation (Fig. 1, Tables 2, S1; −B + 2Ca treatment). Some of these results also support the suggestion by Koshiba et al. (2010) that Ca2+ influx plays a role in B-deprivation stress signalling. Accordingly, what is reported here agrees with those previous reports where application of a higher dose of Ca2+ to low-B N. tabacum BY-2 cells also affected the B-responsive gene expression. The M. truncatula genes with expression restored following Ca2+ application included most genes involved in cell cycle regulation, signal transduction and stress-responsive genes, enzymes involved in cell wall synthesis, several leghaemoglobins and nodule-specific cysteine-rich nucleotides (NCRs). However, although some NCRs govern terminal bacteroid differentiation (Van de Velde et al., 2010), the expression of other NCRs and nodulins or structural cell wall proteins were not restored to normal levels by the addition of Ca2+. This correlates with our previous report that Ca2+ can prevent abnormal nodule tissue differentiation under B deficiency but not the alteration of cell wall structure or aberrant bacteriod differentiation (Redondo-Nieto et al., 2003).
Overall, the results show that B nutrition affects the expression of a majority of genes during nodule organogenesis. To our knowledge, there is no other nutrient deficiency, including Ca2+, that results in such an alteration of gene expression. It has been proposed that the disturbed pectic network triggers low B-stress signalling, leading to reactive oxygen species (ROS) production and cell death in cultured tobacco BY-2 cells (Koshiba et al., 2009). However, as mentioned earlier, symptoms of B deficiency appeared in nodules earlier than in shoots or roots (Bolaños et al., 1994), and B deficiency apparently did not elicit ROS production and oxidative damage in nodules (Reguera et al., 2010c). Moreover, short-term studies performed by our group on B-deficient nodule development have shown that expression of some of the genes related to the symbiotic process, in particular those induced early after inoculation with rhizobia, were also affected (Redondo-Nieto et al., 2001; Reguera et al., 2009, 2010b). Furthermore, B deficiency also negatively regulates the physiology and development of living forms without cell walls, including animals (Rowe & Eckhert, 1999), and therefore it is unlikely that altered cell wall stability in low-B nodules is the main or only cause of this loss of gene regulation. Why then is there such a far-reaching effect? The key role of B on animal physiology is consistent with the fact that low B induced the mitogen-activated protein kinase (MAPK) pathway in cultured animal cells and that mammalian cell lines mutated in B transporters stop developing and proliferating (Park et al., 2004, 2005). The finding that B in bacteria is a signalling molecule (Chen et al., 2002) and that borate compounds can interact with regulatory proteins (see several examples in Golbach & Wimmer, 2007) admits the exciting possibility that B also exerts its action through interaction with transcription factors, hence explaining such a wide alteration of gene expression, as was hypothesized by González-Fontes et al. (2008). Supporting this hypothesis, Kasajima et al. (2010) have suggested that WRKY6 is a transcription factor involved in response to B deficiency in Arabidopsis.
Other possibilities come from the fact that several components of plant cell signalling and signal transduction pathways during organogenesis processes are known to be affected by B deficiency. Several studies on B effects in plant and animal development and metabolism point to a role for B in extracellular matrices and/or membrane functions (Brown et al., 2002). There is a well-known relationship linking B, phytohormones and development (Wang et al., 2006; Martín-Rejano et al., 2011), albeit this seems to be insufficient to explain the high B requirement for nodule organogenesis previously reported (Redondo-Nieto et al., 2003). Boron has been described as a modulator of host plant–rhizobia interactions by interacting with legume arabinogalactan proteins important for nodule infection and development (Reguera et al., 2010a). Moreover, several reports described abnormal glycosylation of glycoproteins apparently involved in cell-to-cell signalling during symbiosome development in nodules (Bolaños et al., 2001; Redondo-Nieto et al., 2007), and a similar role for B during animal organogenesis has been hypothesized (Redondo-Nieto et al., 2008). In a preliminary study (Bolaños et al., 2011), we have detected altered overall protein N glycosylation as a very early response to B deprivation during legume nodule and Arabidopsis root or shoot apical meristem development. This consisted of abnormal accumulation of high-mannose-type N-glycans, typical for the endoplasmic reticulum. As described earlier, B deficiency led to uncontrolled cell proliferation and aberrant cell differentiation, and interestingly, accumulation of high-mannose N-glycans is characteristic of some breast cancer lines (de Leoz et al., 2011). Therefore, the role of B in glycosylation mechanisms important for organogenesis has to be explored in more detail.
Moreover, using this M. truncatula nodule organogenesis model, an approach to identify molecules involved in cell signalling, other than glycans or transcription factors, affected by B and/or able to interact with borate could be designed. An exciting report by Ricardo et al. (2004) proposed that borate minerals could play a crucial role in an early ‘RNA world’ of life on Earth by stabilizing cyclic ribose; therefore, the structural and functional stability of microRNAs by borate is a possibility that should be tested. Also, inositides or adenylates, which are potential targets of B (Ralston & Hunt, 2001; Bolaños et al., 2004), could be part of signalling pathways affected by B deficiency. The fact that Ca2+ prevents altered expression of most genes affected by B deficiency suggests an effect of B on Ca2+-mediated signalling pathways. In this regard, it has been demonstrated that boric acid can prevent proliferation of some tumour cells by affecting the release of Ca2+ by cyclic ADP ribose (cADPr), and that this effect is based on its binding capacity to NAD+, the substrate of ADP ribosyl cyclase (Barranco et al., 2008). Therefore, in addition to exploring a primary interaction of B with transcription factors or with signalling and regulatory molecules such as glycans or microRNAs, it will be interesting to determine whether B deficiency affects Ca2+ influx (as suggested by Koshiba et al., 2010) and/or also interferes with Ca2+-releasing mechanisms that are dependent on adenylates or inositides. Studies like this will shed new light on the role of B in the regulation of gene expression and on the effect of the B–Ca2+ relationship on gene expression.