Flavonols affect the interrelated glucosinolate and camalexin biosynthetic pathways in Arabidopsis thaliana.

Flavonols are structurally and functionally diverse biomolecules involved in plant biotic and abiotic stress tolerance, pollen development, and inhibition of auxin transport. However, their effects on global gene expression and signaling pathways are unclear. To explore the roles of flavonol metabolites in signaling, we performed comparative transcriptome and targeted metabolite profiling of seedlings from the flavonol-deficient Arabidopsis loss-of-function mutant flavonol synthase1 (fls1) with and without exogenous supplementation of flavonol derivatives (kaempferol, quercetin, and rutin). RNA-seq results indicated that flavonols modulate various biological and metabolic pathways, with significant alterations in camalexin and aliphatic glucosinolate synthesis. Flavonols negatively regulated camalexin biosynthesis but appeared to promote the accumulation of aliphatic glucosinolates via transcription factor-mediated up-regulation of biosynthesis genes. Interestingly, upstream amino acid biosynthesis genes involved in methionine and tryptophan synthesis were altered under flavonol deficiency and exogenous supplementation. Quercetin treatment significantly up-regulated aliphatic glucosinolate biosynthesis genes compared with kaempferol and rutin. In addition, expression and metabolite analysis of the transparent testa7 mutant, which lacks hydroxylated flavonol derivatives, clarified the role of quercetin in the glucosinolate biosynthesis pathway. This study elucidates the molecular mechanisms by which flavonols interfere with signaling pathways, their molecular targets, and the multiple biological activities of flavonols in plants.

TaGPX1-D overexpression provides salinity and osmotic stress tolerance in Arabidopsis.

Glutathione peroxidases (GPXs) are known to play an essential role in guarding cells against oxidative stress by catalyzing the reduction of hydrogen peroxide and organic hydroperoxides. The current study aims functional characterization of the TaGPX1-D gene of bread wheat (Triticum aestivum) for salinity and osmotic stress tolerance. To achieve this, we initially performed the spot assays of TaGPX1-D expressing yeast cells. The growth of recombinant TaGPX1-D expressing yeast cells was notably higher than the control cells under stress conditions. Later, we generated transgenic Arabidopsis plants expressing the TaGPX1-D gene and investigated their tolerance to various stress conditions. The transgenic plants exhibited improved tolerance to both salinity and osmotic stresses compared to the wild-type plants. The higher germination rates, increased antioxidant enzymes activities, improved chlorophyll, carotenoid, proline and relative water contents, and reduced hydrogen peroxide and MDA levels in the transgenic lines supported the stress tolerance mechanism. Overall, this study demonstrated the role of TaGPX1-D in abiotic stress tolerance, and it can be used for improving the tolerance of crops to environmental stressors, such as salinity and osmotic stress in future research.

Expression of TaNCL2-A ameliorates cadmium toxicity by increasing calcium and enzymatic antioxidants activities in arabidopsis.

Cadmium (Cd) is a heavy metal that occurs naturally in the environment and is toxic to both animals and plants. The impact of Cd toxicity is shown to be reduced by the exogenous application of calcium (Ca) in crop plants. The sodium/calcium exchanger-like (NCL) protein is involved in Ca enrichment in the cytoplasm by transporting it from the vacuole in the exchange of cytosolic sodium (Na). However, it has not been utilized to ameliorate the Cd toxicity, to date. An elevated expression of TaNCL2-A gene in the root and shoot tissues of bread wheat seedlings, and a higher growth rate of recombinant yeast cells, suggested its role in Cd stress response. The TaNCL2-A expressing transgenic Arabidopsis lines exhibited significant Cd tolerance with increased Ca (∼10-fold) accumulation. The proline content and antioxidant enzymes activities were increased while oxidative stress-related molecules such as H2O2 and MDA were reduced in the transgenic lines. In addition, the growth and yield parameters of transgenic lines such as seed germination rate, root length, leaf biomass, leaf area index, rosette diameter, leaf length and width, and silique count, along with various physiological indicators like chlorophyll, carotenoid, and relative water contents were also improved in comparison to the control plants. Further, the transgenic lines exhibited significant salinity and osmotic stress tolerance, as well. Taken together, these results suggested that the TaNCL2-A could mitigate Cd toxicity along with salinity and osmotic stress. This gene may also be utilized for phytoremediation and Cd sequestration in future studies.

The R2R3-MYB gene family in Cicer arietinum: genome-wide identification and expression analysis leads to functional characterization of proanthocyanidin biosynthesis regulators in the seed coat.

MAIN CONCLUSION: We identified 119 typical CaMYB encoding genes and reveal the major components of the proanthocyanidin regulatory network. CaPARs emerged as promising targets for genetic engineering toward improved agronomic traits in C. arietinum. Chickpea (Cicer arietinum) is among the eight oldest crops and has two main types, i.e., desi and kabuli, whose most obvious difference is the color of their seeds. We show that this color difference is due to differences in proanthocyanidin content of seed coats. Using a targeted approach, we performed in silico analysis, metabolite profiling, molecular, genetic, and biochemical studies to decipher the transcriptional regulatory network involved in proanthocyanidin biosynthesis in the seed coat of C. arietinum. Based on the annotated C. arietinum reference genome sequence, we identified 119 typical CaMYB encoding genes, grouped in 32 distinct clades. Two CaR2R3-MYB transcription factors, named CaPAR1 and CaPAR2, clustering with known proanthocyanidin regulators (PARs) were identified and further analyzed. The expression of CaPAR genes correlated well with the expression of the key structural proanthocyanidin biosynthesis genes CaANR and CaLAR and with proanthocyanidin levels. Protein-protein interaction studies suggest the in vivo interaction of CaPAR1 and CaPAR2 with the bHLH-type transcription factor CaTT8. Co-transfection analyses using Arabidopsis thaliana protoplasts showed that the CaPAR proteins form a MBW complex with CaTT8 and CaTTG1, able to activate the promoters of CaANR and CaLAR in planta. Finally, transgenic expression of CaPARs in the proanthocyanidin-deficient A. thaliana mutant tt2-1 leads to complementation of the transparent testa phenotype. Taken together, our results reveal main components of the proanthocyanidin regulatory network in C. arietinum and suggest that CaPARs are relevant targets of genetic engineering toward improved agronomic traits.

Novel insight on ferric ions addition to mitigate recalcitrant formation during thermal-alkali hydrolysis to enhance biomethanation.

Thermal-chemical pre-treatment has proven to facilitate the solubilization of organics and improvement in biogas generation from the organic fraction of municipal solid waste (OFMSW). However, the production of recalcitrant is inevitable when OFMSW is pretreated at high temperatures and alkali dosage. This study develops a strategy to use Fe3+ to reduce the formation of recalcitrant compounds, i.e., 5-HydroxyMethyl Furfural (5-HMF), furfurals, and humic acids (HA) during thermal-alkali pre-treatment. It was postulated that the formation of the recalcitrant compound during pre-treatment can be reduced by Fe3+ dosing to oxidize intermediates of Maillard reactions. A decrease in 5-HMF (45-49%) and furfurals (54-66%) was observed during Fe3+ (optimum dose: 10 mg/L) mediated thermal-alkali pre-treatment owing to the Lewis acid behavior of FeCl3. The Fe3+ mediated assays show a substantial improvement in VS removal (28%) and biogas yield, i.e., 31% (292 mL/gVSadded) in 150 °C + 3 g/L NaOH, 34% (316 mL/gVSadded) in 175 °C + 3 g/L NaOH, and 36% (205 mL/gVSadded) in 200 °C + 3 g/L NaOH assays, over their respective controls (no Fe3+ dosing). The reducing property of Fe3+ rendered a low ORP (-345 mV) in the system than control, which is beneficial to the anaerobic microbiome. Electrical conductivity (EC) also shows a three-fold increase in Fe3+ mediated assays over control, promoting direct interspecies electron transfer (DIET) amongst microbes involved in the electrical syntrophy. The score plot and loading plots from principal component analysis (PCA) showed that the results obtained by supplementing 10 mg/L Fe3+ at 150, 175, and 200 °C were significantly different. The correlation of the operational parameters was also mutually correlated. This work provides a techno-economically and environmentally feasible option to mitigate the formation of recalcitrant compounds and enhance biogas production in downstream AD by improving the degradability of pretreated substrate.

Carbon based conductive materials mediated recalcitrant toxicity mitigation during anaerobic digestion of thermo-chemically pre-treated organic fraction of municipal solid waste.

High-temperature thermal pretreatment alone or in conjugation with chemical pretreatment (highly acidic or alkaline) produced recalcitrant compounds, which inhibits the anaerobic digestion (AD) process performance. This study aims to develop a strategy to use carbon-based conductive materials to mitigate the recalcitrant toxicity and enhance the methane generation in the downstream AD. The formation of recalcitrant compounds, mainly the furan derivatives, i.e., furfural and 5-HydroxyMethyl furfurals (5-HMF) during thermo-chemical pretreatment of OFMSW at 150 °C, 175 °C, 200 °C with 3 g/L-NaOH dose, and the alleviation of their inhibitory effects by adding 25 g/L of each of granular activated carbon (GAC) and granular biochar (GBC) during mesophilic AD were studied. The addition of conductive materials resulted in the highest biogas yield of 462 mL/gVSadded (GAC) and 449 mL/gVSadded (GBC) for 175°C-3g/L-NaOH pretreatment, which was >45% higher over control. The highest improvement of >65% in biogas yield was observed for 200°C-3g/L-NaOH pretreatment despite the lower biogas yield. The conductive materials amended digester shows a significant decrease in the 5-HMF and furfurals concertation. The highest reduction in 5-HMF (44%) and furfural (51%) concentrations were observed for 200°C-3g/L-NaOH pretreatment, and 25 g/L GBC amended tests. The score plots from the principal component analysis (PCA) of the characterization of the digestate showed that the data were significant, whereas the loading plots depicted the correlation of different experimental parameters studied (like fate of recalcitrant, biogas yield and other parameters post AD of OFMSW when aided with conductive materials). Application of regression models in all the batch assays depicted that a lag phase of 2-4 days was observed in Modified Gompertz Model (MGM), 4-5 days in Logistic Model (LM) and a rapid hydrolysis was proven with the value of hydrolysis coefficient being between 0.003 and 0.029 from the first-order (FO) model.

Exploration of glutathione reductase for abiotic stress response in bread wheat (Triticum aestivum L.).

KEY MESSAGE: A total of seven glutathione reductase (GR) genes were identified in Triticum aestivum, which were used for comparative structural characterization, phylogenetic analysis and expression profiling with the GR genes of other cereal plants. The modulated gene expression and enzyme activity revealed the role of GRs in abiotic stress response in T. aestivum. Glutathione reductase (GR) is an enzymatic antioxidant that converts oxidized glutathione (GSSG) into reduced glutathione (GSH) through the ascorbate-glutathione cycle. In this study, a total of seven GR genes forming two homeologous groups were identified in the allohexaploid genome of bread wheat (Triticum aestivum). Besides, we identified three GR genes in each Aegilops tauschii, Brachypodium distachyon, Triticum urartu and Sorghum bicolor, which were used for comparative characterization. Phylogenetic analysis revealed the clustering of GR proteins into two groups; class I and class II, which were predicted to be localized in cytoplasm and chloroplast, respectively. The exon-intron and conserved motif patterns were almost conserved in each group, in which a maximum of 10 and 17 exons were present in chloroplastic and cytoplasmic GRs, respectively. The protein structure analysis confirmed the occurrence of conserved pyridine nucleotide disulfide oxidoreductase (Pyr_redox) and pyridine nucleotide disulfide oxidoreductase dimerization (Pyr_redox_dim) domains in each GR. The active site of GR proteins consisted of two conserved cysteine residues separated by four amino acid residues. Promoter analysis revealed the occurrence of growth and stress-related cis-active elements. Tissue-specific expression profiling suggested the involvement of GRs in both vegetative and reproductive tissue development in various plants. The differential expression of TaGR genes and enhanced GR enzyme activity suggested their roles under drought, heat, salt and arsenic stress. Interaction of GRs with other proteins and chemical compounds of the ascorbate-glutathione cycle revealed their coordinated functioning. The current study will provide a foundation for the validation of the precise role of each GR gene in future studies.

Molecular characterization revealed the role of catalases under abiotic and arsenic stress in bread wheat (Triticum aestivum L.).

Catalases are crucial antioxidant enzymes that reduce the excessive level of H2O2 caused by various environmental stresses and metal toxicity and hence protect the plant cells. In this study, a total of ten TaCAT genes, forming three homeologous groups, were identified in the genome of bread wheat (Triticum aestivum L.) and named as per the wheat gene symbolization guidelines. The identified catalases were characterized for various structural and physicochemical features. The proximal active-site (F(D/A)RERIPERVVHAKGASA) and heme-ligand (R(I/V)F(S/A)Y(A/S)DTQ) signature motifs, catalytic residues and peroxisomal targeting peptides were found conserved. Phylogenetic analysis clustered TaCATs into three classes, which showed conserved functional specialization based on their tissue specific expression. Modulated spatio-temporal expression of various TaCAT genes and alteration in total catalase enzyme activity during heat, drought, salt and arsenic (AsIII and AsV) treatment suggested their roles in abiotic stress response and arsenic tolerance. Molecular cloning and overexpression of TaCAT3-B gene in Escherichia coli showed tolerance against heat, drought, salt and varied concentrations of AsIII and AsV treatments. The results further confirmed their role in stress tolerance and recommended that these genes can be used in future stress management strategies for the development of abiotic and arsenic stress resistant transgenic crops.

Genome-wide characterization and expression analysis suggested diverse functions of the mechanosensitive channel of small conductance-like (MSL) genes in cereal crops.

Mechanosensitive ion channels are pore-forming transmembrane proteins that allow ions to move down their electrochemical gradient in response to mechanical stimuli. They participate in many plant developmental processes including the maintenance of plastid shape, pollen tube growth, etc. Herein, a total of 11, 10, 6, 30, 9, and 8 MSL genes were identified in Aegilops tauschii, Hordeum vulgare, Sorghum bicolor, Triticum aestivum, Triticum urartu, and Zea mays, respectively. These genes were located on various chromosomes of their respective cereal, while MSLs of T. urartu were found on scaffolds. The phylogenetic analysis, subcellular localization, and sequence homology suggested clustering of MSLs into two classes. These genes consisted of cis-regulatory elements related to growth and development, responsive to light, hormone, and stress. Differential expression of various MSL genes in tissue developmental stages and stress conditions revealed their precise role in development and stress responses. Altered expression during CaCl2 stress suggested their role in Ca2+ homeostasis and signaling. The co-expression analysis suggested their interactions with other genes involved in growth, defense responses etc. A comparative expression profiling of paralogous genes revealed either retention of function or pseudo-functionalization. The present study unfolded various characteristics of MSLs in cereals, which will facilitate their in-depth functional characterization in future studies.

Molecular characterization of ascorbate peroxidase (APX) and APX-related (APX-R) genes in Triticum aestivum L.

Ascorbate peroxidases (APXs) are heme-dependent H2O2 scavenging enzymes involved in myriad biological processes. Herein, a total of 21 TaAPX and six TaAPX-R genes were identified from the A, B and D sub-genomes of Triticum aestivum L. The occurrence of three paralogous gene pairs with unequal evolutionary rate suggested functional divergence. The phylogenetic analysis formed four distinct clades having conserved gene and protein architecture, and sub-cellular localization. The tertiary structure analysis revealed the presence of helices and coils and residues involved in ligand binding. Transcriptional profiling of each TaAPX and TaAPX-R gene suggested their specific role during development and stress response. Modulated transcript expression and APX enzyme activity during various stress conditions indicated their role in stress response. Interaction analyses suggested their association with other genes, miRNAs and various legends. The present study reported numerous features of these genes, and may provide a platform for their detailed functional characterization in future studies.

Thaumatin-like protein kinases: Molecular characterization and transcriptional profiling in five cereal crops.

Thaumatin-like protein kinases (TLPKs) are defense related proteins having antimicrobial property. Herein, we identified two TLPKs in the genome of Brachypodium distachyon and Oryza sativa, four in Hordeum vulgare and Sorghum bicolor, and 16 in Triticum aestivum. All the TLPKs were located at only one chromosome in each plant except T. aestivum, where they were located on chromosome 2 and chromosome 3. Paralogous analysis suggested the occurrence of one duplication event (DE) in each B. distachyon and O. sativa, two in H. vulgare while four DEs in T. aestivum genome during the evolution of TLPKs. The majority of TLPKs were intron less, while a few contains one or two introns. The introns were found in each 0, 1 and 2 phase. Protein structure analysis suggested the occurrence of a thaumatin and a kinase domain with a transmembrane (TM) helix in each TLPK. Further, a thaumatin family signature motif "GX[GF]XCXT[GA]DCX(1,2)GX(2,3)C", a "REDDD" motif and 16 cysteine residues were found conserved in the majority of TLPKs. Expression analysis indicated variable expression of TLPKs in various tissues of different cereal crops. They were high expressing in reproductive tissues in B. distachyon, while in leaves in T. aestivum. Modulated expression of TaTLPKs in the presence of fungal pathogen, and heat, drought and salt stress in T. aestivum suggested their roles in stress response. Co-expression analysis showed interaction of TLPKs with various development and stress related genes. The results indicated diverse roles of TLPKs, which can be utilized for the development of eco-friendly pest resistant crops in future.

Genomic dissection and transcriptional profiling of Cysteine-rich receptor-like kinases in five cereals and functional characterization of TaCRK68-A.

Cysteine-rich receptor-like kinases (CRK) constitute one of the largest subfamily of receptor-like kinases, which play crucial roles in plant development and stress response. In total, 43, 37, 36, 38 and 170 CRK genes including duplicated genes were identified in the genome of Brachypodium distachyon, Hordeum vulgare, Oryza sativa, Sorghum bicolor and Triticum aestivum, respectively. These CRK proteins were tightly clustered into four phylogenetic groups and exhibited close syntenic relationship among orthologous genes. Majority of CRK proteins contain a transmembrane domain for plasma membrane localization. The organization of exon/intron, domains and motifs were variably conserved. Tissue-specific expression suggested the involvement of certain CRK genes in plant development. Modulated expression revealed their specific stress-responsive functions. Co-expression and interaction analysis indicated their role in signaling. Ks value and divergence time analysis suggested duplication of TaCRK genes before the hybridization of T. aestivum sub-genomes. Expression comparison of duplicated TaCRK genes revealed functional retention, neofunctionalization or pseudo-functionalization. Recombinant expression of a stress-responsive gene TaCRK68-A in Escherichia coli and Saccharomyces cerevisiae displayed enhanced tolerance against heat, drought, cold and salinity stresses. The study suggested vital functions of CRKs during development and stresses, and provides the basis for functional characterization of each gene in future studies.

Gene architecture and expression analyses provide insights into the role of glutathione peroxidases (GPXs) in bread wheat (Triticum aestivum L.).

Glutathione peroxidases (GPXs) are redox sensor proteins that maintain a steady-state of H2O2 in plant cells. They exhibit distinct sub-cellular localization and have diverse functionality in response to different stimuli. In this study, a total of 14 TaGPX genes and three splice variants were identified in the genome of Triticum aestivum and evaluated for various physicochemical properties. The TaGPX genes were scattered on the various chromosomes of the A, B, and D sub-genomes and clustered into five homeologous groups based on high sequence homology. The majority of genes were derived from the B sub-genome and localized on chromosome 2. The intron-exon organization, motif and domain architecture, and phylogenetic analyses revealed the conserved nature of TaGPXs. The occurrence of both development-related and stress-responsive cis-acting elements in the promoter region, the differential expression of these genes during various developmental stages, and the modulation of expression in the presence of biotic and abiotic stresses suggested their diverse role in T. aestivum. The majority of TaGPX genes showed higher expression in various leaf developmental stages. However, TaGPX1-A1 was upregulated in the presence of each abiotic stress treatment. A co-expression analysis revealed the interaction of TaGPXs with numerous development and stress-related genes, which indicated their vital role in numerous biological processes. Our study revealed the opportunities for further characterization of individual TaGPX proteins, which might be useful in designing future crop improvement strategies.

Survey of High Throughput RNA-Seq Data Reveals Potential Roles for lncRNAs during Development and Stress Response in Bread Wheat.

Long non-coding RNAs (lncRNAs) are a family of regulatory RNAs that play essential role in the various developmental processes and stress responses. Recent advances in sequencing technology and computational methods enabled identification and characterization of lncRNAs in certain plant species, but they are less known in Triticum aestivum (bread wheat). Herein, we analyzed 52 RNA seq data (>30 billion reads) and identified 44,698 lncRNAs in T. aestivum genome, which were characterized in comparison to the coding sequences (mRNAs). Similar to the mRNAs, lncRNAs were also derived from each sub-genome and chromosome, and showed tissue developmental stage specific and differential expression, as well. The modulated expression of lncRNAs during abiotic stresses like heat, drought, and salt indicated their putative role in stress response. The co-expression of lncRNAs with vital mRNAs including various transcription factors and enzymes involved in Abscisic acid (ABA) biosynthesis, and gene ontology mapping inferred their regulatory roles in numerous biological processes. A few lncRNAs were predicted as precursor (19 lncRNAs), while some as target mimics (1,047 lncRNAs) of known miRNAs involved in various regulatory functions. The results suggested numerous functions of lncRNAs in T. aestivum, and unfolded the opportunities for functional characterization of individual lncRNA in future studies.

Ca2+/Cation Antiporters (CaCA): Identification, Characterization and Expression Profiling in Bread Wheat (Triticum aestivum L.).

The Ca2+/cation antiporters (CaCA) superfamily proteins play vital function in Ca2+ ion homeostasis, which is an important event during development and defense response. Molecular characterization of these proteins has been performed in certain plants, but they are still not characterized in Triticum aestivum (bread wheat). Herein, we identified 34 TaCaCA superfamily proteins, which were classified into TaCAX, TaCCX, TaNCL, and TaMHX protein families based on their structural organization and evolutionary relation with earlier reported proteins. Since the T. aestivum comprises an allohexaploid genome, TaCaCA genes were derived from each A, B, and D subgenome and homeologous chromosome (HC), except chromosome-group 1. Majority of genes were derived from more than one HCs in each family that were considered as homeologous genes (HGs) due to their high similarity with each other. These HGs showed comparable gene and protein structures in terms of exon/intron organization and domain architecture. Majority of TaCaCA proteins comprised two Na_Ca_ex domains. However, TaNCLs consisted of an additional EF-hand domain with calcium binding motifs. Each TaCaCA protein family consisted of about 10 transmembrane and two α-repeat regions with specifically conserved signature motifs except TaNCL, which had single α-repeat. Variable expression of most of the TaCaCA genes during various developmental stages suggested their specified role in development. However, constitutively high expression of a few genes like TaCAX1-A and TaNCL1-B indicated their role throughout the plant growth and development. The modulated expression of certain genes during biotic (fungal infections) and abiotic stresses (heat, drought, salt) suggested their role in stress response. Majority of TaCCX and TaNCL family genes were found highly affected during various abiotic stresses. However, the role of individual gene needs to be established. The present study unfolded the opportunity for detail functional characterization of TaCaCA proteins and their utilization in future crop improvement programs.