Elemental Composition and Implications on Brown Rice Flour Biofortified with Selenium.

Rice (Oryza sativa L.) is one of the most economically and socially important cereals in the world. Several strategies such as biofortification have been developed in a way eco-friendly and sustainable to enhance crop productivity. This study implemented an agronomic itinerary in Ariete and Ceres rice varieties in experimental fields using the foliar application of selenium (Se) to increase rice nutritional value. At strategic phases of the plant's development (at the end of booting, anthesis, and at the milky grain stage), they were sprayed with sodium selenate (Na2SeO4) and sodium selenite (Na2SeO3). In the first foliar application plants were sprayed with 500 g Se·ha-1 and in the remaining two foliar applications were sprayed with 300 g Se·ha-1. The effects of Se in the level of micro and macronutrients in brown grains, the localization of Se in these grains, and the subsequent quality parameters such as colorimetric characteristics and total protein were considered. After grain harvesting, the application of selenite showed the highest enrichment in all grain with levels reaching 17.06 µg g-1 Se and 14.28 µg g-1 Se in Ariete and Ceres varieties, respectively. In the Ceres and Ariete varieties, biofortification significantly affected the K and P contents. Regarding Ca, a clear trend prevailed suggesting that Se antagonizes the uptake of it, while for the remaining elements in general (except Mn) no significant differences were noted. Protein content increased with selenite treatment in the Ariete variety but not in Ceres. Therefore, it was possible to conclude, without compromising quality, that there was an increase in the nutritional content of Se in brown rice grain.

Are Portuguese Cowpea Genotypes Adapted to Drought? Phenological Development and Grain Quality Evaluation.

Along with population growth, global climate change represents a critical threat to agricultural production, compromising the goal of achieving food and nutrition security for all. It is urgent to create sustainable and resilient agri-food systems capable of feeding the world without debilitating the planet. The Food and Agriculture Organization of the United Nations (FAO) refers to pulses as a superfood, as one of the most nutritious crops with high health benefits. Considered to be low-cost, many can be produced in arid lands and have an extended shelf-life. Their cultivation helps reduce greenhouse gases and increases carbon sequestration, also improving soil fertility. Cowpea, Vigna unguiculata (L.) Walp. is particularly drought tolerant, with a wide diversity of landraces adapted to different environments. Considering the importance of knowing and valuing the genetic variability of this species in Portugal, this study assessed the impact of drought on four landraces of cowpea (L1 to L4) from different regions of the country and a national commercial variety (CV) as a reference. The development and evaluation of morphological characteristics were monitored in response to terminal drought (imposed during the reproductive phase), and its effects were evaluated on the yield and quality of the produced grain, namely the weight of 100 grains, color, protein content, and soluble sugars. Under drought conditions, the landraces L1 and L2 showed early maturation as a strategy to avoid water deficit. Morphological alteration of the aerial part of the plants was evident in all genotypes, with a rapid reduction in the number of leaves and a reduction in the number of flowers and pods by between 44 and 72%. The parameters of grain quality, the weight of 100 grains, color, protein, and soluble sugars did not vary significantly, except for sugars of the raffinose family that is associated with the adaptive mechanisms of plants to drought. The performance and maintenance of the evaluated characteristics reflect the adaptation acquired in the past by exposure to the Mediterranean climate, highlighting the potential agronomic and genetic value, still little exploited, that could contribute to production stability, preserved nutritional value, and food safety under water stress.

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.

Phenotypic Diversity of Seminal Root Traits in Bread Wheat Germplasm from Different Origins.

Bread wheat (Triticum aestivum L.) is a major staple crop, and more adapted varieties are needed to ensure productivity under unpredictable stress scenarios resulting from climate changes. In the development of new genotypes, root system traits are essential since roots have a key function in water and nutrient uptake, and root architecture determines the plant's ability to spatially explore the soil resources. Genetic variation in wheat root system may be assessed at the early stages of development. This study evaluates in vitro and at the seedling stage, the genetic diversity of root growth angle (RGA), seminal root number (SRN), and radicle length (RadL) in 30 bread wheat genotypes from different origins and belonging to distinct evolutive or breeding groups. SRN and RadL were analyzed at 1, 2, 3 and 6 days after sowing (DAS) and RGA was measured through the angle between the first pair of seminal roots. A large variability was found in RGA values that ranged from 63° to 122°. Although differences were found between genotypes within the same groups, the narrower angles tended to occur among landraces, while the higher RGA values were observed in advanced lines and Australian varieties. Differences were also observed as regards the SRN (1.0-3.0, 2.7-4.7, 3.2-5.0 and 4.4-6.3 at 1, 2, 3 and 6 DAS, respectively) and RadL (0.1-1.5, 2.1-5.0, 4.0-7.5 and 5.1-13.7 cm at 1, 2, 3 and 6 DAS, respectively). Genetic variability in root traits at seedling stage allows more rapid selection of genotypes better adapted to environmental and soil constraints, necessary to Portuguese Wheat Breeding Program. It will also contribute to the definition of wheat ideotypes with improved performance under Mediterranean climate conditions.

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.

Foliar Spraying of Solanum tuberosum L. with CaCl2 and Ca(NO3)2: Interactions with Nutrients Accumulation in Tubers.

Calcium is essential for plants, yet as its mobility is limited, the understanding of the rate of Ca2+ accumulation and deposition in tissues of tubers, as well as the interactions with other critical nutrients prompted this study. To assess the interactions and differential accumulation of micro and macronutrients in the tissues of tubers, Solanum tuberosum L. varieties Agria and Rossi were cultivated and, after the beginning of tuberization, four foliar sprayings (at 8-10 day intervals) with CaCl2 (3 and 6 kg ha-1) or Ca(NO3)2 (2 and 4 kg ha-1) solutions were performed. It was found that both fertilizers increased Ca accumulation in tubers (mostly in the parenchyma tissues located in the center of the equatorial region). The functioning of the photosynthetic apparatus was not affected until the 3rd application but was somewhat affected when approaching the end of the crop cycle (after the 4th application), although the lower dose of CaCl2 seemed to improve the photochemical use of energy, particularly when compared with the greater dose of Ca(NO3)2. Still, none of these impacts modified tuber height and diameter. Following the increased accumulation of Ca, in the tubers of both varieties, the mean contents of P, K, Na, Fe, and Zn revealed different accumulation patterns. Moreover, accumulation of K, Fe, Mn, and Zn prevailed in the epidermis, displaying a contrasting pattern relative to Ca. Therefore, Ca accumulation revealed a heterogeneous trend in the different regions analyzed, and Ca enrichment of tubers altered the accumulation of other nutrients.

Cowpea Physiological Responses to Terminal Drought-Comparison between Four Landraces and a Commercial Variety.

Cowpea (Vigna unguiculata) is a robust legume; nevertheless, yield is always affected by drought, especially when it occurs during reproductive growth and seed filling. Considered a key crop in the effort to attain food security, and a suitable crop for a scenario of climate change, modern disregard for cowpea landraces is particularly detrimental as it causes genetic variability loss, compromising breeding efforts. To contribute to the evaluation of the cowpea germplasm, four Portuguese landraces (L1, L2, L3, L4) were compared with a commercial variety (CV) to evaluate their physiological responses to terminal drought and their inter-variation on productivity, under semi-controlled conditions. Despite no differences in relative water content (RWC) between the CV and the landraces under water deficit (WD), differences in leaf water potential (Ψ) defined the CV as having an isohydric control of stomata in contrast with anisohydric control for landraces. There was an identical decrease in the photosynthetic rate for all plants under stress, caused by both stomatal and non-stomatal limitations, namely, damages at the level of photosystem II as indicated by fluorescence measurements. Instantaneous water use efficiency (iWUE) was improved with stress in L1 and L3. Maintenance of higher relative chlorophyll content for longer periods in the CV revealed a stay-green phenotype. The slim differences observed in terms of stomatal control, iWUE and progression of senescence between the CV and the landraces under WD led to quite important differences in terms of productivity, as inferred from improved yield (number of pods and number of grains per plant). This is a clear result of pragmatic on-farm selection. On one hand it shows that small differences in stomatal responses or water saving strategies under WD may lead to desirable outcomes and should therefore be considered during breeding. On the other hand, it suggests that other traits could be explored in view of drought adaptation. These results highlight the need to preserve and characterize as many genetic pools as possible within a species.

Grain Composition and Quality in Portuguese Triticum aestivum Germplasm Subjected to Heat Stress after Anthesis.

Bread wheat (Triticum aestivum) is a major crop worldwide, and it is highly susceptible to heat. In this work, grain production and composition were evaluated in Portuguese T. aestivum germplasm (landraces and commercial varieties), which was subjected to heat after anthesis (grain filling stage). Heat increased the test weight (TW) in Nabão, Grécia and Restauração, indicating an improved flour-yield potential. Mocho de Espiga Branca (MEB) and Transmontano (T94) showed higher thousand-kernel weight (TKW). Gentil Rosso presented increased soluble sugars, which are yeast substrates in the bread-making process. Ardila stood out for its protein increase under heat. Overall SDS was unaffected by higher temperature, but increased in T94, indicating a better dough elasticity for bread-making purposes. Under heat, lipid content was maintained in most genotypes, being endogenous fatty acids (FAs) key players in fresh bread quality. Lipid unsaturation, evaluated through the double bond index (DBI), also remained unaffected in most genotypes, suggesting a lower flour susceptibility to lipoperoxidation. In Grécia, heat promoted a higher abundance of monounsaturated oleic (C18:1) and polyunsaturated linoleic (C18:2) acids, which are essential fatty acids in the human diet. This work highlighted a great variability in most parameters both under control conditions or in response to heat during grain filling. Cluster analysis of traits revealed a lower susceptibility to heat during grain filling in Ardila, Restauração, and Ruivo, in contrast to MEQ, which seems to be more differentially affected at this stage. Characterization and identification of more favorable features under adverse environments may be relevant for agronomic, industrial, or breeding purposes, in view of a better crop adaptation to changing climate and an improved crop sustainability in agricultural systems more prone to heat stress.

Wheat Crop under Waterlogging: Potential Soil and Plant Effects.

Inundation, excessive precipitation, or inadequate field drainage can cause waterlogging of cultivated land. It is anticipated that climate change will increase the frequency, intensity, and unpredictability of flooding events. This stress affects 10-15 million hectares of wheat every year, resulting in 20-50% yield losses. Since this crop greatly sustains a population's food demands, providing ca. 20% of the world's energy and protein diets requirements, it is crucial to understand changes in soil and plant physiology under excess water conditions. Variations in redox potential, pH, nutrient availability, and electrical conductivity of waterlogged soil will be addressed, as well as their impacts in major plant responses, such as root system and plant development. Waterlogging effects at the leaf level will also be addressed, with a particular focus on gas exchanges, photosynthetic pigments, soluble sugars, membrane integrity, lipids, and oxidative stress.

Elemental composition, total fatty acids, soluble sugar content and essential oils of flowers and leaves of Moringa oleifera cultivated in Southern Portugal.

The evaluation of the elemental content of moringa leaves and flowers by Energy Dispersive X-Ray Fluorescence Spectrometry revealed that the leaves are a good source of some macro (Ca and K) and micronutrients (Mn) beyond the presence of important polyunsaturated fatty acids (PUFAs), essential in human nutrition. Total soluble sugars prevail in the flowers which may be linked to insect attraction and the pollination process. M. oleifera leaves, flowers and seeds essential oils (EOs) were isolated by hydrodistillation. Gas chromatography and gas chromatography-mass spectrometry analysis (GC-MS) showed EOs dominated by alkanes and fatty acids in diverse ratios in the analyzed plant parts. The nutritional characterization of M. oleifera cultivated in Portugal showed some important nutrients to human physiology. Further studies will allow determining if its consumption may overcome the nutritional imbalances of daily modern households, preventing the emergence of hypertension and diabetes.

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.

Implications of nighttime temperature on metamitron impacts on the photosynthetic machinery functioning of Malus x domestica Borkh.

Metamitron (MET) is a fruitlet thinning compound for apple trees, needing better understanding of its action on leaf energy metabolism, depending on nighttime temperature. A trial under environmental controlled conditions was set with 'Golden Reinders' potted trees, under 25/7.5 and 25/15 °C (diurnal/nighttime temperature), with (MET, 247.5 ppm) or without (CTR) application, and considering the monitoring of photosynthetic and respiration components from day 1 (D1) to 14 (D14). Net photosynthesis (Pn) decline promoted by MET after D1 was not stomatal related. Instead, non-stomatal constraints, reflected on the photosynthetic capacity (Amax), included a clear photosystem (PS) II inhibition (but barely of PSI), as shown by severe reductions in thylakoid electron transport at PSII level, maximal (Fv/Fm) and actual (Fv'/Fm') PSII photochemical efficiencies, estimate of quantum yield of linear electron transport (Y(II)), and the rise in PSII photoinhibition status (Fs/Fm' and PIChr) and uncontrolled energy dissipation (Y(NO)). To Pn inhibition also contributed the impact in RuBisCO along the entire experiment, regardless of night temperature, here reported for the first time. Globally, MET impact on the photosynthetic parameters was usually greater under 7.5 °C, with maximal impacts between D4 and D7, probably associated to a less active metabolism at lower temperature. Cellular energy metabolism was further impaired under 7.5 °C, through moderate inhibition of NADH-dependent malate dehydrogenase (MDH) and pyruvate kinase (PK) enzymes involved in respiration, in contrast with the increase of dark respiration in MET 7.5 until D7. The lower impact on PK and MDH under 15 °C and a likely global higher active metabolism at that temperature would agree with the lowest sucrose levels in MET 15 at D4 and D7. Our findings showed that MET alters the cell energy machinery in a temperature dependent manner, affecting the sucrose balance mainly at 15 °C, justifying the observed greater thinning potential.

Can Foliar Pulverization with CaCl2 and Ca(NO3)2 Trigger Ca Enrichment in Solanum tuberosum L. Tubers?

This study aimed to assess the efficiency of Ca enrichment in tubers of three genotypes of Solanum tuberosum L., through foliar spraying with CaCl2 and Ca(NO3)2 solutions. In this context, soil heterogeneity of three potato-growing fields, as well as the implications of Ca accumulation among tissues and some quality parameters were assessed. Three potato varieties (Agria, Picasso and Rossi) were grown in three production fields and during the life cycle, four pulverizations with calcium chloride (3 and 6 kg ha-1) or calcium nitrate (0.5, 2 and 4 kg ha-1) were applied. For screening the potential phytotoxicity, using Agria as a test system, the potential synthesis of photoassimilates was determined, and it was found that after the 3rd Ca application, leaf gas exchanges were moderately (net photosynthesis), to strongly (stomatal conductance) affected, although without impact on Ca accumulation in tubers. At harvest, the average Ca biofortification index varied between 5-40%, 40-35% and 4.3-13% in Agria, Picasso and Rossi, respectively. Moreover, the equatorial region of the tubers in general showed that Ca accumulation prevailed in the epidermis and, in some cases, in inner areas of the potato tubers. Biofortified tubers with Ca also showed some significant changes in total soluble solids and colorimetric parameters. It is concluded that Ca enrichment of potato tubers through foliar spraying complemented the xylem mass flow of Ca from roots, through phloem redistribution. Both fertilizers showed similar efficiency, but Rossi revealed a lower index of Ca accumulation, eventually due to different metabolic characteristics. Although affected by Ca enrichment, potato tubers maintained a high quality for industrial processing.

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.

Quantification and Tissue Localization of Selenium in Rice (Oryza sativa L., Poaceae) Grains: A Perspective of Agronomic Biofortification.

In worldwide production, rice is the second-most-grown crop. It is considered a staple food for many populations and, if naturally enriched in Se, has a huge potential to reduce nutrient deficiencies in foodstuff for human consumption. This study aimed to develop an agronomic itinerary for Se biofortification of Oryza sativa L. (Poaceae) and assess potential physicochemical deviations. Trials were implemented in rice paddy field with known soil and water characteristics and two genotypes resulting from genetic breeding (OP1505 and OP1509) were selected for evaluation. Plants were sprayed at booting, anthesis and milky grain phases with two different foliar fertilizers (sodium selenate and sodium selenite) at different concentrations (25, 50, 75 and 100 g Se·ha-1). After grain harvesting, the application of selenate showed 4.9-7.1 fold increases, whereas selenite increased 5.9-8.4-fold in OP1509 and OP1505, respectively. In brown grain, it was found that in the highest treatment selenate or selenite triggered much higher Se accumulation in OP1505 relatively to OP1509, and that no relevant variation was found with selenate or selenite spraying in each genotype. Total protein increased exponentially in OP1505 genotype when selenite was applied, and higher dosage of Se also increased grain weight and total protein content. It was concluded that, through agronomic biofortification, rice grain can be enriched with Se without impairing its quality, thus highlighting its value in general for the industry and consumers with special needs.

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.

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.

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.

Colonization and beneficial effects on annual ryegrass by mixed inoculation with plant growth promoting bacteria.

Multi-strain inoculants have increased potential to accomplish a diversity of plant needs, mainly attributed to its multi-functionality. This work evaluated the ability of a mixture of three bacteria to colonize and induce a beneficial response on the pasture crop annual ryegrass. Pseudomonas G1Dc10 and Paenibacillus G3Ac9 were previously isolated from annual ryegrass and were selected for their ability to perform multiple functions related to plant growth promotion. Sphingomonas azotifigens DSMZ 18530T was included due to nitrogen fixing ability. The effects of the bacterial mixture were assessed in gnotobiotic plant inoculation assays and compared with single and dual inoculation treatments. Triple inoculation with 3×108 bacteria significantly increased plant dry weight and leaf pigments, indicating improved photosynthetic performance. Plant lipid biosynthesis was enhanced by 65%, mainly due to the rise of linolenic acid, an omega-3 fatty acid with high dietary value. Electrolyte leakage, an indicator of plant membrane stability under stress, was decreased pointing to a beneficial effect by inoculation. Plants physiological condition was more favoured by triple inoculation than by single, although benefits on biomass were only evident relative to non-inoculated plants. The colonization behaviour and coexistence in plant tissues were assessed using FISH and GFP-labelling, combined with confocal microscopy and a cultivation-based approach for quantification. The three strains occupied the same sites, localizing preferentially along root hairs and in stem epidermis. Endophytic colonization was observed as bacteria entered root and stem inner tissues. This study reveals the potential of this mixture of strains for biofertilization, contributing to improve crop productivity and nutritional value.

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.