Specificity of Membrane-Associated J-Domain Protein, Caj1, in Amphotericin B Tolerance in Budding Yeast.

Hsp70:J-domain protein (JDP) machineries play pivotal roles in maintaining cellular proteostasis and governing various aspects of fungal physiology. While Hsp70 is known for its involvement in conferring tolerance to diverse antifungal drugs, the specific contribution of JDPs remains unclear. In this study, we examined the sensitivity of cytosolic JDP deletion strains of budding yeast to amphotericin B (AmB), a polyene antifungal agent widely utilized in fungal disease treatment due to its ability to disrupt the fungal plasma membrane (PM). Deleting Caj1, a PM-associated class II JDP, heightened susceptibility to AmB, and the protection conferred by Caj1 against AmB necessitated both its N-terminal J-domain and C-terminal lipid binding domain. Moreover, Caj1 deficiency compromised PM integrity as evidenced by increased phosphate efflux and exacerbated AmB sensitivity, particularly at elevated temperatures. Notably, phytosphingosine (PHS) addition as well as overexpression of PMP3, a positive PM integrity regulator, significantly rescued AmB sensitivity of caj1Δ cells. Our results align with the notion that Caj1 associates with the PM and cooperates with Hsp70 to regulate PM proteostasis, thereby influencing PM integrity in budding yeast. Loss of Caj1 function at the PM compromises PM protein quality control, thereby rendering yeast cells more susceptible to AmB.

Meta-QTL and ortho analysis unravels the genetic architecture and key candidate genes for cold tolerance at seedling stage in rice.

Rice, a critical cereal crop, grapples with productivity challenges due to its inherent sensitivity to low temperatures, primarily during the seedling and booting stages. Recognizing the polygenic complexity of cold stress signaling in rice, a meta-analysis was undertaken, focusing on 20 physiological traits integral to cold tolerance. This initiative allowed the consolidation of genetic data from 242 QTLs into 58 meta-QTLs, thereby significantly constricting the genetic and physical intervals, with 84% of meta-QTLs (MQTLs) being reduced to less than 2 Mb. The list of 10,505 genes within these MQTLs, was further refined utilizing expression datasets to pinpoint 46 pivotal genes exhibiting noteworthy differential regulation during cold stress. The study underscored the presence of several TFs such as WRKY, NAC, CBF/DREB, MYB, and bHLH, known for their roles in cold stress response. Further, ortho-analysis involving maize, barley, and Arabidopsis identified OsWRKY71, among others, as a prospective candidate for enhancing cold tolerance in diverse crop plants. In conclusion, our study delineates the intricate genetic architecture underpinning cold tolerance in rice and propounds significant candidate genes, offering crucial insights for further research and breeding strategies focused on fortifying crops against cold stress, thereby bolstering global food resilience. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01412-1.

Identification of subfunctionalized aggregate-remodeling J-domain proteins in Arabidopsis thaliana.

J-domain proteins (JDPs) are critical components of the cellular protein quality control machinery, playing crucial roles in preventing the formation and, solubilization of cytotoxic protein aggregates. Bacteria, yeast, and plants additionally have large, multimeric heat shock protein 100 (Hsp100)-class disaggregases that resolubilize protein aggregates. JDPs interact with aggregated proteins and specify the aggregate-remodeling activities of Hsp70s and Hsp100s. However, the aggregate-remodeling properties of plant JDPs are not well understood. Here we identify eight orthologs of Sis1 (an evolutionarily conserved Class II JDP of budding yeast) in Arabidopsis thaliana with distinct aggregate-remodeling functionalities. Six of these JDPs associate with heat-induced protein aggregates in vivo and co-localize with Hsp101 at heat-induced protein aggregate centers. Consistent with a role in solubilizing cytotoxic protein aggregates, an atDjB3 mutant had defects in both solubilizing heat-induced aggregates and acquired thermotolerance as compared with wild-type seedlings. Next, we used yeast prions as protein aggregate models to show that the six JDPs have distinct aggregate-remodeling properties. Results presented in this study, as well as findings from phylogenetic analysis, demonstrate that plants harbor multiple, evolutionarily conserved JDPs with capacity to process a variety of protein aggregate conformers induced by heat and other stressors.

J-like protein family of Arabidopsis thaliana: the enigmatic cousins of J-domain proteins.

KEY MESSAGE: J-like proteins (JLPs) are emerging as ancillaries to the cellular chaperone network. They modulate functions of Hsp70:J-domain protein (JDP) systems in novel ways thereby having key roles in diverse plant processes. J-domain proteins (JDPs) form an obligate co-chaperone partnership with Hsp70s with their highly conserved J-domain to steer protein quality control processes in the cell. The HPD motif between helix II and helix III of the J-domain is crucial for JDP's interaction with Hsp70s. According to the most recent classification, J-like proteins (JLPs) form an extended class of the JDP family possessing a degenerate J-domain with the HPD motif non-conservatively replaced by other amino acid residues and hence are not able to interact with Hsp70s. Considering this most updated and acceptable JLP classification, we identified 21 JLPs in Arabidopsis thaliana that share a structurally conserved J-like domain (JLD), but lack the HPD motif. Analysis of publicly available gene expression data as well as real-time quantitative PCR performed for a few selected JLPs implicated some of these proteins in growth, development and stress response. Here, we summarize the current state of knowledge on plant JLPs and their involvement in vital plant cellular/metabolic processes, including chloroplast division, mitochondrial protein import and flowering. Finally, we propose possible modes of action for these highly elusive proteins and other DnaJ-related proteins (DNAJRs) in regulating the Hsp70 chaperone network.

Expansion of the evolutionarily conserved network of J-domain proteins in the Arabidopsis mitochondrial import complex.

KEY MESSAGE: We report that discriminate interaction between the expanded mitochondrial chaperone network and variability in their expression might determine their functional specificities and impart robustness to mitochondrial import processes in plants. Mitochondrial Hsp70 (mtHsp70), the central component of the pre-sequence associated motor (PAM) complex, is crucial for the import of proteins to the mitochondrial matrix. Activity of mtHsp70 is regulated by a heterodimeric complex of two J-domain proteins (JDPs), Pam18 and Pam16. Compared to other eukaryotes, plants harbor multiple copies of these JDPs, which posit that plants have an increasingly complex mtHsp70: JDP network in their mitochondrial matrix. Here, we show that although highly similar in sequence, some of the plant JDPs are functionally different. Protein: protein interaction studies including yeast two-hybrid and Bimolecular Fluorescence Complementation revealed that while all the AtPam18s interacted with AtPam16s, the strengths of these promiscuous interactions are variable. Further, down-regulation of AtPAM16L affected seed germination, even in the presence of its seemingly identical paralog, AtPAM16. Knockdown of AtPAM16L caused reduction in mitochondrial number and deregulation of several mitochondrial genes, suggesting towards a specific role of AtPam16L in maintaining mitochondrial homeostasis, especially under stress conditions. Our findings suggest that variations in the spatio-temporal expression, accompanied by discriminate interactions between the JDPs, might be defining the functional specificity of the mtHsp70 co-chaperone machinery and providing resilience to mitochondrial import processes in plants, especially under stress conditions.

Dosage sensitivity of JDPs, a valuable tool for understanding their function: a case study on Caj1 overexpression-mediated filamentous growth in budding yeast.

J-domain proteins (JDPs) partner with Hsp70s to oversee proper synthesis, folding, transport and turnover of proteins in the cell. In any subcellular compartment, often multiple JDPs collaborate with a single Hsp70 to perform a variety of functions. Being co-localized, JDPs may exhibit complex genetic and physical interactions with each other, their clients as well as the Hsp70 partners. Even though most JDPs are highly specialized, redundancy between them is possible, making their functional analysis challenging. In the absence of assayable deletion phenotypes, protein overexpression appears to be a powerful alternative strategy to study JDP function. Here, we show that high levels of Caj1, one of the cytosolic JDPs, cause filamentous growth and G2/M arrest in yeast cells. Mutation in the critical HPD motif in the J-domain of Caj1 completely abolished these phenotypes, suggesting that Hsp70 co-chaperone function is important for the dominant-negative phenotypes exhibited by Caj1 overexpression. In this paper, we discuss the possible underlying mechanisms responsible for the pleiotropic phenotypes displayed by Caj1 overexpression in the light of current models proposed for dosage-sensitive genes (DSGs). Finally, we present generalized mechanisms of JDP overexpression-mediated dominant-negative phenotypes in budding yeast.

Design, synthesis and biological evaluation of some tetrazole acetamide derivatives as novel non-carboxylic PTP1B inhibitors.

A series of ten N-(3-(1H-tetrazole-5-yl)phenyl)acetamide derivatives (NM-07 to NM-16) designed from a lead molecule identified previously in our laboratory were synthesized and evaluated for protein tyrosine phosphatase 1B (PTP1B) inhibitory activity. Among the synthesized molecules, NM-14, a 5-Cl substituted benzothiazole analogue elicited significant PTP1B inhibition with an IC50 of 1.88 µM against reference standard suramin (IC50 ≥ 10 µM). Furthermore, this molecule also showed good in vivo antidiabetic activity which was comparable to that of standard antidiabetic drugs metformin and glimepiride. Overall, the results of the study clearly reveal that the reported tetrazole derivatives especially NM-14 are valuable prototypes for the development of novel non-carboxylic inhibitors of PTP1B with antidiabetic potential.

Synthesis and biological evaluation of some N-(3-(1H-tetrazol-5-yl) phenyl)acetamide derivatives as novel non-carboxylic PTP1B inhibitors designed through bioisosteric modulation.

Described herein is the synthesis and biological evaluation of a series of non-carboxylic inhibitors of Protein Tyrosine Phosphatase 1B designed using bioisosteric replacement strategy. Six N-(3-(1H-tetrazol-5-yl)phenyl)acetamide derivatives designed employing the aforementioned strategy were synthesized and screened for PTP1B inhibitory activity. Among the synthesized compounds, compound NM-03 exhibited the most potent inhibitory activity with IC50 value of 4.48 µM. Docking studies with NM-03 revealed the key interactions with desired amino acids in the binding site of PTP1B. Furthermore, compound NM-03 also elicited good in vivo activity. Taken together, the results of this study establish N-(3-(1H-tetrazole-5-yl)phenyl)-2-(benzo[d]oxazol-2-ylthio)acetamide (NM-03) as a valuable lead molecule with great potential for PTP1B inhibitor development targeting diabetes.

Nucleoid localization of Hsp40 Mdj1 is important for its function in maintenance of mitochondrial DNA.

Faithful replication and propagation of mitochondrial DNA (mtDNA) is critical for cellular respiration. Molecular chaperones, ubiquitous proteins involved in protein folding and remodeling of protein complexes, have been implicated in mtDNA transactions. In particular, cells lacking Mdj1, an Hsp40 co-chaperone of Hsp70 in the mitochondrial matrix, do not maintain functional mtDNA. Here we report that the great majority of Mdj1 is associated with nucleoids, DNA-protein complexes that are the functional unit of mtDNA transactions. Underscoring the importance of Hsp70 chaperone activity in the maintenance of mtDNA, an Mdj1 variant having an alteration in the Hsp70-interacting J-domain does not maintain mtDNA. However, a J-domain containing fragment expressed at the level that Mdj1 is normally present is not competent to maintain mtDNA, suggesting a function of Mdj1 beyond that carried out by its J-domain. Nevertheless, loss of mtDNA function upon Mdj1 depletion is retarded when the J-domain, is overexpressed. Analysis of Mdj1 variants revealed a correlation between nucleoid association and DNA maintenance activity, suggesting that localization is functionally important. We found that Mdj1 has DNA binding activity and that variants retaining DNA-binding activity also retained nucleoid association. Together, our results are consistent with a model in which Mdj1, tethered to the nucleoid via DNA binding, thus driving a high local concentration of the Hsp70 machinery, is important for faithful DNA maintenance and propagation.

Sequential duplications of an ancient member of the DnaJ-family expanded the functional chaperone network in the eukaryotic cytosol.

Across eukaryotes, Hsp70-based chaperone machineries display an underlying unity in their sequence, structure, and biochemical mechanism of action, while working in a myriad of cellular processes. In good part, this extraordinary functional versatility is derived from the ability of a single Hsp70 to interact with an array of J-protein cochaperones to form a functional chaperone network. Among J-proteins, the DnaJ-type is the most prevalent, being present in all three kingdoms and in several different compartments of eukaryotic cells. However, because these ancient DnaJ-type proteins diverged at the base of the eukaryotic phylogeny, little is understood about the evolutionary basis of their diversification and thus the functional expansion of the chaperone network. Here, we report results of evolutionary and experimental analyses of two more recent members of the cytosolic DnaJ family of Saccharomyces cerevisiae, Xdj1 and Apj1, which emerged by sequential duplications of the ancient YDJ1 in Ascomycota. Sequence comparison and molecular modeling revealed that both Xdj1 and Apj1 maintained a domain organization similar to that of multifunctional Ydj1. However, despite these similarities, both Xdj1 and Apj1 evolved highly specialized functions. Xdj1 plays a unique role in the translocation of proteins from the cytosol into mitochondria. Apj1's specialized role is related to degradation of sumolyated proteins. Together these data provide the first clear example of cochaperone duplicates that evolved specialized functions, allowing expansion of the chaperone functional network, while maintaining the overall structural organization of their parental gene.

Arabidopsis Dph4 is an Hsp70 Cochaperone with Iron-Binding Properties.

J-domain proteins (JDPs) are obligate cochaperones of Hsp70s with a wide range of functions in protein homeostasis. Although the J-domain is required for the stimulation of Hsp70s ATPase activity, the functional specificity of JDPs is governed by domains or regions other than the J-domain. Jjj3/Dph4, a class III JDP, is required for diphthamide (DPH) biosynthesis in eukaryotes, including yeast and mammals. Dph4 has a conserved N-terminal J-domain and an uncharacterized C-terminal domain containing a signature CSL zinc finger motif. Previously, we showed that the Dph4 ortholog in Arabidopsis thaliana (atDjC13/AtJjj3/AtDph4) could restore DPH biosynthesis in yeast jjj3Δ mutant in a J-domain-dependent manner. Here, we characterize the C-terminal CSL motif of AtDph4 using yeast genetic and biochemical approaches. The CSL motif of AtDph4 is essential for DPH biosynthesis, and like human Dph4, AtDph4 showed distinct iron-binding activity, which is not present in its yeast counterpart. ScDph4 and AtDph4 proteins exhibit distinct iron-binding capabilities, as evidenced by UV-vis spectrophotometry, SEM-EDS (energy-dispersive spectroscopy function on the scanning electron microscope) and electron paramagnetic resonance (EPR) spectra analyses. Collectively, our data suggests that beyond their role as an Hsp70 cochaperone, Dph4 homologues in complex eukaryotes may have iron-binding abilities, indicating a potential role in iron-sulfur cluster assembly and iron homeostasis.

Unique characteristics of the J-domain proximal regions of Hsp70 cochaperone Apj1 in prion propagation/elimination and its overlap with Sis1 function.

J-domain proteins (JDPs) are obligate cochaperones of Hsp70s. The Class A JDP Apj1 of the yeast cytosol has an unusually complex region between the N-terminal J-domain and the substrate binding region-often called the Grich or GF region in Class A and B JDPs because of its typical abundance of glycine. The N-terminal 161-residue Apj1 fragment is known to be sufficient for Apj1 function in prion curing, driven by the overexpression of Hsp104. Further analyzing the N-terminal segment of Apj1, we found that a 90-residue fragment that includes the 70-residue J-domain and the adjacent 12-residue glutamine/alanine (Q/A) segment is sufficient for curing. Furthermore, the 121-residue fragment that includes the Grich region was sufficient to not only sustain the growth of cells lacking the essential Class B JDP Sis1 but also enabled the maintenance of several prions normally dependent on Sis1 for propagation. A J-domain from another cytosolic JDP could substitute for the Sis1-related functions but not for Apj1 in prion curing. Together, these results separate the functions of JDPs in prion biology and underscore the diverse functionality of multi-domain cytosolic JDPs in yeast.

J-domain proteins: From molecular mechanisms to diseases.

J-domain proteins (JDPs) are the largest family of chaperones in most organisms, but much of how they function within the network of other chaperones and protein quality control machineries is still an enigma. Here, we report on the latest findings related to JDP functions presented at a dedicated JDP workshop in Gdansk, Poland. The report does not include all (details) of what was shared and discussed at the meeting, because some of these original data have not yet been accepted for publication elsewhere or represented still preliminary observations at the time.

Arabidopsis plants overexpressing additional copies of heat shock protein Hsp101 showed high heat tolerance and endo-gene silencing.

Hsp101 chaperone is vital for survival of plants under heat stress. We generated transgenic Arabidopsis thaliana (Arabidopsis) lines with extra copies of Hsp101 gene using diverse approaches. Arabidopsis plants transformed with rice Hsp101 cDNA driven by Arabidopsis Hsp101 promoter (IN lines) showed high heat tolerance while the plants transformed with rice Hsp101 cDNA driven by CaMV35S promoter (C lines) were like wild type plants in heat stress response. Transformation of Col-0 plants with 4633 bp Hsp101 genomic fragment (GF lines) from A. thaliana containing both its coding and the regulatory sequence resulted in mostly over-expressor (OX) lines and a few under-expressor (UX) lines of Hsp101. OX lines showed enhanced heat tolerance while the UX lines were overly heat sensitive. In UX lines, silencing of not only Hsp101 endo-gene was noted but also transcript of choline kinase (CK2) was silenced. Previous work established that in Arabidopsis, CK2 and Hsp101 are convergent gene pairs sharing a bidirectional promoter. The elevated AtHsp101 protein amount in most GF and IN lines was accompanied by lowered CK2 transcript levels under HS. We observed increased methylation of the promoter and gene sequence region in UX lines; however, methylation was lacking in OX lines.

Volleying plasma membrane proteins from birth to death: Role of J-domain proteins.

The function, stability, and turnover of plasma membrane (PM) proteins are crucial for cellular homeostasis. Compared to soluble proteins, quality control of plasma membrane proteins is extremely challenging. Failure to meet the high quality control standards is detrimental to cellular and organismal health. J-domain proteins (JDPs) are among the most diverse group of chaperones that collaborate with other chaperones and protein degradation machinery to oversee cellular protein quality control (PQC). Although fragmented, the available literature from different models, including yeast, mammals, and plants, suggests that JDPs assist PM proteins with their synthesis, folding, and trafficking to their destination as well as their degradation, either through endocytic or proteasomal degradation pathways. Moreover, some JDPs interact directly with the membrane to regulate the stability and/or functionality of proteins at the PM. The deconvoluted picture emerging is that PM proteins are relayed from one JDP to another throughout their life cycle, further underscoring the versatility of the Hsp70:JDP machinery in the cell.

A targeted analysis of cellular chaperones reveals contrasting roles for heat shock protein 70 in flock house virus RNA replication.

Cytosolic chaperones are a diverse group of ubiquitous proteins that play central roles in multiple processes within the cell, including protein translation, folding, intracellular trafficking, and quality control. These cellular proteins have also been implicated in the replication of numerous viruses, although the full extent of their involvement in viral replication is unknown. We have previously shown that the heat shock protein 40 (hsp40) chaperone encoded by the yeast YDJ1 gene facilitates RNA replication of flock house virus (FHV), a well-studied and versatile positive-sense RNA model virus. To further explore the roles of chaperones in FHV replication, we examined a panel of 30 yeast strains with single deletions of cytosolic proteins that have known or hypothesized chaperone activity. We found that the majority of cytosolic chaperone deletions had no impact on FHV RNA accumulation, with the notable exception of J-domain-containing hsp40 chaperones, where deletion of APJ1 reduced FHV RNA accumulation by 60%, while deletion of ZUO1, JJJ1, or JJJ2 markedly increased FHV RNA accumulation, by 4- to 40-fold. Further studies using cross complementation and double-deletion strains revealed that the contrasting effects of J domain proteins were reproduced by altering expression of the major cytosolic hsp70s encoded by the SSA and SSB families and were mediated in part by divergent effects on FHV RNA polymerase synthesis. These results identify hsp70 chaperones as critical regulators of FHV RNA replication and indicate that cellular chaperones can have both positive and negative regulatory effects on virus replication.

Specificity of the J-protein Sis1 in the propagation of 3 yeast prions.

Yeast prions, such as [PSI(+)], [RNQ(+)], and [URE3], are heritable elements formed by proteins capable of acquiring self-perpetuating conformations. Their propagation is dependent on fragmentation of the amyloid protein complexes formed to generate the additional seeds necessary for conversion of nascent soluble protein to the prion conformation. We report that, in addition to its known role in [RNQ(+)] propagation, Sis1, a J-protein cochaperone of Hsp70 Ssa, is also specifically required for propagation of [PSI(+)] and [URE3]. Whereas both [RNQ(+)] and [URE3] are cured rapidly upon SIS1 repression, [PSI(+)] loss is markedly slower. This disparity cannot be explained simply by differences in seed number, as [RNQ(+)] and [PSI(+)] are lost with similar kinetics upon inhibition of Hsp104, a remodeling protein required for propagation of all yeast prions. Rather, in the case of [PSI(+)], our results are consistent with the partial impairment, rather than the complete abolition, of fragmentation of prion complexes upon Sis1 depletion. We suggest that a common set of molecular chaperones, the J-protein Sis1, the Hsp70 Ssa, and the AAA+ ATPase Hsp104, act sequentially in the fragmentation of all yeast prions, but that the threshold of Sis1 activity required for each prion varies.

Tyrosine-Templated Dual-Component Silver Nanomaterials Exhibit Photoluminescence and Versatile Antimicrobial Properties through ROS Generation.

The role of small molecules in the preparation of metal nanomaterials generates considerable interest in the fields from materials science to interdisciplinary sciences. In this study, a small amino acid, l-tyrosine (Tyr), has been used as a ligand precursor for the preparation of silver nanomaterials (AgNMs) comprising a dual system: smaller silver nanoclusters (responsible exclusively for the photophysical properties) and larger silver nanoparticles (responsible exclusively for the antimicrobial properties). The luminescent properties of this AgNM system substantiate the role played by Tyr as a capping and a reducing agent outside the protein environment. An interesting feature of this report is the promising antimicrobial properties of the AgNMs against Saccharomyces cerevisiae, Candida albicans, Escherichia coli, and Bacillus cereus cell lines. The importance of this work is that this investigation demonstrates the combating ability of our AgNM system against pathogenic strains (C. albicans and B. cereus) as well. Moreover, the mechanistic aspects of the antimicrobial activity of the AgNMs were elucidated using various methods, such as propidium iodide staining, monitoring reactive oxygen species generation, leakage of proteins, DNA cleavage, etc. We propose that AgNM-mediated cytotoxicity in S. cerevisiae stems from the generation of singlet oxygen (1O2) species that create oxidative stress, disrupting the cell membrane and thereby resulting in leakage of proteins from the cells. This study can pave the way toward elucidating the role of a small molecule, Tyr, in the formation of NMs and describes the use of new NMs in potential antimicrobial applications.

Noscapine alleviates cerebral damage in ischemia-reperfusion injury in rats.

With high unmet medical needs, stroke remains an intensely focused research area. Although noscapine is a neuroprotective agent, its mechanism of action in ischemic-reperfusion (I-R) injury is yet to be ascertained. We investigated the effect of noscapine on the molecular mechanisms of cell damage using yeast, and its neuroprotection on cerebral I-R injury in rats. Yeast, both wild-type and Δtrx2 strains, was evaluated for cell growth and viability, and oxidative stress to assess the noscapine effect at 8, 10, 12, and 20 µg/ml concentrations. The neuroprotective activity of noscapine (5 and 10 mg/kg; po for 8 days) was investigated in rats using middle cerebral artery occlusion-induced I-R injury. Infarct volume, neurological deficit, oxidative stress, myeloperoxidase activity, and histological alterations were determined in I-R rats. In vitro yeast assays exhibited significant antioxidant activity and enhanced cell tolerance against oxidative stress after noscapine treatment. Similarly, noscapine pretreatment significantly reduced infarct volume and edema in the brain. The neurological deficit was decreased and oxidative stress biomarkers, superoxide dismutase activity and glutathione levels, were significantly increased while lipid peroxidation showed significant decrease in comparison to vehicle-treated I-R rats. Myeloperoxidase activity, an indicator of inflammation, was also reduced significantly in treated rats; histological changes were attenuated with noscapine. The study demonstrates the protective effect of noscapine in yeast and I-R rats by improving cell viability and attenuating neuronal damage, respectively. This protective activity of noscapine could be attributed to potent free radical scavenging and inhibition of inflammation in cerebral ischemia-reperfusion injury.

Chymotrypsin protease inhibitor gene family in rice: Genomic organization and evidence for the presence of a bidirectional promoter shared between two chymotrypsin protease inhibitor genes.

Protease inhibitors play important roles in stress and developmental responses of plants. Rice genome contains 17 putative members in chymotrypsin protease inhibitor (ranging in size from 7.21 to 11.9 kDa) gene family with different predicted localization sites. Full-length cDNA encoding for a putative subtilisin-chymotrypsin protease inhibitor (OCPI2) was obtained from Pusa basmati 1 (indica) rice seedlings. 620 bp-long OCPI2 cDNA contained 219 bp-long ORF, coding for 72 amino acid-long 7.7 kDa subtilisin-chymotrypsin protease inhibitor (CPI) cytoplasmic protein. Expression analysis by semi-quantitative RT-PCR analysis showed that OCPI2 transcript is induced by varied stresses including salt, ABA, low temperature and mechanical injury in both root and shoot tissues of the seedlings. Transgenic rice plants produced with OCPI2 promoter-gus reporter gene showed that this promoter directs high salt- and ABA-regulated expression of the GUS gene. Another CPI gene (OCPI1) upstream to OCPI2 (with 1126 bp distance between the transcription initiation sites of the two genes; transcription in the reverse orientation) was noted in genome sequence of rice genome. A vector that had GFP and GUS reporter genes in opposite orientations driven by 1881 bp intergenic sequence between the OCPI2 and OCPI1 (encompassing the region between the translation initiation sites of the two genes) was constructed and shot in onion epidermal cells by particle bombardment. Expression of both GFP and GUS from the same epidermal cell showed that this sequence represents a bidirectional promoter. Examples illustrating gene pairs showing co-expression of two divergent neighboring genes sharing a bidirectional promoter have recently been extensively worked out in yeast and human systems. We provide an example of a gene pair constituted of two homologous genes showing co-expression governed by a bidirectional promoter in rice.