Leaf transcriptome profiling of contrasting sugarcane genotypes for drought tolerance under field conditions.

Drought is the most detrimental abiotic stress to sugarcane production. Nevertheless, transcriptomic analyses remain scarce for field-grown plants. Here we performed comparative transcriptional profiling of two contrasting sugarcane genotypes, 'IACSP97-7065' (drought-sensitive) and 'IACSP94-2094' (drought-tolerant) grown in a drought-prone environment. Physiological parameters and expression profiles were analyzed at 42 (May) and 117 (August) days after the last rainfall. The first sampling was done under mild drought (soil water potential of -60 kPa), while the second one was under severe drought (soil water potential of -75 kPa). Microarray analysis revealed a total of 622 differentially expressed genes in both sugarcane genotypes under mild and severe drought stress, uncovering about 250 exclusive transcripts to 'IACSP94-2094' involved in oxidoreductase activity, transcriptional regulation, metabolism of amino acids, and translation. Interestingly, the enhanced antioxidant system of 'IACSP94-2094' may protect photosystem II from oxidative damage, which partially ensures stable photochemical activity even after 117 days of water shortage. Moreover, the tolerant genotype shows a more extensive set of responsive transcription factors, promoting the fine-tuning of drought-related molecular pathways. These results help elucidate the intrinsic molecular mechanisms of a drought-tolerant sugarcane genotype to cope with ever-changing environments, including prolonged water deficit, and may be useful for plant breeding programs.

Reference genes for gene expression studies targeting sugarcane infected with Sugarcane mosaic virus (SCMV).

OBJECTIVE: The selection of reference genes in sugarcane under Sugarcane mosaic virus (SCMV) infection has not been reported and is indispensable to get reliable reverse transcription quantitative PCR (RT-qPCR) results for validation of transcriptome analysis. In this regard, seven potential reference genes were tested by RT-qPCR and ranked according to their stability using BestKeeper, NormFinder and GeNorm algorithms, and RefFinder WEB-based software in an experiment performed with samples from two sugarcane cultivars contrasting for SCMV resistance, when mechanically inoculated with a severe SCMV strain and using mock inoculated plant controls. RESULTS: The genes Uridylate kinase (UK) and Ubiquitin-conjugating enzyme 18 (UBC18) were the most stable according to GeNorm algorithm and the Pearson correlation coefficients with the BestKeeper index. On the other hand, ribosomal protein L35-4 (RPL1), Actin (ACT) and Ubiquitin1 (UBQ1) were the least stable genes for all algorithms tested.

Biomass Accumulation and Cell Wall Structure of Rice Plants Overexpressing a Dirigent-Jacalin of Sugarcane (ShDJ) Under Varying Conditions of Water Availability.

A sugarcane gene encoding a dirigent-jacalin, ShDJ, was induced under drought stress. To elucidate its biological function, we integrated a ShDJ-overexpression construction into the rice Nipponbare genome via Agrobacterium-mediated transformation. Two transgenic lines with a single copy gene in T0 were selected and evaluated in both the T1 and T4 generations. Transgenic lines had drastically improved survival rate under water deficit conditions, at rates close to 100%, while WT did not survive. Besides, transgenic lines had improved biomass production and higher tillering under water deficit conditions compared with WT plants. Reduced pectin and hemicellulose contents were observed in transgenic lines compared with wild-type plants under both well-watered and water deficit conditions, whereas cellulose content was unchanged in line #17 and reduced in line #29 under conditions of low water availability. Changes in lignin content under water deficit were only observed in line #17. However, improvements in saccharification were found in both transgenic lines along with changes in the expression of OsNTS1/2 and OsMYB58/63 secondary cell wall biosynthesis genes. ShDJ-overexpression up-regulated the expression of the OsbZIP23, OsGRAS23, OsP5CS, and OsLea3 genes in rice stems under well-watered conditions. Taken together, our data suggest that ShDJ has the potential for improving drought tolerance, plant biomass accumulation, and saccharification efficiency.

Reference genes for normalization of qPCR assays in sugarcane plants under water deficit.

BACKGROUND: Sugarcane (Saccharum spp.) is the main raw material for sugar and ethanol production. Among the abiotic stress, drought is the main one that negatively impact sugarcane yield. Although gene expression analysis through quantitative PCR (qPCR) has increased our knowledge about biological processes related to drought, gene network that mediates sugarcane responses to water deficit remains elusive. In such scenario, validation of reference gene is a major requirement for successful analyzes involving qPCR. RESULTS: In this study, candidate genes were tested for their suitable as reference genes for qPCR analyses in two sugarcane cultivars with varying drought tolerance. Eight candidate reference genes were evaluated in leaves sampled in plants subjected to water deficit in both field and greenhouse conditions. In addition, five genes were evaluated in shoot roots of plants subjected to water deficit by adding PEG8000 to the nutrient solution. NormFinder and RefFinder algorithms were used to identify the most stable gene(s) among genotypes and under different experimental conditions. Both algorithms revealed that in leaf samples, UBQ1 and GAPDH genes were more suitable as reference genes, whereas GAPDH was the best reference one in shoot roots. CONCLUSION: Reference genes suitable for sugarcane under water deficit were identified, which would lead to a more accurate and reliable analysis of qPCR. Thus, results obtained in this study may guide future research on gene expression in sugarcane under varying water conditions.

Spatial distribution of AT- and GC-rich DNA within interphase cell nuclei of Triatoma infestans Klug.

Heterochromatin bodies in single- and multichromocentered interphase cell nuclei of Triatoma infestans, a vector of Chagas disease, have been suggested to contain AT-rich DNA, based on their positive response to Q-banding and Hoechst 33248 treatment. No information exists on whether GC-rich DNA is also present in these nuclei and whether it plays a role on chromatin condensation. Considering that methodologies more precise than those previously used to determine DNA base composition in situ are currently available, and that the spatial distribution of chromatin areas differing in composition in interphase cell nuclei of different species is a matter of interest, the localization of AT- and GC-rich DNA in T. infestans nuclei is revisited here. The methodologies used included DAPI/AMD and CMA(3)/Distamycin differential staining, Feulgen-DNA image analysis following Msp I and Hpa II enzymatic digestion, 5-methylcytidine immunodetection, AgNOR response, confocal microscopy, and the 5-aza-2'-deoxycytidine (5-AZA) demethylation assay. The results identified the presence of AT-rich/GC-poor DNA in chromocenters and evenly distributed AT and GC sequences in euchromatin. A GC-rich DNA zone encircling the chromocenters was also found but it could not be associated with NOR regions. To corroborate the DNA AT-richness in T. infestans nuclei, bioinformatic analyses were also performed. Methylated cytosine was evident at some points of the chromocenters' edge in single- and multichromocentered nuclei and at the euchromatin of multichromocentered nuclei and could be transiently affected by the 5-AZA treatment. The present results suggest that in the particular case of chromocenters of the hemipteran T. infestans, cytosine methylation is not a relevant factor involved in chromatin condensation.