Arabidopsis plasma membrane intrinsic protein (AtPIP2;1) is implicated in a salinity conditional influence on seed germination

Published Online 6/6/2023 OnlinePublDynamic changes in aquaporin gene expression occur during seed germination. One example is the ~30-fold increase in Arabidopsis thaliana PIP2;1 transcripts within 24 h of seed imbibition. To investigate whether AtPIP2;1 can influence seed germination wild-type Columbia-0, single (Atpip2;1) and double (Atpip2;1-Atpip2;2) loss-of-function mutants, along with transgenic 2x35S::AtPIP2;1 over-expressing (OE) lines and null-segregant controls, were examined. The various genotypes were germinated in control and saline (75 mM NaCl treatment) conditions and tested for germination efficiency, imbibed seed maximum cross sectional (MCS) area, imbibed seed mass, and seed Na+ and K+ content. Seed lacking functional AtPIP2;1 and/or AtPIP2;2 proteins or constitutively over-expressing AtPIP2;1, had delayed germination in saline conditions relative to wild-type and null-segregant seed, respectively. Exposure to saline germination conditions resulted in Atpip2;1 mutants having greater imbibed seed mass and less accumulated Na+ than wild-type, whereas lines over-expressing AtPIP2;1 had reduced imbibed seed mass and greater seed K+ content than null-segregant control seed. The results imply a role for AtPIP2;1 in seed germination processes, whether directly through its capacity for water and ion transport or H2O2 signalling, or indirectly through potentially triggering dynamic differential regulation of other aquaporins expressed during germination. Future research will aid in dissecting the aquaporin functions influencing germination and may lead to novel solutions for optimising germination in sub-optimal conditions, such as saline soils.Phan Thi Thanh Hoai, Jiaen Qiu, Michael Groszmann, Annamaria De Rosa, Stephen D. Tyerman and Caitlin S. Byr

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

PUBLISHED 19 January 2023Introduction: 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 AQPbiology 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.Michael Groszmann, Annamaria De Rosa, Weihua Chen, Jiaen Qiu, Samantha A. McGaughey, Caitlin S. Byrt and John R. Evan

Overexpression of HvCslF6 in barley grain alters carbohydrate partitioning plus transfer tissue and endosperm development

In cereal grain sucrose is converted into storage carbohydrates; mainly starch, fructan and (1,3;1,4)-β-glucan (MLG). Previously, endosperm-specific overexpression of the HvCslF6 gene in hull-less barley resulted in high MLG and low starch content in mature grains. Morphological changes included inwardly elongated aleurone cells, irregular cell shapes of peripheral endosperm and smaller starch granules of starchy endosperm. Here we explored the physiological basis for these defects by investigating how changes in carbohydrate composition of developing grain impact mature grain morphology. Augmented MLG coincided with increased levels of soluble carbohydrates in the cavity and endosperm at the storage phase. Transcript levels of genes relating to cell wall, starch, sucrose and fructan metabolism were perturbed in all tissues. The cell walls of endosperm transfer cells (ETC) in transgenic grain were thinner and showed reduced mannan labelling relative to wild type. At the early storage phase rupture of the non-uniformly developed ETC and disorganization of adjacent endosperm cells was observed. Soluble sugars accumulated in the developing grain cavity, suggesting a disturbance of carbohydrate flow from the cavity towards the endosperm, resulting in a shrunken mature grain phenotype. Our findings demonstrate the importance of regulating carbohydrate partitioning in maintenance of grain cellularization and filling processes.Wai Li Lim, Helen M. Collins, Caitlin S. Byrt, Jelle Lahnstein, Neil J. Shirley, Matthew K. Aubert, Matthew R. Tucker, Manuela Peukert, Andrea Matros, and Rachel A. Burto

C4 plants as biofuel feedstocks: optimising biomass production and feedstock quality from a lignocellulosic perspective

The main feedstocks for bioethanol are sugarcane (Saccharum officinarum) and maize (Zea mays), both of which are C4 grasses, highly efficient at converting solar energy into chemical energy, and both are food crops. As the systems for lignocellulosic bioethanol production become more efficient and cost effective, plant biomass from any source may be used as a feedstock for bioethanol production. Thus, a move away from using food plants to make fuel is possible, and sources of biomass such as wood from forestry and plant waste from cropping may be used. However, the bioethanol industry will need a continuous and reliable supply of biomass that can be produced at a low cost and with minimal use of water, fertilizer and arable land. As many C4 plants have high light, water and nitrogen use efficiency, as compared with C3 species, they are ideal as feedstock crops. We consider the productivity and resource use of a number of candidate plant species, and discuss biomass ‘quality’, that is, the composition of the plant cell wall.Caitlin Byrt, Christopher P.L. Grof and Robert T. Furban

Regulating root aquaporin function in response to changes in salinity

Rapid changes in soil salinity present plants with an osmotic stress. If the concentration of salt in the soil water is too high then water may flow from the plant roots back into the soil. As part of an adaptive response, the regulation of proteins associated with water transport, such as aquaporins, is altered. The change in regulation of aquaporins in roots in response to salt treatments occurs at transcriptional and post‐translational levels. Cell specific changes in aquaporin transcript levels, protein abundance, subcellular localisation, protein phosphorylation, and protein:protein interactions have been observed. It has recently been revealed that a subset of plant aquaporins can function both as water and as ion channels. Both Arabidopsis PIP2;1 and PIP2;2 function as water and ion channels in heterologous systems. AtPIP2;1 and AtPIP2;2 ionic conductance was carried by sodium (Na⁺), indicating that a subset of plant PIP2s could have a role in Na+ transport in plants. We show quantitatively how AtPIP2;1 may account for the calcium (Ca²⁺) and pH sensitive Arabidopsis root non‐selective cation channel. Salt treatments are known to result in a change in AtPIP2;1 phosphorylation, and in Arabidopsis roots this is associated with internalisation of AtPIP2;1 into pre‐vacuolar compartments. Here we review how the regulation of aquaporins, in particular PIP2s, changes in root tissues in response to salt treatments.Samantha A. McGaughey, Jiaen Qiu, Stephen D. Tyerman and Caitlin S. Byr

Osmotic adjustment and energy limitations to plant growth in saline soil

Plant roots must exclude almost all of the Na+ and Cl- in saline soil while taking up water, otherwise these ions would build up to high concentrations in leaves. Plants evaporate c. 50 times more water than they retain, so 98% exclusion would result in shoot NaCl concentrations equal to that of the external medium. Taking up just 2% of the NaCl allows a plant to osmotically adjust the Na+ and Cl- in vacuoles, while organic solutes provide the balancing osmotic pressure in the cytoplasm. We quantify the costs of this exclusion by roots, the regulation of Na+ and Cl- transport through the plant, and the costs of osmotic adjustment with organic solutes in roots.Rana Munns, John B. Passioura, Timothy D. Colmer, Caitlin S. Byr

Roles of membrane transporters: connecting the dots from sequence to phenotype

Background: Plant membrane transporters are involved in diverse cellular processes underpinning plant physiology, such as nutrient acquisition, hormone movement, resource allocation, exclusion or sequestration of various solutes from cells and tissues, and environmental and developmental signalling. A comprehensive characterization of transporter function is therefore key to understanding and improving plant performance. Scope and conclusions: In this review, we focus on the complexities involved in characterizing transporter function and the impact that this has on current genomic annotations. Specific examples are provided that demonstrate why sequence homology alone cannot be relied upon to annotate and classify transporter function, and to show how even single amino acid residue variations can influence transporter activity and specificity. Misleading nomenclature of transporters is often a source of confusion in transporter characterization, especially for people new to or outside the field. Here, to aid researchers dealing with interpretation of large data sets that include transporter proteins, we provide examples of transporters that have been assigned names that misrepresent their cellular functions. Finally, we discuss the challenges in connecting transporter function at the molecular level with physiological data, and propose a solution through the creation of new databases. Further fundamental in-depth research on specific transport (and other) proteins is still required; without it, significant deficiencies in large-scale data sets and systems biology approaches will persist. Reliable characterization of transporter function requires integration of data at multiple levels, from amino acid residue sequence annotation to more in-depth biochemical, structural and physiological studies.Rakesh David, Caitlin S. Byrt, Stephen D. Tyerman, Matthew Gilliham, and Stefanie Weg

Adaptable and multifunctional ion-conducting aquaporins

Aquaporins function as water and neutral solute channels, signaling hubs, disease virulence factors, and metabolon components. We consider plant aquaporins that transport ions compared to some animal counterparts. These are candidates for important, as yet unidentified, cation and anion channels in plasma, tonoplast, and symbiotic membranes. For those individual isoforms that transport ions, water, and gases, the permeability spans 12 orders of magnitude. This requires tight regulation of selectivity via protein interactions and posttranslational modifications. A phosphorylation-dependent switch between ion and water permeation in AtPIP2;1 might be explained by coupling between the gates of the four monomer water channels and the central pore of the tetramer. We consider the potential for coupling between ion and water fluxes that could form the basis of an electroosmotic transducer. A grand challenge in understanding the roles of ion transporting aquaporins is their multifunctional modes that are dependent on location, stress, time, and development. Expected final online publication date for the Annual Review of Plant Biology, Volume 72 is May 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.Stephen D. Tyerman, Samantha A. McGaughey, Jiaen Qiu, Andrea J. Yool and Caitlin S. Byr

Exploring aquaporin functions during changes in leaf water potential

Maintenance of optimal leaf tissue humidity is important for plant productivity and food security. Leaf humidity is influenced by soil and atmospheric water availability, by transpiration and by the coordination of water flux across cell membranes throughout the plant. Flux of water and solutes across plant cell membranes is influenced by the function of aquaporin proteins. Plants have numerous aquaporin proteins required for a multitude of physiological roles in various plant tissues and the membrane flux contribution of each aquaporin can be regulated by changes in protein abundance, gating, localisation, posttranslational modifications, protein:protein interactions and aquaporin stoichiometry. Resolving which aquaporins are candidates for influencing leaf humidity and determining how their regulation impacts changes in leaf cell solute flux and leaf cavity humidity is challenging. This challenge involves resolving the dynamics of the cell membrane aquaporin abundance, aquaporin sub-cellular localisation and location-specific post-translational regulation of aquaporins in membranes of leaf cells during plant responses to changes in water availability and determining the influence of cell signalling on aquaporin permeability to a range of relevant solutes, as well as determining aquaporin influence on cell signalling. Here we review recent developments, current challenges and suggest open opportunities for assessing the role of aquaporins in leaf substomatal cavity humidity regulation.Caitlin S. Byrt, Rose Y. Zhang, Isobel Magrath, Kai Xun Chan, Annamaria De Rosa, and Samantha McGaughe

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.Phan T. T. Hoai, Stephen D. Tyerman ... Matthew Tucker ... Jiaen Qiu ... Michael Groszmann and Caitlin S. Byrt ... et al