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.

Monocyclic Components and Photosynthetic Damage Caused by Myrtle Rust in Guava Leaves.

Austropuccinia psidii, the causal agent of myrtle rust, was, for many years, restricted to the Americas, but since reaching Hawaii in 2005, the pathogen has expanded its global range exponentially. In Brazil, myrtle rust is the main fungal disease in guava plants. Despite this, there are few studies on guava rust epidemiology. The objectives of this study were to quantify the monocyclic components of rust and to evaluate the photosynthetic damage caused by A. psidii in young and old leaves of 'Paluma' guava. The monocyclic components of guava rust and gas exchange in healthy or inoculated (105 ml-1 urediniospores of A. psidii) leaves were quantified over time. Additionally, young leaves were inoculated with varying concentrations of A. psidii inoculum, and leaf gas exchange and chlorophyll fluorescence were measured at 25 days postinoculation. The relationship between the relative CO2 assimilation of a diseased leaf (Px) and a healthy leaf (Po) is related to disease severity (x) by Px/Po = (1 - x)ß. The density of lesions, disease severity, and urediniospore production were high in young leaves, averaging 58 lesions cm-2, 50% leaf area diseased, and 2.5 × 104 urediniospores per lesion, respectively. Rust symptoms were not observed in old leaves, and resistance to infection did not cause any photosynthetic cost to these leaves. On young leaves, ß was 2.13, indicating a reduction on CO2 assimilation at green tissues from symptomatic leaves. Our data revealed that photosynthesis reduction in diseased guava leaves was caused by biochemical and photochemical damage rather than by stomatal limitation.

Extracellular and Intracellular NO Detection in Plants by Diaminofluoresceins.

Many assays focus on determining NO content within plant tissues to assess the actual concentration that impacts on cellular processes. Diaminofluorescein fluorescent dyes (DAFs) have been very widely used by plant scientists to reveal likely sites of NO production inside and outside cells. In general, DAFs dyes react with N2O3, a byproduct of NO oxidation, resulting in fluorescence. It is initially available in the form of diacetate (DAF-2DA), which allowed the ready absorption by the cells. The diacetate group is removed by cell esterases leaving the membrane impermeable to DAF-2 and available for N2O3 nitration to generate the highly fluorescent triazole (DAF-2T). Here, we describe two methods for detection of NO by fluorescence, one for NO extracellular detection by DAF-2 and the other one for NO intracellular detection, in this case using DAF-2DA.

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.

Structural and functional changes in coffee trees after 4 years under free air CO2 enrichment.

Background and Aims: Climate forecasts suggest that [CO2] in the atmosphere will continue to increase. Structural and ecophysiological responses to elevated air [CO2] (e[CO2]) in tree species are contradictory due to species-dependent responses and relatively short-term experiments. It was hypothesized that long-term exposure (4 year) to e[CO2] would change canopy structure and function of Coffea arabica trees. Methods: Coffee plants were grown in a FACE (free air CO2 enrichment) facility under two air [CO2]: actual and elevated (actual + approx. 200 µL CO2 L-1). Plants were codified following the VPlants methodology to obtain coffee mock-ups. Plant canopies were separated into three 50 cm thick layers over a vertical profile to evaluate their structure and photosynthesis, using functional-structural plant modelling. Key Results: Leaf area was strongly reduced on the bottom and upper canopy layers, and increased soil carbon concentration suggested changes in carbon partitioning of coffee trees under e[CO2]. Increased air [CO2] stimulated stomatal conductance and leaf photosynthesis at the middle and upper canopy layers, increasing water-use efficiency. Under e[CO2], plants showed reduced diameter of the second-order axes and higher investment in the youngest third to fifth-order axes. Conclusions: The responses of Arabica coffee grown under long-term exposure to e[CO2] integrated structural and functional modifications, which balanced leaf area loss through improvements in leaf and whole-plant photosynthesis.

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.

Physiological and transcriptional analyses of developmental stages along sugarcane leaf.

BACKGROUND: Sugarcane is one of the major crops worldwide. It is cultivated in over 100 countries on 22 million ha. The complex genetic architecture and the lack of a complete genomic sequence in sugarcane hamper the adoption of molecular approaches to study its physiology and to develop new varieties. Investments on the development of new sugarcane varieties have been made to maximize sucrose yield, a trait dependent on photosynthetic capacity. However, detailed studies on sugarcane leaves are scarce. In this work, we report the first molecular and physiological characterization of events taking place along a leaf developmental gradient in sugarcane. RESULTS: Photosynthetic response to CO2 indicated divergence in photosynthetic capacity based on PEPcase activity, corroborated by activity quantification (both in vivo and in vitro) and distinct levels of carbon discrimination on different segments along leaf length. Additionally, leaf segments had contrasting amount of chlorophyll, nitrogen and sugars. RNA-Seq data indicated a plethora of biochemical pathways differentially expressed along the leaf. Some transcription factors families were enriched on each segment and their putative functions corroborate with the distinct developmental stages. Several genes with higher expression in the middle segment, the one with the highest photosynthetic rates, were identified and their role in sugarcane productivity is discussed. Interestingly, sugarcane leaf segments had a different transcriptional behavior compared to previously published data from maize. CONCLUSION: This is the first report of leaf developmental analysis in sugarcane. Our data on sugarcane is another source of information for further studies aiming to understand and/or improve C4 photosynthesis. The segments used in this work were distinct in their physiological status allowing deeper molecular analysis. Although limited in some aspects, the comparison to maize indicates that all data acquired on one C4 species cannot always be easily extrapolated to other species. However, our data indicates that some transcriptional factors were segment-specific and the sugarcane leaf undergoes through the process of suberizarion, photosynthesis establishment and senescence.

Exogenous sucrose supply changes sugar metabolism and reduces photosynthesis of sugarcane through the down-regulation of Rubisco abundance and activity.

Photosynthetic modulation by sugars has been known for many years, but the biochemical and molecular comprehension of this process is lacking. We studied how the exogenous sucrose supplied to leaves could affect sugar metabolism in leaf, sheath and stalk and inhibit photosynthesis in four-month old sugarcane plants. Exogenous sucrose 50mM sprayed on attached leaves strongly impaired the net CO2 assimilation (PN) and decreased the instantaneous carboxylation efficiency (PN/Ci), suggesting that the impairment in photosynthesis was caused by biochemical restrictions. The photosystem II activity was also affected by excess sucrose as indicated by the reduction in the apparent electron transport rate, effective quantum yield and increase in non-photochemical quenching. In leaf segments, sucrose accumulation was related to increases in the activities of soluble acid and neutral invertases, sucrose synthase and sucrose phosphate synthase, whereas the contents of fructose increased and glucose slightly decreased. Changes in the activities of sucrose hydrolyzing and synthesizing enzymes in leaf, sheath and stalk and sugar profile in intact plants were not enough to identify which sugar(s) or enzyme(s) were directly involved in photosynthesis modulation. However, exogenous sucrose was able to trigger down-regulation in the Rubisco abundance, activation state and enzymatic activity. Despite the fact that PN/Ci had been notably decreased by sucrose, in vitro activity and abundance of PEPCase did not change, suggesting an in vivo modulation of this enzyme. The data reveal that sucrose and/or other derivative sugars in leaves inhibited sugarcane photosynthesis by down-regulation of Rubisco synthesis and activity. Our data also suggest that sugar modulation was not exerted by a feedback mechanism induced by the accumulation of sugars in immature sugarcane stalk.

Looking for the physiological role of anthocyanins in the leaves of Coffea arabica.

The aim of this study was to determine which anthocyanins are related to the purple coloration of young leaves in Coffea arabica var. Purpurascens and assess their impact on photosynthesis as compared to C. arabica var. Catuaí, with green leaves. Two delphinidin glicosides were identified and histological cross-sections showed they were located throughout the adaxial epidermis in young leaves, disappearing as the leaves mature. Regardless the irradiance level, the photosynthetic performance of Purpurascens leaves did not differ from that observed in leaves of the Catuaí variety, providing no evidence that anthocyanins improve photosynthetic performance in coffee plants. To analyze the photoprotective action of anthocyanins, we evaluated the isomerization process for chlorogenic acids (CGAs) in coffee leaves exposed to UV-B radiation. No differences were observed in the total concentration of phenolic compounds in either variety before or after the UV treatment; however, we observed less degradation of CGA isomers in the Purpurascens leaves and a relative increase of cis-5-caffeoylquinic acid, a positional isomer of one of the most abundant form of CQA in coffee leaves, trans-5-caffeoylquinic acid, suggesting a possible protective role for anthocyanins in this purple coffee variety.

Photosynthetic changes and protective mechanisms against oxidative damage subjected to isolated and combined drought and heat stresses in Jatropha curcas plants.

Photosynthetic changes and protective mechanisms against oxidative damage were evaluated in Jatropha curcas leaves subjected to drought and heat stresses, both individually and combined, in order to elucidate the synergistic and antagonistic mechanisms involved with these abiotic factors. Both the drought and heat stresses caused significant damage to the leaf membrane integrity and lipid peroxidation, and the combination of these stresses greatly enhanced these physiological disturbances. The leaf CO(2) assimilation rate, stomatal conductance and instantaneous carboxylation efficiency (P(N)/C(I)) were significantly decreased in all plants subjected to stressful conditions in comparison to unstressed plants (reference). In contrast, a reduction in photochemical activity was observed only in plants exposed to drought and drought+heat conditions. Catalase (CAT), ascorbate peroxidase (APX) and superoxide dismutase (SOD) activities were stimulated only under heat stress, whereas APX activity was increased in all treated plants in comparison to the references. Moreover, the leaf H(2)O(2) content was increased similarly under all studied stresses. However, the balance of reduced and oxidized ascorbate did not show significant differences between reference and stressed plants. Although J. curcas plants acclimated to the studied stresses, they did not present an efficient mechanism for protection against drought-induced oxidative stress, especially when at high temperatures. However, heat-treated plants triggered an efficient enzymatic antioxidant system of reactive oxygen species scavenging and an effective protection against photochemical damages. The combination of drought and heat most significantly impaired the photosynthetic assimilation of CO(2) and the photochemical activity. These results indicate that drought greatly disturbs photosystem II activity and oxidative metabolism and that these negative effects are strongly stimulated by heat stress. The data also evidence that the combination of heat and drought triggers an intricate response involving antagonistic and synergistic interactions.

Descascamento de laranja 'Pêra' em função da duração do tratamento hidrotérmico / Peeling of 'Pera' sweet orange related to the duration of the hydrothermal treatment

Os objetivos deste trabalho foram adequar a tecnologia de descascamento de laranja 'Pêra' pelo uso do tratamento hidrotérmico e avaliar sua influência na atividade respiratória e nas características físico-químicas, microbiológicas e sensoriais dos frutos. O tempo de descascamento, o rendimento em frutos comercializáveis e a temperatura interna dos frutos durante o tratamento também foram avaliados. O tratamento consistiu em colocar os frutos em banho-maria a 50°C por 10, 15, 20, 25 e 30 minutos. Em seguida, os frutos foram descascados retirando-se a parte peduncular com a faca e, posteriormente, o flavedo e o albedo foram retirados manualmente. Os frutos sem tratamento hidrotérmico foram considerados controle. Os frutos foram armazenados durante seis dias a 5°C. O tratamento hidrotérmico alterou a atividade respiratória dos frutos somente nas primeiras horas após o processamento. A temperatura interna dos frutos, após 30 minutos de tratamento, atingiu no máximo 35°C. Não ocorreram alterações nas características físico-químicas e microbiológicas dos frutos. O tratamento não alterou o sabor, melhorou a aparência, diminuiu em até 78 por cento o tempo de descascamento e aumentou o rendimento em frutos comercializáveis. Conclui-se que o tratamento hidrotérmico de 10 a 30 minutos pode ser utilizado como técnica de descascamento para laranja 'Pêra'.

The objective of this research was to adapt the technology of peeling of 'Pera' sweet orange for the use of hydrothermal treatment, and to evaluate its influence in the respiratory activity and physicochemical, microbiologic and sensorial characteristics of fruits. Peeling time, marketable fruits yield and internal fruit temperature during the treatment were also evaluated. The hydrothermal treatment consisted of water-bath fruits at 50°C for 10, 15, 20, 25 e 30 minutes. The fruits were peeled by first opening cut the peduncle region with a knife and then, the flavedo and albedo were removed, manually. Fruits with no hydrothermal treatment were considerate as control. The fruits were stored at 5°C by six days. Hydrothermal treatment caused changes in respiratory activity just in first hours after processing. Internal fruit temperature after 30 minutes of treatment reached up to 35°C. There were no changes in the physicochemical and microbiologic characteristics of the fruits. The treatment did not change the flavor and improved the appearance. The treatment decreased in until 78 percent the peeling time of the treated fruits and it increased marketable fruits yield. In conclusion, the hydrothermal treatment from 10 to 30 minutes at 50°C can be used as peeling technique for 'Pera' sweet orange.