Exploring the microbiome of coffee plants: Implications for coffee quality and production.

Coffee stands as one of the world's most popular beverages, and its quality undergoes the influence of numerous pre- and post-harvest procedures. These encompass genetic variety, cultivation environment, management practices, harvesting methods, and post-harvest processing. Notably, microbial communities active during fermentation hold substantial sway over the ultimate quality and sensory characteristics of the final product. The interaction between plants and microorganisms assumes critical significance, with specific microbes assuming pivotal roles in coffee plant growth, fruit development, and, subsequently, the fruit's quality. Microbial activities can synthesize or degrade compounds that influence the sensory profile of the beverage. However, studies on the metabolic products generated by various coffee-related microorganisms and their chemical functionality, especially in building sensory profiles, remain scarce. The primary aim of this study was to conduct a literature review, based on a narrative methodology, on the current understanding of the plant-microorganism interaction in coffee production. Additionally, it aimed to explore the impacts of microorganisms on plant growth, fruit production, and the fermentation processes, directly influencing the ultimate quality of the coffee beverage. Articles were sourced from ScienceDirect, Scopus, Web of Science, and Google Scholar using specific search terms such as "coffee microorganisms", "microorganisms-coffee interactions", "coffee fermentation", "coffee quality", and 'coffee post-harvest processing". The articles used were published in English between 2000 and 2023. Selection criteria involved thoroughly examining articles to ensure their inclusion was based on results about the contribution of microorganisms to both the production and quality of the coffee beverage. The exploration of microorganisms associated with the coffee plant and its fruit presents opportunities for bioprospecting, potentially leading to targeted fermentations via starter cultures, consequently generating new profiles. This study synthesizes existing data on the current understanding of the coffee-associated microbiome, its functionalities within ecosystems, the metabolic products generated by microorganisms, and their impacts on fermentation processes and grain and beverage quality. It highlights the importance of plant-microorganism interactions in the coffee production chain.

Accumulation of Nutrients and the Relation between Fruit, Grain, and Husk of Coffee Robusta Cultivated in Brazilian Amazon.

Coffee genotypes cultivated in the Amazonian region have been gaining increasing prominence in Brazilian plantations. This study aimed to quantify nutrient accumulation in the fruits, grains, and husks of Robusta coffee genotypes cultivated in the Brazilian Amazon and estimate genetic diversity. The experiment was conducted in Alta Floresta D'Oeste-Rondônia, Brazil. To assess nutrient accumulation, fresh fruits were collected. These were dried, processed, separated into grains and husks, and subjected to chemical analysis. Nutrient accumulation in fruits, grains, and husks, as well as the grain/husk ratio, underwent analysis of variance through the F-test (p < 0.01. For each evaluated trait, the experimental coefficient of 337 variation (CVe), genetic coefficient of variation (CVg), and genotypic determination coefficient (H2) were also estimated. Variability was observed among Robusta coffee genotypes, with VP06, AS4, and AS10 being the most dissimilar. LB080 had the lowest dry fruit weight and the lowest percentage of grains in relation to husks. ZD156 accumulated more K in the grains, while VP06 and AS10 were the genotypes that accumulated more nutrients in the husks. Nutrients N, K, Ca, and P are accumulated in larger quantities, necessitating the calibration of mineral fertilization dosages and distribution.

Genomic Evaluation of Coffea arabica and Its Wild Relative Coffea racemosa in Mozambique: Settling Resilience Keys for the Coffee Crop in the Context of Climate Change.

Climate change is negatively affecting the coffee value chain, with a direct effect on approximately 100 million people from 80 countries. This has been attributed to the high vulnerability of the two-mainstream species, Coffea arabica and Coffea canephora, to extreme weather events, with notable uneven increases in market prices. Taking into account the narrow genetic plasticity of the commercial coffee cultivars, wild-relatives and underutilized Coffea species are valuable genetic resources. In this work, we have assessed the occurrence of Coffea species in to understand the degree of genetic relationships between Coffea species in the country, as well as the patterns of genetic diversity, differentiation, and genetic structure. Only one wild species was found, C. racemosa, which showed a high level of genetic separation with C. arabica, based on plastid, as well as SSR and SNP analysis. C. arabica presented low levels of diversity likely related to their autogamous nature, while the allogamous C. racemosa presented higher levels of diversity and heterozygosity. The analysis of the functional pathways based on SNPs suggests that the stress signaling pathways are more robust in this species. This novel approach shows that it is vital to introduce more resilient species and increase genomic diversity in climate-smart practices.

Nutritional Balance and Genetic Diversity of Coffea canephora Genotypes.

Our objectives were to study characteristics of the fruit (weight, percentage of husk/grain), to determine the concentration and accumulation of nutrients in the fruits, grain and husk, and to verify the existence of genetic diversity in Coffea canephora genotypes. The experiment was conducted with 20 genotypes in a four-year-old plantation, in a randomized block design with four replications and five plants per plot. The fruits were oven-dried, depulped (husk separated from the grain) and sent to a laboratory for nutritional analysis. Macronutrients N and K were the most accumulated/exported in fruits, respectively. In addition, the different genotype control cycles influenced the accumulation of nutrients in the fruits. There was genetic diversity among the 20 C. canephora genotypes, studied for the characteristics of concentration and percentage of grain/straw nutrients in the fruit. Genotypes 2, 8 and 13 were the ones with the greatest genetic distance, consequently they are the most dissimilar when compared to the other genotypes. Genotypes 8 and 1 stand out for having a higher proportion of fruit weight in relation to grains. Therefore, they are the genotypes that need a smaller amount of fruit to produce 1000 kg of ground coffee.

Overexpression of Water-Responsive Genes Promoted by Elevated CO2 Reduces ROS and Enhances Drought Tolerance in Coffea Species.

Drought is a major constraint to plant growth and productivity worldwide and will aggravate as water availability becomes scarcer. Although elevated air [CO2] might mitigate some of these effects in plants, the mechanisms underlying the involved responses are poorly understood in woody economically important crops such as Coffea. This study analyzed transcriptome changes in Coffea canephora cv. CL153 and C. arabica cv. Icatu exposed to moderate (MWD) or severe water deficits (SWD) and grown under ambient (aCO2) or elevated (eCO2) air [CO2]. We found that changes in expression levels and regulatory pathways were barely affected by MWD, while the SWD condition led to a down-regulation of most differentially expressed genes (DEGs). eCO2 attenuated the impacts of drought in the transcripts of both genotypes but mostly in Icatu, in agreement with physiological and metabolic studies. A predominance of protective and reactive oxygen species (ROS)-scavenging-related genes, directly or indirectly associated with ABA signaling pathways, was found in Coffea responses, including genes involved in water deprivation and desiccation, such as protein phosphatases in Icatu, and aspartic proteases and dehydrins in CL153, whose expression was validated by qRT-PCR. The existence of a complex post-transcriptional regulatory mechanism appears to occur in Coffea explaining some apparent discrepancies between transcriptomic, proteomic, and physiological data in these genotypes.

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.

Multi-Environment and Multi-Year Bayesian Analysis Approach in Coffee canephora.

This work aimed to use the Bayesian approach to discriminate 43 genotypes of Coffea canephora cv. Conilon, which were cultivated in two producing regions to identify the most stable and productive genotypes. The experiment was a randomized block design with three replications and seven plants per plot, carried out in the south of Bahia and the north of Espírito Santo, environments with different climatic conditions, and evaluated during four harvests. The proposed Bayesian methodology was implemented in R language, using the MCMCglmm package. This approach made it possible to find great genetic divergence between the materials, and detect significant effects for both genotype, environment, and year, but the hyper-parametrized models (block effect) presented problems of singularity and convergence. It was also possible to detect a few differences between crops within the same environment. With a model with lower residual, it was possible to recommend the most productive genotypes for both environments: LB1, AD1, Peneirão, Z21, and P2.

Coffea canephora: Heterotic Crosses Indicated by Molecular Approach.

The genus Coffea comprises more than 100 species, of which the most commercially important are Coffea arabica and Coffea canephora. The latter is a self-incompatible plant with high natural genetic variability. The detection of polymorphism at the DNA level by molecular markers allowed significant progress with the selection of superior plants. The objective of this study was the molecular characterization of C. canephora using microsatellite markers. To this end, a population of forty-four C. canephora genotypes and one C. arabica genotype, was evaluated with 21 primers. These primers identified 61 alleles in the population and between 2 and 5 alleles per locus. The information index indicated a high level of polymorphism of the analyzed markers. According to the observed and expected heterozygosity, the genetic diversity in the population is high. The overall inbreeding coefficient of the population detected high heterozygosity and zero inbreeding within this population. Genetic diversity among the accessions was also evaluated by the unweighted pair-group method based on arithmetic averages (UPGMA). Six groups were formed based on Mojena's cutting rule and three using the Bayesian approach. These results confirmed the existence of genetic diversity, genetic variability and a potential for selection in future breeding efforts involving the 45 genotypes studied.

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.

Adaptability and stability of Coffea canephora to dynamic environments using the Bayesian approach.

The objective of this work was to use the Bayesian approach, modeling the interaction of coffee genotypes with the environment, using a bisegmented regression to identify stable and adapted genotypes. A group of 43 promising genotypes of Coffea canephora was chosen. The genotypes were arranged in a randomized block design with three replications of seven plants each. The experimental plot was harvested four years in the study period, according to the maturation cycle of each genotype. The proposed Bayesian methodology was implemented in the free program R using rstanarm and coda packages. It was possible to use previous information on coffee genotypes as prior information on parameter distributions of an Adaptability and Stability model, which allowed obtaining shorter credibility intervals and good evidence of low bias in the model by the determination coefficient. After fine adjustments in the approach, it was possible to make inferences about the significant GxE interaction and to discriminate the coffee genotypes regarding production, adaptability, and stability. This is still a new approach for perennials, and since it allows more accurate estimates it can be advantageous when planning breeding programs. The Z21 genotype is recommended to compose part of selected genetic material for highly technical farmers, as it responds very well to the favorable environment, being one of the most productive and with excellent stability.

Microclimate and development of Coffea canephora intercropped with Carica papaya: measures to mitigate climate change

Intercropped systems with Conilon coffee might provide a better environment for coffee production. The aim of this study was to assess the microclimate and development of Conilon coffee intercropped with papaya trees. Papaya was planted with spacing of 3.20 x 2.40 m. The coffee trees were planted after eight months, with spacing of 3.20 x 1.60 m, in-between papaya trees (in the same row). The measurements were taken 0, 40 and 80 cm away from the coffee plants, both in the north and south direction. Concomitantly, an adjoining full sunlight coffee system (not intercropped) was also assessed. The measurements included atmospheric parameters (temperature, irradiance, and relative humidity) and vegetative parameters for the coffee plants (leaf area, relative chlorophyll index, length of plagiotropic branches, length of orthotopic branches and number of nodes) in three periods of the year. The intercropped system of Conilon coffee and papaya trees led to a decrease in both irradiance and temperature, and higher means of relative humidity during daytime in all the periods assessed, which contributes to a better environment for coffee cultivation. The shadow provided by papaya trees in the coffee plants contributed to a higher leaf area but did not affect neither the growth of both plagiotropic and orthotopic branches, nor the number of nodes and the etiolation. The intercropped system of Conilon coffee and papaya trees may be potentially used as a farming system to mitigate climate change.

Concentration of Nutrients in Leaves, Flowers, and Fruits of Genotypes of Coffea canephora.

It is important to consider the nutritional demand among genotypes of the same species to achieve high yields. Thus, the objective of this study was to determine the concentration of nutrients in leaves, flowers, grains, and fruit straw in conilon coffee genotypes. The experiment was carried out under a randomized block design, with three replications and the evaluation of nine genotypes. Leaf collections were carried out every three months, from August 2019 to May 2020. Flowers were collected at flowering in July 2019 and fruits in June 2020, upon complete maturation of the genotypes. The materials were dried in an oven and sent for laboratory analysis to determine the nutritional content. Data were subjected to analysis of variance followed by a comparison of means and estimates of genetic parameters and clustering using the hierarchical method (UPGMA). The nutrients found in the highest concentrations in the evaluated plant organs were N and K for macronutrients and Fe for micronutrients. For the leaves, the concentrations of the main nutrients were high in the first and reduced in the last evaluated periods, possibly due to mobilization to the fruits. Considering all the plant tissues evaluated, the order of concentration of macronutrients and micronutrients was N > K > Ca > P = Mg = S and Fe > B > Mn > Cu > Zn, respectively. For a nutritional diagnosis, it is important to take comparisons of the genetic diversity and evaluation periods into consideration.

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.

Soil patterns and foliar standards for two cocoa clones in the States of Espírito Santo and Bahia, Brazil / Padrões de solo e normas foliares para dois clones de cacau cultivados nos Estados do Espírito Santo e Bahia, Brasil

ABSTRACT: The objective of this research was to establish soil patterns and foliar standards for clonal crops of cocoa CCN51 and PS1319 in the Atlantic regions within the northern Espírito Santo and southern Bahia States. Samples were collected from September to October 2015, in 45 high productive plots, being 23 plots of clone CCN51 and 22 of clone PS1319. Sufficiency ranges for soil and foliar standards were recommended for cocoa clones CCN51 and PS1319 planted in the Northern Espírito Santo and Southern Bahia states. Differences in the contents of foliar macronutrients K, Ca, Mg, S and micronutrients Fe and Mn, were verified between clones CCN51 and PS1319. Differences reported for standards from different geographical regions confirmed that these standards need to be regional and specific for different cocoa clones.

RESUMO: O objetivo deste trabalho foi estabelecer padrões de solo e padrões foliares para culturas clonais de cacau CCN51 e PS1319 nas regiões do Atlântico no norte do Espírito Santo e no sul da Bahia. As amostras foram coletadas de setembro a outubro de 2015, em 45 parcelas de alta produtividade, sendo 23 parcelas do clone CCN51 e 22 do clone PS1319. As faixas de suficiência para solo e padrões foliares foram sugeridas para os clones de cacau CCN51 e PS1319 plantados no norte do Espírito Santo e no sul da Bahia. Diferenças nos teores dos macronutrientes foliares K, Ca, Mg, S e micronutrientes Fe e Mn foram verificadas entre os clones CCN51 e PS1319. As diferenças relatadas para padrões de diferentes regiões geográficas permitem confirmar que esses padrões precisam ser regionais e específicos para diferentes clones de cacau.

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.