The importance of species-specific and temperature-sensitive parameterisation of A/Ci models: A case study using cotton (Gossypium hirsutum L.) and the automated 'OptiFitACi' R-package.

Leaf gas exchange measurements are an important tool for inferring a plant's photosynthetic biochemistry. In most cases, the responses of photosynthetic CO2 assimilation to variable intercellular CO2 concentrations (A/Ci response curves) are used to model the maximum (potential) rate of carboxylation by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, Vcmax) and the rate of photosynthetic electron transport at a given incident photosynthetically active radiation flux density (PAR; JPAR). The standard Farquhar-von Caemmerer-Berry model is often used with default parameters of Rubisco kinetic values and mesophyll conductance to CO2 (gm) derived from tobacco that may be inapplicable across species. To study the significance of using such parameters for other species, here we measured the temperature responses of key in vitro Rubisco catalytic properties and gm in cotton (Gossypium hirsutum cv. Sicot 71) and derived Vcmax and J2000 (JPAR at 2000 µmol m-2 s-1 PAR) from cotton A/Ci curves incrementally measured at 15°C-40°C using cotton and other species-specific sets of input parameters with our new automated fitting R package 'OptiFitACi'. Notably, parameterisation by a set of tobacco parameters produced unrealistic J2000:Vcmax ratio of <1 at 25°C, two- to three-fold higher estimates of Vcmax above 15°C, up to 2.3-fold higher estimates of J2000 and more variable estimates of Vcmax and J2000, for our cotton data compared to model parameterisation with cotton-derived values. We determined that errors arise when using a gm,25 of 2.3 mol m-2 s-1 MPa-1 or less and Rubisco CO2-affinities in 21% O2 (KC 21%O2) at 25°C outside the range of 46-63 Pa to model A/Ci responses in cotton. We show how the A/Ci modelling capabilities of 'OptiFitACi' serves as a robust, user-friendly, and flexible extension of 'plantecophys' by providing simplified temperature-sensitivity and species-specificity parameterisation capabilities to reduce variability when modelling Vcmax and J2000.

Rubisco deactivation and chloroplast electron transport rates co-limit photosynthesis above optimal leaf temperature in terrestrial plants.

Net photosynthetic CO2 assimilation rate (An) decreases at leaf temperatures above a relatively mild optimum (Topt) in most higher plants. This decline is often attributed to reduced CO2 conductance, increased CO2 loss from photorespiration and respiration, reduced chloroplast electron transport rate (J), or deactivation of Ribulose-1,5-bisphosphate Carboxylase Oxygenase (Rubisco). However, it is unclear which of these factors can best predict species independent declines in An at high temperature. We show that independent of species, and on a global scale, the observed decline in An with rising temperatures can be effectively accounted for by Rubisco deactivation and declines in J. Our finding that An declines with Rubisco deactivation and J supports a coordinated down-regulation of Rubisco and chloroplast electron transport rates to heat stress. We provide a model that, in the absence of CO2 supply limitations, can predict the response of photosynthesis to short-term increases in leaf temperature.

A high-throughput yeast approach to characterize aquaporin permeabilities: Profiling the Arabidopsis PIP aquaporin sub-family.

Introduction: Engineering membrane transporters to achieve desired functionality is reliant on availability of experimental data informing structure-function relationships and intelligent design. Plant aquaporin (AQP) isoforms are capable of transporting diverse substrates such as signaling molecules, nutrients, metalloids, and gases, as well as water. AQPs can act as multifunctional channels and their transport function is reliant on many factors, with few studies having assessed transport function of specific isoforms for multiple substrates. Methods: High-throughput yeast assays were developed to screen for transport function of plant AQPs, providing a platform for fast data generation and cataloguing of substrate transport profiles. We applied our high-throughput growth-based yeast assays to screen all 13 Arabidopsis PIPs (AtPIPs) for transport of water and several neutral solutes: hydrogen peroxide (H2O2), boric acid (BA), and urea. Sodium (Na+) transport was assessed using elemental analysis techniques. Results: All AtPIPs facilitated water and H2O2 transport, although their growth phenotypes varied, and none were candidates for urea transport. For BA and Na+ transport, AtPIP2;2 and AtPIP2;7 were the top candidates, with yeast expressing these isoforms having the most pronounced toxicity response to BA exposure and accumulating the highest amounts of Na+. Linking putative AtPIP isoform substrate transport profiles with phylogenetics and gene expression data, enabled us to align possible substrate preferences with known and hypothesized biological roles of AtPIPs. Discussion: This testing framework enables efficient cataloguing of putative transport functionality of diverse AQPs at a scale that can help accelerate our understanding of AQP biology through big data approaches (e.g. association studies). The principles of the individual assays could be further adapted to test additional substrates. Data generated from this framework could inform future testing of AQP physiological roles, and address knowledge gaps in structure-function relationships to improve engineering efforts.

Enhancing crop yields through improvements in the efficiency of photosynthesis and respiration.

The rate with which crop yields per hectare increase each year is plateauing at the same time that human population growth and other factors increase food demand. Increasing yield potential ( Y p ) of crops is vital to address these challenges. In this review, we explore a component of Y p that has yet to be optimised - that being improvements in the efficiency with which light energy is converted into biomass ( ε c ) via modifications to CO2 fixed per unit quantum of light (α), efficiency of respiratory ATP production ( ε prod ) and efficiency of ATP use ( ε use ). For α, targets include changes in photoprotective machinery, ribulose bisphosphate carboxylase/oxygenase kinetics and photorespiratory pathways. There is also potential for ε prod to be increased via targeted changes to the expression of the alternative oxidase and mitochondrial uncoupling pathways. Similarly, there are possibilities to improve ε use via changes to the ATP costs of phloem loading, nutrient uptake, futile cycles and/or protein/membrane turnover. Recently developed high-throughput measurements of respiration can serve as a proxy for the cumulative energy cost of these processes. There are thus exciting opportunities to use our growing knowledge of factors influencing the efficiency of photosynthesis and respiration to create a step-change in yield potential of globally important crops.

A cross-scale analysis to understand and quantify the effects of photosynthetic enhancement on crop growth and yield across environments.

Photosynthetic manipulation provides new opportunities for enhancing crop yield. However, understanding and quantifying the importance of individual and multiple manipulations on the seasonal biomass growth and yield performance of target crops across variable production environments is limited. Using a state-of-the-art cross-scale model in the APSIM platform we predicted the impact of altering photosynthesis on the enzyme-limited (Ac ) and electron transport-limited (Aj ) rates, seasonal dynamics in canopy photosynthesis, biomass growth, and yield formation via large multiyear-by-location crop growth simulations. A broad list of promising strategies to improve photosynthesis for C3 wheat and C4 sorghum were simulated. In the top decile of seasonal outcomes, yield gains were predicted to be modest, ranging between 0% and 8%, depending on the manipulation and crop type. We report how photosynthetic enhancement can affect the timing and severity of water and nitrogen stress on the growing crop, resulting in nonintuitive seasonal crop dynamics and yield outcomes. We predicted that strategies enhancing Ac alone generate more consistent but smaller yield gains across all water and nitrogen environments, Aj enhancement alone generates larger gains but is undesirable in more marginal environments. Large increases in both Ac and Aj generate the highest gains across all environments. Yield outcomes of the tested manipulation strategies were predicted and compared for realistic Australian wheat and sorghum production. This study uniquely unpacks complex cross-scale interactions between photosynthesis and seasonal crop dynamics and improves understanding and quantification of the potential impact of photosynthesis traits (or lack of it) for crop improvement research.

Hormone regulation of thrombospondin-1 mRNA in porcine granulosa cells in vitro.

Thrombospondin-1 (THBS1) is involved in the process of angiogenesis and is down-regulated by insulin-like growth factor 1 (IGF1) in porcine granulosa cells (GC), but what other hormones regulate GC THBS1 and its role in follicular growth is unclear. Thus, six experiments were conducted to determine the influence of other hormones on THBS1 gene expression in porcine GC, and to determine if THBS1 mRNA changes during follicular development. For Exp. 1-5, small (1-5 mm) follicles from ovaries of abattoir gilts were aspirated, GC collected and treated with FSH, IGF1, fibroblast growth factor 9 (FGF9), Sonic hedgehog (SHH), estradiol, cortisol, and/or prostaglandin E2 (PGE2). FSH, IGF1 and FGF9 each decreased (P < 0.05) THBS1 mRNA abundance. Alone, PGE2 increased (P < 0.05) THBS1 mRNA abundance. PGE2 significantly attenuated the FSH-induced inhibition of THBS1 mRNA expression. Estradiol, cortisol, and SHH had no effect on THBS1 mRNA abundance. In Exp. 6, small (1-3 mm), medium (4-6 mm) and large (7-14 mm) follicles were aspirated to measure abundance of THBS1 mRNA in GC which did not differ (P > 0.10) between small and medium-sized follicles but was threefold greater (P < 0.05) in large compared to small or medium follicles. We hypothesize that the inhibitory effects of FSH, IGF1 and FGF9 on the antiangiogenic gene THBS1 could contribute to promoting angiogenesis in the developing follicle, while stimulation of THBS1 mRNA by PGE2 may help reduce angiogenesis during the preovulatory period when PGE2 and THBS1 mRNA are at their greatest levels.

Contrasting anatomical and biochemical controls on mesophyll conductance across plant functional types.

Mesophyll conductance (gm ) limits photosynthesis by restricting CO2 diffusion between the substomatal cavities and chloroplasts. Although it is known that gm is determined by both leaf anatomical and biochemical traits, their relative contribution across plant functional types (PFTs) is still unclear. We compiled a dataset of gm measurements and concomitant leaf traits in unstressed plants comprising 563 studies and 617 species from all major PFTs. We investigated to what extent gm limits photosynthesis across PFTs, how gm relates to structural, anatomical, biochemical, and physiological leaf properties, and whether these relationships differ among PFTs. We found that gm imposes a significant limitation to photosynthesis in all C3 PFTs, ranging from 10-30% in most herbaceous annuals to 25-50% in woody evergreens. Anatomical leaf traits explained a significant proportion of the variation in gm (R2 > 0.3) in all PFTs except annual herbs, in which gm is more strongly related to biochemical factors associated with leaf nitrogen and potassium content. Our results underline the need to elucidate mechanisms underlying the global variability of gm . We emphasise the underestimated potential of gm for improving photosynthesis in crops and identify modifications in leaf biochemistry as the most promising pathway for increasing gm in these species.

Phenotypic variation in photosynthetic traits in wheat grown under field versus glasshouse conditions.

Recognition of the untapped potential of photosynthesis to improve crop yields has spurred research to identify targets for breeding. The CO2-fixing enzyme Rubisco is characterized by a number of inefficiencies, and frequently limits carbon assimilation at the top of the canopy, representing a clear target for wheat improvement. Two bread wheat lines with similar genetic backgrounds and contrasting in vivo maximum carboxylation activity of Rubisco per unit leaf nitrogen (Vc,max,25/Narea) determined using high-throughput phenotyping methods were selected for detailed study from a panel of 80 spring wheat lines. Detailed phenotyping of photosynthetic traits in the two lines using glasshouse-grown plants showed no difference in Vc,max,25/Narea determined directly via in vivo and in vitro methods. Detailed phenotyping of glasshouse-grown plants of the 80 wheat lines also showed no correlation between photosynthetic traits measured via high-throughput phenotyping of field-grown plants. Our findings suggest that the complex interplay between traits determining crop productivity and the dynamic environments experienced by field-grown plants needs to be considered in designing strategies for effective wheat crop yield improvement when breeding for particular environments.

Mesophyll conductance is unaffected by expression of Arabidopsis PIP1 aquaporins in the plasmalemma of Nicotiana.

In plants with C3 photosynthesis, increasing the diffusion conductance for CO2 from the substomatal cavity to chloroplast stroma (mesophyll conductance) can improve the efficiencies of both CO2 assimilation and photosynthetic water use. In the diffusion pathway from substomatal cavity to chloroplast stroma, the plasmalemma and chloroplast envelope membranes impose a considerable barrier to CO2 diffusion, limiting photosynthetic efficiency. In an attempt to improve membrane permeability to CO2, and increase photosynthesis in tobacco, we generated transgenic lines in Nicotiana tabacum L. cv Petite Havana carrying either the Arabidopsis PIP1;2 (AtPIP1;2) or PIP1;4 (AtPIP1;4) gene driven by the constitutive dual 2x35S CMV promoter. From a collection of independent T0 transgenics, two T2 lines from each gene were characterized, with western blots confirming increased total aquaporin protein abundance in the AtPIP1;2 tobacco lines. Transient expression of AtPIP1;2-mGFP6 and AtPIP1;4-mGFP6 fusions in Nicotiana benthamiana identified that both AtPIP1;2 and AtPIP1;4 localize to the plasmalemma. Despite achieving ectopic production and correct localization, gas exchange measurements combined with carbon isotope discrimination measurements detected no increase in mesophyll conductance or CO2 assimilation rate in the tobacco lines expressing AtPIP. We discuss the complexities associated with trying to enhance gm through modified aquaporin activity.

Wheat physiology predictor: predicting physiological traits in wheat from hyperspectral reflectance measurements using deep learning.

BACKGROUND: The need for rapid in-field measurement of key traits contributing to yield over many thousands of genotypes is a major roadblock in crop breeding. Recently, leaf hyperspectral reflectance data has been used to train machine learning models using partial least squares regression (PLSR) to rapidly predict genetic variation in photosynthetic and leaf traits across wheat populations, among other species. However, the application of published PLSR spectral models is limited by a fixed spectral wavelength range as input and the requirement of separate custom-built models for each trait and wavelength range. In addition, the use of reflectance spectra from the short-wave infrared region requires expensive multiple detector spectrometers. The ability to train a model that can accommodate input from different spectral ranges would potentially make such models extensible to more affordable sensors. Here we compare the accuracy of prediction of PLSR with various deep learning approaches and an ensemble model, each trained and tested using previously published data sets. RESULTS: We demonstrate that the accuracy of PLSR to predict photosynthetic and related leaf traits in wheat can be improved with deep learning-based and ensemble models without overfitting. Additionally, these models can be flexibly applied across spectral ranges without significantly compromising accuracy. CONCLUSION: The method reported provides an improved prediction of wheat leaf and photosynthetic traits from leaf hyperspectral reflectance and do not require a full range, high cost leaf spectrometer. We provide a web service for deploying these algorithms to predict physiological traits in wheat from a variety of spectral data sets, with important implications for wheat yield prediction and crop breeding.

Wah Soon Chow, a teacher, a friend and a colleague.

To finish this special issue, some friends, colleagues and students of Prof. Chow (Emeritus Professor, the Research School of Biology, the Australian National University) have written small tributes to acknowledge not only his eminent career but to describe his wonderful personality.

A consensus on the Aquaporin Gene Family in the Allotetraploid Plant, Nicotiana tabacum.

Aquaporins (AQPs) are membrane-spanning channel proteins with exciting applications for plant engineering and industrial applications. Translational outcomes will be improved by better understanding the extensive diversity of plant AQPs. However, AQP gene families are complex, making exhaustive identification difficult, especially in polyploid species. The allotetraploid species of Nicotiana tabacum (Nt; tobacco) plays a significant role in modern biological research and is closely related to several crops of economic interest, making it a valuable platform for AQP research. Recently, De Rosa et al., (2020) and Ahmed et al., (2020), concurrently reported on the AQP gene family in tobacco, establishing family sizes of 76 and 88 members, respectively. The discrepancy highlights the difficulties of characterizing large complex gene families. Here, we identify and resolve the differences between the two studies, clarify gene models, and yield a consolidated collection of 84 members that more accurately represents the complete NtAQP family. Importantly, this consensus NtAQP collection will reduce confusion and ambiguity that would inevitably arise from having two different descriptive studies and sets of NtAQP gene names. This report also serves as a case study, highlighting and discussing variables to be considered and refinements required to ensure comprehensive gene family characterizations, which become valuable resources for examining the evolution and biological functions of genes.

Stomatal, mesophyll conductance, and biochemical limitations to photosynthesis during induction.

The dynamics of leaf photosynthesis in fluctuating light affects carbon gain by plants. Mesophyll conductance (gm) limits CO2 assimilation rate (A) under the steady state, but the extent of this limitation under non-steady-state conditions is unknown. In the present study, we aimed to characterize the dynamics of gm and the limitations to A imposed by gas diffusional and biochemical processes under fluctuating light. The induction responses of A, stomatal conductance (gs), gm, and the maximum rate of RuBP carboxylation (Vcmax) or electron transport (J) were investigated in Arabidopsis (Arabidopsis thaliana (L.)) and tobacco (Nicotiana tabacum L.). We first characterized gm induction after a change from darkness to light. Each limitation to A imposed by gm, gs and Vcmax or J was significant during induction, indicating that gas diffusional and biochemical processes limit photosynthesis. Initially, gs imposed the greatest limitation to A, showing the slowest response under high light after long and short periods of darkness, assuming RuBP-carboxylation limitation. However, if RuBP-regeneration limitation was assumed, then J imposed the greatest limitation. gm did not vary much following short interruptions to light. The limitation to A imposed by gm was the smallest of all the limitations for most of the induction phase. This suggests that altering induction kinetics of mesophyll conductance would have little impact on A following a change in light. To enhance the carbon gain by plants under naturally dynamic light environments, attention should therefore be focused on faster stomatal opening or activation of electron transport.

Uncovering candidate genes involved in photosynthetic capacity using unexplored genetic variation in Spring Wheat.

To feed an ever-increasing population we must leverage advances in genomics and phenotyping to harness the variation in wheat breeding populations for traits like photosynthetic capacity which remains unoptimized. Here we survey a diverse set of wheat germplasm containing elite, introgression and synthetic derivative lines uncovering previously uncharacterized variation. We demonstrate how strategic integration of exotic material alleviates the D genome genetic bottleneck in wheat, increasing SNP rate by 62% largely due to Ae. tauschii synthetic wheat donors. Across the panel, 67% of the Ae. tauschii donor genome is represented as introgressions in elite backgrounds. We show how observed genetic variation together with hyperspectral reflectance data can be used to identify candidate genes for traits relating to photosynthetic capacity using association analysis. This demonstrates the value of genomic methods in uncovering hidden variation in wheat and how that variation can assist breeding efforts and increase our understanding of complex traits.

Facilitators and barriers to physical activity participation experienced by Aboriginal and Torres Strait Islander adults: a mixed methods systematic review protocol.

OBJECTIVE: To synthesize the existing research about physical activity and sport facilitators and barriers experienced by Aboriginal and Torres Strait Islander adults in Australia. INTRODUCTION: Physical activity and sport have cultural importance for First Nations peoples. Achieving health and broader benefits from physical activity and sport is impacted by experiences of both facilitators and barriers to participation. Identifying how to facilitate participation and overcome barriers to physical activity and sport is important to develop strategies to increase physical activity levels and sport participation among Aboriginal and Torres Strait Islander adults. Several studies have examined physical activity and sport facilitators and barriers experienced by Aboriginal and Torres Strait Islander adults, and collective synthesis of these studies can provide a more comprehensive understanding of their findings. INCLUSION CRITERIA: This mixed methods systematic review will consider studies that include Aboriginal and Torres Strait Islander peoples aged 18 years and over from any setting or region of Australia. Studies will be considered if they report on facilitators and barriers to physical activity and/or sport participation. METHODS: Eleven databases will be searched, as well as gray literature sources, and a selection of websites containing resources relevant to physical activity participation for Aboriginal and Torres Strait Islander adults. Studies published in English will be included. No date limits will be set. After screening the titles and abstracts of identified citations, potentially relevant studies will be retrieved in full. Study selection, critical appraisal, data extraction, and data synthesis will be undertaken according to the convergent integrated approach to mixed methods reviews. SYSTEMATIC REVIEW REGISTRATION NUMBER: PROSPERO CRD42020162134.

Effect of leaf temperature on the estimation of photosynthetic and other traits of wheat leaves from hyperspectral reflectance.

A growing number of leaf traits can be estimated from hyperspectral reflectance data. These include structural and compositional traits, such as leaf mass per area (LMA) and nitrogen and chlorophyll content, but also physiological traits such a Rubisco carboxylation activity, electron transport rate, and respiration rate. Since physiological traits vary with leaf temperature, how does this impact on predictions made from reflectance measurements? We investigated this with two wheat varieties, by repeatedly measuring each leaf through a sequence of temperatures imposed by varying the air temperature in a growth room. Leaf temperatures ranging from 20 °C to 35 °C did not alter the estimated Rubisco capacity normalized to 25 °C (Vcmax25), or chlorophyll or nitrogen contents per unit leaf area. Models estimating LMA and Vcmax25/N were both slightly influenced by leaf temperature: estimated LMA increased by 0.27% °C-1 and Vcmax25/N increased by 0.46% °C-1. A model estimating Rubisco activity closely followed variation associated with leaf temperature. Reflectance spectra change with leaf temperature and therefore contain a temperature signal.

Mesophyll conductance: walls, membranes and spatial complexity.

A significant resistance to CO2 diffusion is imposed by mesophyll tissue inside leaves. Mesophyll resistance, rm (or its reciprocal, mesophyll conductance, gm ), reduces the rate at which Rubisco can fix CO2 , increasing the water and nitrogen costs of carbon acquisition. gm varies in proportion to the surface area of chloroplasts exposed to intercellular airspace per unit leaf area. It also depends on the thickness and effective porosity of the cell wall and the CO2 permeabilities of membranes. As no measurements exist for the effective porosity of mesophyll cell walls, and CO2 permeability values are too low to account for observed rates of CO2 assimilation, conclusions from modelling must be treated with caution. There is great variation in the mesophyll resistance per unit chloroplast area for a given cell wall thickness, which may reflect differences in effective porosity. While apparent gm can vary with CO2 and irradiance, the underlying conductance at the cellular level may remain unchanged. Dynamic changes in apparent gm arise for spatial reasons and because chloroplasts differ in their photosynthetic composition and operate in different light environments. Measurements of the temperature sensitivity of membrane CO2 permeability are urgently needed to explain variation in temperature responses of gm .

Physical activity and sport participation characteristics of Indigenous children registered in the Active Kids voucher program in New South Wales.

OBJECTIVES: Investigate sociodemographic factors associated with physical activity and sport participation among Indigenous children registered in the New South Wales (NSW) government-funded Active Kids voucher program in 2018, including comparison with non-Indigenous children. DESIGN: Cross-sectional study. METHODS: The Active Kids voucher program aims to support the cost of children's sport and physical activities. All children aged 5-18 years in NSW are eligible for a voucher. To register, parent/carers report child sociodemographic characteristics, physical activity, sport participation and optional height and weight. Regression models were used to determine which sociodemographic characteristics were associated with meeting physical activity guidelines and sport participation for Indigenous and non-Indigenous children. RESULTS: Of the 671,375 children aged 5-18 years, 36,129 (5.4%) were Indigenous. More Indigenous children than non-Indigenous children met the physical activity guidelines before registering in the Active Kids program. Indigenous children had greater odds of meeting physical activity guidelines across all socio-economic quartiles. Among non-Indigenous children, odds reduced with social disadvantage. Indigenous children (38%) were less likely to participate in organised physical activity and sport sessions at least twice a week compared to non-Indigenous children (43%). Indigenous children living in major cities had higher sport participation levels compared with those living in outer regional and remote areas. CONCLUSIONS: The Active Kids voucher program achieved population representative reach among Indigenous children, whose physical activity levels were higher than non-Indigenous children across all socioeconomic quartiles. The program has potential to supplement Indigenous children's physical activity levels using organised sessions and reduce sport drop-out among older children.

Genome-wide identification and characterisation of Aquaporins in Nicotiana tabacum and their relationships with other Solanaceae species.

BACKGROUND: Cellular membranes are dynamic structures, continuously adjusting their composition, allowing plants to respond to developmental signals, stresses, and changing environments. To facilitate transmembrane transport of substrates, plant membranes are embedded with both active and passive transporters. Aquaporins (AQPs) constitute a major family of membrane spanning channel proteins that selectively facilitate the passive bidirectional passage of substrates across biological membranes at an astonishing 108 molecules per second. AQPs are the most diversified in the plant kingdom, comprising of five major subfamilies that differ in temporal and spatial gene expression, subcellular protein localisation, substrate specificity, and post-translational regulatory mechanisms; collectively providing a dynamic transportation network spanning the entire plant. Plant AQPs can transport a range of solutes essential for numerous plant processes including, water relations, growth and development, stress responses, root nutrient uptake, and photosynthesis. The ability to manipulate AQPs towards improving plant productivity, is reliant on expanding our insight into the diversity and functional roles of AQPs. RESULTS: We characterised the AQP family from Nicotiana tabacum (NtAQPs; tobacco), a popular model system capable of scaling from the laboratory to the field. Tobacco is closely related to major economic crops (e.g. tomato, potato, eggplant and peppers) and itself has new commercial applications. Tobacco harbours 76 AQPs making it the second largest characterised AQP family. These fall into five distinct subfamilies, for which we characterised phylogenetic relationships, gene structures, protein sequences, selectivity filter compositions, sub-cellular localisation, and tissue-specific expression. We also identified the AQPs from tobacco's parental genomes (N. sylvestris and N. tomentosiformis), allowing us to characterise the evolutionary history of the NtAQP family. Assigning orthology to tomato and potato AQPs allowed for cross-species comparisons of conservation in protein structures, gene expression, and potential physiological roles. CONCLUSIONS: This study provides a comprehensive characterisation of the tobacco AQP family, and strengthens the current knowledge of AQP biology. The refined gene/protein models, tissue-specific expression analysis, and cross-species comparisons, provide valuable insight into the evolutionary history and likely physiological roles of NtAQPs and their Solanaceae orthologs. Collectively, these results will support future functional studies and help transfer basic research to applied agriculture.