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.

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.

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.

The Peptide Hormone Receptor CEPR1 Functions in the Reproductive Tissue to Control Seed Size and Yield.

The interaction of C-TERMINALLY ENCODED PEPTIDES (CEPs) with CEP RECEPTOR1 (CEPR1) controls root growth and development, as well as nitrate uptake, but has no known role in determining yield. We used physiological, microscopic, molecular, and grafting approaches to demonstrate a reproductive tissue-specific role for CEPR1 in controlling yield and seed size. Independent Arabidopsis (Arabidopsis thaliana) cepr1 null mutants showed disproportionately large reductions in yield and seed size relative to their decreased vegetative growth. These yield defects correlated with compromised reproductive development predominantly in female tissues, as well as chlorosis, and the accumulation of anthocyanins in cepr1 reproductive tissues. The thinning of competing reproductive organs to improve source-to-sink ratios in cepr1, along with reciprocal bolt-grafting experiments, demonstrated that CEPR1 acts locally in the reproductive bolt to control yield and seed size. CEPR1 is expressed throughout the vasculature of reproductive organs, including in the chalazal seed coat, but not in other seed tissues. This expression pattern implies that CEPR1 controls yield and seed size from the maternal tissue. The complementation of cepr1 mutants with transgenic CEPR1 rescued the yield and other phenotypes. Transcriptional analyses of cepr1 bolts showed alterations in the expression levels of several genes of the CEP-CEPR1 and nitrogen homeostasis pathways. This transcriptional profile was consistent with cepr1 bolts being nitrogen deficient and with a reproductive tissue-specific function for CEP-CEPR1 signaling. The results reveal a local role for CEPR1 in the maternal reproductive tissue in determining seed size and yield, likely via the control of nitrogen delivery to the reproductive sinks.

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.

Senescence and Defense Pathways Contribute to Heterosis.

Hybrids are used extensively in agriculture due to their superior performance in seed yield and plant growth, yet the molecular mechanisms underpinning hybrid performance are not well understood. Recent evidence has suggested that a decrease in basal defense response gene expression regulated by reduced levels of salicylic acid (SA) may be important for vigor in certain hybrid combinations. Decreasing levels of SA in the Arabidopsis (Arabidopsis thaliana) accession C24 through the introduction of the SA catabolic enzyme salicylate1 hydroxylase (NahG) increases plant size, phenocopying the large-sized C24/Landsberg erecta (Ler) F1 hybrids. C24♀ × Ler♂ F1 hybrids and C24 NahG lines shared differentially expressed genes and pathways associated with plant defense and leaf senescence including decreased expression of SA biosynthetic genes and SA response genes. The expression of TL1 BINDING TRANSCRIPTION FACTOR1, a key regulator in resource allocation between growth and defense, was decreased in both the F1 hybrid and the C24 NahG lines, which may promote growth. Both C24 NahG lines and the F1 hybrids showed decreased expression of the key senescence-associated transcription factors WRKY53, NAC-CONTAINING PROTEIN29, and ORESARA1 with a delayed onset of senescence compared to C24 plants. The delay in senescence resulted in an extension of the photosynthetic period in the leaves of F1 hybrids compared to the parental lines, potentially allowing each leaf to contribute more resources toward growth.

Phosphorylation influences water and ion channel function of AtPIP2;1.

The phosphorylation state of two serine residues within the C-terminal domain of AtPIP2;1 (S280, S283) regulates its plasma membrane localization in response to salt and osmotic stress. Here, we investigated whether the phosphorylation state of S280 and S283 also influence AtPIP2;1 facilitated water and cation transport. A series of single and double S280 and S283 phosphomimic and phosphonull AtPIP2;1 mutants were tested in heterologous systems. In Xenopus laevis oocytes, phosphomimic mutants AtPIP2;1 S280D, S283D, and S280D/S283D had significantly greater ion conductance for Na+ and K+ , whereas the S280A single phosphonull mutant had greater water permeability. We observed a phosphorylation-dependent inverse relationship between AtPIP2;1 water and ion transport with a 10-fold change in both. The results revealed that phosphorylation of S280 and S283 influences the preferential facilitation of ion or water transport by AtPIP2;1. The results also hint that other regulatory sites play roles that are yet to be elucidated. Expression of the AtPIP2;1 phosphorylation mutants in Saccharomyces cerevisiae confirmed that phosphorylation influences plasma membrane localization, and revealed higher Na+ accumulation for S280A and S283D mutants. Collectively, the results show that phosphorylation in the C-terminal domain of AtPIP2;1 influences its subcellular localization and cation transport capacity.

Deciphering aquaporin regulation and roles in seed biology.

Seeds are the typical dispersal and propagation units of angiosperms and gymnosperms. Water movement into and out of seeds plays a crucial role from the point of fertilization through to imbibition and seed germination. A class of membrane intrinsic proteins called aquaporins (AQPs) assist with the movement of water and other solutes within seeds. These highly diverse and abundant proteins are associated with different processes in the development, longevity, imbibition, and germination of seed. However, there are many AQPs encoded in a plant's genome and it is not yet clear how, when, or which AQPs are involved in critical stages of seed biology. Here we review the literature to examine the evidence for AQP involvement in seeds and analyse Arabidopsis seed-related transcriptomic data to assess which AQPs are likely to be important in seed water relations and explore additional roles for AQPs in seed biology.

Twenty-four-nucleotide siRNAs produce heritable trans-chromosomal methylation in F1 Arabidopsis hybrids.

Hybrid Arabidopsis plants undergo epigenetic reprogramming producing decreased levels of 24-nt siRNAs and altered patterns of DNA methylation that can affect gene expression. Driving the changes in methylation are the processes trans-chromosomal methylation (TCM) and trans-chromosomal demethylation (TCdM). In TCM/TCdM the methylation state of one allele is altered to resemble the other allele. We show that Pol IV-dependent sRNAs are required to establish TCM events. The changes in DNA methylation and the associated changes in sRNA levels in the F1 hybrid can be maintained in subsequent generations and affect hundreds of regions in the F2 epigenome. The inheritance of these altered epigenetic states varies in F2 individuals, resulting in individuals with genetically identical loci displaying different epigenetic states and gene expression profiles. The change in methylation at these regions is associated with the presence of sRNAs. Loci without any sRNA activity can have altered methylation states, suggesting that a sRNA-independent mechanism may also contribute to the altered methylation state of the F1 and F2 generations.

Hybrid mimics and hybrid vigor in Arabidopsis.

F1 hybrids can outperform their parents in yield and vegetative biomass, features of hybrid vigor that form the basis of the hybrid seed industry. The yield advantage of the F1 is lost in the F2 and subsequent generations. In Arabidopsis, from F2 plants that have a F1-like phenotype, we have by recurrent selection produced pure breeding F5/F6 lines, hybrid mimics, in which the characteristics of the F1 hybrid are stabilized. These hybrid mimic lines, like the F1 hybrid, have larger leaves than the parent plant, and the leaves have increased photosynthetic cell numbers, and in some lines, increased size of cells, suggesting an increased supply of photosynthate. A comparison of the differentially expressed genes in the F1 hybrid with those of eight hybrid mimic lines identified metabolic pathways altered in both; these pathways include down-regulation of defense response pathways and altered abiotic response pathways. F6 hybrid mimic lines are mostly homozygous at each locus in the genome and yet retain the large F1-like phenotype. Many alleles in the F6 plants, when they are homozygous, have expression levels different to the level in the parent. We consider this altered expression to be a consequence of transregulation of genes from one parent by genes from the other parent. Transregulation could also arise from epigenetic modifications in the F1. The pure breeding hybrid mimics have been valuable in probing the mechanisms of hybrid vigor and may also prove to be useful hybrid vigor equivalents in agriculture.

Hormone-regulated defense and stress response networks contribute to heterosis in Arabidopsis F1 hybrids.

Plant hybrids are extensively used in agriculture to deliver increases in yields, yet the molecular basis of their superior performance (heterosis) is not well understood. Our transcriptome analysis of a number of Arabidopsis F1 hybrids identified changes to defense and stress response gene expression consistent with a reduction in basal defense levels. Given the reported antagonism between plant immunity and growth, we suggest that these altered patterns of expression contribute to the greater growth of the hybrids. The altered patterns of expression in the hybrids indicate decreases to the salicylic acid (SA) biosynthesis pathway and increases in the auxin [indole-3-acetic acid (IAA)] biosynthesis pathway. SA and IAA are hormones known to control stress and defense responses as well as plant growth. We found that IAA-targeted gene activity is frequently increased in hybrids, correlating with a common heterotic phenotype of greater leaf cell numbers. Reduced SA concentration and target gene responses occur in the larger hybrids and promote increased leaf cell size. We demonstrated the importance of SA action to the hybrid phenotype by manipulating endogenous SA concentrations. Increasing SA diminished heterosis in SA-reduced hybrids, whereas decreasing SA promoted growth in some hybrids and phenocopied aspects of hybrid vigor in parental lines. Pseudomonas syringae infection of hybrids demonstrated that the reductions in basal defense gene activity in these hybrids does not necessarily compromise their ability to mount a defense response comparable to the parents.

Carbon dioxide and water transport through plant aquaporins.

Aquaporins are channel proteins that function to increase the permeability of biological membranes. In plants, aquaporins are encoded by multigene families that have undergone substantial diversification in land plants. The plasma membrane intrinsic proteins (PIPs) subfamily of aquaporins is of particular interest given their potential to improve plant water relations and photosynthesis. Flowering plants have between 7 and 28 PIP genes. Their expression varies with tissue and cell type, through development and in response to a variety of factors, contributing to the dynamic and tissue specific control of permeability. There are a growing number of PIPs shown to act as water channels, but those altering membrane permeability to CO2 are more limited. The structural basis for selective substrate specificities has not yet been resolved, although a few key amino acid positions have been identified. Several regions important for dimerization, gating and trafficking are also known. PIP aquaporins assemble as tetramers and their properties depend on the monomeric composition. PIPs control water flux into and out of veins and stomatal guard cells and also increase membrane permeability to CO2 in mesophyll and stomatal guard cells. The latter increases the effectiveness of Rubisco and can potentially influence transpiration efficiency.

Inheritance of Trans Chromosomal Methylation patterns from Arabidopsis F1 hybrids.

Hybridization in plants leads to transinteractions between the parental genomes and epigenomes that can result in changes to both 24 nt siRNA and cytosine methylation ((m)C) levels in the hybrid. In Arabidopsis the principle processes altering the hybrid methylome are Trans Chromosomal Methylation (TCM) and Trans Chromosomal deMethylation (TCdM) in which the (m)C pattern of a genomic segment attains the same (m)C pattern of the corresponding segment on the other parental chromosome. We examined two loci that undergo TCM/TCdM in the Arabidopsis C24/Landsberg erecta (Ler) F1 hybrids, which show patterns of inheritance dependent on the properties of the particular donor and recipient chromosomal segments. At At1g64790 the TCM- and TCdM-derived (m)C patterns are maintained in the F2 generation but are transmitted in outcrosses or backcrosses only by the C24 genomic segment. At a region between and adjacent to At3g43340 and At3g43350, the originally unmethylated Ler genomic segment receives the C24 (m)C pattern in the F1, which is then maintained in backcross plants independent of the presence of the parental C24 segment. In backcrosses to an unmethylated Ler allele, the newly methylated F1 Ler segment may act as a TCM source in a process comparable to paramutation in maize. TCM-derived (m)C patterns are associated with reduced expression of both At3g43340 and At3g43350 in F1 and F2 plants, providing support for such events influencing the transcriptome. The inheritance of the F1 (m)C patterns and the segregation of other genetic and epigenetic determinants may contribute to the reduced hybrid vigor in the F2 and subsequent generations.

The role of epigenetics in hybrid vigour.

Hybrid vigour, or heterosis, refers to the increased yield and biomass of hybrid offspring relative to the parents. Although this has been exploited in plants for agriculture and horticulture, the molecular and cellular mechanisms underlying hybrid vigour are largely unknown. Genetic analyses show that there are a large number of quantitative trait loci (QTLs) that contribute to the heterotic phenotype, indicating that it is a complex phenomenon. Gene expression in hybrids is regulated by the interactions of the two parental epigenetic systems and the underlying genomes. Increasing understanding of the interplay of small RNA (sRNA) molecules, DNA methylation, and histone marks provides new opportunities to define the basis of hybrid vigour and to understand why F1 heterosis is not passed on to subsequent generations. We discuss recent findings that suggest the existence of several pathways that alter DNA methylation patterns, which may lead to transcriptional changes resulting in the heterotic phenotype.

Epigenetic Changes in Hybrids.

Genome-wide approaches to the study of hybrid vigor have identified epigenetic changes in the hybrid nucleus in Arabidopsis (Arabidopsis thaliana), maize (Zea mays), and rice (Oryza sativa). DNA methylation associated with 24-nucleotide small interfering RNAs exhibits transallelic effects in hybrids of Arabidopsis and other species. Some of the transmethylation changes are inherited and some affect gene expression. Hybrids have larger leaves than those of the parents and have increases in cell size and number. The increased leaf size results in a greater photosynthetic capacity, which may support the increased vegetative and reproductive yields of the F1 hybrids. Genes and metabolic pathways that have altered expression relative to the parents include loci involved in responses to hormones and to biotic and abiotic stress. Whereas epigenetically induced changes in gene expression may contribute to hybrid vigor, the link between the transcriptional changes and the hybrid phenotype is not confirmed. Recurrent selection of high yielding F1 lines from the F2/F3 of a number of crops has fixed heterosis yields in pure breeding lines. These hybrid-like lines may have valuable applications in crop systems.

Intraspecific Arabidopsis hybrids show different patterns of heterosis despite the close relatedness of the parental genomes.

Heterosis is important for agriculture; however, little is known about the mechanisms driving hybrid vigor. Ultimately, heterosis depends on the interactions of specific alleles and epialleles provided by the parents, which is why hybrids can exhibit different levels of heterosis, even within the same species. We characterize the development of several intraspecific Arabidopsis (Arabidopsis thaliana) F1 hybrids that show different levels of heterosis at maturity. We identify several phases of heterosis beginning during embryogenesis and culminating in a final phase of vegetative maturity and seed production. During each phase, the hybrids show different levels and patterns of growth, despite the close relatedness of the parents. For instance, during the vegetative phases, the hybrids develop larger leaves than the parents to varied extents, and they do so by exploiting increases in cell size and cell numbers in different ratios. Consistent with this finding, we observed changes in the expression of genes known to regulate leaf size in developing rosettes of the hybrids, with the patterns of altered expression differing between combinations. The data show that heterosis is dependent on changes in development throughout the growth cycle of the hybrid, with the traits of mature vegetative biomass and reproductive yield as cumulative outcomes of heterosis at different levels, tissues, and times of development.

Trans chromosomal methylation in Arabidopsis hybrids.

The heterotic hybrid offspring of Arabidopsis accessions C24 and Landsberg erecta have altered methylomes. Changes occur most frequently at loci where parental methylation levels are different. There are context-specific biases in the nonadditive methylation patterns with (m)CG generally increased and (m)CHH decreased relative to the parents. These changes are a result of two main mechanisms, Trans Chromosomal Methylation and Trans Chromosomal deMethylation, where the methylation level of one parental allele alters to resemble that of the other parent. Regions of altered methylation are enriched around genic regions and are often correlated with changes in siRNA levels. We identified examples of genes with altered expression likely to be due to methylation changes and suggest that in crosses between the C24 and Ler accessions, epigenetic controls can be important in the generation of altered transcription levels that may contribute to the increased biomass of the hybrids.

Changes in 24-nt siRNA levels in Arabidopsis hybrids suggest an epigenetic contribution to hybrid vigor.

Intraspecific hybrids between the Arabidopsis thaliana accessions C24 and Landsberg erecta have strong heterosis. The reciprocal hybrids show a decreased level of 24-nt small RNA (sRNA) relative to the parents with the decrease greatest for those loci where the parents had markedly different 24-nt sRNA levels. The genomic regions with reduced 24-nt sRNA levels were largely associated with genes and their flanking regions indicating a potential effect on gene expression. We identified several examples of genes with altered 24-nt sRNA levels that showed correlated changes in DNA methylation and expression levels. We suggest that such epigenetically generated differences in gene activity may contribute to hybrid vigor and that the epigenetic diversity between ecotypes provides increased allelic (epi-allelic) variability that could contribute to heterosis.

Clinical effectiveness of drop-in mental health services at paediatric hospitals: A non-randomised multi-site study for children and young people and their families - study protocol.

BACKGROUND: Despite the high prevalence of mental health difficulties in children and young people with long-term health conditions (LTCs), these difficulties and experiences are often overlooked and untreated. Previous research demonstrated the effectiveness of psychological support provided via a drop-in mental health centre located in a paediatric hospital. The aim of this prospective non-randomised single-arm multi-centre interventional study is to determine the clinical effectiveness of drop-in mental health services when implemented at paediatric hospitals in England. METHODS: It is hypothesised that families who receive psychological interventions through the drop-in services will show improved emotional and behavioural symptoms. Outcomes will be measured at baseline and at 6-month follow-up. The primary outcome is the difference in the total difficulties score on the Strengths and Difficulties Questionnaire (SDQ) reported by parent or child at 6 months. Secondary outcomes include self and parent reported Paediatric Quality of Life Inventory (PedsQL), self-reported depression (PHQ-9) and anxiety measures (GAD-7) and family satisfaction (CSQ-8). DISCUSSION: This trial aims to determine the clinical effectiveness of providing psychological support in the context of LTCs through drop-in mental health services at paediatric hospitals in England. These findings will contribute to policies and practice addressing mental health needs in children and young people with other long-term health conditions. TRIAL REGISTRATION: ISRCTN15063954, Registered on 9 December 2022.

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.