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The aim of this study was to identify the correlation between photochemical efficiency and candidate genes expression to elucidate the drought tolerance mechanisms in coffee progenies (Icatu Vermelho IAC 3851-2 x Catimor UFV 1602-215) previously identified as tolerant in field conditions. Four progenies (2, 5, 12 and 15) were evaluated under water-deficit conditions (water deficit imposed 8 months after transplanting seedlings to the pots) and under irrigated system. Evaluations of physiological parameters and expression of candidate genes for drought tolerance were performed. Progeny 5 showed capacity to maintain water potential, which contributed to lower qP variation between irrigated and deficit conditions. However, the increases of qN and NPQ in response to stress indicate that this progeny is photochemically responsive to small variations of am protecting the photosystem and maintaining qP. Data obtained for progeny 12 indicated a lower water status maintenance capacity, but with increased qN and NPQ providing maintenance of the PSII and ETR parameters. A PCA analysis revealed that the genes coding regulatory proteins, ABA-synthesis, cellular protectors, isoforms of ascorbate peroxidase clearly displayed a major response to drought stress and discriminated the progenies 5 and 12 which showed a better photochemical response. The genes CaMYB1, CaERF017, CaEDR2, CaNCED, CaAPX1, CaAPX5, CaGolS3, CaDHN1 and CaPYL8a were up-regulated in the arabica coffee progenies with greater photochemical efficiency under deficit and therefore contributing to efficiency of the photosynthesis in drought tolerant progenies Made available in DSpace on 2021-07-20T15:01:13Z (GMT). No. of bitstreams: 1 Photochemical-efciency-correlated.pdf: 1599792 bytes, checksum: 8fcd09d5ca7177dd144b3a900116a490 (MD5) Previous issue date: 2021

The employment of biotechnology-based approaches such as somatic embryogenesis has been applied to several plants including Coffea sp. Despite the economic importance of this genus, few information about the role of key regulatory genes in somatic embryogenesis in coffee is available. This work provides information about ABI3 and VAL2 genes performance by RT-qPCR during indirect somatic embryogenesis of Coffea arabica. To achieve this, bioinformatics analysis was performed to identify the genes of the B3 superfamily in the coffee genome. The cell suspensions lines presented similar histological and regeneration patterns, yielding of up to 6.6 embryos per 1 mg of embryogenic aggregates at 7 months. We have identified possible orthologs for VAL2 (Cc06g00410) and ABI3 (Cc01g17380) as well as the other members belonging to superfamily B3. The CaABI3 expression was higher in dedifferentiated competent cells for somatic embryogenesis as compared with non-embryogenic calli. Whereas the expression of VAL2 gene is more active in cotyledonary embryos and plantlets, showing its clear performance in the embryogenesis late stages. The present study suggests that CaABI3 gene could be potentially used as a biomarker for embryogenic process improvement. The good plantlets development obtained from the protocol used may be a reflection of the high expression of CaVAL2 in cotyledonary embryos and plantlets. The activity of CaABI3 is correlated to embryogenic potential with highly expressed in embryogenic masses and expression of the VAL2 gene is increased at the end of the embryogenic process.

International audience; Climate change is posing a major challenge to coffee production worldwide leading to a need for the development of coffee cultivars with increased drought tolerance. In several plant species, the use of DREB genes in crop improvement has achieved promising results to desiccation tolerance engineering. Recent studies reported CcDREB1D specific patterns of expression in Coffea canephora and functional evidence of this gene involvement in drought stress responses. However, knowledge on natural diversity of this gene is largely unknown. In this context, this study aimed at evaluating the sequence variability of the DREB1D gene in several Coffea genotypes. Nucleotide variation in promoters and coding regions of this gene were evaluated in a population consisting of 38 genotypes of C. canephora, C. arabica and C. eugenioides, most of them characterized by different phenotypes (tolerance vs. susceptibility) in relation to drought. The genetic diversity of the loci revealed different haplotypes for the promoter and coding regions. In particular, our findings suggest association between drought tolerance and the genetic variations on DREB1D promoter regions, but not with those from its corresponding coding regions. Gene expression studies revealed up-regulated expression of DREB1D gene upon drought mainly in leaves of drought-tolerant clones of C. canephora, and in response to drought, high, and low temperatures in leaves of C. arabica, suggesting a key role of this gene in coffee responses to abiotic stress.

Plant growth is related to the amount of nutrients that it can acquire and it can be limited by poor root development and nutrient uptake, and low efficiency of nutrient use. Nutrients are not distributed evenly throughout the soil around roots, causing plants to put in action the functioning of the various local and systemic signaling pathways to coordinate adaptative responses to the most diverse types of stresses, among them nutritional limitation. A coordinated array of long-distance signaling mechanisms integrates the communication between shoots and roots, which provide photoassimilates and nutrients, respectively. The use of techniques to study signaling among the most diverse plant species has gained prominence. The main techniques for studying nutrient signaling between shoots and roots are split-root, grafting and girdling. The split-root system is characterized by separating a single root system in two independent compartments, with a common shoot. Split-root has been used for analyzing transcriptional, biochemical, and physiological changes in roots in response to nutritional challenges. Grafting has stood out as an important method concerning signaling mechanisms studies. It still follows the basic principle of cutting and fitting two parts (scion = bud and rootstock = root) from different plants, which merge, grow and develop as a single plant. Most recent studies have been in detecting long-distance signals, their transport, and the factors involved in signaling mechanisms, with particular attention to the role of phytohormones, RNAs, peptides, and proteins. Girdling is an important technique widely used in fruit trees. It consists of the removal of a strip of bark from branches or trunk, affecting different stages of reproductive growth, including flowering and fruiting, development, ripening, and nutritional quality of fruits. The increase in the amount of carbohydrates above the cut induces the expression of genes involved in the metabolism of carbohydrates, favoring the respiratory potential of the stem and branches, as well as the leaf mass per area. The techniques reviewed here allow the monitoring and study of long-distance signaling and relate it to plant development through simple, efficient, and reproducible approaches. They permit researchers to make inferences about the existence, direction, and intensity of the cross-talk between long-distance signaling and nutrients, suggesting that mobile signals and their interactions with individual nutrients exist.

International audience; The aim of this work was to study the regulation of coffee DREB-like genes in leaves of C. arabica subjected to cold, heat, low relative humidity, exogenous abscisic acid and high light stress, as well as in leaves and roots of drought-tolerant and drought-susceptible clones of Coffea canephora subjected to water limitation. In C. arabica, CaERF017 was the most expressed gene under low temperatures and relative humidity, while low humidity and high temperatures up-regulated the expression of CaERF053 and CaERF014, respectively. Under water limitation, CcDREB1B, CcRAP2.4, CcERF027, CcDREB1D and CcTINY were the most expressed genes mainly in leaves of drought-tolerant C. canephora. On the other hand, expression of the CcERF016, CcRAP2.4 and CcDREB2F genes was highly up-regulated under water limitation in the roots of drought-susceptible C. canephora clone 22. We previously reported fine-tuned regulation of CcDREB1D promoter haplotypes (HP15, HP16 and HP17) in transgenic C. arabica subjected to low humidity. Here, we investigated the regulation of these haplotypes under high light, cold, heat, and abscisic acid (ABA) stress. In apical buds and leaf guard cells, GUS-stained percentages were higher in pHP16L-transformed plants subjected to low humidity, high light and ABA stress than in pHP17L- and pHP15L-transformed plants. We also reported up-regulated expression of the endogenous CaDREB1D gene for both the cold and low humidity in leaves of pHP16L-transformed C. arabica suggesting a key role of this gene in controlling the responses of coffee plants to abiotic stress probably through an ABA-dependent pathway.

The present study aimed to evaluate the quality of cell suspension of Coffea arabica cv. Catigua at four different ages by morphological and gene expression analysis. For this purpose, cell suspension samples were collected at 30, 45, 60 and 75 days. Morphological analysis revealed the presence of embryogenic regions in all samples, and after 60 days, non-embryogenic regions were also identified. The genic analyses revealed that as the cells were transferred to a new medium a change in both their physical and chemical conditions was noticed that caused stress. The decrease in SERK and BBM genes expression after 75th day may also be due to the non-embryogenic regions, characterized by large, elongated and vacuolated cells that were observed in the periphery of embryogenic regions, starting from 60 days of culture.

The aim of this work was to study the regulation of coffee DREB-like genes in leaves of C. arabica subjected to cold, heat, low relative humidity, exogenous abscisic acid and high light stress, as well as in leaves and roots of drought-tolerant and drought-susceptible clones of Coffea canephora subjected to water limitation. In C. arabica, CaERF017 was the most expressed gene under low temperatures and relative humidity, while low humidity and high temperatures up-regulated the expression of CaERF053 and CaERF014, respectively. Under water limitation, CcDREB1B, CcRAP2.4, CcERF027, CcDREB1D and CcTINY were the most expressed genes mainly in leaves of drought-tolerant C. canephora. On the other hand, expression of the CcERF016, CcRAP2.4 and CcDREB2F genes was highly up-regulated under water limitation in the roots of drought-susceptible C. canephora clone 22. We previously reported fine-tuned regulation of CcDREB1D promoter haplotypes (HP15, HP16 and HP17) in transgenic C. arabica subjected to low humidity. Here, we investigated the regulation of these haplotypes under high light, cold, heat, and abscisic acid (ABA) stress. In apical buds and leaf guard cells, GUS-stained percentages were higher in pHP16L-transformed plants subjected to low humidity, high light and ABA stress than in pHP17L- and pHP15L-transformed plants. We also reported up-regulated expression of the endogenous CaDREB1D gene for both the cold and low humidity in leaves of pHP16L-transformed C. arabica suggesting a key role of this gene in controlling the responses of coffee plants to abiotic stress probably through an ABA-dependent pathway.

Made available in DSpace on 2019-06-19T01:15:26Z (GMT). No. of bitstreams: 1 Silicoandinvivo.pdf: 2006646 bytes, checksum: fdb4ea1794f6c0ea9912ad66c8647e24 (MD5) Previous issue date: 2019

Fine-tuning of DREB1D expression in stomatal guard cells under water deficit is mediated differentially by promoter haplotypes from sensitive and tolerant coffee genotypes., Despite the importance of the DREB1D gene (also known as CBF4) in plant responses to water deficit and cold stress, studies analysing its regulation by transgenic approaches are lacking. In the current work, a functional study of three CcDREB1D promoter haplotypes (named HP15, HP16 and HP17) isolated from drought-tolerant and drought-sensitive clones of Coffea canephora was carried out in plants of C. arabica stably transformed by Agrobacterium tumefaciens by analysing their ability to regulate the expression of the uidA reporter gene in response to water deficit mimicked by polyethylene glycol (−2.0 MPa) and low relative humidity treatments. A deletion analysis of their corresponding 5′-upstream regions revealed increased specificity of β-glucuronidase activity in the polyethylene glycol and low relative humidity treatments, with high expression in leaf mesophyll and guard cells in full-length constructs. RT-qPCR assays also revealed that the HP16 haplotype (specific to clone tolerant to water deficit) had stronger and earlier activity compared with the HP15 and HP17 haplotypes. As most of the cis-regulatory elements involved in ABA-dependent and -independent networks, tissue specificity and light regulation are common to these haplotypes, we propose that their organization, as well as the nucleic acid polymorphisms present outside these boxes, may play a role in modulating activities of DREB1D promoters in guard cells.