Genome-wide identification and multiple abiotic stress transcript profiling of potassium transport gene homologs in Sorghum bicolor.

Potassium (K+) is the most abundant cation that plays a crucial role in various cellular processes in plants. Plants have developed an efficient mechanism for the acquisition of K+ when grown in K+ deficient or saline soils. A total of 47 K+ transport gene homologs (27 HAKs, 4 HKTs, 2 KEAs, 9 AKTs, 2 KATs, 2 TPCs, and 1 VDPC) have been identified in Sorghum bicolor. Of 47 homologs, 33 were identified as K+ transporters and the remaining 14 as K+ channels. Chromosome 2 has been found as the hotspot of K+ transporters with 9 genes. Phylogenetic analysis revealed the conservation of sorghum K+ transport genes akin to Oryza sativa. Analysis of regulatory elements indicates the key roles that K+ transport genes play under different biotic and abiotic stress conditions. Digital expression data of different developmental stages disclosed that expressions were higher in milk, flowering, and tillering stages. Expression levels of the genes SbHAK27 and SbKEA2 were higher during milk, SbHAK17, SbHAK11, SbHAK18, and SbHAK7 during flowering, SbHAK18, SbHAK10, and 23 other gene expressions were elevated during tillering inferring the important role that K+ transport genes play during plant growth and development. Differential transcript expression was observed in different tissues like root, stem, and leaf under abiotic stresses such as salt, drought, heat, and cold stresses. Collectively, the in-depth genome-wide analysis and differential transcript profiling of K+ transport genes elucidate their role in ion homeostasis and stress tolerance mechanisms.

Advances in Plant Metabolomics and Its Applications in Stress and Single-Cell Biology.

In the past two decades, the post-genomic era envisaged high-throughput technologies, resulting in more species with available genome sequences. In-depth multi-omics approaches have evolved to integrate cellular processes at various levels into a systems biology knowledge base. Metabolomics plays a crucial role in molecular networking to bridge the gaps between genotypes and phenotypes. However, the greater complexity of metabolites with diverse chemical and physical properties has limited the advances in plant metabolomics. For several years, applications of liquid/gas chromatography (LC/GC)-mass spectrometry (MS) and nuclear magnetic resonance (NMR) have been constantly developed. Recently, ion mobility spectrometry (IMS)-MS has shown utility in resolving isomeric and isobaric metabolites. Both MS and NMR combined metabolomics significantly increased the identification and quantification of metabolites in an untargeted and targeted manner. Thus, hyphenated metabolomics tools will narrow the gap between the number of metabolite features and the identified metabolites. Metabolites change in response to environmental conditions, including biotic and abiotic stress factors. The spatial distribution of metabolites across different organs, tissues, cells and cellular compartments is a trending research area in metabolomics. Herein, we review recent technological advancements in metabolomics and their applications in understanding plant stress biology and different levels of spatial organization. In addition, we discuss the opportunities and challenges in multiple stress interactions, multi-omics, and single-cell metabolomics.

Altered chromatin conformation and transcriptional regulation in watermelon following genome doubling.

Polyploidy has played a crucial role in plant evolution, development and function. Synthetic autopolyploid represents an ideal system to investigate the effects of polyploidization on transcriptional regulation. In this study, we deciphered the impact of genome duplication at phenotypic and molecular levels in watermelon. Overall, 88% of the genes in tetraploid watermelon followed a >1:1 dosage effect, and accordingly, differentially expressed genes were largely upregulated. In addition, a great number of hypomethylated regions (1688) were identified in an isogenic tetraploid watermelon. These differentially methylated regions were localized in promoters and intergenic regions and near transcriptional start sites of the identified upregulated genes, which enhances the importance of methylation in gene regulation. These changes were reflected in sophisticated higher-order chromatin structures. The genome doubling caused switching of 108 A and 626 B compartments that harbored genes associated with growth, development and stress responses.

Improvement of Postharvest Quality of Plum (Prunus domestica L.) Using Polysaccharide-Based Edible Coatings.

Polysaccharide-based edible coatings are served as an attractive preservation method for postharvest maintenance of most fruits. The current study examined the effect of carboxymethylcellulose (CMC)- and pectin (Pec)- based edible coatings on titratable acidity (TA), firmness; vitamin C (vit C); total soluble solids (TSS); pH; total phenolics; anthocyanin and flavonoid contents; total antioxidant capacity (based on 1,1-Diphenyl-2-picryl-hydrazyl hydrate (DPPH)); the activities of peroxidase (POD), polyphenol oxidase (PPO) and polygalacturonase (PG) enzymes; and weight loss during cold storage. The results showed that each coating and their combinations caused positive effects in all measured parameters except weight loss. The applied coatings preserved firmness and improved total phenols, anthocyanin and flavonoid contents, antioxidant capacity and POD activity. In addition, TSS decreased and pH values remained more or less stable with the coating application. The coatings delayed TA and vitamin C loss, and decreased enzymatic activities such as PPO and PG. It could be stated that CMC at 1% and Pec at 1.5% separately demonstrated the best results for most of the measured parameters; and 0.5% Pec + 1.5% CMC could be considered as the best combination. In conclusion, application of CMC, Pec, or their combinations would be considered as an interesting approach to improve postharvest quality characteristics of plum fruit.

Proteomics, physiological, and biochemical analysis of cross tolerance mechanisms in response to heat and water stresses in soybean.

Water stress (WS) and heat stress (HS) have a negative effect on soybean plant growth and crop productivity. Changes in the physiological characteristics, proteome, and specific metabolites investigated on molecular and cellular functions were studied in two soybean cultivars exposed to different heat and water stress conditions independently and in combination. Leaf protein composition was studied using 2-DE and complemented with MALDI TOF mass spectrometry. While the two cultivars displayed genetic variation in response to water and heat stress, thirty-nine proteins were significantly altered in their relative abundance in response to WS, HS and combined WS+HS in both cultivars. A majority of these proteins were involved in metabolism, response to heat and photosynthesis showing significant cross-tolerance mechanisms. This study revealed that MED37C, a probable mediator of RNA polymerase transcription II protein, has potential interacting partners in Arabidopsis and signified the marked impact of this on the PI-471938 cultivar. Elevated activities in antioxidant enzymes indicate that the PI-471938 cultivar can restore the oxidation levels and sustain the plant during the stress. The discovery of this plant's development of cross-stress tolerance could be used as a guide to foster ongoing genetic modifications in stress tolerance.

Sodium Chloride Induced Stress Responses of Antioxidative Activities in Leaves and Roots of Pistachio Rootstock.

Salinity substantially affects plant growth and crop productivity worldwide. Plants adopt several biochemical mechanisms including regulation of antioxidant biosynthesis to protect themselves against the toxic effects induced by the stress. One-year-old pistachio rootstock exhibiting different degrees of salinity tolerance were subjected to sodium chloride induced stress to identify genetic diversity among cultivated pistachio rootstock for their antioxidant responses, and to determine the correlation of these enzymes to salinity stress. Leaves and roots were harvested following NaCl-induced stress. The results showed that a higher concentration of NaCl treatment induced oxidative stress in the leaf tissue and to a lesser extent in the roots. Both tissues showed an increase in ascorbate peroxidase, superoxide dismutase, catalase, glutathione reductase, peroxidase, and malondialdehyde. Responses of antioxidant enzymes were cultivar dependent, as well as temporal and dependent on the salinity level. Linear and quadratic regression model analysis revealed significant correlation of enzyme activities to salinity treatment in both tissues. The variation in salinity tolerance reflected their capabilities in orchestrating antioxidant enzymes at the roots and harmonized across the cell membranes of the leaves. This study provides a better understanding of root and leaf coordination in regulating the antioxidant enzymes to NaCl induced oxidative stress.

Transcriptome changes in reciprocal grafts involving watermelon and bottle gourd reveal molecular mechanisms involved in increase of the fruit size, rind toughness and soluble solids.

KEY MESSAGE: Transcriptome landscape reveals the molecular mechanisms involved in the improvement of fruit traits by the grafting of watermelon and bottle gourd. Grafting has been used as a sustainable alternative for watermelon breeding to control soil-borne pathogens and to increase tolerance to various abiotic stresses. However, some reports have shown that grafting can negatively affect the quality of fruits. Despite several field studies on the effects of grafting on fruit quality, the regulation of this process at the molecular level has not been revealed. The aim of this study was to elucidate various molecular mechanisms involved in different tissues of heterografted watermelon and bottle gourd plants. Grafting with bottle gourd rootstock increased the size and rind thickness of watermelon fruits, whereas that with watermelon rootstock produced bottle gourd fruits with higher total soluble solid content and thinner rinds. Correspondingly, genes related to ripening, softening, cell wall strengthening, stress response and disease resistance were differentially expressed in watermelon fruits. Moreover, genes associated mainly with sugar metabolism were differentially expressed in bottle gourd fruits. RNA-seq revealed more than 400 mobile transcripts across the heterografted sets. More than half of these were validated from PlaMoM, a database for plant mobile macromolecules. In addition, some of these mobile transcripts contained a transfer RNA-like structure. Other RNA motifs were also enriched in these transcripts, most with a biological role based on GO analysis. This transcriptome study provided a comprehensive understanding of various molecular mechanisms underlying grafted tissues in watermelon.

Enhanced tolerance of industrial hemp (Cannabis sativa L.) plants on abandoned mine land soil leads to overexpression of cannabinoids.

Industrial activities have a detrimental impact on the environment and health when high concentrations of pollutants are released. Phytoremediation is a natural method of utilizing plants to remove contaminants from the soil. The goal of this study was to investigate the ability of Cannabis sativa L. to sustainably grow and remediate abandoned coal mine land soils in Pennsylvania. In this study, six different varieties of industrial hemp (Fedora 17, Felina 32, Ferimon, Futura 75, Santhica 27, and USO 31) were grown on two different contaminated soil types and two commercial soils (Miracle-Gro Potting Mix and PRO-MIX HP Mycorrhizae High Porosity Grower Mix). Plants growing in all soil types were exposed to two environmental conditions (outside and in the greenhouse). Seed germination response and plant height indicated no significant differences among all hemp varieties grown in different soils, however on an average, the height of the plants grown in the greenhouse exceeded that of the plants grown outdoors. In addition, heavy metal analysis of Arsenic, Lead, Nickel, Mercury, and Cadmium was performed. The concentration of Nickel was 2.54 times greater in the leaves of hemp grown in mine land soil outdoors when compared to greenhouse conditions. No differences were found between expression of heavy metal transporter genes. Secondary metabolite analysis of floral buds from hemp grown in mine land soil displayed a significant increase in the total Cannabidiol content (2.16%, 2.58%) when compared to Miracle-Gro control soil (1.08%, 1.6%) for outdoors and in the greenhouse, respectively. Molecular analysis using qRT-PCR indicated an 18-fold increase in the expression of the cannabidiolic acid synthase gene in plants grown on mine land soil. The data indicates a high tolerance to heavy metals as indicated from the physiological and metabolites analysis.

Comparative leaf proteomics of drought-tolerant and -susceptible peanut in response to water stress.

UNLABELLED: Water stress (WS) predisposes peanut plants to fungal infection resulting in pre-harvest aflatoxin contamination. Major changes during water stress including oxidative stress, lead to destruction of photosynthetic apparatus and other macromolecules within cells. Two peanut cultivars with diverse drought tolerance characteristics were subjected to WS, and their leaf proteome was compared using two-dimensional electrophoresis complemented with MALDI-TOF/TOF mass spectrometry. Ninety-six protein spots were differentially abundant to water stress in both cultivars that corresponded to 60 non-redundant proteins. Protein interaction prediction analysis suggests that 42 unique proteins showed interactions in tolerant cultivar while 20 showed interactions in the susceptible cultivar, activating other proteins in directed system response networks. Four proteins: glutamine ammonia ligase, chitin class II, actin isoform B, and beta tubulin, involved in metabolism, defense and cellular biogenesis, are unique in tolerant cultivar and showed positive interactions with other proteins. In addition, four proteins: serine/threonine protein phosphate PP1, choline monooxygenase, peroxidase 43, and SNF1-related protein kinase regulatory subunit beta-2, that play a role as cryoprotectants through signal transduction, were induced in drought tolerant cultivar following WS. Eleven interologs of these proteins were found in Arabidopsis interacting with several proteins and it is believed that similar mechanisms/pathways exist in peanut. SIGNIFICANCE: Peanuts (Arachis hypogaea L.) are a major source of plant protein grown in subtropical and tropical regions of the world. Pre-harvest aflatoxin contamination is a major problem that affects peanut crop yield and food safety. Poor understanding of molecular and cellular mechanisms associated with aflatoxin resistance is largely responsible for the lack of progress in elucidating a process/methodology for reducing aflatoxin contamination in peanuts. Drought perturbs the invasion of the aflatoxin producing fungus and thus affects the quality and yield of peanut. Therefore, more studies involving the effects of drought stress to determine the molecular changes will enhance our understanding of the key metabolic pathways involved in the combined stresses. The changes associated with the biotic and abiotic interactions within the peanut will be used to determine the metabolic pathways involved in the stress tolerance. This research would be beneficial in identifying the tolerant molecular signatures and promoting food safety and consumer health through breeding superior quality peanut cultivars.

Analysis of genetic diversity and population structure of peanut cultivars and breeding lines from China, India and the US using simple sequence repeat markers.

Cultivated peanut is grown worldwide as rich-source of oil and protein. A broad genetic base is needed for cultivar improvement. The objectives of this study were to develop highly informative simple sequence repeat (SSR) markers and to assess the genetic diversity and population structure of peanut cultivars and breeding lines from different breeding programs in China, India and the US. A total of 111 SSR markers were selected for this study, resulting in a total of 472 alleles. The mean values of gene diversity and polymorphic information content (PIC) were 0.480 and 0.429, respectively. Country-wise analysis revealed that alleles per locus in three countries were similar. The mean gene diversity in the US, China and India was 0.363, 0.489 and 0.47 with an average PIC of 0.323, 0.43 and 0.412, respectively. Genetic analysis using the STRUCTURE divided these peanut lines into two populations (P1, P2), which was consistent with the dendrogram based on genetic distance (G1, G2) and the clustering of principal component analysis. The groupings were related to peanut market types and the geographic origin with a few admixtures. The results could be used by breeding programs to assess the genetic diversity of breeding materials to broaden the genetic base and for molecular genetics studies.

Overexpression of a Plasma Membrane Bound Na+/H+ Antiporter-Like Protein (SbNHXLP) Confers Salt Tolerance and Improves Fruit Yield in Tomato by Maintaining Ion Homeostasis.

A Na+/H+ antiporter-like protein (NHXLP) was isolated from Sorghum bicolor L. (SbNHXLP) and validated by overexpressing in tomato for salt tolerance. Homozygous T2 transgenic lines when evaluated for salt tolerance, accumulated low Na+ and displayed enhanced salt tolerance compared to wild-type plants (WT). This is consistent with the amiloride binding assay of the protein. Transgenics exhibited higher accumulation of proline, K+, Ca2+, improved cambial conductivity, higher PSII, and antioxidative enzyme activities than WT. Fluorescence imaging results revealed lower Na+ and higher Ca2+ levels in transgenic roots. Co-immunoprecipitation experiments demonstrate that SbNHXLP interacts with a Solanum lycopersicum cation proton antiporter protein2 (SlCHX2). qRT-PCR results showed upregulation of SbNHXLP and SlCHX2 upon treatment with 200 mM NaCl and 100 mM potassium nitrate. SlCHX2 is known to be involved in K+ acquisition, and the interaction between these two proteins might help to accumulate more K+ ions, and thus maintain ion homeostasis. These results strongly suggest that plasma membrane bound SbNHXLP involves in Na+ exclusion, maintains ion homeostasis in transgenics in comparison with WT and alleviates NaCl stress.

Proteome Biomarkers in Xylem Reveal Pierce's Disease Tolerance in Grape.

Pierce's disease (PD) is a significant threat to grape cultivation and industry. The disease caused by bacterium Xylella fastidiosa clogs xylem vessels resulting in wilting of the plant. PD-tolerant grape genotypes are believed to produce certain novel components in xylem tissue that help them to combat invading pathogens. Research has been aimed at characterizing the uniquely expressed xylem proteins by PD-tolerant genotypes. The objectives were to i) compare and characterize Vitis xylem proteins differentially expressed in PD-tolerant and PD-susceptible cultivars and, ii) identify xylem proteins uniquely expressed in PD-tolerant genotypes. A high throughput two-dimensional gel electrophoresis of xylem proteins from three Vitis species identified more than 200 proteins with pls 3.0 to 9.0 and molecular weights of 20 to 75 kDa. The differentially expressed proteins were then excised and analyzed with MALDI/TOF mass spectrometer. The mass spectra were collected and protein identification was performed against the Viridiplantae database using Matrix Science algorithm. Proteins were mapped to the universal protein resource to study gene ontology. Comparative analysis of the xylem proteome of three species indicated the highest number of proteins in muscadine grape, followed by Florida hybrid bunch and bunch grape. These proteins were all associated with disease resistance, energy metabolism, protein processing and degradation, biosynthesis, stress related functions, cell wall biogenesis, signal transduction, and ROS detoxification. Furthermore, ß-1, 3-glucanase, 10-deacetyl baccatin III-10-O-acetyl transferase-like, COP9, and aspartyl protease nepenthesin precursor proteins were found to be uniquely expressed in PD-tolerant muscadine grape, while they are absent in PD-susceptible bunch grape. Data suggests that muscadine and Florida hybrid bunch grapes express novel proteins in xylem to overcome pathogen attack while bunch grape lacks this capability, making them susceptible to PD.

iTRAQ-based quantitative proteomics of developing and ripening muscadine grape berry.

Grapes are among the widely cultivated fruit crops in the world. Grape berries like other nonclimacteric fruits undergo a complex set of dynamic, physical, physiological, and biochemical changes during ripening. Muscadine grapes are widely cultivated in the southern United States for fresh fruit and wine. To date, changes in the metabolites composition of muscadine grapes have been well documented; however, the molecular changes during berry development and ripening are not fully known. The aim of this study was to investigate changes in the berry proteome during ripening in muscadine grape cv. Noble. Isobaric tags for relative and absolute quantification (iTRAQ) MS/MS was used to detect statistically significant changes in the berry proteome. A total of 674 proteins were detected, and 76 were differentially expressed across four time points in muscadine berry. Proteins obtained were further analyzed to provide information about its potential functions during ripening. Several proteins involved in abiotic and biotic stimuli and sucrose and hexose metabolism were upregulated during berry ripening. Quantitative real-time PCR analysis validated the protein expression results for nine proteins. Identification of vicilin-like antimicrobial peptides indicates additional disease tolerance proteins are present in muscadines for berry protection during ripening. The results provide new information for characterization and understanding muscadine berry proteome and grape ripening.

Analysis of peanut leaf proteome.

Peanut (Arachis hypogaea) is one of the most important sources of plant protein. Current selection of genotypes requires molecular characterization of available populations. Peanut genome database has several EST cDNAs which can be used to analyze gene expression. Analysis of proteins is a direct approach to define function of their associated genes. Proteome analysis linked to genome sequence information is critical for functional genomics. However, the available protein expression data is extremely inadequate. Proteome analysis of peanut leaf was conducted using two-dimensional gel electrophoresis in combination with sequence identification using MALDI/TOF to determine their identity and function related to growth, development and responses to stresses. Peanut leaf proteins were resolved into 300 polypeptides with pI values between 3.5 and 8.0 and relative molecular masses from 12 to 100 kDa. A master leaf polypeptide profile was generated based on the consistently expressed protein pattern. Proteins present in 205 spots were identified using GPS software and Viridiplantae database (NCBI). Identity of some of these proteins included RuBisCO, glutamine synthetase, glyoxisomal malate dehydrogenase, oxygen evolving enhancer protein and tubulin. Bioinformatical analyses showed that there are 133 unique protein identities. They were categorized into 10 and 8 groups according to their cellular compartmentalization and biological functionality, respectively. Enzymes necessary for carbohydrate metabolism and photosynthesis dominated in the set of identified proteins. The reference map derived from a drought-tolerant cv.Vemana should serve as the basis for further investigations of peanut physiology such as detection of expressed changes due to biotic and abiotic stresses, plant development. Furthermore, the leaf proteome map will lead to development of protein markers for cultivar identification at seedling stage of the plant. Overall, this study will contribute to improve our understanding of plant genetics and metabolism, and overall assist in the selection and breeding programs geared toward crop improvement.

Characterization of unique and differentially expressed proteins in anthracnose-tolerant Florida hybrid bunch grapes.

Anthracnose is a major disease in Florida hybrid bunch grapes, caused by a fungus viz. Elsinoe ampelina. Florida hybrid bunch grapes are grown in southeastern USA for their superior wine characteristics. However, the effect of anthracnose on grape productivity and wine quality is a major concern to grape growers. Our research is aimed at determining biochemical basis of anthracnose tolerance in Florida hybrid bunch grape. Leaf samples were collected from the plants infected with E. ampelina at different periods and analyzed for differential protein expression using high throughput two-dimensional gel electrophoresis. Among the 32 differentially expressed leaf proteins, two were uniquely expressed in tolerant genotypes in response to E. ampelina infection. These proteins were identified as mitochondrial adenosine triphosphate synthase and glutamine synthetase, which are known to play a major role in carbohydrate metabolism and defense. Several proteins including ribulose 1-5 bisphosphate-carboxylase involved in photosynthesis were found to be suppressed in susceptible genotypes compared to tolerant genotypes following E. ampelina infection. The results indicate that the anthracnose-tolerant genotypes have the ability to up-regulate and induce new proteins upon infection to defend the invasion of the pathogen as well as maintain the normal regulatory processes.

Efficient protocol for isolation of functional RNA from different grape tissue rich in polyphenols and polysaccharides for gene expression studies

Extraction of RNA from plant tissue containing high levels of polyphenols and polysaccharides is tedious and difficult in grapes. Although several protocols have been published for plant RNA isolation, most have failed to yield high quality RNA in sufficient quantity from mature and diseased grape tissue. We describe a protocol for isolating intact and high quality RNA from various grape tissues as evident by high A260/A280 absorbance ratio (1.8 to 1.9) and electrophoretic profile on denaturing formaldehyde agarose gel. On an average, 205 µg RNA per g of fresh tissue were obtained using this modified protocol. RNA quality was further assessed through RT-PCR, differential display RT-PCR and subtractive hybridization, and found to be suitable for molecular studies.