In the context of precision agriculture, high-throughput phenotyping (HTP) aims to rapidly and effectively identify factors that affect crop yield, enabling timely and appropriate interventions. However, interpreting data from HTP remains challenging. We performed a proximal red-green-blue (RGB)-based HTP on several tomato genotypes exposed to abiotic stress (drought) or biotic stress induced by tomato spotted wilt virus (TSWV), Pseudopyrenochaeta lycopersici (corky root rot; CRR), or Meloidogyne incognita (root-knot nematode; RKN). We aimed to determine if RGB-based HTP is effectively able to: a) distinguish the effects of biotic from abiotic stress; b) differentiate resistant/tolerant from susceptible genotypes. Our HTP data analysis produced 12 morphometric and eight colorimetric indices. Principal Component Analysis (PCA; P ​< ​0.0001; 83 ​% variation explained by three PCs) showed that factors such as shoot area solidity and certain color-based indices, including the senescence index and green area, effectively differentiated biotic from abiotic stress. Morphometric parameters, including plant height, projected shoot area, and convex hull area, proved to be applicable for identifying the stress status regardless of the type of stress. HTP effectively distinguished the genotype resistant to TSWV from the susceptible ones. This task was more challenging for below-ground stresses like CRR and RKN. Different profiles of HTP indices were observed among the genotypes assayed for drought tolerance, indicating variability in their ability to withstand drought conditions. In conclusion, our findings highlight the value of RGB-based HTP as a tool for precision farming of tomatoes, enabling the identification of both biotic and abiotic stressors.

Water deficit negatively impacts crop productivity and quality. Plants face these challenges by adjusting biological processes and molecular functions according to the intensity and duration of the stress. The cultivated potato (Solanum tuberosum) is considered sensitive to water deficit, thus breeding efforts are needed to enhance its resilience. To capture novel functional information and gene regulatory networks, we carried out mass spectrometry-based proteomics in potato cell suspensions exposed to abrupt or stepwise osmotic stresses. Both forms of stress triggered significant alterations in protein expression, though with divergent response mechanisms. Stress response pathways orchestrated by key proteins enrolled in primary and secondary metabolism, antioxidant processes, transcriptional and translational machinery and chromatin organization were found in adapted cells. Target metabolites and reactive oxygen species levels were quantified to associate functional outcomes with the proteome study. Remarkably, we also showed that adapted cells tolerate an array of diverse conditions, including anoxia, salt and heat stress. Finally, the expression patterns of genes encoding selected differentially expressed proteins were investigated in potato plants subjected to either drought or salt stress. Collectively, our findings reveal the complex cellular strategies of osmotic stress adaptation, identifying new fundamental genes that could enhance potato resilience.

Despite intense research towards the understanding of abiotic stress adaptation in tomato, the physiological adjustments and transcriptome modulation induced by combined salt and low nitrate (low N) conditions remain largely unknown. Here, three traditional tomato genotypes were grown under long-term single and combined stresses throughout a complete growth cycle. Physiological, molecular, and growth measurements showed extensive morphophysiological modifications under combined stress compared to the control, and single stress conditions, resulting in the highest penalty in yield and fruit size.The mRNA sequencing performed on both roots and leaves of genotype TRPO0040 indicated that the transcriptomic signature in leaves under combined stress conditions largely overlapped that of the low N treatment, whereas root transcriptomes were highly sensitive to salt stress. Differentially expressed genes were functionally interpreted using GO and KEGG enrichment analysis, which confirmed the stress and the tissue-specific changes. We also disclosed a set of genes underlying the specific response to combined conditions, including ribosome components and nitrate transporters, in leaves, and several genes involved in transport and response to stress in roots. Altogether, our results provide a comprehensive understanding of above- and below-ground physiological and molecular responses of tomato to salt stress and low N treatment, alone or in combination.

Tomato is a horticultural crop of high economic and nutritional value. Suboptimal environmental conditions, such as limited water and nutrient availability, cause severe yield reductions. Thus, selection of genotypes requiring lower inputs is a goal for the tomato breeding sector. We screened 10 tomato varieties exposed to water deficit, low nitrate or a combination of both. Biometric, physiological and molecular analyses revealed different stress responses among genotypes, identifying T270 as severely affected, and T250 as tolerant to the stresses applied. Investigation of transcriptome changes caused by combined stress in roots and leaves of these two genotypes yielded a low number of differentially expressed genes (DEGs) in T250 compared to T270, suggesting that T250 tailors changes in gene expression to efficiently respond to combined stress. By contrast, the susceptible tomato activated approximately one thousand and two thousand genes in leaves and roots respectively, indicating a more generalized stress response in this genotype. In particular, developmental and stress-related genes were differentially expressed, such as hormone responsive factors and transcription factors. Analysis of differential alternative splicing (DAS) events showed that combined stress greatly affects the splicing landscape in both genotypes, highlighting the important role of AS in stress response mechanisms. In particular, several stress and growth-related genes as well as transcription and splicing factors were differentially spliced in both tissues. Taken together, these results reveal important insights into the transcriptional and post-transcriptional mechanisms regulating tomato adaptation to growth under reduced water and nitrogen inputs.

RNA splicing is a fundamental mechanism contributing to the definition of the cellular protein population in any given environmental condition. DRT111/SFPS is a splicing factor previously shown to interact with phytochromeB and characterized for its role in splicing of pre-mRNAs involved in photomorphogenesis. Here, we show that DRT111 interacts with Arabidopsis Splicing Factor 1 (SF1), involved in 3’ splicing site recognition. Double and triple mutant analysis shows that DRT111 controls splicing of ABI3 and acts upstream of the splicing factor SUPPRESSOR OF ABI3-5 (SUA). DRT111 is highly expressed in seeds and stomata of Arabidopsis and is induced by long-term treatments with polyethylene glycol and ABA. DRT111 knock-out mutants are defective in ABA-induced stomatal closure and are hypersensitive to ABA during seed germination. Conversely, DRT111 over-expressing plants show ABA hyposensitive seed germination. RNAseq experiments show that in dry seeds, DRT111 controls expression and splicing of genes involved in response to osmotic stress and ABA, light signaling and mRNA splicing, including targets of ABSCISIC ACID INSENSITIVE3 (ABI3) and PHYTOCHROME INTERACTING FACTORs (PIFs). Consistently, expression of the germination inhibitor SOMNUS, induced by ABI3 and PIF1 is up-regulated in imbibed seeds of drt111-2 mutants. Altogether, these results indicate that DRT111 controls sensitivity to abscisic acid (ABA) during seed development, germination and stomatal movements and constitutes a point of integration of the ABA- and light-regulated pathways to control seed germination. One Sentence Summary Arabidopsis splicing factor DRT111/SFPS is required for ABA-mediated responses in seeds

RNA splicing is a fundamental mechanism contributing to the definition of the cellular protein population in any given environmental condition. DNA-DAMAGE REPAIR/TOLERATION PROTEIN111 (DRT111)/SPLICING FACTOR FOR PHYTOCHROME SIGNALING is a splicing factor previously shown to interact with phytochrome B and characterized for its role in splicing of pre-mRNAs involved in photomorphogenesis. Here, we show that DRT111 interacts with Arabidopsis (Arabidopsis thaliana) Splicing Factor1, involved in 3 ' splicing site recognition. Double- and triple-mutant analysis shows that DRT111 controls splicing ofABI3and acts upstream of the splicing factor SUPPRESSOR OF ABI3-ABI5.DRT111is highly expressed in seeds and stomata of Arabidopsis and is induced by long-term treatments of polyethylene glycol and abscisic acid (ABA).DRT111knock-out mutants are defective in ABA-induced stomatal closure and are hypersensitive to ABA during seed germination. Conversely,DRT111overexpressing plants show ABA-hyposensitive seed germination. RNA-sequencing experiments show that in dry seeds, DRT111 controls expression and splicing of genes involved in osmotic-stress and ABA responses, light signaling, and mRNA splicing, including targets of ABSCISIC ACID INSENSITIVE3 (ABI3) and PHYTOCHROME INTERACTING FACTORs (PIFs). Consistently, expression of the germination inhibitorSOMNUS, induced by ABI3 and PIF1, is upregulated in imbibed seeds ofdrt111-2mutants. Together, these results indicate that DRT111 controls sensitivity to ABA during seed development, germination, and stomatal movements, and integrates ABA- and light-regulated pathways to control seed germination. Arabidopsis DNA-DAMAGE REPAIR/TOLERATION PROTEIN111/SPLICING FACTOR FOR PHYTOCHROME SIGNALING regulates pre-mRNA splicing in seeds, affecting ABA-mediated responses.

Abscisic acid (ABA) plays an important role in various aspects of plant growth and development, including adaptation to stresses, fruit development and ripening. In seeds, ABA participates through its core signaling components in dormancy instauration, longevity determination, and inhibition of germination in unfavorable environmental conditions such as high soil salinity. Here, we show that seed germination in pepper was delayed but only marginally reduced by ABA or NaCl with respect to control treatments. Through a similarity search, pepper orthologs of ABA core signaling components PYL (PYRABACTIN RESISTANCE1-LIKE), PP2C (PROTEIN PHOSPHATASE2C), and SnRK2 (SUCROSE NONFERMENTING1 (SNF1)-RELATED PROTEIN KINASE2) genes were identified. Gene expression analyses of selected members showed a low abundance of PYL and SnRK2 transcripts in dry seeds compared to other tissues, and an up-regulation at high concentrations of ABA and/or NaCl for both positive and negative regulators of ABA signaling. As expected, in hydroponically-grown seedlings exposed to NaCl, only PP2C encoding genes were up-regulated. Yeast two hybrid assays performed among putative pepper core components and with Arabidopsis thaliana orthologs confirmed the ability of the identified proteins to function in ABA signaling cascade, with the exception of a CaABI isoform cloned from seeds. BiFC assay in planta confirmed some of the interactions obtained in yeast. Altogether, our results indicate that a low expression of perception and signaling components in pepper seeds might contribute to explain the observed high percentages of seed germination in the presence of ABA. These results might have direct implications on the improvement of seed longevity and vigor, a bottleneck in pepper breeding.

Modulation of growth in response to environmental cues is a fundamental aspect of plant adaptation to abiotic stresses. TIP41 (TAP42 INTERACTING PROTEIN OF 41 kDa) is the Arabidopsis thaliana orthologue of proteins isolated in mammals and yeast that participate in the Target-of-Rapamycin (TOR) pathway, which modifies cell growth in response to nutrient status and environmental conditions. Here, we characterized the function of TIP41 in Arabidopsis. Expression analyses showed that TIP41 is constitutively expressed in vascular tissues, and is induced following long-term exposure to NaCl, polyethylene glycol and abscisic acid (ABA), suggesting a role of TIP41 in adaptation to abiotic stress. Visualization of a fusion protein with yellow fluorescent protein indicated that TIP41 is localized in the cytoplasm and the nucleus. Abolished expression of TIP41 results in smaller plants with a lower number of rosette leaves and lateral roots, and an increased sensitivity to treatments with chemical TOR inhibitors, indicating that TOR signalling is affected in these mutants. In addition, tip41 mutants are hypersensitive to ABA at germination and seedling stage, whereas over-expressing plants show higher tolerance. Several TOR- and ABA-responsive genes are differentially expressed in tip41, including iron homeostasis, senescence and ethylene-associated genes. In yeast and mammals, TIP41 provides a link between the TOR pathway and the protein phosphatase 2A (PP2A), which in plants participates in several ABA-mediated mechanisms. Here, we showed an interaction of TIP41 with the catalytic subunit of PP2A. Taken together, these results offer important insights into the function of Arabidopsis TIP41 in the modulation of plant growth and ABA responses.

Water-limiting conditions affect dramatically plant growth and development and, ultimately, yield of potato plants (Solanum tuberosum L.). Therefore, understanding the mechanisms underlying the response to water deficit is of paramount interest to obtain drought tolerant potato varieties. Herein, potato 10K cDNA array slides were used to profile transcriptomic changes of two potato cell populations under abrupt (shocked cells) or gradual exposure (adapted cells) to polyethylene glycol (PEG)-mediated water stress. Data analysis identified >1000 differentially expressed genes (DEGs) in our experimental conditions. Noteworthy, our microarray study also suggests that distinct gene networks underlie the cellular response to shock or gradual water stress. On the basis of our experimental findings, it is possible to speculate that DEGs identified in shocked cells participate in early protective and sensing mechanisms to environmental insults, while the genes whose expression was modulated in adapted cells are directly involved in the acquisition of a new cellular homeostasis to cope with water stress conditions. To validate microarray data obtained for potato cells, the expression analysis of 21 selected genes of interest was performed by Real-Time Quantitative Reverse Transcription PCR (qRT-PCR). Intriguingly, the expression levels of these transcripts in 4-week old potato plants exposed to long-term water-deficit. qRT-PCR analysis showed that several genes were regulated similarly in potato cells cultures and tissues exposed to drought, thus confirming the efficacy of our simple experimental system to capture important genes involved in osmotic stress response. Highlighting the differences in gene expression between shock-like and adaptive response, our findings could contribute to the discussion on the biological function of distinct gene networks involved in the response to abrupt and gradual adaptation to water deficit.

Improving crop performance under water-limiting conditions is essential for achieving environmentally sustainable food production. This requires significant progress in both the identification and characterization of key genetic and physiological processes involved in water uptake and loss. Plants regulate water uptake and loss through both developmental and environmental responses. These responses include: root morphology and architecture, cuticle development, stomatal development, and guard cell movements in response to the environment. Genes controlling root traits and stomatal development and guard cell movements strongly impact water use efficiency (WUE), and represent the best targets for molecular breeding programs. This article provides an overview of the complex networks of genes involved in water uptake and loss. These traits represent novel opportunities and strategies for genetic improvement of WUE and drought tolerance in crops.

BACKGROUND:Drought is a major constraint for plant growth and crop productivity that is receiving an increased attention due to global climate changes. Chloroplasts act as environmental sensors, however, only partial information is available on stress-induced mechanisms within plastids. Here, we investigated the chloroplast response to a severe drought treatment and a subsequent recovery cycle in tomato through physiological, metabolite and proteomic analyses.RESULTS:Under stress conditions, tomato plants showed stunted growth, and elevated levels of proline, abscisic acid (ABA) and late embryogenesis abundant gene transcript. Proteomics revealed that water deficit deeply affects chloroplast protein repertoire (31 differentially represented components), mainly involving energy-related functional species. Following the rewatering cycle, physiological parameters and metabolite levels indicated a recovery of tomato plant functions, while proteomics revealed a still ongoing adjustment of the chloroplast protein repertoire, which was even wider than during the drought phase (54 components differentially represented). Changes in gene expression of candidate genes and accumulation of ABA suggested the activation under stress of a specific chloroplast-to-nucleus (retrograde) signaling pathway and interconnection with the ABA-dependent network.CONCLUSIONS:Our results give an original overview on the role of chloroplast as enviromental sensor by both coordinating the expression of nuclear-encoded plastid-localised proteins and mediating plant stress response. Although our data suggest the activation of a specific retrograde signaling pathway and interconnection with ABA signaling network in tomato, the involvement and fine regulation of such pathway need to be further investigated through the development and characterization of ad hoc designed plant mutants.

Tomato is a major crop in the Mediterranean basin, where the cultivation in the open field is often vulnerable to drought. In order to adapt and survive to naturally occurring cycles of drought stress and recovery, plants employ a coordinated array of physiological, biochemical, and molecular responses. Transcriptomic studies on tomato responses to drought and subsequent recovery are few in number. As the search for novel traits to improve the genetic tolerance to drought increases, a better understanding of these responses is required. To address this need we designed a study in which we induced two cycles of prolonged drought stress and a single recovery by rewatering in tomato. In order to dissect the complexity of plant responses to drought, we analyzed the physiological responses (stomatal conductance, CO2 assimilation, and chlorophyll fluorescence), abscisic acid (ABA), and proline contents. In addition to the physiological and metabolite assays, we generated transcriptomes for multiple points during the stress and recovery cycles. Cluster analysis of differentially expressed genes (DEGs) between the conditions has revealed potential novel components in stress response. The observed reduction in leaf gas exchanges and efficiency of the photosystem PSII was concomitant with a general down-regulation of genes belonging to the photosynthesis, light harvesting, and photosystem I and II category induced by drought stress. Gene ontology (GO) categories such as cell proliferation and cell cycle were also significantly enriched in the down-regulated fraction of genes upon drought stress, which may contribute to explain the observed growth reduction. Several histone variants were also repressed during drought stress, indicating that chromatin associated processes are also affected by drought. As expected, ABA accumulated after prolonged water deficit, driving the observed enrichment of stress related GOs in the up-regulated gene fractions, which included transcripts putatively involved in stomatal movements. This transcriptomic study has yielded promising candidate genes that merit further functional studies to confirm their involvement in drought tolerance and recovery. Together, our results contribute to a better understanding of the coordinated responses taking place under drought stress and recovery in adult plants of tomato.

Salt and drought stress severely reduce plant growth and crop productivity worldwide. The identification of genes underlying stress response and tolerance is the subject of intense research in plant biology. Through microarray analyses, we previously identified in potato (Solanum tuberosum) StRGGA, coding for an Arginine Glycine Glycine (RGG) box-containing RNA-binding protein, whose expression was specifically induced in potato cell cultures gradually exposed to osmotic stress. Here, we show that the Arabidopsis (Arabidopsis thaliana) ortholog, AtRGGA, is a functional RNA-binding protein required for a proper response to osmotic stress. AtRGGA gene expression was up-regulated in seedlings after long-term exposure to abscisic acid (ABA) and polyethylene glycol, while treatments with NaCl resulted in AtRGGA down-regulation. AtRGGA promoter analysis showed activity in several tissues, including stomata, the organs controlling transpiration. Fusion of AtRGGA with yellow fluorescent protein indicated that AtRGGA is localized in the cytoplasm and the cytoplasmic perinuclear region. In addition, the rgga knockout mutant was hypersensitive to ABA in root growth and survival tests and to salt stress during germination and at the vegetative stage. AtRGGA-overexpressing plants showed higher tolerance to ABA and salt stress on plates and in soil, accumulating lower levels of proline when exposed to drought stress. Finally, a global analysis of gene expression revealed extensive alterations in the transcriptome under salt stress, including several genes such as ASCORBATE PEROXIDASE2, GLUTATHIONE S-TRANSFERASE TAU9, and several SMALL AUXIN UPREGULATED RNA-like genes showing opposite expression behavior in transgenic and knockout plants. Taken together, our results reveal an important role of AtRGGA in the mechanisms of plant response and adaptation to stress.

For plant cells in the early phases of water stress exposure, the genes induced under such conditions play a key role in detecting and responding to water deficit. In this study, potato cell suspensions were used as a simplified model system to dissect early molecular changes upon low water potential. In particular, the cDNA-amplified fragment length polymorphism approach was used to capture genes rapidly activated in potato cell cultures in response to water deficit induced by short-term exposure (up to 1 h) to polyethylene glycol. Selective amplifications with 38 primer combinations allowed the visualization of about 167 transcript-derived fragments (TDFs) differentially expressed upon exposure to low water potential. The gene expression pattern of 18 up-regulated genes was further investigated by semi-quantitative reverse transcriptase polymerase chain reaction analysis. Sequencing and similarity analysis revealed that TDFs present homologies chiefly with proteins involved in chaperone activity and protein degradation (hsps, proteinase precursor), in protein synthesis (elongation factor, ribosomal proteins) and in the ROS scavenging pathway (phenylalanine ammonia-lyase, peroxidase). Our findings might contribute to describe the potential role of genes activated in the early phases of plant response to drought.

When experiencing water deicit, glycophyte plants undergo physiological and biochemical changes aimed at limiting cellular damages and rescuing a new cellular homeostasis. Discriminate irreversible cell injury from adaptive rearrangements to water stress, is quite critical since only the latter plant response is compatible with active growth and development sustaining, ultimately, plant yield. An up-dated view of the molecular basis of cellular response to drought stress and of key functions activated able to protect cellular processes is summarized in the paper. It also reported a functional approach developed to identify genes with a crucial role in the adaptation to water deicit based on the systematic comparison of potato cell populations exposed abruptly or gradually to PEG-induced low water potential. Gradually adapted cells were characterized by distinctive metabolic adaptations (proline accumulation, changes in membrane lipid composition, de novo synthesis, etc) which enable them to grow actively at non-permissive water stress conditions. Differential gene expression in response to shock or gradually increasing water deicit was monitored by microarray technology, using the 1K TIGR potato cDNA array. More than 100 genes belonging to different functional categories were up-regulated in response to stress conditions. However, only a few induced genes were common to both cell populations, conirming that different gene networks mediate shock or long-term response to water deicit.

RNA-binding proteins (RBPs) govern many aspects of RNA metabolism, including pre-mRNA processing, transport, stability/decay and translation. Although relatively few plant RNA-binding proteins have been characterized genetically and biochemically, more than 200 RBP genes have been predicted in Arabidopsis and rice genomes, suggesting that they might serve specific plant functions. Besides their role in normal cellular functions, RBPs are emerging also as an interesting class of proteins involved in a wide range of post-transcriptional regulatory events that are important in providing plants with the ability to respond rapidly to changes in environmental conditions. Here, we review the most recent results and evidence on the functional role of RBPs in plant adaptation to various unfavourable environmental conditions and their contribution to enhance plant tolerance to abiotic stresses, with special emphasis on osmotic and temperature stress.

Potato cDNA microarray slides (TIGR 1 K) were used to compare changes in gene expression of two potato cell populations subjected to shock or gradual exposure to PEG-mediated water stress. A total of 180 transcripts were found to be statistically up- or down-regulated in PEG-shocked or gradually stressed cells, compared to untreated control cells. Up-regulated genes in shocked cells were prevalently involved in carbohydrate metabolism, cellular communication, and signal transduction. In cells stressed gradually, the most represented induced genes were those involved in signal transduction, in response to environmental signals and in the regulation of transcription and translation machineries, including the heat shock protein 90-2, ribosomal and RNA-binding proteins. The expression of selected up-regulated genes was also tested in potato plants subjected to slow soil-drying conditions. Interestingly, the rgga gene, encoding an RNA-binding protein, was strongly induced in both leaves and roots of water-stressed potato plants, supporting its putative function in adaptive mechanisms to water stress. Pointing to differences in gene expression between shock-like and adaptive response, our findings might contribute to the controversial debate on the biological function of stress-responsive genes in cell damage repair or in restoring homeostasis and elaborating longer term responses for adaptation.