Comprehensive RNAseq analysis for identification of genes expressed under heat stress in lentil.

Lentils are highly sensitive to abrupt increases in temperature during the mid to late reproductive stages, leading to severe biomass and seed yield reduction. Therefore, we carried out an RNAseq analysis between IG4258 (heat tolerant) and IG3973 (heat sensitive) lentil genotypes at the reproductive stage under both normal and heat stress conditions in the field. It resulted in 209,549 assembled transcripts and among these 161,809 transcripts had coding regions, of which 94,437 transcripts were annotated. The differential gene expression analysis showed upregulation of 678 transcripts and downregulation of 680 transcripts between the tolerant and sensitive genotypes at the early reproductive stage. While 76 transcripts were upregulated and 47 transcripts were downregulated at the late reproductive stage under heat stress conditions. The validation of 12 up-or downregulated transcripts through RT-PCR corresponded well with the expression analysis data of RNAseq, with a correlation of R2  = 0.89. Among these transcripts, the DN364_c1_g1_i9 and DN2218_c0_g1_i5 transcripts encoded enzymes involved in the tryptophan pathway, indicating that tryptophan biosynthesis plays a role under heat stress in lentil. Moreover, KEGG pathways enrichment analysis identified transcripts associated with genes encoding proteins/regulating factors related to different metabolic pathways including signal transduction, fatty acid biosynthesis, rRNA processing, ribosome biogenesis, gibberellin (GA) biosynthesis, and riboflavin biosynthesis. This analysis also identified 6852 genic-SSRs leading to the development of 4968 SSR primers that are potential genomic resources for molecular mapping of heat-tolerant genes in lentil.

Candidate genes of flavonoid biosynthesis in Selaginella bryopteris (L.) Baker identified by RNA-Seq.

In the present study, de novo transcriptome analysis of Selaginella bryopteris in frond and root was performed to understand the regulation of flavonoid (FL) biosynthesis. High-quality data of 5.84 and 5.86 Gb was generated for frond and root, respectively, that assembled into 94,713 and 81,567 transcripts. A total of 87,471 and 73,395 unigenes were obtained from frond and root, respectively. A total of 41,267 and 31,048 CDS of frond and root, respectively, were annotated by BLASTX, which showed maximum hits against S. moellendorffii. Out of 11,285 differentially expressed genes, a total of 5639 genes were found to be down-regulated and 5628 genes up-regulated in frond as compared to those in root. In silico analysis of expression of genes in frond as compared to that in root was done for those related to phenylpropanoid (PP)/FL biosynthesis along with transcription factors (TFs) after DESeq and MapMan-based information. Results showed that genes of PP/FL biosynthesis pathway namely SbCHS, SbCHI, SbF3H, SbF3'H, SbDFR, SbUF3GT, SbCCOAMT, and SbCATOMT and TFs (SbMYB1, SbMYB2, SbMYB3, SbBHLH1, and SbWD40-5) were up-regulated in frond in comparison to those in root. Further, this in silico expression data was validated by RT-PCR analysis which showed predominant expression of most of these genes in frond and indicated their importance in the biosynthesis of flavonoids in S. bryopteris. A total of 9074 simple sequence repeats (SSRs) were also identified for frond and 3811 SSRs for root; these can be used for experimental validation.

Intron-mediated alternative splicing of Arabidopsis P5CS1 and its association with natural variation in proline and climate adaptation.

Drought-induced proline accumulation is widely observed in plants but its regulation and adaptive value are not as well understood. Proline accumulation of the Arabidopsis accession Shakdara (Sha) was threefold less than that of Landsberg erecta (Ler) and quantitative trait loci mapping identified a reduced function allele of the proline synthesis enzyme Δ(1)-pyrroline-5-carboxylate synthetase1 (P5CS1) as a basis for the lower proline of Sha. Sha P5CS1 had additional TA repeats in intron 2 and a G-to-T transversion in intron 3 that were sufficient to promote alternative splicing and production of a nonfunctional transcript lacking exon 3 (exon 3-skip P5CS1). In Sha, and additional accessions with the same intron polymorphisms, the nonfunctional exon 3-skip P5CS1 splice variant constituted as much as half of the total P5CS1 transcript. In a larger panel of Arabidopsis accessions, low water potential-induced proline accumulation varied by 10-fold and variable production of exon 3-skip P5CS1 among accessions was an important, but not the sole, factor underlying variation in proline accumulation. Population genetic analyses suggest that P5CS1 may have evolved under positive selection, and more extensive correlation of exon 3-skip P5CS1 production than proline abundance with climate conditions of natural accessions also suggest a role of P5CS1 in local adaptation to the environment. These data identify a unique source of alternative splicing in plants, demonstrate a role of exon 3-skip P5CS1 in natural variation of proline metabolism, and suggest an association of P5CS1 and its alternative splicing with environmental adaptation.

A dominant repressor version of the tomato Sl-ERF.B3 gene confers ethylene hypersensitivity via feedback regulation of ethylene signaling and response components.

Ethylene Response Factors (ERFs) are downstream components of the ethylene signal transduction pathway, although their role in ethylene-dependent developmental processes remains poorly understood. As the ethylene-inducible tomato Sl-ERF.B3 has been shown previously to display a strong binding affinity to GCC-box-containing promoters, its physiological significance was addressed here by a reverse genetics approach. However, classical up- and down-regulation strategies failed to give clear clues to its roles in planta, probably due to functional redundancy among ERF family members. Expression of a dominant repressor ERF.B3-SRDX version of Sl-ERF.B3 in the tomato resulted in pleiotropic ethylene responses and vegetative and reproductive growth phenotypes. The dominant repressor etiolated seedlings displayed partial constitutive ethylene response in the absence of ethylene and adult plants exhibited typical ethylene-related alterations such as leaf epinasty, premature flower senescence and accelerated fruit abscission. The multiple symptoms related to enhanced ethylene sensitivity correlated with the altered expression of ethylene biosynthesis and signaling genes and suggested the involvement of Sl-ERF.B3 in a feedback mechanism that regulates components of ethylene production and response. Moreover, Sl-ERF.B3 was shown to modulate the transcription of a set of ERFs and revealed the existence of a complex network interconnecting different ERF genes. Overall, the study indicated that Sl-ERF.B3 had a critical role in the regulation of multiple genes and identified a number of ERFs among its primary targets, consistent with the pleiotropic phenotypes displayed by the dominant repression lines.

Expression of phytochelatin synthase from aquatic macrophyte Ceratophyllum demersum L. enhances cadmium and arsenic accumulation in tobacco.

UNLABELLED: Phytochelatin synthase (PCS), the key enzyme involved in heavy metal detoxification and accumulation has been used from various sources to develop transgenic plants for the purpose of phytoremediation. However, some of the earlier studies provided contradictory results. Most of the PCS genes were isolated from plants that are not potential metal accumulators. In this study, we have isolated PCS gene from Ceratophyllum demersum cv. L. (CdPCS1), a submerged rootless aquatic macrophyte, which is considered as potential accumulator of heavy metals. The CdPCS1 cDNA of 1,757 bp encodes a polypeptide of 501 amino acid residues and differs from other known PCS with respect to the presence of a number of cysteine residues known for their interaction with heavy metals. Complementation of cad1-3 mutant of Arabidopsis deficient in PC (phytochelatin) biosynthesis by CdPCS1 suggests its role in the synthesis of PCs. Transgenic tobacco plants expressing CdPCS1 showed several-fold increased PC content and precursor non-protein thiols with enhanced accumulation of cadmium (Cd) and arsenic (As) without significant decrease in plant growth. We conclude that CdPCS1 encodes functional PCS and may be part of metal detoxification mechanism of the heavy metal accumulating plant C. demersum. KEY MESSAGE: Heterologous expression of PCS gene from C. demersum complements Arabidopsis cad1-3 mutant and leads to enhanced accumulation of Cd and As in transgenic tobacco.

Comprehensive genome-wide identification, characterization, and expression profiling of MATE gene family in Nicotiana tabacum.

The transporters belonging to the MATE family are involved in the transportation of diverse ligands, including metal ions and small organic molecules, and, therefore, play an important role in plant biology. Our genome-wide analysis led to the identification of 138 MATE genes in N. tabacum, which were grouped into four major phylogenetic clades. The expression of several NtMATE genes was reported to be differential in different tissues, namely young leaf, mature leaf, stem, root, and mature flower. The upstream regions of the NtMATE genes were predicted to contain several cis-acting elements associated with hormonal, developmental, and stress responses. Some of the genes were found to display induced expression following methyl jasmonate treatment. The co-expression analysis revealed 126 candidate transcription factor genes that might be involved in the transcriptional regulation of 21 NtMATE genes. Certain MATE genes (NtMATE81, NtMATE82, NtMATE88, and NtMATE89) were predicted to be targeted by micro RNAs (nta-miR167a, nta-miR167b, nta-miR167c, nta-miR167d and nta-miR167e). The computational analysis of MATE transporters provided insights into the key amino acid residues involved in the binding of the alkaloids. Further, the putative function of some of the NtMATE transporters was also revealed. The present study develops a solid foundation for the functional characterization of MATE transporter genes in N. tabacum.

Heterologous expression of Ceratophyllum demersum phytochelatin synthase, CdPCS1, in rice leads to lower arsenic accumulation in grain.

Recent studies have identified rice (Oryza sativa) as a major dietary source of inorganic arsenic (As) and poses a significant human health risk. The predominant model for plant detoxification of heavy metals is complexation of heavy metals with phytochelatins (PCs), synthesized non-translationally by PC synthase (PCS) and compartmentalized in vacuoles. In this study, in order to restrict As in the rice roots as a detoxification mechanism, a transgenic approach has been followed through expression of phytochelatin synthase, CdPCS1, from Ceratophyllum demersum, an aquatic As-accumulator plant. CdPCS1 expressing rice transgenic lines showed marked increase in PCS activity and enhanced synthesis of PCs in comparison to non-transgenic plant. Transgenic lines showed enhanced accumulation of As in root and shoot. This enhanced metal accumulation potential of transgenic lines was positively correlated to the content of PCs, which also increased several-fold higher in transgenic lines. However, all the transgenic lines accumulated significantly lower As in grain and husk in comparison to non-transgenic plant. The higher level of PCs in transgenic plants relative to non-transgenic presumably allowed sequestering and detoxification of higher amounts of As in roots and shoots, thereby restricting its accumulation in grain.

Factors associated with smoking in HIV-infected patients and potential barriers to cessation.

Smoking is common in patients with HIV and is associated with increased morbidity and mortality. With the goal of targeting future cessation interventions, we sought to identify factors associated with smoking status, readiness and confidence in cessation, and success in quitting. As part of a larger study in New York City assessing predictors of chronic obstructive pulmonary disease (COPD), we enrolled HIV-infected subjects at least 35 years of age without known asthma or COPD. Current smokers received detailed tobacco history, and smoking status was assessed by chart review at 3 and 6 months post-enrollment. Two hundred subjects were enrolled (29% current smokers, 31.5% never smokers, 39.5% former smokers, mean age of 49, 84% male, 64% had AIDS, and 97% were receiving antiretroviral therapy). Current smokers had higher unemployment and increased rates of other substance use than former smokers or never smokers. In multivariate analysis, being unemployed and having used inhalant drugs were associated with current smoking. Substance abuse history was not correlated with readiness to quit or patient estimated cessation. Lower education was associated with decreased readiness to quit. Follow-up smoking status for baseline current smokers was available for 47/58 enrollees at 6 months; 4 (9%) stopped smoking completely, and 17 (36%) decreased the number of packs-per-day. Smoking and concomitant substance abuse is common in HIV, and special attention should be given to this issue, in addition to a patient's readiness to quit, when implementing tobacco cessation protocols, especially in busy urban HIV care centers.

Expression of Ceratophyllum demersum phytochelatin synthase, CdPCS1, in Escherichia coli and Arabidopsis enhances heavy metal(loid)s accumulation.

Phytochelatin synthase (PCS) gene encoding key enzyme for heavy metal detoxification and accumulation has been characterised from different sources and used to develop a technology for bioremediation. Past efforts provided limited success and contradictory results. Therefore, functional characterisation of PCS gene from new sources into different target systems is considered as an important task in the area of bioremediation. Earlier, we isolated and functionally characterised PCS gene from an aquatic macrophyte Ceratophyllum demersum L., a metal accumulator aquatic plant. Expression of this gene, CdPCS1, in tobacco enhanced PC synthesis and metal accumulation of transgenic tobacco plants. In the present study, we have expressed CdPCS1 in more diverse systems, Escherichia coli and Arabidopsis, and studied growth and metal accumulation of transgenic organisms. The expression of CdPCS1 in E. coli offered tolerance against cadmium as well as higher accumulation accompanied with PCS1 activity. The expression of CdPCS1 in Arabidopsis showed a significant enhanced accumulation of heavy metal(loid)s in aerial parts without significant difference in growth parameters in comparison to wild-type Arabidopsis plants. Our study suggests that CdPCS1 can be utilised for enhancing bioremediation potential of different organisms using biotechnological approaches.