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.

Protective Responses at the Biochemical and Molecular Level Differ between a Coffea arabica L. Hybrid and Its Parental Genotypes to Supra-Optimal Temperatures and Elevated Air [CO2].

Climate changes with global warming associated with rising atmospheric [CO2] can strongly impact crop performance, including coffee, which is one of the most world's traded agricultural commodities. Therefore, it is of utmost importance to understand the mechanisms of heat tolerance and the potential role of elevated air CO2 (eCO2) in the coffee plant response, particularly regarding the antioxidant and other protective mechanisms, which are crucial for coffee plant acclimation. For that, plants of Coffea arabica cv. Geisha 3, cv. Marsellesa and their hybrid (Geisha 3 × Marsellesa) were grown for 2 years at 25/20 °C (day/night), under 400 (ambient CO2, aCO2) or 700 µL (elevated CO2, eCO2) CO2 L-1, and then gradually submitted to a temperature increase up to 42/30 °C, followed by recovery periods of 4 (Rec4) and 14 days (Rec14). Heat (37/28 °C and/or 42/30 °C) was the major driver of the response of the studied protective molecules and associated genes in all genotypes. That was the case for carotenoids (mostly neoxanthin and lutein), but the maximal (α + ß) carotenes pool was found at 37/28 °C only in Marsellesa. All genes (except VDE) encoding for antioxidative enzymes (catalase, CAT; superoxide dismutases, CuSODs; ascorbate peroxidases, APX) or other protective proteins (HSP70, ELIP, Chape20, Chape60) were strongly up-regulated at 37/28 °C, and, especially, at 42/30 °C, in all genotypes, but with maximal transcription in Hybrid plants. Accordingly, heat greatly stimulated the activity of APX and CAT (all genotypes) and glutathione reductase (Geisha3, Hybrid) but not of SOD. Notably, CAT activity increased even at 42/30 °C, concomitantly with a strongly declined APX activity. Therefore, increased thermotolerance might arise through the reinforcement of some ROS-scavenging enzymes and other protective molecules (HSP70, ELIP, Chape20, Chape60). Plants showed low responsiveness to single eCO2 under unstressed conditions, while heat promoted changes in aCO2 plants. Only eCO2 Marsellesa plants showed greater contents of lutein, the pool of the xanthophyll cycle components (V + A + Z), and ß-carotene, compared to aCO2 plants at 42/30 °C. This, together with a lower CAT activity, suggests a lower presence of H2O2, likely also associated with the higher photochemical use of energy under eCO2. An incomplete heat stress recovery seemed evident, especially in aCO2 plants, as judged by the maintenance of the greater expression of all genes in all genotypes and increased levels of zeaxanthin (Marsellesa and Hybrid) relative to their initial controls. Altogether, heat was the main response driver of the addressed protective molecules and genes, whereas eCO2 usually attenuated the heat response and promoted a better recovery. Hybrid plants showed stronger gene expression responses, especially at the highest temperature, when compared to their parental genotypes, but altogether, Marsellesa showed a greater acclimation potential. The reinforcement of antioxidative and other protective molecules are, therefore, useful biomarkers to be included in breeding and selection programs to obtain coffee genotypes to thrive under global warming conditions, thus contributing to improved crop sustainability.

Zinc Biofortification in Vitis vinifera: Implications for Quality and Wine Production.

Nowadays, there is a growing concern about micronutrient deficits in food products, with agronomic biofortification being considered a mitigation strategy. In this context, as Zn is essential for growth and maintenance of human health, a workflow for the biofortification of grapes from the Vitis vinifera variety Fernão Pires, which contains this nutrient, was carried out considering the soil properties of the vineyard. Additionally, Zn accumulation in the tissues of the grapes and the implications for some quality parameters and on winemaking were assessed. Vines were sprayed three times with ZnO and ZnSO4 at concentrations of 150, 450, and 900 g ha-1 during the production cycle. Physiological data were obtained through chlorophyll a fluorescence data, to access the potential symptoms of toxicity. At harvest, treated grapes revealed significant increases of Zn concentration relative to the control, being more pronounced for ZnO and ZnSO4 in the skin and seeds, respectively. After winemaking, an increase was also found regarding the control (i.e., 1.59-fold with ZnSO4-450 g ha-1). The contents of the sugars and fatty acids, as well as the colorimetric analyses, were also assessed, but significant variations were not found among treatments. In general, Zn biofortification increased with ZnO and ZnSO4, without significantly affecting the physicochemical characteristics of grapes.

Will Casuarina glauca Stress Resilience Be Maintained in the Face of Climate Change?

Actinorhizal plants have been regarded as promising species in the current climate change context due to their high tolerance to a multitude of abiotic stresses. While combined salt-heat stress effects have been studied in crop species, their impact on the model actinorhizal plant, Casuarina glauca, has not yet been fully addressed. The effect of single salt (400 mM NaCl) and heat (control at 26/22 °C, supra optimal temperatures at 35/22 °C and 45/22 °C day/night) conditions on C. glauca branchlets was characterised at the physiological level, and stress-induced metabolite changes were characterised by mass spectrometry-based metabolomics. C. glauca could withstand single salt and heat conditions. However, the harshest stress condition (400 mM NaCl, 45 °C) revealed photosynthetic impairments due to mesophyll and membrane permeability limitations as well as major stress-specific differential responses in C and N metabolism. The increased activity of enzymatic ROS scavengers was, however, revealed to be sufficient to control the plant oxidative status. Although C. glauca could tolerate single salt and heat stresses, their negative interaction enhanced the effects of salt stress. Results demonstrated that C. glauca responses to combined salt-heat stress could be explained as a sum of the responses from each single applied stress.

Can Elevated Air [CO2] Conditions Mitigate the Predicted Warming Impact on the Quality of Coffee Bean?

Climate changes, mostly related to high temperature, are predicted to have major negative impacts on coffee crop yield and bean quality. Recent studies revealed that elevated air [CO2] mitigates the impact of heat on leaf physiology. However, the extent of the interaction between elevated air [CO2] and heat on coffee bean quality was never addressed. In this study, the single and combined impacts of enhanced [CO2] and temperature in beans of Coffea arabica cv. Icatu were evaluated. Plants were grown at 380 or 700 µL CO2 L-1 air, and then submitted to a gradual temperature rise from 25°C up to 40°C during ca. 4 months. Fruits were harvested at 25°C, and in the ranges of 30-35 or 36-40°C, and bean physical and chemical attributes with potential implications on quality were then examined. These included: color, phenolic content, soluble solids, chlorogenic, caffeic and p-coumaric acids, caffeine, trigonelline, lipids, and minerals. Most of these parameters were mainly affected by temperature (although without a strong negative impact on bean quality), and only marginally, if at all, by elevated [CO2]. However, the [CO2] vs. temperature interaction strongly attenuated some of the negative impacts promoted by heat (e.g., total chlorogenic acids), thus maintaining the bean characteristics closer to those obtained under adequate temperature conditions (e.g., soluble solids, caffeic and p-coumaric acids, trigonelline, chroma, Hue angle, and color index), and increasing desirable features (acidity). Fatty acid and mineral pools remained quite stable, with only few modifications due to elevated air [CO2] (e.g., phosphorous) and/or heat. In conclusion, exposure to high temperature in the last stages of fruit maturation did not strongly depreciate bean quality, under the conditions of unrestricted water supply and moderate irradiance. Furthermore, the superimposition of elevated air [CO2] contributed to preserve bean quality by modifying and mitigating the heat impact on physical and chemical traits of coffee beans, which is clearly relevant in a context of predicted climate change and global warming scenarios.

Rice calcium-dependent protein kinase OsCPK17 targets plasma membrane intrinsic protein and sucrose-phosphate synthase and is required for a proper cold stress response.

Calcium-dependent protein kinases (CDPKs) are involved in plant tolerance mechanisms to abiotic stresses. Although CDPKs are recognized as key messengers in signal transduction, the specific role of most members of this family remains unknown. Here, we test the hypothesis that OsCPK17 plays a role in rice cold stress response by analysing OsCPK17 knockout, silencing and overexpressing rice lines under low temperature. Altered OsCPK17 gene expression compromises cold tolerance performance, without affecting the expression of key cold stress-inducible genes. A comparative phosphoproteomic approach led to the identification of six potential in vivo OsCPK17 targets, which are associated with sugar and nitrogen metabolism, and with osmotic regulation. To test direct interaction, in vitro kinase assays were performed, showing that the sucrose-phosphate synthase OsSPS4 and the aquaporin OsPIP2;1/OsPIP2;6 are phosphorylated by OsCPK17 in a calcium-dependent manner. Altogether, our data indicates that OsCPK17 is required for a proper cold stress response in rice, likely affecting the activity of membrane channels and sugar metabolism.

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.

Is salt stress tolerance in Casuarina glauca Sieb. ex Spreng. associated with its nitrogen-fixing root-nodule symbiosis? An analysis at the photosynthetic level.

Casuarina glauca is an actinorhizal tree which establishes root-nodule symbiosis with N2-fixing Frankia bacteria. This plant is commonly found in saline zones and is widely used to remediate marginal soils and prevent desertification. The nature of its ability to survive in extreme environments and the extent of Frankia contribution to stress tolerance remain unknown. Thus, we evaluated the ability of C. glauca to cope with salt stress and the influence of the symbiosis on this trait. To this end, we analysed the impact of salt on plant growth, mineral contents, water relations, photosynthetic-related parameters and non-structural sugars in nodulated vs. non-nodulated plants. Although the effects on photosynthesis and stomatal conductance started to become measurable in the presence of 200 mM NaCl, photochemical (e.g., photosynthetic electron flow) and biochemical (e.g., activity of photosynthetic enzymes) parameters were only strongly impaired when NaCl levels reached 600 mM. These results indicate the maintenance of high tissue hydration under salt stress, probably associated with enhanced osmotic potential. Furthermore, the maintenance of photosynthetic assimilation potential (A(max)), together with the increase in the quantum yield of down-regulated energy dissipation of PSII (Y(NPQ)), suggested a down-regulation of photosynthesis instead of photo-damaging effects. A comparison of the impact of increasing NaCl levels on the activities of photosynthetic (RubisCO and ribulose-5 phosphate kinase) and respiratory (pyruvate kinase and NADH-dependent malate dehydrogenase) enzymes vs. photosynthetic electron flow and fluorescence parameters, revealed that biochemical impairments are more limiting than photochemical damage. Altogether, these results indicate that, under controlled conditions, C. glauca tolerates high NaCl levels and that this capacity is linked to photosynthetic adjustments.

Flower abscission in Vitis vinifera L. triggered by gibberellic acid and shade discloses differences in the underlying metabolic pathways.

Understanding abscission is both a biological and an agronomic challenge. Flower abscission induced independently by shade and gibberellic acid (GAc) sprays was monitored in grapevine (Vitis vinifera L.) growing under a soilless greenhouse system during two seasonal growing conditions, in an early and late production cycle. Physiological and metabolic changes triggered by each of the two distinct stimuli were determined. Environmental conditions exerted a significant effect on fruit set as showed by the higher natural drop rate recorded in the late production cycle with respect to the early cycle. Shade and GAc treatments increased the percentage of flower drop compared to the control, and at a similar degree, during the late production cycle. The reduction of leaf gas exchanges under shade conditions was not observed in GAc treated vines. The metabolic profile assessed in samples collected during the late cycle differently affected primary and secondary metabolisms and showed that most of the treatment-resulting variations occurred in opposite trends in inflorescences unbalanced in either hormonal or energy deficit abscission-inducing signals. Particularly concerning carbohydrates metabolism, sucrose, glucose, tricarboxylic acid metabolites and intermediates of the raffinose family oligosaccharides pathway were lower in shaded and higher in GAc samples. Altered oxidative stress remediation mechanisms and indolacetic acid (IAA) concentration were identified as abscission signatures common to both stimuli. According to the global analysis performed, we report that grape flower abscission mechanisms triggered by GAc application and C-starvation are not based on the same metabolic pathways.

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.