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Chloroplast ascorbate peroxidases exert an important role in the maintenance of hydrogen peroxide levels in chloroplasts by using ascorbate as the specific electron donor. In this work, we performed a functional study of the stromal APX in rice (OsAPX7) and demonstrated that silencing of OsAPX7 did not impact plant growth, redox state, or photosynthesis parameters. Nevertheless, when subjected to drought stress, silenced plants (APX7i) show a higher capacity to maintain stomata aperture and photosynthesis performance, resulting in a higher tolerance when compared to non-transformed plants. RNA-seq analyses indicate that the silencing of OsAPX7 did not lead to changes in the global expression of genes related to reactive oxygen species metabolism. In addition, the drought-mediated induction of several genes related to the proteasome pathway and the down-regulation of genes related to nitrogen and carotenoid metabolism was impaired in APX7i plants. During drought stress, APX7i showed an up-regulation of genes encoding flavonoid and tyrosine metabolism enzymes and a down-regulation of genes related to phytohormones signal transduction and nicotinate and nicotinamide metabolism. Our results demonstrate that OsAPX7 might be involved in signaling transduction pathways related to drought stress response, contributing to the understanding of the physiological role of chloroplast APX isoforms in rice. [ABSTRACT FROM AUTHOR]

Simple Summary: The climatic changes pose important threats to agriculture. Plants are sessile organisms and need to cope with different environmental conditions throughout their lifespan. Most responses involve different signaling molecules, including reactive oxygen species (ROS). Due to their high reactivity, ROS are also an oxidative threat to the cell. Consequently, plant cells display elaborated defenses against oxidative stress. The ascorbate-glutathione cycle is the main antioxidant pathway in photosynthetic organisms and is composed by the enzymes ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR). Here, the APX, MDAR, DHAR and GR genes from castor bean, cassava, jatropha and rubber tree were identified and classified. This classification allowed the prediction of their subcellular localization within plant cells, such as cytosol, peroxisomes, chloroplasts, and mitochondria. Our analysis also contributes to understanding the evolutionary history of these genes. The expression pattern of ascorbate-glutathione cycle genes in castor bean submitted to drought reveals changes in leaves and roots. Altogether, these data contribute to uncovering the regulation of ROS metabolism during stress response in castor bean, which is highly tolerant to drought. Ascorbate peroxidase (APX), Monodehydroascorbate Reductase (MDAR), Dehydroascorbate Reductase (DHAR) and Glutathione Reductase (GR) enzymes participate in the ascorbate-glutathione cycle, which exerts a central role in the antioxidant metabolism in plants. Despite the importance of this antioxidant system in different signal transduction networks related to development and response to environmental stresses, the pathway has not yet been comprehensively characterized in many crop plants. Among different eudicotyledons, the Euphorbiaceae family is particularly diverse with some species highly tolerant to drought. Here the APX, MDAR, DHAR, and GR genes in Ricinus communis, Jatropha curcas, Manihot esculenta, and Hevea brasiliensis were identified and characterized. The comprehensive phylogenetic and genomic analyses allowed the classification of the genes into different classes, equivalent to cytosolic, peroxisomal, chloroplastic, and mitochondrial enzymes, and revealed the duplication events that contribute to the expansion of these families within plant genomes. Due to the high drought stress tolerance of Ricinus communis, the expression patterns of ascorbate-glutathione cycle genes in response to drought were also analyzed in leaves and roots, indicating a differential expression during the stress. Altogether, these data contributed to the characterization of the expression pattern and evolutionary analysis of these genes, filling the gap in the proposed functions of core components of the antioxidant mechanism during stress response in an economically relevant group of plants. [ABSTRACT FROM AUTHOR]

Ascorbate peroxidases (APXs) are heme peroxidases that remove hydrogen peroxide in different subcellular compartments with concomitant ascorbate cycling. Here, we analysed and discussed phylogenetic and molecular features of the APX family. Ancient APX originated as a soluble stromal enzyme, and early during plant evolution, acquired both chloroplast‐targeting and mitochondrion‐targeting sequences and an alternative splicing mechanism whereby it could be expressed as a soluble or thylakoid membrane‐bound enzyme. Later, independent duplication and neofunctionalization events in some angiosperm groups resulted in individual genes encoding stromal, thylakoidal and mitochondrial isoforms. These data reaffirm the complexity of plant antioxidant defenses that allow diverse plant species to acquire new means to adapt to changing environmental conditions. [ABSTRACT FROM AUTHOR]

Key message: MdoDHN11 acts in the nucellus layer to protect the embryo and the endosperm from limited water availability during apple seed development. Dehydrins (DHNs) are protective proteins related to several plant developmental responses that involve dehydration such as seed desiccation and abiotic stresses. In apple (Malus × domestica Borkh.), the seed-specific MdoDHN11 was suggested to play important roles against dehydration during seed development. However, this hypothesis has not yet been evaluated. Within this context, several experiments were performed to functionally characterize MdoDHN11. In situ hybridization analysis during apple seed development showed that MdoDHN11 expression is confined to a maternal tissue called nucellus, a central mass of parenchyma between the endosperm and the testa. The MdoDHN11 protein was localized in the cytosol and nucleus. Finally, transgenic Arabidopsis plants expressing MdoDHN11 were generated and exposed to a severe water-deficit stress, aiming to mimic a situation that can occurs during seed development. All transgenic lines showed increased tolerance to water deficit in relation to wild-type plants. Taken together, our results provide evidences that MdoDHN11 plays important roles during apple seed development by protecting the embryo and the endosperm from limited water availability, and the mechanism of action probably involves the interaction of MdoDHN11 with proteins and other components in the cell. [ABSTRACT FROM AUTHOR]

Galactinol synthase (GolS) is a key enzyme in the biosynthetic pathway of raffinose family oligosaccharides (RFOs), which play roles in carbon storage, signal transduction, and osmoprotection. The present work assessed the evolutionary history of GolS genes across the Rosaceae using several bioinformatic tools. Apple (Malus × domestica) GolS genes were transcriptionally characterized during bud dormancy, in parallel with galactinol and raffinose measurements. Additionally, MdGolS2, a candidate to regulate seasonal galactinol and RFO content during apple bud dormancy, was functionally characterized in Arabidopsis. Evolutionary analyses revealed that whole genome duplications have driven GolS gene evolution and diversification in Rosaceae speciation. The strong purifying selection identified in duplicated GolS genes suggests that differential gene expression might define gene function better than protein structure. Interestingly, MdGolS2 was differentially expressed during bud dormancy, concomitantly with the highest galactinol and raffinose levels. One of the intrinsic adaptive features of bud dormancy is limited availability of free water; therefore, we generated transgenic Arabidopsis plants expressing MdGolS2. They showed higher galactinol and raffinose contents and increased tolerance to water deficit. Our results suggest that MdGolS2 is the major GolS responsible for RFO accumulation during apple dormancy, and these carbohydrates help to protect dormant buds against limited water supply. [ABSTRACT FROM AUTHOR]

Despite the wide appreciation of seedless grapes, little is known about the molecular mechanisms that drive the stenospermocarpic seedless-type phenotype in grapevine. In order to address the molecular mechanisms that control seedlessness in grapevine, our study aimed to characterize VviAGL11, a class D MADS-box transcription factor gene that has been proposed as the major candidate gene involved in Vitis vinifera seed morphogenesis. VviAGL11 allelic variations in seeded and seedless grapevine cultivars were determined, and its correlations with allele-specific steady-state mRNA levels were investigated. VviAGL11 relative expression was significantly higher in seeds at 2, 4, and 6 weeks after fruit set, whereas in the seedless grape its transcript levels were extremely low in all stages analyzed. In situ hybridization revealed transcript accumulation specifically in the dual endotesta layer of the seeds, which is responsible for elongation and an increase of cell number, a necessary step to determine the lignification and the final seed size. No hybridization signals were visible in the seedless grapevine tissues, and a morphoanatomical analysis showed an apparent loss of identity of the endotesta layer of the seed traces. Ectopic expression of VviAGL11 in the Arabidopsis SEEDSTICK mutant background restored the wild-type phenotype and confirmed the direct role of VviAGL11 in seed morphogenesis, suggesting that depletion of its expression is responsible for the erroneous development of a highly essential seed layer, therefore culminating in the typical apirenic phenotype. [ABSTRACT FROM AUTHOR]

The physiological role of plant mitochondrial glutathione peroxidases is scarcely known. This study attempted to elucidate the role of a rice mitochondrial isoform (GPX1) in photosynthesis under normal growth and salinity conditions. GPX1 knockdown rice lines (GPX1s) were tested in absence and presence of 100 mM NaCl for 6 d. Growth reduction of GPX1s line under non-stressful conditions, compared with non-transformed (NT) plants occurred in parallel to increased H2O2 and decreased GSH contents. These changes occurred concurrently with photosynthesis impairment, particularly in Calvin cycle's reactions, since photochemical efficiency did not change. Thus, GPX1 silencing and downstream molecular/metabolic changes modulated photosynthesis differentially. In contrast, salinity induced reduction in both phases of photosynthesis, which were more impaired in silenced plants. These changes were associated with root morphology alterations but not shoot growth. Both studied lines displayed increased GPX activity but H2O2 content did not change in response to salinity. Transformed plants exhibited lower photorespiration, water use efficiency and root growth, indicating that GPX1 could be important to salt tolerance. Growth reduction of GPX1s line might be related to photosynthesis impairment, which in turn could have involved a cross talk mechanism between mitochondria and chloroplast originated from redox changes due to GPX1 deficiency. [ABSTRACT FROM AUTHOR]

The Lesion Simulating Disease (LSD) genes encode a family of zinc finger proteins that play a role in programmed cell death (PCD) and other biological processes, such as plant growth and photosynthesis. In the present study, we report the reconstruction of the evolutionary history of the LSD gene family in Viridiplantae. Phylogenetic analysis revealed that the monocot and eudicot genes were distributed along the phylogeny, indicating that the expansion of the family occurred prior to the diversification between these clades. Sequences encoding proteins that present one, two, or three LSD domains formed separate groups. The secondary structure of these different LSD proteins presented a similar composition, with the β-sheets being their main component. The evolution by gene duplication was identified only to the genes that contain three LSD domains, which generated proteins with equal structure. Moreover, genes encoding proteins with one or two LSD domains evolved as single-copy genes and did not result from loss or gain in LSD domains. These results were corroborated by synteny analysis among regions containing paralogous/orthologous genes in Glycine max and Populus trichocarpa. The Ka/Ks ratio between paralogous/orthologous genes revealed that a subfunctionalization process possibly could be occurring with the LSD genes, explaining the involvement of LSD members in different biological processes, in addition to the negative regulation of PCD. This study presents important novelty in the evolutionary history of the LSD family and provides a basis for future research on individual LSD genes and their involvement in important pathway networks in plants. [ABSTRACT FROM AUTHOR]

Key message: The work describes an ASR knockdown transcriptomic analysis by deep sequencing of rice root seedlings and the transactivation of ASR cis-acting elements in the upstream region of a MIR gene. Abstract: MicroRNAs are key regulators of gene expression that guide post-transcriptional control of plant development and responses to environmental stresses. ASR (ABA, Stress and Ripening) proteins are plant-specific transcription factors with key roles in different biological processes. In rice, ASR proteins have been suggested to participate in the regulation of stress response genes. This work describes the transcriptomic analysis by deep sequencing two libraries, comparing miRNA abundance from the roots of transgenic ASR5 knockdown rice seedlings with that of the roots of wild-type non-transformed rice seedlings. Members of 59 miRNA families were detected, and 276 mature miRNAs were identified. Our analysis detected 112 miRNAs that were differentially expressed between the two libraries. A predicted inverse correlation between miR167abc and its target gene (LOC_Os07g29820) was confirmed using RT-qPCR. Protoplast transactivation assays showed that ASR5 is able to recognize binding sites upstream of the MIR167a gene and drive its expression in vivo. Together, our data establish a comparative study of miRNAome profiles and is the first study to suggest the involvement of ASR proteins in miRNA gene regulation. [ABSTRACT FROM AUTHOR]

Reactive oxygen species ( ROS) are signaling molecules that regulate plant development and responses to stresses. Mitochondria are the source of most ROS in heterotrophic cells, and mitochondrial complex I and complex III are regarded as the main sites of ROS production in plant mitochondria. Recent studies have demonstrated that succinate dehydrogenase ( SDH) also contributes to mitochondrial ROS production. However, the ability of SDH to generate ROS in plants is unclear. The aim of this study was to evaluate the role of SDH in mitochondrial ROS production., Our results demonstrated that SDH is a direct source of ROS in Arabidopsis thaliana and Oryza sativa, and the induction of ROS production by specific SDH inhibitors impaired plant growth. In addition, this effect was accompanied by the down-regulation of cell cycle genes and the up-regulation of stress-related genes., However, the partial inhibition of SDH by a competitive inhibitor decreased ROS production, which was associated with increased shoot and root growth, and prevented the down-regulation of cell cycle genes and the induction of stress-related genes by noncompetitive inhibitors., In conclusion, SDH plays an important role in ROS production, being a direct source of ROS in plant mitochondria and regulating plant development and stress responses. [ABSTRACT FROM AUTHOR]

Dehydrins (DHN) are proteins involved in plant adaptive responses to abiotic stresses, mainly dehydration. Several studies in perennial crops have linked bud dormancy progression, a process characterized by the inability to initiate growth from meristems under favorable conditions, with DHN gene expression. However, an in-depth characterization of DHNs during bud dormancy progression is still missing. An extensive in silico characterization of the apple DHN gene family was performed. Additionally, we used five different experiments that generated samples with different dormancy status, including genotypes with contrasting dormancy traits, to analyze how DHN genes are being regulated during bud dormancy progression in apple by real-time quantitative polymerase chain reaction (RT-qPCR). Duplication events took place in the diversification of apple DHN family. Additionally, MdDHN genes presented tissue- and bud dormant-specific expression patterns. Our results indicate that MdDHN genes are highly divergent in function, with overlapping levels, and that their expressions are fine-tuned by the environment during the dormancy process in apple. [ABSTRACT FROM AUTHOR]

Key message: MicroRNAs have higher expression stability than protein-coding genes in B. napus seeds and are therefore good reference genes for miRNA and mRNA RT-qPCR analysis. Abstract: Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) has become the 'gold standard' to gain insight into function of genes. However, the accuracy of the technique depends on appropriate reference genes for quantification analysis in different experimental conditions. Accumulation of microRNAs (miRNAs) has also been studied by RT-qPCR, but there are no reference genes currently validated for normalization of Brassica napus miRNA expression data. In this study, we selected 43 B. napus miRNAs and 18 previously validated mRNA reference genes. The expression stability of the candidate reference genes was evaluated in different tissue samples (stages of seed development, flowers, and leaves) using geNorm, NormFinder, and RefFinder analysis. The best-ranked reference genes for expression studies during seed development (miR167-1_2, miR11-1, miR159-1 and miR168-1) were used to asses the expression of miR03-1. Since candidate miRNAs showed higher expression stability than protein-coding genes in most of the tested conditions, the expression profile of DGAT1 gene was compared when normalized by the four most stable miRNAs reference genes and by the four most stable mRNA reference genes. The expected expression pattern of DGAT1 during seed development was achieved with the use of miRNA as reference genes. In conclusion, the most stable miRNA reference genes can be employed in the normalization of RT-qPCR quantification of miRNAs and protein-coding genes. This work is the first to perform a comprehensive survey of the stability of miRNA reference genes in B. napus and provides guidelines to obtain more accurate RT-qPCR results in B. napus seeds studies. [ABSTRACT FROM AUTHOR]

The physiological role of peroxisomal ascorbate peroxidases ( pAPX) is unknown; therefore, we utilized pAPX4 knockdown rice and catalase ( CAT) inhibition to assess its role in CAT compensation under high photorespiration. pAPX4 knockdown induced co-suppression in the expression of pAPX3. The rice mutants exhibited metabolic changes such as lower CAT and glycolate oxidase ( GO) activities and reduced glyoxylate content; however, APX activity was not altered. CAT inhibition triggered different changes in the expression of CAT, APX and glutathione peroxidase ( GPX) isoforms between non-transformed ( NT) and silenced plants. These responses were associated with alterations in APX, GPX and GO activities, suggesting redox homeostasis differences. The glutathione oxidation-reduction states were modulated differently in mutants, and the ascorbate redox state was greatly affected in both genotypes. The pAPX suffered less oxidative stress and photosystem II ( PSII) damage and displayed higher photosynthesis than the NT plants. The improved acclimation exhibited by the pAPX plants was indicated by lower H2 O2 accumulation, which was associated with lower GO activity and glyoxylate content. The suppression of both pAPXs and/or its downstream metabolic and molecular effects may trigger favourable antioxidant and compensatory mechanisms to cope with CAT deficiency. This physiological acclimation may involve signalling by peroxisomal H2 O2, which minimized the photorespiration. [ABSTRACT FROM AUTHOR]

Background Drought is by far the most important environmental factor contributing to yield losses in crops, including soybeans [Glycine max (L.) Merr.]. To address this problem, a gene that encodes an osmotin-like protein isolated from Solanum nigrum var. americanum (SnOLP) driven by the UBQ3 promoter from Arabidopsis thaliana was transferred into the soybean genome by particle bombardment. Results Two independently transformed soybean lines expressing SnOLP were produced. Segregation analyses indicated single-locus insertions for both lines. qPCR analysis suggested a single insertion of SnOLP in the genomes of both transgenic lines, but one copy of the hpt gene was inserted in the first line and two in the second line. Transgenic plants exhibited no remarkable phenotypic alterations in the seven analyzed generations. When subjected to water deficit, transgenic plants performed better than the control ones. Leaf physiological measurements revealed that transgenic soybean plants maintained higher leaf water potential at predawn, higher net CO2 assimilation rate, higher stomatal conductance and higher transpiration rate than non-transgenic plants. Grain production and 100-grain weight were affected by water supply. Decrease in grain productivity and 100-grain weight were observed for both transgenic and non-transgenic plants under water deficit; however, it was more pronounced for non-transgenic plants. Moreover, transgenic lines showed significantly higher 100-grain weight than non-transgenic plants under water shortage. Conclusions This is the first report showing that expression of SnOLP in transgenic soybeans improved physiological responses and yield components of plants when subjected to water deficit, highlighting the potential of this gene for biotechnological applications. [ABSTRACT FROM AUTHOR]

Background Many previous studies have shown that soybean WRKY transcription factors are involved in the plant response to biotic and abiotic stresses. Phakopsora pachyrhizi is the causal agent of Asian Soybean Rust, one of the most important soybean diseases. There are evidences that WRKYs are involved in the resistance of some soybean genotypes against that fungus. The number of WRKY genes already annotated in soybean genome was underrepresented. In the present study, a genome-wide annotation of the soybean WRKY family was carried out and members involved in the response to P. pachyrhizi were identified. Results As a result of a soybean genomic databases search, 182 WRKY-encoding genes were annotated and 33 putative pseudogenes identified. Genes involved in the response to P. pachyrhizi infection were identified using superSAGE, RNA-Seq of microdissected lesions and microarray experiments. Seventy-five genes were differentially expressed during fungal infection. The expression of eight WRKY genes was validated by RT-qPCR. The expression of these genes in a resistant genotype was earlier and/or stronger compared with a susceptible genotype in response to P. pachyrhizi infection. Soybean somatic embryos were transformed in order to overexpress or silence WRKY genes. Embryos overexpressing a WRKY gene were obtained, but they were unable to convert into plants. When infected with P. pachyrhizi, the leaves of the silenced transgenic line showed a higher number of lesions than the wild-type plants. Conclusions The present study reports a genome-wide annotation of soybean WRKY family. The participation of some members in response to P. pachyrhizi infection was demonstrated. The results contribute to the elucidation of gene function and suggest the manipulation of WRKYs as a strategy to increase fungal resistance in soybean plants. [ABSTRACT FROM AUTHOR]

Apple ( Malus × domestica Borkh.) is the most important deciduous tree fruit crop grown around the world. Comparisons of gene expression profiles from different tissues, conditions or cultivars are valuable scientific tools to better understand the gene expression changes behind important silvicultural and nutritional traits. However, the accuracy of techniques employed to access gene expression is dependent on the evaluation of stable reference genes for data normalization to avoid statistical significance undue or incorrect conclusions. The objective of this work was to select the best genes to be used as references for gene expression studies in apple trees by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Vegetative and reproductive tissues of the apple 'Gala' cultivar were evaluated during their seasonal cycle of growth and dormancy. The expression of 23 traditional housekeeping genes or genes suggested as constitutive by microarray data was investigated. Tested combinations of primers allowed the specific amplification and the generation of suitable efficiency curves for gene expression studies by RT-qPCR. Gene stability was determined by two different statistical descriptors, geNorm and NormFinder. The known variable PAL gene expression was used to validate selected normalizers. Results obtained allowed us to conclude that MDH, SAND, THFS, TMp1 and WD40 are the best reference genes to accurately normalize the relative transcript abundances using RT-qPCR in various tissues of apple. [ABSTRACT FROM AUTHOR]

Tung oil, the major product of tung tree ( Vernicia fordii) seeds, is one of the highest quality oils for industrial applications and has been considered for the production of biodiesel. Considering the poor agronomical traits of this crop, efforts have been made to breed tung trees for a higher fruit yield and oil property modification for biodiesel use or to engineer plants to produce a higher tung oil yield. However, these efforts have been hampered by a lack of molecular information, as there is no available genome and identified and characterized transcripts of this tree are scarce. Furthermore, there are still many knowledge gaps regarding tung oil biosynthesis. To provide a comprehensive and accurate foundation for molecular studies of tung tree, we present here a reference transcriptome dataset of mature tung seeds. A set of 43,081,927 reads were assembled into 47,585 unigenes. A homology search using blastx against the GenBank nonredundant protein database and the Swiss-Prot database resulted in the annotation of 96 and 81 % of these unigenes, respectively. We also systematically arranged a series of unigenes potentially associated with oil biosynthesis and degradation and examined the expression profile of a subset of those genes in samples from five different stages of seed development, providing a valuable source of genes and transcriptional information related to these pathways. This study represents the first large-scale transcriptome annotation of tung tree and will be useful in tung breeding for oil properties and other agronomical traits. [ABSTRACT FROM AUTHOR]

The production of temperate fruit crops depends on plant developmental processes, primarily the shift from the juvenile phase to the reproductive phase, dormancy transitions and flowering. Apple tree ( Malus × domestica Borkh.) development is regulated by chilling temperatures, which are required for bud dormancy progression. The apple cultivar Castel Gala is a spontaneous mutation of "Gala Standard". "Castel Gala" is characterized by a 50 % decrease in the chilling requirement (CR) for dormancy release, which results in an earlier budbreak. This work explores the contrasting phenotypes of these cultivars using suppression subtractive hybridization (SSH). From 1,019 unigenes identified by SSH, we selected 28 candidate genes putatively associated with dormancy cycling. Reverse transcription-quantitative polymerase chain reaction was used to validate the differential expression profiles and to transcriptionally characterize these genes in three distinct apple cultivars ("Castel Gala", "Royal Gala" and "Fuji Standard") during a cycle comprising growth to dormancy. Of the 28 candidate genes analyzed, 17 confirmed the differences in expression predicted by SSH. Seasonal transcript accumulation during the winter was observed for several genes, with higher steady-state mRNA levels maintained longer in cultivars with a high CR. The transcription profiles suggest that these genes may be associated with dormancy establishment and maintenance. Of the 17 candidate genes, transcripts coding for dormancy-associated MADS-box (DAM), dehydrins, GAST1, LTI65, NAC, HTA8, HTA12 and RAP2.12-like proteins displayed major differences in gene expression between cultivars through the winter. These genes were therefore considered good candidates for key roles in the dormancy process in apple trees. [ABSTRACT FROM AUTHOR]

The biochemical mechanisms underlying the involvement of cytosolic ascorbate peroxidases (cAPXs) in photosynthesis are still unknown. In this study, rice plants doubly silenced in these genes (APX1/2) were exposed to moderate light (ML) and high light (HL) to assess the role of cAPXs in photosynthetic efficiency. APX1/2 mutants that were exposed to ML overexpressed seven and five proteins involved in photochemical activity and photorespiration, respectively. These plants also increased the pheophytin and chlorophyll levels, but the amount of five proteins that are important for Calvin cycle did not change. These responses in mutants were associated with Rubisco carboxylation rate, photosystem II (PSII) activity and potential photosynthesis, which were similar to non-transformed plants. The upregulation of photochemical proteins may be part of a compensatory mechanism for APX1/2 deficiency but apparently the finer-control for photosynthesis efficiency is dependent on Calvin cycle proteins. Conversely, under HL the mutants employed a different strategy, triggering downregulation of proteins related to photochemical activity, Calvin cycle and decreasing the levels of photosynthetic pigments. These changes were associated to strong impairment in PSII activity and Rubisco carboxylation. The upregulation of some photorespiratory proteins was maintained under that stressful condition and this response may have contributed to photoprotection in rice plants deficient in cAPXs. The data reveal that the two cAPXs are not essential for photosynthesis in rice or, alternatively, the deficient plants are able to trigger compensatory mechanisms to photosynthetic acclimation under ML and HL conditions. These mechanisms involve differential regulation in protein expression related to photochemistry, Calvin cycle and photorespiration. [ABSTRACT FROM AUTHOR]

MicroRNAs, or miRNAs, are endogenously encoded small RNAs that play a key role in diverse plant biological processes. Jatropha curcas L. has received significant attention as a potential oilseed crop for the production of renewable oil. Here, a sRNA library of mature seeds and three mRNA libraries from three different seed development stages were generated by deep sequencing to identify and characterize the miRNAs and pre-miRNAs of J. curcas. Computational analysis was used for the identification of 180 conserved miRNAs and 41 precursors (pre-miRNAs) as well as 16 novel pre-miRNAs. The predicted miRNA target genes are involved in a broad range of physiological functions, including cellular structure, nuclear function, translation, transport, hormone synthesis, defense, and lipid metabolism. Some pre-miRNA and miRNA targets vary in abundance between the three stages of seed development. A search for sequences that produce siRNA was performed, and the results indicated that J. curcas siRNAs play a role in nuclear functions, transport, catalytic processes and disease resistance. This study presents the first large scale identification of J. curcas miRNAs and their targets in mature seeds based on deep sequencing, and it contributes to a functional understanding of these miRNAs. [ABSTRACT FROM AUTHOR]

The Lesion Simulating Disease ( LSD) genes encode a family of zinc finger proteins that are reported to play an important role in the hypersensitive response and programmed cell death (PCD) that are caused by biotic and abiotic stresses. In the present study, 117 putative LSD family members were identified in Viridiplantae. Genes with one, two, or three conserved LSD domains were identified. Proteins with three LSD domains were highly represented in the species analyzed and were present in basal organisms. Proteins with two LSD domains were identified only in the Embryophyte clade, and proteins possessing one LSD domain were highly represented in grass species. Expression analyses of Glycine max LSD ( GmLSD) genes were performed by real-time quantitative polymerase chain reaction. The results indicated that GmLSD genes are not ubiquitously expressed in soybean organs and that their expression patterns are instead organ-dependent. The expression of the majority of GmLSD genes is modulated in soybean during Phakopsora pachyrhizi infection. In addition, the expression of some GmLSD genes is modulated in plants under dehydration stress. These results suggest the involvement of GmLSD genes in the response of soybean to both biotic and abiotic stresses. [ABSTRACT FROM AUTHOR]

ABSTRACT Among cereal crops, rice is considered the most tolerant to aluminium (Al). However, variability among rice genotypes leads to remarkable differences in the degree of Al tolerance for distinct cultivars. A number of studies have demonstrated that rice plants achieve Al tolerance through an unknown mechanism that is independent of root tip Al exclusion. We have analysed expression changes of the rice ASR gene family as a function of Al treatment. The gene ASR5 was differentially regulated in the Al-tolerant rice ssp. Japonica cv. Nipponbare. However, ASR5 expression did not respond to Al exposure in Indica cv. Taim rice roots, which are highly Al sensitive. Transgenic plants carrying RNAi constructs that targeted the ASR genes were obtained, and increased Al susceptibility was observed in T1 plants. Embryogenic calli of transgenic rice carrying an ASR5-green fluorescent protein fusion revealed that ASR5 was localized in both the nucleus and cytoplasm. Using a proteomic approach to compare non-transformed and ASR-RNAi plants, a total of 41 proteins with contrasting expression patterns were identified. We suggest that the ASR5 protein acts as a transcription factor to regulate the expression of different genes that collectively protect rice cells from Al-induced stress responses. [ABSTRACT FROM AUTHOR]

The soybean ubiquitous urease (encoded by GmEu4) is responsible for recycling metabolically derived urea. Additional biological roles have been demonstrated for plant ureases, notably in toxicity to other organisms. However, urease enzymatic activity is not related to its toxicity. The role of GmEu4 in soybean susceptibility to fungi was investigated in this study. A differential expression pattern of GmEu4 was observed in susceptible and resistant genotypes of soybeans over the course of a Phakopsora pachyrhizi infection, especially 24 h after infection. Twenty-nine adult, transgenic soybean plants, representing six independently transformed lines, were obtained. Although the initial aim of this study was to overexpress GmEu4, the transgenic plants exhibited GmEu4 co-suppression and decreased ureolytic activity. The growth of Rhizoctonia solani, Phomopsis sp., and Penicillium herguei in media containing a crude protein extract from either transgenic or non-transgenic leaves was evaluated. The fungal growth was higher in the protein extracts from transgenic urease-deprived plants than in extracts from non-transgenic controls. When infected by P. pachyrhizi uredospores, detached leaves of urease-deprived plants developed a significantly higher number of lesions, pustules and erupted pustules than leaves of non-transgenic plants containing normal levels of the enzyme. The results of the present work show that the soybean plants were more susceptible to fungi in the absence of urease. It was not possible to overexpress active GmEu4. For future work, overexpression of urease fungitoxic peptides could be attempted as an alternative approach. [ABSTRACT FROM AUTHOR]

ABSTRACT Current studies, particularly in Arabidopsis, have demonstrated that mutants deficient in cytosolic ascorbate peroxidases (APXs) are susceptible to the oxidative damage induced by abiotic stress. In contrast, we demonstrate here that rice mutants double silenced for cytosolic APXs (APx1/2s) up-regulated other peroxidases, making the mutants able to cope with abiotic stress, such as salt, heat, high light and methyl viologen, similar to non-transformed (NT) plants. The APx1/2s mutants exhibited an altered redox homeostasis, as indicated by increased levels of H2O2 and ascorbate and glutathione redox states. Both mutant and NT plants exhibited similar photosynthesis (CO2 assimilation and photochemical efficiency) under both normal and stress conditions. Overall, the antioxidative compensatory mechanism displayed by the mutants was associated with increased expression of OsGpx genes, which resulted in higher glutathione peroxidase (GPX) activity in the cytosolic and chloroplastic fractions. The transcript levels of OsCatA and OsCatB and the activities of catalase (CAT) and guaiacol peroxidase (GPOD; type III peroxidases) were also up-regulated. None of the six studied isoforms of OsApx were up-regulated under normal growth conditions. Therefore, the deficiency in cytosolic APXs was effectively compensated for by up-regulation of other peroxidases. We propose that signalling mechanisms triggered in rice mutants could be distinct from those proposed for Arabidopsis. [ABSTRACT FROM AUTHOR]

n Agrobacterium-mediated transformation procedure for soybean [ Glycine max L. Merrill] proliferating somatic embryos is here described. The A grobacterium tumefaciens LBA4404 strain harboring pTOK233, pCAMBIA1390- olp or pH7WG2D wrky plasmids was used to mediate gene transfer into the plant genome. Prior to Agrobacterium inoculation, proliferative soybean embryogenic clusters were microwounded by DNA-free tungsten particle bombardment. Three independent transformation experiments were performed. In Experiment I, 26 transgenic plants were obtained from a unique clone of cv Bragg, while 580 plants were recovered from 105 clones of cv IAS5. In Experiment II, a single hygromycin-resistant clone of cv BRSMG68 Vencedora was recovered and gave rise to five plants. In Experiment III, 19 plants of cv Bragg and 48 plants of IAS5 were recovered, representing five and 14 independent transformation events, respectively. PCR and Southern analyses confirmed the transgenes' integration into plant genomes. Transgenic plants were fertile. They flowered, set pods and seeds. Transgene segregation in two T progenies fits the Mendelian pattern (3:1 transgenic:non-transgenic plants). This is the first report of transgenic fertile soybean plants obtained from somatic embryogenic tissues transformed by the system that combines DNA-free particle bombardment and Agrobacterium. [ABSTRACT FROM AUTHOR]

Aerobic organisms evolved a complex antioxidant system, which protect the cells against oxidative damage caused by partially reduced oxygen intermediates, also known as reactive oxygen species. In plants, ascorbate peroxidases (EC, 1.11.1.11) catalyze the conversion of H2O2 to H2O, using ascorbate as the specific electron donor in this enzymatic reaction. Previously, eight APx genes were identified in the rice ( Oryza sativa L.) genome through in silico analysis: two cytosolic isoforms, two putative peroxisomal isoforms, and four putative chloroplastic ones. Using gene-specific probes, we confirmed the presence of the eight APx genes in the rice genome by Southern blot hybridization. Transcript accumulation analysis showed specific expression patterns for each member of the APx family according to developmental stage and in response to salt stress, revealing the complexity of the antioxidant system in plants. Finally, the subcellular localization of rice APx isoforms was determined using GFP-fusion proteins in BY-2 tobacco cells. In agreement with the initial prediction, OSAPX3 was localized in the peroxisomes. On the other hand, the OSAPX6-GFP fusion protein was found in mitochondria of the BY-2 cells, in contrast to the chloroplastic location predicted by sequence analysis. Our findings reveal the functional diversity of the rice APx genes and suggest complementation and coordination of the antioxidant defenses in different cellular compartments during development and abiotic stress. [ABSTRACT FROM AUTHOR]

Ascorbate peroxidase (APx) is a class I peroxidase that catalyzes the conversion of H2O2 to H2O and O2 using ascorbate as the specific electron donor. This enzyme has a key function in scavenging reactive oxygen species (ROS) and the protection against toxic effects of ROS in higher plants, algae, andEuglena. Here we report the identification of an APx multigene family in rice and propose a molecular evolutionary relationship between the diverse APx isoforms. In rice, the APx gene family has eight members, which encode two cytosolic, two putative peroxisomal, and four chloroplastic isoforms, respectively. Phylogenetic analyses were conducted using all APx protein sequences available in the NCBI databases. The results indicate that the different APx isoforms arose by a complex evolutionary process involving several gene duplications. The structural organization of APx genes also reflects this process and provides evidence for a close relationship among proteins located in the same subcellular compartment. A molecular evolutionary pathway, in which cytosolic and peroxisomal isoforms diverged early from chloroplastic ones, is proposed. [ABSTRACT FROM AUTHOR]

Salinity alters general metabolic processes and enzymatic activities, causing increased production of reactive oxygen species (ROS). Expression of antioxidant defense genes would, in turn, be triggered to defend the cell against oxidative damage. We report that salt disturbed antioxidant metabolism in maize seedlings, causing detrimental effects on the growth and development of maize plantlets, increased hydrogen peroxide production and altered antioxidant activities and transcripts profiles. Excessive ROS levels were accompanied by increased catalase (CAT) activity in photosynthesizing shoots, along with induction of mRNA accumulation. Increased accumulation of superoxide dismutase (SOD) transcripts was also observed although no significant changes in total SOD enzymatic activity and isozyme profiles were detected. Higher salt concentrations (above 0.25 M NaCl) were highly detrimental to the plants, causing arrested growth and severe wilting, among other effects. Histochemical detection of H[sub 2]O[sub 2] by 3,3-diaminobenzidine (DAB) staining indicated a collapse of the leaf veins, with hydrogen peroxide leaking to neighboring cells. In agreement to these observations, Sod1, Sod2, Sod4, Sod4A, as well as all Cat transcripts were severely inhibited in plants exposed to high salt concentrations. [ABSTRACT FROM AUTHOR]

The genus Phytomonas is responsible for many diseases in different crop plant species. The finding that chitin is an exposed cell surface polysaccharide in Phytomonas franc¸ai and the observation that chitinases can inhibit fungal growth raises expectations about the potential effect of plant chitinases on the P. franc¸ai cell membrane surface. The plant chitinases Urtica dioica agglutinin (UDA) and Arabidopsis thaliana Chia4 (ATCHIT4) proteins were over-expressed in bacteria and the interaction between these proteins and P. franc¸ai surface was analyzed by immunocytochemistry. We showed that UDA and ATCHIT4 proteins can interact with surface-exposed chitin from P. franc¸ai. [Copyright &y& Elsevier]

To investigate how PvChi4 promoter regulates gene expression during plant development and in response to stress, we have introduced promoter-GUS transcriptional fusion constructs in transgenic Arabidopsis thaliana and tobacco plants. GUS activity was not detected in aerial vegetative organs of transgenic plants carrying the 2189 bp promoter. However, gus expression was activated at the parenchyma and epidermis of leaves and stem, when these transgenic tobacco plants were submitted to heat shock and UV irradiation. The non-stressed plants showed promoter activity in the meristematic region of lateral roots and in reproductive organs. In both species, the promoter was active in pollen grains and ovaries. In tobacco anthers, gus expression was observed in the middle layer. In transgenic plants harbouring promoters of 1445 bp, 1199 bp, 501 bp and 416 bp, GUS activity has been detected, in addition to the meristematic region of lateral roots, at the apical meristem, and in the phloem and epidermic tissues of leaves, stems and petioles. These analyses suggest that all cis-acting elements controlling the tissue expression of PvChi4 promoter are located 416 bp upstream of the gene. A putative negative regulatory element that is inactivated during heat shock or UV treatments was also identified between 2189 and 1445 bp. GUS activity was not detected in globular embryos, but is present in the radicle region at the heart-shaped stage. During transition from heart-shaped to torpedo, the activity was extended until the hypocotyl. This expression pattern was maintained until embryo maturity, when staining was detected in the whole embryo, with lower intensity in the cotyledon region. These results corroborate the current idea that this class of chitinase may have some functional role on plant development in addition to the stress response. [ABSTRACT FROM AUTHOR]

Legume nodules formed by diazotrophic microorganisms are active sites for biological nitrogen fixation (BNF). In tropical regions, a significant part of N supply for soybean, peanut and bean crops is derived from BNF, which is nevertheless often limited by high temperature stress. In contrast, cowpea nodules are very resistant to high temperatures. To understand the molecular bases of thermotolerance during BNF under heat stress, we have used cDNA-amplified fragment length polymorphism experiments to identify differentially expressed transcripts from cowpea nodules subjected to heat shock treatment. The expression profiles obtained showed approximately 600 bands, 55 up-regulated and nine corresponding to genes repressed by heat stress. Twenty transcript-derived fragments were isolated, cloned and sequenced. The Vigna unguiculata nodule and stress response transcripts present similarities to those that encode low molecular weight heat shock proteins, wound-induced proteins, disease resistance protein, and xylan endohydrolase isoenzyme, as well as different housekeeping genes. The differential expression of 15 genes was confirmed by using Northern blot or reverse Northern hybridization experiments. [Copyright &y& Elsevier]

Simple Summary: Glutathione peroxidases (GPxs) are considered as one of the main antioxidant enzymes, which reduce peroxides into less toxic compounds. This family of enzymes is found in most eukaryotic organisms, but it is highly divergent regarding its structure, catalytic mechanism, and substrate usage. Furthermore, it is still unclear how these enzymes are dispersed in the animal kingdom. Through robust phylogenetic and sequence analyses, we show that all GPx genes originated from a common ancestor and evolved independently across different kingdoms. In Metazoa, GPx genes expanded into three main groups before the rise of bilaterian animals, and they were further expanded in vertebrates. These expansions allowed GPx enzymes to diversify, not only structurally, but also functionally. Our study contributes to the understanding of how this abundant class of antioxidant enzymes evolved. The evolution of GPxs appears to be a continuous process, leading to the diversification of their functions. There is large diversity among glutathione peroxidase (GPx) enzymes regarding their function, structure, presence of the highly reactive selenocysteine (SeCys) residue, substrate usage, and reducing agent preference. Moreover, most vertebrate GPxs are very distinct from non-animal GPxs, and it is still unclear if they came from a common GPx ancestor. In this study, we aimed to unveil how GPx evolved throughout different phyla. Based on our phylogenetic trees and sequence analyses, we propose that all GPx encoding genes share a monomeric common ancestor and that the SeCys amino acid was incorporated early in the evolution of the metazoan kingdom. In addition, classical GPx and the cysteine-exclusive GPx07 have been present since non-bilaterian animals, but they seem to have been lost throughout evolution in different phyla. Therefore, the birth-and-death of GPx family members (like in other oxidoreductase families) seems to be an ongoing process, occurring independently across different kingdoms and phyla. [ABSTRACT FROM AUTHOR]

Ascorbate peroxidases (APX) are class I members of the Peroxidase-Catalase superfamily, a large group of evolutionarily related but rather divergent enzymes. Through mining in public databases, unusual subsets of APX homologs were identified, disclosing the existence of two yet uncharacterized families of peroxidases named ascorbate peroxidase-related (APX-R) and ascorbate peroxidase-like (APX-L). As APX, APX-R harbor all catalytic residues required for peroxidatic activity. Nevertheless, proteins of this family do not contain residues known to be critical for ascorbate binding and therefore cannot use it as an electron donor. On the other hand, APX-L proteins not only lack ascorbate-binding residues, but also every other residue known to be essential for peroxidase activity. Through a molecular phylogenetic analysis performed with sequences derived from basal Archaeplastida, the present study discloses the existence of hybrid proteins, which combine features of these three families. The results here presented show that the prevalence of hybrid proteins varies among distinct groups of organisms, accounting for up to 33% of total APX homologs in species of green algae. The analysis of this heterogeneous group of proteins sheds light on the origin of APX-R and APX-L and suggests the occurrence of a process characterized by the progressive deterioration of ascorbate-binding and catalytic sites towards neofunctionalization. [ABSTRACT FROM AUTHOR]

An abstract of the article "Investigating the expression pattern of the OsAPx1 gene promoter in rice" by Julio Garighan and colleagues is presented.