World coffee production has faced increasing challenges associated with ongoing climatic changes. Several studies, which have been almost exclusively based on temperature increase, have predicted extensive reductions (higher than half by 2,050) of actual coffee cropped areas. However, recent studies showed that elevated [CO2] can strongly mitigate the negative impacts of heat stress at the physiological and biochemical levels in coffee leaves. In addition, it has also been shown that coffee genotypes can successfully cope with temperatures above what has been traditionally accepted. Altogether, this information suggests that the real impact of climate changes on coffee growth and production could be significantly lower than previously estimated. Gene expression studies are an important tool to unravel crop acclimation ability, demanding the use of adequate reference genes. We have examined the transcript stability of 10 candidate reference genes to normalize RT-qPCR expression studies using a set of 24 cDNAs from leaves of three coffee genotypes (CL153, Icatu, and IPR108), grown under 380 or 700 μL CO2 L−1, and submitted to increasing temperatures from 25/20°C (day/night) to 42/34°C. Samples were analyzed according to genotype, [CO2], temperature, multiple stress interaction ([CO2], temperature) and total stress interaction (genotype, [CO2], and temperature). The transcript stability of each gene was assessed through a multiple analytical approach combining the Coeficient of Variation method and three algorithms (geNorm, BestKeeper, NormFinder). The transcript stability varied according to the type of stress for most genes, but the consensus ranking obtained with RefFinder, classified MDH as the gene with the highest mRNA stability to a global use, followed by ACT and S15, whereas α-TUB and CYCL showed the least stable mRNA contents. Using the coffee expression profiles of the gene encoding the large-subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RLS), results from the in silico aggregation and experimental validation of the best number of reference genes showed that two reference genes are adequate to normalize RT-qPCR data. Altogether, this work highlights the importance of an adequate selection of reference genes for each single or combined experimental condition and constitutes the basis to accurately study molecular responses of Coffea spp. in a context of climate changes and global warming.

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Temperature increase assumes a prominent role in the context of expected climate change because of its significant impact on plant metabolism. High temperature can affect the carbon-assimilation pathway at both stomatal and non-stomatal levels, mainly through stomatal closure and photochemical and biochemical limitations. In general, however, plants have some ability to trigger acclimation mechanisms to cope with stressful conditions, especially if the limitations are imposed in a gradual manner during seasonal change. This study aims at evaluating changes at stomatal and photochemical levels in Coffea arabica and C. canephora under exposure to mild temperature (spring) and high temperature (summer). Potted plants were maintained in a greenhouse, watered to field capacity and subject to natural variations of light, temperature and relative humidity. In C. arabica, exposure to summer conditions decreased photosynthetic rates (A), stomatal conductance (gs) and stomatal density and increased intrinsic water-use efficiency (iWUE) compared with spring values, whereas C. canephora plants maintained similar values in both seasons. However, C. canephora presented lower A and gs during spring than C. arabica. Because photosynthetic capacity (Amax), photosynthetic performance index and membrane permeability were similar between genotypes and seasons, and maximum quantum yield (Fv/Fm) and photosynthetic pigments were not affected in C. arabica in summer, we conclude that under high temperature conditions, stomatal closure imposes the major limitation on C. arabica photosynthesis in summer. Finally, both coffee genotypes were able to avoid damage to photochemistry pathway under supra-optimal temperatures.

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Diagnosing foliar nutritional status is essential for fertilizer recommendations and for the identification of nutrient imbalances. This study aimed to verify genetic diversity and establish mean standards (leaf nutrient contents; LNCs) and relationships among leaf nutrients (LNC relationships; LNCRs) in seven conilon coffee genotypes during both pre-flowering and bean-filling stages. Twenty crops from several cities in the northern region of Espírito Santo State, Brazil, with crop yield either equal to or greater than 100 bags per hectare (during two harvests) were assessed. A total of 140 samples were collected during each evaluation period for quantification of leaf nutrient contents (N, P, K, Ca, Mg, S, Fe, Zn, Cu, Mn, and B). The Ward procedure, a hierarchical genetic clustering method, was used to quantify the genetic diversity among genotypes. To examine differences between the LNCs and LNCRs, F-and Scott-Knott tests were used. LNCs and LNCRs showed significant differences among the conilon coffee genotypes during the evaluation periods. Additionally, the 8V, 10V,and 12V genotypes exhibited the highest values for most of the nutrients, especially for N, P, and Cu. Therefore, the clustering method revealed genetic diversity among genotypes for standard leaf nutrient levels, implying that leaf diagnosis could be specific to each genotype and phenological stage.