The aim of this study was to investigate the molecular mechanisms underlying drought acclimation in coffee plants by the identification of candidate genes (CGs) using different approaches. The first approach used the data generated during the Brazilian Coffee expressed sequence tag (EST) project to select 13 CGs by an in silico analysis (electronic northern). The second approach was based on screening macroarrays spotted with plasmid DNA (coffee ESTs) with separate hybridizations using leaf cDNA probes from drought-tolerant and susceptible clones of Coffea canephora var. Conilon, grown under different water regimes. This allowed the isolation of seven additional CGs. The third approach used two-dimensional gel electrophoresis to identify proteins displaying differential accumulation in leaves of drought-tolerant and susceptible clones of C. canephora. Six of them were characterized by MALDI-TOF-MS/MS (matrix-assisted laser desorption-time of flight-tandem mass spectrometry) and the corresponding proteins were identified. Finally, additional CGs were selected from the literature, and quantitative real-time polymerase chain reaction (qPCR) was performed to analyse the expression of all identified CGs. Altogether, >40 genes presenting differential gene expression during drought acclimation were identified, some of them showing different expression profiles between drought-tolerant and susceptible clones. Based on the obtained results, it can be concluded that factors involved a complex network of responses probably involving the abscisic signalling pathway and nitric oxide are major molecular determinants that might explain the better efficiency in controlling stomata closure and transpiration displayed by drought-tolerant clones of C. canephora.

O objetivo deste trabalho foi comparar os graus de resistência à mosca-branca (Bemisia argentifolii) e ao ácaro-rajado (Tetranychus urticae) de híbridos de tomateiro resultantes do cruzamento entre linhagens com alto teor de zingibereno (ZGB) e linhagens com alto teor de acilaçúcar (AA), em contraste com as linhagens parentais e testemunhas comerciais. Foram avaliadas linhagens com altos teores de AA, linhagens com alto teor de ZGB, híbridos duplos heterozigotos ZGB+AA, híbridos heterozigotos para ZGB e híbridos heterozigotos para AA. Os acessos selvagens PI-127826 e LA-716 foram utilizados como testemunhas para alto teor de ZGB e AA, respectivamente, e os genótipos Débora Max e TOM-684 foram utilizados como testemunhas para baixo teor de ambos os aleloquímicos. Os genótipos foram submetidos ao teste de resistência à mosca-branca e ao teste de repelência ao ácaro. Os genótipos duplos heterozigotos apresentaram graus de resistência à mosca-branca superiores aos das testemunhas comerciais e inferiores aos das linhagens com alto ZGB ou com alto AA. Os genótipos duplos heterozigotos apresentaram maior repelência ao ácaro, em relação às testemunhas comerciais, e repelência semelhante à das linhagens com alto ZGB ou com alto AA. Não foi observado efeito sinérgico entre ZGB e AA nos genótipos duplos heterozigotos quanto à resistência à mosca-branca e repelência ao ácaro.

The effects of water deficit on photochemical parameters and expression of several candidate genes were investigated in drought-tolerant clone 73 of Coffea canephora submitted to slowly imposed water limitation. Under irrigation, this clone showed low values of stomatal conductance (g s) and of CO 2 assimilation rates (A) suggesting that it had a great efficiency in controlling stomatal closure and transpiration. After water withdrawal, this clone reached a −3.0 MPa after 15 days without irrigation and showed a slow decrease in the pre-dawn leaf water potential. Under drought, the suppression of A was accompanied by maintenance of photochemical quenching (q P) and internal to ambient CO 2 concentration (Ci/Ca) ratios as well as by a decrease of non-photochemical quenching (q N). This is confirmed by the transport rate/CO 2 assimilation (ETR/A) rates that suggested the participation of an alternative electron sink protecting the photosynthetic apparatus against photoinhibition. At the transcriptomic level, high up-regulation of genes encoding for a dehydrin (CcDH3), an ascorbate peroxidase (CcAPX1), a prephenate-dehydrogenase like protein (CcPDH1) and a non-symbiotic haemoglobin (CcNSH1) was also observed upon drought suggesting a strong induction of antioxidant and osmoprotection systems in this clone. High expression levels of gene-encoding ABA receptors (CcPYL3 and CcPYL7) under water limitation were also observed suggesting the involvement of the ABA signaling pathway in response to drought. All these results where compared to those previously obtained for drought-tolerant clones 14 and 120. Our results demonstrated the existence of different mechanisms amongst the drought-tolerant coffee clones regarding water deficit. Keywords Candidate gene. Coffea canephora. Drought. Gene expression. Real-time quantitative PCR. Water potential Abbreviations CG Candidate gene qPCR Quantitative polymerase chain reaction

BackgroundIn higher plants, the inhibition of photosynthetic capacity under drought is attributable to stomatal and non-stomatal (i.e., photochemical and biochemical) effects. In particular, a disruption of photosynthetic metabolism and Rubisco regulation can be observed. Several studies reported reduced expression of the RBCS genes, which encode the Rubisco small subunit, under water stress.ResultsExpression of the RBCS1 gene was analysed in the allopolyploid context of C. arabica, which originates from a natural cross between the C. canephora and C. eugenioides species. Our study revealed the existence of two homeologous RBCS1 genes in C. arabica: one carried by the C. canephora sub-genome (called CaCc) and the other carried by the C. eugenioides sub-genome (called CaCe). Using specific primer pairs for each homeolog, expression studies revealed that CaCe was expressed in C. eugenioides and C. arabica but was undetectable in C. canephora. On the other hand, CaCc was expressed in C. canephora but almost completely silenced in non-introgressed ("pure") genotypes of C. arabica. However, enhanced CaCc expression was observed in most C. arabica cultivars with introgressed C. canephora genome. In addition, total RBCS1 expression was higher for C. arabica cultivars that had recently introgressed C. canephora genome than for "pure" cultivars. For both species, water stress led to an important decrease in the abundance of RBCS1 transcripts. This was observed for plants grown in either greenhouse or field conditions under severe or moderate drought. However, this reduction of RBCS1 gene expression was not accompanied by a decrease in the corresponding protein in the leaves of C. canephora subjected to water withdrawal. In that case, the amount of RBCS1 was even higher under drought than under unstressed (irrigated) conditions, which suggests great stability of RBCS1 under adverse water conditions. On the other hand, for C. arabica, high nocturnal expression of RBCS1 could also explain the accumulation of the RBCS1 protein under water stress. Altogether, the results presented here suggest that the content of RBCS was not responsible for the loss of photosynthetic capacity that is commonly observed in water-stressed coffee plants.ConclusionWe showed that the CaCe homeolog was expressed in C. eugenioides and non-introgressed ("pure") genotypes of C. arabica but that it was undetectable in C. canephora. On the other hand, the CaCc homeolog was expressed in C. canephora but highly repressed in C. arabica. Expression of the CaCc homeolog was enhanced in C. arabica cultivars that experienced recent introgression with C. canephora. For both C. canephora and C. arabica species, total RBCS1 gene expression was highly reduced with WS. Unexpectedly, the accumulation of RBCS1 protein was observed in the leaves of C. canephora under WS, possibly coming from nocturnal RBCS1 expression. These results suggest that the increase in the amount of RBCS1 protein could contribute to the antioxidative function of photorespiration in water-stres...

O objetivo deste trabalho foi determinar alterações fisiológicas e de tolerância à seca em clones de café Conilon (Coffea canephora) contrastantes quanto à sensibilidade ao deficit hídrico. Foram avaliadas as enxertias recíprocas entre os clones 109A, sensível ao deficit hídrico, e 120, tolerante - 120/109A, 120/120, 109A/120, 109A/109A -, além de seus respectivos pés-francos. As plantas foram cultivadas em vasos de 12 L em casa de vegetação. Após seis meses, metade das plantas foi submetida ao deficit hídrico por meio da suspensão da irrigação, até que as folhas atingissem o potencial hídrico de antemanhã de -3,0 MPa. Quando o clone 120 foi usado como porta-enxerto, as plantas apresentaram sistema radicular mais profundo, mas com menor massa, retardaram por mais tempo a desidratação celular das folhas e apresentaram maior eficiência no uso da água. Sob seca severa, os teores de amido e sacarose decresceram em todos os tratamentos, enquanto os teores de glicose, frutose, aminoácidos totais e prolina aumentaram, particularmente nos tratamentos 109A pé-franco, 109A/109A e 120/109A. Essas plantas apresentaram menor eficiência no uso da água. O acúmulo de solutos não foi associado à tolerância à seca. O uso de porta-enxertos tolerantes à seca contribui para a maior tolerância das plantas ao deficit hídrico.

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A água é um dos fatores limitantes ao desenvolvimento de plantas cítricas, o que faz da reposição hídrica uma prática comum, em plantios onde a sua distribuição é escassa. O gel hidroretentor tem sido uma das tecnologias disponíveis de fornecimento de água para plantas, podendo ser uma alternativa no plantio de mudas cítricas, contribuindo para o uso racional da água. Neste trabalho, foi avaliada a eficiência do hidrogel como alternativa para minimizar os efeitos do défice hídrico, em mudas de cultivares apirênicas de tangerineiras ('Ortanique', 'Okitsu'e 'Clemenules') e de laranjeiras ('Navelina', 'Navelate' e 'Lanelate'), todas enxertadas sobre Poncirus trifoliata. O experimento foi conduzido em casa-de-vegetação, em delineamento de blocos casualizados, no qual plantas com hidrogel foram comparadas a plantas sob irrigação convencional e a plantas submetidas a estresse hídrico, em fatorial triplo. Foram determinadas as taxas de assimilação líquida de carbono, condutância estomática, transpiração e razão entre concentrações interna e externa de CO2. Verificou-se que o efeito do hidrogel na manutenção do status hídrico de mudas cítricas é variável e dependente de mecanismos fisiológicos de resposta ao défice hídrico. As mudas de 'Ortanique'e 'Navelate'não responderam à aplicação de hidrogel. Este promoveu recuperação e manutenção do status hídrico das mudas de 'Okitsu', 'Clemenules', 'Navelina' e 'Lanelate', porém, estas cultivares foram sensíveis a variações no status hídrico, apresentando redução considerável nas trocas gasosas.

Climate change greatly influences coffee production, especially in areas infested with plant‐parasitic nematodes. In this study, coffee genotypes showed differences in their morphological and physiological characteristics when subjected to a water deficit and parasitism by Meloidogyne paranaensis. The cultivar IPR 100 had the largest superficial and volumetric root system area, even when parasitized. The two progenies (MG 0179‐1 and MG 0179‐3) and the cultivar Catuaí IAC 62 had a similar surface area (p < .05) when parasitized. However, the root surface area and volume of MG 0179‐3 increased by 96% and 400%, respectively, when parasitized by M. paranaensis. On the other hand, Catuaí IAC 62 had a 31% reduction in root surface area. Catuai 62 and IPR 100 showed higher sensitivity to drought when parasitized because of the increased photochemical sensitivity and reduction in photochemical quenching. In MG 0179‐1 and MG 0179‐3, an increase in non‐photochemical quenching occurred in response to stress, indicating that these progenies use a photochemical response to protect photosystem II. In this work, MG 0179‐3, which is resistant to M. paranaensis, was remarkable because, interestingly, the infestation caused an increase in its root surface area. In addition, MG 0179‐3 had relatively good photochemical performance under water deficit and M. paranaensis parasitism.