Swarup Roy Choudhury

Comparative quantitative proteomics analysis of the ABA response of roots of drought-sensitive and drought-tolerant wheat varieties identifies proteomic signatures of drought adaptability.

Wheat is one of the most highly cultivated cereals in the world. Like other cultivated crops, wheat production is significantly affected by abiotic stresses such as drought. Multiple wheat varieties suitable for different geographical regions of the world have been developed that are adapted to different environmental conditions; however, the molecular basis of such adaptations remains unknown in most cases. We have compared the quantitative proteomics profile of the roots of two different wheat varieties, Nesser (drought-tolerant) and Opata (drought-sensitive), in the absence and presence of abscisic acid (ABA, as a proxy for drought). A labeling LC-based quantitative proteomics approach using iTRAQ was applied to elucidate the changes in protein abundance levels. Quantitative differences in protein levels were analyzed for the evaluation of inherent differences between the two varieties as well as the overall and variety-specific effect of ABA on the root proteome. This study reveals the most elaborate ABA-responsive root proteome identified to date in wheat. A large number of proteins exhibited inherently different expression levels between Nesser and Opata. Additionally, significantly higher numbers of proteins were ABA-responsive in Nesser roots compared with Opata roots. Furthermore, several proteins showed variety-specific regulation by ABA, suggesting their role in drought adaptation.

Characterization of transcriptional profiles of MA-ACS1 and MA-ACO1 genes in response to ethylene, auxin, wounding, cold and different photoperiods during ripening in banana fruit

The ripening-specific genes MA-ACS1 (Musa acuminata ACC synthase1) and MA-ACO1 (M. acuminata ACC oxidase 1) are regulated in response to a wide variety of factors. Here, we have studied the differential transcript accumulation pattern and protein levels of MA-ACS1 and MA-ACO1 genes in response to ethylene, auxin, wounding and low temperature in preclimacteric banana fruit. We have shown that exogenous application of ethylene and auxin induced the expression of MA-ACS1, while MA-ACO1 showed marginal expression following ethylene treatment in preclimacteric stage. Auxin did not induce MA-ACO1 expression. Thus, auxin-treated banana fruits showed lower ethylene production rate as compared to ethylene-treated fruits. Conversely, wounding and cold treatment down-regulated the expression of both the genes and thus inhibited ethylene production. Furthermore, we have detected a GCC-box putative ethylene-responsive element (ERE)- and an auxin-responsive element (ARE)-specific DNA-binding activity in the banana pulp and studied the ethylene and auxin responsive characteristics of the GCC-box and ARE (TGTCTC) containing synthetic promoter fragments. In addition, we have detected an enhanced ethylene production rate and expression level of MA-ACS1 and MA-ACO1 genes along with a strong GCC-box-specific DNA-binding activity following exposure to constant dark period for 8d at the preclimacteric stage. Together, our study provides interesting information about the regulation of expression of MA-ACS1 and MA-ACO1 genes in response to various factors during ripening in banana fruit, which may have physiological relevance concerning ethylene biosynthesis during post-harvest conditions.

Constitutive or seed-specific overexpression of Arabidopsis G-protein γ subunit 3 (AGG3) results in increased seed and oil production and improved stress tolerance in Camelina sativa

Heterotrimeric G-proteins consisting of Gα, Gβ and Gγ subunits play an integral role in mediating multiple signalling pathways in plants. A novel, recently identified plant-specific Gγ protein, AGG3, has been proposed to be an important regulator of organ size and mediator of stress responses in Arabidopsis, whereas its potential homologs in rice are major quantitative trait loci for seed size and panicle branching. To evaluate the role of AGG3 towards seed and oil yield improvement, the gene was overexpressed in Camelina sativa, an oilseed crop of the Brassicaceae family. Analysis of multiple homozygous T4 transgenic Camelina lines showed that constitutive overexpression of AGG3 resulted in faster vegetative as well as reproductive growth accompanied by an increase in photosynthetic efficiency. Moreover, when expressed constitutively or specifically in seed tissue, AGG3 was found to increase seed size, seed mass and seed number per plant by 15%-40%, effectively resulting in significantly higher oil yield per plant. AGG3 overexpressing Camelina plants also exhibited improved stress tolerance. These observations draw a strong link between the roles of AGG3 in regulating two critical yield parameters, seed traits and plant stress responses, and reveal an effective biotechnological tool to dramatically increase yield in agricultural crops.

Differential transcriptional regulation of banana sucrose phosphate synthase gene in response to ethylene, auxin, wounding, low temperature and different photoperiods during fruit ripening and functional analysis of banana SPS gene promoter

Sucrose phosphate synthase (SPS) (EC 2.3.1.14) is the key regulatory component in sucrose formation in banana (Musa acuminata subgroup Cavendish, cv Giant governor) fruit during ripening. This report illustrates differential transcriptional responses of banana SPS gene following ethylene, auxin, wounding, low temperature and different photoperiods during ripening in banana fruit. Whereas ethylene strongly stimulated SPS transcript accumulation, auxin and cold treatment only marginally increased the abundance of SPS mRNA level, while wounding negatively regulated SPS gene expression. Conversely, SPS transcript level was distinctly increased by constant exposure to white light. Protein level, enzymatic activity of SPS and sucrose synthesis were substantially increased by ethylene and increased exposure to white light conditions as compared to other treatments. To further study the transcriptional regulation of SPS in banana fruit, the promoter region of SPS gene was cloned and some cis-acting regulatory elements such as a reverse GCC-box ERE, two ARE motifs (TGTCTC), one LTRE (CCGAA), a GAGA-box (GAGA…) and a GATA-box LRE (GATAAG) were identified along with the TATA and CAAT-box. DNA–protein interaction studies using these cis-elements indicated a highly specific cis–trans interaction in the banana nuclear extract. Furthermore, we specifically studied the light responsive characteristics of GATA-box containing synthetic as well as native banana SPS promoter. Transient expression assays using banana SPS promoter have also indicated the functional importance of the SPS promoter in regulating gene expression. Together, these results provide insights into the transcriptional regulation of banana SPS gene in response to phytohormones and other environmental factors during fruit ripening.

Characterization of differential ripening pattern in association with ethylene biosynthesis in the fruits of five naturally occurring banana cultivars and detection of a GCC-box-specific DNA-binding protein.

MA-ACS1 and MA-ACO1 are the two major ripening genes in banana and play crucial role in the regulation of ethylene production during ripening. Here, we report a comparative ripening pattern in five different naturally occurring banana cultivars namely Cavendish (AAA), Rasthali (AAB), Kanthali (AB), Poovan (AAB) and Monthan (ABB), which have distinct genome composition. We found a distinct variation in the climacteric ethylene production and in-vivo ACC oxidase activity level during the ripening stages in the five cultivars. We identified the cDNAs for MA-ACS1 and MA-ACO1 from the five cultivars and studied the transcript accumulation patterns of the two genes, which correlated well with the differential timing in the expression of these two genes during ripening. The GCC-box is one of the ethylene-responsive elements (EREs) found in the promoters of many ethylene-inducible genes. We have identified a GCC-box motif (putative ERE) in the promoters of MA-ACS1 and MA-ACO1 in banana cultivars. DNA–protein interaction studies revealed the presence of a GCC-box-specific DNA-binding activity in the fruit nuclear extract and such DNA-binding activity was enhanced following ethylene treatment. South-Western blotting revealed a 25-kDa nuclear protein that binds specifically to GCC-box DNA in the climacteric banana fruit. Together, these results indicate the probable involvement of the GCC-box motif as the cis-acting ERE in the regulation of MA-ACS1 and MA-ACO1 during ripening in banana fruits via binding of specific ERE-binding protein.

Specific Subunits of Heterotrimeric G Proteins Play Important Roles during Nodulation in Soybean

Subunits of heterotrimeric G proteins are positive regulators of nodule formation in soybean. Interactions with nod factor receptor NFR1 implicates plant G proteins in a yet unexplored signaling network. Heterotrimeric G proteins comprising Gα, Gβ, and Gγ subunits regulate many fundamental growth and development processes in all eukaryotes. Plants possess a relatively limited number of G-protein components compared with mammalian systems, and their detailed functional characterization has been performed mostly in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa). However, the presence of single Gα and Gβ proteins in both these species has significantly undermined the complexity and specificity of response regulation in plant G-protein signaling. There is ample pharmacological evidence for the role of G proteins in regulation of legume-specific processes such as nodulation, but the lack of genetic data from a leguminous species has restricted its direct assessment. Our recent identification and characterization of an elaborate G-protein family in soybean (Glycine max) and the availability of appropriate molecular-genetic resources have allowed us to directly evaluate the role of G-protein subunits during nodulation. We demonstrate that all G-protein genes are expressed in nodules and exhibit significant changes in their expression in response to Bradyrhizobium japonicum infection and in representative supernodulating and nonnodulating soybean mutants. RNA interference suppression and overexpression of specific G-protein components results in lower and higher nodule numbers, respectively, validating their roles as positive regulators of nodule formation. Our data further show preferential usage of distinct G-protein subunits in the presence of an additional signal during nodulation. Interestingly, the Gα proteins directly interact with the soybean nodulation factor receptors NFR1α and NFR1β, suggesting that the plant G proteins may couple with receptors other than the canonical heptahelical receptors common in metazoans to modulate signaling.

Characterization of an AGAMOUS-like MADS Box Protein, a Probable Constituent of Flowering and Fruit Ripening Regulatory System in Banana

The MADS-box family of genes has been shown to play a significant role in the development of reproductive organs, including dry and fleshy fruits. In this study, the molecular properties of an AGAMOUS like MADS box transcription factor in banana cultivar Giant governor (Musa sp, AAA group, subgroup Cavendish) has been elucidated. We have detected a CArG-box sequence binding AGAMOUS MADS-box protein in banana flower and fruit nuclear extracts in DNA-protein interaction assays. The protein fraction in the DNA-protein complex was analyzed by mass spectrometry and using this information we have obtained the full length cDNA of the corresponding protein. The deduced protein sequence showed ∼95% amino acid sequence homology with MA-MADS5, a MADS-box protein described previously from banana. We have characterized the domains of the identified AGAMOUS MADS-box protein involved in DNA binding and homodimer formation in vitro using full-length and truncated versions of affinity purified recombinant proteins. Furthermore, in order to gain insight about how DNA bending is achieved by this MADS-box factor, we performed circular permutation and phasing analysis using the wild type recombinant protein. The AGAMOUS MADS-box protein identified in this study has been found to predominantly accumulate in the climacteric fruit pulp and also in female flower ovary. In vivo and in vitro assays have revealed specific binding of the identified AGAMOUS MADS-box protein to CArG-box sequence in the promoters of major ripening genes in banana fruit. Overall, the expression patterns of this MADS-box protein in banana female flower ovary and during various phases of fruit ripening along with the interaction of the protein to the CArG-box sequence in the promoters of major ripening genes lead to interesting assumption about the possible involvement of this AGAMOUS MADS-box factor in banana fruit ripening and floral reproductive organ development.

Quantitative proteomics-based analysis supports a significant role of GTG proteins in regulation of ABA response in Arabidopsis roots.

Abscisic acid (ABA) is proposed to be perceived by multiple receptors in plants. We have previously reported on the role of two GPCR-type G-proteins (GTG proteins) as plasma membrane-localized ABA receptors in Arabidopsis thaliana. However, due to the presence of multiple transmembrane domains, detailed structural and biochemical characterization of GTG proteins remains limited. Since ABA induces substantial changes in the proteome of plants, a labeling LC-based quantitative proteomics approach was applied to elucidate the global effects and possible downstream targets of GTG1/GTG2 proteins. Quantitative differences in protein abundance between wild-type and gtg1gtg2 were analyzed for evaluation of the effect of ABA on the root proteome and its dependence on the presence of functional GTG1/GTG2 proteins. The results presented in this study reveal the most comprehensive ABA-responsive root proteome reported to date in Arabidopsis. Notably, the majority of ABA-responsive proteins required the presence of GTG proteins, supporting their key role in ABA signaling. These observations were further confirmed by additional experiments. Overall, comparison of the ABA-dependent protein abundance changes in wild-type versus gtg1gtg2 provides clues to their possible links with some of the well-established effectors of the ABA signaling pathways and their role in mediating phytohormone cross-talk.

Molecular characterization and differential expression of β-1,3-glucanase during ripening in banana fruit in response to ethylene, auxin, ABA, wounding, cold and light-dark cycles

Abstractβ-1,3-Glucanases (E.C. 3.2.1.39) are widely distributed enzyme among bacteria, fungi, and higher plants. Analyses of accumulation levels of β-1,3-glucanase protein in various tissues in banana have clearly indicated abundance of β-1,3-glucanase protein accumulation in ripe pulp tissue. After cloning of β-1,3-glucanase from banana pulp (cultivar Cavendish), we have carried out an in silico analysis to investigate the sequential, structural, and phylogenetic characteristics of the putative banana β-1,3-glucanase protein. As like other ripening specific genes, β-1,3-glucanase is regulated in response to a wide variety of factors. Therefore, we have analyzed the transcript accumulation pattern and protein levels of β-1,3-glucanase in response to ethylene, auxin, ABA, wounding and, low temperature in preclimacteric banana fruit. Expression profile analyses have indicated that whereas exogenous application of ethylene strongly stimulated β-1,3-glucanase transcript accumulation, ABA partially induced the expression of the gene. On the other hand, wound treatment did not induce β-1,3-glucanase expression. Conversely, auxin and cold treatment negatively regulated β-1,3-glucanase gene expression and thus inhibited glucanase activity. In addition, β-1,3-glucanase transcript level was markedly decreased by constant exposure to white light. Protein level and enzymatic activity of β-1,3-glucanase were substantially increased with considerable decrease in fruit firmness by ethylene treatment and reduced exposure to white light conditions as compared with other treatments. Together, the overall study of β-1,3-glucanase expression pattern, glucanase activity, and changes in fruit firmness during ripening in various conditions suggest the possible physiological function of β-1,3-glucanase in fruit pulp softening.

AtPolλ, A Homolog of Mammalian DNA Polymerase λ in Arabidopsis thaliana, is Involved in the Repair of UV-B Induced DNA Damage Through the Dark Repair Pathway

Plants are constantly exposed to a wide range of environmental genotoxic stress factors including obligatory exposure to UV radiation in sunlight. Here, we report the functional characterization of a DNA repair protein, AtPolλ, a homolog of mammalian DNA polymerase λ in Arabidopsis, in relation to its role in repair of UV-B-induced DNA damage during early stages of seedling development. The abundance of the AtPolλ transcript and the protein levels were distinctly increased in response to UV-B irradiation in 6-day-old wild-type seedlings. Growth of atpolλ mutant seedlings, deficient in AtPolλ expression, was more sensitive to UV-B radiation compared with wild-type plants when seeds were exposed to UV-B radiation before germination. The atpolλ mutants showed accumulation of relatively higher amounts of DNA lesions than wild-type plants following UV-B exposure and were less proficient in repair of UV-induced DNA damage. Increased accumulation of AtPolλ protein in UV-B-irradiated 6-day-old wild-type seedlings during the dark recovery period has indicated a possible role for the protein in repair of UV-B-induced lesions in the dark. Overexpression of AtPolλ in the atpolλ mutant line partially complemented the repair proficiency of UV-B-induced DNA damage. In vitro repair synthesis assays using whole-cell extracts from the wild-type and atpolλ mutant line have further demonstrated the role of AtPolλ in repair synthesis of UV-B-damaged DNA in the dark through an excision repair mechanism. Overall, our results have indicated the possible involvement of AtPolλ in a plants response for repair of UV-B-mediated DNA damage during seedling development.