Glutathione imparts stress tolerance against Alternaria brassicicola infection via miRNA mediated gene regulation.

Glutathione (GSH) is well known to play a crucial role in imparting resistance against various pathogen invasions. Nevertheless, the role of GSH in regulating miRNA-mediated defense response is yet to be explored. To decipher the GSH-mediated regulation of miRNA expression during necrotrophic infection in Arabidopsis thaliana, wild-type Col-0 and AtECS1, the transgenic line exhibiting enhanced GSH content, were infected with necrotrophic pathogen Alternaria brassicicola. AtECS1 plants exhibited enhanced resistance as compared to wild-type. MiRNA next-generation sequencing (NGS) was performed to compare the miRNA expression in Col-0 and AtECS1 leaves. Under control condition, differentially expressed 96 known miRNAs and 17 novel miRNAs viz. ath-miR8167f, ath-miR1886.3, ath-miR3932b-5p, etc. were identified. However, under infected condition, 73 known and 43 novel differentially expressed miRNAs viz. ath-miR5652, ath-miR160b, ath-miR865-5p, etc. were identified. Functional annotation and enrichment analysis revealed that several miRNAs that target defense-related genes like leucine-rich repeat protein kinase, MYB transcription factors, TCP8, etc. were down regulated in the AtECS1 line, which, in turn, relieves the repression of their target gene expression, leading to resistance against infection. Together, the present investigation suggests that GSH plays a decisive role in modulating the miRNA-mediated regulation of defense-related genes during pathogen invasion.

Glutathione regulates transcriptional activation of iron transporters via S-nitrosylation of bHLH factors to modulate subcellular iron homoeostasis.

Glutathione (GSH) is known to regulate iron (Fe) deficiency response in plants but its involvement in modulating subcellular Fe homoeostasis remains elusive. In this study, we report that the GSH-deficient mutants, cad2-1 and pad2-1 displayed increased sensitivity to Fe deficiency with significant downregulation of the vacuolar Fe exporters, AtNRAMP3 and AtNRAMP4, and the chloroplast Fe importer, AtPIC1. Moreover, the pad2-1 mutant accumulated higher Fe levels in vacuoles but lower Fe levels in chloroplasts compared to wild type (Columbia ecotype [Col-0]) under Fe limited conditions. Exogenous GSH treatment enhanced chloroplast Fe contents in Col-0 but failed to do so in the nramp3nramp4 double mutants demonstrating that GSH plays a role in modulating subcellular Fe homoeostasis. Pharmacological experiments, mutant analysis, and promoter assays revealed that this regulation involves the transcriptional activation of Fe transporter genes by a GSH-S-nitrosoglutathione (GSNO) module. The Fe responsive bHLH transcription factors (TFs), AtbHLH29, AtbHLH38, and AtbHLH101 were found to interact with the promoters of these genes, which were, in turn, activated via S-nitrosylation (SNO). Taken together, the present study highlights the role of the GSH-GSNO module in regulating subcellular Fe homoeostasis by transcriptional activation of the Fe transporters AtNRAMP3, AtNRAMP4, and AtPIC1 via SNO of bHLH TFs during Fe deficiency.

Rice lectin protein r40c1 imparts drought tolerance by modulating S-adenosylmethionine synthase 2, stress-associated protein 8 and chromatin-associated proteins.

Lectin proteins play an important role in biotic and abiotic stress responses in plants. Although the rice lectin protein Osr40c1 has been reported to be regulated by drought stress, the mechanism of its drought tolerance activity has not been studied so far. In this study, it is shown that expression of the Osr40c1 gene correlates with the drought tolerance potential of various rice cultivars. Transgenic rice plants overexpressing Osr40c1 were significantly more tolerant to drought stress than the wild-type plants. Furthermore, ectopic expression of the Osr40c1 gene in tobacco yielded a similar result. Interestingly, the protein displayed a nucleo-cytoplasmic localization and was found to interact with a number of drought-responsive proteins such as S-adenosylmethionine synthase 2 (OsSAM2), stress-associated protein 8 (OsSAP8), DNA-binding protein MNB1B (OsMNB1B), and histone 4 (OsH4). Silencing of each of these protein partners led to drought sensitivity in otherwise tolerant Osr40c1-expressing transgenic tobacco lines indicating that these partners were crucial for the Osr40c1-mediated drought tolerance in planta. Moreover, the association of Osr40c1 with these partners occurred specifically under drought stress forming a multi-protein complex. Together, our findings delineate a novel role of Osr40c1 in imparting drought tolerance by regulating OsMNB1B, OsSAM2, and OsH4 proteins, which presumably enables OsSAP8 to induce downstream gene expression.

Long non-coding RNAs: Fine-tuning the developmental responses in plants.

Plant developmental biology is associated with various gene regulatory pathways involved in different phases of their life cycle. In course of development, growth and differentiation of different organs in plants are regulated by specific sets of gene expression. With the advances in genomic and bioinformatic techniques, particularly high-throughput sequencing technology, many transcriptional units with no protein-coding potential have been discovered. Previously thought to be the dark matters of genome, long non-coding RNAs (lncRNAs) are gradually gaining importance as crucial players in gene regulation during different developmental phases. Some lncRNAs, showing complementarity to microRNAs (miRNAs), are used as endogenous target mimics of specific miRNA family. A number of lncRNAs can also act as natural antisense transcripts to attenuate the expression of coding genes. Although lncRNA-mediated regulations have extensively been studied in animals, plant lncRNA research is still in its initial phase. The present review highlights the regulatory mechanism and different physiological aspects of lncRNAs in plant development. In plants, lncRNAs are found to be associated with a number of plant developmental functions such as lateral root development, vernalization, photomorphogenesis, pollen development, fiber development and nodulation. Understanding these potent roles of lncRNAs in plant development can further provide novel tools for crop improvement programs in future.

Membrane proteome profiling of Mentha arvensis leaves in response to Alternaria alternata infection identifies crucial candidates for defense response.

The leaf spot disease of Mentha arvensis, caused by Alternaria alternata, is a devastating foliar disease worldwide and leads to considerable economic losses. In this investigation, 2-dimensional gel electrophoresis (2-DE) was used to identify the membrane proteins potentially involved in M. arvensis - A. alternata interaction. Membrane proteins, isolated from leaves of control and infected plants, were analyzed by 2-DE and identified using mass spectrometry (MALDI TOF-TOF MS/MS). Our analysis identified 21 differentially expressed membrane proteins including several interesting receptors and channel proteins. Of these identified proteins, 34% were found to be involved in plant defense responses. Leucine-rich repeat family protein/ protein kinase family protein which plays critical role in stress response and nucleotide-binding site-leucine-rich repeat (NBS-LRR) which is involved in detecting the advent of pathogen on plant surface were identified to be up-regulated in our study. Interestingly, AKT1-like potassium channel protein which is known to play a crucial role in maintaining ion homeostasis within the cell was also upregulated in the infected sample. In addition, ADP ribolysation factor (ARF)-GTPase activating domain containing protein, a membrane trafficking protein, was also up-regulated in the current study. Protein-protein interaction network analysis followed by functional enrichment revealed that transmembrane ion transport-related proteins represented a major class in this network followed by nucleic acid binding proteins and proteins with kinase activities respectively. Together, our investigation identified several key defense-related proteins which are crucial sensors for detecting pathogen invasion and can serve as a potential resource to understand disease resistance mechanism in mint.

Transcriptome analysis of Arabidopsis mutants suggests a crosstalk between ABA, ethylene and GSH against combined cold and osmotic stress.

The involvement of ethylene and abscisic acid in providing stress tolerance and defence response to plants is widely recognized. However, little is known about the cross-talk between glutathione with ethylene and abscisic acid to combat stress in planta. Here, transcriptome analysis of combined cold and osmotic stress treated Arabidopsis mutants were carried out to elucidate the crosstalk between the abscisic acid, ethylene and glutathione. Microarray experiment revealed the differential regulation of about 2313 and 4131 transcripts in ein2 (ethylene insensitive mutant) and aba1.6 (abscisic acid mutant) respectively. Functional analysis exposed common down-regulated stress and defence, secondary metabolite biosynthesis viz. phenylpropanoid, lignin and flavonols, redox and transcription factors related genes in ein2, aba1.6 and pad2.1 (glutathione mutant) in response to combined stress treatment. The reduced glutathione content was less in stress treated mutants in comparison to Col-0. Again, selective down-regulated transcripts in stress treated mutants were noted up-regulated after glutathione feeding. Some of the important differentially expressed genes were also validated by comparative proteomics analysis of stress treated mutants. In summary, our results suggested the role of ethylene and abscisic acid in inducing stress-responsive genes and proteins by activating glutathione biosynthesis to combat abiotic stress conditions in plant system.

Transcriptome-wide identification and characterization of CAD isoforms specific for podophyllotoxin biosynthesis from Podophyllum hexandrum.

Podophyllotoxin (ptox) is a therapeutically important lignan derived from Podophyllum hexandrum and is used as a precursor for the synthesis of anticancer drugs etoposide, teniposide and etopophose. In spite of its enormous economic significance, genomic information on this endangered medicinal herb is scarce. We have performed de novo transcriptome analysis of methyl jasmonate (MeJA)-treated P. hexandrum cell cultures exhibiting enhanced ptox accumulation. The results revealed the maximum up-regulation of several isoforms of cinnamyl alcohol dehydrogenase (CAD). CAD catalyzes the synthesis of coniferyl alcohol and sinapyl alcohol from coniferaldehyde (CAld) and sinapaldehyde respectively. Coniferyl alcohol can produce both lignin and lignan while sinapyl alcohol produces only lignin. To isolate the CAD isoforms favoring ptox, we deduced full length cDNA sequences of four CAD isoforms: PhCAD1, PhCAD2, PhCAD3 and PhCAD4 from the contigs of the transcriptome data. In vitro enzyme assays indicated a higher affinity for CAld over sinapaldehyde for each isoform. In silico molecular docking analyses also suggested that PhCAD3 has a higher binding preference with CAld over sinapaldehyde, followed by PhCAD4, PhCAD2, and PhCAD1, respectively. The transgenic cell cultures overexpressing these isoforms independently revealed that PhCAD3 favored the maximum accumulation of ptox as compared to lignin followed by PhCAD4 and PhCAD2, whereas, PhCAD1 favored both equally. Together, our study reveals transcriptome-wide identification and characterization of ptox specific CAD isoforms from P. hexandrum. It provides a useful resource for future research not only on the ptox biosynthetic pathway but on overall P. hexandrum, an endangered medicinal herb with immense therapeutic importance.

Glutathione Regulates 1-Aminocyclopropane-1-Carboxylate Synthase Transcription via WRKY33 and 1-Aminocyclopropane-1-Carboxylate Oxidase by Modulating Messenger RNA Stability to Induce Ethylene Synthesis during Stress.

Glutathione (GSH) plays a fundamental role in plant defense-signaling network. Recently, we have established the involvement of GSH with ethylene (ET) to combat environmental stress. However, the mechanism of GSH-ET interplay still remains unexplored. Here, we demonstrate that GSH induces ET biosynthesis by modulating the transcriptional and posttranscriptional regulations of its key enzymes, 1-aminocyclopropane-1-carboxylate synthase (ACS) and 1-aminocyclopropane-1-carboxylate oxidase (ACO). Transgenic Arabidopsis (Arabidopsis thaliana) plants with enhanced GSH content (AtECS) exhibited remarkable up-regulation of ACS2, ACS6, and ACO1 at transcript as well as protein levels, while they were down-regulated in the GSH-depleted phytoalexin deficient2-1 (pad2-1) mutant. We further observed that GSH induced ACS2 and ACS6 transcription in a WRKY33-dependent manner, while ACO1 transcription remained unaffected. On the other hand, the messenger RNA stability for ACO1 was found to be increased by GSH, which explains our above observations. In addition, we also identified the ACO1 protein to be a subject for S-glutathionylation, which is consistent with our in silico data. However, S-glutathionylation of ACS2 and ACS6 proteins was not detected. Further, the AtECS plants exhibited resistance to necrotrophic infection and salt stress, while the pad2-1 mutant was sensitive. Exogenously applied GSH could improve stress tolerance in wild-type plants but not in the ET-signaling mutant ethylene insensitive2-1, indicating that GSH-mediated resistance to these stresses occurs via an ET-mediated pathway. Together, our investigation reveals a dual-level regulation of ET biosynthesis by GSH during stress.

Changes in the proteome of pad2-1, a glutathione depleted Arabidopsis mutant, during Pseudomonas syringae infection.

The involvement of glutathione (GSH) in plant defense against pathogen invasion is an established fact. However, the molecular mechanism conferring this tolerance remains to be explored. Here, proteomic analysis of pad2-1, an Arabidopsis thaliana GSH-depleted mutant, in response to Pseudomonas syringae infection has been performed to explore the intricate position of GSH in defense against biotrophic pathogens. The pad2-1 mutant displayed severe susceptibility to P. syringae infection compared to the wild-type (Col-0) thus re-establishing a fundamental role of GSH in defense. Apart from general up-accumulation of energy metabolism-related protein-species in both infected Col-0 and pad2-1, several crucial defense-related protein-species were identified to be differentially accumulated. Leucine-rich repeat-receptor kinase (LRR-RK) and nucleotide-binding site-leucine-rich repeat resistance protein (NBS-LRR), known to play a pioneering role against pathogen attack, were only weakly up-accumulated in pad2-1 after infection. Transcriptional and post-transcriptional regulators like MYB-P1 and glycine-rich repeat RNA-binding protein (GRP) and several other stress-related protein-species like heat shock protein 17 (HSP17) and glutathione-S-transferase (GST) were also identified to be differentially regulated in pad2-1 and Col-0 in response to infection. Together, the present investigation reveals that the optimum GSH-level is essential for the efficient activation of plant defense signaling cascades thus conferring resistance to pathogen invasion.

Plant microRNAs: master regulator of gene expression mechanism.

Several signaling molecules critically regulate the physiological responses in plants. Among them, miRNAs, generally 21-24 nucleotides long, are widely distributed in different plant species and play as key signaling intermediates in diverse physiological responses. The mature miRNAs are synthesized from MIR genes by RNA polymerase II and processed by Dicer-like (DCL) protein family members associated with some accessory protein molecules. The processed miRNAs are transported to the cytoplasm from the nucleus by specific group of transporters and incorporated into RNA-induced silencing complex (RISC) for specific mRNA cleavage. MicroRNAs can suppress the diverse gene expression, depending on the sequence complementarity of the target transcript except of its own gene. Besides, miRNAs can modulate the gene expression by DNA methylation and translational inhibition of the target transcript. Different classes of DCLs and Argonaute proteins (AGOs) help the miRNAs-mediated gene silencing mechanism in plants.

Transcriptomic profiling of Arabidopsis thaliana mutant pad2.1 in response to combined cold and osmotic stress.

The contribution of glutathione (GSH) in stress tolerance, defense response and antioxidant signaling is an established fact. In this study transcriptome analysis of pad2.1, an Arabidopsis thaliana mutant, after combined osmotic and cold stress treatment has been performed to explore the intricate position of GSH in the stress and defense signaling network in planta. Microarray data revealed the differential regulation of about 1674 genes in pad2.1 amongst which 973 and 701 were significantly up- and down-regulated respectively. Gene enrichment, functional pathway analysis by DAVID and MapMan analysis identified various stress and defense related genes viz. members of heat shock protein family, peptidyl prolyl isomerase (PPIase), thioredoxin peroxidase (TPX2), glutathione-S-transferase (GST), NBS-LRR type resistance protein etc. as down-regulated. The expression pattern of the above mentioned stress and defense related genes and APETALA were also validated by comparative proteomic analysis of combined stress treated Col-0 and pad2.1. Functional annotation noted down-regulation of UDP-glycosyl transferase, 4-coumarate CoA ligase 8, cinnamyl alcohol dehydrogenase 4 (CAD4), ACC synthase and ACC oxidase which are the important enzymes of phenylpropanoid, lignin and ethylene (ET) biosynthetic pathway respectively. Since the only difference between Col-0 (Wild type) and pad2.1 is the content of GSH, so, this study suggested that in addition to its association with specific stress responsive genes and proteins, GSH provides tolerance to plants by its involvement with phenylpropanoid, lignin and ET biosynthesis under stress conditions.

Multistep involvement of glutathione with salicylic acid and ethylene to combat environmental stress.

The role of glutathione (GSH) in plant defense is an established fact. However, the association of GSH with other established signaling molecules within the defense signaling network remains to be evaluated. Previously we have shown that GSH is involved in defense signaling network likely through NPR1-dependent salicylic acid (SA)-mediated pathway. In this study, to gain further insight, we developed chloroplast-targeted gamma-glutamylcysteine synthetase (γ-ECS) overexpressed transgenic Nicotiana tabacum (NtGp line) and constructed a forward subtracted cDNA (suppression subtractive hybridization (SSH)) library using NtGp line as a tester. Interestingly, in addition to SA-related transcripts like pathogenesis-related protein 1a (PR1a) and SAR8.2 m/2l, 1-aminocyclopropane-1-carboxylate oxidase (ACC oxidase), a key enzyme of ethylene (ET) biosynthesis, was identified in the SSH library. Besides, transcription factors like WRKY transcription factor 3 (WRKY3), WRKY1 and ethylene responsive factor 4 (ERF4), associated with SA and ET respectively, were also identified thus suggesting an interplay of GSH with ET and SA. Furthermore, proteomic profiling of NtGp line, performed by employing two-dimensional gel electrophoresis (2-DE), corroborated with the transcriptomic profile and several defense-related proteins like serine/threonine protein kinase, and heat shock 70 protein (HSP70) were identified with increased accumulation. Fascinatingly, induction of 1-aminocyclopropane-1-carboxylate synthase (ACC synthase) was also noted thus demonstrating the active involvement of GSH with ET. Protein gel blot analysis confirmed the enhanced accumulation of ACC oxidase in NtGp line. Together, our data revealed that GSH is involved in the synergistic multiple steps crosstalk through ET as well as SA to combat environmental stress.

Proteomic profiling of γ-ECS overexpressed transgenic Nicotiana in response to drought stress.

The contribution of Glutathione (GSH) in drought stress tolerance is an established fact. However, the proteins which are directly or indirectly related to the increased level of GSH in response to drought stress are yet to be known. To explore this, here, transgenic tobacco plants (NtGp11) overexpressing gamma-glutamylcysteine synthetase (γ-ECS) was tested for tolerance against drought stress. NtGp11 conferred tolerance to drought stress by increased germination rate, water retention, water recovery, chlorophyll, and proline content compared with wild-type plants. Semi-quantitative RT-PCR analysis revealed that the transcript levels of stress-responsive genes were higher in NtGp11 compared with wild-type in response to drought stress. Two-dimensional gel electrophoresis (2-DE) coupled with MALDI TOF-TOF MS/MS analysis has been used to identify 43 differentially expressed proteins in response to drought in wild-type and NtGp11 plants. The results demonstrated the up-accumulation of 58.1% of proteins among which 36%, 24%, and 20% of them were related to stress and defense, carbon metabolism and energy metabolism categories, respectively. Taken together, our results demonstrated that GSH plays an important role in combating drought stress in plants by inducing stress related genes and proteins like HSP70, chalcone synthase, glutathione peroxidase, thioredoxin peroxidase, ACC oxidase, and heme oxygenase I.

De novo transcriptome analysis using 454 pyrosequencing of the Himalayan Mayapple, Podophyllum hexandrum.

BACKGROUND: The Himalayan or Indian Mayapple (Podophyllum hexandrum Royle) produces podophyllotoxin, which is used in the production of semisynthetic anticancer drugs. High throughput transcriptome sequences or genomic sequence data from the Indian Mayapple are essential for further understanding of the podophyllotoxin biosynthetic pathway. RESULTS: 454 pyrosequencing of a P. hexandrum cell culture normalized cDNA library generated 2,667,207 raw reads and 1,503,232 high quality reads, with an average read length of 138 bp. The denovo assembly was performed by Newbler using default and optimized parameters. The optimized parameter generated 40, 380 assembled sequences, comprising 12,940 contigs and 27,440 singlets which resulted in better assembly as compared to default parameters. BLASTX analysis resulted in the annotation of 40,380 contigs/singlet using a cut-off value of ≤ 1E-03. High similarity to Medicago truncatula using optimized parameters and to Populus trichocarpa using default parameters was noted. The Kyoto encyclopedia of genes and genomes (KEGG) analysis using KEGG Automatic Annotation Server (KAAS) combined with domain analysis of the assembled transcripts revealed putative members of secondary metabolism pathways that may be involved in podophyllotoxin biosynthesis. A proposed schematic pathway for phenylpropanoids and podophyllotoxin biosynthesis was generated. Expression profiling was carried out based on fragments per kilobase of exon per million fragments (FPKM). 1036 simple sequence repeats were predicted in the P. hexandrum sequences. Sixty-nine transcripts were mapped to 99 mature and precursor microRNAs from the plant microRNA database. Around 961 transcripts containing transcription factor domains were noted. High performance liquid chromatography analysis showed the peak accumulation of podophyllotoxin in 12-day cell suspension cultures. A comparative qRT-PCR analysis of phenylpropanoid pathway genes identified in the present data was performed to analyze their expression patterns in 12-day cell culture, callus and rhizome. CONCLUSIONS: The present data will help the identification of the potential genes and transcription factors involved in podophyllotoxin biosynthesis in P. hexandrum. The assembled transcripts could serve as potential candidates for marker discovery and conservation, which should form the foundations for future endeavors.

Changes in leaf proteome profile of Arabidopsis thaliana in response to salicylic acid.

Salicylic acid (SA) has been implicated in determining the outcome of interactions between many plants and their pathogens. Global changes in response to this phytohormone have been observed at the transcript level, but little is known of how it induces changes in protein abundance. To this end we have investigated the effect of 1 mM SA on soluble proteins of Arabidopsis thaliana leaves by proteomic analysis. An initial study at transcript level has been performed on temporal landscape, which revealed that induction of most of the SA-responsive genes occurs within 3 to 6 h post treatment (HPT) and the expression peaked within 24 HPT. Two-dimensional gel electrophoresis (2-DE) coupled with MALDI-TOF MS/MS analysis has been used to identify differentially expressed proteins and 63 spots have been identified successfully. This comparative proteomic profiling of SA treated leaves versus control leaves demonstrated the changes of many defence related proteins like pathogenesis related protein 10a (PR10a), diseaseresistance- like protein, putative late blight-resistance protein, WRKY4, MYB4, etc. along with gross increase in the rate of energy production, while other general metabolism rate is slightly toned down, presumably signifying a transition from 'normal mode' to 'defence mode'.

Leaf proteome profiling of transgenic mint infected with Alternaria alternata.

The genus Mentha has been widely used in food, flavor, culinary, cosmetic and pharmaceutical industries. Substantial damage to this crop happened regularly due to environmental stresses like metal toxicity and pathogen attack. Here, an approach has been taken to raise transgenic mint over-expressing γ-glutamyl-cysteine synthetase (γ-ECS), the rate-limiting enzyme of GSH biosynthesis, resulted enhanced GSH content and its in planta expression confers significant tolerance towards abiotic/biotic stresses viz. metal toxicity - Cd, Zn as well as against infection of Alternaria alternata and Rhizoctonia solani. A differential proteomic analysis through 2-DE and MALDI TOF-TOF MSMS was performed to focus on the altered abundance of functionally important protein species in control and infected transgenic mint. Results showed a significant variation in the protein profile of the infected transgenic plant as compared to the wild/control transgenic counterpart. In addition to protein species related to stress and defense, redox regulation, transcription factors and energy & metabolism, protein species related to signaling and gene regulation as well as cell division also showed differential accumulation in infected transgenic. Hence, proteomics can be used as a tool to decipher the mechanism of action of GSH in providing tolerance against a necrotrophic fungus, A. alternata in transgenic mint. BIOLOGICAL SIGNIFICANCE: The reported work describes a comparative proteomics of non-model unsequenced plants like Mentha. There is a comparative protein profile between transgenic and its wild counterparts under control and infected condition. The work has an impact in crop proteomics and also tries to explain the application of proteomic approach to decipher the mechanism by which a foreign metabolite mediates stress tolerance in plant under control and infected condition. This article is part of a Special Issue entitled: Translational Plant Proteomics.