Comparative analysis of wild-type and chloroplast MCU-deficient plants reveals multiple consequences of chloroplast calcium handling under drought stress.

Introduction: Chloroplast calcium homeostasis plays an important role in modulating the response of plants to abiotic and biotic stresses. One of the greatest challenges is to understand how chloroplast calcium-permeable pathways and sensors are regulated in a concerted manner to translate specific information into a calcium signature and to elucidate the downstream effects of specific chloroplast calcium dynamics. One of the six homologs of the mitochondrial calcium uniporter (MCU) was found to be located in chloroplasts in the leaves and to crucially contribute to drought- and oxidative stress-triggered uptake of calcium into this organelle. Methods: In the present study we integrated comparative proteomic analysis with biochemical, genetic, cellular, ionomic and hormone analysis in order to gain an insight into how chloroplast calcium channels are integrated into signaling circuits under watered condition and under drought stress. Results: Altogether, our results indicate for the first time a link between chloroplast calcium channels and hormone levels, showing an enhanced ABA level in the cmcu mutant already in well-watered condition. Furthermore, we show that the lack of cMCU results in an upregulation of the calcium sensor CAS and of enzymes of chlorophyll synthesis, which are also involved in retrograde signaling upon drought stress, in two independent KO lines generated in Col-0 and Col-4 ecotypes. Conclusions: These observations point to chloroplasts as important signaling hubs linked to their calcium dynamics. Our results obtained in the model plant Arabidopsis thaliana are discussed also in light of our limited knowledge regarding organellar calcium signaling in crops and raise the possibility of an involvement of such signaling in response to drought stress also in crops.

Gene regulatory networks shape developmental plasticity of root cell types under water extremes in rice.

Understanding how roots modulate development under varied irrigation or rainfall is crucial for development of climate-resilient crops. We established a toolbox of tagged rice lines to profile translating mRNAs and chromatin accessibility within specific cell populations. We used these to study roots in a range of environments: plates in the lab, controlled greenhouse stress and recovery conditions, and outdoors in a paddy. Integration of chromatin and mRNA data resolves regulatory networks of the following: cycle genes in proliferating cells that attenuate DNA synthesis under submergence; genes involved in auxin signaling, the circadian clock, and small RNA regulation in ground tissue; and suberin biosynthesis, iron transporters, and nitrogen assimilation in endodermal/exodermal cells modulated with water availability. By applying a systems approach, we identify known and candidate driver transcription factors of water-deficit responses and xylem development plasticity. Collectively, this resource will facilitate genetic improvements in root systems for optimal climate resilience.

Innovation, conservation, and repurposing of gene function in root cell type development.

Plant species have evolved myriads of solutions, including complex cell type development and regulation, to adapt to dynamic environments. To understand this cellular diversity, we profiled tomato root cell type translatomes. Using xylem differentiation in tomato, examples of functional innovation, repurposing, and conservation of transcription factors are described, relative to the model plant Arabidopsis. Repurposing and innovation of genes are further observed within an exodermis regulatory network and illustrate its function. Comparative translatome analyses of rice, tomato, and Arabidopsis cell populations suggest increased expression conservation of root meristems compared with other homologous populations. In addition, the functions of constitutively expressed genes are more conserved than those of cell type/tissue-enriched genes. These observations suggest that higher order properties of cell type and pan-cell type regulation are evolutionarily conserved between plants and animals.

Chloroplast Calcium Signaling in the Spotlight.

Calcium has long been known to regulate the metabolism of chloroplasts, concerning both light and carbon reactions of photosynthesis, as well as additional non photosynthesis-related processes. In addition to undergo Ca2+ regulation, chloroplasts can also influence the overall Ca2+ signaling pathways of the plant cell. Compelling evidence indicate that chloroplasts can generate specific stromal Ca2+ signals and contribute to the fine tuning of cytoplasmic Ca2+ signaling in response to different environmental stimuli. The recent set up of a toolkit of genetically encoded Ca2+ indicators, targeted to different chloroplast subcompartments (envelope, stroma, thylakoids) has helped to unravel the participation of chloroplasts in intracellular Ca2+ handling in resting conditions and during signal transduction. Intra-chloroplast Ca2+ signals have been demonstrated to occur in response to specific environmental stimuli, suggesting a role for these plant-unique organelles in transducing Ca2+-mediated stress signals. In this mini-review we present current knowledge of stimulus-specific intra-chloroplast Ca2+ transients, as well as recent advances in the identification and characterization of Ca2+-permeable channels/transporters localized at chloroplast membranes. In particular, the potential role played by cMCU, a chloroplast-localized member of the mitochondrial calcium uniporter (MCU) family, as component of plant environmental sensing is discussed in detail, taking into account some specific structural features of cMCU. In summary, the recent molecular identification of some players of chloroplast Ca2+ signaling has opened new avenues in this rapidly developing field and will hopefully allow a deeper understanding of the role of chloroplasts in shaping physiological responses in plants.

A Meta-Analysis of Comparative Transcriptomic Data Reveals a Set of Key Genes Involved in the Tolerance to Abiotic Stresses in Rice.

Several environmental factors, such as drought, salinity, and extreme temperatures, negatively affect plant growth and development, which leads to yield losses. The tolerance or sensitivity to abiotic stressors are the expression of a complex machinery involving molecular, biochemical, and physiological mechanisms. Here, a meta-analysis on previously published RNA-Seq data was performed to identify the genes conferring tolerance to chilling, osmotic, and salt stresses, by comparing the transcriptomic changes between tolerant and susceptible rice genotypes. Several genes encoding transcription factors (TFs) were identified, suggesting that abiotic stress tolerance involves upstream regulatory pathways. A gene co-expression network defined the metabolic and signalling pathways with a prominent role in the differentiation between tolerance and susceptibility: (i) the regulation of endogenous abscisic acid (ABA) levels, through the modulation of genes that are related to its biosynthesis/catabolism, (ii) the signalling pathways mediated by ABA and jasmonic acid, (iii) the activity of the "Drought and Salt Tolerance" TF, involved in the negative regulation of stomatal closure, and (iv) the regulation of flavonoid biosynthesis by specific MYB TFs. The identified genes represent putative key players for conferring tolerance to a broad range of abiotic stresses in rice; a fine-tuning of their expression seems to be crucial for rice plants to cope with environmental cues.

A chloroplast-localized mitochondrial calcium uniporter transduces osmotic stress in Arabidopsis.

Chloroplasts are integral to sensing biotic and abiotic stress in plants, but their role in transducing Ca2+-mediated stress signals remains poorly understood1,2. Here we identify cMCU, a member of the mitochondrial calcium uniporter (MCU) family, as an ion channel mediating Ca2+ flux into chloroplasts in vivo. Using a toolkit of aequorin reporters targeted to chloroplast stroma and the cytosol in cMCU wild-type and knockout lines, we provide evidence that stress-stimulus-specific Ca2+ dynamics in the chloroplast stroma correlate with expression of the channel. Fast downstream signalling events triggered by osmotic stress, involving activation of the mitogen-activated protein kinases (MAPK) MAPK3 and MAPK6, and the transcription factors MYB60 and ethylene-response factor 6 (ERF6), are influenced by cMCU activity. Relative to wild-type plants, cMCU knockouts display increased resistance to long-term water deficit and improved recovery on rewatering. Modulation of stromal Ca2+ in specific processing of stress signals identifies cMCU as a component of plant environmental sensing.

Salt tolerance in indica rice cell cultures depends on a fine tuning of ROS signalling and homeostasis.

Among cereal crops, salinity tolerance is rare and complex. Multiple genes control numerous pathways, which constitute plant's response to salinity. Cell cultures act as model system and are useful to investigate the salinity response which can possibly mimic a plant's response to stress. In the present study two indica rice varieties, KS-282 and Super Basmati which exhibited contrasting sodium chloride (NaCl) stress response were used to establish cell cultures. The cell cultures showed a contrasting response to salt stress at 100 mM NaCl. High level of intracellular hydrogen peroxide (H2O2) and nitric oxide (NO) were observed in sensitive cell culture for prolonged period as compared to the tolerant cells in which an extracellular H2O2 burst along with controlled intracellular H2O2 and NO signal was seen. To evaluate the role of NO in inducing cell death under salt stress, cell death percentage (CDP) was measured after 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) pre-treatment. CDP was reduced significantly in both tolerant and sensitive cell cultures emphasizing NO's possible role in programmed cell death. Expression analysis of apoplastic NADPH oxidase, i.e. OsRbohA and recently characterised OSCA family members i.e. OsOSCA 1.2 and OsOSCA 3.1 was done. Intracellular H2O2/NO levels displayed an interplay between Ca2+ influx and ROS/RNS signal. Detoxifying enzyme (i.e. ascorbate peroxidase and catalase) activity was considerably higher in tolerant KS-282 while the activity of superoxide dismutase was significantly prominent in the sensitive cells triggering greater oxidative damage owing to the prolonged presence of intracellular H2O2. Salt stress and ROS responsive TFs i.e. OsSERF1 and OsDREB2A were expressed exclusively in the tolerant cells. Similarly, the expression of genes involved in maintaining high [K+]/[Na+] ratio was considerably higher and earlier in the tolerant variety. Overall, we suggest that a control over ROS production, and an increase in the expression of genes important for potassium homeostasis play a dynamic role in salinity tolerance in rice cell cultures.

H2O2 Signature and Innate Antioxidative Profile Make the Difference Between Sensitivity and Tolerance to Salt in Rice Cells.

Salt tolerance is a complex trait that varies between and within species. H2O2 profiles as well as antioxidative systems have been investigated in the cultured cells of rice obtained from Italian rice varieties with different salt tolerance. Salt stress highlighted differences in extracellular and intracellular H2O2 profiles in the two cell cultures. The tolerant variety had innate reactive oxygen species (ROS) scavenging systems that enabled ROS, in particular H2O2, to act as a signal molecule rather than a damaging one. Different intracellular H2O2 profiles were also observed: in tolerant cells, an early and narrow peak was detected at 5 min; while in sensitive cells, a large peak was associated with cell death. Likewise, the transcription factor salt-responsive ethylene responsive factor 1 (TF SERF1), which is known for being regulated by H2O2, showed a different expression profile in the two cell lines. Notably, similar H2O2 profiles and cell fates were also obtained when exogenous H2O2 was produced by glucose/glucose oxidase (GOX) treatment. Under salt stress, the tolerant variety also exhibited rapid upregulation of K+ transporter genes in order to deal with K+/Na+ impairment. This upregulation was not detected in the presence of oxidative stress alone. The importance of the innate antioxidative profile was confirmed by the protective effect of experimentally increased glutathione in salt-treated sensitive cells. Overall, these results underline the importance of specific H2O2 signatures and innate antioxidative systems in modulating ionic and redox homeostasis for salt stress tolerance.

Fast Regulation of Hormone Metabolism Contributes to Salt Tolerance in Rice (Oryzasativa spp. Japonica, L.) by Inducing Specific Morpho-Physiological Responses.

Clear evidence has highlighted a role for hormones in the plant stress response, including salt stress. Interplay and cross-talk among different hormonal pathways are of vital importance in abiotic stress tolerance. A genome-wide transcriptional analysis was performed on leaves and roots of three-day salt treated and untreated plants of two Italian rice varieties, Baldo and Vialone Nano, which differ in salt sensitivity. Genes correlated with hormonal pathways were identified and analyzed. The contents of abscisic acid, indoleacetic acid, cytokinins, and gibberellins were measured in roots, stems, and leaves of seedlings exposed for one and three days to salt stress. From the transcriptomic analysis, a huge number of genes emerged as being involved in hormone regulation in response to salt stress. The expression profile of genes involved in biosynthesis, signaling, response, catabolism, and conjugation of phytohormones was analyzed and integrated with the measurements of hormones in roots, stems, and leaves of seedlings. Significant changes in the hormone levels, along with differences in morphological responses, emerged between the two varieties. These results support the faster regulation of hormones metabolism in the tolerant variety that allows a prompt growth reprogramming and the setting up of an acclimation program, leading to specific morpho-physiological responses and growth recovery.

Transcriptome and Cell Physiological Analyses in Different Rice Cultivars Provide New Insights Into Adaptive and Salinity Stress Responses.

Salinity tolerance has been extensively investigated in recent years due to its agricultural importance. Several features, such as the regulation of ionic transporters and metabolic adjustments, have been identified as salt tolerance hallmarks. Nevertheless, due to the complexity of the trait, the results achieved to date have met with limited success in improving the salt tolerance of rice plants when tested in the field, thus suggesting that a better understanding of the tolerance mechanisms is still required. In this work, differences between two varieties of rice with contrasting salt sensitivities were revealed by the imaging of photosynthetic parameters, ion content analysis and a transcriptomic approach. The transcriptomic analysis conducted on tolerant plants supported the setting up of an adaptive program consisting of sodium distribution preferentially limited to the roots and older leaves, and in the activation of regulatory mechanisms of photosynthesis in the new leaves. As a result, plants resumed grow even under prolonged saline stress. In contrast, in the sensitive variety, RNA-seq analysis revealed a misleading response, ending in senescence and cell death. The physiological response at the cellular level was investigated by measuring the intracellular profile of H2O2 in the roots, using a fluorescent probe. In the roots of tolerant plants, a quick response was observed with an increase in H2O2 production within 5 min after salt treatment. The expression analysis of some of the genes involved in perception, signal transduction and salt stress response confirmed their early induction in the roots of tolerant plants compared to sensitive ones. By inhibiting the synthesis of apoplastic H2O2, a reduction in the expression of these genes was detected. Our results indicate that quick H2O2 signaling in the roots is part of a coordinated response that leads to adaptation instead of senescence in salt-treated rice plants.

Targeted Next-Generation Sequencing Identification of Mutations in Disease Resistance Gene Analogs (RGAs) in Wild and Cultivated Beets.

Resistance gene analogs (RGAs) were searched bioinformatically in the sugar beet (Beta vulgaris L.) genome as potential candidates for improving resistance against different diseases. In the present study, Ion Torrent sequencing technology was used to identify mutations in 21 RGAs. The DNA samples of ninety-six individuals from six sea beets (Beta vulgaris L. subsp. maritima) and six sugar beet pollinators (eight individuals each) were used for the discovery of single-nucleotide polymorphisms (SNPs). Target amplicons of about 200 bp in length were designed with the Ion AmpliSeq Designer system in order to cover the DNA sequences of the RGAs. The number of SNPs ranged from 0 in four individuals to 278 in the pollinator R740 (which is resistant to rhizomania infection). Among different groups of beets, cytoplasmic male sterile lines had the highest number of SNPs (132) whereas the lowest number of SNPs belonged to O-types (95). The principal coordinates analysis (PCoA) showed that the polymorphisms inside the gene Bv8_184910_pkon (including the CCCTCC sequence) can effectively differentiate wild from cultivated beets, pointing at a possible mutation associated to rhizomania resistance that originated directly from cultivated beets. This is unlike other resistance sources that are introgressed from wild beets. This gene belongs to the receptor-like kinase (RLK) class of RGAs, and is associated to a hypothetical protein. In conclusion, this first report of using Ion Torrent sequencing technology in beet germplasm suggests that the identified sequence CCCTCC can be used in marker-assisted programs to differentiate wild from domestic beets and to identify other unknown disease resistance genes in beet.

Pathway Inspector: a pathway based web application for RNAseq analysis of model and non-model organisms.

SUMMARY: Pathway Inspector is an easy-to-use web application helping researchers to find patterns of expression in complex RNAseq experiments. The tool combines two standard approaches for RNAseq analysis: the identification of differentially expressed genes and a topology-based analysis of enriched pathways. Pathway Inspector is equipped with ad hoc interactive graphical interfaces simplifying the discovery of modulated pathways and the integration of the differentially expressed genes in the corresponding pathway topology. AVAILABILITY AND IMPLEMENTATION: Pathway Inspector is available at the website http://admiral.fmach.it/PI and has been developed in Python, making use of the Django Web Framework. CONTACT: Contact:paolo.fontana@fmach.it

Physiological Characterization of a Plant Mitochondrial Calcium Uniporter in Vitro and in Vivo.

Over the recent years, several proteins that make up the mitochondrial calcium uniporter complex (MCUC) mediating Ca2+uptake into the mitochondrial matrix have been identified in mammals, including the channel-forming protein MCU. Although six MCU gene homologs are conserved in the model plant Arabidopsis (Arabidopsis thaliana) in which mitochondria can accumulate Ca2+, a functional characterization of plant MCU homologs has been lacking. Using electrophysiology, we show that one isoform, AtMCU1, gives rise to a Ca2+-permeable channel activity that can be observed even in the absence of accessory proteins implicated in the formation of the active mammalian channel. Furthermore, we provide direct evidence that AtMCU1 activity is sensitive to the mitochondrial calcium uniporter inhibitors Ruthenium Red and Gd3+, as well as to the Arabidopsis protein MICU, a regulatory MCUC component. AtMCU1 is prevalently expressed in roots, localizes to mitochondria, and its absence causes mild changes in Ca2+ dynamics as assessed by in vivo measurements in Arabidopsis root tips. Plants either lacking or overexpressing AtMCU1 display root mitochondria with altered ultrastructure and show shorter primary roots under restrictive growth conditions. In summary, our work adds evolutionary depth to the investigation of mitochondrial Ca2+ transport, indicates that AtMCU1, together with MICU as a regulator, represents a functional configuration of the plant mitochondrial Ca2+ uptake complex with differences to the mammalian MCUC, and identifies a new player of the intracellular Ca2+ regulation network in plants.

Eliciting the Functional Taxonomy from protein annotations and taxa.

The advances of omics technologies have triggered the production of an enormous volume of data coming from thousands of species. Meanwhile, joint international efforts like the Gene Ontology (GO) consortium have worked to provide functional information for a vast amount of proteins. With these data available, we have developed FunTaxIS, a tool that is the first attempt to infer functional taxonomy (i.e. how functions are distributed over taxa) combining functional and taxonomic information. FunTaxIS is able to define a taxon specific functional space by exploiting annotation frequencies in order to establish if a function can or cannot be used to annotate a certain species. The tool generates constraints between GO terms and taxa and then propagates these relations over the taxonomic tree and the GO graph. Since these constraints nearly cover the whole taxonomy, it is possible to obtain the mapping of a function over the taxonomy. FunTaxIS can be used to make functional comparative analyses among taxa, to detect improper associations between taxa and functions, and to discover how functional knowledge is either distributed or missing. A benchmark test set based on six different model species has been devised to get useful insights on the generated taxonomic rules.

Calcium Flux across Plant Mitochondrial Membranes: Possible Molecular Players.

Plants, being sessile organisms, have evolved the ability to integrate external stimuli into metabolic and developmental signals. A wide variety of signals, including abiotic, biotic, and developmental stimuli, were observed to evoke specific spatio-temporal Ca(2+) transients which are further transduced by Ca(2+) sensor proteins into a transcriptional and metabolic response. Most of the research on Ca(2+) signaling in plants has been focused on the transport mechanisms for Ca(2+) across the plasma- and the vacuolar membranes as well as on the components involved in decoding of cytoplasmic Ca(2+) signals, but how intracellular organelles such as mitochondria are involved in the process of Ca(2+) signaling is just emerging. The combination of the molecular players and the elicitors of Ca(2+) signaling in mitochondria together with newly generated detection systems for measuring organellar Ca(2+) concentrations in plants has started to provide fruitful grounds for further discoveries. In the present review we give an updated overview of the currently identified/hypothesized pathways, such as voltage-dependent anion channels, homologs of the mammalian mitochondrial uniporter (MCU), LETM1, a plant glutamate receptor family member, adenine nucleotide/phosphate carriers and the permeability transition pore (PTP), that may contribute to the transport of Ca(2+) across the outer and inner mitochondrial membranes in plants. We briefly discuss the relevance of the mitochondrial Ca(2+) homeostasis for ensuring optimal bioenergetic performance of this organelle.

Alternative splicing-mediated targeting of the Arabidopsis GLUTAMATE RECEPTOR3.5 to mitochondria affects organelle morphology.

Since the discovery of 20 genes encoding for putative ionotropic glutamate receptors in the Arabidopsis (Arabidopsis thaliana) genome, there has been considerable interest in uncovering their physiological functions. For many of these receptors, neither their channel formation and/or physiological roles nor their localization within the plant cells is known. Here, we provide, to our knowledge, new information about in vivo protein localization and give insight into the biological roles of the so-far uncharacterized Arabidopsis GLUTAMATE RECEPTOR3.5 (AtGLR3.5), a member of subfamily 3 of plant glutamate receptors. Using the pGREAT vector designed for the expression of fusion proteins in plants, we show that a splicing variant of AtGLR3.5 targets the inner mitochondrial membrane, while the other variant localizes to chloroplasts. Mitochondria of knockout or silenced plants showed a strikingly altered ultrastructure, lack of cristae, and swelling. Furthermore, using a genetically encoded mitochondria-targeted calcium probe, we measured a slightly reduced mitochondrial calcium uptake capacity in the knockout mutant. These observations indicate a functional expression of AtGLR3.5 in this organelle. Furthermore, AtGLR3.5-less mutant plants undergo anticipated senescence. Our data thus represent, to our knowledge, the first evidence of splicing-regulated organellar targeting of a plant ion channel and identify the first cation channel in plant mitochondria from a molecular point of view.

A thylakoid-located two-pore K+ channel controls photosynthetic light utilization in plants.

The size of the light-induced proton motive force (pmf) across the thylakoid membrane of chloroplasts is regulated in response to environmental stimuli. Here, we describe a component of the thylakoid membrane, the two-pore potassium (K(+)) channel TPK3, which modulates the composition of the pmf through ion counterbalancing. Recombinant TPK3 exhibited potassium-selective channel activity sensitive to Ca(2+) and H(+). In Arabidopsis plants, the channel is found in the thylakoid stromal lamellae. Arabidopsis plants silenced for the TPK3 gene display reduced growth and altered thylakoid membrane organization. This phenotype reflects an impaired capacity to generate a normal pmf, which results in reduced CO2 assimilation and deficient nonphotochemical dissipation of excess absorbed light. Thus, the TPK3 channel manages the pmf necessary to convert photochemical energy into physiological functions.