Edit Ábrahám

Differential expression of two P5CS genes controlling proline accumulation during salt‐stress requires ABA and is regulated by ABA1, ABI1 and AXR2 in Arabidopsis

Proline is a common compatible osmolyte in higher plants. Proline accumulation in response to water stress and salinity is preceded by a rapid increase of the mRNA level of delta 1-pyrroline-5-carboxylate synthase (P5CS) controlling the rate-limiting step of glutamate-derived proline biosynthesis. P5CS is encoded by two differentially regulated genes in Arabidopsis. Gene AtP5CS1 mapped to chromosome 2-78.5 is expressed in most plant organs, but silent in dividing cells. Gene AtP5CS2 located close to marker m457 on chromosome 3-101.3 contributes 20-40% of total P5CS mRNA in plant tissues, but is solely responsible for the synthesis of abundant P5CS mRNA in rapidly dividing cell cultures. Accumulation of AtP5CS transcripts is regulated in a tissue specific manner and inducible by drought, salinity, ABA, and to a lesser extent by auxin. Induction of AtP5CS1 mRNA accumulation in salt-treated seedlings involves an immediate early transcriptional response regulated by ABA signalling that is not inhibited by cycloheximide, but abolished by the deficiency of ABA biosynthesis in the aba1 Arabidopsis mutant. However, inhibition of protein synthesis by cycloheximide prevents the induction of AtP5CS2 mRNA accumulation, and blocks further increase of AtP5CS1 mRNA levels during the second, slow phase of salt-induction. Mutations abi1 and axr2, affecting ABA-perception in Arabidopsis, reduce the accumulation of both AtP5CS mRNAs during salt-stress, whereas ABA-signalling functions defined by the abi2 and abi3 mutations have no effect on salt-induction of the AtP5CS genes.

Light-dependent induction of proline biosynthesis by abscisic acid and salt stress is inhibited by brassinosteroid in Arabidopsis.

Osmotic stress-induced accumulation of proline, an important protective osmolyte in higher plants, is dependent on the expression of Δ1-pyrroline-5-carboxylate synthase (P5CS) and proline dehydrogenase (PDH) enzymes that catalyze the rate-limiting steps of proline biosynthesis and degradation, respectively. Proline metabolism is modulated by differential regulation of organ specific expression of PDH and duplicated P5CS genes in Arabidopsis. Stimulation of proline synthesis by abscisic acid (ABA) and salt stress correlates with a striking activation of P5CS1 expression. By contrast, P5CS2 is only weakly induced, whereas PDH is inhibited to different extent by ABA and salt stress in shoots and roots of light-grown plants. Proline accumulation and light-dependent induction of P5CS1 by ABA and salt stress is inhibited in dark-adapted plants. During dark adaptation P5CS2 is also down-regulated, whereas PDH expression is significantly enhanced in shoots. The inhibitory effect of dark adaptation on P5CS1 is mimicked by the steroid hormone brassinolide. However, brassinolide fails to stimulate PDH, and inhibits P5CS2 only in shoots. Proline accumulation and induction of P5CS1 transcription are simultaneously enhanced in the ABA-hypersensitive prl1 and brassinosteroid-deficient det2 mutants, whereas P5CS2 shows enhanced induction by ABA and salt only in the det2 mutant. In comparison, the prl1 mutation reduces the basal level of PDH expression, whereas the det2 mutation enhances the inhibition of PDH by ABA. Regulation of P5CS1 expression thus appears to play a principal role in controlling proline accumulation stimulated by ABA and salt stress in Arabidopsis.

Functional identification of Arabidopsis stress regulatory genes using the Controlled cDNA Overexpression System

Responses to environmental stresses in higher plants are controlled by a complex web of abscisic acid (ABA)-dependent and independent signaling pathways. To perform genetic screens for identification of novel Arabidopsis (Arabidopsis thaliana) loci involved in the control of abiotic stress responses, a complementary DNA (cDNA) expression library was created in a Gateway version of estradiol-inducible XVE binary vector (controlled cDNA overexpression system [COS]). The COS system was tested in three genetic screens by selecting for ABA insensitivity, salt tolerance, and activation of a stress-responsive ADH1-LUC (alcohol dehydrogenase-luciferase) reporter gene. Twenty-seven cDNAs conferring dominant, estradiol-dependent stress tolerance phenotype, were identified by polymerase chain reaction amplification and sequence analysis. Several cDNAs were recloned into the XVE vector and transformed recurrently into Arabidopsis, to confirm that the observed conditional phenotypes were due to their estradiol-dependent expression. Characterization of a cDNA conferring insensitivity to ABA in germination assays has identified the coding region of heat shock protein HSP17.6A suggesting its implication in ABA signal transduction. Screening for enhanced salt tolerance in germination and seedling growth assays revealed that estradiol-controlled overexpression of a 2-alkenal reductase cDNA confers considerable level of salt insensitivity. Screening for transcriptional activation of stress- and ABA-inducible ADH1-LUC reporter gene has identified the ERF/AP2-type transcription factor RAP2.12, which sustained high-level ADH1-LUC bioluminescence, enhanced ADH1 transcription rate, and increased ADH enzyme activity in the presence of estradiol. These data illustrate that application of the COS cDNA expression library provides an efficient strategy for genetic identification and characterization of novel regulators of abiotic stress responses.

Methods for determination of proline in plants.

Accumulation of proline in higher plants is an indication of disturbed physiological condition, triggered by biotic or abiotic stress condition. Free proline content can increase upon exposure of plants to drought, salinity, cold, heavy metals, or certain pathogens. Determination of free proline levels is a useful assay to monitor physiological status and to assess stress tolerance of higher plants. Here we describe three methods suitable for determination of free proline content. The isatin paper assay is simple and is suitable to assay proline content in large number of samples. The colorimetric measurement is quantitative and provides reliable data about proline content. The HPLC-based amino acid analysis can be employed when concentration of all amino acids has to be compared.

Loss of the nodule-specific cysteine rich peptide, NCR169, abolishes symbiotic nitrogen fixation in the Medicago truncatula dnf7 mutant

Significance In certain legume–rhizobia symbioses, the host plant is thought to control the terminal differentiation of its bacterial partner leading to nitrogen fixation. In Medicago truncatula, over 600 genes coding for nodule-specific cysteine-rich (NCR) peptides are expressed during nodule development and have been implicated in bacteroid differentiation. Up to now it was generally assumed that most of these peptides, if not all, act redundantly. By demonstrating that deletion of a single member of the NCR gene family can result in an ineffective symbiotic phenotype, we show that specific NCR peptides can have essential, non-redundant roles in controlling bacterial differentiation and symbiotic nitrogen fixation. Host compatible rhizobia induce the formation of legume root nodules, symbiotic organs within which intracellular bacteria are present in plant-derived membrane compartments termed symbiosomes. In Medicago truncatula nodules, the Sinorhizobium microsymbionts undergo an irreversible differentiation process leading to the development of elongated polyploid noncultivable nitrogen fixing bacteroids that convert atmospheric dinitrogen into ammonia. This terminal differentiation is directed by the host plant and involves hundreds of nodule specific cysteine-rich peptides (NCRs). Except for certain in vitro activities of cationic peptides, the functional roles of individual NCR peptides in planta are not known. In this study, we demonstrate that the inability of M. truncatula dnf7 mutants to fix nitrogen is due to inactivation of a single NCR peptide, NCR169. In the absence of NCR169, bacterial differentiation was impaired and was associated with early senescence of the symbiotic cells. Introduction of the NCR169 gene into the dnf7-2/NCR169 deletion mutant restored symbiotic nitrogen fixation. Replacement of any of the cysteine residues in the NCR169 peptide with serine rendered it incapable of complementation, demonstrating an absolute requirement for all cysteines in planta. NCR169 was induced in the cell layers in which bacteroid elongation was most pronounced, and high expression persisted throughout the nitrogen-fixing nodule zone. Our results provide evidence for an essential role of NCR169 in the differentiation and persistence of nitrogen fixing bacteroids in M. truncatula.

Cell-cycle control as a target for calcium, hormonal and developmental signals: the role of phosphorylation in the retinoblastoma-centred pathway

BACKGROUND During the life cycle of plants, both embryogenic and post-embryogenic growth are essentially based on cell division and cell expansion that are under the control of inherited developmental programmes modified by hormonal and environmental stimuli. Considering either stimulation or inhibition of plant growth, the key role of plant hormones in the modification of cell division activities or in the initiation of differentiation is well supported by experimental data. At the same time there is only limited insight into the molecular events that provide linkage between the regulation of cell-cycle progression and hormonal and developmental control. Studies indicate that there are several alternative ways by which hormonal signalling networks can influence cell division parameters and establish functional links between regulatory pathways of cell-cycle progression and genes and protein complexes involved in organ development. SCOPE An overview is given here of key components in plant cell division control as acceptors of hormonal and developmental signals during organ formation and growth. Selected examples are presented to highlight the potential role of Ca(2+)-signalling, the complex actions of auxin and cytokinins, regulation by transcription factors and alteration of retinoblastoma-related proteins by phosphorylation. CONCLUSIONS Auxins and abscisic acid can directly influence expression of cyclin, cyclin-dependent kinase (CDK) genes and activities of CDK complexes. D-type cyclins are primary targets for cytokinins and over-expression of CyclinD3;1 can enhance auxin responses in roots. A set of auxin-activated genes (AXR1-ARGOS-ANT) controls cell number and organ size through modification of CyclinD3;1 gene expression. The SHORT ROOT (SHR) and SCARECROW (SCR) transcriptional factors determine root patterning by activation of the CYCD6;1 gene. Over-expression of the EBP1 gene (plant homologue of the ErbB-3 epidermal growth factor receptor-binding protein) increased biomass by auxin-dependent activation of both D- and B-type cyclins. The direct involvement of auxin-binding protein (ABP1) in the entry into the cell cycle and the regulation of leaf size and morphology is based on the transcriptional control of D-cyclins and retinoblastoma-related protein (RBR) interacting with inhibitory E2FC transcriptional factor. The central role of RBRs in cell-cycle progression is well documented by a variety of experimental approaches. Their function is phosphorylation-dependent and both RBR and phospho-RBR proteins are present in interphase and mitotic phase cells. Immunolocalization studies showed the presence of phospho-RBR protein in spots of interphase nuclei or granules in mitotic prophase cells. The Ca(2+)-dependent phosphorylation events can be accomplished by the calcium-dependent, calmodulin-independent or calmodulin-like domain protein kinases (CDPKs/CPKs) phosphorylating the CDK inhibitor protein (KRP). Dephosphorylation of the phospho-RBR protein by PP2A phosphatase is regulated by a Ca(2+)-binding subunit.

Host-secreted antimicrobial peptide enforces symbiotic selectivity in Medicago truncatula

Significance Nitrogen is a limiting factor for plant growth. Most crops obtain their nitrogen through the use of nitrogen-based fertilizers, which is costly, and also causes environmental pollution. Legumes, however, have the unique ability to fix atmospheric nitrogen through symbioses with nitrogen-fixing bacteria. Although legumes can be nodulated by indigenous soil bacteria, nitrogen fixation efficiency differs significantly depending on host and bacterial genotypes. Understanding the genetic mechanisms that underlie this specificity will allow for optimizing symbiotic partnerships with improved symbiotic performance. We report that specific nodule-specific cysteine-rich (NCR) peptides negatively regulate symbiotic persistence in a strain-specific manner in Medicago truncatula. This finding offers a strategy to improve nitrogen fixation efficiency through selection or manipulation of NCR alleles that favor specific bacterial strains. Legumes engage in root nodule symbioses with nitrogen-fixing soil bacteria known as rhizobia. In nodule cells, bacteria are enclosed in membrane-bound vesicles called symbiosomes and differentiate into bacteroids that are capable of converting atmospheric nitrogen into ammonia. Bacteroid differentiation and prolonged intracellular survival are essential for development of functional nodules. However, in the Medicago truncatula–Sinorhizobium meliloti symbiosis, incompatibility between symbiotic partners frequently occurs, leading to the formation of infected nodules defective in nitrogen fixation (Fix−). Here, we report the identification and cloning of the M. truncatula NFS2 gene that regulates this type of specificity pertaining to S. meliloti strain Rm41. We demonstrate that NFS2 encodes a nodule-specific cysteine-rich (NCR) peptide that acts to promote bacterial lysis after differentiation. The negative role of NFS2 in symbiosis is contingent on host genetic background and can be counteracted by other genes encoded by the host. This work extends the paradigm of NCR function to include the negative regulation of symbiotic persistence in host–strain interactions. Our data suggest that NCR peptides are host determinants of symbiotic specificity in M. truncatula and possibly in closely related legumes that form indeterminate nodules in which bacterial symbionts undergo terminal differentiation.

Immunodetection of retinoblastoma-related protein and its phosphorylated form in interphase and mitotic alfalfa cells

Plant retinoblastoma-related (RBR) proteins are primarily considered as key regulators of G1/S phase transition, with functional roles in a variety of cellular events during plant growth and organ development. Polyclonal antibody against the C-terminal region of the Arabidopsis RBR1 protein also specifically recognizes the alfalfa 115 kDa MsRBR protein, as shown by the antigen competition assay. The MsRBR protein was detected in all cell cycle phases, with a moderate increase in samples representing G2/M cells. Antibody against the human phospho-pRb peptide (Ser807/811) cross-reacted with the same 115 kDa MsRBR protein and with the in vitro phosphorylated MsRBR protein C-terminal fragment. Phospho-MsRBR protein was low in G1 cells. Its amount increased upon entry into the S phase and remained high during the G2/M phases. Roscovitine treatment abolished the activity of alfalfa MsCDKA1;1 and MsCDKB2;1, and the phospho-MsRBR protein level was significantly decreased in the treated cells. Colchicine block increased the detected levels of both forms of MsRBR protein. Reduced levels of the MsRBR protein in cells at stationary phase or grown in hormone-free medium can be a sign of the division-dependent presence of plant RBR proteins. Immunolocalization of the phospho-MsRBR protein indicated spots of variable number and size in the labelled interphase nuclei and high signal intensity of nuclear granules in prophase. Structures similar to phospho-MsRBR proteins cannot be recognized in later mitotic phases. Based on the presented western blot and immunolocalization data, the possible involvement of RBR proteins in G2/M phase regulation in plant cells is discussed.

Synchronization of Medicago sativa Cell Suspension Culture

Deepening our knowledge on the regulation of the plant cell division cycle depends on techniques that allow for the enrichment of cell populations in defined cell cycle phases. Synchronization of cell division can be achieved using different plant tissues; however, well-established cell suspension cultures provide the largest amount of biological sample for further analysis. Here we describe the methodology of the establishment, propagation, and analysis of a Medicago sativa suspension culture that can be used for efficient synchronization of the cell division and also the application and removal of hydroxyurea blocking agent. A novel method is used for the estimation of cell portion that enters S phase during the assay. The protocol can be used in the case of other species as well.

Structure, Function and Regulation of ATP5CS Genes in Arabidopsis

ABSTRACTProline is a common compatible osmolyte in higher plants. Proline accumulation in response to water stress and salinity is preceded by a rapid increase of the mRNA level of Δ1-pyrroline-5-carboxylate synthase (P5CS) controlling the rate-limiting step of glutamate-derived proline biosynthesis. P5CS is encoded by two differentially regulated genes in Arabidopsis. Gene AtP5CS1 mapped to chromosome 2–78.5 is expressed in most plant organs, but silent in dividing cells. Gene AtP5CS2 located close to marker m457 on chromosome 3–101.3, and is responsible for the synthesis of abundant P5CS mRNA in dividing cells. Accumulation of AtP5CS transcripts is regulated in a tissue specific manner and inducible by drought, salinity, ABA, and to lesser extent by auxin. Induction of AtP5CS1 mRNA accumulation in salt-treated seedlings involves an immediate early transcriptional response regulated by ABA signaling. Inhibition of protein synthesis by cycloheximide affects the induction of AtP5CS mRNA accumulation. Mutat...