Sujit Roy

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.

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.

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.

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.

Characterization of cultivar differences in β-1,3 glucanase gene expression, glucanase activity and fruit pulp softening rates during fruit ripening in three naturally occurring banana cultivars.

Abstractβ-1,3 glucanase (E.C.3.2.1.39) is the key enzyme involved in the hydrolytic cleavage of 1,3 β-D glucosidic linkages in β-1,3 glucans. This work describes a comparative analysis of expression patterns of β-1,3 glucanase gene in relation to changes in fruit pulp softening rates in three banana cultivars, Rasthali (AAB), Kanthali (AB), and Monthan (ABB). Analysis of transcript and protein levels of β-1,3 glucanase gene during ripening revealed differential timing in expression of the gene which correlated well with the variation in enzymatic activity of glucanase and fruit pulp softening rates in the three cultivars. Exogenously applied ethylene strongly induced β-1,3 glucanase expression during the early ripening days in Rasthali, while the expression of the gene was marginally stimulated following ethylene treatment in preclimacteric Kanthali fruit. Conversely, in Monthan, β-1,3 glucanase expression was very low throughout the ripening stages, and ethylene treatment did not induce the expression of the gene in this cultivar. Analysis of glucanase activity using protein extracts from unripe and ripe fruit of Monthan with crude cell wall polysaccharide fractions (used as substrate) indicated that the natural substrate for glucanase remained almost unutilized in this cultivar due to low in vivo glucanase activity. Furthermore, the recombinant β-1,3 glucanase protein, overexpressed in E. coli, showed requirement for substrates with contiguous β-1,3 linkages for optimal activity. Overall, our results provide new information on the expression profile of β-1,3glucanase gene in connection with the pattern of changes in fruit firmness at the physiological and molecular levels during ripening in three banana cultivars.

Sequential, Structural, and Phylogenetic Study of BRCT Module in Plants

Abstract The BRCT (Breast Cancer Carboxyl Terminus) domain is widely distributed in proteins involved in DNA metabolism and cell cycle regulation. In most of the representative members of the BRCT family, this domain is usually comprising of about 90–100 amino acid residues and generally present as single motif or in tandem repeats. Although the members of BRCT family share little sequence similarity, structural studies have demonstrated a relatively conserved structure of two or three a-helices surrounding the central β-sheets. This report illustrates an in silico analysis with the aim of understanding the sequential, structural, and phylogenetic features of BRCT domain in higher plant genome. Based on database searches 25 BRCT domain containing proteins were identified and many of them were found to be involved in multiple DNA damage repair pathways. We have further combined the homology modeling in order to address the structure-function relations of BRCT domain in connection with DNA damage repair mechanism in plants.

Involvement of AtPolλ in the Repair of High Salt- and DNA Cross-Linking Agent-Induced Double Strand Breaks in Arabidopsis

DNA Pol λ participates in the repair of double strand breaks induced by high salinity and DNA cross-linking agent in Arabidopsis seedlings, demonstrating the importance of Pol λ in the double strand break repair signaling network in higher plant genome. DNA polymerase λ (Pol λ) is the sole member of family X DNA polymerase in plants and plays a crucial role in nuclear DNA damage repair. Here, we report the transcriptional up-regulation of Arabidopsis (Arabidopsis thaliana) AtPolλ in response to abiotic and genotoxic stress, including salinity and the DNA cross-linking agent mitomycin C (MMC). The increased sensitivity of atpolλ knockout mutants toward high salinity and MMC treatments, with higher levels of accumulation of double strand breaks (DSBs) than wild-type plants and delayed repair of DSBs, has suggested the requirement of Pol λ in DSB repair in plants. AtPolλ overexpression moderately complemented the deficiency of DSB repair capacity in atpolλ mutants. Transcriptional up-regulation of major nonhomologous end joining (NHEJ) pathway genes KU80, X-RAY CROSS COMPLEMENTATION PROTEIN4 (XRCC4), and DNA Ligase4 (Lig4) along with AtPolλ in Arabidopsis seedlings, and the increased sensitivity of atpolλ-2/atxrcc4 and atpolλ-2/atlig4 double mutants toward high salinity and MMC treatments, indicated the involvement of NHEJ-mediated repair of salinity- and MMC-induced DSBs. The suppressed expression of NHEJ genes in atpolλ mutants suggested complex transcriptional regulation of NHEJ genes. Pol λ interacted directly with XRCC4 and Lig4 via its N-terminal breast cancer-associated C terminus (BRCT) domain in a yeast two-hybrid system, while increased sensitivity of BRCT-deficient Pol λ-expressing transgenic atpolλ-2 mutants toward genotoxins indicated the importance of the BRCT domain of AtPolλ in mediating the interactions for processing DSBs. Our findings provide evidence for the direct involvement of DNA Pol λ in the repair of DSBs in a plant genome.

An insight into the biological functions of family X-DNA polymerase in DNA replication and repair of plant genome

Recently we have reported the characterization of a novel single subunit 62-kDa polypeptide with ddNTP-sensitive DNA polymerase activity from the developing seeds of mungbean (Vigna radiata). The protein showed higher expression and activity level during nuclear endoreduplication stages of mungbean seeds and similarity with mammalian DNA polymerase in many physicochemical properties.1 The enzyme was found to specifically interact with PCNA (proliferating cell nuclear antigen),2 and expressed in both meristematic and meiotic tissues. Functional assays have demonstrated binding of the enzyme to normal and mismatched DNA substrates and with fidelity DNA synthesis in moderately processive mode, suggesting probable involvement of the enzyme in both replication and recombination.3 Here we have discussed the position of mungbean DNA polymerase as a homologue of DNA Pol λone of the newly identified member of family-X DNA polymerase in plants and illustrated the functional relevance of this enzyme in maintaining the coordination between DNA replication and repair in plant genome.