Drought-induced fiber water release and xylem embolism susceptibility of intact balsam poplar saplings.

Balsam poplar (Populus balsamifera L.) is a widespread tree species in North America with significant ecological and economic value. However, little is known about the susceptibility of saplings to drought-induced embolism and its link to water release from surrounding xylem fibers. Questions remain regarding localized mechanisms that contribute to the survival of saplings in vivo of this species under drought. Using X-ray micro-computed tomography on intact saplings of genotypes Gillam-5 and Carnduff-9, we found that functional vessels are embedded in a matrix of water-filled fibers under well-watered conditions in both genotypes. However, water-depleted fibers started to appear under moderate drought stress while vessels remained water-filled in both genotypes. Drought-induced xylem embolism susceptibility was comparable between genotypes, and a greater frequency of smaller diameter vessels in GIL-5 did not increase embolism resistance in this genotype. Despite having smaller vessels and a total vessel number that was comparable to CAR-9, stomatal conductance was generally higher in GIL-5 compared to CAR-9. In conclusion, our in vivo data on intact saplings indicate that differences in embolism susceptibility are negligible between GIL-5 and CAR-9, and that fiber water release should be considered as a mechanism that contributes to the maintenance of vessel functional status in saplings of balsam poplar experiencing their first drought event.

A workflow for segmenting soil and plant X-ray computed tomography images with deep learning in Google's Colaboratory.

X-ray micro-computed tomography (X-ray µCT) has enabled the characterization of the properties and processes that take place in plants and soils at the micron scale. Despite the widespread use of this advanced technique, major limitations in both hardware and software limit the speed and accuracy of image processing and data analysis. Recent advances in machine learning, specifically the application of convolutional neural networks to image analysis, have enabled rapid and accurate segmentation of image data. Yet, challenges remain in applying convolutional neural networks to the analysis of environmentally and agriculturally relevant images. Specifically, there is a disconnect between the computer scientists and engineers, who build these AI/ML tools, and the potential end users in agricultural research, who may be unsure of how to apply these tools in their work. Additionally, the computing resources required for training and applying deep learning models are unique, more common to computer gaming systems or graphics design work, than to traditional computational systems. To navigate these challenges, we developed a modular workflow for applying convolutional neural networks to X-ray µCT images, using low-cost resources in Google's Colaboratory web application. Here we present the results of the workflow, illustrating how parameters can be optimized to achieve best results using example scans from walnut leaves, almond flower buds, and a soil aggregate. We expect that this framework will accelerate the adoption and use of emerging deep learning techniques within the plant and soil sciences.

Structural and functional leaf diversity lead to variability in photosynthetic capacity across a range of Juglans regia genotypes.

Similar to other cropping systems, few walnut cultivars are used as scion in commercial production. Germplasm collections can be used to diversify cultivar options and hold potential for improving crop productivity, disease resistance and stress tolerance. In this study, we explored the anatomical and biochemical bases of photosynthetic capacity and response to water stress in 11 Juglans regia accessions in the U.S. department of agriculture, agricultural research service (USDA-ARS) National Clonal Germplasm. Net assimilation rate (An ) differed significantly among accessions and was greater in lower latitudes coincident with higher stomatal and mesophyll conductances, leaf thickness, mesophyll porosity, gas-phase diffusion, leaf nitrogen and lower leaf mass and stomatal density. High CO2 -saturated assimilation rates led to increases in An under diffusional and biochemical limitations. Greater An was found in lower-latitude accessions native to climates with more frost-free days, greater precipitation seasonality and lower temperature seasonality. As expected, water stress consistently impaired photosynthesis with the highest % reductions in lower-latitude accessions (A3, A5 and A9), which had the highest An under well-watered conditions. However, An for A3 and A5 remained among the highest under dehydration. J. regia accessions, which have leaf structural traits and biochemistry that enhance photosynthesis, could be used as commercial scions or breeding parents to enhance productivity.

Desiccation of the leaf mesophyll and its implications for CO2 diffusion and light processing.

Leaves balance CO2 and radiative absorption while maintaining water transport to maximise photosynthesis. Related species with contrasting leaf anatomy can provide insights into inherent and stress-induced links between structure and function for commonly measured leaf traits for important crops. We used two walnut species with contrasting mesophyll anatomy to evaluate these integrated exchange processes under non-stressed and drought conditions using a combination of light microscopy, X-ray microCT, gas exchange, hydraulic conductance, and chlorophyll distribution profiles through leaves. Juglans regia had thicker palisade mesophyll, higher fluorescence in the palisade, and greater low-mesophyll porosity that were associated with greater gas-phase diffusion (gIAS ), stomatal and mesophyll (gm ) conductances and carboxylation capacity. More and highly-packed mesophyll cells and bundle sheath extensions (BSEs) in Juglans microcarpa led to higher fluorescence in the spongy and in proximity to the BSEs. Both species exhibited drought-induced reductions in mesophyll cell volume, yet the associated increases in porosity and gIAS were obscured by declines in biochemical activity that decreased gm . Inherent differences in leaf anatomy between the species were linked to differences in gas exchange, light absorption and photosynthetic capacity, and drought-induced changes in leaf structure impacted performance via imposing species-specific limitations to light absorption, gas exchange and hydraulics.

A meta-analysis of mesophyll conductance to CO2 in relation to major abiotic stresses in poplar species.

Mesophyll conductance (gm) determines the diffusion of CO2 from the substomatal cavities to the site of carboxylation in the chloroplasts and represents a critical component of the diffusive limitation of photosynthesis. In this study, we evaluated the average effect sizes of different environmental constraints on gm in Populus spp., a forest tree model. We collected raw data of 815 A-Ci response curves from 26 datasets to estimate gm, using a single curve-fitting method to alleviate method-related bias. We performed a meta-analysis to assess the effects of different abiotic stresses on gm. We found a significant increase in gm from the bottom to the top of the canopy that was concomitant with the increase of maximum rate of carboxylation and light-saturated photosynthetic rate (Amax). gm was positively associated with increases in soil moisture and nutrient availability, but was insensitive to increasing soil copper concentration and did not vary with atmospheric CO2 concentration. Our results showed that gm was strongly related to Amax and to a lesser extent to stomatal conductance (gs). Moreover, a negative exponential relationship was obtained between gm and specific leaf area, which may be used to scale-up gm within the canopy.

Modification of a gas exchange system to measure active and passive chlorophyll fluorescence simultaneously under field conditions.

Solar-induced fluorescence (SIF) is a promising tool to estimate photosynthesis across scales; however, there has been limited research done at the leaf level to investigate the relationship between SIF and photosynthesis. To help bridge this gap, a LI-COR LI-6800 gas exchange instrument was modified with a visible-near-infrared (VIS-NIR) spectrometer to measure active and passive fluorescence simultaneously. The system was adapted by drilling a hole into the bottom plate of the leaf chamber and inserting a fibre-optic to measure passive steady-state fluorescence (F t , λ , analogous to SIF) from the abaxial surface of a leaf. This new modification can concurrently measure gas exchange, passive fluorescence and active fluorescence over the same leaf area and will allow researchers to measure leaf-level F t , λ in the field to validate tower-based and satellite measurements. To test the modified instrument, measurements were performed on leaves of well-watered and water-stressed walnut plants at three light levels and a constant air temperature. Measurements on these same plants were also conducted using a similarly modified Walz GFS-3000 gas exchange instrument to compare results. We found a positive linear correlation between F t , λ measurements from the modified LI-6800 and GFS-3000 instruments. We also report a positive linear relationship between F t , λ and normalized steady-state chlorophyll fluorescence (F t /F o ) from the pulse-amplitude modulation (PAM) fluorometer of the LI-6800 system. Accordingly, this modification will inform the link between spectrally resolved F t , λ and gas exchange-leading to improved interpretation of how remotely sensed SIF tracks changes in the light reactions of photosynthesis.

Emerging roles for carbonic anhydrase in mesophyll conductance and photosynthesis.

Carbonic anhydrase (CA) is an abundant protein in most photosynthesizing organisms and higher plants. This review paper considers the physiological importance of the more abundant CA isoforms in photosynthesis, through their effects on CO2 diffusion and other processes in photosynthetic organisms. In plants, CA has multiple isoforms in three different families (α, ß and γ) and is mainly known to catalyze the CO2↔HCO3- equilibrium. This reversible conversion has a clear role in photosynthesis, primarily through sustaining the CO2 concentration at the site of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Despite showing the same major reaction mechanism, the three main CA families are evolutionarily distinct. For different CA isoforms, cellular localization and total gene expression as a function of developmental stage are predicted to determine the role of each family in relation to the net assimilation rate. Reaction-diffusion modeling and observational evidence support a role for CA activity in reducing resistance to CO2 diffusion inside mesophyll cells by facilitating CO2 transfer in both gas and liquid phases. In addition, physical and/or biochemical interactions between CAs and other membrane-bound compartments, for example aquaporins, are suggested to trigger a CO2 -sensing response by stomatal movement. In response to environmental stresses, changes in the expression level of CAs and/or stimulated deactivation of CAs may correspond with lower photosynthetic capacity. We suggest that further studies should focus on the dynamics of the relationship between the activity of CAs (with different subcellular localization, abundance and gene expression) and limitations due to CO2 diffusivity through the mesophyll and supply of CO2 to photosynthetic reactions.

Blue light differentially represses mesophyll conductance in high vs low latitude genotypes of Populus trichocarpa Torr. & Gray.

To explore what role chloroplast positioning might have in relation to latitudinal variation in mesophyll conductance (gm) of Populus trichocarpa Torr. & Gray (black cottonwood), we examined photosynthetic response to different blue light treatments in six representative genotypes (three northern and three southern). The proportion of blue (B) to red light was varied from 0:100, 10:90, 20:80, 40:60, and 60:40 while keeping the total photosynthetic photon flux density constant. Mesophyll conductance was estimated by monitoring chlorophyll fluorescence in combination with gas exchange. Compared to the control (10% B), gm was significantly lower with increasing blue light. Consistent with a change in chloroplast positioning, there was a simultaneous but reversible decrease in chlorophyll content index (CCI), as measured by foliar greenness, while the extracted, actual chlorophyll content (ACC) remained unchanged. Blue-light-induced decreases in gm and CCI were greater in northern genotypes than in southern genotypes, both absolutely and proportionally, consistent with their inherently higher photosynthetic rate. Treatment of leaves with cytochalasin D, an inhibitor of actin-based chloroplast motility, reduced both CCI and ACC but had no effect on the CCI/ACC ratio and fully blocked any effect of blue light on CCI. Cytochalasin D reduced gm by ∼56% under 10% B, but did not block the effect of 60% B on gm, which was reduced a further 20%. These results suggest that the effect of high blue light on gm is at least partially independent of chloroplast repositioning. High blue light reduced carbonic anhydrase activity by 20% (P<0.05), consistent with a possible reduction in protein-mediated facilitation of CO2 diffusion.

Substantial role for carbonic anhydrase in latitudinal variation in mesophyll conductance of Populus trichocarpa Torr. & Gray.

In Populus trichocarpa (black cottonwood), net photosynthesis (An ) varies with latitude and, in northern genotypes, is supported by higher stomatal conductance (gs ). We report here a parallel cline in mesophyll conductance (gm ) and link this variation to carbonic anhydrase (CA) activity. Using concurrent carbon isotope discrimination and chlorophyll fluorescence methods, we examined the effects of acetazolamide, an inhibitor of CA, on gm in six representative genotypes (three from either end of the north-south cline). Acetazolamide reduced CA activity, gm , gs , chloroplast CO2 concentration (Cc ) and An at normal CO2 (400 µmol mol-1 ), the latter being reversible at saturating CO2 . Absolute reductions in An , gm and CA activity were greater in northern genotypes than in southern genotypes (P < 0.025) but percent reductions were similar. In contrast, northern genotypes showed lower percent reduction in Cc compared to southern genotypes (P < 0.025). The northern genotypes had greater CA activity relative to both leaf area (two-fold) and mass (1.8-fold) (P < 0.016). The relationship between CA activity and gm was similar whether the variation was inherent or inhibitor induced. We suggest that greater CA activity contributes to higher gm in northern P. trichocarpa genotypes, but other diffusion pathway components may also be involved.