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Genes associated with translation and oxidative phosphorylation as components of the translational response in nodulated and water-restricted soybean.

BACKGROUND: Soybean primarily acquires nitrogen through symbiosis with nitrogen-fixing bacteria. Water deficit (WD) is a major stress limiting crop yield. Nodulation may enhance drought tolerance in legumes by modulating nitrogen and hormone metabolism, osmotic adjustment, and antioxidant defenses; however, the molecular basis underlying the differential WD responses between N-fix and N-fed plants remain unclear. Translational control of gene expression is a key regulatory mechanism during stress. RESULTS: We compared the transcriptome and translatome of soybean roots from N-fix and N-fed plants exposed to WD across four combined treatments. N-fix plants under WD exhibited more complex responses in terms of total differentially expressed genes (DEGs) compared to N-fed plants. This increased complexity was also evident among translationally regulated DEGs and differentially expressed transcription factors, whose involvement in WD responses of N-fix plants is novel. Co-expression network analysis identified modules associated with core biological processes encompassing nodulation, WD, and notably, their interplay was particularly prominent in Module 1, which was enriched in genes related to ribosomal protein synthesis and oxidative phosphorylation (OXPHOS). Guilt-by-Association analysis enabled the prediction of novel functions for differentially expressed, uncharacterized hub genes related to stress and/or nodulation responses. CONCLUSIONS: Translational regulation of genes involved in OXPHOS and translation initiation emerged as a central response in N-fix plants under WD. These findings reveal distinct molecular adaptations in N-fix soybean roots facing WD and highlight translational control as a key regulatory layer. We also identified promising candidate genes-including transcription factors and uncharacterized hub genes under translational regulation-that represent potential targets for improving drought tolerance in legumes once validated functionally.

Glycine max↗

enod40, a gene expressed during nodule organogenesis, codes for a non-translatable RNA involved in plant growth.

Rhizobium meliloti can interact symbiotically with Medicago plants, thereby inducing root nodules. However, certain Medicago plants can form nodules spontaneously, in the absence of rhizobia. A differential screening was performed using spontaneous nodule versus root cDNAs from Medicago sativa ssp. varia. Transcripts of a differentially expressed clone, Msenod40, were detected in all differentiating cells of nodule primordia and spontaneous nodules, but were absent in fully differentiated cells. Msenod40 showed homology to a soybean early nodulin gene, Gmenod40, although no significant open reading frame (ORF) or coding capacity was found in the Medicago sequence. Furthermore, in the sequences of cDNAs and a genomic clone (Mtenod40) isolated from Medicago truncatula, a species containing a unique copy of this gene, no ORFs were found either. In vitro translation of purified Mtenod40 transcripts did not reveal any protein product. Evaluation of the RNA secondary structure indicated that both msenod40 and Gmenod40 transcripts showed a high degree of stability, a property shared with known non-coding RNAs. The Mtenod40 RNA was localized in the cytoplasm of cells in the nodule primordium. Infection with Agrobacterium tumefaciens strains bearing antisense constructs of Mtenod40 arrested callus growth of Medicago explants, while overexpressing Mtenod40 embryos developed into teratomas. These data suggest that the enod40 genes might have a role in plant development, acting as 'riboregulators', a novel class of untranslated RNAs associated with growth control and differentiation.

Amino Acid Sequence↗

Calcium spiking in plant root hairs responding to Rhizobium nodulation signals.

SUMMARY: Rhizobium lipochitooligosaccharide signal molecules stimulate multiple responses in legume host plants, including changes in host gene expression, cell growth, and mitoses leading to root nodule development. The basis for signal transduction in the plant is not known. We examined cytoplasmic free calcium in host root hairs using calcium-sensitive reporter dyes. Image analysis of injected dyes revealed localized periodic spikes in cytoplasmic calcium levels that ensued after a characteristic lag following signal application. Structural features of the signal molecules required to cause nodulation responses in alfalfa are also essential for stimulating calcium spiking. A nonnodulating alfalfa mutant is defective in calcium spiking, consistent with the possibility that this mutant is blocked in an early stage of nodulation signal perception.

Calcium↗

A Krüppel-like zinc finger protein is involved in nitrogen-fixing root nodule organogenesis.

Mechanisms regulating plant host differentiation of the nitrogen-fixing root nodules remain mostly unknown. Sinorhizobium meliloti induces this process in Medicago sativa in which the Mszpt2-1 gene is expressed in vascular bundles of roots and nodules. This gene codes for a Krüppel-like zinc finger protein, a class of transcription factors involved in many animal developmental processes. Expression of Mszpt2-1 in yeast cells conferred osmotic tolerance. Antisense plants grew normally but developed nonfunctional nodules, in which differentiation of the nitrogen-fixing zone and bacterial invasion were arrested. Hence, a vascular bundle-associated Krüppel-like gene is required for the formation of the central nitrogen-fixing zone of the root nodule.

Amino Acid Sequence↗

A novel nodule-enhanced gene encoding a putative universal stress protein from Astragalus sinicus.

A nodule-enhanced gene, AsD243, was identified from infected roots of Astragalus sinicus using suppressive subtractive hybridization (SSH). It encodes a 20-kD protein related to the bacterial universal stress protein family (Usp). Sequence analysis showed that AsD243 is highly similar to the bacterial MJ0577-type of ATP-binding Usp proteins, which have been proposed to function as a molecular switch. Expression analyses revealed that AsD243 was transcribed in all plant organs, and progressively during all stages of nodulation. Its transcripts increased significantly at 7 days after inoculation, which is 2 days later than the onset of leghemoglobin expression in A. sinicus nodules. AsD243 was expressed more strongly in mature roots than in young roots regardless of inoculation status. We suggest that the AsD243 may have other functions in plant processes besides nodulation.

Amino Acid Sequence↗

A cytokinin perception mutant colonized by Rhizobium in the absence of nodule organogenesis.

In legumes, Nod-factor signaling by rhizobia initiates the development of the nitrogen-fixing nodule symbiosis, but the direct cell division stimulus that brings about nodule primordia inception in the root cortex remains obscure. We showed that Lotus japonicus plants homozygous for a mutation in the HYPERINFECTED 1 (HIT1) locus exhibit abundant infection-thread formation but fail to initiate timely cortical cell divisions in response to rhizobial signaling. We demonstrated that the corresponding gene encodes a cytokinin receptor that is required for the activation of the nodule inception regulator Nin and nodule organogenesis.

Alleles↗

Toxic effects of low concentrations of Cu on nodulation of cowpea (Vigna unguiculata).

Although Cu is phytotoxic at Cu(2+) activities as low as 1-2 microM, the effect of Cu(2+) on the nodulation of legumes has received little attention. The effect of Cu(2+) on nodulation of cowpea (Vigna unguiculata (L.) Walp. cv. Caloona) was examined in a dilute solution culture system utilising a cation exchange resin to buffer solution Cu(2+). The nodulation process was more sensitive to increasing Cu(2+) activities than both shoot and root growth; whilst a Cu(2+) activity of 1.0 microM corresponded to a 10% reduction in the relative yield of the shoots and roots, a Cu(2+) activity of 0.2 microM corresponded to a 10% reduction in nodulation. This reduction in nodulation with increasing Cu(2+) activity was associated with an inhibition of root hair formation in treatments containing > or =0.77 microM Cu(2+), rather than to a reduction in the size of the Rhizobium population.

Copper↗

Cell cycle regulation in the course of nodule organogenesis in Medicago.

The molecular mechanisms of de novo meristem formation, cell differentiation and the integration of the cell cycle machinery into appropriate stages of the developmental programmes are still largely unknown in plants. Legume root nodules, which house nitrogen-fixing rhizobia, are unique plant organs and their development may serve as a model for organogenetic processes in plants. Nodules form and are essential for the plant only under limitation of combined nitrogen in the soil. Moreover, their development is triggered by external mitogenic signals produced by their symbiotic partners, the rhizobia. These signals, the lipochitooligosaccharide Nod factors, act as host-specific morphogens and induce the re-entry of root cortical cells into mitotic cycles. Maintenance of cell division activity leads to the formation of a persistent nodule meristem from which cells exit continuously and enter the nodule differentiation programme, involving multiple cycles of endoreduplication and enlargement of nuclear and cell volumes. While the small diploid 2C cells remain uninfected, the large polyploid cells can be invaded and, after completing the differentiation programme, host the nitrogen-fixing bacteroids. This review summarizes the present knowledge on cell cycle reactivation and meristem formation in response to Nod factors and reports on a novel plant cell cycle regulator that can switch mitotic cycles to differentiation programmes.

Cell Cycle↗

Arbuscular mycorrhizal fungi colonize nonfixing root nodules of several legume species.

Many legumes form tripartite symbiotic associations with rhizobia and arbuscular mycorrhizal fungi (AMF). Rhizobia are located in root nodules and provide the plant with fixed atmospheric nitrogen, while AMF colonize plant roots and deliver several essential nutrients to the plant. Recent studies showed that AMF are also associated with root nodules. This might point to interactions between AMF and rhizobia inside root nodules. Here, we test whether AMF colonize root nodules in various plant-AMF combinations. We also test whether nodules that are colonized by AMF fix nitrogen. Using microscopy, we observed that AMF colonized the root nodules of three different legume species. The AMF colonization of the nodules ranged from 5% to 74% and depended on plant species, AMF identity and nutrient availability. However, AMF-colonized nodules were not active, that is, they did not fix nitrogen. The results suggest that AMF colonize old senescent nodules after nitrogen fixation has stopped, although it is also possible that AMF colonization of nodules inhibits nitrogen fixation.

Fabaceae↗

Role of cellulose fibrils and exopolysaccharides of Rhizobium leguminosarum in attachment to and infection of Vicia sativa root hairs.

Infection and subsequent nodulation of legume host plants by the root nodule symbiote Rhizobium leguminosarum usually require attachment of the bacteria to root-hair tips. Bacterial cellulose fibrils have been shown to be involved in this attachment process but appeared not to be essential for successful nodulation. Detailed analysis of Vicia sativa root-hair infection by wild-type Rhizobium leguminosarum RBL5523 and its cellulose fibril-deficient celE mutant showed that wild-type bacteria infected elongated growing root hairs, whereas cellulose-deficient bacteria infected young emerging root hairs. Exopolysaccharide-deficient strains that retained the ability to produce cellulose fibrils could also infect elongated root hairs but infection thread colonization was defective. Cellulose-mediated agglutination of these bacteria in the root-hair curl appeared to prevent entry into the induced infection thread. Infection experiments with V sativa roots and an extracellular polysaccharide (EPS)- and cellulose-deficient double mutant showed that cellulose-mediated agglutination of the EPS-deficient bacteria in the infection thread was now abolished and that infection thread colonization was partially restored. Interestingly, in this case, infection threads were initiated in root hairs that originated from the cortical cell layers of the root and not in epidermal root hairs. Apparently, surface polysaccharides of R. leguminosarum, such as cellulose fibrils, are determining factors for infection of different developmental stages of root hairs.

Bacterial Adhesion↗

Evolution and Expression Divergence of Legume PAL Genes Suggest Associations with Drought Response and Root Nodule Development.

Comparative genomic analyses provide insight into the mechanisms underlying gene-family evolution and crop adaptation. Here, we used the legume phenylalanine ammonia-lyase (PAL) gene family as a model and integrated pan-genomic, phylogenetic, molecular evolutionary, duplication-mode, and transcriptomic analyses, while developing GFtool for gene family identification. Across 45 genomes, we identified 302 PAL genes and classified them into five Groups. Groups 1-3 represented ancient lineages shared with outgroups, whereas Groups 4 and 5 were legume-specific. Molecular-clock analyses placed the divergence of Group 2 near the Paleocene-Eocene transition, while Groups 4 and 5 diversified from the middle Eocene to the early Oligocene. WGD/segmental duplication broadly contributed to PAL copy-number expansion, whereas tandem duplication was enriched in Group 5 of Papilionoideae. Group 2 genes showed drought-induced expression, whereas Group 5 genes were associated with early root nodule development. GFtool provides a scalable framework for gene-family studies.

Fabaceae↗

LjnsRING, a novel RING finger protein, is required for symbiotic interactions between Mesorhizobium loti and Lotus japonicus.

Nodule-specific (nodulin) genes are thought to play crucial roles during establishment of the nitrogen-fixing symbiosis between legume plants and Rhizobium bacteria. On the basis of a gene expression database for early stages of the nodulation process of Lotus japonicus, previously constructed by a cDNA macroarray analysis, we identified a novel nodulin gene, LjnsRING, which encodes a protein with a typical RING-H2 finger domain that is well conserved in a number of plant E3 ubiquitin ligases. LjnsRING transcripts were almost exclusively expressed in nodules, and very low expression was detected in roots and shoots. RNA interference (RNAi) knockdown of LjnsRING by hairy root transformation caused impaired root growth together with abortion of nodule formation. Examination with lacZ-labeled Mesorhizobium loti indicated that infection thread formation in the RNAi transgenic hairy roots was significantly inhibited. Analysis using a chimeric gene of LjnsRING promoter and beta-glucuronidase (GUS) coding region demonstrated that LjnsRING transcription in nodules was restricted to the infected cells. These results suggest the requirement for LjnsRING in rhizobial infection and the subsequent nodule formation process.

Amino Acid Sequence↗

Proteolysis during Development and Senescence of Effective and Plant Gene-Controlled Ineffective Alfalfa Nodules.

Plant-controlled ineffective root nodules, conditioned by the in1 gene in Medicago sativa L. cv Saranac, undergo premature senescence and have reduced levels of many late nodulins. To ascertain which factors contribute to premature senescence, we have evaluated proteolysis as it occurs throughout the development of ineffective Saranac (in1Sa) and effective Saranac nodules. Cysteine protease activities with acidic pH optimum and enzyme proteins were present in both genotypes. We found that acidic protease activity was low in effective Saranac nodules throughout their development. In contrast, by 2 weeks after inoculation, acid protease activity of in1Sa nodules was severalfold higher than that of Saranac nodules and remained high until the experiment was terminated 8 weeks later. This increase in protease enzyme activity correlated with an increase in protease protein amounts. Increased protease activity and amount in in1Sa nodules was correlated with a decrease in nodule soluble protein. The time at which in1Sa nodules initially showed increased protease activity corresponded to when symbiosis deteriorated. High levels of phosphoenolpyruvate carboxylase (PEPC) protein were expressed in effective nodules by 12 d after inoculation and expression was associated with low proteolytic enzyme activity. In contrast, although PEPC was expressed in in1Sa nodules, PEPC protein was not found 12 d after inoculation and thereafter. Acidic protease from in1Sa nodules could also degrade purified leghemoglobin. These data indicate that premature senescence and low levels of late nodulins in in1Sa nodules can be correlated in part with increased proteolysis.

Journal Article↗

Morphological variation, histology, histochemistry and nutrient contents of Frankia root nodules in Casuarina equisetifolia.

Root nodules were collected from the existing plantation of Casuarina equisetifolia from three different agroclimatic zones of Tamil Nadu, India. Morphological variation, histology, histochemistry and nutrient contents of the nodules were studied. The nodules were found to be simple, dichotomously branched and coralloid. The stele in simple and branched nodules reached up to the tip of the nodules. Structure of Frankia nodules was described using histochemical methods to elucidate the nature of micro-symbiont within the nodules. The nodule consisted of a distinct periderm enclosing the cortex. The cortex showed patches of infected cells interspersed among the uninfected ones. Inside the cortex was a stele bound by an endodermis, which contained high amount of tannin. The micro-symbiont was observed in the nodule tisues in hyphal form and spore packed sporocorp was also seen in the cortex. The histochemical tests revealed the presence of proteins, insoluble polysaccharides and polyphenols.

Biomass↗

CRISPR/Cas9-mediated knockout of PsLykX gene of pea (Pisum sativum L.) leads to loss of symbiotic nodules.

Pea (Pisum sativum L.) symbiosis with nodule bacteria supplying plants with additional nitrogen is a very specific plant-microbial interaction. Mutual recognition of the partners occurs through perception of bacterial signal molecules (Nod factors) by plant receptors, enabling bacterial entry via root hairs and formation of nitrogen-fixing nodules. The pea gene Sym2, described but not yet cloned, exists in different allelic forms defining the symbiotic specificity, and is therefore thought to encode a Nod factor receptor. The PsLykX gene is a strong candidate for the Sym2, since its alleles coincide with high or low symbiotic specificity; however, to date, no genetic evidence has been obtained for a role of PsLykX in symbiosis. Here, we knocked-out the PsLykX in European pea cultivar Caméor using Agrobacterium-mediated hairy root transformation and CRISPR-Cas9 editing. The roots with editing events confirmed by sequencing lost the ability to form nodules, providing direct functional evidence that PsLykX is essential, at least, for the symbiosis between pea cultivar Caméor and Rhizobium ruizarguesonis RCAM1026.

Pisum sativum↗

Weather and nodule mediated variations in delta 13C and delta 15N values in field-grown soybean (Glycine max L.) with special interest in the analyses of xylem fluids.

The nodulating soybean (Enrei) and its non-nodulating mutant (EN 1282) were grown in outdoor plots for 2 years (1994: extraordinary dry, high temperature, 1995: ordinary year). Carbon and nitrogen accumulation, delta 13C and delta 15N values in plant parts and xylem fluids and delta 15N values in the water-extractable soil N were analysed throughout the growing period. Plant growth in 1994 was rapid during the early growth stages, but no pods were produced. In 1995, plant growth was normal and pods were formed. The delta 13C values of the leaves were less negative in 1994 than in 1995 and the nodulated plants showed less negative delta 13C values than non-nodulated plants in both years. The delta 13C values of the leaves during the vegetative phase were positively correlated to the leaf N concentrations. Leaf N concentrations in their turn were influenced by nodulation and weather conditions and/or soil available N. The delta 15N values in the plants and xylem fluids were lower in the nodulated soybean than in non-nodulated soybean in both years, and estimates of the contribution of N2 fixation in nodulated plants based on plant top delta 15N values were 7-14% in 1994 and 37-63% in 1995. The delta 13C values of xylem fluids did not differ between nodulated and non-nodulated plants. Thus, the expected contribution by phosphopenolpyruvate carboxylase-mediated CO2 fixation in the root nodules to plant C-incorporation could not have been significant.

Carbon↗