Validation of Novel Reference Genes for Reverse Transcription Quantitative Real-Time PCR in Drought-Stressed Sugarcane

One of the most challenging aspects of RT-qPCR data analysis is the identification of reliable reference genes. Ideally, they should be neither induced nor repressed under different experimental conditions. To date, few reference genes have been adequately studied for sugarcane (Saccharum spp.) using statistical approaches. In this work, six candidate genes (αTUB, GAPDH, H1, SAMDC, UBQ, and 25S rRNA) were tested for gene expression normalization of sugarcane root tissues from drought-tolerant and -sensitive accessions after continuous dehydration (24 h). By undergoing different approaches (GeNorm, NormFinder, and BestKeeper), it was shown that most of them could be used in combinations for normalization purposes, with the exception of SAMDC. Nevertheless three of them (H1, αTUB, and GAPDH) were considered the most reliable reference genes. Their suitability as reference genes validated the expression profiles of two targets (AS and PFPα1), related to SuperSAGE unitags, in agreement with results revealed by previous in silico analysis. The other two sugarcane unitags (ACC oxidase and PIP1-1), after salt stress (100 mM NaCl), presented their expressions validated in the same way. In conclusion, these reference genes will be useful for dissecting gene expression in sugarcane roots under abiotic stress, especially in transcriptomic studies using SuperSAGE or RNAseq approaches

The Transcriptional Modulation of Inositols and Raffinose Family Oligosaccharides Pathways in Plants — An (A)Biotic Stress Perspective

Among the multifunctional molecules that participate in processes of plant tolerance/resistance to stresses, inositol (Ins) and its derivatives (phosphorylated, methylated, oxygenated, and Raffinose Family Oligosaccharides) have attracted the attention of researchers. These compounds represent versatile and dynamic signaling molecules and osmolytes in all eukaryotes. Due to the impacts related to Ins and its derivatives in a plant cell, assays have been conducted to understand how these biomolecules affect plant physiology. Thus, overexpression or knockout of Ins-related genes has been shown as interesting strategies for generating more efficient plants capable of growing under stress conditions. In this chapter, studies using molecular tools are presented, and the impacts of their results are discussed based on the plant stress tolerance/resistance. Furthermore, an informative panel is provided with transcriptional modulation of genes related to Ins and its derivatives expressed in plants under stress. There is a gap involving about two dozen enzymes associated with the synthesis of Ins-related compounds that have not been adequately studied, and they represent an area of high biotechnological potential

Análise in Silico de Subfamílias de Aquaporinas em Cana-de-açúcar (Saccharum spp.) sob Condições de Déficit Hídrico via Tecnologia SuperSAGE

O presente trabalho, pioneiro em abordar especificamente o tema aquaporinas em transcriptomas de cana-de-açúcar, permitiu detectar representantes das quatro subfamílias de aquaporinas descritas em vegetais superiores (PIP, TIP, SIP e NIP), apresentando 43 isoformas distintas, oriundas de quatro bibliotecas SuperSAGE de raízes de genótipos tolerantes e sensíveis à seca. As subfamílias de aquaporinas com maiores níveis de transcritos em cana-de-açúcar foram PIP e TIP. Já as subfamílias SIP e NIP, além de menos abundantes, foram também menos responsivas ao estresse de supressão de rega (24 h). Ao menos 10 potenciais alvos distintos de isoformas de aquaporinas e suas respectivas unitags foram considerados promissores para estudos futuros, com potencial para desenvolvimento de marcadores moleculares para uso no melhoramento genético. Desses, quatro mostraram respostas exclusivas e diferencialmente significativas e divergentes entre os bulks de genótipos tolerantes e sensíveis quando comparado nas condições de estresse e controle. Tomando por base a expressão dessas isoformas nos genótipos tolerantes, a maioria foi reprimida sob estresse (SoPIP2-4, SoPIP2-6, OsPIP2-4), com exceção da SsPIP1-1 (induzida). Foi também proposto um protocolo para validar a expressão das unitags via RTqPCR, bem como pares de primers (5) funcionais como genes de referência, além de dois outros propostos para aquaporinas, tendo-se validado os dados de SuperSAGE para os alvos SsPIP1-1 e SoPIP1-3/PIP1-4, com genótipos pertencentes aos bulks tolerante ou sensível ao estresse

GROWTH AND SEED YIELD OF SUNFLOWER ON SOIL FERTILIZED WITH CASSAVA WASTEWATER

Cassava wastewater is the liquid residue of the cassava flour processing and its application to the soil as fertilizer for agricultural crops is a good alternative to sources of nutrients for plants. In this context, the objective of this work was to evaluate the growth and seed yield of sunflower on soil fertilized with cassava wastewater. A randomized block experimental design with four replications was used, with six cassava wastewater rates (0, 8.5, 17, 34, 68 and 136 m3 ha-1) and six plant sampling times (15, 30, 45, 60, 75 and 90 days after sowing), using the sunflower cultivar Helio-250. The evaluated variables were total leaf area, leaf area index, leaf area ratio, specific leaf area, leaf weight ratio, shoot dry mass and sunflower seed yield. The use of cassava wastewater rate of 136 m3 ha-1 increases the seed yield, leaf area, leaf area index and shoot dry mass, and results in lower leaf area ratio, specific leaf area and leaf weight ratio of sunflower (cv. Helio-250) crops

Production components of sunflower plants irrigated with treated domestic wastewater and drinking water in semiarid region

ABSTRACT This study aimed to evaluate production components of sunflower (Helianthus annuus L.) plants irrigated with domestic wastewaters treated by different processes, with two irrigation depths. The experiment was carried out in Ibimirim, state of Pernambuco, Brazil, using a randomized block design in a 4×2 factorial arrangement, with four replications. The treatments consisted of four irrigation water types (domestic wastewater treated by UASB reactor - DW1; domestic wastewater treated by digestion decanter and anaerobic filtration - DW2; domestic wastewater treated by anaerobic filtration - DW3; and public water - PW4) and two irrigation depths (equal to the crop evapotranspiration - ID1; and 20% higher than the crop evapotranspiration - ID2). The production components-plant fresh and dry weights, 1000-achene weight, capitulum internal diameter, achene yield, oil content, and oil yield-were evaluated at the end of the crop cycle. The irrigation with treated domestic wastewater improved all variables analyzed when compared to the drinking water, especially when using DW2 and DW3 with ID1

Validation of novel reference genes for reverse transcription quantitative real-time PCR in drought-stressed sugarcane

One of the most challenging aspects of RT-qPCR data analysis is the identification of reliable reference genes. Ideally, they should be neither induced nor repressed under different experimental conditions. To date, few reference genes have been adequately studied for sugarcane (Saccharum spp.) using statistical approaches. In this work, six candidate genes (αTUB, GAPDH, H1, SAMDC, UBQ, and 25S rRNA) were tested for gene expression normalization of sugarcane root tissues from drought-tolerant and -sensitive accessions after continuous dehydration (24 h). By undergoing different approaches (GeNorm, NormFinder, and BestKeeper), it was shown that most of them could be used in combinations for normalization purposes, with the exception of SAMDC. Nevertheless three of them (H1, αTUB, and GAPDH) were considered the most reliable reference genes. Their suitability as reference genes validated the expression profiles of two targets (AS and PFPα1), related to SuperSAGE unitags, in agreement with results revealed by previous in silico analysis. The other two sugarcane unitags (ACC oxidase and PIP1-1), after salt stress (100 mM NaCl), presented their expressions validated in the same way. In conclusion, these reference genes will be useful for dissecting gene expression in sugarcane roots under abiotic stress, especially in transcriptomic studies using SuperSAGE or RNAseq approaches

Expression analysis of sugarcane aquaporin genes under water deficit

The present work is a pioneer study specifically addressing the aquaporin transcripts in sugarcane transcriptomes. Representatives of the four aquaporin subfamilies (PIP, TIP, SIP, and NIP), already described for higher plants, were identified. Forty-two distinct aquaporin isoforms were expressed in four HT-SuperSAGE libraries from sugarcane roots of drought-tolerant and -sensitive genotypes, respectively. At least 10 different potential aquaporin isoform targets and their respective unitags were considered to be promising for future studies and especially for the development of molecular markers for plant breeding. From those 10 isoforms, four (SoPIP2-4, SoPIP2-6, OsPIP2-4, and SsPIP1-1) showed distinct responses towards drought, with divergent expressions between the bulks from tolerant and sensitive genotypes, when they were compared under normal and stress conditions. Two targets (SsPIP1-1 and SoPIP1-3/PIP1-4) were selected for validation via RT-qPCR and their expression patterns as detected by HT-SuperSAGE were confirmed. The employed validation strategy revealed that different genotypes share the same tolerant or sensitive phenotype, respectively, but may use different routes for stress acclimation, indicating the aquaporin transcription in sugarcane to be potentially genotype-specific

Bridging the Gap: Combining Genomics and Transcriptomics Approaches to Understand <i>Stylosanthes scabra</i>, an Orphan Legume from the Brazilian Caatinga

Stylosanthes scabra is a scientifically orphaned legume found in the Brazilian Caatinga biome (a semi-arid environment). This work utilized omics approaches to investigate some ecophysiological aspects of stress tolerance/resistance in S. scabra, study its genomic landscape, and predict potential metabolic pathways. Considering its high-confidence conceptual proteome, 1694 (~2.6%) proteins were associated with resistance proteins, some of which were found in soybean QTL regions that confer resistance to Asian soybean rust. S. scabra was also found to be a potential source of terpenes, as biosynthetic gene clusters associated with terpene biosynthesis were identified in its genome. The analysis revealed that mobile elements comprised approximately 59% of the sequenced genome. In the remaining 41% of the sections, some of the 22,681 protein-coding gene families were categorized into two informational groups: those that were specific to S. scabra and those that expanded significantly compared to their immediate ancestor. Biological process enrichment analyses indicated that these gene families play fundamental roles in the adaptation of S. scabra to extreme environments. Additionally, phylogenomic analysis indicated a close evolutionary relationship between the genera Stylosanthes and Arachis. Finally, this study found a high number (57) of aquaporin-encoding loci in the S. scabra genome. RNA-Seq and qPCR data suggested that the PIP subfamily may play a key role in the species’ adaptation to water deficit conditions. Overall, these results provide valuable insights into S. scabra biology and a wealth of gene/transcript information for future legume omics studies