Transcriptomic Analyses Reveal That Coffea arabica and Coffea canephora Have More Complex Responses under Combined Heat and Drought than under Individual Stressors.

Increasing exposure to unfavorable temperatures and water deficit imposes major constraints on most crops worldwide. Despite several studies regarding coffee responses to abiotic stresses, transcriptome modulation due to simultaneous stresses remains poorly understood. This study unravels transcriptomic responses under the combined action of drought and temperature in leaves from the two most traded species: Coffea canephora cv. Conilon Clone 153 (CL153) and C. arabica cv. Icatu. Substantial transcriptomic changes were found, especially in response to the combination of stresses that cannot be explained by an additive effect. A large number of genes were involved in stress responses, with photosynthesis and other physiologically related genes usually being negatively affected. In both genotypes, genes encoding for protective proteins, such as dehydrins and heat shock proteins, were positively regulated. Transcription factors (TFs), including MADS-box genes, were down-regulated, although responses were genotype-dependent. In contrast to Icatu, only a few drought- and heat-responsive DEGs were recorded in CL153, which also reacted more significantly in terms of the number of DEGs and enriched GO terms, suggesting a high ability to cope with stresses. This research provides novel insights into the molecular mechanisms underlying leaf Coffea responses to drought and heat, revealing their influence on gene expression.

Uncovering the wide protective responses in Coffea spp. leaves to single and superimposed exposure of warming and severe water deficit.

Climate changes boosted the frequency and severity of drought and heat events, with aggravated when these stresses occur simultaneously, turning crucial to unveil the plant response mechanisms to such harsh conditions. Therefore, plant responses/resilience to single and combined exposure to severe water deficit (SWD) and heat were assessed in two cultivars of the main coffee-producing species: Coffea arabica cv. Icatu and C. canephora cv. Conilon Clone 153 (CL153). Well-watered plants (WW) were exposed to SWD under an adequate temperature of 25/20°C (day/night), and thereafter submitted to a gradual increase up to 42/30°C, and a 14-d recovery period (Rec14). Greater protective response was found to single SWD than to single 37/28°C and/or 42/30°C (except for HSP70) in both cultivars, but CL153-SWD plants showed the larger variations of leaf thermal imaging crop water stress index (CWSI, 85% rise at 37/28°C) and stomatal conductance index (IG, 66% decline at 25/20°C). Both cultivars revealed great resilience to SWD and/or 37/28°C, but a tolerance limit was surpassed at 42/30°C. Under stress combination, Icatu usually displayed lower impacts on membrane permeability, and PSII function, likely associated with various responses, usually mostly driven by drought (but often kept or even strengthened under SWD and 42/30°C). These included the photoprotective zeaxanthin and lutein, antioxidant enzymes (superoxide dismutase, Cu,Zn-SOD; ascorbate peroxidase, APX), HSP70, arabinose and mannitol (involving de novo sugar synthesis), contributing to constrain lipoperoxidation. Also, only Icatu showed a strong reinforcement of glutathione reductase activity under stress combination. In general, the activities of antioxidative enzymes declined at 42/30°C (except Cu,Zn-SOD in Icatu and CAT in CL153), but HSP70 and raffinose were maintained higher in Icatu, whereas mannitol and arabinose markedly increased in CL153. Overall, a great leaf plasticity was found, especially in Icatu that revealed greater responsiveness of coordinated protection under all experimental conditions, justifying low PIChr and absence of lipoperoxidation increase at 42/30°C. Despite a clear recovery by Rec14, some aftereffects persisted especially in SWD plants (e.g., membranes), relevant in terms of repeated stress exposure and full plant recovery to stresses.

High-resolution shotgun proteomics reveals that increased air [CO2] amplifies the acclimation response of coffea species to drought regarding antioxidative, energy, sugar, and lipid dynamics.

As drought threatens crop productivity it is crucial to characterize the defense mechanisms against water deficit and unveil their interaction with the expected rise in the air [CO2]. For that, plants of Coffea canephora cv. Conilon Clone 153 (CL153) and C. arabica cv. Icatu grown under 380 (aCO2) or 700 µL L-1 (eCO2) were exposed to moderate (MWD) and severe (SWD) water deficits. Responses were characterized through the activity and/or abundance of a selected set of proteins associated with antioxidative (e.g., Violaxanthin de-epoxidase, Superoxide dismutase, Ascorbate peroxidases, Monodehydroascorbate reductase), energy/sugar (e.g., Ferredoxin-NADP reductase, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, sucrose synthase, mannose-6-phosphate isomerase, Enolase), and lipid (Lineolate 13S-lipoxygenase) processes, as well as with other antioxidative (ascorbate) and protective (HSP70) molecules. MWD caused small changes in both genotypes regardless of [CO2] level while under the single imposition to SWD, only Icatu showed a global reinforcement of most studied proteins supporting its tolerance to drought. eCO2 alone did not promote remarkable changes but strengthened a robust multi-response under SWD, even supporting the reversion of impacts already observed by CL153 at aCO2. In the context of climate changes where water constraints and [CO2] levels are expected to increase, these results highlight why eCO2 might have an important role in improving drought tolerance in Coffea species.

Intrinsic non-stomatal resilience to drought of the photosynthetic apparatus in Coffea spp. is strengthened by elevated air [CO2].

Growing water restrictions associated with climate changes constitute daunting challenges to crop performance. This study unveils the impacts of moderate (MWD) or severe (SWD) water deficit, and their interaction with air [CO2], on the photosynthetic apparatus of Coffea canephora Pierre ex A. Froehner cv. Conilon Clone 153 (CL153) and Coffea arabica L. cv. Icatu. Seven year-old potted plants grown under 380 (aCO2) or 700 µl l -1 (eCO2) [CO2] gradually reached predawn water potentials between -1.6 and -2.1 MPa (MWD), and below -3.5 MPa (SWD). Under drought, stomata closure was chiefly related to abscisic acid (ABA) rise. Increasing drought severity progressively affected gas exchange and fluorescence parameters in both genotypes, with non-stomatal limitations becoming gradually dominating, especially regarding the photochemical and biochemical components of CL153 SWD plants. In contrast, Icatu plants were highly tolerant to SWD, with minor, if any, negative impacts on the potential photosynthetic functioning and components (e.g., Amax, Fv/Fm, electron carriers, photosystems (PSs) and ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO) activities). Besides, drought-stressed Icatu plants displayed increased abundance of a large set of proteins associated with the photosynthetic apparatus (PSs, light-harvesting complexes, cyclic electron flow, RuBisCO activase) regardless of [CO2]. Single eCO2 did not promote stomatal and photosynthetic down-regulation in both genotypes. Instead, eCO2 increased photosynthetic performance, moderately reinforced photochemical (PSs activity, electron carriers) and biochemical (RuBisCO, ribulose-5-phosphate kinase) components, whereas photoprotective mechanisms and protein abundance remained mostly unaffected. In both genotypes, under MWD, eCO2 superimposition delayed stress severity and promoted photosynthetic functioning with lower energy dissipation and PSII impacts, whereas stomatal closure was decoupled from increases in ABA. In SWD plants, most impacts on the photosynthetic performance were reduced by eCO2, especially in the moderately drought affected CL153 genotype, although maintaining RuBisCO as the most sensitive component, deserving special breeder's attention to improve coffee sustainability under future climate scenarios.

Resilient and Sensitive Key Points of the Photosynthetic Machinery of Coffea spp. to the Single and Superimposed Exposure to Severe Drought and Heat Stresses.

This study unveils the single and combined drought and heat impacts on the photosynthetic performance of Coffea arabica cv. Icatu and C. canephora cv. Conilon Clone 153 (CL153). Well-watered (WW) potted plants were gradually submitted to severe water deficit (SWD) along 20 days under adequate temperature (25/20°C, day/night), and thereafter exposed to a gradual temperature rise up to 42/30°C, followed by a 14-day water and temperature recovery. Single drought affected all gas exchanges (including Amax ) and most fluorescence parameters in both genotypes. However, Icatu maintained Fv/Fm and RuBisCO activity, and reinforced electron transport rates, carrier contents, and proton gradient regulation (PGR5) and chloroplast NADH dehydrogenase-like (NDH) complex proteins abundance. This suggested negligible non-stomatal limitations of photosynthesis that were accompanied by a triggering of protective cyclic electron transport (CEF) involving both photosystems (PSs). These findings contrasted with declines in RuBisCO and PSs activities, and cytochromes (b559 , f, b563 ) contents in CL153. Remarkable heat tolerance in potential photosynthetic functioning was detected in WW plants of both genotypes (up to 37/28°C or 39/30°C), likely associated with CEF in Icatu. Yet, at 42/30°C the tolerance limit was exceeded. Reduced Amax and increased Ci values reflected non-stomatal limitations of photosynthesis, agreeing with impairments in energy capture (F0 rise), PSII photochemical efficiency, and RuBisCO and Ru5PK activities. In contrast to PSs activities and electron carrier contents, enzyme activities were highly heat sensitive. Until 37/28°C, stresses interaction was largely absent, and drought played the major role in constraining photosynthesis functioning. Harsher conditions (SWD, 42/30°C) exacerbated impairments to PSs, enzymes, and electron carriers, but uncontrolled energy dissipation was mitigated by photoprotective mechanisms. Most parameters recovered fully between 4 and 14 days after stress relief in both genotypes, although some aftereffects persisted in SWD plants. Icatu was more drought tolerant, with WW and SWD plants usually showing a faster and/or greater recovery than CL153. Heat affected both genotypes mostly at 42/30°C, especially in SWD and Icatu plants. Overall, photochemical components were highly tolerant to heat and to stress interaction in contrast to enzymes that deserve special attention by breeding programs to increase coffee sustainability in climate change scenarios.

Agronomic performance and genetic divergence between genotypes of Manihot esculenta.

The morphoagronomic characterization of 12 genotypes of M. esculenta was performed during the 2013/2014 and 2014/2015 crop years. The 12 genotypes were planted in a randomized block design, with four replicates per genotype. Number of tuberous roots per plant, weight of tuberous roots, root yield, total plant weight, harvest index, plant height, height of first branch, number of shoots, stem diameter, number of buds, leaf dry weight and petiole length were evaluated. Genotypes "Camuquem" and "Goiás" were the most productive, and "Amarela" and "Gema de Ovo" were the most divergent. Seventy percent of genetic diversity was due to petiole length (22.86%), root yield (19.20%), weight of tuberous roots (14.89%) and number of buds (13.72%). Overall, the present results indicate a broad genetic basis for the evaluated genotypes, so that such genetic variation benefits the plant breeding for future scenarios Further studies of the evaluated genotypes should be performed under environmental limitations, using biochemical and molecular tools to identify markers for genetic improvement.

Genetic diversity of promising 'conilon' coffee clones based on morpho-agronomic variables.

Knowledge of the genetic variability of a population is essential to guide its preservation and maintenance in addition to increasing the efficiency of genetic breeding programs. On this basis, this study was conducted to evaluate the genetic diversity of Coffea canephora genotypes using multivariate statistical procedures applied to a set of morpho-agronomic variables. The materials employed in this study constitute a crop located in Vila Valério - ES, Brazil, where the genotypes are arranged in a randomized-blocks experimental design with four replicates. Significant differences were detected by the F test at the 1% or 5% probability levels among the genotypes for all evaluated traits, demonstrating heterogeneity of genetic constitution in the studied population, which is favorable to breeding, as it indicates the possibility to identify superior and divergent individuals. Based on the generalized Mahalanobis distance, the most divergent combinations were obtained between genotypes 23 and 10 (256.43) and 23 and 17 (250.09). The clusters formed by Tocher's optimization and the UPGMA hierarchical method agreed, both similarly grouping the genotypes into three clusters. Of the analyzed traits, mature fruit weight (19.08%), yield (15.50%), plant diameter (12.42%), and orthotropic-shoot internode length (10.94%) were the most efficient to explain the dissimilarity among the genotypes.

Selection and Validation of Reference Genes for Accurate RT-qPCR Data Normalization in Coffea spp. under a Climate Changes Context of Interacting Elevated [CO2] and Temperature.

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.

Protective Response Mechanisms to Heat Stress in Interaction with High [CO2] Conditions in Coffea spp.

Modeling studies have predicted that coffee crop will be endangered by future global warming, but recent reports highlighted that high [CO2] can mitigate heat impacts on coffee. This work aimed at identifying heat protective mechanisms promoted by CO2 in Coffea arabica (cv. Icatu and IPR108) and Coffea canephora cv. Conilon CL153. Plants were grown at 25/20°C (day/night), under 380 or 700 µL CO2 L(-1), and then gradually submitted to 31/25, 37/30, and 42/34°C. Relevant heat tolerance up to 37/30°C for both [CO2] and all coffee genotypes was observed, likely supported by the maintenance or increase of the pools of several protective molecules (neoxanthin, lutein, carotenes, α-tocopherol, HSP70, raffinose), activities of antioxidant enzymes, such as superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), catalase (CAT), and the upregulated expression of some genes (ELIP, Chaperonin 20). However, at 42/34°C a tolerance threshold was reached, mostly in the 380-plants and Icatu. Adjustments in raffinose, lutein, ß-carotene, α-tocopherol and HSP70 pools, and the upregulated expression of genes related to protective (ELIPS, HSP70, Chape 20, and 60) and antioxidant (CAT, CuSOD2, APX Cyt, APX Chl) proteins were largely driven by temperature. However, enhanced [CO2] maintained higher activities of GR (Icatu) and CAT (Icatu and IPR108), kept (or even increased) the Cu,Zn-SOD, APX, and CAT activities, and promoted a greater upregulation of those enzyme genes, as well as those related to HSP70, ELIPs, Chaperonins in CL153, and Icatu. These changes likely favored the maintenance of reactive oxygen species (ROS) at controlled levels and contributed to mitigate of photosystem II photoinhibition at the highest temperature. Overall, our results highlighted the important role of enhanced [CO2] on the coffee crop acclimation and sustainability under predicted future global warming scenarios.

Long-term elevated air [CO2 ] strengthens photosynthetic functioning and mitigates the impact of supra-optimal temperatures in tropical Coffea arabica and C. canephora species.

The tropical coffee crop has been predicted to be threatened by future climate changes and global warming. However, the real biological effects of such changes remain unknown. Therefore, this work aims to link the physiological and biochemical responses of photosynthesis to elevated air [CO2 ] and temperature in cultivated genotypes of Coffea arabica L. (cv. Icatu and IPR108) and Coffea canephora cv. Conilon CL153. Plants were grown for ca. 10 months at 25/20°C (day/night) and 380 or 700 µl CO2 l(-1) and then subjected to temperature increase (0.5°C day(-1) ) to 42/34°C. Leaf impacts related to stomatal traits, gas exchanges, C isotope composition, fluorescence parameters, thylakoid electron transport and enzyme activities were assessed at 25/20, 31/25, 37/30 and 42/34°C. The results showed that (1) both species were remarkably heat tolerant up to 37/30°C, but at 42/34°C a threshold for irreversible nonstomatal deleterious effects was reached. Impairments were greater in C. arabica (especially in Icatu) and under normal [CO2 ]. Photosystems and thylakoid electron transport were shown to be quite heat tolerant, contrasting to the enzymes related to energy metabolism, including RuBisCO, which were the most sensitive components. (2) Significant stomatal trait modifications were promoted almost exclusively by temperature and were species dependent. Elevated [CO2 ], (3) strongly mitigated the impact of temperature on both species, particularly at 42/34°C, modifying the response to supra-optimal temperatures, (4) promoted higher water-use efficiency under moderately higher temperature (31/25°C) and (5) did not provoke photosynthetic downregulation. Instead, enhancements in [CO2 ] strengthened photosynthetic photochemical efficiency, energy use and biochemical functioning at all temperatures. Our novel findings demonstrate a relevant heat resilience of coffee species and that elevated [CO2 ] remarkably mitigated the impact of heat on coffee physiology, therefore playing a key role in this crop sustainability under future climate change scenarios.

Phospholipids profile in chloroplasts of Coffea spp. genotypes differing in cold acclimation ability.

Environmental temperature change may induce modifications in membrane lipid properties and composition, which account for different physiological responses among plant species. Coffee plants, as many tropical species, are particularly sensitive to cold, but genotypes can present differences that can be exploited to improve crop management and breeding. This work intended to highlight the changes promoted by low non-freezing temperatures (chilling) in phospholipid (PL) composition of chloroplast membranes of genotypes from two Coffea species, Coffea arabica cv. Catuaí (moderately tolerant) and Coffea canephora cv. Conilon (Clone 153, more susceptible), and relate them with cold sensitivity differences. Such evaluation was performed considering a gradual temperature decrease, chilling (4 °C) exposure and a recovery period under rewarming conditions. Catuaí presented an earlier acclimation response than Clone 153 (CL 153). It displayed a higher metabolic activity during acclimation (total fatty acids and total PL increases) and chilling (phosphatidylglycerol increases), and an overall better recovery. Catuaí also showed the highest phosphatidylglycerol unsaturation (higher double bond index) after chilling, in contrast with CL 153 (gradual unsaturation decrease). Higher unsaturation degree in Catuaí than in CL 153 was also observed for phosphatidylcholine and phosphatidylinositol, resulting, mainly, from raises in unsaturated C18:2 and C18:3. It is suggested that an enhanced PL synthesis and turnover induced by a gradual cold exposure, as well as unsaturation increases in major PL classes, is related to decreased Catuaí susceptibility to low temperatures and strongly contributes to sustain photosynthetic activity in this genotype under chilling conditions, as reported in previous work by this team.