Metabolic Background, Not Photosynthetic Physiology, Determines Drought and Drought Recovery Responses in C3 and C2 Moricandias.

Distinct photosynthetic physiologies are found within the Moricandia genus, both C3-type and C2-type representatives being known. As C2-physiology is an adaptation to drier environments, a study of physiology, biochemistry and transcriptomics was conducted to investigate whether plants with C2-physiology are more tolerant of low water availability and recover better from drought. Our data on Moricandia moricandioides (Mmo, C3), M. arvensis (Mav, C2) and M. suffruticosa (Msu, C2) show that C3 and C2-type Moricandias are metabolically distinct under all conditions tested (well-watered, severe drought, early drought recovery). Photosynthetic activity was found to be largely dependent upon the stomatal opening. The C2-type M. arvensis was able to secure 25-50% of photosynthesis under severe drought as compared to the C3-type M. moricandioides. Nevertheless, the C2-physiology does not seem to play a central role in M. arvensis drought responses and drought recovery. Instead, our biochemical data indicated metabolic differences in carbon and redox-related metabolism under the examined conditions. The cell wall dynamics and glucosinolate metabolism regulations were found to be major discriminators between M. arvensis and M. moricandioides at the transcription level.

Transcriptomic Leaf Profiling Reveals Differential Responses of the Two Most Traded Coffee Species to Elevated [CO2].

As atmospheric [CO2] continues to rise to unprecedented levels, understanding its impact on plants is imperative to improve crop performance and sustainability under future climate conditions. In this context, transcriptional changes promoted by elevated CO2 (eCO2) were studied in genotypes from the two major traded coffee species: the allopolyploid Coffea arabica (Icatu) and its diploid parent, C. canephora (CL153). While Icatu expressed more genes than CL153, a higher number of differentially expressed genes were found in CL153 as a response to eCO2. Although many genes were found to be commonly expressed by the two genotypes under eCO2, unique genes and pathways differed between them, with CL153 showing more enriched GO terms and metabolic pathways than Icatu. Divergent functional categories and significantly enriched pathways were found in these genotypes, which altogether supports contrasting responses to eCO2. A considerable number of genes linked to coffee physiological and biochemical responses were found to be affected by eCO2 with the significant upregulation of photosynthetic, antioxidant, and lipidic genes. This supports the absence of photosynthesis down-regulation and, therefore, the maintenance of increased photosynthetic potential promoted by eCO2 in these coffee genotypes.

Stress cross-response of the antioxidative system promoted by superimposed drought and cold conditions in Coffea spp.

The understanding of acclimation strategies to low temperature and water availability is decisive to ensure coffee crop sustainability, since these environmental conditions determine the suitability of cultivation areas. In this context, the impacts of single and combined exposure to drought and cold were evaluated in three genotypes of the two major cropped species, Coffea arabica cv. Icatu, Coffea canephora cv. Apoatã, and the hybrid Obatã. Crucial traits of plant resilience to environmental stresses have been examined: photosynthesis, lipoperoxidation and the antioxidant response. Drought and/or cold promoted leaf dehydration, which was accompanied by stomatal and mesophyll limitations that impaired leaf C-assimilation in all genotypes. However, Icatu showed a lower impact upon stress exposure and a faster and complete photosynthetic recovery. Although lipoperoxidation was increased by drought (Icatu) and cold (all genotypes), it was greatly reduced by stress interaction, especially in Icatu. In fact, although the antioxidative system was reinforced under single drought and cold exposure (e.g., activity of enzymes as Cu,Zn-superoxide dismutase, ascorbate peroxidase, APX, glutathione reductase and catalase, CAT), the stronger increases were observed upon the simultaneous exposure to both stresses, which was accompanied with a transcriptional response of some genes, namely related to APX. Complementary, non-enzyme antioxidant molecules were promoted mostly by cold and the stress interaction, including α-tocopherol (in C. arabica plants), ascorbate (ASC), zeaxanthin, and phenolic compounds (all genotypes). In general, drought promoted antioxidant enzymes activity, whereas cold enhanced the synthesis of both enzyme and non-enzyme antioxidants, the latter likely related to a higher need of antioxidative capability when enzyme reactions were probably quite repressed by low temperature. Icatu showed the wider antioxidative capability, with the triggering of all studied antioxidative molecules by drought (except CAT), cold, and, particularly, stress interaction (except ASC), revealing a clear stress cross-tolerance. This justified the lower impacts on membrane lipoperoxidation and photosynthetic capacity under stress interaction conditions, related to a better ROS control. These findings are also relevant to coffee water management, showing that watering in the cold season should be largely avoided.

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.

A comprehensive assessment of the transcriptome of cork oak (Quercus suber) through EST sequencing.

BACKGROUND: Cork oak (Quercus suber) is one of the rare trees with the ability to produce cork, a material widely used to make wine bottle stoppers, flooring and insulation materials, among many other uses. The molecular mechanisms of cork formation are still poorly understood, in great part due to the difficulty in studying a species with a long life-cycle and for which there is scarce molecular/genomic information. Cork oak forests are of great ecological importance and represent a major economic and social resource in Southern Europe and Northern Africa. However, global warming is threatening the cork oak forests by imposing thermal, hydric and many types of novel biotic stresses. Despite the economic and social value of the Q. suber species, few genomic resources have been developed, useful for biotechnological applications and improved forest management. RESULTS: We generated in excess of 7 million sequence reads, by pyrosequencing 21 normalized cDNA libraries derived from multiple Q. suber tissues and organs, developmental stages and physiological conditions. We deployed a stringent sequence processing and assembly pipeline that resulted in the identification of ~159,000 unigenes. These were annotated according to their similarity to known plant genes, to known Interpro domains, GO classes and E.C. numbers. The phylogenetic extent of this ESTs set was investigated, and we found that cork oak revealed a significant new gene space that is not covered by other model species or EST sequencing projects. The raw data, as well as the full annotated assembly, are now available to the community in a dedicated web portal at http://www.corkoakdb.org. CONCLUSIONS: This genomic resource represents the first trancriptome study in a cork producing species. It can be explored to develop new tools and approaches to understand stress responses and developmental processes in forest trees, as well as the molecular cascades underlying cork differentiation and disease response.

Sustained photosynthetic performance of Coffea spp. under long-term enhanced [CO2].

Coffee is one of the world's most traded agricultural products. Modeling studies have predicted that climate change will have a strong impact on the suitability of current cultivation areas, but these studies have not anticipated possible mitigating effects of the elevated atmospheric [CO2] because no information exists for the coffee plant. Potted plants from two genotypes of Coffea arabica and one of C. canephora were grown under controlled conditions of irradiance (800 µmol m(-2) s(-1)), RH (75%) and 380 or 700 µL CO2 L(-1) for 1 year, without water, nutrient or root development restrictions. In all genotypes, the high [CO2] treatment promoted opposite trends for stomatal density and size, which decreased and increased, respectively. Regardless of the genotype or the growth [CO2], the net rate of CO2 assimilation increased (34-49%) when measured at 700 than at 380 µL CO2 L(-1). This result, together with the almost unchanged stomatal conductance, led to an instantaneous water use efficiency increase. The results also showed a reinforcement of photosynthetic (and respiratory) components, namely thylakoid electron transport and the activities of RuBisCo, ribulose 5-phosphate kinase, malate dehydrogenase and pyruvate kinase, what may have contributed to the enhancements in the maximum rates of electron transport, carboxylation and photosynthetic capacity under elevated [CO2], although these responses were genotype dependent. The photosystem II efficiency, energy driven to photochemical events, non-structural carbohydrates, photosynthetic pigment and membrane permeability did not respond to [CO2] supply. Some alterations in total fatty acid content and the unsaturation level of the chloroplast membranes were noted but, apparently, did not affect photosynthetic functioning. Despite some differences among the genotypes, no clear species-dependent responses to elevated [CO2] were observed. Overall, as no apparent sign of photosynthetic down-regulation was found, our data suggest that Coffea spp. plants may successfully cope with high [CO2] under the present experimental conditions.

Biochemical and molecular characterization of the antioxidative system of Coffea sp. under cold conditions in genotypes with contrasting tolerance.

Low positive temperature (chilling) is frequently linked to the promotion of oxidative stress conditions, and is of particular importance in the coffee plant due to its severe impact on growth, development, photosynthesis and production. Nevertheless, some acclimation ability has been reported within the Coffea genus, and is possibly related to oxidative stress control. Using an integrated biochemical and molecular approach, the characterization of the antioxidative system of genotypes with different cold acclimation abilities was performed. Experiments were carried out using 1.5-year-old coffee seedlings of Coffea canephora cv. Apoatã, C. arabica cv. Catuaí, C. dewevrei and 2 hybrids, Icatu (C. arabicaxC. canephora) and Piatã (C. dewevreixC. arabica) subjected to a gradual cold treatment and a recovery period. Icatu showed the greatest ability to control oxidative stress, as reflected by the enhancement of several antioxidative components (Cu,Zn-SOD and APX activities; ascorbate, alpha-tocopherol and chlorogenic acids (CGAs) contents) and lower reactive oxygen species contents (H(2)O(2) and OH). Gene expression studies show that GRed, DHAR and class III and IV chitinases might also be involved in the cold acclimation ability of Icatu. Catuaí showed intermediate acclimation ability through the reinforcement of some antioxidative molecules, usually to a lesser extent than that observed in Icatu. On the other hand, C. dewevrei showed the poorest response in terms of antioxidant accumulation, and also showed the greatest increase in OH values. The difference in the triggering of antioxidative traits supports the hypothesis of its importance to cold (and photoinhibition) tolerance in Coffea sp. and could provide a useful probe to identify tolerant genotypes.

Cloning and characterisation of a basic IAA oxidase associated with root induction in Vitis vinifera.

Changes in apoplastic peroxidases during auxin-induced in vitro rooting of cultured grapevine (Vitis vinifera L. cv. Touriga) stems have been studied. The largest increase in peroxidase activity (EC 1.11.1.7) was associated with the early stages of root initiation and could be attributed to an increase in activity of an apoplastic 36 kDa cationic peroxidase (PxB2). Relative to other peroxidases, PxB2 demonstrated high indole-3-acetic acid (IAA) oxidase activity and apparently contributed the majority of potential IAA oxidase activity in rooting tissues. The distribution of this peroxidase in developing roots additionally associates it with early phases of growth restriction. PxB2 was purified from cell wall extracts prepared from the basal 1 cm of rooting stems. Microsequencing and subsequent cloning of its corresponding 3' truncated cDNA (encoding 255 amino acids of the mature protein) revealed it to have a typical class III peroxidase structure. The results suggest that this class III peroxidase with IAA oxidase activity is important for the control of IAA levels during root initiation and development.