Exploring plant responses to abiotic stress by contrasting spectral signature changes.

In this study, daily changes over a short period and diurnal progression of spectral reflectance at the leaf level were used to identify spring wheat genotypes (Triticum aestivum L.) susceptible to adverse conditions. Four genotypes were grown in pots experiments under semi-controlled conditions in Chile and Spain. Three treatments were applied: i) control (C), ii) water stress (WS), and iii) combined water and heat shock (WS+T). Spectral reflectance, gas exchange and chlorophyll fluorescence measurements were performed on flag leaves for three consecutive days at anthesis. High canopy temperature ( H CT ) genotypes showed less variability in their mean spectral reflectance signature and chlorophyll fluorescence, which was related to weaker responses to environmental fluctuations. While low canopy temperature ( L CT ) genotypes showed greater variability. The genotypes spectral signature changes, in accordance with environmental fluctuation, were associated with variations in their stomatal conductance under both stress conditions (WS and WS+T); L CT genotypes showed an anisohydric response compared that of H CT , which was isohydric. This approach could be used in breeding programs for screening a large number of genotypes through proximal or remote sensing tools and be a novel but simple way to identify groups of genotypes with contrasting performances.

Different Metabolic Roles for Alternative Oxidase in Leaves of Palustrine and Terrestrial Species.

The alternative oxidase pathway (AOP) is associated with excess energy dissipation in leaves of terrestrial plants. To address whether this association is less important in palustrine plants, we compared the role of AOP in balancing energy and carbon metabolism in palustrine and terrestrial environments by identifying metabolic relationships between primary carbon metabolites and AOP in each habitat. We measured oxygen isotope discrimination during respiration, gas exchange, and metabolite profiles in aerial leaves of ten fern and angiosperm species belonging to five families organized as pairs of palustrine and terrestrial species. We performed a partial least square model combined with variable importance for projection to reveal relationships between the electron partitioning to the AOP (τa) and metabolite levels. Terrestrial plants showed higher values of net photosynthesis (AN) and τa, together with stronger metabolic relationships between τa and sugars, important for water conservation. Palustrine plants showed relationships between τa and metabolites related to the shikimate pathway and the GABA shunt, to be important for heterophylly. Excess energy dissipation via AOX is less crucial in palustrine environments than on land. The basis of this difference resides in the contrasting photosynthetic performance observed in each environment, thus reinforcing the importance of AOP for photosynthesis.

Intracultivar genetic diversity in grapevine: Water use efficiency variability within cv. Grenache.

The selection of genotypes best adapted to environmental conditions has traditionally focused on agronomic and grape composition parameters. However, to classify the genotypes most adapted to climate change conditions, the aim must be to focus on the ecophysiological responses that will ultimately determine their performance. The variability in water use efficiency of 13 Grenache genotypes over three-seasons was assessed under field conditions at leaf, grape and plant level. Results showed a significant effect of genotype at all three levels, and despite the large interannual variability there was a remarkable consistency among levels. Furthermore, using genotype-specific regressions it was possible to identify significant differences in the intrinsic water use efficiency response of each genotype as a function of the vine water status. The relationship between net photosynthesis and stomatal conductance, as well as carbon isotope discrimination in grapes, were also confirmed as reliable physiological indicators for selecting grapevine genotypes to future environmental conditions. Therefore, the proposed multi-level methodology was useful to quantify the intracultivar variability and the identification of more and less efficient genotypes within Grenache.

Cell wall thickness and composition are related to photosynthesis in Antarctic mosses.

Cell wall thickness (Tcw ) has been proposed as an important anatomical trait that could determine photosynthesis through land plants' phylogeny, bryophytes being the plant group presenting the thickest walls and the lowest photosynthetic rates. Also, it has recently been suggested that cell wall composition may have the potential to influence both thickness and mesophyll conductance (gm ), representing a novel trait that could ultimately affect photosynthesis. However, only a few studies in spermatophytes have demonstrated this issue. In order to explore the role of cell wall composition in determining both Tcw and gm in mosses, we tested six species grown under field conditions in Antarctica. We performed gas exchange and chlorophyll fluorescence measurements, an anatomical characterization, and a quantitative analysis of cell wall main composition (i.e., cellulose, hemicelluloses and pectins) in these six species. We found the photosynthetic rates to vary between the species, and they also presented differences in anatomical characteristics and in cell wall composition. Whilst gm correlated negatively with Tcw and pectins content, a positive relationship between Tcw and pectins emerged, suggesting that pectins could contribute to determine cell wall porosity. Although our results do not allow us to provide conclusive statements, we suggest for the first time that cell wall composition-with pectins playing a key role-could strongly influence Tcw and gm in Antarctic mosses, ultimately defining photosynthesis.

Leaf anatomy does not explain apparent short-term responses of mesophyll conductance to light and CO2 in tobacco.

Mesophyll conductance to CO2 (gm ), a key photosynthetic trait, is strongly constrained by leaf anatomy. Leaf anatomical parameters such as cell wall thickness and chloroplast area exposed to the mesophyll intercellular airspace have been demonstrated to determine gm in species with diverging phylogeny, leaf structure and ontogeny. However, the potential implication of leaf anatomy, especially chloroplast movement, on the short-term response of gm to rapid changes (i.e. seconds to minutes) under different environmental conditions (CO2 , light or temperature) has not been examined. The aim of this study was to determine whether the observed rapid variations of gm in response to variations of light and CO2 could be explained by changes in any leaf anatomical arrangements. When compared to high light and ambient CO2 , the values of gm estimated by chlorophyll fluorescence decreased under high CO2 and increased at low CO2 , while it decreased with decreasing light. Nevertheless, no changes in anatomical parameters, including chloroplast distribution, were found. Hence, the gm estimated by analytical models based on anatomical parameters was constant under varying light and CO2 . Considering this discrepancy between anatomy and chlorophyll fluorescence estimates, it is concluded that apparent fast gm variations should be due to artefacts in its estimation and/or to changes in the biochemical components acting on diffusional properties of the leaf (e.g. aquaporins and carbonic anhydrase).

Whole-Plant Water Use in Field Grown Grapevine: Seasonal and Environmental Effects on Water and Carbon Balance.

Water scarcity is a main challenge in vineyards sustainability in most of the grapevine areas now and even more in near future due to climatic change perspectives. In consequence, water use efficiency (WUE) measurements are of the highest interest to improve the sustainability of this crop. The vast majority of WUE measurements relays on measurements of leaf carbon and water fluxes at leaf-level. However, less data are available at the whole-plant level, and for the moment those data are not totally coincident with conclusions reached at leaf scale. In this study, we used whole-plant chambers able to enclose an entire plant of 12 years old to measure at the same time water and carbon fluxes under realistic field grown conditions. The main objectives were to identify the technical issues interfering the whole-plant measurements and track the environmental and other abiotic factors that can affect water and carbon balance, i.e., WUE at the whole-plant scale. To achieve those objectives, we measured whole-plant water and carbon fluxes in grapevine exposed to two different water regimes at three phenological stages [pea size (July), ripening (August), and harvest (September)]. In September, measurements were repeated under high CO2 to also check its effect at the whole-plant scale. The results indicate that water and carbon fluxes are well coordinated under both water availability treatments. Under drought conditions, both fluxes were drastically reduced, but surprisingly the estimated WUE resulted not improved but decreased, contrarily to what is shown at the leaf scale. The phenology (September) also strongly decreased both water and carbon fluxes when compared to measurements in July. We hypostatized that harvest load respiration rates could have an important weight on the whole-plant net carbon exchange (NCE). Finally, high CO2 measurements, after correction for leaks, indicated an increase of whole-plant NCE as well as increased whole-plant WUE, as expected. Several technical issues were identified, like 1/instability of [CO2] during the night period that prevent robust estimation of whole-plant respiration and 2/condensation during last night and sun-rise hours which may affect the estimation of daily plant transpiration.

Physiological performance of transplastomic tobacco plants overexpressing aquaporin AQP1 in chloroplast membranes.

The leaf mesophyll CO2 conductance and the concentration of CO2 within the chloroplast are major factors affecting photosynthetic performance. Previous studies have shown that the aquaporin NtAQP1 (which localizes to the plasma membrane and chloroplast inner envelope membrane) is involved in CO2 permeability in the chloroplast. Levels of NtAQP1 in plants genetically engineered to overexpress the protein correlated positively with leaf mesophyll CO2 conductance and photosynthetic rate. In these studies, the nuclear transformation method used led to changes in NtAQP1 levels in the plasma membrane and the chloroplast inner envelope membrane. In the present work, NtAQP1 levels were increased up to 16-fold in the chloroplast membranes alone by the overexpression of NtAQP1 from the plastid genome. Despite the high NtAQP1 levels achieved, transplastomic plants showed lower photosynthetic rates than wild-type plants. This result was associated with lower Rubisco maximum carboxylation rate and ribulose 1,5-bisphosphate regeneration. Transplastomic plants showed reduced mesophyll CO2 conductance but no changes in chloroplast CO2 concentration. The absence of differences in chloroplast CO2 concentration was associated with the lower CO2 fixation activity of the transplastomic plants. These findings suggest that non-functional pores of recombinant NtAQP1 may be produced in the chloroplast inner envelope membrane.

Differential coordination of stomatal conductance, mesophyll conductance, and leaf hydraulic conductance in response to changing light across species.

Stomatal conductance (gs ) and mesophyll conductance (gm ) represent major constraints to photosynthetic rate (A), and these traits are expected to coordinate with leaf hydraulic conductance (Kleaf ) across species, under both steady-state and dynamic conditions. However, empirical information about their coordination is scarce. In this study, Kleaf , gas exchange, stomatal kinetics, and leaf anatomy in 10 species including ferns, gymnosperms, and angiosperms were investigated to elucidate the correlation of H2 O and CO2 diffusion inside leaves under varying light conditions. Gas exchange, Kleaf , and anatomical traits varied widely across species. Under light-saturated conditions, the A, gs , gm , and Kleaf were strongly correlated across species. However, the response patterns of A, gs , gm , and Kleaf to varying light intensities were highly species dependent. Moreover, stomatal opening upon light exposure of dark-adapted leaves in the studied ferns and gymnosperms was generally faster than in the angiosperms; however, stomatal closing in light-adapted leaves after darkening was faster in angiosperms. The present results show that there is a large variability in the coordination of leaf hydraulic and gas exchange parameters across terrestrial plant species, as well as in their responses to changing light.

Genes and gene clusters related to genotype and drought-induced variation in saccharification potential, lignin content and wood anatomical traits in Populus nigra.

Wood is a renewable resource that can be employed for the production of second generation biofuels by enzymatic saccharification and subsequent fermentation. Knowledge on how the saccharification potential is affected by genotype-related variation of wood traits and drought is scarce. Here, we used three Populus nigra L. genotypes from habitats differing in water availability to (i) investigate the relationships between wood anatomy, lignin content and saccharification and (ii) identify genes and co-expressed gene clusters related to genotype and drought-induced variation in wood traits and saccharification potential. The three poplar genotypes differed in wood anatomy, lignin content and saccharification potential. Drought resulted in reduced cambial activity, decreased vessel and fiber lumina, and increased the saccharification potential. The saccharification potential was unrelated to lignin content as well as to most wood anatomical traits. RNA sequencing of the developing xylem revealed that 1.5% of the analyzed genes were differentially expressed in response to drought, while 67% differed among the genotypes. Weighted gene correlation network analysis identified modules of co-expressed genes correlated with saccharification potential. These modules were enriched in gene ontology terms related to cell wall polysaccharide biosynthesis and modification and vesicle transport, but not to lignin biosynthesis. Among the most strongly saccharification-correlated genes, those with regulatory functions, especially kinases, were prominent. We further identified transcription factors whose transcript abundances differed among genotypes, and which were co-regulated with genes for biosynthesis and modifications of hemicelluloses and pectin. Overall, our study suggests that the regulation of pectin and hemicellulose metabolism is a promising target for improving wood quality of second generation bioenergy crops. The causal relationship of the identified genes and pathways with saccharification potential needs to be validated in further experiments.

Rapid responses of mesophyll conductance to changes of CO2 concentration, temperature and irradiance are affected by N supplements in rice.

Photosynthesis in C3 plants is significantly limited by mesophyll conductance (gm ), which can vary with leaf anatomical traits and nitrogen (N) supplements. Several studies have investigated the response of gm to N supplements; however, none examined the implications of N supplements on the response of gm to rapid environmental changes. Here we investigated the effect of N supplement on gm and the response of gm to change of CO2 , temperature and irradiance in rice. High N supplement (HN) increased mesophyll cell wall surface area and chloroplast surface area exposed to intercellular airspace per leaf area, and reduced cell wall thickness. These changes resulted in increased gm . The gm of leaves with HN was more sensitive to changes in CO2 concentration, temperature and irradiance. The difference in leaf structural features between low N supplement and HN indicates that a rapid change in gm is related to the regulation of diffusion through biological membranes rather than leaf structural features. These results will contribute to an understanding of the determinants of gm response to rapid changes in environmental factors.

Opportunities for improving leaf water use efficiency under climate change conditions.

WUEi (intrinsic water use efficiency) is a complex (multi)-trait, that depends on several physiological processes, driving plant productivity and its relation with a changing environment. Climatic change predictions estimate increases in temperature and drought in the semi-arid regions, rendering improved water use efficiency is a mandatory objective to maintain the current global food supply. The aims of this review were (i) to identify through a meta-analysis the leaf traits mostly related to intrinsic water use efficiency (WUEi, the ratio between A - net photosynthesis and gs - stomatal conductance), based on a newly compiled dataset covering more than 200 species/varieties and 106 genus of C3 plants (ii) to describe the main potential targets for WUEi improvement via biotechnological manipulations and (iii) to introduce emergent and innovative technologies including UAVs (Unmanned Aerial Vehicles) to scale up levels from leaf to whole plant water status. We confirmed that increases in gm/gs and Vcmax/gs ratios are systematically related with increases in WUEi maintained across species, habitats, and environmental conditions. Other emergent opportunities to improve WUEi are described such as the relationship between photosynthesis and respiration and their link with metabolomics. Finally, we outline our hypothesis that we are observing the advent of a "smart" agriculture, wherein new technologies, such as UAVs equipped with remote sensors will rapidly facilitate an efficient water use regulating the irrigation schedule and determination, under field conditions, of cultivars with improved water use efficiency. We, therefore, conclude that the multi-disciplinary challenge toward WUE has only just begun.

Diffusional conductances to CO2 as a target for increasing photosynthesis and photosynthetic water-use efficiency.

A key objective for sustainable agriculture and forestry is to breed plants with both high carbon gain and water-use efficiency (WUE). At the level of leaf physiology, this implies increasing net photosynthesis (A N) relative to stomatal conductance (g s). Here, we review evidence for CO2 diffusional constraints on photosynthesis and WUE. Analyzing past observations for an extensive pool of crop and wild plant species that vary widely in mesophyll conductance to CO2 (g m), g s, and foliage A N, it was shown that both g s and g m limit A N, although the relative importance of each of the two conductances depends on species and conditions. Based on Fick's law of diffusion, intrinsic WUE (the ratio A N/g s) should correlate on the ratio g m/g s, and not g m itself. Such a correlation is indeed often observed in the data. However, since besides diffusion A N also depends on photosynthetic capacity (i.e., V c,max), this relationship is not always sustained. It was shown that only in a very few cases, genotype selection has resulted in simultaneous increases of both A N and WUE. In fact, such a response has never been observed in genetically modified plants specifically engineered for either reduced g s or enhanced g m. Although increasing g m alone would result in increasing photosynthesis, and potentially increasing WUE, in practice, higher WUE seems to be only achieved when there are no parallel changes in g s. We conclude that for simultaneous improvement of A N and WUE, genetic manipulation of g m should avoid parallel changes in g s, and we suggest that the appropriate trait for selection for enhanced WUE is increased g m/g s.

Mesophyll diffusion conductance to CO2: an unappreciated central player in photosynthesis.

Mesophyll diffusion conductance to CO(2) is a key photosynthetic trait that has been studied intensively in the past years. The intention of the present review is to update knowledge of g(m), and highlight the important unknown and controversial aspects that require future work. The photosynthetic limitation imposed by mesophyll conductance is large, and under certain conditions can be the most significant photosynthetic limitation. New evidence shows that anatomical traits, such as cell wall thickness and chloroplast distribution are amongst the stronger determinants of mesophyll conductance, although rapid variations in response to environmental changes might be regulated by other factors such as aquaporin conductance. Gaps in knowledge that should be research priorities for the near future include: how different is mesophyll conductance among phylogenetically distant groups and how has it evolved? Can mesophyll conductance be uncoupled from regulation of the water path? What are the main drivers of mesophyll conductance? The need for mechanistic and phenomenological models of mesophyll conductance and its incorporation in process-based photosynthesis models is also highlighted.

Is mesophyll conductance to CO2 in leaves of three Eucalyptus species sensitive to short-term changes of irradiance under ambient as well as low O2?

Mesophyll conductance to CO2 (g m) limits the diffusion of CO2 to the sites of carboxylation, and may respond rapidly (within minutes) to abiotic factors. Using three Eucalyptus species, we tested the rapid response of g m to irradiance under 21% and 1% O2. We used simultaneous measurements of leaf gas exchange and discrimination against 13CO2 with a tuneable diode laser absorption spectrometer. Measurements under 1% O2 were used to limit uncertainties due to 13C-12C fractionation occurring during photorespiration. Switching irradiance from 600 to 200µmolm-2s-1 led to a ≈60% decrease of g m within minutes in all species under both 21% O2 and 1% O2. The g m response to irradiance is unlikely to be a computation artefact since using different values for the parameters of the discrimination model changed the absolute values of g m but did not affect the relative response to irradiance. Simulations showed that possible rapid changes of any parameter were unable to explain the observed variations of g m with irradiance, except for13C-12C fractionation during carboxylation (b), which, in turn, is dependent on the fraction of leaf C assimilated by phospho-enol pyruvate carboxylase (PEPc) (ß). g m apparently increased by ≈30% when O2 was switched from 21% to 1% O2. Again, possible changes of ß with O2 could explain this apparent g m response to O2. Nevertheless, large irradiance or O2-induced changes in ß would be required to fully explain the observed changes in g m, reinforcing the hypothesis that g m is responsive to irradiance and possibly also to O2.

Mesophyll conductance to CO2, assessed from online TDL-AS records of ¹³CO2 discrimination, displays small but significant short-term responses to CO2 and irradiance in Eucalyptus seedlings.

Mesophyll conductance (g(m)) is now recognized as an important limiting process for photosynthesis, as it results in a significant decrease of CO(2) diffusion from substomatal cavities where water evaporation occurs, to chloroplast stroma. Over the past decade, an increasing number of studies proposed that g(m) can vary in the short term (e.g. minutes), but these variations are still controversial, especially those potentially induced by changing CO(2) and irradiance. In this study, g(m) data estimated with online (13)C discrimination recorded with a tunable diode laser absorption spectrometer (TDL-AS) during leaf gas exchange measurements, and based on the single point method, are presented. The data were obtained with three Eucalyptus species. A 50% decrease in g(m) was observed when the CO(2) mole fraction was increased from 300 µmol mol(-1) to 900 µmol mol(-1), and a 60% increase when irradiance was increased from 200 µmol mol(-1) to 1100 µmol mol(-1) photosynthetic photon flux density (PPFD). The relative contribution of respiration and photorespiration to overall (13)C discrimination was also estimated. Not taking this contribution into account may lead to a 50% underestimation of g(m) but had little effect on the CO(2)- and irradiance-induced changes. In conclusion, (i) the observed responses of g(m) to CO(2) and irradiance were not artefactual; (ii) the respiratory term is important to assess absolute values of g(m) but has no impact on the responses to CO(2) and PPFD; and (iii) increasing irradiance and reducing the CO(2) mole fraction results in rapid increases in g(m) in Eucalyptus seedlings.

Mechanism of water-stress induced cavitation in conifers: bordered pit structure and function support the hypothesis of seal capillary-seeding.

Resistance to water-stress induced cavitation is an important indicator of drought tolerance in woody species and is known to be intimately linked to the anatomy of the xylem. However, the actual mechanical properties of the pit membrane are not well known and the exact mode of air-seeding by which cavitation occurs is still uncertain. We examined the relationship between cavitation resistance and bordered pit structure and function in 40 coniferous species. Xylem pressure inducing 50% loss of hydraulic conductance (P(50), a proxy for cavitation resistance) varied widely among species, from -2.9 to -11.3 MPa. The valve effect of the pit membrane, measured as a function of margo flexibility and torus overlap, explained more variation in cavitation-resistance than simple anatomical traits such as pit membrane, pit aperture or torus size. Highly cavitation resistant species exhibited both a high flexibility of the margo and a large overlap between the torus and the pit aperture, allowing the torus to tightly seal the pit aperture. Our results support the hypothesis of seal capillary-seeding as the most likely mode of air-seeding, and suggest that the adhesion of the torus to the pit border may be the main determinant of cavitation resistance in conifers.