Root microbiota drive direct integration of phosphate stress and immunity.

Plants live in biogeochemically diverse soils with diverse microbiota. Plant organs associate intimately with a subset of these microbes, and the structure of the microbial community can be altered by soil nutrient content. Plant-associated microbes can compete with the plant and with each other for nutrients, but may also carry traits that increase the productivity of the plant. It is unknown how the plant immune system coordinates microbial recognition with nutritional cues during microbiome assembly. Here we establish that a genetic network controlling the phosphate stress response influences the structure of the root microbiome community, even under non-stress phosphate conditions. We define a molecular mechanism regulating coordination between nutrition and defence in the presence of a synthetic bacterial community. We further demonstrate that the master transcriptional regulators of phosphate stress response in Arabidopsis thaliana also directly repress defence, consistent with plant prioritization of nutritional stress over defence. Our work will further efforts to define and deploy useful microbes to enhance plant performance.

Wide-ranging transcriptome remodelling mediated by alternative polyadenylation in response to abiotic stresses in Sorghum.

Alternative polyadenylation (APA) regulates diverse developmental and physiological processes through its effects on gene expression, mRNA stability, translatability, and transport. Sorghum is a major cereal crop in the world and, despite its importance, not much is known about the role of post-transcriptional regulation in mediating responses to abiotic stresses in Sorghum. A genome-wide APA analysis unveiled widespread occurrence of APA in Sorghum in response to drought, heat, and salt stress. Abiotic stress treatments incited changes in poly(A) site choice in a large number of genes. Interestingly, abiotic stresses led to the re-directing of transcriptional output into non-productive pathways defined by the class of poly(A) site utilized. This result revealed APA to be part of a larger global response of Sorghum to abiotic stresses that involves the re-direction of transcriptional output into non-productive transcriptional and translational pathways. Large numbers of stress-inducible poly(A) sites could not be linked with known, annotated genes, suggestive of the existence of numerous unidentified genes whose expression is strongly regulated by abiotic stresses. Furthermore, we uncovered a novel stress-specific cis-element in intronic poly(A) sites used in drought- and heat-stressed plants that might play an important role in non-canonical poly(A) site choice in response to abiotic stresses.

The polyadenylation factor FIP1 is important for plant development and root responses to abiotic stresses.

Root development and its response to environmental changes is crucial for whole plant adaptation. These responses include changes in transcript levels. Here, we show that the alternative polyadenylation (APA) of mRNA is important for root development and responses. Mutations in FIP1, a component of polyadenylation machinery, affects plant development, cell division and elongation, and response to different abiotic stresses. Salt treatment increases the amount of poly(A) site usage within the coding region and 5' untranslated regions (5'-UTRs), and the lack of FIP1 activity reduces the poly(A) site usage within these non-canonical sites. Gene ontology analyses of transcripts displaying APA in response to salt show an enrichment in ABA signaling, and in the response to stresses such as salt or cadmium (Cd), among others. Root growth assays show that fip1-2 is more tolerant to salt but is hypersensitive to ABA or Cd. Our data indicate that FIP1-mediated alternative polyadenylation is important for plant development and stress responses.

Noncanonical Alternative Polyadenylation Contributes to Gene Regulation in Response to Hypoxia.

Stresses from various environmental challenges continually confront plants, and their responses are important for growth and survival. One molecular response to such challenges involves the alternative polyadenylation of mRNA. In plants, it is unclear how stress affects the production and fate of alternative mRNA isoforms. Using a genome-scale approach, we show that in Arabidopsis thaliana, hypoxia leads to increases in the number of mRNA isoforms with polyadenylated 3' ends that map to 5'-untranslated regions (UTRs), introns, and protein-coding regions. RNAs with 3' ends within protein-coding regions and introns were less stable than mRNAs that end at 3'-UTR poly(A) sites. Additionally, these RNA isoforms were underrepresented in polysomes isolated from control and hypoxic plants. By contrast, mRNA isoforms with 3' ends that lie within annotated 5'-UTRs were overrepresented in polysomes and were as stable as canonical mRNA isoforms. These results indicate that the generation of noncanonical mRNA isoforms is an important feature of the abiotic stress response. The finding that several noncanonical mRNA isoforms are relatively unstable suggests that the production of non-stop and intronic mRNA isoforms may represent a form of negative regulation in plants, providing a conceptual link with mechanisms that generate these isoforms (such as alternative polyadenylation) and RNA surveillance.

SPX1 is a phosphate-dependent inhibitor of Phosphate Starvation Response 1 in Arabidopsis.

To cope with growth in low-phosphate (Pi) soils, plants have evolved adaptive responses that involve both developmental and metabolic changes. Phosphate Starvation Response 1 (PHR1) and related transcription factors play a central role in the control of Pi starvation responses (PSRs). How Pi levels control PHR1 activity, and thus PSRs, remains to be elucidated. Here, we identify a direct Pi-dependent inhibitor of PHR1 in Arabidopsis, SPX1, a nuclear protein that shares the SPX domain with yeast Pi sensors and with several Pi starvation signaling proteins from plants. Double mutation of SPX1 and of a related gene, SPX2, resulted in molecular and physiological changes indicative of increased PHR1 activity in plants grown in Pi-sufficient conditions or after Pi refeeding of Pi-starved plants but had only a limited effect on PHR1 activity in Pi-starved plants. These data indicate that SPX1 and SPX2 have a cellular Pi-dependent inhibitory effect on PHR1. Coimmunoprecipitation assays showed that the SPX1/PHR1 interaction in planta is highly Pi-dependent. DNA-binding and pull-down assays with bacterially expressed, affinity-purified tagged SPX1 and ΔPHR1 proteins showed that SPX1 is a competitive inhibitor of PHR1 binding to its recognition sequence, and that its efficiency is highly dependent on the presence of Pi or phosphite, a nonmetabolizable Pi analog that can repress PSRs. The relative strength of the SPX1/PHR1 interaction is thus directly influenced by Pi, providing a link between Pi perception and signaling.

WRKY6 transcription factor restricts arsenate uptake and transposon activation in Arabidopsis.

Stress constantly challenges plant adaptation to the environment. Of all stress types, arsenic was a major threat during the early evolution of plants. The most prevalent chemical form of arsenic is arsenate, whose similarity to phosphate renders it easily incorporated into cells via the phosphate transporters. Here, we found that arsenate stress provokes a notable transposon burst in plants, in coordination with arsenate/phosphate transporter repression, which immediately restricts arsenate uptake. This repression was accompanied by delocalization of the phosphate transporter from the plasma membrane. When arsenate was removed, the system rapidly restored transcriptional expression and membrane localization of the transporter. We identify WRKY6 as an arsenate-responsive transcription factor that mediates arsenate/phosphate transporter gene expression and restricts arsenate-induced transposon activation. Plants therefore have a dual WRKY-dependent signaling mechanism that modulates arsenate uptake and transposon expression, providing a coordinated strategy for arsenate tolerance and transposon gene silencing.

Catalytic mechanism and kinetics of malate dehydrogenase.

Malate dehydrogenase (MDH) is a ubiquitous and central enzyme in cellular metabolism, found in all kingdoms of life, where it plays vital roles in the cytoplasm and various organelles. It catalyzes the reversible NAD+-dependent reduction of L-malate to oxaloacetate. This review describes the reaction mechanism for MDH and the effects of mutations in and around the active site on catalytic activity and substrate specificity, with a particular focus on the loop that encloses the active site after the substrates have bound. While MDH exhibits selectivity for its preferred substrates, mutations can alter the specificity of MDH for each cosubstrate. The kinetic characteristics and similarities of a variety of MDH isozymes are summarized, and they illustrate that the KM values are consistent with the relative concentrations of the substrates in cells. As a result of its existence in different cellular environments, MDH properties vary, making it an attractive model enzyme for studying enzyme activity and structure under different conditions.

Diversity in oat potential immunogenicity: basis for the selection of oat varieties with no toxicity in coeliac disease.

BACKGROUND AND AIMS: Coeliac disease (CD) is triggered by an abnormal reaction to gluten. Peptides resulting from partially digested gluten of wheat, barley or rye cause inflammation of the small intestinal mucosa. Previous contradictory studies suggest that oats may trigger the abnormal immunological response in patients with CD. Monoclonal antibodies (moAbs) against the main immunotoxic 33-mer peptide (A1 and G12) react strongly against wheat, barley and rye but have less reactivity against oats. The stated aim of this study is to test whether this observed reactivity could be related to the potential toxicity of oats for patients with CD. METHODS: In the present study, different oat varieties, controlled for their purity and by their distinct protein pattern, were used to examine differences in moAb G12 recognition by ELISA and western blot. Immunogenicity of oat varieties was determined by 33-mer concentration, T cell proliferation and interferon γ production. RESULTS: Three groups of oat cultivars reacting differently against moAb G12 could be distinguished: a group with considerable affinity, a group showing slight reactivity and a third with no detectable reactivity. The immunogenicity of the three types of oats as well as that of a positive and negative control was determined with isolated peripheral blood mononuclear T cells from patients with CD by measurement of cell proliferation and interferon γ release. A direct correlation of the reactivity with G12 and the immunogenicity of the different prolamins was observed. CONCLUSIONS: The results showed that the reactivity of the moAb G12 is proportional to the potential immunotoxicity of the cereal cultivar. These differences may explain the different clinical responses observed in patients suffering from CD and open up a means to identify immunologically safe oat cultivars, which could be used to enrich a gluten-free diet.

Alternative Polyadenylation and Salicylic Acid Modulate Root Responses to Low Nitrogen Availability.

Nitrogen (N) is probably the most important macronutrient and its scarcity limits plant growth, development and fitness. N starvation response has been largely studied by transcriptomic analyses, but little is known about the role of alternative polyadenylation (APA) in such response. In this work, we show that N starvation modifies poly(A) usage in a large number of transcripts, some of them mediated by FIP1, a component of the polyadenylation machinery. Interestingly, the number of mRNAs isoforms with poly(A) tags located in protein-coding regions or 5'-UTRs significantly increases in response to N starvation. The set of genes affected by APA in response to N deficiency is enriched in N-metabolism, oxidation-reduction processes, response to stresses, and hormone responses, among others. A hormone profile analysis shows that the levels of salicylic acid (SA), a phytohormone that reduces nitrate accumulation and root growth, increase significantly upon N starvation. Meta-analyses of APA-affected and fip1-2-deregulated genes indicate a connection between the nitrogen starvation response and salicylic acid (SA) signaling. Genetic analyses show that SA may be important for preventing the overgrowth of the root system in low N environments. This work provides new insights on how plants interconnect different pathways, such as defense-related hormonal signaling and the regulation of genomic information by APA, to fine-tune the response to low N availability.

Pipecolic acid confers systemic immunity by regulating free radicals.

Pipecolic acid (Pip), a non-proteinaceous product of lysine catabolism, is an important regulator of immunity in plants and humans alike. In plants, Pip accumulates upon pathogen infection and has been associated with systemic acquired resistance (SAR). However, the molecular mechanisms underlying Pip-mediated signaling and its relationship to other known SAR inducers remain unknown. We show that in plants, Pip confers SAR by increasing levels of the free radicals, nitric oxide (NO), and reactive oxygen species (ROS), which act upstream of glycerol-3-phosphate (G3P). Plants defective in NO, ROS, G3P, or salicylic acid (SA) biosynthesis accumulate reduced Pip in their distal uninfected tissues although they contain wild-type-like levels of Pip in their infected leaves. These data indicate that de novo synthesis of Pip in distal tissues is dependent on both SA and G3P and that distal levels of SA and G3P play an important role in SAR. These results also suggest a unique scenario whereby metabolites in a signaling cascade can stimulate each other's biosynthesis depending on their relative levels and their site of action.

Novel signals in the regulation of Pi starvation responses in plants: facts and promises.

Plants have evolved numerous adaptive developmental and metabolic responses to cope with growth in conditions of limited phosphate (Pi). Regulation of these Pi starvation responses (PSR) at the organism level involves not only cellular Pi perception in different organs, but also inter-organ communication of Pi levels via systemic signaling. Here we summarize recent discoveries on Pi starvation sensing and signaling, with special emphasis on structure-function studies that showed a role for inositol polyphosphates (InsP) as intracellular Pi signals, and on genomic studies that identified a large number of mRNAs with inter-organ mobility, which provide an immense source of potential systemic signals in the control of PSR and other responses.

Plasmodesmata Localizing Proteins Regulate Transport and Signaling during Systemic Acquired Immunity in Plants.

Systemic acquired resistance (SAR) in plants is mediated by the signaling molecules azelaic acid (AzA), glycerol-3-phosphate (G3P), and salicylic acid (SA). Here, we show that AzA and G3P transport occurs via the symplastic route, which is regulated by channels known as plasmodesmata (PD). In contrast, SA moves via the extracytosolic apoplast compartment. We found that PD localizing proteins (PDLP) 1 and 5 were required for SAR even though PD permeability in pdlp1 and 5 mutants was comparable to or higher than wild-type plants, respectively. Furthermore, PDLP function was required in the recipient cell, suggesting regulatory function in SAR. Interestingly, overexpression of PDLP5 drastically reduced PD permeability, yet also impaired SAR. PDLP1 interacted with AZI1 (lipid transfer-like protein required for AzA- and G3P-induced SAR) and contributed to its intracellular partitioning. Together, these results reveal the transport routes of SAR chemical signals and highlight the regulatory role of PD-localizing proteins in SAR.

Genome-wide analysis of distribution of RNA polymerase II isoforms using ChIP-seq.

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a powerful technique for genome-wide profiling of DNA-binding proteins in vivo. ChIP has been used to study diverse nuclear processes such as transcription regulation, at specific loci as well as across the entire genome. In this report, a protocol is described for the application of ChIP to the genome-wide analysis of the distribution of different RNA polymerase II forms. The method makes use of the possibility to crosslink proteins to the DNA, to which they bind in vivo. Specific RNA-Pol II-DNA complexes can then be purified by immunoprecipitation using a specific antibody against the DNA-binding protein of interest, and the associated DNA fragments recovered and analyzed.

Genome expansion of Arabis alpina linked with retrotransposition and reduced symmetric DNA methylation.

Despite evolutionary conserved mechanisms to silence transposable element activity, there are drastic differences in the abundance of transposable elements even among closely related plant species. We conducted a de novo assembly for the 375 Mb genome of the perennial model plant, Arabis alpina. Analysing this genome revealed long-lasting and recent transposable element activity predominately driven by Gypsy long terminal repeat retrotransposons, which extended the low-recombining pericentromeres and transformed large formerly euchromatic regions into repeat-rich pericentromeric regions. This reduced capacity for long terminal repeat retrotransposon silencing and removal in A. alpina co-occurs with unexpectedly low levels of DNA methylation. Most remarkably, the striking reduction of symmetrical CG and CHG methylation suggests weakened DNA methylation maintenance in A. alpina compared with Arabidopsis thaliana. Phylogenetic analyses indicate a highly dynamic evolution of some components of methylation maintenance machinery that might be related to the unique methylation in A. alpina.

Comparative transcriptomic analysis of salt adaptation in roots of contrasting Medicago truncatula genotypes.

Evolutionary diversity can be driven by the interaction of plants with different environments. Molecular bases involved in ecological adaptations to abiotic constraints can be explored using genomic tools. Legumes are major crops worldwide and soil salinity is a main stress affecting yield in these plants. We analyzed in the Medicago truncatula legume the root transcriptome of two genotypes having contrasting responses to salt stress: TN1.11, sampled in a salty Tunisian soil, and the reference Jemalong A17 genotype. TN1.11 plants show increased root growth under salt stress as well as a differential accumulation of sodium ions when compared to A17. Transcriptomic analysis revealed specific gene clusters preferentially regulated by salt in root apices of TN1.11, notably those related to the auxin pathway and to changes in histone variant isoforms. Many genes encoding transcription factors (TFs) were also differentially regulated between the two genotypes in response to salt. Among those selected for functional studies, overexpression in roots of the A17 genotype of the bHLH-type TF most differentially regulated between genotypes improved significantly root growth under salt stress. Despite the global complexity of the differential transcriptional responses, we propose that an increase in this bHLH TF expression may be linked to the adaptation of M. truncatula to saline soil environments.

Molecular and immunological characterization of gluten proteins isolated from oat cultivars that differ in toxicity for celiac disease.

A strict gluten-free diet (GFD) is the only currently available therapeutic treatment for patients with celiac disease (CD). Traditionally, treatment with a GFD has excluded wheat, barley and rye, while the presence of oats is a subject of debate. The most-recent research indicates that some cultivars of oats can be a safe part of a GFD. In order to elucidate the toxicity of the prolamins from oat varieties with low, medium, and high CD toxicity, the avenin genes of these varieties were cloned and sequenced, and their expression quantified throughout the grain development. At the protein level, we have accomplished an exhaustive characterization and quantification of avenins by RP-HPLC and an analysis of immunogenicity of peptides present in prolamins of different oat cultivars. Avenin sequences were classified into three different groups, which have homology with S-rich prolamins of Triticeae. Avenin proteins presented a lower proline content than that of wheat gliadin; this may contribute to the low toxicity shown by oat avenins. The expression of avenin genes throughout the development stages has shown a pattern similar to that of prolamins of wheat and barley. RP-HPLC chromatograms showed protein peaks in the alcohol-soluble and reduced-soluble fractions. Therefore, oat grains had both monomeric and polymeric avenins, termed in this paper gliadin- and glutenin-like avenins. We found a direct correlation between the immunogenicity of the different oat varieties and the presence of the specific peptides with a higher/lower potential immunotoxicity. The specific peptides from the oat variety with the highest toxicity have shown a higher potential immunotoxicity. These results suggest that there is wide range of variation of potential immunotoxicity of oat cultivars that could be due to differences in the degree of immunogenicity in their sequences.

Significant differences in coeliac immunotoxicity of barley varieties.

SCOPE: The only treatment available for coeliac disease (CD) is a strict diet in which the intake of wheat, barley, rye, or oats is avoided. Barley is a major cereal crop, grown mainly for its use in brewing, and it has high nutritional value. The identification of varieties with a reduced toxicity profile may contribute to improve the diet, the quality of life and health of CD patients. METHODS AND RESULTS: Searching for harmless barleys, we investigated accessions of malting and wild barley, used for developing new cultivated cereals. The CD toxicity profile of barleys was screened using G12 antibody and cell proliferation and IFN-γ release from peripheral blood mononuclear cells and intestinal biopsies from CD patients. We found a direct correlation between the reactivity with G12 and the immunogenicity of the different barleys. CONCLUSION: The malting barleys were less immunogenic, with reduced levels of toxic gluten, and were possibly less harmful to CD patients. Our findings could raise the prospect of breeding barley species with low levels of harmful gluten, and the attractive goal of developing nontoxic barley cultivars, always taking into account the Codex standard for foods for special dietary use for persons intolerant to gluten.

A novel plant leucine-rich repeat receptor kinase regulates the response of Medicago truncatula roots to salt stress.

In plants, a diverse group of cell surface receptor-like protein kinases (RLKs) plays a fundamental role in sensing external signals to regulate gene expression. Roots explore the soil environment to optimize their growth via complex signaling cascades, mainly analyzed in Arabidopsis thaliana. However, legume roots have significant physiological differences, notably their capacity to establish symbiotic interactions. These major agricultural crops are affected by environmental stresses such as salinity. Here, we report the identification of a leucine-rich repeat RLK gene, Srlk, from the legume Medicago truncatula. Srlk is rapidly induced by salt stress in roots, and RNA interference (RNAi) assays specifically targeting Srlk yielded transgenic roots whose growth was less inhibited by the presence of salt in the medium. Promoter-beta-glucuronidase fusions indicate that this gene is expressed in epidermal root tissues in response to salt stress. Two Srlk-TILLING mutants also failed to limit root growth in response to salt stress and accumulated fewer sodium ions than controls. Furthermore, early salt-regulated genes are downregulated in Srlk-RNAi roots and in the TILLING mutant lines when submitted to salt stress. We propose a role for Srlk in the regulation of the adaptation of M. truncatula roots to salt stress.

Differential expression of the TFIIIA regulatory pathway in response to salt stress between Medicago truncatula genotypes.

Soil salinity is one of the most significant abiotic stresses for crop plants, including legumes. These plants can establish root symbioses with nitrogen-fixing soil bacteria and are able to grow in nitrogen-poor soils. Medicago truncatula varieties show diverse adaptive responses to environmental conditions, such as saline soils. We have compared the differential root growth of two genotypes of M. truncatula (108-R and Jemalong A17) in response to salt stress. Jemalong A17 is more tolerant to salt stress than 108-R, regarding both root and nodulation responses independently of the nitrogen status of the media. A dedicated macroarray containing 384 genes linked to stress responses was used to compare root gene expression during salt stress in these genotypes. Several genes potentially associated with the contrasting cellular responses of these plants to salt stress were identified as expressed in the more tolerant genotype even in the absence of stress. Among them, a homolog of the abiotic stress-related COLD-REGULATEDA1 gene and a TFIIIA-related transcription factor (TF), MtZpt2-1, known to regulate the former gene. Two MtZpt2 TFs (MtZpt2-1 and MtZpt2-2) were found in Jemalong A17 plants and showed increased expression in roots when compared to 108-R. Overexpression of these TFs in the sensitive genotype 108-R, but not in Jemalong A17, led to increased root growth under salt stress, suggesting a role for this pathway in the adaptive response to salt stress of these M. truncatula genotypes.

Identification of regulatory pathways involved in the reacquisition of root growth after salt stress in Medicago truncatula.

Root growth and function are determined by the action of environmental stresses through specific genes that adapt root development to these restrictive conditions. We have defined in vitro conditions affecting the growth and recovery of Medicago truncatula roots after a salt stress. A dedicated macroarray containing 384 genes, based on a large-scale subtractive hybridization approach, was constructed and used to analyze gene expression during salt stress and recovery of root growth from this stress. Several potential regulatory genes were identified as being linked to this recovery process: a novel RNA-binding protein, a small G-protein homologous to ROP9, a receptor-like kinase, two TF IIIA-like and an AP2-like transcription factors (TF), MtZpt2-1, MtZpt2-2 and MtAp2, and a histidine kinase associated with cytokinin transduction pathways. The two ZPT2-type TFs were also rapidly induced by cold stress in roots. By analyzing transgenic M. truncatula plants showing reduced expression levels of both TFs and affected in their capacity to recover root growth after a salt stress, we identified potential target genes that were either activated or repressed in these plants. Overexpression of MtZpt2-1 in roots conferred salt tolerance and affected the expression of three putative targets in the predicted manner: a cold-regulated A (CORA) homolog, a flower-promoting factor (FPF1) homolog and an auxin-induced proline-rich protein (PRP) gene. Hence, regulatory networks depending on TFIIIA-like transcription factors are involved in the control of root adaptation to salt stress.