RESUMO
To ensure reproductive success, flowering plants produce an excess of pollen to fertilize a limited number of ovules. Pollen grains mature into two distinct subpopulations - those that display high metabolic activity and elevated reactive oxygen species (ROS) levels immediately after hydration (high-ROS/active), and those that maintain an extended period of dormancy with low metabolic activity (low-ROS/inactive/arrested/dormant). We propose that the dormant pollen serves as a backup to provide a second chance for successful fertilization when the 'first wave' of pollen encounters an unpredictable growth condition such as heat stress. This model provides a framework for considering the role of dormancy in reproductive stress tolerance as well as strategies for mitigating pollen thermovulnerability to daytime and night-time warming that is associated with global climate change.
Assuntos
Pólen , Polinização , Resposta ao Choque Térmico , Óvulo Vegetal , Espécies Reativas de Oxigênio/metabolismo , Sementes/metabolismoRESUMO
KEY MESSAGE: Arabidopsis pollen transcriptome analysis revealed new intergenic transcripts of unknown function, many of which are long non-coding RNAs, that may function in pollen-specific processes, including the heat stress response. The male gametophyte is the most heat sensitive of all plant tissues. In recent years, long noncoding RNAs (lncRNAs) have emerged as important components of cellular regulatory networks involved in most biological processes, including response to stress. While examining RNAseq datasets of developing and germinating Arabidopsis thaliana pollen exposed to heat stress (HS), we identified 66 novel and 246 recently annotated intergenic expressed loci (XLOCs) of unknown function, with the majority encoding lncRNAs. Comparison with HS in cauline leaves and other RNAseq experiments indicated that 74% of the 312 XLOCs are pollen-specific, and at least 42% are HS-responsive. Phylogenetic analysis revealed that 96% of the genes evolved recently in Brassicaceae. We found that 50 genes are putative targets of microRNAs and that 30% of the XLOCs contain small open reading frames (ORFs) with homology to protein sequences. Finally, RNAseq of ribosome-protected RNA fragments together with predictions of periodic footprint of the ribosome P-sites indicated that 23 of these ORFs are likely to be translated. Our findings indicate that many of the 312 unknown genes might be functional and play a significant role in pollen biology, including the HS response.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Resposta ao Choque Térmico/genética , Filogenia , Pólen/genéticaRESUMO
Sexual reproduction in flowering plants depends on the fitness of the male gametophyte during fertilization. Because pollen development is highly sensitive to hot and cold temperature extremes, reliable methods to evaluate pollen viability are important for research into improving reproductive heat stress (HS) tolerance. Here, we describe an approach to rapidly evaluate pollen viability using a reactive oxygen species (ROS) probe dichlorodihydrofluorescein diacetate (i.e. H2 DCFDA-staining) coupled with flow cytometry. In using flow cytometry to analyze mature pollen harvested from Arabidopsis and tomato flowers, we discovered that pollen distributed bimodally into 'low-ROS' and 'high-ROS' subpopulations. Pollen germination assays following fluorescence-activated cell sorting revealed that the high-ROS pollen germinated with a frequency that was 35-fold higher than the low-ROS pollen, supporting a model in which a significant fraction of a flower's pollen remains in a low metabolic or dormant state even after hydration. The ability to use flow cytometry to quantify ROS dynamics within a large pollen population was shown by dose-dependent alterations in DCF-fluorescence in response to oxidative stress or antioxidant treatments. HS treatments (35°C) increased ROS levels, which correlated with a ~60% reduction in pollen germination. These results demonstrate the potential of using flow cytometry-based approaches to investigate metabolic changes during stress responses in pollen.
Assuntos
Adaptação Fisiológica/fisiologia , Flores/fisiologia , Resposta ao Choque Térmico/fisiologia , Pólen/fisiologia , Polinização/fisiologia , Arabidopsis/citologia , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Sobrevivência Celular/fisiologia , Citometria de Fluxo , Flores/citologia , Flores/metabolismo , Solanum lycopersicum/citologia , Solanum lycopersicum/metabolismo , Solanum lycopersicum/fisiologia , Estresse Oxidativo/fisiologia , Pólen/citologia , Pólen/metabolismo , Tubo Polínico/citologia , Tubo Polínico/metabolismo , Tubo Polínico/fisiologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Protein phosphorylation regulates numerous cellular processes. Identifying the substrates and protein kinases involved is vital to understand how these important posttranslational modifications modulate biological function in eukaryotic cells. Pyrophosphatases catalyze the hydrolysis of inorganic phosphate (PPi) to inorganic phosphate Pi, driving biosynthetic reactions; they are essential for low cytosolic inorganic phosphate. It was suggested recently that posttranslational regulation of Family I soluble inorganic pyrophosphatases (sPPases) may affect their activity. We previously demonstrated that two pollen-expressed sPPases, Pr-p26.1a and Pr-p26.1b, from the flowering plant Papaver rhoeas were inhibited by phosphorylation. Despite the potential significance, there is a paucity of data on sPPase phosphorylation and regulation. Here, we used liquid chromatographic tandem mass spectrometry to map phosphorylation sites to the otherwise divergent amino-terminal extensions on these pollen sPPases. Despite the absence of reports in the literature on mapping phosphorylation sites on sPPases, a database survey of various proteomes identified a number of examples, suggesting that phosphorylation may be a more widely used mechanism to regulate these enzymes. Phosphomimetic mutants of Pr-p26.1a/b significantly and differentially reduced PPase activities by up to 2.5-fold at pH 6.8 and 52% in the presence of Ca2+ and hydrogen peroxide over unmodified proteins. This indicates that phosphoregulation of key sites can inhibit the catalytic responsiveness of these proteins in concert with key intracellular events. As sPPases are essential for many metabolic pathways in eukaryotic cells, our findings identify the phosphorylation of sPPases as a potential master regulatory mechanism that could be used to attenuate metabolism.
Assuntos
Pirofosfatase Inorgânica/metabolismo , Papaver/enzimologia , Proteínas de Plantas/metabolismo , Pólen/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Cálcio/metabolismo , Cálcio/farmacologia , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Peróxido de Hidrogênio/farmacologia , Concentração de Íons de Hidrogênio , Pirofosfatase Inorgânica/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Mutação , Oxidantes/farmacologia , Papaver/genética , Fosforilação , Filogenia , Proteínas de Plantas/genética , Pólen/genética , Proteínas Quinases/classificação , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Solubilidade , Especificidade por Substrato , Espectrometria de Massas em TandemRESUMO
Over 13% of all genes in the Arabidopsis thaliana genome encode for proteins classified as having a completely unknown function, with the function of >30% of the Arabidopsis proteome poorly characterized. Although empirical data in the form of mRNA and proteome profiling experiments suggest that many of these proteins play an important role in different biological processes, their functional characterization remains one of the major challenges in modern biology. To expand the annotation of genes with unknown function involved in the response of Arabidopsis to different environmental stress conditions, we selected 1007 such genes and tested the response of their corresponding homozygous T-DNA insertional mutants to salinity, oxidative, osmotic, heat, cold and hypoxia stresses. Depending on the specific abiotic stresses tested, 12-31% of mutants had an altered stress-response phenotype. Interestingly, 832 out of 1007 mutants showed tolerance or sensitivity to more than one abiotic stress treatment, suggesting that genes of unknown function could play an important role in abiotic stress-response signaling, or general acclimation mechanisms. Further analysis of multiple stress-response phenotypes within different populations of mutants revealed interesting links between acclimation to heat, cold and oxidative stresses, as well as between sensitivity to ABA, osmotic, salinity, oxidative and hypoxia stresses. Our findings provide a significant contribution to the biological characterization of genes with unknown function in Arabidopsis and demonstrate that many of these genes play a key role in the response of plants to abiotic stresses.
Assuntos
Arabidopsis/genética , Arabidopsis/fisiologia , Genes de Plantas/genética , Ensaios de Triagem em Larga Escala , Estresse Fisiológico/genética , Ácido Abscísico/farmacologia , Adaptação Fisiológica/efeitos dos fármacos , Adaptação Fisiológica/genética , DNA Bacteriano/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Mutagênese Insercional/efeitos dos fármacos , Mutagênese Insercional/genética , Mutação/genética , Fenótipo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Plântula/efeitos dos fármacos , Plântula/genética , Estresse Fisiológico/efeitos dos fármacosRESUMO
Reactive oxygen species play a key role in the response of plants to abiotic stress conditions. Their level is controlled in Arabidopsis thaliana by a large network of genes that includes the H(2)O(2)-scavenging enzymes cytosolic ascorbate peroxidase (APX) 1 and 2. Although the function of APX1 has been established under different growth conditions, genetic evidence for APX2 function, as well as for the mode of cooperation between APX1 and APX2, is very limited. This study characterized the response of Arabidopsis mutants deficient in APX1, APX2, and APX1/APX2 to heat, salinity, light, and oxidative stresses. The findings reveal that deficiency in APX2 resulted in a decreased tolerance to light stress, as well as an enhanced tolerance to salinity and oxidative stresses. Interestingly, plants lacking APX2 were more sensitive to heat stress at the seedling stage, but more tolerant to heat stress at the reproductive stage. Cooperation between APX1 and APX2 was evident during oxidative stress, but not during light, salinity, or heat stress. The findings demonstrate a role for APX2 in the response of plants to light, heat, salinity, and oxidative stresses. The finding that plants lacking APX2 produced more seeds under prolonged heat stress conditions suggests that redundant mechanisms activated in APX2-deficient plants during heat stress play a key role in the protection of reproductive tissues from heat-related damage. This finding is very important because heat-associated damage to reproductive tissues in different crops is a major cause for yield loss in agriculture production worldwide.
Assuntos
Arabidopsis/enzimologia , Arabidopsis/fisiologia , Ascorbato Peroxidases/deficiência , Citosol/enzimologia , Temperatura Alta , Sementes/crescimento & desenvolvimento , Estresse Fisiológico , Adaptação Fisiológica/efeitos dos fármacos , Adaptação Fisiológica/genética , Adaptação Fisiológica/efeitos da radiação , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ascorbato Peroxidases/genética , Ascorbato Peroxidases/metabolismo , Citosol/efeitos dos fármacos , Citosol/efeitos da radiação , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Técnicas de Inativação de Genes , Peróxido de Hidrogênio/metabolismo , Luz , Mutação/genética , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Estresse Oxidativo/efeitos da radiação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodução/efeitos dos fármacos , Reprodução/efeitos da radiação , Plântula/efeitos dos fármacos , Plântula/fisiologia , Plântula/efeitos da radiação , Sementes/efeitos dos fármacos , Sementes/efeitos da radiação , Cloreto de Sódio/farmacologia , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Estresse Fisiológico/efeitos da radiaçãoRESUMO
Over one-quarter of all plant genes encode proteins of unknown function that can be further classified as proteins with obscure features (POFs), which lack currently defined motifs or domains, or proteins with defined features, which contain at least one previously defined domain or motif. Although empirical data in the form of transcriptome and proteome profiling suggest that many of these proteins play important roles in plants, their functional characterization remains one of the main challenges in modern biology. To begin the functional annotation of proteins with unknown function, which are involved in the oxidative stress response of Arabidopsis (Arabidopsis thaliana), we generated transgenic Arabidopsis plants that constitutively expressed 23 different POFs (four of which were specific to Arabidopsis) and 18 different proteins with defined features. All were previously found to be expressed in response to oxidative stress in Arabidopsis. Transgenic plants were tested for their tolerance to oxidative stress imposed by paraquat or t-butyl hydroperoxide, or were subjected to osmotic, salinity, cold, and heat stresses. More than 70% of all expressed proteins conferred tolerance to oxidative stress. In contrast, >90% of the expressed proteins did not confer enhanced tolerance to the other abiotic stresses tested, and approximately 50% rendered plants more susceptible to osmotic or salinity stress. Two Arabidopsis-specific POFs, and an Arabidopsis and Brassica-specific protein of unknown function, conferred enhanced tolerance to oxidative stress. Our findings suggest that tolerance to oxidative stress involves mechanisms and pathways that are unknown at present, including some that are specific to Arabidopsis or the Brassicaceae.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Estresse Oxidativo , Adaptação Fisiológica , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Oxirredução , Plantas Geneticamente Modificadas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
In plant cells, the vacuole functions as a major calcium store. Although a calmodulin-regulated Ca2+-ATPase (ACA4) is known to be present in prevacuolar compartments, the presence of an ACA-type Ca2+-ATPase in the mature vacuole of a plant cell has not been verified. Here we provide evidence that ACA11 localizes to the vacuole membrane. ACA11 tagged with GFP was expressed in stable transgenic plants, and visualized in root cells and protoplasts by confocal microscopy. A Ca2+-ATPase function for ACA11 was confirmed by complementation of yeast mutants. A calmodulin binding domain was identified within the first 37 residues of the N-terminal autoinhibitory region.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , ATPases Transportadoras de Cálcio/metabolismo , Raízes de Plantas/enzimologia , Vacúolos/enzimologia , Arabidopsis/citologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , ATPases Transportadoras de Cálcio/genética , Calmodulina/metabolismo , Ativação Enzimática/fisiologia , Teste de Complementação Genética , Microscopia Confocal , Raízes de Plantas/citologia , Raízes de Plantas/genética , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Estrutura Terciária de Proteína/fisiologia , Protoplastos/citologia , Protoplastos/enzimologia , Saccharomyces cerevisiae/genética , Vacúolos/genéticaRESUMO
Abscisic acid (ABA) signal transduction has been proposed to utilize cytosolic Ca(2+) in guard cell ion channel regulation. However, genetic mutants in Ca(2+) sensors that impair guard cell or plant ion channel signaling responses have not been identified, and whether Ca(2+)-independent ABA signaling mechanisms suffice for a full response remains unclear. Calcium-dependent protein kinases (CDPKs) have been proposed to contribute to central signal transduction responses in plants. However, no Arabidopsis CDPK gene disruption mutant phenotype has been reported to date, likely due to overlapping redundancies in CDPKs. Two Arabidopsis guard cell-expressed CDPK genes, CPK3 and CPK6, showed gene disruption phenotypes. ABA and Ca(2+) activation of slow-type anion channels and, interestingly, ABA activation of plasma membrane Ca(2+)-permeable channels were impaired in independent alleles of single and double cpk3cpk6 mutant guard cells. Furthermore, ABA- and Ca(2+)-induced stomatal closing were partially impaired in these cpk3cpk6 mutant alleles. However, rapid-type anion channel current activity was not affected, consistent with the partial stomatal closing response in double mutants via a proposed branched signaling network. Imposed Ca(2+) oscillation experiments revealed that Ca(2+)-reactive stomatal closure was reduced in CDPK double mutant plants. However, long-lasting Ca(2+)-programmed stomatal closure was not impaired, providing genetic evidence for a functional separation of these two modes of Ca(2+)-induced stomatal closing. Our findings show important functions of the CPK6 and CPK3 CDPKs in guard cell ion channel regulation and provide genetic evidence for calcium sensors that transduce stomatal ABA signaling.
Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Canais de Cálcio/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Canais Iônicos/metabolismo , Proteínas Quinases/metabolismo , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Epiderme Vegetal/citologia , Epiderme Vegetal/enzimologia , Proteínas Quinases/genética , Transdução de SinaisRESUMO
Serial analysis of gene expression provides quantitative and comprehensive expression profiling in a given cell population. In our efforts to define the genes overexpressed in carcinoma of the stomach, we performed serial analysis of gene expression analyses on dissected neoplastic and normal gastric epithelia. We identified 91,334 expressed tags, including 26,633 that were unique. The 20 most up-regulated genes (P < 0.01) in gastric cancer (GC) compared with normal gastric epithelia included several keratins that are specific for epithelial cells such as keratin 6A, 13, and 17. Interestingly, five calcium-binding proteins (S100A2, S100A7, S100A8, S100A9, and S100A10) were overexpressed. Quantitative real-time PCR on primary GC samples demonstrated overexpression of S100A2 in 18 of 20 tumors (90%). The other calcium-binding proteins were overexpressed in 25-45% of the GC samples that we studied. Our results indicate that S100A proteins may be important for gastric tumorigenesis. Additional investigations are required to elucidate the biological role of calcium-binding proteins in cancer.
Assuntos
Proteínas S100/biossíntese , Proteínas S100/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Perfilação da Expressão Gênica , Humanos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Numerous studies have shown that transcription factors are important in regulating plant responses to environmental stress. However, specific functions for most of the genes encoding transcription factors are unclear. In this study, we used mRNA profiles generated from microarray experiments to deduce the functions of genes encoding known and putative Arabidopsis transcription factors. The mRNA levels of 402 distinct transcription factor genes were examined at different developmental stages and under various stress conditions. Transcription factors potentially controlling downstream gene expression in stress signal transduction pathways were identified by observed activation and repression of the genes after certain stress treatments. The mRNA levels of a number of previously characterized transcription factor genes were changed significantly in connection with other regulatory pathways, suggesting their multifunctional nature. The expression of 74 transcription factor genes responsive to bacterial pathogen infection was reduced or abolished in mutants that have defects in salicylic acid, jasmonic acid, or ethylene signaling. This observation indicates that the regulation of these genes is mediated at least partly by these plant hormones and suggests that the transcription factor genes are involved in the regulation of additional downstream responses mediated by these hormones. Among the 43 transcription factor genes that are induced during senescence, 28 of them also are induced by stress treatment, suggesting extensive overlap responses to these stresses. Statistical analysis of the promoter regions of the genes responsive to cold stress indicated unambiguous enrichment of known conserved transcription factor binding sites for the responses. A highly conserved novel promoter motif was identified in genes responding to a broad set of pathogen infection treatments. This observation strongly suggests that the corresponding transcription factors play general and crucial roles in the coordinated regulation of these specific regulons. Although further validation is needed, these correlative results provide a vast amount of information that can guide hypothesis-driven research to elucidate the molecular mechanisms involved in transcriptional regulation and signaling networks in plants.