Expanding collaborative autoethnography into the world of natural science for transdisciplinary teams.

Wicked problems such as climate change and the COVID-19 pandemic require authentically transdisciplinary approaches to achieving effective collaboration. There exist several research approaches for identifying the components and interactions of complex problems; however, collaborative autoethnography provides an empirical way to collect and analyze self-reflection that leads to transformative change. Here, we present a case study of collaborative autoethnography, applied as a tool to transform research practice among a group of natural and social scientists, by constructively revealing and resolving deep, often unseen, disciplinary divides. We ask, "How can natural and social scientists genuinely accept, respect, and share one another's approaches to work on the wicked problems that need to be solved?" This study demonstrates how disciplinary divisions can be successfully bridged by open-minded and committed collaborators who are prepared to recognize the academic bias they bring to their research and use this as a platform of strength.

Mesophyll conductance exerts a significant limitation on photosynthesis during light induction.

Past studies have established mesophyll diffusion conductance to CO2 (gm ) as a variable and significant limitation to plant photosynthesis under steady-state conditions. However, the role of gm in influencing photosynthesis (A) during the transient period of light induction is largely unknown. We combined gas exchange measurements with laser-enabled carbon isotope discrimination measurements to assess gm during photosynthetic induction, using Arabidopsis as the measurement species. Our measurements revealed three key findings: (1) we found that the rate at which gm approached steady state during induction was not necessarily faster than the induction rate of the carboxylation process, contradictory to what has been suggested in previous studies; (2) gm displayed a strong and consistent coordination with A under both induction and steady-state settings, hinting that the mechanism driving gm -A coupling does not require physiological stability as a prerequisite; and (3) photosynthetic limitation analysis of our data revealed that when integrated over the entire induction period, the relative limitation of A imposed by gm can be as high as > 35%. The present study provides the first demonstration of the important role of gm in limiting CO2 assimilation during photosynthetic induction, thereby pointing to a need for more research attention to be devoted to gm in future induction studies.

Open source 3D phenotyping of chickpea plant architecture across plant development.

BACKGROUND: Being able to accurately assess the 3D architecture of plant canopies can allow us to better estimate plant productivity and improve our understanding of underlying plant processes. This is especially true if we can monitor these traits across plant development. Photogrammetry techniques, such as structure from motion, have been shown to provide accurate 3D reconstructions of monocot crop species such as wheat and rice, yet there has been little success reconstructing crop species with smaller leaves and more complex branching architectures, such as chickpea. RESULTS: In this work, we developed a low-cost 3D scanner and used an open-source data processing pipeline to assess the 3D structure of individual chickpea plants. The imaging system we developed consists of a user programmable turntable and three cameras that automatically captures 120 images of each plant and offloads these to a computer for processing. The capture process takes 5-10 min for each plant and the majority of the reconstruction process on a Windows PC is automated. Plant height and total plant surface area were validated against "ground truth" measurements, producing R2 > 0.99 and a mean absolute percentage error < 10%. We demonstrate the ability to assess several important architectural traits, including canopy volume and projected area, and estimate relative growth rate in commercial chickpea cultivars and lines from local and international breeding collections. Detailed analysis of individual reconstructions also allowed us to investigate partitioning of plant surface area, and by proxy plant biomass. CONCLUSIONS: Our results show that it is possible to use low-cost photogrammetry techniques to accurately reconstruct individual chickpea plants, a crop with a complex architecture consisting of many small leaves and a highly branching structure. We hope that our use of open-source software and low-cost hardware will encourage others to use this promising technique for more architecturally complex species.

The effects on isotopic composition of leaf water and transpiration of adding a gas-exchange cuvette.

An expression was earlier derived for the non-steady state isotopic composition of a leaf when the composition of the water entering the leaf was not necessarily the same as that of the water being transpired (Farquhar and Cernusak 2005). This was relevant to natural conditions because the associated time constant is typically sufficiently long to ensure that the leaf water composition and fluxes of the isotopologues are rarely steady. With the advent of laser-based measurements of isotopologues, leaves have been enclosed in cuvettes and time courses of fluxes recorded. The enclosure modifies the time constant by effectively increasing the resistance to the one-way gross flux out of the stomata because transpiration increases the vapour concentration within the chamber. The resistance is increased from stomatal and boundary layer in series, to stomata, boundary layer and chamber resistance, where the latter is given by the ratio of leaf area to the flow rate out of the chamber. An apparent change in concept from one-way to net flux, introduced by Song, Simonin, Loucos and Barbour (2015) is resolved, and shown to be unnecessary, but the value of their data is reinforced.

Understanding airspace in leaves: 3D anatomy and directional tortuosity.

The leaf intercellular airspace is a tortuous environment consisting of cells of different shapes, packing densities, and orientation, all of which have an effect on the travelling distance of molecules from the stomata to the mesophyll cell surfaces. Tortuosity, the increase in displacement over the actual distance between two points, is typically defined as encompassing the whole leaf airspace, but heterogeneity in pore dimensions and orientation between the spongy and palisade mesophyll likely result in heterogeneity in tortuosity along different axes and would predict longer traveling distance along the path of least tortuosity, such as vertically within the columnar cell matrix of the palisade layer. Here, we compare a previously established geometric method to a random walk approach, novel for this analysis in plant leaves, in four different Eucalyptus species. The random walk method allowed us to quantify directional tortuosity across the whole leaf profile, and separately for the spongy and palisade mesophyll. For all species tortuosity was higher in the palisade mesophyll than the spongy mesophyll and horizontal (parallel to the epidermis) tortuosity was consistently higher than vertical (from epidermis to epidermis) tortuosity. We demonstrate that a random walk approach improves on previous geometric approaches and is valuable for investigating CO2 and H2 O transport within leaves.

Can hydraulic design explain patterns of leaf water isotopic enrichment in C3 plants?

H2 18 O enrichment develops when leaves transpire, but an accurate generalized mechanistic model has proven elusive. We hypothesized that leaf hydraulic architecture may affect the degree to which gradients in H2 18 O develop within leaves, influencing bulk leaf stable oxygen isotope enrichment (ΔL ) and the degree to which the Péclet effect is relevant in leaves. Leaf hydraulic design predicted the relevance of a Péclet effect to ΔL in 19 of the 21 species tested. Leaves with well-developed hydraulic connections between the vascular tissue and the epidermal cells through bundle sheath extensions and clear distinctions between palisade and spongy mesophyll layers (while the mesophyll is hydraulically disconnected) may have velocities of the transpiration stream such that gradients in H2 18 O develop and are expressed in the mesophyll. In contrast, in leaves where the vascular tissue is hydraulically disconnected from the epidermal layers, or where all mesophyll cells are well connected to the transpiration stream, velocities within the liquid transport pathways may be low enough that gradients in H2 18 O are very small. Prior knowledge of leaf hydraulic design allows informed selection of the appropriate ΔL modelling framework.

No evidence of homeostatic regulation of leaf temperature in Eucalyptus parramattensis trees: integration of CO2 flux and oxygen isotope methodologies.

Thermoregulation of leaf temperature (Tleaf ) may foster metabolic homeostasis in plants, but the degree to which Tleaf is moderated, and under what environmental contexts, is a topic of debate. Isotopic studies inferred the temperature of photosynthetic carbon assimilation to be a constant value of c. 20°C; by contrast, leaf biophysical theory suggests a strong dependence of Tleaf on environmental drivers. Can this apparent disparity be reconciled? We continuously measured Tleaf and whole-crown net CO2 uptake for Eucalyptus parramattensis trees growing in field conditions in whole-tree chambers under ambient and +3°C warming conditions, and calculated assimilation-weighted leaf temperature (TL-AW ) across 265 d, varying in air temperature (Tair ) from -1 to 45°C. We compared these data to TL-AW derived from wood cellulose δ18 O. Tleaf exhibited substantial variation driven by Tair , light intensity, and vapor pressure deficit, and Tleaf was strongly linearly correlated with Tair with a slope of c. 1.0. TL-AW values calculated from cellulose δ18 O vs crown fluxes were remarkably consistent; both varied seasonally and in response to the warming treatment, tracking variation in Tair . The leaves studied here were nearly poikilothermic, with no evidence of thermoregulation of Tleaf towards a homeostatic value. Importantly, this work supports the use of cellulose δ18 O to infer TL-AW , but does not support the concept of strong homeothermic regulation of Tleaf.

Identification of quantitative trait loci for dynamic and steady-state photosynthetic traits in a barley mapping population.

Enhancing the photosynthetic induction response to fluctuating light has been suggested as a key target for improvement in crop breeding programmes, with the potential to substantially increase whole-canopy carbon assimilation and contribute to crop yield potential. Rubisco activation may be the main physiological process that will allow us to achieve such a goal. In this study, we assessed the phenotype of Rubisco activation rate in a doubled haploid (DH) barley mapping population [131 lines from a Yerong/Franklin (Y/F) cross] after a switch from moderate to saturating light. Rates of Rubisco activation were found to be highly variable across the mapping population, with a median activation rate of 0.1 min-1 in the slowest genotype and 0.74 min-1 in the fastest genotype. A unique quantitative trait locus (QTL) for Rubisco activation rate was identified on chromosome 7H. This is the first report on the identification of a QTL for Rubisco activation rate in planta and the discovery opens the door to marker-assisted breeding to improve whole-canopy photosynthesis of barley. This also suggests that genetic factors other than the previously characterized Rubisco activase (RCA) isoforms on chromosome 4H control Rubisco activity. Further strength is given to this finding as this QTL co-localized with QTLs identified for steady-state photosynthesis and stomatal conductance. Several other distinct QTLs were identified for these steady-state traits, with a common overlapping QTL on chromosome 2H, and distinct QTLs for photosynthesis and stomatal conductance identified on chromosomes 4H and 5H, respectively. Future work should aim to validate these QTLs under field conditions so that they can be used to aid plant breeding efforts.

The role of leaf water potential in the temperature response of mesophyll conductance.

Variation in temperature (T) is usually accompanied by changes in leaf water potential (Ψleaf ), which may influence mesophyll conductance (gm ). However, the effects of Ψleaf on gm have not yet been considered in models of the gm response to temperature. Temperature responses of gm and Ψleaf and the response of gm to Ψleaf were studied in rice (Oryza sativa) and wheat (Triticum aestivum), and then an empirical model of Ψleaf was incorporated into an existing gm -T model. In wheat, Ψleaf was dramatically decreased with increasing T, whereas in rice Ψleaf was less sensitive or insensitive to T. Without taking Ψleaf into account, gm for wheat showed no response to T. However, at a given Ψleaf , gm was significantly higher at high temperature compared with low. After incorporating the function of Ψleaf into the gm -T model, we suggest that the gm -T relationship can be influenced by the activation and deactivation energy for membrane permeability, Ψleaf gradient between temperatures, and the sensitivity of gm to Ψleaf , below a threshold (Ψleaf,0 ). The data presented here suggest that Ψleaf plays an important role in the gm -T relationship and should be considered in future studies related to the temperature response of gm and photosynthesis.

Cell and chloroplast anatomical features are poorly estimated from 2D cross-sections.

Leaf function is intimately related to the size, shape, abundance and position of cells and chloroplasts. Anatomy has long been assessed and quantified in two dimensions with 3D structure inferred from 2D micrographs. Serial block face scanning electron microscopy (SBF-SEM) was used to reconstruct 95 cells and 1173 chloroplasts from three wheat and nine chickpea leaves (three samples each from three chickpea genotypes). Wheat chloroplast volume was underestimated by 61% in mesophyll cells and 45% in bundle sheath cells from 2D micrographs, whereas chickpea mesophyll chloroplast volume was underestimated by 60% using simple geometrical models. Models of chickpea spongy and palisade cells both under- and overestimated surface area and volume by varying degrees. These models did not adequately capture irregular shapes such as flattening of chloroplasts or lobed spongy mesophyll cells. It is concluded that simple geometrical models to estimate chloroplast and cell 3D volume and surface area from 2D micrographs are inadequate, and that SBF-SEM has strong potential to contribute to improved understanding of leaf form and function.

The temperature response of mesophyll conductance, and its component conductances, varies between species and genotypes.

The temperature response of mesophyll conductance to CO2 diffusion (gm) has been shown to vary considerably between species but remains poorly understood. Here, we tested the hypothesis that increases in chloroplast surface area with increasing temperature, due to the formation of chloroplast protrusions, caused observed positive responses of gm to temperature. We found no evidence of chloroplast protrusions. Using simultaneous measurements of carbon and oxygen isotope discrimination during photosynthesis to separate total gm (gm13) into cell wall and plasma membrane conductance (gm18) and chloroplast membrane conductance (gcm) components, we explored the temperature response in genotypes of soybean and barley, and sunflower plants grown at differing CO2 concentrations. Differences in the temperature sensitivity of gm18 were found between genotypes and between plants grown at differing CO2 concentration but did not relate to measured anatomical features such as chloroplast surface area or cell wall thickness. The closest fit of modelled gm13 to estimated values was found when cell wall thickness was allowed to decline at higher temperatures and transpiration rates, but it remains to be tested if this decline is realistic. The temperature response of gcm (calculated from the difference between 1/gm13 and 1/gm18) varied between barley genotypes, and was best fitted by an optimal response in sunflower. Taken together, these results indicate that gm is a highly complex trait with unpredictable sensitivity to temperature that varies between species, between genotypes within a single species, with growth environment, between replicate leaves, and even with age for an individual leaf.

The response of mesophyll conductance to short- and long-term environmental conditions in chickpea genotypes.

. Mesophyll conductance (g m) has been shown to vary between genotypes of a number of species and with growth environments, including nitrogen availability, but understanding of g m variability in legumes is limited. We might expect g m in legumes to respond differently to limited nitrogen availability, due to their ability to fix atmospheric N2. Using online stable carbon isotope discrimination method, we quantified genetic variability in g m under ideal conditions, investigated g m response to N source (N2-fixation or inorganic N) and determined the effects of N source and water availability on the rapid response of g m to photosynthetic photon flux density (PPFD) and radiation wavelength in three genotypes of chickpea (Cicer arietinum). Genotypes varied 2-fold in g m under non-limiting environments. N-fed plants had higher g m than N2-fixing plants in one genotype, while g m in the other two genotypes was unaffected. g m response to PPFD was altered by N source in one of three genotypes, in which the g m response to PPFD was statistically significant in N-fed plants but not in N2-fixing plants. There was no clear effect of moderate water stress on the g m response to PPFD and radiation wavelength. Genotypes of a single legume species differ in the sensitivity of g m to both long- and short-term environmental conditions, precluding utility in crop breeding programmes.

Embracing 3D Complexity in Leaf Carbon-Water Exchange.

Leaves are a nexus for the exchange of water, carbon, and energy between terrestrial plants and the atmosphere. Research in recent decades has highlighted the critical importance of the underlying biophysical and anatomical determinants of CO2 and H2O transport, but a quantitative understanding of how detailed 3D leaf anatomy mediates within-leaf transport has been hindered by the lack of a consensus framework for analyzing or simulating transport and its spatial and temporal dynamics realistically, and by the difficulty of measuring within-leaf transport at the appropriate scales. We discuss how recent technological advancements now make a spatially explicit 3D leaf analysis possible, through new imaging and modeling tools that will allow us to address long-standing questions related to plant carbon-water exchange.

Leaf day respiration: low CO2 flux but high significance for metabolism and carbon balance.

Contents 986 I. 987 II. 987 III. 988 IV. 991 V. 992 VI. 995 VII. 997 VIII. 998 References 998 SUMMARY: It has been 75 yr since leaf respiratory metabolism in the light (day respiration) was identified as a low-flux metabolic pathway that accompanies photosynthesis. In principle, it provides carbon backbones for nitrogen assimilation and evolves CO2 and thus impacts on plant carbon and nitrogen balances. However, for a long time, uncertainties have remained as to whether techniques used to measure day respiratory efflux were valid and whether day respiration responded to environmental gaseous conditions. In the past few years, significant advances have been made using carbon isotopes, 'omics' analyses and surveys of respiration rates in mesocosms or ecosystems. There is substantial evidence that day respiration should be viewed as a highly dynamic metabolic pathway that interacts with photosynthesis and photorespiration and responds to atmospheric CO2 mole fraction. The view of leaf day respiration as a constant and/or negligible parameter of net carbon exchange is now outdated and it should now be regarded as a central actor of plant carbon-use efficiency.

Leaf hydraulic conductance and mesophyll conductance are not closely related within a single species.

Stomata represent one resistor in a series of resistances for carbon and water exchange between the leaf and the atmosphere; the remaining resistors occurring within the leaf, commonly represented as mesophyll conductance to CO2 , gm , and leaf hydraulic conductance, kLeaf . Recent studies have proposed that gm and kLeaf may be coordinated across species because of shared pathways. We assessed the correlation between gm and kLeaf within cotton, under growth CO2 partial pressure and irradiance treatments and also with short-term variation in irradiance and humidity. gm was estimated using two isotopic techniques that allowed partitioning of total gm (Δ13 C-gm ) into cell wall plus plasma membrane conductance (Δ18 O-gm ) and chloroplast membrane conductance (gcm ). A weak correlation was found between Δ13 C-gm and kLeaf only when measured under growth conditions. However, Δ18 O-gm was related to kLeaf under both short-term environmental variation and growth conditions. Partitioning gm showed that gcm was not affected by short-term changes in irradiance or correlated with kLeaf , but was strongly reduced at high growth CO2 partial pressure. Thus, simultaneous measurements of gm , kLeaf and gcm suggest independent regulation of carbon and water transport across the chloroplast membrane with limited coordinated regulation across the cell wall and plasma membrane.

Understanding regulation of leaf internal carbon and water transport using online stable isotope techniques.

83 I. 83 II. 84 III. 84 IV. 85 V. 86 VI. 86 VII. 86 87 References 87 SUMMARY: The balance of carbon dioxide (CO2 ) and water vapour exchange between leaves and the atmosphere is strongly controlled by stomatal conductance. However, the influence of transport processes within leaves has recently been gaining prominence. Stable isotope techniques are at the forefront of understanding transport within leaves and the recent development of online, real-time optical isotope analysers has paved the way for new questions to be asked. In this insight, I outline these new techniques and the questions they can potentially address, including assessing possible coordination between mesophyll conductance to CO2 and leaf hydraulic conductance.

The response of mesophyll conductance to nitrogen and water availability differs between wheat genotypes.

Increased mesophyll conductance (gm) has been suggested as a target for selection for high productivity and high water-use efficiency in crop plants, and genotypic variability in gm has been reported in several important crop species. However, effective selection requires an understanding of how gm varies with growth conditions, to ensure that the ranking of genotypes is consistent across environments. We assessed the genotypic variability in gm and other leaf gas exchange traits, as well as growth and biomass allocation for six wheat genotypes under different water and nitrogen availabilities. The wheat genotypes differed in their response of gm to growth conditions, resulting in genotypic differences in the mesophyll limitation to photosynthesis and a significant increase in the mesophyll limitation to photosynthesis under drought. In this experiment, leaf intrinsic water-use efficiency was more closely related to stomatal conductance than to mesophyll conductance, and stomatal limitation to photosynthesis increased more in some genotypes than in others in response to drought. Screening for gm should be carried out under a range of growth conditions.

Leaf water oxygen isotope measurement by direct equilibration.

The oxygen isotope composition of leaf water imparts a signal to a range of molecules in the atmosphere and biosphere, but has been notoriously difficult to measure in studies requiring a large number of samples as a consequence of the labour-intensive extraction step. We tested a method of direct equilibration of water in fresh leaf samples with CO2 , and subsequent oxygen isotope analysis on an optical spectrometer. The oxygen isotope composition of leaf water measured by the direct equilibration technique was strongly linearly related to that of cryogenically extracted leaf water in paired samples for a wide range of species with differing anatomy, with an R(2) of 0.95. The somewhat more enriched values produced by the direct equilibration method may reflect lack of full equilibration with unenriched water in the vascular bundles, but the strong relationship across a wide range of species suggests that this difference can be adequately corrected for using a simple linear relationship.