MYB4, a member of R2R3-subfamily of MYB transcription factor functions as a repressor of key genes involved in flavonoid biosynthesis and repair of UV-B induced DNA double strand breaks in Arabidopsis.

Plants accumulate flavonoids as part of UV-B acclimation, while a high level of UV-B irradiation induces DNA damage and leads to genome instability. Here, we show that MYB4, a member of the R2R3-subfamily of MYB transcription factor plays important role in regulating plant response to UV-B exposure through the direct repression of the key genes involved in flavonoids biosynthesis and repair of DNA double-strand breaks (DSBs). Our results demonstrate that MYB4 inhibits seed germination and seedling establishment in Arabidopsis following UV-B exposure. Phenotype analyses of atmyb4-1 single mutant line along with uvr8-6/atmyb4-1, cop1-6/atmyb4-1, and hy5-215/atmyb4-1 double mutants indicate that MYB4 functions downstream of UVR8 mediated signaling pathway and negatively affects UV-B acclimation and cotyledon expansion. Our results indicate that MYB4 acts as transcriptional repressor of two key flavonoid biosynthesis genes, including 4CL and FLS, via directly binding to their promoter, thus reducing flavonoid accumulation. On the other hand, AtMYB4 overexpression leads to higher accumulation level of DSBs along with repressed expression of several key DSB repair genes, including AtATM, AtKU70, AtLIG4, AtXRCC4, AtBRCA1, AtSOG1, AtRAD51, and AtRAD54, respectively. Our results further suggest that MYB4 protein represses the expression of two crucial DSB repair genes, AtKU70 and AtXRCC4 through direct binding with their promoters. Together, our results indicate that MYB4 functions as an important coordinator to regulate plant response to UV-B through transcriptional regulation of key genes involved in flavonoids biosynthesis and repair of UV-B induced DNA damage.

Modification of G-protein biochemistry and its effect on plant/environment interaction.

Heterotrimeric GTP-binding proteins comprised of Gα, Gß and Gγ subunits are key regulators of a multitude of signaling pathways in eukaryotes. In plants, G-proteins are currently a focus of intense research due to their involvement in modulation of many agronomically important traits such as seed yield, organ size, abscisic acid (ABA)-dependent signaling and stress responses, plant defense responses, symbiosis and nitrogen use efficiency. The mechanistic details of G-protein biochemistry in modulating these processes in plants remain largely unknown. Although the core G-protein components and their activation/deactivation chemistries are broadly conserved throughout eukaryotic evolution, their regulation seems to have been rewired in plants to meet specific needs. Plant G-proteins may be spontaneously active and/or are regulated by phosphorylation-dependent changes, by the activity of lipid second messengers such as phospholipases, or may even have nucleotide-exchange independent regulation. Regardless of these deviations from the established norm, the biochemical properties of plant G-proteins are key to affecting plant phenotypes and responses. Detailed characterization of such activities, in vitro and in planta, will pave the way for precise manipulation of these proteins for future agricultural needs.

Nucleolar GTP-Binding Protein 1-2 (NOG1-2) Interacts with Jasmonate-ZIMDomain Protein 9 (JAZ9) to Regulate Stomatal Aperture during Plant Immunity.

Plant defense responses at stomata and apoplast are the most important early events during plant⁻bacteria interactions. The key components of stomatal defense responses have not been fully characterized. A GTPase encoding gene, NOG1-2, which is required for stomatal innate immunity against bacterial pathogens, was recently identified. Functional studies in Arabidopsis revealed that NOG1-2 regulates guard cell signaling in response to biotic and abiotic stimulus through jasmonic acid (JA)- and abscisic acid (ABA)-mediated pathways. Interestingly, in this study, Jasmonate-ZIM-domain protein 9 (JAZ9) was identified to interact with NOG1-2 for the regulation of stomatal closure. Upon interaction, JAZ9 reduces GTPase activity of NOG1-2. We explored the role of NOG1-2 binding with JAZ9 for COI1-mediated JA signaling and hypothesized that its function may be closely linked to MYC2 transcription factor in the regulation of the JA-signaling cascade in stomatal defense against bacterial pathogens. Our study provides valuable information on the function of a small GTPase, NOG1-2, in guard cell signaling and early plant defense in response to bacterial pathogens.

The small GTPase, nucleolar GTP-binding protein 1 (NOG1), has a novel role in plant innate immunity.

Plant defense responses at stomata and apoplast are the most important early events during plant-bacteria interactions. The key components for the signaling of stomatal defense and nonhost resistance have not been fully characterized. Here we report the newly identified small GTPase, Nucleolar GTP-binding protein 1 (NOG1), functions for plant immunity against bacterial pathogens. Virus-induced gene silencing of NOG1 compromised nonhost resistance in N. benthamiana and tomato. Comparative genomic analysis showed that two NOG1 copies are present in all known plant species: NOG1-1 and NOG1-2. Gene downregulation and overexpression studies of NOG1-1 and NOG1-2 in Arabidopsis revealed the novel function of these genes in nonhost resistance and stomatal defense against bacterial pathogens, respectively. Specially, NOG1-2 regulates guard cell signaling in response to biotic and abiotic stimuli through jasmonic acid (JA)- and abscisic acid (ABA)-mediated pathways. The results here provide valuable information on the new functional role of small GTPase, NOG1, in guard cell signaling and early plant defense in response to bacterial pathogens.

Phosphorylation-Dependent Regulation of G-Protein Cycle during Nodule Formation in Soybean.

Signaling pathways mediated by heterotrimeric G-protein complexes comprising Gα, Gß, and Gγ subunits and their regulatory RGS (Regulator of G-protein Signaling) protein are conserved in all eukaryotes. We have shown that the specific Gß and Gγ proteins of a soybean (Glycine max) heterotrimeric G-protein complex are involved in regulation of nodulation. We now demonstrate the role of Nod factor receptor 1 (NFR1)-mediated phosphorylation in regulation of the G-protein cycle during nodulation in soybean. We also show that during nodulation, the G-protein cycle is regulated by the activity of RGS proteins. Lower or higher expression of RGS proteins results in fewer or more nodules, respectively. NFR1 interacts with RGS proteins and phosphorylates them. Analysis of phosphorylated RGS protein identifies specific amino acids that, when phosphorylated, result in significantly higher GTPase accelerating activity. These data point to phosphorylation-based regulation of G-protein signaling during nodule development. We propose that active NFR1 receptors phosphorylate and activate RGS proteins, which help maintain the Gα proteins in their inactive, trimeric conformation, resulting in successful nodule development. Alternatively, RGS proteins might also have a direct role in regulating nodulation because overexpression of their phospho-mimic version leads to partial restoration of nodule formation in nod49 mutants.

Measurement of GTP-binding and GTPase activity of heterotrimeric Gα proteins.

Heterotrimeric G-proteins are important signaling intermediates in all eukaryotes. These proteins link signal perception by a cell surface localized receptor to the downstream effectors of a given signaling pathways. The minimal core of the heterotrimeric G-protein complex consists of Gα, Gß, and Gγ subunits, the G protein coupled receptor (GPCR) and the regulator of G-protein signaling (RGS) proteins. Signal transduction by heterotrimeric G-proteins is controlled by the distinct biochemical activities of Gα protein, which binds and hydrolyses GTP. Evaluation of the rate of GTP binding, the rate of GTP hydrolysis, and the rate of GTP/GDP exchange on Gα protein are required to better understand the mechanistic aspects of heterotrimeric G-protein signaling, which remains significantly limited for the plant G-proteins. Here we describe the optimized methods for measurement of the distinct biochemical activities of the Arabidopsis Gα protein.

Involvement of AtPolλ in the repair of high salt- and DNA cross-linking agent-induced double strand breaks in Arabidopsis.

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.

Specific subunits of heterotrimeric G proteins play important roles during nodulation in soybean.

Heterotrimeric G proteins comprising Gα, Gß, 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 Gß 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 NFR1ß, suggesting that the plant G proteins may couple with receptors other than the canonical heptahelical receptors common in metazoans to modulate signaling.

C-terminal phosphorylation is essential for regulation of ethylene synthesizing ACC synthase enzyme.

The genetic and molecular biological studies mainly in Arabidopsis and in some other plants have begun to uncover the various components of ripening signaling pathway in plants. Although transcriptional regulation of major ripening genes have been studied in detail, information on role of phosphorylation in regulating the activity and stability of core ripening pathway associated proteins in relation to ethylene biosynthesis during fruit ripening is still limited. Recently we have demonstrated the evidence for post-translational regulation of MA-ACS1 (Musa acuminata ACC synthase 1), the rate limiting step enzyme regulating ripening ethylene production in banana, through phosphorylation at the C-terminal Ser 476 and 479 residues by a 41-kDa Ser/Thr protein kinase. (1) Here we have further discussed role of protein phosphorylation in regulation of stability and activity of ACS enzymes and the mechanistic and evolutionary perspective of phosphorylation pattern of Type I ACC synthase enzymes.

The RGS proteins add to the diversity of soybean heterotrimeric G-protein signaling.

Regulator of G-protein signaling (RGS) proteins are a family of highly diverse, multifunctional proteins that function primarily as GTPase accelerating proteins (GAPs). RGS proteins increase the rate of GTP hydrolysis by Gα proteins and essentially regulate the duration of active signaling. Recently, we have identified two chimeric RGS proteins from soybean and reported their distinct GAP activities on individual Gα proteins. A single amino acid substitution (Alanine 357 to Valine) of RGS2 is responsible for differential GAP activity. Surprisingly, most monocot plant genomes do not encode for a RGS protein homolog. Here we discuss the soybean RGS proteins in the context of their evolution in plants, their relatedness to non-plant RGS protein homologs and the effect they might have on the heterotrimeric G-protein signaling mechanisms. We also provide experimental evidence to show that the interaction interface between plant RGS and Gα proteins is different from what is predicted based on mammalian models.

A Ser/Thr protein kinase phosphorylates MA-ACS1 (Musa acuminata 1-aminocyclopropane-1-carboxylic acid synthase 1) during banana fruit ripening.

1-Aminocyclopropane-1-carboxylic acid synthase (ACS) catalyzes the rate-limiting step in ethylene biosynthesis during ripening. ACS isozymes are regulated both transcriptionally and post-translationally. However, in banana, an important climacteric fruit, little is known about post-translational regulation of ACS. Here, we report the post-translational modification of MA-ACS1 (Musa acuminata ACS1), a ripening inducible isozyme in the ACS family, which plays a key role in ethylene biosynthesis during banana fruit ripening. Immunoprecipitation analyses of phospholabeled protein extracts from banana fruit using affinity-purified anti-MA-ACS1 antibody have revealed phosphorylation of MA-ACS1, particularly in ripe fruit tissue. We have identified the induction of a 41-kDa protein kinase activity in pulp at the onset of ripening. The 41-kDa protein kinase has been identified as a putative protein kinase by MALDI-TOF/MS analysis. Biochemical analyses using partially purified protein kinase fraction from banana fruit have identified the protein kinase as a Ser/Thr family of protein kinase and its possible involvement in MA-ACS1 phosphorylation during ripening. In vitro phosphorylation analyses using synthetic peptides and site-directed mutagenized recombinant MA-ACS1 have revealed that serine 476 and 479 residues at the C-terminal region of MA-ACS1 are phosphorylated. Overall, this study provides important novel evidence for in vivo phosphorylation of MA-ACS1 at the molecular level as a possible mechanism of post-translational regulation of this key regulatory protein in ethylene signaling pathway in banana fruit during ripening.

Functional analysis of light-regulated promoter region of AtPolλ gene.

Genetic and molecular analyses mainly in Arabidopsis and in some other plants have demonstrated involvement of light signaling in cell cycle regulation. In this report, we show light-mediated activation of the promoter of AtPolλ gene, a homolog of mammalian DNA polymerase λ in Arabidopsis thaliana and an important component of DNA damage repair/recombination machinery in plants. Analyses of the light-mediated promoter activity using various deletion versions of AtPolλ promoter in transformed Arabidopsis and tobacco (Nicotiana tabaccum) plants indicate that a 130-bp promoter region between -536 and -408 of AtPolλ promoter is essential for light-induced regulation of AtPolλ expression. DNA-protein interaction studies reveal that an ATCT-motif and AE-box light-responsive elements in the light-regulated promoter region confer light responsiveness of AtPolλ promoter. DNA-binding analysis has identified a 63-kDa trans-acting protein factor which showed specific binding to ATCT-motif, while another trans-acting factor of ~52 kDa was found to bind specifically to both ATCT and AE-box sequences. The 52-kDa protein has been identified as B3-domain transcription factor by MALDI-TOF/MS analysis. Overall, our results provide novel information on the role of light signaling in regulation of expression of an important component of DNA repair machinery in plants.

Conventional and novel Gγ protein families constitute the heterotrimeric G-protein signaling network in soybean.

Heterotrimeric G-proteins comprised of Gα, Gß and Gγ proteins are important signal transducers in all eukaryotes. The Gγ protein of the G-protein heterotrimer is crucial for its proper targeting at the plasma membrane and correct functioning. Gγ proteins are significantly smaller and more diverse than the Gα and Gß proteins. In model plants Arabidopsis and rice that have a single Gα and Gß protein, the presence of two canonical Gγ proteins provide some diversity to the possible heterotrimeric combinations. Our recent analysis of the latest version of the soybean genome has identified ten Gγ proteins which belong to three distinct families based on their C-termini. We amplified the full length cDNAs, analyzed their detailed expression profile by quantitative PCR, assessed their localization and performed yeast-based interaction analysis to evaluate interaction specificity with different Gß proteins. Our results show that ten Gγ genes are retained in the soybean genome and have interesting expression profiles across different developmental stages. Six of the newly identified proteins belong to two plant-specific Gγ protein families. Yeast-based interaction analyses predict some degree of interaction specificity between different Gß and Gγ proteins. This research thus identifies a highly diverse G-protein network from a plant species. Homologs of these novel proteins have been previously identified as QTLs for grain size and yield in rice.

Understanding DNA repair and recombination in higher plant genome: information from genome-wide screens in Arabidopsis and rice.

Recently we have reported the in silico identification and in depth analysis of genes potentially involve in DNA repair and recombination (DRR) in two fully sequenced higher plant genomes, Arabidopsis and rice. In spite of strong conservation of DRR gene along with all three domain of life, we found some peculiar difference in presence and function of DRR genes in plants. Beside the eukaryotic homologs, several prokaryotes specific genes were also found to be well conserved in both plant genomes. Several functionally important DRR gene duplications were found in Arabidopsis, which do not occur in rice. In spite of the fact that same DRR protein functions in different DNA repair pathways, we found that proteins belonging to the nucleotide excision repair (NER) pathway were relatively more conserved than proteins needed for the other DRR pathways. Identified DRR gene were found to reside in nucleus mainly while gene drain in between nucleus and cell organelles were also found in some cases. Here, we have discussed the peculiar features of DRR genes in higher plant genomes.

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 plant's response for repair of UV-B-mediated DNA damage during seedling development.

An insight into the sequential, structural and phylogenetic properties of banana 1-aminocyclopropane-1-carboxylate synthase 1 and study of its interaction with pyridoxal-5'-phosphate and aminoethoxyvinylglycine.

In banana, ethylene production for ripening is accompanied by a dramatic increase in 1-aminocyclopropane-1-carboxylate (ACC) content, transcript level of Musa acuminata ACC synthase 1 (MA-ACS1) and the enzymatic activity of ACC synthase 1 at the onset of the climacteric period. MA-ACS1 catalyses the conversion of S-adenosyl-L-methionine (SAM) to ACC, the key regulatory step in ethylene biosynthesis. Multiple sequence alignments of 1-aminocyclopropane-1-carboxylate synthase (ACS) amino acid sequences based on database searches have indicated that MA-ACS1 is a highly conserved protein across the plant kingdom. This report describes an in silico analysis to provide the first important insightful information about the sequential, structural and phylogenetic characteristics of MA-ACS1. The three-dimensional structure of MA-ACS1, constructed based on homology modelling, in combination with the available data enabled a comparative mechanistic analysis of MA-ACS1 to explain the catalytic roles of the conserved and non-conserved active site residues. We have further demonstrated that, as in apple and tomato, banana- ACS1 (MA-ACS1) forms a homodimer and a complex with cofactor pyridoxal-5'-phosphate (PLP) and inhibitor aminoethoxyvinylglycine (AVG). We have also predicted that the residues from the PLP-binding pocket, essential for ligand binding, are mostly conserved across the MA-ACS1 structure and the competitive inhibitor AVG binds at a location adjacent to PLP.

DNA repair and recombination in higher plants: insights from comparative genomics of Arabidopsis and rice.

BACKGROUND: The DNA repair and recombination (DRR) proteins protect organisms against genetic damage, caused by environmental agents and other genotoxic agents, by removal of DNA lesions or helping to abide them. RESULTS: We identified genes potentially involved in DRR mechanisms in Arabidopsis and rice using similarity searches and conserved domain analysis against proteins known to be involved in DRR in human, yeast and E. coli. As expected, many of DRR genes are very similar to those found in other eukaryotes. Beside these eukaryotes specific genes, several prokaryotes specific genes were also found to be well conserved in plants. In Arabidopsis, several functionally important DRR gene duplications are present, which do not occur in rice. Among DRR proteins, we found that proteins belonging to the nucleotide excision repair pathway were relatively more conserved than proteins needed for the other DRR pathways. Sub-cellular localization studies of DRR gene suggests that these proteins are mostly reside in nucleus while gene drain in between nucleus and cell organelles were also found in some cases. CONCLUSIONS: The similarities and dissimilarities in between plants and other organisms' DRR pathways are discussed. The observed differences broaden our knowledge about DRR in the plants world, and raises the potential question of whether differentiated functions have evolved in some cases. These results, altogether, provide a useful framework for further experimental studies in these organisms.

Understanding the molecular mechanism of transcriptional regulation of banana Sucrose phosphate synthase (SPS) gene during fruit ripening: an insight into the functions of various cis-acting regulatory elements.

Recently, we have reported the characterization of promoter region of Sucrose phosphate synthase (SPS) gene in banana and investigated the role of some cis-elements/motifs, present in the promoter of SPS, in the transcriptional regulation of the gene. DNA-protein interaction studies have demonstrated the presence of specific trans-acting factors which showed specific interactions with ethylene, auxin, low temperature and light responsive elements in regulating SPS transcription. Transient expression analyses have demonstrated the functional significance of the various cis-acting regulatory elements present in banana SPS promoter in regulating SPS expression during ripening. (1) Here, we have further discussed the possible role of these regulatory sequences in the regulation of transcriptional network and comment on their function in relation to sucrose metabolism during banana fruit ripening.

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 beta in many physicochemical properties. The enzyme was found to specifically interact with PCNA (proliferating cell nuclear antigen), 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. Here we have discussed the position of mungbean DNA polymerase as a homologue of DNA Pol lambda, 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.