Comparative transcriptome analyses revealed different heat stress responses in pigeonpea (Cajanus cajan) and its crop wild relatives.

KEY MESSAGE: Comparative transcriptome analyses accompanied by biochemical assays revealed high variability in heat stress response in Cajanus species. Among the studied species, C. scarabaeoides was the most thermotolerant followed by C. cajanifolius, C. cajan, and C. acutifolius. Pigeonpea is one of the climate-resilient grain legumes. Though the optimum temperature for cultivated pigeonpea is ~ 25-35 °C, its wild relatives grow in temperatures ranging between 18 and 45 °C. To gain insight into molecular mechanisms responsible for the heat stress tolerance in pigeonpea, we conducted time-series transcriptome analysis of one pigeonpea cultivar (Cajanus cajan) and two wild relatives, Cajanus acutifolius, and Cajanus scarabaeoides subjected to heat stress at 42 ± 2 ºC for 30 min and 3 h. A total of 9521, 12,447, and 5282 identified transcripts were differentially expressed in C. cajan, C. acutifolius, and C. scarabaeoides, respectively. In this study, we observed that a significant number of genes undergo alternative splicing in a species-specific pattern during heat stress. Gene expression profiling analysis, histochemical assay, chlorophyll content, and electrolyte leakage assay showed that C. scarabaeoides has adaptive features for heat stress tolerance. The gene set enrichment analyses of differentially expressed genes in these Cajanus species during heat stress revealed that oxidoreductase activity, transcription factor activity, oxygen-evolving complex, photosystem-II, thylakoid, phenylpropanoid biosynthetic process, secondary metabolic process, and flavonoid biosynthetic process were highly affected. The histochemical assay showed more lipid peroxidation in C. acutifolius compared to other Cajanus species inferring the presence of higher quantities of polyunsaturated fatty acids in the plasma membrane which might have led to severe damage of membrane-bound organelles like chloroplast, and high electrolyte leakage during heat stress. This study paves the way for the identification of candidate genes, which can be useful for the development of thermo-tolerant pigeonpea cultivars.

Genome wide identification and characterization of small heat shock protein gene family in pigeonpea and their expression profiling during abiotic stress conditions.

Small heat shock proteins as large multigene family are present ubiquitously among Archaea to Eukaryota. The sHSPs are molecular chaperones that maintain the proper protein folding and disaggregation of denatured proteins during stress conditions. In the present study, out of identified 38 sHSPs in the pigeonpea genome, the 20 are distributed across seven chromosomes while the remaining are located on unassembled scaffolds. These Cc_sHSPs are classified into 16 subfamilies. The cytoplasmic class-II is the largest sub-family with five Cc_sHSPs. The gene structure analysis revealed that Cc_sHSP genes specifically containing no or very few introns. The promoter analysis revealed the presence of various cis-acting elements responsible for developmental, biotic, and abiotic stress specific-induction of Cc_sHSPs. A total of one segmental duplication and four tandem duplication events are identified for Cc_sHSPs. The qRT-PCR based expression analysis of all 38 Cc_sHSP genes was conducted for diverse abiotic stress conditions. The Cc_sHSP genes are highly induced by heat, drought, cold, and salt stresses indicating a key role in mitigating the various abiotic stress responses. The divergence time of paralogous Cc_sHSPs ranged from 8.66 to 191.82 MYA. The present study can be a strong basis for the functional characterization of Cc_sHSPs.

Interaction of drug metabolizing cytochrome P450 2D6 poor metabolizers with cytochrome P450 2C9 and 2C19 genotypes modify the susceptibility to head and neck cancer and treatment response.

The present case-control study attempted to investigate the association of poor metabolizer (PM) genotypes of cytochrome P450 2D6 (CYP2D6*4 and CYP2D6*10) with squamous cell carcinoma of head and neck (HNSCC) and treatment response in patients receiving chemotherapy or combination of chemo- and radiotherapy. Cases with the PM genotypes of CYP2D6 displayed a significantly increased risk for HNSCC as compared to wild type genotypes. The risk was found to further increase in cases (up to 4.8) carrying combination of PM genotypes of CYP2D6, CYP2C9 (CYP2C9*2) or CYP2C19 (CYP2C19*2), suggesting that synergism amongst the PM genotypes of drug metabolizing CYPs leads to impairment in the detoxification of the tobacco carcinogens. A small increase in the risk in tobacco (chewers or smokers) or alcohol users in cases with CYP2D6*4 allele while no change or even a small decrease in risk in cases with CYP2D6*10 allele when compared to non-tobacco or alcohol users have suggested that CYP2D6 genotypes alone do not appear to interact significantly with environmental risk factors in modifying the susceptibility to HNSCC. Furthermore, most of the cases carrying PM genotypes of CYP2D6 did not respond to the treatment. Moreover, higher prevalence of non-responders among cases carrying combination of CYP2D6*4 or CYP2D6*4, CYP2C9*2 and CYP2C19*2 have demonstrated that interaction of PM genotypes may not only significantly modify the susceptibility to HNSCC but also the treatment response.

Association of poor metabolizers of cytochrome P450 2C19 with head and neck cancer and poor treatment response.

A case-control study consisting of 300 patients and an equal number of healthy controls was carried out to investigate the association of polymorphism in cytochrome P450 2C19 (CYP2C19), which results in poor and extensive metabolizers (PMs and EMs) genotypes, with squamous cell carcinoma of head and neck (HNSCC) and treatment response in patients receiving combination of chemo-radiotherapy. A higher frequency of CYP2C19 2 variants was observed in the cases resulting in significantly higher risk to HNSCC (Ad OR 3.36, 95% CI 1.94-5.82, p-value<0.05). The PM genotype of CYP2C19 3 was also found to be slightly increased in the cases, though the increase in risk was not significant when analyzed by multivariate logistic regression model. Tobacco chewing amongst the cases resulted in almost 13-fold increase in the risk with CYP2C19 2 (OR: 12.39) and 3-fold with CYP2C19 3 genotype (OR: 2.90) when compared to the tobacco chewers amongst the controls. Likewise, cigarette smoking in the cases increased the risk approximately 9-fold and 3-fold with CYP2C19 2 (OR: 8.93) and CYP2C19 3 (OR: 2.18) genotypes respectively when compared to smokers amongst the controls. Similar increase in risk was associated with alcohol use amongst the cases carrying variant genotypes of CYP2C19 2 (OR: 7.75) or CYP2C19 3 (OR: 2.60), demonstrating the importance of gene-environment interaction in modifying susceptibility to HNSCC. Interestingly, patients with PMs of CYP2C19 (CYP2C19 2 and CYP2C19 3) exhibited little response to the respective chemotherapy than the patients carrying wild-type genotype demonstrating that functional enzyme deficiencies due to polymorphism in CYPs may not only be important in modifying the susceptibility to HNSCC but also in determining chemotherapeutic response.

Association of polymorphism in cytochrome P450 2C9 with susceptibility to head and neck cancer and treatment outcome.

The present case-control study involving 750 cases and equal number of healthy controls investigates the association of polymorphism in cytochrome P450 2C9 (CYP2C9) with head and neck squamous cell carcinoma (HNSCC) and response in patients receiving chemotherapy or combination of radio-chemotherapy. The frequency of heterozygous or homozygous genotypes of CYP2C9*2 & CYP2C9*3, which leads to the poor metabolizer (PM) genotype was significantly higher in HNSCC cases when compared to the healthy controls resulting in significantly increased risk in the cases. Tobacco use in the form of tobacco smoking or tobacco chewing was found to increase the risk several fold in cases when compared to the non-tobacco users. Likewise, alcohol intake in cases with variant genotypes of CYP2C9*2 or CYP2C9*3 also significantly increased the HNSCC risk in cases when compared to non-alcohol users. Further, majority of the cases carrying variant alleles of both CYP2C9*2 or CYP2C9*3 were found to respond poorly to the chemotherapy or combination of radio-chemotherapy. The data suggests a significant association of the CYP2C9 polymorphism with HNSCC and treatment outcome.