Exploring natural genetic diversity in a bread wheat multi-founder population: Dual imaging of photosynthesis and stomatal kinetics.

A better understanding of crop phenotype under dynamic environmental conditions will help inform the development of new cultivars with superior adaptation to constantly changing field conditions. Recent research has shown that optimising photosynthetic and stomatal conductance traits holds promise for improved crop performance. However, standard phenotyping tools such as gas-exchange systems are limited by their throughput. In this work, a novel approach based on a bespoke gas-exchange chamber allowing combined measurement of the quantum yield of photosystem II (PSII) with an estimation of stomatal conductance via thermal imaging, was used to phenotype a range of bread wheat (Triticum aestivum L.) genotypes, that were a sub-set of a multi-founder experimental population. Datasets were further supplemented by measurement of photosynthetic capacity and stomatal density. First, we showed that measurement of stomatal traits using our dual imaging system compared to standard IRGA methods showed good agreement between the two methods (R2=0.86) for the rapidity of stomatal opening (Ki), with the dual-imager method resulting in less intra-genotype variation. Using the dual-imaging methods, and traditional approaches we found broad and significant variation in key traits, including photosynthetic CO2 uptake at saturating light and ambient CO2 concentration (Asat), photosynthetic CO2 uptake at saturating light and elevated CO2 concentration (Amax), the maximum velocity of Rubisco for carboxylation (Vcmax), time for stomatal opening (Ki), and leaf evaporative cooling. Anatomical analysis revealed significant variation in flag leaf adaxial stomatal density. Associations between traits highlighted significant relationships between leaf evaporative cooling, leaf stomatal conductance under low (gsmin) and high (gsmax) light intensity, and the operating efficiency of PSII (Fq'/Fm'), highlighting the importance of stomatal conductance and stomatal rapidity in maintaining optimal leaf temperature for photosynthesis in wheat. Additionally, gsmin and gsmax were positively associated, indicating that potential combination of preferable traits (i.e. inherently high gsmax, low Ki and maintained leaf evaporative cooling) are present in wheat. This work highlights for the first time the effectiveness of thermal imaging in screening dynamic stomatal conductance in a large panel of wheat genotypes. The wide phenotypic variation observed suggested the presence of exploitable genetic variability in bread wheat for dynamic stomatal conductance traits and photosynthetic capacity for targeted optimisation within future breeding programs.

Olive (Olea europaea L.) reproductive biology: implications for yield, compatibility conundrum, environmental constraints.

Olive (Olea europaea L.) is an important Mediterranean tree species with a longstanding history of cultivation, boasting a diverse array of local cultivars. While traditional olive orchards are valued for their cultural and aesthetic significance, they often face economic sustainability challenges in the modern context. The success of both traditional and newly introduced cultivars (e.g. those obtained by crossbreeding) is hindered by self-incompatibility, a prevalent issue for this species that results in low fruit set when limited genetic diversity is present. Further, biological, environmental, and agronomic factors have been shown to interlink in shaping fertilization patterns, hence impacting on the final yield. Climatic conditions during pollination, such as excessive rainfall or high temperatures, can further exacerbate the problem. In this work, we provide an overview of the various complex and multifaceted factors that trigger the phenomenon of sub-optimal fruit set in olive trees. Through this work, we aim to provide a comprehensive understanding of the interplay among these factors, shedding light on potential mechanisms and pathways that contribute to the observed outcomes in the context of self-incompatibility of olive. This review aims to contribute to the development of sustainable olive production systems and the preservation of this vital component of Mediterranean culture.

Nitrogen control of transpiration in grapevine.

Transpiration per unit of leaf area is the end-product of the root-to-leaf water transport within the plant, and it is regulated by a series of morpho-physiological resistances and hierarchical signals. The rate of water transpired sustains a series of processes such as nutrient absorption and leaf evaporative cooling, with stomata being the end-valves that maintain the optimal water loss under specific degrees of evaporative demand and soil moisture conditions. Previous work provided evidence of a partial modulation of water flux following nitrogen availability linking high nitrate availability with tight stomatal control of transpiration in several species. In this work, we tested the hypothesis that stomatal control of transpiration, among others signals, is partially modulated by soil nitrate ( NO 3 - ) availability in grapevine, with reduced NO 3 - availability (alkaline soil pH, reduced fertilization, and distancing NO 3 - source) associated with decreased water-use efficiency and higher transpiration. We observed a general trend when NO 3 - was limiting with plants increasing either stomatal conductance or root-shoot ratio in four independent experiments with strong associations between leaf water status, stomatal behavior, root aquaporins expression, and xylem sap pH. Carbon and oxygen isotopic signatures confirm the proximal measurements, suggesting the robustness of the signal that persists over weeks and under different gradients of NO 3 - availability and leaf nitrogen content. Nighttime stomatal conductance was unaffected by NO 3 - manipulation treatments, while application of high vapor pressure deficit conditions nullifies the differences between treatments. Genotypic variation for transpiration increase under limited NO 3 - availability was observed between rootstocks indicating that breeding (e.g., for high soil pH tolerance) unintentionally selected for enhanced mass flow nutrient acquisition under restrictive or nutrient-buffered conditions. We provide evidence of a series of specific traits modulated by NO 3 - availability and suggest that NO 3 - fertilization is a potential candidate for optimizing grapevine water-use efficiency and root exploration under the climate-change scenario.

Longer epidermal cells underlie a quantitative source of variation in wheat flag leaf size.

The wheat flag leaf is the main contributor of photosynthetic assimilates to developing grains. Understanding how canopy architecture strategies affect source strength and yield will aid improved crop design. We used an eight-founder population to investigate the genetic architecture of flag leaf area, length, width and angle in European wheat. For the strongest genetic locus identified, we subsequently created a near-isogenic line (NIL) pair for more detailed investigation across seven test environments. Genetic control of traits investigated was highly polygenic, with colocalisation of replicated quantitative trait loci (QTL) for one or more traits identifying 24 loci. For QTL QFll.niab-5A.1 (FLL5A), development of a NIL pair found the FLL5A+ allele commonly conferred a c. 7% increase in flag and second leaf length and a more erect leaf angle, resulting in higher flag and/or second leaf area. Increased FLL5A-mediated flag leaf length was associated with: (1) longer pavement cells and (2) larger stomata at lower density, with a trend for decreased maximum stomatal conductance (Gsmax ) per unit leaf area. For FLL5A, cell size rather than number predominantly determined leaf length. The observed trade-offs between leaf size and stomatal morphology highlight the need for future studies to consider these traits at the whole-leaf level.

Leaf trait plasticity and site-specific environmental variability modulate the severity of visible foliar ozone symptoms in Viburnum lantana.

The assessment of Visible Foliar Symptoms (VFS) is commonly adopted by forest monitoring programs to evaluate ozone impact on vegetation. The occurrence of ozone VFS may differ among individuals of the same species at the same site, and within leaves of the same individual. The aim of this study was to identify site and plant characteristics as well as functional leaf traits associated with the occurrence and severity of VFS in Viburnum lantana (an ozone-sensitive species) and at the scale of an individual site. V. lantana plants growing at one site of the ViburNeT monitoring network (Trentino, North Italy) experiencing high ozone levels were surveyed in relation to 1) sun exposure, 2) shading effect from neighbor vegetation, 3) plant height and 4) presence and severity of VFS. Leaves from three different sections of each plant were subjected to a phenotypic characterization of leaf area, dry weight, specific leaf area (SLA), chlorophyll content (ChlSPAD), percentage of VFS, and adaxial and abaxial trichome density (Tr). We showed that plants at high irradiation levels had significantly lower SLA (p<0.05), higher Tr (p<0.01) and greater ChlSPAD (p<0.01) when compared to shaded and/or west- and north-exposed plants, thus indicating a strong influence of site-specific characteristics on leaf trait plasticity. Similar differences were observed for taller vs. shorter plants and apical vs. basal branches (p<0.05). Ozone-induced VFS at leaf level were associated with lower SLA (p<0.001) and higher Tr in the abaxial leaf surface (p<0.05). Both leaf traits showed significant differences also within the south and east exposed plant category, thus suggesting the increase in leaf thickness and Tr as a potential adaptive strategy under multiple stress conditions. Our results provide evidence of a strong relationship between VFS, leaf traits and site-specific variables, offering new insights for interpreting data on the impact of ozone on vegetation.

VvEPFL9-1 Knock-Out via CRISPR/Cas9 Reduces Stomatal Density in Grapevine.

Epidermal Patterning Factor Like 9 (EPFL9), also known as STOMAGEN, is a cysteine-rich peptide that induces stomata formation in vascular plants, acting antagonistically to other epidermal patterning factors (EPF1, EPF2). In grapevine there are two EPFL9 genes, EPFL9-1 and EPFL9-2 sharing 82% identity at protein level in the mature functional C-terminal domain. In this study, CRISPR/Cas9 system was applied to functionally characterize VvEPFL9-1 in 'Sugraone', a highly transformable genotype. A set of plants, regenerated after gene transfer in embryogenic calli via Agrobacterium tumefaciens, were selected for evaluation. For many lines, the editing profile in the target site displayed a range of mutations mainly causing frameshift in the coding sequence or affecting the second cysteine residue. The analysis of stomata density revealed that in edited plants the number of stomata was significantly reduced compared to control, demonstrating for the first time the role of EPFL9 in a perennial fruit crop. Three edited lines were then assessed for growth, photosynthesis, stomatal conductance, and water use efficiency in experiments carried out at different environmental conditions. Intrinsic water-use efficiency was improved in edited lines compared to control, indicating possible advantages in reducing stomatal density under future environmental drier scenarios. Our results show the potential of manipulating stomatal density for optimizing grapevine adaptation under changing climate conditions.

Natural variation in stomatal dynamics drives divergence in heat stress tolerance and contributes to seasonal intrinsic water-use efficiency in Vitis vinifera (subsp. sativa and sylvestris).

Stomata control CO2 uptake for photosynthesis and water loss through transpiration, thus playing a key role in leaf thermoregulation, water-use efficiency (iWUE), and plant productivity. In this work, we investigated the relationship between several leaf traits and hypothesized that stomatal behavior to fast (i.e. minutes) environmental changes co-determines, along with steady-state traits, the physiological response of grapevine to the surrounding fluctuating environment over the growing season. No relationship between iWUE, heat stress tolerance, and stomatal traits was observed in field-grown grapevine, suggesting that other physiological mechanisms are involved in determining leaf evaporative cooling capacity and the seasonal ratio of CO2 uptake (A) to stomatal conductance (gs). Indeed, cultivars that in the field had an unexpected combination of high iWUE but low sensitivity to thermal stress displayed a quick stomatal closure to light, but a sluggish closure to increased vapor pressure deficit (VPD) levels. This strategy, aiming both at conserving water under a high to low light transition and in prioritizing evaporative cooling under a low to high VPD transition, was mainly observed in the cultivars Regina and Syrah. Moreover, cultivars with different known responses to soil moisture deficit or high air VPD (isohydric versus anisohydric) had opposite behavior under fluctuating environments, with the isohydric cultivar showing slow stomatal closure to reduced light intensity but quick temporal responses to VPD manipulation. We propose that stomatal behavior to fast environmental fluctuations can play a critical role in leaf thermoregulation and water conservation under natural field conditions in grapevine.

Interspecific and intraspecific phenotypic diversity for drought adaptation in bioenergy Arundo species.

Biomass crops are commonly grown in low-grade land and selection of drought-tolerant accessions is of major importance to sustain productivity. In this work, we assess phenotypic variation under different environmental scenarios in a series of accessions of Arundo donax, and contrast it with two closely related species, Arundo donaciformis and Arundo plinii. Gas-exchange and stomatal anatomy analysis showed an elevated photosynthetic capacity in A. plinii compared to A. donax and A. donaciformis with a significant intraspecific variation in A. donax. The three species showed significantly contrasting behaviour of transpiration under developing water stress and increasing vapour pressure deficit (VPD), with A. donax being the most conservative while A. plinii showed an elevated degree of insensitivity to environmental cues. Under optimal conditions, A. donax had the highest estimated leaf area (projected leaf area) and plant dry weight although a significant reduction under water stress was observed for A. donax and A. donaciformis accessions while no differences were recorded for A. plinii between optimal growing conditions (well-watered [WW]) and reduced soil water availability (water-stressed [WS]). A. donax displayed a markedly conservative water use behaviour but elevated sensitivity of biomass accumulation under stress conditions. By contrast, in A. plinii, biomass and transpiration were largely insensitive to WS and increasing VPD, though biomass dry weight under optimal conditions was significantly lower than A. donax. We provide evidence of interspecific phenotypic variation within the Arundo genus while the intraspecific phenotypic plasticity may be exploited for further selection of superior clones under disadvantageous environmental conditions. The extensive trade-off between water use and biomass accumulation present in the three species under stress conditions provides a series of novel traits to be exploited in the selection of superior clones adapted to different environmental scenarios. Non-destructive approaches are provided to screen large populations for water-stress-tolerant A. donax clones.

Leaf Monoterpene Emission Limits Photosynthetic Downregulation under Heat Stress in Field-Grown Grapevine.

Rising temperature is among the most remarkably stressful phenomena induced by global climate changes with negative impacts on crop productivity and quality. It has been previously shown that volatiles belonging to the isoprenoid family can confer protection against abiotic stresses. In this work, two Vitis vinifera cv. 'Chardonnay' clones (SMA130 and INRA809) differing due to a mutation (S272P) of the DXS gene encoding for 1-deoxy-D-xylulose-5-phosphate (the first dedicated enzyme of the 2C-methyl-D-erythritol-4-phosphate (MEP) pathway) and involved in the regulation of isoprenoids biosynthesis were investigated in field trials and laboratory experiments. Leaf monoterpene emission, chlorophyll fluorescence and gas-exchange measurements were assessed over three seasons at different phenological stages and either carried out in in vivo or controlled conditions under contrasting temperatures. A significant (p < 0.001) increase in leaf monoterpene emission was observed in INRA809 when plants were experiencing high temperatures and over two experiments, while no differences were recorded for SMA130. Significant variation was observed for the rate of leaf CO2 assimilation under heat stress, with INRA809 maintaining higher photosynthetic rates and stomatal conductance values than SMA130 (p = 0.003) when leaf temperature increased above 30 °C. At the same time, the maximum photochemical quantum yield of PSII (Fv/Fm) was affected by heat stress in the non-emitting clone (SMA130), while the INRA809 showed a significant resilience of PSII under elevated temperature conditions. Consistent data were recorded between field seasons and temperature treatments in controlled environment conditions, suggesting a strong influence of monoterpene emission on heat tolerance under high temperatures. This work provides further insights on the photoprotective role of isoprenoids in heat-stressed Vitis vinifera, and additional studies should focus on unraveling the mechanisms underlying heat tolerance on the monoterpene-emitter grapevine clone.

Shoot Characterization of Isoprene and Ocimene-Emitting Transgenic Arabidopsis Plants under Contrasting Environmental Conditions.

Isoprenoids are among the most abundant biogenic volatile compounds (VOCs) emitted by plants, and mediate both biotic and abiotic stress responses. Here, we provide for the first time a comparative analysis of transgenic Arabidopsis lines constitutively emitting isoprene and ocimene. Transgenic lines and Columbia-0 (Col-0) Arabidopsis were characterized under optimal, water stress, and heat stress conditions. Under optimal conditions, the projected leaf area (PLA), relative growth rate, and final dry weight were generally higher in transgenics than Col-0. These traits were associated to a larger photosynthetic capacity and CO2 assimilation rate at saturating light. Isoprene and ocimene emitters displayed a moderately higher stress tolerance than Col-0, showing higher PLA and gas-exchange traits throughout the experiments. Contrasting behaviors were recorded for the two overexpressors under water stress, with isoprene emitters showing earlier stomatal closure (conservative behavior) than ocimene emitters (non-conservative behavior), which might suggest different induced strategies for water conservation and stress adaptation. Our work indicates that (i) isoprene and ocimene emitters resulted in enhanced PLA and biomass under optimal and control conditions and that (ii) a moderate stress tolerance is induced when isoprene and ocimene are constitutively emitted in Arabidopsis, thus providing evidence of their role as a potential preferable trait for crop improvement.

Photosynthesis in non-foliar tissues: implications for yield.

Photosynthesis is currently a focus for crop improvement. The majority of this work has taken place and been assessed in leaves, and limited consideration has been given to the contribution that other green tissues make to whole-plant carbon assimilation. The major focus of this review is to evaluate the impact of non-foliar photosynthesis on carbon-use efficiency and total assimilation. Here we appraise and summarize past and current literature on the substantial contribution of different photosynthetically active organs and tissues to productivity in a variety of different plant types, with an emphasis on fruit and cereal crops. Previous studies provide evidence that non-leaf photosynthesis could be an unexploited potential target for crop improvement. We also briefly examine the role of stomata in non-foliar tissues, gas exchange, maintenance of optimal temperatures and thus photosynthesis. In the final section, we discuss possible opportunities to manipulate these processes and provide evidence that Triticum aestivum (wheat) plants genetically manipulated to increase leaf photosynthesis also displayed higher rates of ear assimilation, which translated to increased grain yield. By understanding these processes, we can start to provide insights into manipulating non-foliar photosynthesis and stomatal behaviour to identify novel targets for exploitation in continuing breeding programmes.

Natural genetic variation in photosynthesis: an untapped resource to increase crop yield potential?

Raising crop yield potential is a major goal to ensure food security for the growing global population. Photosynthesis is the primary determinant of crop productivity and any gain in photosynthetic CO2 assimilation per unit of leaf area (A) has the potential to increase yield. Significant intraspecific variation in A is known to exist in various autotrophic organs that represent an unexploited target for crop improvement. However, the large number of factors that influence photosynthetic rates often makes it difficult to measure or estimate A under dynamic field conditions (i.e. fluctuating light intensities or temperatures). This complexity often results in photosynthetic capacity, rather than realized photosynthetic rates being used to assess natural variation in photosynthesis. Here we review the work on natural variation in A, the different factors determining A and their interaction in yield formation. A series of drawbacks and perspectives are presented for the most common analyses generally used to estimate A. The different yield components and their determination based on different photosynthetic organs are discussed with a major focus on potential exploitation of various traits for crop improvement. To conclude, an example of different possibilities to increase yield in wheat through enhancing A is illustrated.

Genotypic, Developmental and Environmental Effects on the Rapidity of gs in Wheat: Impacts on Carbon Gain and Water-Use Efficiency.

Stomata are the primary gatekeepers for CO2 uptake for photosynthesis and water loss via transpiration and therefore play a central role in crop performance. Although stomatal conductance (gs ) and assimilation rate (A) are often highly correlated, studies have demonstrated an uncoupling between A and gs that can result in sub-optimal physiological processes in dynamic light environments. Wheat (Triticum aestivum L.) is exposed to changes in irradiance due to leaf self-shading, moving clouds and shifting sun angle to which both A and gs respond. However, stomatal responses are generally an order of magnitude slower than photosynthetic responses, leading to non-synchronized A and gs responses that impact CO2 uptake and water use efficiency ( iWUE). Here we phenotyped a panel of eight wheat cultivars (estimated to capture 80% of the single nucleotide polymorphism variation in North-West European bread wheat) for differences in the speed of stomatal responses (to changes in light intensity) and photosynthetic performance at different stages of development. The impact of water stress and elevated [CO2] on stomatal kinetics was also examined in a selected cultivar. Significant genotypic variation was reported for the time constant for stomatal opening (Ki, P = 0.038) and the time to reach 95% steady state A (P = 0.045). Slow gs opening responses limited A by ∼10% and slow closure reduced iWUE, with these impacts found to be greatest in cultivars Soissons, Alchemy and Xi19. A decrease in stomatal rapidity (and thus an increase in the limitation of photosynthesis) (P < 0.001) was found during the post-anthesis stage compared to the early booting stage. Reduced water availability triggered stomatal closure and asymmetric stomatal opening and closing responses, while elevated atmospheric [CO2] conditions reduced the time for stomatal opening during a low to high light transition, thus suggesting a major environmental effect on dynamic stomatal kinetics. We discuss these findings in terms of exploiting various traits to develop ideotypes for specific environments, and suggest that intraspecific variation in the rapidity of stomatal responses could provide a potential unexploited breeding target to optimize the physiological responses of wheat to dynamic field conditions.

Exploiting natural variation and genetic manipulation of stomatal conductance for crop improvement.

Rising global temperatures and more frequent episodes of drought are expected to drive reductions in crop yield, therefore new avenues for improving crop productivity must be exploited. Stomatal conductance (gs) balances plant CO2 uptake and water loss, therefore, greatly impacting the cumulative rate of photosynthesis and water use over the growing season, which are key determinants of crop yield and productivity. Considerable natural variation exists in stomatal anatomy, biochemistry and behavioural characteristics that impact on the kinetics and magnitude of gs and thus gaseous exchange between the plant and atmosphere. Exploiting these differences in stomatal traits could provide novel breeding targets for new crop varieties that are potentially more water use efficient and have the ability to maintain and/or maximize yield in a range of diverse environments. Here we provide an overview of variation in stomatal traits and the impact these have on gs behaviour, as well as the potential to exploit such variation and genetic manipulation for crop improvement.

Rising CO2 from historical concentrations enhances the physiological performance of Brassica napus seedlings under optimal water supply but not under reduced water availability.

The productivity of many important crops is significantly threatened by water shortage, and the elevated atmospheric CO2 can significantly interact with physiological processes and crop responses to drought. We examined the effects of three different CO2 concentrations (historical ~300 ppm, ambient ~400 ppm and elevated ~700 ppm) on physiological traits of oilseed rape (Brassica napus L.) seedlings subjected to well-watered and reduced water availability. Our data show (1) that, as expected, increasing CO2 level positively modulates leaf photosynthetic traits, leaf water-use efficiency and growth under non-stressed conditions, although a pronounced acclimation of photosynthesis to elevated CO2 occurred; (2) that the predicted elevated CO2 concentration does not reduce total evapotranspiration under drought when compared with present (400 ppm) and historical (300 ppm) concentrations because of a larger leaf area that does not buffer transpiration; and (3) that accordingly, the physiological traits analysed decreased similarly under stress for all CO2 concentrations. Our data support the hypothesis that increasing CO2 concentrations may not significantly counteract the negative effect of increasing drought intensity on Brassica napus performance.