Delaying chloroplast turnover increases water-deficit stress tolerance through the enhancement of nitrogen assimilation in rice.

Abiotic stress-induced senescence in crops is a process particularly affecting the photosynthetic apparatus, decreasing photosynthetic activity and inducing chloroplast degradation. A pathway for stress-induced chloroplast degradation that involves the CHLOROPLAST VESICULATION (CV) gene was characterized in rice (Oryza sativa) plants. OsCV expression was up-regulated with the age of the plants and when plants were exposed to water-deficit conditions. The down-regulation of OsCV expression contributed to the maintenance of the chloroplast integrity under stress. OsCV-silenced plants displayed enhanced source fitness (i.e. carbon and nitrogen assimilation) and photorespiration, leading to water-deficit stress tolerance. Co-immunoprecipitation, intracellular co-localization, and bimolecular fluorescence demonstrated the in vivo interaction between OsCV and chloroplastic glutamine synthetase (OsGS2), affecting source-sink relationships of the plants under stress. Our results would indicate that the OsCV-mediated chloroplast degradation pathway is involved in the regulation of nitrogen assimilation during stress-induced plant senescence

Ion channels in vacuoles from halophytes and glycophytes

AbstractThe electrical properties of the vacuolar membrane (tonoplast) of a halophyte, sugar beet, and a glycophyte, tomato, have been investigated using the patch-clamp technique [(1981) Pflügers Arch. 391, 85–100]. Voltage-dependent ion channels were analyzed using isolated membrane patches. Both species displayed channel activities which were nonselective between sodium and potassium. Beet tonoplast channels displayed inward rectification (65 pS and 10 pS for negative and positive potentials, respectively), while tomato tonoplast channels showed a constant conductance (25 pS) in the range −80 to +80 mV potentials. The observed low channel conductance at positive potentials in halophytes would prevent a significant loss of the Na+ accumulated in the vacuole through the operation of the Na+/H+ antiport [(1987) Physiol. Plant. 69, 731–734], while channel rectification in glycophytes would have no physiological significance

Ethylene Response of Plum ACC Synthase 1 (ACS1) Promoter is Mediated through the Binding Site of Abscisic Acid Insensitive 5 (ABI5).

The enzyme 1-amino-cyclopropane-1-carboxylic acid synthase (ACS) participates in the ethylene biosynthesis pathways and it is tightly regulated transcriptionally and post-translationally. Notwithstanding its major role in climacteric fruit ripening, the transcriptional regulation of ACS during ripening is not fully understood. We studied fruit ripening in two Japanese plum cultivars, the climacteric Santa Rosa (SR) and its non-climacteric bud sport mutant, Sweet Miriam (SM). As the two cultivars show considerable difference in ACS expression, they provide a good system for the study of the transcriptional regulation of the gene. To investigate the differential transcriptional regulation of ACS1 genes in the SR and SM, their promoter region, which showed only minor sequence differences, was isolated and used to identify the binding of transcription factors interacting with specific ACS1 cis-acting elements. Three transcription factors (TFs), abscisic acid-insensitive 5 (ABI5), GLABRA 2 (GL2), and TCP2, showed specific binding to the ACS1 promoter. Synthetic DNA fragments containing multiple cis-acting elements of these TFs fused to β-glucuronidase (GUS), showed the ABI5 binding site mediated ethylene and abscisic acid (ABA) responses of the promoter. While TCP2 and GL2 showed constant and similar expression levels in SM and SR fruit during ripening, ABI5 expression in SM fruits was lower than in SR fruits during advanced fruit ripening states. Overall, the work demonstrates the complex transcriptional regulation of ACS1

Two NHX-type transporters from Helianthus tuberosus improve the tolerance of rice to salinity and nutrient deficiency stress.

The NHX-type cation/H+ transporters in plants have been shown to mediate Na+ (K+ )/H+ exchange for salinity tolerance and K+ homoeostasis. In this study, we identified and characterized two NHX homologues, HtNHX1 and HtNHX2 from an infertile and salinity tolerant species Helianthus tuberosus (cv. Nanyu No. 1). HtNHX1 and HtNHX2 share identical 5'- and 3'-UTR and coding regions, except for a 342-bp segment encoding 114 amino acids (L272 to Q385 ) which is absent in HtNHX2. Both hydroponics and soil culture experiments showed that the expression of HtNHX1 or HtNHX2 improved the rice tolerance to salinity. Expression of HtNHX2, but not HtNHX1, increased rice grain yield, harvest index, total nutrient uptake under K+ -limited salt-stress or general nutrient deficiency conditions. The results provide a novel insight into NHX function in plant mineral nutrition

Beyond osmolytes and transporters: novel plant salt-stress tolerance-related genes from transcriptional profiling data

With recent advancements in DNA-chip technology, requisite software development and support and progress in related aspects of plant molecular biology, it is now possible to comprehensively analyze the expression of complete genomes. Global transcript profiling shows that in plants, salt-stress response involves simultaneous up and downregulation of a large number of genes. This analysis further suggests that apart from the transcripts that govern synthesis of osmolytes and ion transporters, two candidate systems that have attracted much of the attention thus far, transcripts encoding for proteins related to the regulation of transcriptional and translational machineries have a distinct role in salt-stress response. In particular, induction of transcripts of specific transcription factors, RNA-binding proteins, ribosomal genes, and translation initiation and elongation factors has recently been noted to be important during salt stress. There is an urgent need to examine cellular functionality of the above putative salt-tolerance-related genes emerging from the transcriptome analysis

Functional specialization of chloroplast vesiculation (CV) duplicated genes from soybean shows partial overlapping roles during stress-induced or natural senescence

Soybean is a globally important legume crop which is highly sensitive to drought. The identification of genes of particular relevance for drought responses provides an important basis to improve tolerance to environmental stress. Chloroplast Vesiculation (CV) genes have been characterized in Arabidopsis and rice as proteins participating in a specific chloroplast-degradation vesicular pathway (CVV) during natural or stress-induced leaf senescence. Soybean genome contains two paralogous genes encoding highly similar CV proteins, CV1 and CV2. In this study, we found that expression of CV1 was differentially upregulated by drought stress in soybean contrasting genotypes exhibiting slow-wilting (tolerant) or fast-wilting (sensitive) phenotypes. CV1 reached higher induction levels in fast-wilting plants, suggesting a negative correlation between CV1 gene expression and drought tolerance. In contrast, autophagy (ATG8) and ATI-PS (ATI1) genes were induced to higher levels in slow-wilting plants, supporting a pro-survival role for these genes in soybean drought tolerance responses. The biological function of soybean CVs in chloroplast degradation was confirmed by analyzing the effect of conditional overexpression of CV2-FLAG fusions on the accumulation of specific chloroplast proteins. Functional specificity of CV1 and CV2 genes was assessed by analyzing their specific promoter activities in transgenic Arabidopsis expressing GUS reporter gene driven by CV1 or CV2 promoters. CV1 promoter responded primarily to abiotic stimuli (hyperosmolarity, salinity and oxidative stress), while the promoter of CV2 was predominantly active during natural senescence. Both promoters were highly responsive to auxin but only CV1 responded to other stress-related hormones, such as ABA, salicylic acid and methyl jasmonate. Moreover, the dark-induced expression of CV2, but not of CV1, was strongly inhibited by cytokinin, indicating similarities in the regulation of CV2 to the reported expression of Arabidopsis and rice CV genes. Finally, we report the expression of both CV1 and CV2 genes in roots of soybean and transgenic Arabidopsis, suggesting a role for the encoded proteins in root plastids. Together, the results indicate differential roles for CV1 and CV2 in development and in responses to environmental stress, and point to CV1 as a potential target for gene editing to improve crop performance under stress without compromising natural development.Agencia Nacional de Investigación e InnovaciónComisión Académica de PosgradosComisión Sectorial de Investigación CientíficaPEDECIBA Biologí

Copper impacts on grape berry cells: uptake and detoxification

Copper has been extensively used as the active principle of fungicides, since the late 1800s when the “Bordeaux mixture” was developed and its spectacular efficiency proved against fungal pathogens such as downy mildew, which is a large threat to winegrowers. Although initially it seemed to improve plant growth in unproductive lands, repeated use of copper-based fungicides has led to the accumulation of large concentrations of this metal ion in vineyard soils and raised concerns regarding phytotoxicity. As major targets for heavy metal stress, plants have developed a number of mechanisms to withstand the elevated metal levels. Such responses include exclusion, chelation and compartmentation of metal ions. Both the mitochondria and plastids are copper sinks, and the vacuole is believed to constitute a copper delivery pathway within the cell, and not just a sequestration compartment, due to the proximity of the tonoplast to the other organelles of the plant cell. In the present study, grape berry cells (cv. Cabernet Sauvignon) were used as a model system to study the effect of copper on cell growth and viability. In the concentration range of 0 (+ the copper chelator BCS) to 100 μM CuSO4 growth was virtually unaffected. However, concentrations from 100 to 500 μM caused a sharp decrease in cell growth. The viability of grown cells decreased with the increase in copper concentration in a dose-dependent manner. Studies with the copper-sensitive fluorescent probe PhenGreen™ SK allowed for the identification of copper sinks in grape berry cells. Furthermore, transport studies were performed in isolated intact protoplasts loaded with this probe. The initial velocities of fluorescence quenching upon addition of copper followed a Michaelis-Menten kinetics, suggesting the involvement of mediated transport with a Km= 0.7 mM. Isolated vacuoles labeled with the pH-dependent fluorescent dye ACMA were used to study copper compartmentation as a mean of metal tolerance. Results showed that CuCl2 dissipates a pre-established pH gradient across the tonoplast suggesting the involvement of a Cu2+/H+ antiport system. Eight putative VvCTr (Vitis vinifera Copper Transporter) genes were identified, among which VvCTr1 was isolated and cloned and its expression is currently being studied.VM is supported by a PhD grant (SFRH/BD/64587/2009). This work was supported by Fundação para a Ciência e a Tecnologia (research project no. PTDC/AGRALI/100636/2008)

Label-free shotgun proteomics and metabolite analysis reveal a significant metabolic shift during citrus fruit development.

Label-free LC-MS/MS-based shot-gun proteomics was used to quantify the differential protein synthesis and metabolite profiling in order to assess metabolic changes during the development of citrus fruits. Our results suggested the occurrence of a metabolic change during citrus fruit maturation, where the organic acid and amino acid accumulation seen during the early stages of development shifted into sugar synthesis during the later stage of citrus fruit development. The expression of invertases remained unchanged, while an invertase inhibitor was up-regulated towards maturation. The increased expression of sucrose-phosphate synthase and sucrose-6-phosphate phosphatase and the rapid sugar accumulation suggest that sucrose is also being synthesized in citrus juice sac cells during the later stage of fruit development

Cytokinin-mediated source ⁄sink modifications improve drought tolerance and increase grain yield in rice under water-stress

Drought is the major environmental factor limiting crop productivity worldwide. We hypothesized that it is possible to enhance drought tolerance by delaying stress-induced senescence through the stress-induced synthesis of cytokinins in crop-plants. We generated transgenic rice (Oryza sativa) plants expressing an isopentenyltransferase (IPT) gene driven by PSARK, a stress- and maturation-induced promoter. Plants were tested for drought tolerance at two yield-sensitive developmental stages: pre- and post-anthesis. Under both treatments, the transgenic rice plants exhibited delayed response to stress with significantly higher grain yield (GY) when compared to wild-type plants. Gene expression analysis revealed a significant shift in expression of hormone-associated genes in the transgenic plants. During water-stress (WS), PSARK::IPT plants displayed increased expression of brassinosteroid-related genes and repression of jasmonate-related genes. Changes in hormone homeostasis were associated with resource(s) mobilization during stress. The transgenic plants displayed differential expression of genes encoding enzymes associated with hormone synthesis and hormone-regulated pathways. These changes and associated hormonal crosstalk resulted in the modification of source ⁄ sink relationships and a stronger sink capacity of the PSARK::IPT plants during WS. As a result, the transgenic plants had higher GY with improved quality (nutrients and starch content)

Salt stress response in rice: genetics, molecular biology, and comparative genomics

Significant progress has been made in unraveling the molecular biology of rice in the past two decades. Today, rice stands as a forerunner amongst the cereals in terms of details known on its genetics. Evidence show that salt tolerance in plants is a quantitative trait. Several traditional cultivars, landraces, and wild types of rice like Pokkali, CSR types, and Porteresia coarctata appear as promising materials for donation of requisite salt tolerance genes. A large number of quantitative trait loci (QTL) have been identified for salt tolerance in rice through generation of recombinant inbred lines and are being mapped using different types of DNA markers. Salt-tolerant transgenic rice plants have been produced using a host of different genes and transcript profiling by micro- and macroarray-based methods has opened the gates for the discovery of novel salt stress mechanisms in rice, and comparative genomics is turning out to be a critical input in this respect. In this paper, we present a comprehensive review of the genetic, molecular biology, and comparative genomics effort towards the generation of salt-tolerant rice. From the data on comprehensive transcript expression profiling of clones representing salt-stress-associated genes of rice, it is shown that transcriptional and translational machineries are important determinants in controlling salt stress response, and gene expression response in tolerant and susceptible rice plants differs mainly in quantitative terms