- Top of page
- Materials and Methods
- Supporting Information
Biological nitrogen fixation is carried out only by prokaryotes in an enzymatic complex called nitrogenase where atmospheric nitrogen (N2) is reduced to ammonia. The importance of this mutualistic interaction arises from the fact that legumes are an important source of protein in the diet of many countries worldwide, and symbiotic fixed nitrogen makes a major contribution to this, accounting for roughly 200 million tons of nitrogen annually (Graham & Vance, 2003; Kouchi et al., 2010). Leguminous plants are the second largest group of food and feed crop species grown globally, and represent the third largest group of angiosperm species. A key developmental process in this remarkable plant–bacteria interaction is the formation on plant roots of novel organs called nodules, wherein rhizobia differentiate into their intracellular nitrogen-fixing form, known as bacteroids. These structures develop after exchange and recognition of signaling molecules between the rhizobial bacteria and the leguminous plant (Gage, 2004; Ferguson et al., 2010).
The ontogeny of nodules has been characterized genetically, biochemically, and at the physiological and molecular levels throughout all stages, leading to successful and efficient symbiosis (Streeter, 1987; Fougère et al., 1991; Salminen & Streeter, 1992; López et al., 2008; Kouchi et al., 2010). Recently, the metabolites produced at the beginning of the rhizobium–legume symbiotic interaction were analyzed by high-throughput methods (metabolome analysis) and a significant induction in the biosynthesis of 166 metabolites was found, corresponding to flavonoids, amino acids, fatty acids, carboxylic acids, and carbohydrates, with trehalose among the metabolites induced to the greatest extent (Brechenmacher et al., 2010). In Phaseolus vulgaris (common bean) interacting with Rhizobium, enhanced germination, quality, and grain yield have been correlated with trehalose content, and a higher tolerance to abiotic stress has been reported (Farías-Rodriguez et al., 1998; Altamirano-Hernández et al., 2007).
Trehalose (α-D-glucopyranosyl-1,1-α-D-glucopyranoside) is a nonreducing disaccharide. Trehalose biosynthetic and degradation pathways are widespread throughout bacteria, archaebacteria, fungi, metazoans (except for vertebrates), and plants (Avonce et al., 2006). Trehalose (and its precursor trehalose-6-phosphate) is a signaling molecule involved in growth, development, and differentiation in plant cells (Paul et al., 2008). There are five reported biosynthetic pathways for trehalose, but only one for degradation. Trehalose degradation is carried out by trehalase, yielding two glucose moieties (Avonce et al., 2006). Although trehalose is commonly detected during the legume–rhizobia symbiosis, from rhizobia recognition by root hairs through nodule development and nitrogen fixation, trehalose metabolism in leguminous plants is still poorly understood (Müller et al., 2001b; Brechenmacher et al., 2010; Vauclare et al., 2010). Moreover, the importance of trehalose metabolism in plant–microbe interactions, such as legume–rhizobium, plant–mycorrhiza, and plant–pathogen interactions, has recently been highlighted (Müller et al., 2001b; Brodmann et al., 2002; Foster et al., 2003; Ocón et al., 2007; Nehls, 2008; Wilson et al., 2010).
In senescent nodules, trehalose becomes the most abundant nonstructural carbohydrate, as evidenced by the fact that the sucrose content was found to undergo a marked (84%) decrease, while up to one-half of the trehalose remained during nodule senescence (Müller et al., 2001b). Nodule trehalose biosynthesis and catabolism seem to be two separate, albeit interconnected, processes, as trehalose in the symbiosomes is not degraded by the trehalase present in the cytosol of the infected cells of the nodules, and the concentrations of trehalose inside the symbiosomes are kept constant throughout nitrogen fixation (Vauclare et al., 2010).
Three approaches have been described to study the role of trehalose in the symbiotic process: addition of validamycin A directly to the nodules to inhibit trehalase enzymatic activity; overexpression of the endogenous trehalose phosphate synthase (TPS) from Rhizobium to enhance trehalose accumulation in Rhizobium before and during symbiotic interaction with leguminous plants; and mutating genes for trehalose assimilation in Sinorhizobium to induce trehalose accumulation in Sinorhizobium. Induction of trehalose accumulation in nodules of leguminous plants through validamycin A addition leads to improvement of tolerance to drought and salinity stress. Overexpression of endogenous rhizobial TPS or mutating trehalose assimilation genes enhances trehalose accumulation in bacteria as well as in bacteroids, leading to a significant increase in nodule biomass and nitrogenase activity and conferring abiotic stress tolerance to the plant (Jensen et al., 2005; Suárez et al., 2008; López et al., 2009).
Trehalose accumulation in the cytosol of infected nodule cells strongly suggests that induction of trehalose synthesis and degradation during nodule development are tightly regulated. Furthermore, trehalose metabolism directly impacts photosynthate partitioning and utilization in sink organs such as nodules, which constantly require carbohydrates to fuel growth, N2 fixation and assimilation (Aeschbacher et al., 1999; Müller et al., 2001a; Xie et al., 2003; Van Dijken et al., 2004). Trehalase transcripts are present in expressed sequenced tag (EST) databases of several leguminous plants during symbiosis (Pontius et al., 2003; Ramírez et al., 2005). In the common bean, only three sequences corresponding to trehalase (CV535700, CV537568, and CV535738) were found, from a total of 112 229 sequences deposited to date in the Common Bean Gene Index (DFCI/Common Bean Gene Index, v4.0, http://compbio.dfci.harvard.edu/tgi/). Interestingly, these ESTs are part of the same contig and were found only in the nodule transcriptome.
Herein, transgenic roots (composite plants) induced by Agrobacterium rhizogenes K599 (Estrada-Navarrete et al., 2007; Blanco et al., 2009; Sánchez-López et al., 2011) were used to assess the role of trehalose in common bean symbiosis by knocking down PvTRE1 transcript abundances by RNAi. Furthermore, since genetic transformation in composite plants is restricted to roots and nodules, we evaluated the systemic effect of nodule trehalose accumulation on the untransformed upper part of the plant. This approach helped us to characterize and directly determine the role of trehalose metabolism in nodules of P. vulgaris, and revealed the tight relationship among nodules and the upper parts (leaves) of the plant during symbiotic interaction.
- Top of page
- Materials and Methods
- Supporting Information
Herein the complete sequence of the trehalase gene (PvTRE1) found in the common bean genome (Phytozome v8.0 and Mazorka) was obtained and compared with homologous plant (GmTRE1) and E. coli (EcTRE) trehalase genes. Prediction of the three-dimensional structure of the encoded protein based on the known structure of the E. coli homolog led us to designate this gene PvTRE1. The structural analysis revealed a high degree of conservation, and moreover, they are grouped (Fig. 1) with other plant trehalases, suggesting a high degree of sequence conservation and perhaps of structure/function identity among plant trehalases, as was reported for trehalases from insects and fungi, and other phylogenetic groups (Doehlemann et al., 2006; Lunn, 2007; Silva et al., 2009; Chen et al., 2010). The PvTRE1 gene structure consists of 10 exons and nine introns, similar to the gene structure of GmTRE1 and MtTRE1, both of which have 11 exons and 10 introns, and different from AtTRE1, which contains six exons and five introns (Fig. S1a). Furthermore, in nodulated roots, the expression of PvTRE1 was restricted mainly to nodules, which resembled the expression of nodulin genes, for example, leghemoglobin A and sucrose synthase. Nodule-enhanced expression of PvTRE1 (Fig. 2a,b) suggests that PvTRE1 could have an active role in carbon metabolism in nodules, as previously suggested for its homolog (GmTRE1) in soybean (Aeschbacher et al., 1999).
PvTRE1 transcript accumulation in roots was lower than that in nodules, but remained constant throughout nodule ontogeny. By contrast, the PvTRE1 transcript accumulation profile in nodules was inversely proportional to trehalose concentration during nodule development, and it is worth noting that the highest PvTRE1 transcript accumulation coincided with the beginning of nodule senescence, correlating with the up-regulation of AtTRE1 during senescence in Arabidopsis (Fernandez et al., 2010). Interestingly, PvSUS1 transcript accumulation paralleled trehalose content (Fig. S2b) and was opposite to accumulation of the PvTRE1 transcript (Fig. 2c). This supports the idea that PvTRE1 regulates trehalose content, as inhibition of trehalase activity by addition of validamycin A in nodules resulted in trehalose accumulation (López et al., 2009). Additionally, it seems that trehalose content influences PvSUS1 expression directly, which is consistent with a previous report that addition of trehalose into nonnodulated soybean roots strongly induces sucrose synthase activity, and trehalase inhibition by validamycin A leads to a decrease in sucrose content (Müller et al., 1995, 1998).
We decided to assess the impact of trehalose on nodule physiology by down-regulating PvTRE1 expression via RNAi in common bean composite plants. The resulting 82% decrease in PvTRE1 transcript (Fig. 3a) accumulation seems to have a higher impact on trehalose content (78% increase; Fig. 5a) than that attained by inhibiting trehalase activity with validamycin A in soybean nodules; this led to a 73% inhibition of enzyme activity but only a 46% increase in trehalose (Müller et al., 1995). In addition to the significant enhancement of trehalose content in PvTRE1-RNAi transgenic nodules, the expression of genes directly involved in growth and carbon metabolism (PvSUS1, PvHXK1, PvTOR, PvSnRK1) and autophagy (PvATG3) were also increased (Fig. 3b), suggesting that the up-regulation was triggered by PvTRE1 down-regulation, which in turn gave rise to a higher trehalose content. The increase in transcript accumulation for sucrose synthase (203%) and hexokinase (134%) reflects an enhancement in carbon assimilation, most probably driven by an increased rate of sucrose degradation, as evidenced by the specific decrease in sucrose in these nodules without any significant changes in soluble fructose and glucose concentrations (Fig. 5a; Müller et al., 1998; Moore et al., 2003). Furthermore, the increase in PvTOR (43%), PvSnRK1 (81%), and PvATG3 (26%) transcript (Fig. 3b) accumulation suggests a direct positive impact on autophagy and cellular cytoplasm recycling, as these genes are actively involved in regulating cell development and protein turnover. Previously, it has been reported that trehalose can induce autophagy (trehalose-dependent autophagy) by a target of rapamycin (TOR)-independent pathway, suggesting that plant and animal cells possess similar mechanisms to promote cellular recycling instead of triggering programmed cell death (Sarkar et al., 2007; Aguib et al., 2009; Jossier et al., 2009; Wang et al., 2009; Casarejos et al., 2011; Hofius et al., 2011).
The accumulation of trehalose, mediated by PvTRE1 down-regulation, triggered sucrose degradation and increased bacterial survival, nodule biomass, nitrogen fixation, and GOGAT transcript accumulation (Fig. 5). The enhanced bacterial proliferation inside nodules could be fueled by the trehalose accumulation and its translocation from the infected cells to Rhizobium. Rhizobia possess two operons and two genes directly involved in trehalose transportation and assimilation (thuEFGK, aglEFGAK, thuA, thuB) mainly induced by trehalose (Jensen et al., 2002, 2005; Ampomah et al., 2008; Schmeisser et al., 2009), suggesting that the trehalose-processing metabolism could be active in bacteroids. Furthermore, the increased trehalose in nodules not only triggered an increase in nodule biomass, but also had a positive impact on trehalose accumulation in leaves. This suggests a systemic effect of trehalose accumulation in nodules, as the plant is only partially genetically modified: only the roots and nodules are transgenic. The trehalose content in leaves was increased by c. 27%, without any detriment to leaf area or leaf DW (net biomass; Fig. 6a,d,e). The increased trehalose in leaves could be a result of the mobilization of this disaccharide from nodules to leaves, as previously reported and suggested to occur in seeds from common bean plants inoculated with Rhizobium (Altamirano-Hernández et al., 2007). Therefore, this could suggest that there is direct bidirectional transport of disaccharides between nodules and leaves in common bean plants during symbiosis. In addition, the accumulation of trehalose caused by down-regulating PvTRE1 in nodules led to an increased proliferation of bacteroids (by almost one order of magnitude), the enhancement of nitrogen fixation (70%) and a higher nodule biomass (threefold). It is worth noting that increased nodule biomass and bacteroid proliferation did not have a negative effect on leaf area or leaf biomass. In supernodulating legumes, a negative impact has been observed on the aerial part of the plant (length and biomass reduced). Interestingly, this increase in nodule number does not lead to higher nitrogen fixation rates (Penmetsa et al., 2003; Schnabel et al., 2010). Thus, PvTRE1 down-regulation in nodules leads to an increase in carbon consumption, reflected in enhanced nodule biomass, and in turn an increased trehalose content in nodules as well as in leaves. Trehalose is known to play a central role in the regulation of sucrose content in plant cells (Lunn, 2008; Paul, 2008; Schluepmann & Paul, 2009). The results obtained in this work suggest that trehalose is able to induce PvSUS1 expression for sucrose degradation, and to regulate the sucrose content in plant cells (Müller et al., 1998; Schluepmann et al., 2003, 2004; Gómez et al., 2006; Ramon & Rolland, 2007). Altogether, the data suggest that by modifying the trehalose content in nodules, it is possible to alter metabolic carbohydrate flux, changing the sink-and-source state of these organs (Schluepmann & Paul, 2009).
Interestingly, changes in the bacteroid number and morphology of the bacteroids inside these nodules, including an edb structure surrounding the bacteroids (Fig. 4i), were observed. These effects could be derived from carbohydrates, as previously reported for another Rhizobium species (Fernandez-Aunión et al., 2010). The modification of the trehalose content in Rhizobium by the overexpression or mutation of a trehalose biosynthesis (OtsA) gene directly affected the distribution of infected cells inside the nodules, and triggered the expression of several genes involved in nitrogen assimilation, carbon assimilation, oxygen transport, and hydrogen peroxide or reactive oxygen species detoxification in common bean during the symbiotic interaction, which gave rise to improvements in grain yield, nitrogen fixation, and stress tolerance (Suárez et al., 2008). These findings strongly suggest that modification of the trehalose content in the nodules triggers physiological alterations that enhance carbon and nitrogen metabolism, as well as bacteroid fitness (greater survival) and nitrogen fixation, which in turn positively impact the symbiotic interaction. Finally, we propose that breeding for the genetic modification of trehalose degradation in nodules could be a valuable mechanism for improving agricultural symbiotic nitrogen fixation.
PvTRE1 down-regulation in common bean nodules was achieved using RNAi technology, giving rise to a 78% increase in trehalose accumulation. The resulting enhancement in nodule biomass and bacterial proliferation strongly supports a central and active role for trehalose in orchestrating carbon metabolism in plant cells. In spite of the nodule biomass increase, there was no detrimental effect on leaf area or leaf biomass. Since PvTRE1 down-regulation has a remarkably positive effect on the symbiosis, this approach could represent a new promising and viable strategy for genetic screening of trehalase mutants that could enhance symbiotic nitrogen fixation.