Measuring stomatal and guard cell metrics for plant physiology and growth using StoManager1.

Automated guard cell detection and measurement are vital for understanding plant physiological performance and ecological functioning in global water and carbon cycles. Most current methods for measuring guard cells and stomata are laborious, time-consuming, prone to bias, and limited in scale. We developed StoManager1, a high-throughput tool utilizing geometrical, mathematical algorithms, and convolutional neural networks to automatically detect, count, and measure over 30 guard cell and stomatal metrics, including guard cell and stomatal area, length, width, stomatal aperture area/guard cell area, orientation, stomatal evenness, divergence, and aggregation index. Combined with leaf functional traits, some of these StoManager1-measured guard cell and stomatal metrics explained 90% and 82% of tree biomass and intrinsic water use efficiency (iWUE) variances in hardwoods, making them substantial factors in leaf physiology and tree growth. StoManager1 demonstrated exceptional precision and recall (mAP@0.5 over 0.96), effectively capturing diverse stomatal properties across over 100 species. StoManager1 facilitates the automation of measuring leaf stomatal and guard cells, enabling broader exploration of stomatal control in plant growth and adaptation to environmental stress and climate change. This has implications for global gross primary productivity (GPP) modeling and estimation, as integrating stomatal metrics can enhance predictions of plant growth and resource usage worldwide. Easily accessible open-source code and standalone Windows executable applications are available on a GitHub repository (https://github.com/JiaxinWang123/StoManager1) and Zenodo (https://doi.org/10.5281/zenodo.7686022).

Labeled temperate hardwood tree stomatal image datasets from seven taxa of Populus and 17 hardwood species.

Machine learning (ML) algorithms have shown potential in automatically detecting and measuring stomata. However, ML algorithms require substantial data to efficiently train and optimize models, but their potential is restricted by the limited availability and quality of stomatal images. To overcome this obstacle, we have compiled a collection of around 11,000 unique images of temperate broadleaf angiosperm tree leaf stomata from various projects conducted between 2015 and 2022. The dataset includes over 7,000 images of 17 commonly encountered hardwood species, such as oak, maple, ash, elm, and hickory, and over 3,000 images of 55 genotypes from seven Populus taxa. Inner_guard_cell_walls and whole_stomata (stomatal aperture and guard cells) were labeled and had a corresponding YOLO label file that can be converted into other annotation formats. With the use of our dataset, users can (1) employ state-of-the-art machine learning models to identify, count, and quantify leaf stomata; (2) explore the diverse range of stomatal characteristics across different types of hardwood trees; and (3) develop new indices for measuring stomata.

Using hyperspectral leaf reflectance to estimate photosynthetic capacity and nitrogen content across eastern cottonwood and hybrid poplar taxa.

Eastern cottonwood (Populus deltoides W. Bartram ex Marshall) and hybrid poplars are well-known bioenergy crops. With advances in tree breeding, it is increasingly necessary to find economical ways to identify high-performing Populus genotypes that can be planted under different environmental conditions. Photosynthesis and leaf nitrogen content are critical parameters for plant growth, however, measuring them is an expensive and time-consuming process. Instead, these parameters can be quickly estimated from hyperspectral leaf reflectance if robust statistical models can be developed. To this end, we measured photosynthetic capacity parameters (Rubisco-limited carboxylation rate (Vcmax), electron transport-limited carboxylation rate (Jmax), and triose phosphate utilization-limited carboxylation rate (TPU)), nitrogen per unit leaf area (Narea), and leaf reflectance of seven taxa and 62 genotypes of Populus from two study plantations in Mississippi. For statistical modeling, we used least absolute shrinkage and selection operator (LASSO) and principal component analysis (PCA). Our results showed that the predictive ability of LASSO and PCA models was comparable, except for Narea in which LASSO was superior. In terms of model interpretability, LASSO outperformed PCA because the LASSO models needed 2 to 4 spectral reflectance wavelengths to estimate parameters. The LASSO models used reflectance values at 758 and 935 nm for estimating Vcmax (R2 = 0.51 and RMSPE = 31%) and Jmax (R2 = 0.54 and RMSPE = 32%); 687, 746, and 757 nm for estimating TPU (R2 = 0.56 and RMSPE = 31%); and 304, 712, 921, and 1021 nm for estimating Narea (R2 = 0.29 and RMSPE = 21%). The PCA model also identified 935 nm as a significant wavelength for estimating Vcmax and Jmax. Therefore, our results suggest that hyperspectral leaf reflectance modeling can be used as a cost-effective means for field phenotyping and rapid screening of Populus genotypes because of its capacity to estimate these physicochemical parameters.

Impacts of prescribed fire on Pinus rigida Mill. in upland forests of the Atlantic Coastal Plain.

A comparative analysis of the impacts of prescribed fire on three upland forest stands in the Northeastern Atlantic Plain, NJ, USA, was conducted. Effects of prescribed fire on water use and gas exchange of overstory pines were estimated via sap-flux rates and photosynthetic measurements on Pinus rigida Mill. Each study site had two sap-flux plots, one experiencing prescribed fire and one control (unburned) plot for comparison before and after the fire. We found that photosynthetic capacity in terms of Rubisco-limited carboxylation rate and intrinsic water-use efficiency was unaffected, while light compensation point and dark respiration rate were significantly lower in the burned vs control plots post-fire. Furthermore, quantum yield in pines in the pine-dominated stands was less affected than pines in the mixed oak/pine stand, as there was an increase in quantum yield in the oak/pine stand post-fire compared with the control (unburned) plot. We attribute this to an effect of forest type but not fire per se. Average daily sap-flux rates of the pine trees increased compared with control (unburned) plots in pine-dominated stands and decreased in the oak/pine stand compared with control (unburned) plots, potentially due to differences in fuel consumption and pre-fire sap-flux rates. Finally, when reference canopy stomatal conductance was analyzed, pines in the pine-dominated stands were more sensitive to changes in vapor pressure deficit (VPD), while stomatal responses of pines in the oak/pine stand were less affected by VPD. Therefore, prescribed fire affects physiological functioning and water use of pines, but the effects may be modulated by forest stand type and fuel consumption pattern, which suggests that these factors may need to be taken into account for forest management in fire-dominated systems.

Resource use and efficiency, and stomatal responses to environmental drivers of oak and pine species in an Atlantic Coastal Plain forest.

Pine-oak ecosystems are globally distributed even though differences in anatomy and leaf habit between many co-occurring oaks and pines suggest different strategies for resource use, efficiency and stomatal behavior. The New Jersey Pinelands contain sandy soils with low water- and nutrient-holding capacity providing an opportunity to examine trade-offs in resource uptake and efficiency. Therefore, we compared resource use in terms of transpiration rates and leaf nitrogen content and resource-use efficiency including water-use efficiency (WUE) via gas exchange and leaf carbon isotopes and photosynthetic nitrogen-use efficiency (PNUE) between oaks (Quercus alba, Q. prinus, Q. velutina) and pines (Pinus rigida, P. echinata). We also determined environmental drivers [vapor pressure deficit (VPD), soil moisture, solar radiation] of canopy stomatal conductance (GS) estimated via sap flow and stomatal sensitivity to light and soil moisture. Net assimilation rates were similar between genera, but oak leaves used about 10% more water and pine foliage contained about 20% more N per unit leaf area. Therefore, oaks exhibited greater PNUE while pines had higher WUE based on gas exchange, although WUE from carbon isotopes was not significantly different. For the environmental drivers of GS, oaks had about 10% lower stomatal sensitivity to VPD normalized by reference stomatal conductance compared with pines. Pines exhibited a significant positive relationship between shallow soil moisture and GS, but only GS in Q. velutina was positively related to soil moisture. In contrast, stomatal sensitivity to VPD was significantly related to solar radiation in all oak species but only pines at one site. Therefore, oaks rely more heavily on groundwater resources but have lower WUE, while pines have larger leaf areas and nitrogen acquisition but lower PNUE demonstrating a trade-off between using water and nitrogen efficiently in a resource-limited ecosystem.

Forest response and recovery following disturbance in upland forests of the Atlantic Coastal Plain.

Carbon and water cycling of forests contribute significantly to the Earth's overall biogeochemical cycling and may be affected by disturbance and climate change. As a larger body of research becomes available about leaf-level, ecosystem and regional scale effects of disturbances on forest ecosystems, a more mechanistic understanding is developing which can improve modeling efforts. Here, we summarize some of the major effects of physical and biogenic disturbances, such as drought, prescribed fire, and insect defoliation, on leaf and ecosystem-scale physiological responses as well as impacts on carbon and water cycling in an Atlantic Coastal Plain upland oak/pine and upland pine forest. During drought, stomatal conductance and canopy stomatal conductance were reduced, however, defoliation increased conductance on both leaf-level and canopy scale. Furthermore, after prescribed fire, leaf-level stomatal conductance was unchanged for pines but decreased for oaks, while canopy stomatal conductance decreased temporarily, but then rebounded the following growing season, thus exhibiting transient responses. This study suggests that forest response to disturbance varies from the leaf to ecosystem level as well as species level and thus, these differential responses interplay to determine the fate of forest structure and functioning post disturbance.

Physiological strategies of co-occurring oaks in a water- and nutrient-limited ecosystem.

Oak species are well suited to water-limited conditions by either avoiding water stress through deep rooting or tolerating water stress through tight stomatal control. In co-occurring species where resources are limited, species may either partition resources in space and/or time or exhibit differing efficiencies in the use of limited resources. Therefore, this study seeks to determine whether two co-occurring oak species (Quercus prinus L. and Quercus velutina Lam.) differ in physiological parameters including photosynthesis, stomatal conductance, water-use (WUE) and nitrogen-use efficiency (NUE), as well as to characterize transpiration and average canopy stomatal responses to climatic variables in a sandy, well-drained and nutrient-limited ecosystem. The study was conducted in the New Jersey Pinelands and we measured sap flux over a 3-year period, as well as leaf gas exchange, leaf nitrogen and carbon isotope concentrations. Both oak species showed relatively steep increases in leaf-specific transpiration at low vapor pressure deficit (VPD) values before maximum transpiration rates were achieved, which were sustained over a broad range in VPD. This suggests tight stomatal control over transpiration in both species, although Q. velutina showed significantly higher leaf-level and canopy-level stomatal conductance than Q. prinus. Average daytime stomatal conductance was positively correlated with soil moisture and both oak species maintained at least 75% of their maximum canopy stomatal conductance at soil moistures in the upper soil layer (0-0.3 m) as low as 0.03 m(3) m(3)(-3). Quercus velutina had significantly higher photosynthetic rates, maximum Rubisco-limited and electron-transport-limited carboxylation rates, dark respiration rates and nitrogen concentration per unit leaf area than Q. prinus. However, both species exhibited similar WUEs and NUEs. Therefore, Q. prinus has a more conservative resource-use strategy, while Q. velutina may need to exploit niches that are locally higher in nutrients and water. Likewise, both species appear to tap deep, stable water sources, highlighting the importance of rooting depth in modeling transpiration and stomatal conductance in many oak ecosystems.

Secondary stem lengthening in palms: response to commentary by Tomlinson and Quinn.

In this response, we address the criticisms put forth by Tomlinson and Quinn (American Journal of Botany 100: 461-464) about our original publication on secondary stem lengthening in Iriartea deltoidea palms (American Journal of Botany 99: 607-613) and find areas on which we may agree. We address our figure of a typical palm vascular bundle; the location, timing, and species where secondary lengthening would likely occur; and our measurement of internodes in various palms as well as our choice of individuals. Our original observations were a novel finding in the field of palm biology, and we invite more research and investigation on this subject.

A comparison of the hydraulic efficiency of a palm species (Iriartea deltoidea) with other wood types.

Palms are an important component of tropical ecosystems, living alongside dicotyledonous trees, even though they have a very different growth pattern and vascular system. As monocots, vessels in palms are located within vascular bundles and, without a vascular cambium that many dicotyledonous trees possess, palms cannot add additional vessels to their vascular system as they get older and taller. This means that hydraulic architecture in palms is more predetermined, which may require a highly efficient hydraulic system. This preset nature, along with the decoupling of hydraulic and mechanical functioning to different cell types, may allow palms to have a more efficient hydraulic system than dicotyledonous trees. Therefore, this study seeks to determine the efficiency of the hydraulic system in the palm Iriartea deltoidea (Ruiz & Pav.) and compare this efficiency with other tree forms. We measured cross-sectional areas of roots, stems and fronds as well as leaf areas of I. deltoidea saplings. Likewise, cross-sections were made and vessel diameters and frequencies measured. This allowed for the calculation of theoretical specific conductivity (K(S,calc)), theoretical leaf-specific conductivity (K(L,calc)), and vessel diameter and vessel number ratios between distal and proximal locations in the palms. Iriartea deltoidea palms were found to have the largest, least frequent vessels that diverged most from the square packing limit (maximum number of vessels that fit into a given area) compared with other major tree forms, and they therefore invested the least space and carbon into water transport structures. Likewise, conduits tapered by ∼1/3 between ranks (root, bole and petiole), which represents an efficient ratio with regard to the trade-offs between safety and efficiency of the conducting system. Conduits also exhibited a high conservation of the sum of the conduit radii cubed (Σr(3)) across ranks, thereby approximating Murray's law patterning. Therefore, our results indicate that the palm I. deltoidea has a very efficient hydraulic system in terms of maintaining a large conducting capacity with a minimal vascular investment. This efficiency may allow palms to compete well with dicotyledonous trees in tropical and subtropical climates but other developmental factors largely restrict palms from regions that experience prolonged freezing temperatures.

Comparison of tissue heat balance- and thermal dissipation-derived sap flow measurements in ring-porous oaks and a pine.

Sap flow measurements have become integral in many physiological and ecological investigations. A number of methods are used to estimate sap flow rates in trees, but probably the most popular is the thermal dissipation (TD) method because of its affordability, relatively low power consumption, and ease of use. However, there have been questions about the use of this method in ring-porous species and whether individual species and site calibrations are needed. We made concurrent measurements of sap flow rates using TD sensors and the tissue heat balance (THB) method in two oak species (Quercus prinus Willd. and Quercus velutina Lam.) and one pine (Pinus echinata Mill.). We also made concurrent measurements of sap flow rates using both 1 and 2-cm long TD sensors in both oak species. We found that both the TD and THB systems tended to match well in the pine individual, but sap flow rates were underestimated by 2-cm long TD sensors in five individuals of the two ring-porous oak species. Underestimations of 20-35% occurred in Q. prinus even when a "Clearwater" correction was applied to account for the shallowness of the sapwood depth relative to the sensor length and flow rates were underestimated by up to 50% in Q. velutina. Two centimeter long TD sensors also underestimated flow rates compared with 1-cm long sensors in Q. prinus, but only at large flow rates. When 2-cm long sensor data in Q. prinus were scaled using the regression with 1-cm long data, daily flow rates matched well with the rates measured by the THB system. Daily plot level transpiration estimated using TD sap flow rates and scaled 1 cm sensor data averaged about 15% lower than those estimated by the THB method. Therefore, these results suggest that 1-cm long sensors are appropriate in species with shallow sapwood, however more corrections may be necessary in ring-porous species.

"Secondary stem lengthening" in the palm Iriartea deltoidea (Arecaceae) provides an efficient and novel method for height growth in a tree form.

PREMISE OF THE STUDY: Although traditionally assumed that all height growth in trees occurs at apical meristems, sequential measurement of internode lengths in the palm Iriartea deltoidea suggested that stems were lengthening long after the differentiation of tissues and far below the apical meristem. This observation is difficult to reconcile with the fact that neither the water-conducting vessels nor the sugar-transporting sieve tube cells are capable of lengthening after differentiation. However, the vascular bundles in palms form a spiral within the stem and could theoretically lengthen if the spiral "straightened". METHODS: We marked stretches of internodes on small and medium-sized palms and measured their lengths over 2 years. Additionally, we collected material from small palms with short internodes and large palms with long internodes and made cross sections to determine the angle of vascular bundles within stems. KEY RESULTS: We found that stems lengthened (up to 12% over 2 years) below the apical meristem in small and medium-sized palms and that the spiral angle in vascular bundles of small palms was significantly larger than at the base of large palms indicating a straightening of the spiral. CONCLUSIONS: These results represent the first determination of "secondary lengthening" in tree stems as well as the most efficient method for height growth in terms of carbon investment. Likewise, elongation of stems allows palms to exhibit plasticity in height growth rates for more rapid growth when short-lived canopy gaps are present than they would have with apical growth alone.

Hydraulic properties of fronds from palms of varying height and habitat.

Because palms grow in highly varying climates and reach considerable heights, they present a unique opportunity to evaluate how environment and plant size impact hydraulic function. We studied hydraulic properties of petioles from palms of varying height from three species: Iriartea deltoidea, a tropical rainforest species; Mauritia flexuosa, a tropical rainforest, swamp species; and Washingtonia robusta, a subtropical species. We measured leaf areas, petiole cross-sectional areas, specific conductivity (K(S)), petiole anatomical properties, vulnerability to embolism and leaf water potentials and calculated petiole Huber values and leaf-specific conductivities (K(L)). Leaf and petiole cross-sectional areas varied widely with height. However, hydraulic properties including Huber values, K(S) and K(L), remained constant. The two palmate species, M. flexuosa and W. robusta, had larger Huber values than I. deltoidea, a pinnately-compound species which exhibited the highest K(S). Metaxylem vessel diameters and vascular bundle densities varied with height in opposing patterns to maintain petiole conductivities. I. deltoidea and W. robusta petioles had similar P(50) values (the point at which 50% of hydraulic conductivity is lost) averaged over all crown heights, but W. robusta exhibited more negative P(50) values in taller palms. Comparison of P (50) values with transpiring midday leaf water potentials, as well as a double-dye staining experiment in a 1-m-tall palm, suggested that a fairly significant amount of embolisms were occurring and refilled on a diurnal basis. Therefore, across palms differing widely in height and growing environments, we found convergence in water transport per unit leaf area (K(L)) with individuals exhibiting differing strategies for achieving this.

Intrinsic and extrinsic hydraulic factors in varying sizes of two Amazonian palm species (Iriartea deltoidea and Mauritia flexuosa) differing in development and growing environment.

PREMISE OF THE STUDY: This study seeks to determine how hydraulic factors vary with ontogeny and whether they begin to limit further height growth in palms. Palms are an attractive group for physiological research because their columnar trunks and simple leaf habit allow key intrinsic and extrinsic hydraulic variables to be estimated more easily than in complex arborescent dicotyledons. • METHODS: We measured various biometric and physiological factors including sap flux, leaf areas, turnover rates, and internode lengths in two Amazonian rainforest species: terra firme Iriartea deltoidea and swamp-adapted Mauritia flexuosa. These two palm species differ markedly in edaphic conditions, leaf type (pinnately compound vs. palmate), and bole development, making physiological comparisons between them important as well. • KEY RESULTS: The species exhibited differing patterns in height growth rate along boles, which appear to relate to their differences in bole development. Growth rates ultimately slowed at the tops of tall palms in both species. We also found a high degree of convergence in total leaf area with height in both species even though they exhibited contrasting patterns in both live frond number and leaf area per frond with height. Sap flux density from leaves was constant with height but four times greater in M. flexuosa than in I. deltoidea. • CONCLUSIONS: Although height growth rates slow considerably in tall palms, neither species shows evidence that hydraulic factors become limiting because they are able to support much greater leaf areas with similar sap flux densities as shorter palms.

Hydraulic architecture and photosynthetic capacity as constraints on release from suppression in Douglas-fir and western hemlock.

We compared hydraulic architecture, photosynthesis and growth in Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco), a shade-intolerant species, and western hemlock (Tsuga heterophylla (Raf.) Sarg.), a shade-tolerant species, to study the temporal pattern of release from suppressive shade. In particular, we sought to determine whether hydraulic architecture or photosynthetic capacity is most important in constraining release. The study was conducted at two sites with mixed stands of 10- to 20-year-old Douglas-fir and western hemlock. At one site, the stand had been thinned allowing release of the understory trees, whereas at the other site, the stand remained unthinned. Douglas-fir had lower height growth (from 1998-2003) and lower relative height growth (height growth from 1998 to 2003/height in 1998) than western hemlock. However, relative height growth of released versus suppressed trees was higher in Douglas-fir (130%) than in western hemlock (65%), indicating that, although absolute height growth was less, Douglas-fir did release from suppression. Release seemed to be constrained initially by a limited photosynthetic capacity in both species. Five years after release, Douglas-fir trees had 14 times the leaf area and 1.5 times the leaf nitrogen concentration (N (area)) of suppressed trees. Needles of released western hemlock trees had about twice the maximum assimilation rate (A (max)) at ambient [CO(2)] as needles of suppressed trees and exhibited no photoinhibition at the highest irradiances. After release, trees increased in leaf area, leaf N concentration and overall photosynthetic capacity. Subsequently, hydraulic architecture appeared to constrain release in Douglas-fir and, to a lesser extent, in western hemlock. Released trees had significantly less negative foliar delta(13)C values than suppressed trees and showed a positive relationship between leaf area:sapwood area ratio (A (L)/A (S)) and delta(13)C, suggesting that trees with more leaf area for a given sapwood area experienced a stomatal limitation on carbon gain. Nonetheless, these changes had no significant effects on leaf specific conductivities of suppressed versus released trees of either species, but leaf specific root conductance was significantly lower in released Douglas-fir.