Gene Expression In Nodules Research Articles

Nitrogen and Phosphorus Signaling and Transport During Legume-Rhizobium Symbiosis.

Nitrogen (N) and phosphorus (P) are the two predominant mineral elements, which are not only essential for plant growth and development in general but also play a key role in symbiotic N fixation in legumes. Legume plants have evolved complex signaling networks to respond to both external and internal levels of these macronutrients to optimize symbiotic N fixation in nodules. Inorganic phosphate (Pi) and nitrate (NO3−) are the two major forms of P and N elements utilized by plants, respectively. Pi starvation and NO3− application both reduce symbiotic N fixation via similar changes in the nodule gene expression and invoke local and long-distance, systemic responses, of which N-compound feedback regulation of rhizobial nitrogenase activity appears to operate under both conditions. Most of the N and P signaling and transport processes have been investigated in model organisms, such as Medicago truncatula, Lotus japonicus, Glycine max, Phaseolus vulgaris, Arabidopsis thaliana, Oryza sativa, etc. We attempted to discuss some of these processes wherever appropriate, to serve as references for a better understanding of the N and P signaling and transport during symbiosis.

MON-516 Quantitative Analysis of Differential Enigma/PDLIM7 Gene Expression in Thyroid Cancer vs. Benign Nodules

Thyroid cancer incidence is rising worldwide. Although fine-needle aspiration biopsy (FNAB) is an accurate modality for evaluating thyroid nodules, up to 25% of FNABs still yield indeterminate results. There is a considerable number of diagnostic thyroidectomies for benign disease as a result of indeterminate FNAB. A more accurate and time-efficient diagnostic approach for analyzing indeterminate thyroid nodules may reduce diagnostic thyroidectomy. Recently, the osteogenic protein, Enigma, has been associated with different cancer types, including thyroid cancer progression and calcification through its interaction with bone morphogenic protein-1 (BMP-1), and tyrosine kinases linked to mitogenic signaling pathways [1, 2]. Our published data on Enigma protein analysis with immunohistochemistry showed promising results in discriminating between malignant versus benign thyroid nodules and demonstrated a correlation with thyroid cancer staging [3]. In this study, we are investigating Enigma at a gene expression level by real-time (RT-qPCR), which is a quantitative and more time-efficient method that requires smaller samples (FNA) than immunohistochemistry. We analyzed Enigma mRNA expression levels to determine if Enigma-qPCR could be used as a diagnostic tool to improve the accuracy of FNAB in both malignant and benign thyroid tissues. We extracted mRNA/DNA/proteins from fresh malignant and benign thyroid nodules using a QIAGEN DNA/RNA/Protein Kit. We ran isolated pure mRNA through Enigma-qPCR. The results showed that the Enigma-mRNA expression was 3-fold higher in malignant as compared to benign thyroid tissues. This finding supports our previous Enigma immunohistochemistry data and shows a relative quantitative difference in Enigma-mRNA expression level between malignant and benign thyroid nodules. We conclude that Enigma-RT-qPCR can be used to effectively determine malignancies in FNAB samples derived from thyroid nodules. This method could potentially enhance the diagnostic accuracy of indeterminate nodules and decrease diagnostic thyroidectomies and associated morbidity.[1] C.R. Jung, J.H. Lim, Y. Choi, D.G. Kim, K.J. Kang, S.M. Noh, D.S. Im, Enigma negatively regulates p53 through MDM2 and promotes tumor cell survival in mice, The Journal of clinical investigation 120(12) (2010) 4493-506.[2] Y.J. Kim, H.J. Hwang, J.G. Kang, C.S. Kim, S.H. Ihm, M.G. Choi, S.J. Lee, Enigma Plays Roles in Survival of Thyroid Carcinoma Cells through PI3K/AKT Signaling and Survivin, Anticancer research 38(6) (2018) 3515-3525.[3] A.A. Firek, M.C. Perez, A. Gonda, L. Lei, I. Munir, A.A. Simental, F.E. Carr, B.J. Becerra, M. De Leon, S. Khan, Pathologic significance of a novel oncoprotein in thyroid cancer progression, Head & neck 39(12) (2017) 2459-2469.

Genome-wide identification and evolutionary analysis of TGA transcription factors in soybean

The gain of function in genes and gene families is a continuous process and is a key factor in understanding gene and genome evolution in plants. TGACG-Binding (TGA) transcription factors (TFs) have long been known for their essential roles in plant defence in Arabidopsis, but their roles in legume symbiosis are yet to be explored. Here, we identified a total of 25 TGA (named GmTGA1-GmTGA25) genes in soybean. Through phylogenetic analysis, we discovered a clade of GmTGA proteins that appear to be legume-specific. Among them, two GmTGAs were unique by possessing the autophagy sequence in their proteins, while the third one was an orphan gene in soybean. GmTGAs were structurally different from AtTGAs, and their expression patterns also differed with the dominant expression of AtTGAs and GmTGAs in aerial and underground parts, respectively. Moreover, twenty-five GmTGAs showed a strong correlation among the gene expression in roots, nodules, and root hairs. The qRT-PCR analysis results revealed that among 15 tested GmTGAs, six were induced and four were suppressed by rhizobia inoculation, while 11 of these GmTGAs were induced by high nitrate. Our findings suggested the important roles of GmTGAs in symbiotic nodulation and in response to nitrogen availability in soybean.

Comparison of Postmarketing Findings vs the Initial Clinical Validation Findings of a Thyroid Nodule Gene Expression Classifier

In the United States, the most used molecular test for the evaluation of cytologically indeterminate thyroid nodules is the Afirma gene expression classifier (GEC). To evaluate the GEC's diagnostic performance through a novel approach to assess whether the findings of the initial validation study are consistent with the results of postmarketing studies. PubMed was systematically searched from inception through October 26, 2017, using the terms gene expression classifier or Afirma or GEC and thyroid. Studies included were those in which the GEC diagnostic performance could be calculated on consecutively resected cytologically indeterminate thyroid nodules. Two observers independently assessed study eligibility and risk of bias using the quality assessment tool for observational cohort and cross-sectional studies of the National Heart, Lung, and Blood Institute. Summary data were extracted by a reviewer and reviewed independently by another. Study authors were contacted if missing data were needed. Data were pooled using a random-effects model. PRISMA and MOOSE guidelines were followed. Evaluation of the linear correlation between the benign call rate (BCR) and the positive predictive value (PPV). Of the 137 retrieved titles, 19 (13.9%) were included, comprising a total of 2568 thyroid nodules. Based on a simulation using the sensitivity and specificity reported in the initial validation study, the observed BCR and PPV values in postmarketing studies would have to be explained by different underlying prevalence rates of cancer (15% vs 30%), which is an impossible event. Furthermore, the overall correlation between BCR and PPV for independent studies fell outside the PPV 95% CI of the initial validation study (95% CI, 0.17-0.32) at the BCR of pooled independent studies (0.45) and was just at the limit of the BCR 95% CI of the initial validation study (95% CI, 0.32-0.45) at the PPV of pooled independent studies (0.45). The diagnostic performance was statistically significantly better for atypia or follicular lesions of undetermined significance (diagnostic odds ratio [DOR], 5.67; 95% CI, 4.23-7.60) compared with follicular neoplasms (DOR, 2.24; 95% CI, 1.45-3.47). The findings suggest that the initial validation study cohort was not representative of the populations in whom the GEC has been used, calling into question its reported diagnostic performance, including its negative predictive value.

Nitric oxide signaling, metabolism and toxicity in nitrogen-fixing symbiosis.

Interactions between legumes and rhizobia lead to the establishment of a symbiotic relationship characterized by the formation of a new organ, the nodule, which facilitates the fixation of atmospheric nitrogen (N2) by nitrogenase through the creation of a hypoxic environment. Significant amounts of nitric oxide (NO) accumulate at different stages of nodule development, suggesting that NO performs specific signaling and/or metabolic functions during symbiosis. NO, which regulates nodule gene expression, accumulates to high levels in hypoxic nodules. NO accumulation is considered to assist energy metabolism within the hypoxic environment of the nodule via a phytoglobin-NO-mediated respiration process. NO is a potent inhibitor of the activity of nitrogenase and other plant and bacterial enzymes, acting as a developmental signal in the induction of nodule senescence. Hence, key questions concern the relative importance of the signaling and metabolic functions of NO versus its toxic action and how NO levels are regulated to be compatible with nitrogen fixation functions. This review analyses these paradoxical roles of NO at various stages of symbiosis, and highlights the role of plant phytoglobins and bacterial hemoproteins in the control of NO accumulation.

A Bacterial Expression Vector Archive (BEVA) for Flexible Modular Assembly of Golden Gate-Compatible Vectors.

We present a Bacterial Expression Vector Archive (BEVA) for the modular assembly of bacterial vectors compatible with both traditional and Golden Gate cloning, utilizing the Type IIS restriction enzyme Esp3I, and have demonstrated its use for Golden Gate cloning in Escherichia coli. Ideal for synthetic biology and other applications, this modular system allows a rapid, low-cost assembly of new vectors tailored to specific tasks. Using the principles outlined here, new modules (e.g., origin of replication for plasmids in other bacteria) can easily be designed for specific applications. It is hoped that this vector construction system will be expanded by the scientific community over time by creation of novel modules through an open source approach. To demonstrate the potential of the system, three example vectors were constructed and tested. The Golden Gate level 1 vector pOGG024 (pBBR1-based broad-host range and medium copy number) was used for gene expression in laboratory-cultured Rhizobium leguminosarum. The Golden Gate level 1 vector pOGG026 (RK2-based broad-host range, lower copy number and stable in the absence of antibiotic selection) was used to demonstrate bacterial gene expression in nitrogen-fixing nodules on pea plant roots formed by R. leguminosarum. Finally, the level 2 cloning vector pOGG216 (RK2-based broad-host range, medium copy number) was used to construct a dual reporter plasmid expressing green and red fluorescent proteins.

Comparative Transcriptomic Analysis of Two Actinorhizal Plants and the Legume Medicago truncatula Supports the Homology of Root Nodule Symbioses and Is Congruent With a Two-Step Process of Evolution in the Nitrogen-Fixing Clade of Angiosperms.

Root nodule symbiosis (RNS) is a symbiotic interaction established between angiosperm hosts and nitrogen-fixing soil bacteria in specialized organs called root nodules. The host plants provide photosynthate and the microsymbionts supply fixed nitrogen. The origin of RNS represents a major evolutionary event in the angiosperms, and understanding the genetic underpinnings of this event is of major economic and agricultural importance. Plants that engage in RNS are restricted to a single angiosperm clade known as the nitrogen-fixing clade (NFC), yet occur in multiple lineages scattered within the NFC. It has been postulated that RNS evolved in two steps: a gain-of-predisposition event occurring at the base of the NFC, followed by a gain-of-function event in each host plant lineage. Here, we first explore the premise that RNS has evolved from a single common background, and then we explore whether a two-step process better explains the evolutionary origin of RNS than either a single-step process, or multiple origins. We assembled the transcriptomes of root and nodule of two actinorhizal plants, Ceanothus thyrsiflorus and Datisca glomerata. Together with the corresponding published transcriptomes of the model legume Medicago truncatula, the gene expression patterns in roots and nodules were compared across the three lineages. We found that orthologs of many genes essential for RNS in the model legumes are expressed in all three lineages, and that the overall nodule gene expression patterns were more similar to each other than expected by random chance, a finding that supports a common evolutionary background for RNS shared by the three lineages. Moreover, phylogenetic analyses suggested that a substantial portion of the genes experiencing selection pressure changes at the base of the NFC also experienced additional changes at the base of each host plant lineage. Our results (1) support the occurrence of an event that led to RNS at the base of the NFC, and (2) suggest a subsequent change in each lineage, most consistent with a two-step origin of RNS. Among several conserved functions identified, strigolactone-related genes were down-regulated in nodules of all three species, suggesting a shared function similar to that shown for arbuscular mycorrhizal symbioses.

NsrA, a Predicted β-Barrel Outer Membrane Protein Involved in Plant Signal Perception and the Control of Secondary Infection in Sinorhizobium meliloti.

An ongoing signal exchange fine-tunes the symbiotic interactions between rhizobia and legumes, ensuring the establishment and maintenance of mutualism. In a recently identified regulatory loop, endosymbiotic Sinorhizobium meliloti exerts negative feedback on root infection in response to unknown plant cues. Upon signal perception, three bacterial adenylate cyclases (ACs) of the inner membrane, namely, CyaD1, CyaD2, and CyaK, synthesize the second messenger cAMP, which, together with the cAMP-dependent Clr transcriptional activator, activates the expression of genes involved in root infection control. The pathway that links signal perception at the surface of the cell to cytoplasmic cAMP production by ACs was thus far unknown. Here we first show that CyaK is the cognate AC for the plant signal, called signal 1, that was observed previously in mature nodule and shoot extracts. We also show that inactivation of the gene immediately upstream of cyaK, nsrA (smb20775), which encodes a β-barrel protein of the outer membrane, abolished signal 1 perception ex planta, whereas nsrA overexpression increased signal 1 responsiveness. Inactivation of the nsrA gene abolished all Clr-dependent gene expression in nodules and led to a marked hyperinfection phenotype on plants, similar to that of a cyaD1 cyaD2 cyaK triple mutant. We suggest that the NsrA protein acts as the (co)receptor for two signal molecules, signal 1 and a hypothetical signal 1', in mature and young nodules that cooperate in controlling secondary infection in S. meliloti-Medicago symbiosis. The predicted topology and domain composition of the NsrA protein hint at a mechanism of transmembrane signaling.IMPORTANCE Symbiotic interactions, especially mutualistic ones, rely on a continuous signal exchange between the symbionts. Here we report advances regarding a recently discovered signal transduction pathway that fine-tunes the symbiotic interaction between S. meliloti and its Medicago host plant. We have identified an outer membrane protein of S. meliloti, called NsrA, that transduces Medicago plant signals to adenylate cyclases in the inner membrane, thereby triggering a cAMP signaling cascade that controls infection. Besides their relevance for the rhizobium-legume symbiosis, these findings shed light on the mechanisms of signal perception and transduction by adenylate cyclases and transmembrane signaling in bacteria.

Characterization of the Symbiotic Nitrogen-Fixing Common Bean Low Phytic Acid (lpa1) Mutant Response to Water Stress.

The common bean (Phaseolus vulgaris L.) low phytic acid (lpa1) biofortified genotype produces seeds with improved nutritional characteristics and does not display negative pleiotropic effects. Here we demonstrated that lpa1 plants establish an efficient nitrogen-fixing symbiosis with Rhizobium etli CE3. The lpa1 nodules showed a higher expression of nodule-function related genes than the nodules of the parental wild type genotype (BAT 93). We analyzed the response to water stress of lpa1 vs. BAT 93 plants grown under fertilized or under symbiotic N2-fixation conditions. Water stress was induced by water withholding (up to 14% soil moisture) to fertilized or R. etli nodulated plants previously grown with normal irrigation. The fertilized lpa1 plants showed milder water stress symptoms during the water deployment period and after the rehydration recovery period when lpa1 plants showed less biomass reduction. The symbiotic water-stressed lpa1 plants showed decreased nitrogenase activity that coincides with decreased sucrose synthase gene expression in nodules; lower turgor weight to dry weight (DW) ratio, which has been associated with higher drought resistance index; downregulation of carbon/nitrogen (C/N)-related and upregulation of stress-related genes. Higher expression of stress-related genes was also observed in bacteroids of stressed lpa1 plants that also displayed very high expression of the symbiotic cbb3 oxidase (fixNd).

Inoculation insensitive promoters for cell type enriched gene expression in legume roots and nodules.

BackgroundEstablishment and maintenance of mutualistic plant–microbial interactions in the rhizosphere and within plant roots involve several root cell types. The processes of host–microbe recognition and infection require complex signal exchange and activation of downstream responses. These molecular events coordinate host responses across root cell layers during microbe invasion, ultimately triggering changes of root cell fates. The progression of legume root interactions with rhizobial bacteria has been addressed in numerous studies. However, tools to globally resolve the succession of molecular events in the host root at the cell type level have been lacking. To this end, we aimed to identify promoters exhibiting cell type enriched expression in roots of the model legume Lotus japonicus, as no comprehensive set of such promoters usable in legume roots is available to date.ResultsHere, we use promoter:GUS fusions to characterize promoters stemming from Arabidopsis, tomato (Lycopersicon esculentum) or L.japonicus with respect to their expression in major cell types of the L.japonicus root differentiation zone, which shows molecular and morphological responses to symbiotic bacteria and fungi. Out of 24 tested promoters, 11 showed cell type enriched activity in L.japonicus roots. Covered cell types or cell type combinations are epidermis (1), epidermis and cortex (2), cortex (1), endodermis and pericycle (2), pericycle and phloem (4), or xylem (1). Activity of these promoters in the respective cell types was stable during early stages of infection of transgenic roots with the rhizobial symbiont of L.japonicus, Mesorhizobium loti. For a subset of five promoters, expression stability was further demonstrated in whole plant transgenics as well as in active nodules.Conclusions11 promoters from Arabidopsis (10) or tomato (1) with enriched activity in major L.japonicus root and nodule cell types have been identified. Root expression patterns are independent of infection with rhizobial bacteria, providing a stable read-out in the root section responsive to symbiotic bacteria. Promoters are available as cloning vectors. We expect these tools to help provide a new dimension to our understanding of signaling circuits and transcript dynamics in symbiotic interactions of legumes with microbial symbionts.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-016-0105-y) contains supplementary material, which is available to authorized users.

Transcriptomic Analysis of Sinorhizobium meliloti and Medicago truncatula Symbiosis Using Nitrogen Fixation-Deficient Nodules.

The bacterium Sinorhizobium meliloti interacts symbiotically with legume plant hosts such as Medicago truncatula to form nitrogen-fixing root nodules. During symbiosis, plant and bacterial cells differentiate in a coordinated manner, resulting in specialized plant cells that contain nitrogen-fixing bacteroids. Both plant and bacterial genes are required at each developmental stage of symbiosis. We analyzed gene expression in nodules formed by wild-type bacteria on six plant mutants with defects in nitrogen fixation. We observed differential expression of 482 S. meliloti genes with functions in cell envelope homeostasis, cell division, stress response, energy metabolism, and nitrogen fixation. We simultaneously analyzed gene expression in M. truncatula and observed differential regulation of host processes that may trigger bacteroid differentiation and control bacterial infection. Our analyses of developmentally arrested plant mutants indicate that plants use distinct means to control bacterial infection during early and late symbiotic stages.

Icariin induced proliferation and differentiation of MC3T3-E1 osteoblasts via ERK and JNK signaling coupled with estrogen receptor

Objective To explore the detailed underlying molecular and signaling mechanisms in the effects of icariin on bone formation by an in vitro cell model. Methods The proliferation of MC3T3-E1 osteoblast-like cells was evaluated by MTT, and gene expression of cell cycle related proteins in MC3T3-E1 cells after icariin treatment was detected by real-time PCR. The phosphorylation of MAPK signals, including ERK, P38, and JNK was determined by Western blot, and then the inhibitors of MAPK signals were used to treat cells with icariin alone or together to determine the role of MAPKs in the process of icariin treatment on MC3T3-E1 cell proliferation. Alkaline phosphatase and Alizarin red staining were used to detect the formation of mineralization nodules, and gene expressions of alkaline phosphatase, type Ⅰ collagen, and osteocalcin in osteoblasts after being treated by icariin were evaluated by real-time PCR. ICI182780, and nilutamide was used to decide the participation of estrogen and androgen receptor signals in the process of icariin treatment on the differentiation and mineralization of MC3T3-E1 cells. Results Treatment with icariin promoted MC3T3-E1 cell growth in a time- and dose-dependent manner. This treatment also revealed that icariin increased the expression of mRNAs encoding both cyclin E and PCNA, positive regulators of cell growth, but decreased levels of mRNAs encoding Cdkn2b, a negative regulator of cell cycle progression. When MC3T3-E1 cells were cultured in a differentiated condition, icariin enhanced mineralized nodule formation and increased the expression of mRNAs encoding alkaline phosphatase, type Ⅰ collagen, and osteocalcin. Treatment with icariin significantly induced phosphorylation of both ERK and JNK and this phosphorylated effect occurred very rapidly within 5 minutes and reached peak at 15 minutes. Furthermore, the stimulated effects of icariin on proliferation and gene expression of cyclin E, PCNA, and Cdkn2b in MC3T3-E1 cells were dramatically attenuated by treatment with both U0126 and SP600125, inhibitors of MAPKs. Interestingly, such stimulating effects of icariin were at least partly reduced by treatment with ICI182780, an inhibitor of estrogen receptor. Icariin induced mineralized nodule formation and gene expression of alkaline phosphatase, type Ⅰ collagen, and osteocalcin in MC3T3-E1 cells were also partly reduced when the cells were treated with ICI182780. Conclusions Our findings indicate that the anabolic effect of icariin on bone formation is, at least partly, mediated through the MAPK signaling pathway in order to modulate osteoblast proliferation and differentiation. (Chin J Endocrinol Metab, 2015, 31: 148-154) Key words: Icariin; Osteoblasts; Estrogen receptor; Mitogen-activated protein kinase

Root developmental programs shape the Medicago truncatula nodule meristem.

Nodules on the roots of legume plants host nitrogen-fixing Rhizobium bacteria. Several lines of evidence indicate that nodules are evolutionarily related to roots. We determined whether developmental control of the Medicago truncatula nodule meristem bears resemblance to that in root meristems through analyses of root meristem-expressed PLETHORA genes. In nodules, MtPLETHORA 1 and 2 are preferentially expressed in cells positioned at the periphery of the meristem abutting nodule vascular bundles. Their expression overlaps with an auxin response maximum and MtWOX5, which is a marker for the root quiescent center. Strikingly, the cells in the central part of the nodule meristem have a high level of cytokinin and display MtPLETHORA 3 and 4 gene expression. Nodule-specific knockdown of MtPLETHORA genes results in a reduced number of nodules and/or in nodules in which meristem activity has ceased. Our nodule gene expression map indicates that the nodule meristem is composed of two distinct domains in which different MtPLETHORA gene subsets are expressed. Our mutant studies show that MtPLETHORA genes function redundantly in nodule meristem maintenance. This indicates that Rhizobium has recruited root developmental programs for nodule formation.

RNA-seq transcriptome profiling reveals that Medicago truncatula nodules acclimate N₂ fixation before emerging P deficiency reaches the nodules.

Legume nodules are plant tissues with an exceptionally high concentration of phosphorus (P), which, when there is scarcity of P, is preferentially maintained there rather than being allocated to other plant organs. The hypothesis of this study was that nodules are affected before the P concentration in the organ declines during whole-plant P depletion. Nitrogen (N₂) fixation and P concentration in various organs were monitored during a whole-plant P-depletion process in Medicago truncatula. Nodule gene expression was profiled through RNA-seq at day 5 of P depletion. Until that point in time P concentration in leaves reached a lower threshold but was maintained in nodules. N₂-fixation activity per plant diverged from that of fully nourished plants beginning at day 5 of the P-depletion process, primarily because fewer nodules were being formed, while the activity of the existing nodules was maintained for as long as two weeks into P depletion. RNA-seq revealed nodule acclimation on a molecular level with a total of 1140 differentially expressed genes. Numerous genes for P remobilization from organic structures were increasingly expressed. Various genes involved in nodule malate formation were upregulated, while genes involved in fermentation were downregulated. The fact that nodule formation was strongly repressed with the onset of P deficiency is reflected in the differential expression of various genes involved in nodulation. It is concluded that plants follow a strategy to maintain N₂ fixation and viable leaf tissue as long as possible during whole-plant P depletion to maintain their ability to react to emerging new P sources (e.g. through active P acquisition by roots).

Localization of phytase transcripts in germinating seeds of the common bean (Phaseolus vulgaris L.).

The work provides the first-time evidence of tissue-specific expression of a phytase gene in the germinating seeds of Phaseolus vulgaris. Phytase enzyme plays a major role in germinating seeds. It is also active during N2 fixation within nodules of legumes. The effect of phosphorus (P) deficiency on phytase gene expression and localization in N2-fixing root nodules has been recently studied in hydroaeroponic culture of Phaseolus vulgaris. In this study, phytase gene transcripts within the germinating seed tissues of the P-inefficient P. vulgaris recombinant inbred line RIL147 were in situ localized with a similar RT-PCR recipe as that used for nodules. Our results show that the phytase gene expression was mainly localized in the outer layers, vascular cells and parenchyma of germinating seeds whereas it was localized in the inner and middle cortex of nodules. Image analysis quantified higher fluorescence intensity of the phytase transcript signal in the seed embryo than in radicles, cotyledons or the nodule cortex. Furthermore, the phytase activity was 22-fold higher in cotyledons (43 nmol min(-1) g(-1) dry weight) than in nodules (2 nmol min(-1) g(-1) dry weight). The K m and V m values of phytase activity in cotyledons were also significantly higher than in nodules. Interestingly, the amplified sequence of cDNA phytase exhibited highest homology with the Glycine max purple acid phosphatase (NM_001289274) 90 % for germinating seed as compared to nodule phytase cDNA displaying 94 % homology with the Glycine max phytase (GQ422774.1). It is concluded that phytase enzymes are likely to vary from seeds to nodules and that phytase enzymes play key roles in the use of organic P or N2 fixation, as it is well known for germination.

An integrated analysis of plant and bacterial gene expression in symbiotic root nodules using laser‐capture microdissection coupled to RNA sequencing

Rhizobium-induced root nodules are specialized organs for symbiotic nitrogen fixation. Indeterminate-type nodules are formed from an apical meristem and exhibit a spatial zonation which corresponds to successive developmental stages. To get a dynamic and integrated view of plant and bacterial gene expression associated with nodule development, we used a sensitive and comprehensive approach based upon oriented high-depth RNA sequencing coupled to laser microdissection of nodule regions. This study, focused on the association between the model legume Medicago truncatula and its symbiont Sinorhizobium meliloti, led to the production of 942million sequencing read pairs that were unambiguously mapped on plant and bacterial genomes. Bioinformatic and statistical analyses enabled in-depth comparison, at a whole-genome level, of gene expression in specific nodule zones. Previously characterized symbiotic genes displayed the expected spatial pattern of expression, thus validating the robustness of our approach. We illustrate the use of this resource by examining gene expression associated with three essential elements of nodule development, namely meristem activity, cell differentiation and selected signaling processes related to bacterial Nod factors and redox status. We found that transcription factor genes essential for the control of the root apical meristem were also expressed in the nodule meristem, while the plant mRNAs most enriched in nodules compared with roots were mostly associated with zones comprising both plant and bacterial partners. The data, accessible on a dedicated website, represent a rich resource for microbiologists and plant biologists to address a variety of questions of both fundamental and applied interest.

An RNA Sequencing Transcriptome Analysis Reveals Novel Insights into Molecular Aspects of the Nitrate Impact on the Nodule Activity of Medicago truncatula

The mechanism through which nitrate reduces the activity of legume nodules is controversial. The objective of the study was to follow Medicago truncatula nodule activity after nitrate provision continuously and to identify molecular mechanisms, which down-regulate the activity of the nodules. Nodule H2 evolution started to decline after about 4 h of nitrate application. At that point in time, a strong shift in nodule gene expression (RNA sequencing) had occurred (1,120 differentially expressed genes). The most pronounced effect was the down-regulation of 127 genes for nodule-specific cysteine-rich peptides. Various other nodulins were also strongly down-regulated, in particular all the genes for leghemoglobins. In addition, shifts in the expression of genes involved in cellular iron allocation and mitochondrial ATP synthesis were observed. Furthermore, the expression of numerous genes for the formation of proteins and glycoproteins with no obvious function in nodules (e.g. germins, patatin, and thaumatin) was strongly increased. This occurred in conjunction with an up-regulation of genes for proteinase inhibitors, in particular those containing the Kunitz domain. The additionally formed proteins might possibly be involved in reducing nodule oxygen permeability. Between 4 and 28 h of nitrate exposure, a further reduction in nodule activity occurred, and the number of differentially expressed genes almost tripled. In particular, there was a differential expression of genes connected with emerging senescence. It is concluded that nitrate exerts rapid and manifold effects on nitrogenase activity. A certain degree of nitrate tolerance might be achieved when the down-regulatory effect on late nodulins can be alleviated.

MMP expression in rheumatoid inflammation: the rs11568818 polymorphism is associated with MMP-7 expression at an extra-articular site

Matrix metalloproteinases (MMPs) contribute to the joint damage in rheumatoid arthritis (RA). Less is known of the involvement of MMPs at extra-articular sites of rheumatoid inflammation. We assessed the relative contribution from MMP-1, MMP-3, MMP-7 and MMP-12 to joint and extra-articular tissue destruction and inflammation by comparing gene expression in joint synovia and subcutaneous rheumatoid nodules from RA patients. Expression of MMP-1 and MMP-3 predominated in synovia, whereas MMP-12 expression was significantly higher in rheumatoid nodules. Markedly higher MMP-7 expression distinguished a subgroup of nodules that featured infiltrating monocyte/macrophage-producing MMP-7 protein. The high MMP-7 expression in nodules was associated with the single-nucleotide polymorphism (SNP) rs11568818 (-181A>G, MMP-7 promoter) and more active inflammation within the nodule lesions. Patients with such nodules had significantly earlier age of RA onset. Our findings indicate that the expression of MMP-1 and MMP-3 occurs relatively independent of the tissue microenvironment with substantial expression also at extra-articular sites. MMP-12 expression reflects the involvement of monocyte/macrophages in rheumatoid inflammation. Evidence for the association between the rs11568818 SNP and increased MMP-7 expression is restricted to nodules, which indicates that consequences of the MMP-7 polymorphism are likely to manifest within aspects of immune/inflammatory activity that are monocyte/macrophage-mediated.

Transcriptomic insights into mechanisms of symbiotic cooperation

Intraspecific genetic variation can affect community structure and ecosystem processes (Bolnick etal. 2011). It can also influence phenotypic expression by genotypes within other species to produce genotype-by-genotype (G×G) interaction (Falconer & Mackay 1996). Evolution of one species drives correlated evolution of others when it causes G×G for fitness (Thompson 2005). However, the mechanisms by which species interact also influence evolutionary outcomes (Kummel & Salant 2006; Golubski & Klausmeier 2010; Akçay & Simms 2011; Grman etal. 2012). To identify genes and putative functional mechanisms underlying G×G interactions, Heath etal. (2012) analysed natural variation in the symbiotic transcriptome of the mutualistic nutritional symbiosis between a legume host Medicago truncatula and the facultative endosymbiotic rhizobium Sinorhizobium meliloti. Using twelve microarrays, the authors simultaneously measured host and symbiont gene expression in root nodules from four factorial pairings of host and symbiont genotypes that produced G×G in host fitness (Fig.1, upper panel). Rhizobium gene expression was influenced by rhizobium and plant genotype and the G×G interaction (Fig.1, lower panel), whereas plant gene expression was influenced primarily by plant genotype. The authors identified rhizobium genes that might contribute to G×G in host plant fitness. Heath etal. (2012) have moved beyond the constraints of single organism analysis towards a more realistic understanding of plants and bacteria as organisms inextricably linked with symbioses that affect even basic patterns of gene expression.

Coevolutionary genetic variation in the legume‐rhizobium transcriptome

Coevolutionary change requires reciprocal selection between interacting species, where the partner genotypes that are favoured in one species depend on the genetic composition of the interacting species. Coevolutionary genetic variation is manifested as genotype × genotype (G × G) interactions for fitness in interspecific interactions. Although quantitative genetic approaches have revealed abundant evidence for G × G interactions in symbioses, the molecular basis of this variation remains unclear. Here we study the molecular basis of G × G interactions in a model legume-rhizobium mutualism using gene expression microarrays. We find that, like quantitative traits such as fitness, variation in the symbiotic transcriptome may be partitioned into additive and interactive genetic components. Our results suggest that plant genetic variation had the largest influence on nodule gene expression and that plant genotype and the plant genotype × rhizobium genotype interaction determine global shifts in rhizobium gene expression that in turn feedback to influence plant fitness benefits. Moreover, the transcriptomic variation we uncover implicates regulatory changes in both species as drivers of symbiotic gene expression variation. Our study is the first to partition genetic variation in a symbiotic transcriptome and illuminates potential molecular routes of coevolutionary change.