Leaf water stable isotopes and water transport outside the xylem.

How water moves through leaves, and where the phase change from liquid to vapour occurs within leaves, remain largely mysterious. Some time ago, we suggested that the stable isotope composition of leaf water may contain information on transport pathways beyond the xylem, through differences in the development of gradients in enrichment within the various pathways. Subsequent testing of this suggestion provided ambiguous results and even questioned the existence of gradients in enrichment within the mesophyll. In this review, we bring together recent theoretical developments in understanding leaf water transport pathways and stable isotope theory to map a path for future work into understanding pathways of water transport and leaf water stable isotope composition. We emphasize the need for a spatially, anatomically and isotopically explicit model of leaf water transport.

An Improved System to Measure Leaf Gas Exchange on Adaxial and Abaxial Surfaces.

Measurement of leaf carbon gain and water loss (gas exchange) in planta is a standard procedure in plant science research for attempting to understand physiological traits related to water use and photosynthesis. Leaves carry out gas exchange through the upper (adaxial) and lower (abaxial) surfaces at different magnitudes, depending on the stomatal density, stomatal aperture, cuticular permeability, etc., of each surface, which we account for in gas exchange parameters such as stomatal conductance. Most commercial devices measure leaf gas exchange by combining the adaxial and abaxial fluxes and calculating bulk gas exchange parameters, missing details of the plant's physiological response on each side. Additionally, the widely used equations to estimate gas exchange parameters neglect the contribution of small fluxes such as cuticular conductance, adding extra uncertainties to measurements performed in water-stress or low-light conditions. Accounting for the gas exchange fluxes from each side of the leaf allows us to better describe plants' physiological traits under different environmental conditions and account for genetic variability. Here, apparatus and materials are presented for adapting two LI-6800 Portable Photosynthesis Systems to work as one gas exchange system to measure adaxial and abaxial gas exchange simultaneously. The modification includes a template script with the equations to account for small fluxes. Instructions are provided for incorporating the add-on script into the device's computational sequence, display, variables, and spreadsheet results. We explain the method to obtain an equation to estimate boundary layer conductance to water for the new setup and how to embed this equation in the devices' calculations using the provided add-on script. The apparatus, methods, and protocols presented here provide a simple adaptation combining two LI-6800s to obtain an improved system to measure leaf gas exchange on adaxial and abaxial surfaces. Graphical overview Figure 1.Diagram of the connection of two LI-6800s.Figure adapted from Márquez et al. (2021).

Dynamics of moisture diffusion and adsorption in plant cuticles including the role of cellulose.

Food production must increase significantly to sustain a growing global population. Reducing plant water loss may help achieve this goal and is especially relevant in a time of climate change. The plant cuticle defends leaves against drought, and so understanding water movement through the cuticle could help future proof our crops and better understand native ecology. Here, via mathematical modelling, we identify mechanistic properties of water movement in cuticles. We model water sorption in astomatous isolated cuticles, utilising three separate pathways of cellulose, aqueous pores and lipophilic. The model compares well to data both over time and humidity gradients. Sensitivity analysis shows that the grouping of parameters influencing plant species variations has the largest effect on sorption, those influencing cellulose are very influential, and aqueous pores less so but still relevant. Cellulose plays a significant role in diffusion and adsorption in the cuticle and the cuticle surfaces.

Ammonia volatilization from senescing leaves of maize.

Ammonia release by plants growing in normal air is reported. Contrary to observations made at high ambient ammonia concentrations, corn plants did not absorb ammonia in normal air but released it as they senesced, even while photosynthesizing actively.

Xylem-tapping mistletoes: water or nutrient parasites?

Most mistletoes parasitize higher plants by tapping the xylem (a conduction tissue) of their hosts. Field observations of diurnal gas exchange parameters and carbon isotope ratios in xylem-tapping mistletoes from three continents support the hypotheses that water use efficiency and carbon isotope composition are related and that mistletoes which are parasitic for water are also nutrient parasites, differing in their water use efficiency relative to that of their hosts on the basis of host nitrogen supply in the transpiration stream.

Macromolecular crowding and its influence on possible reaction mechanisms in photosynthetic electron flow.

The diffusion of plastoquinol and its binding to the Qo site of the cyt bf complex in the course of photosynthetic electron transport was studied by following the sigmoidal flash-induced re-reduction kinetics of P700 after previous oxidation of the intersystem electron carriers. The data resulting from these experiments were matched with a simulation of electron transport using Monte Carlo techniques. The simulation was able to account for the experimental observations. Two different extreme cases of reaction mechanism at the Qo site were compared: a diffusion limited collisional mechanism and a non-diffusion limited tight binding mechanism. Assuming a tight binding mechanism led to best matches due to the high protein density in thylakoids. The varied parameters resulted in values well within the range of published data. The results emphasise the importance of structural characteristics of thylakoids in models of electron transport.

Dependence of plastoquinol diffusion on the shape, size, and density of integral thylakoid proteins.

The diffusion of plastoquinol in the chloroplast thylakoid membrane is modelled using Monte Carlo techniques. The integral proteins are seen as obstacles to diffusion, and features of percolation theory emerge. Thus, the diffusion coefficient diminishes with increasing distance and there is a critical threshold of protein concentration, above which the long-range diffusion coefficient is zero. The area occupied by proteins in vivo is assessed and appears to be around this threshold, as determined from calculations assuming randomly distributed noninteracting proteins. Slight changes in the protein arrangement lead to pronounced changes in diffusion behaviour under such conditions. Mobility of the proteins increases the protein occupancy threshold, while boundary lipids impermeable to PQ diffusion decrease it. Further, the obstruction of plastoquinone/plastoquinol binding sites in a random arrangement is evaluated.

Reduction of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase by Antisense RNA in the C4 Plant Flaveria bidentis Leads to Reduced Assimilation Rates and Increased Carbon Isotope Discrimination.

Transgenic Flaveria bidentis (a C4 species) plants with an antisense gene directed against the mRNA of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) were used to examine the relationship between the CO2 assimilation rate, Rubisco content, and carbon isotope discrimination. Reduction in the amount of Rubisco in the transgenic plants resulted in reduced CO2 assimilation rates and increased carbon isotope discrimination of leaf dry matter. The H2O exchange was similar in transgenic and wild-type plants, resulting in higher ratios of intercellular to ambient CO2 partial pressures. Carbon isotope discrimination was measured concurrently with CO2 and H2O exchange on leaves of the control plants and T1 progeny with a 40% reduction in Rubisco. From the theory of carbon isotope discrimination in the C4 species, we conclude that the reduction in the Rubisco content in the transgenic plants has led to an increase in bundle-sheath CO2 concentration and CO2 leakage from the bundle sheath; however, some down-regulation of the C4 cycle also occurred.

CO2 and Water Vapor Exchange across Leaf Cuticle (Epidermis) at Various Water Potentials.

Cuticular properties affect the gas exchange of leaves, but little is known about how much CO2 and water vapor cross the cuticular barrier or whether low water potentials affect the process. Therefore, we measured the cuticular conductances for CO2 and water vapor in grape (Vitis vinifera L.) leaves having various water potentials. The lower leaf surface was sealed to force all gas exchange through the upper surface, which was stoma-free. In this condition both gases passed through the cuticle, and the CO2 conductance could be directly determined from the internal mole fraction of CO2 near the compensation point, the external mole fraction of CO2, and the CO2 flux. The cuticle allowed small amounts of CO2 and water vapor to pass through, indicating that gas exchange occurs in grape leaves no matter how tightly the stomata are closed. However, the CO2 conductance was only 5.7% of that for water vapor. This discrimination against CO2 markedly affected calculations of the mole fraction of CO2 in leaves as stomatal apertures decreased. When the leaf dehydrated, the cuticular conductance to water vapor decreased, and transpiration and assimilation diminished. This dehydration effect was largest when turgor decreased, which suggests that cuticular gas exchange may have been influenced by epidermal stretching.

"Rolled-upness": phenotyping leaf rolling in cereals using computer vision and functional data analysis approaches.

BACKGROUND: The flag leaf of a wheat (Triticum aestivum L.) plant rolls up into a cylinder in response to drought conditions and then unrolls when leaf water relations improve. This is a desirable trait for extending leaf area duration and improving grain size particularly under drought. But how do we quantify this phenotype so that different varieties of wheat or different treatments can be compared objectively since this phenotype can easily be confounded with inter-genotypic differences in root-water uptake and/or transpiration at the leaf level if using traditional methods? RESULTS: We present a new method to objectively test a range of lines/varieties/treatments for their propensity of leaves to roll. We have designed a repeatable protocol and defined an objective measure of leaf curvature called "rolled-upness" which minimises confounding factors in the assessment of leaf rolling in grass species. We induced leaf rolling by immersing leaf strips in an osmoticum of known osmotic pressure. Using micro-photographs of individual leaf cross-sections at equilibrium in the osmoticum, two approaches were used to quantify leaf rolling. The first was to use some properties of the convex hull of the leaf cross-section. The second was to use cubic smoothing splines to approximate the transverse leaf shape mathematically and then use a statistic derived from the splines for comparison. Both approaches resulted in objective measurements that could differentiate clearly between breeding lines and varieties contrasting genetically in their propensity for leaf rolling under water stress. The spline approach distinguished between upward and downward curvature and allowed detailed properties of the rolling to be examined, such as the position on the strip where maximum curvature occurs. CONCLUSIONS: A method applying smoothing splines to skeletonised images of transverse wheat leaf sections enabled objective measurements of inter-genotypic variation for hydronastic leaf rolling in wheat. Mean-curvature of the leaf cross-section was the measure selected to discriminate between genotypes, as it was straightforward to calculate and easily construed. The method has broad applicability and provides an avenue to genetically dissect the trait in cereals.

On the progressive enrichment of the oxygen isotopic composition of water along a leaf.

A model has been derived for the enrichment of heavy isotopes of water in leaves, including progressive enrichment along the leaf. In the model, lighter water is preferentially transpired leaving heavier water to diffuse back into the xylem and be carried further along the leaf. For this pattern to be pronounced, the ratio of advection to diffusion (Péclet number) has to be large in the longitudinal direction, and small in the radial direction. The progressive enrichment along the xylem is less than that occurring at the sites of evaporation in the mesophyll, depending on the isolation afforded by the radial Péclet number. There is an upper bound on enrichment, and effects of ground tissue associated with major veins are included. When transpiration rate is spatially nonuniform, averaging of enrichment occurs more naturally with transpiration weighting than with area-based weighting. This gives zero average enrichment of transpired water, the modified Craig-Gordon equation for average enrichment at the sites of evaporation and the Farquhar and Lloyd (In Stable Isotopes and Plant Carbon-Water Relations, pp. 47-70. Academic Press, New York, USA, 1993) prediction for mesophyll water. Earlier results on the isotopic composition of evolved oxygen and of retro-diffused carbon dioxide are preserved if these processes vary in parallel with transpiration rate. Parallel variation should be indicated approximately by uniform carbon isotope discrimination across the leaf.

A global survey of carbon isotope discrimination in plants from high altitude.

Carbon 13/12 isotope ratios have been determined from leaves of a hundred C3 plant species (or ecotypes) from all major mountain ranges of the globe, avoiding drought stressed areas. A general increase in 13C content was found with increasing altitude, i.e. overall discrimination against the heavy isotope is reduced at high elevation. The steepest decline of discrimination is observed in taxa typically ranging to highest elevations (e.g. the genus Ranunculus). Mean δ 13C for all samples collected between 2500 and 5600 m altitude is-26.15‰ compared to the lowland average of-28.80‰ (P<0.001). Forbs from highest elevations reach-24‰. According to theory of 13C discrimination this indicates decreasing relative limitation of carbon uptake by carboxylation. In other words, we estimate that the ratio of internal to external partial pressure of CO2 (p i /p a )in leaves of high elevation plants is lower than in leaves of low altitude. These results confirm recent gas exchange analyses in high and low elevation plants.

Carbon isotope discrimination by plants follows latitudinal and altitudinal trends.

In an earlier paper we provided evidence that carbon isotope discrimination during photosynthesis of terrestrial C3 plants decreases with altitude, and it was found that this was associated with greater carboxylation efficiency at high altitudes. Changing partial pressures of CO2 and O2 and changing temperature are possible explanations, since influences of moisture and light were reduced to a minimum by selective sampling. Here we analyse plants sampled using the same criteria, but from high and low altitudes along latitudinal gradients from the equator to the polar ends of plant distribution. These data should permit separation of the pressure and temperature components (Fig. 1). Only leaves of fully sunlit, non-water-stressed, herbaceous C3 plants are compared. The survey covers pressure differences of 400 mbar (ca. 5000 m) and 78 degrees of latitude (ca 25 K of mean temperature of growth period). When habitats of similar low temperature (i.e. high altitude at low latitude and low altitude at polar latitude) are compared, discrimination increases towards the pole (with decreasing altitude and thus increasing atmospheric pressure). Latitudinally decreasing temperature at almost constant atmospheric pressure (samples from low altitude) is associated with a decrease in discrimination. So, polar low-altitude plants have δ13C values half way between humid tropical lowland and tropical alpine plants. It is unlikely that latitudinal changes of the light regime had an effect, since low and high altitude plants show contrasting latitudinal trends in δ13C although local altitudinal differences in overall light consumption were small. These results suggest that both temperature and atmospheric pressure are responsible for the altitudinal trends in 13C discrimination. Temperature effects may partly be related to increased leaf thickness (within the same leaf type) in cold environments. Theoretical considerations and laboratory experiments suggest that it is the oxygen partial pressure that is responsible for the pressure related change in discrimination. The study also provided results of practical significance for the use of carbon isotope data. Within a community of C3 plants, discrimination in species of similar life form, exposed to similar light, water and ambient CO2 conditions ranges over 4‰, with standard deviations for 10-30 species of ±0.6 to 1.2‰. This natural variation has to be taken into account by using a sufficient sample size and standardization of sampling in any attempt at ecological site characterization using carbon isotope data. Evidence of a pronounced genotypic component of this variation in 13C discrimination in wild C3 plant species is provided. Correlations with dry matter partitioning, mesophyll thickness and nitrogen content are also present.

Effect of salinity and humidity on δ13C value of halophytes-Evidence for diffusional isotope fractionation determined by the ratio of intercellular/atmospheric partial pressure of CO2 under different environmental conditions.

Seedlings of two mangrove species, Avicennia marina and Aegiceras corniculatum, were grown in a range of salinities and humidities in controlled environment chambers, and Phaseolus vulgaris plants were grown in the glasshouse. The fractionation of carbon isotopes in the three species was correlated with the ratio of intercellular and ambient partial pressures of CO2. The results are consistent with fractionation being due both to diffusion in air and to carboxylation in the leaf. It was concluded that the latter process discriminates against 13CO2 relative to 12CO2 by about 27‰.

Carbon fixation in eucalypts in the field : Analysis of diurnal variations in photosynthetic capacity.

The rate of CO2 assimilation at light saturation and an intercellular CO2 concentration of 350 µl l-1 (photosynthetic capacity), measured in leaves of Eucalyptus pauciflora, E. behriana, E. delegatensis and Acacia melanoxylon, declined over the course of cloudless days under naturally varying environmental conditions as well as under constant optimal conditions for high CO2 uptake. Since the capacity did not recover during the light period, it was different from the "midday depression" of gas exchange. The change appeared to be caused neither by the diurnal variation of total leaf water potential, by photoinhibition of redox-reaction centres in photosystems nor by changes in the intrinsic properties of Ribulose-bisphosphate carboxylase-oxygenase. The decline was more pronounced in winter than in summer. It was related to the duration of illumination or the cumulative carbon gain. It was reversible in the following dark phase, and it did not occur on changeable days with short peaks of high light.Despite the decline in photosynthetic capacity, the initial slope of the CO2 response of net photosynthesis, as obtained at low intercellular CO2 concentrations, remained constant during the day, but declined at night when photosynthetic capacity recovered. In all cases stomatal conductance varied in parallel with photosynthetic capacity. The relevance of changes in photosynthetic capacity for the intercellular CO2 concentration is discussed.

Mistletoes: a hypothesis concerning morphological and chemical avoidance of herbivory.

Leaves from many misletoe species in Australia strongly resemble those of their hosts. This cryptic mimicry has been hypothesized to be a means of reducing the likelihood of mistletoe herbivory by vertebrates. Leaf Kjeldahl nitrogen contents (a measure of reduced nitrogen and thus amines, amino acids and protein levels) of mistletoes and their hosts were measured on 48 mimetic and nonmimetic host-parasite pairs to evaluate hypotheses concerning the significance of crysis versus noncrypsis. The hypothesis that mistletoes mimicking host leaves should have higher leaf nitrogen levels than their hosts is supported; they may be gaining a selective advantage through crypsis (reduced herbivory). The second hypothesis that mistletoes which do not mimic their hosts should have lower leaf nitrogen levels than their hosts is also supported; they may be gaining a selective advantage through noncrypsis (reduced herbivory resulting from visual advertisement of their reduced nutritional status).

Variation in branchlet delta13C in relation to branchlet nitrogen concentration and growth in 8-year-old hoop pine families (Araucaria cunninghamii) in subtropical Australia.

Carbon isotope composition (delta13C) of branchlet tissue at nine canopy positions, and nitrogen concentration (N(mass)) at four canopy positions, were assessed in 8-year-old hoop pine (Araucaria cunninghamii Ait. ex D. Don) trees from 23 half-sib families, grown in six blocks of a progeny test in southeastern Queensland, Australia. There was considerable variation among sampling positions, families and blocks in both delta13C and N(mass). The delta13C was positively related to N(mass) only for samples from the upper outer crown (P < 0.005). Phenotypic correlations existed between tree growth and canopy delta13C. Branchlet delta13C of the inner and lower outer crown was positively related (P < 0.037) to tree height, but delta13C in branchlets of the upper outer crown was not related to tree height, or was related negatively (P < 0.045). There were significant differences in delta13C between hoop pine families for six canopy positions (upper canopy positions as well as lower canopy positions on the northern side), with heritabilities greater than 0.40. The significance of these findings is discussed in relation to water and light competition within the tree canopy of hoop pine.

Quantitative trait loci for carbon isotope discrimination are repeatable across environments and wheat mapping populations.

Wheat productivity is commonly limited by a lack of water essential for growth. Carbon isotope discrimination (Delta), through its negative relationship with transpiration efficiency, has been used in selection of higher wheat yields in breeding for rainfed environments. The potential also exists for selection of increased Delta for improved adaptation to irrigated and high rainfall environments. Selection efficiency of Delta would be enhanced with a better understanding of its genetic control. Three wheat mapping populations (Cranbrook/Halberd, Sunco/Tasman and CD87/Katepwa) containing between 161 and 190 F(1)-derived, doubled-haploid progeny were phenotyped for Delta and agronomic traits in 3-5 well-watered environments. The range for Delta was large among progeny (c. 1.2-2.3 per thousand), contributing to moderate-to-high single environment (h (2) = 0.37-0.91) and line-mean (0.63-0.86) heritabilities. Transgressive segregation was large and genetic control complex with between 9 and 13 Delta quantitative trait loci (QTL) identified in each cross. The Delta QTL effects were commonly small, accounting for a modest 1-10% of the total additive genetic variance, while a number of chromosomal regions appeared in two or more populations (e.g. 1BL, 2BS, 3BS, 4AS, 4BS, 5AS, 7AS and 7BS). Some of the Delta genomic regions were associated with variation in heading date (e.g. 2DS, 4AS and 7AL) and/or plant height (e.g. 1BL, 4BS and 4DS) to confound genotypic associations between Delta and grain yield. As a group, high Delta progeny were significantly (P < 0.10-0.01) taller and flowered earlier but produced more biomass and grain yield in favorable environments. After removing the effect of height and heading date, strong genotypic correlations were observed for Delta and both yield and biomass across populations (r (g) = 0.29-0.57, P < 0.05) as might be expected for the favorable experimental conditions. Thus selection for Delta appears beneficial in increasing grain yield and biomass in favorable environments. However, care must be taken to avoid confounding genotypic differences in Delta with stature and development time when selecting for improved biomass and yield especially in environments experiencing terminal droughts. Polygenic control and small size of individual QTL for Delta may reduce the potential for QTL in marker-assisted selection for improved yield of wheat.

The influence of protein-protein interactions on the organization of proteins within thylakoid membranes.

The influence of attractive protein-protein interactions on the organization of photosynthetic proteins within the thylakoid membrane was investigated. Protein-protein interactions were simulated using Monte Carlo techniques and the influence of different interaction energies was examined. It was found that weak interactions led to protein clusters whereas strong interactions led to ramified chains. An optimum curve for the relationship between interaction energy and the number of contact sites emerged. With increasing particle densities the effect decreased. In a mixture of interacting and noninteracting particles the distance between the noninteracting particles was increased and there seemed to be much more free space around them. In thylakoids, this could lead to a more homogeneous distribution of the noninteracting but rate-limiting cytochrome bf complexes. Due to the increased free space between cytochrome bf, obstruction of binding sites--occurring unavoidably in a random distribution--may be drastically reduced. Furthermore, protein-protein interactions in thylakoids may lead to a decrease in plastoquinone diffusion.