Roles of Aquaporins in Setaria viridis Stem Development and Sugar Storage

Setaria viridis is a C4 grass used as a model for bioenergy feedstocks. The elongating internodes in developing S. viridis stems grow from an intercalary meristem at the base, and progress acropetally toward fully expanded cells that store sugar. During stem development and maturation, water flow is a driver of cell expansion and sugar delivery. As aquaporin proteins are implicated in regulating water flow, we analyzed elongating and mature internode transcriptomes to identify putative aquaporin encoding genes that had particularly high transcript levels during the distinct stages of internode cell expansion and maturation. We observed that SvPIP2;1 was highly expressed in internode regions undergoing cell expansion, and SvNIP2;2 was highly expressed in mature sugar accumulating regions. Gene co-expression analysis revealed SvNIP2;2 expression was highly correlated with the expression of five putative sugar transporters expressed in the S. viridis internode. To explore the function of the proteins encoded by SvPIP2;1 and SvNIP2;2, we expressed them in Xenopus laevis oocytes and tested their permeability to water. SvPIP2;1 and SvNIP2;2 functioned as water channels in X. laevis oocytes and their permeability was gated by pH. Our results indicate that SvPIP2;1 may function as a water channel in developing stems undergoing cell expansion and SvNIP2;2 is a candidate for retrieving water and possibly a yet to be determined solute from mature internodes. Future research will investigate whether changing the function of these proteins influences stem growth and sugar yield in S. viridis

Sucrose transporters of higher plants

Recent advances have provided new insights into how sucrose is moved from sites of synthesis to sites of utilisation or storage in sink organs. Sucrose transporters play a central role, as they orchestrate sucrose allocation both intracellularly and at the whole plant level. Sucrose produced in mesophyll cells of leaves may be effluxed into the apoplasm of mesophyll or phloem parenchyma cells by a mechanism that remains elusive, but experimentally consistent with facilitated transport or energy-dependent sucrose/H⁺ antiport. From the apoplasm, sucrose/H⁺ symporters transport sucrose across the plasma membrane of cells making up the sieve element/companion cell (SE/CC) complex, the long distance conduits of the phloem. Phloem unloading of sucrose in key sinks such as developing seeds involves two sequential transport steps, sucrose efflux followed by sucrose influx. Besides plasma membrane specific sucrose transporters, sucrose transporters on the tonoplast contribute to the capacity for elevated sucrose accumulation in storage organs such as sugar beet roots or sugarcane culms. Except for several sucrose facilitators from seed coats of some leguminous plants all sucrose transporters cloned to date, including recently identified vacuolar sucrose transporters, have been characterised as sucrose/H⁺ symporters. Transporters functioning to efflux sucrose into source or sink apoplasms as well as those supporting sucrose/H⁺ antiport on tonoplasts, remain to be identified. Sucrose transporter expression and activity is tightly regulated at the transcriptional, post-transcriptional as well as post-translational levels. Light quality and phytohormones play essential regulatory roles and the sucrose molecule itself functions as a signal

Profiling of the Differential Abundance of Drought and Salt Stress-Responsive MicroRNAs Across Grass Crop and Genetic Model Plant Species

In recent years, it has become readily accepted among interdisciplinary agriculturalists that the current global crop yield to land capability ratio is significantly insufficient to achieve food security for the predicted population of 9.5 billion individuals by the year 2050. This issue is further compounded by the: (1) food versus biofuel debate; (2) decreasing availability of arable land; (3) required reductions to the extensive and ongoing environmental damage caused by either poor agricultural practices or agriculture expansion, and; (4) increasingly unfavorable (duration and severity) crop cultivation conditions that accompany man-made climate change, driven by ever-expanding urbanization and its associated industrial practices. Mounting studies are repeatedly highlighting the critical importance of linking genotypes to agronomically beneficial phenotypes and/or using a molecular approach to help address this global crisis, as “simply” clearing the remaining natural ecosystems of the globe for the cultivation of additional, non-modified crops is not efficient, nor is this practice sustainable. The majority of global food crop production is sourced from a small number of members of the Poaceae family of grasses, namely; maize (Zea mays L.), wheat (Triticum aestivum L.) and rice (Oryza sativa L.). It is, therefore, of significant concern that all three of these Poaceae grass species are susceptible to a range of abiotic stresses, including drought and salt stress. Highly conserved among monocotyledonous and dicotyledonous plant species, microRNAs (miRNAs) are now well-established master regulators of gene expression, influencing all aspects of plant development, mediating defense responses against pathogens and adaptation to environmental stress. Here we investigate the variation in the abundance profiles of six known abiotic stress-responsive miRNAs, following exposure to salt and drought stress across these three key Poaceae grass crop species as well as to compare these profiles to those obtained from the well-established genetic model plant species, Arabidopsis thaliana (L.) Heynh. Additionally, we outline the variables that are the most likely primary contributors to instances of differential miRNA abundance across the assessed species following drought or salt stress exposure, specifically; (1) identifying variations in the experimental conditions and/or methodology used to assess miRNA abundance, and; (2) the distribution of regulatory transcription factor binding sites within the putative promoter region of a MICRORNA (MIR) gene that encodes the highly conserved, stress-responsive miRNA. We also discuss the emerging role that non-conserved, species-specific miRNAs play in mediating a plant’s response to drought or salt stress

Sucrose partitioning between vascular bundles and storage parenchyma in the sugarcane stem: A potential role for the ShSUT1 sucrose transporter

A transporter with homology to the SUT/SUC family of plant sucrose transporters was isolated from a sugarcane (Saccharum hybrid) stem cDNA library. The gene, designated ShSUT1, encodes a protein of 517 amino acids, including 12 predicted membrane-spanning domains and a large central cytoplasmic loop. ShSUT1 was demonstrated to be a functional sucrose transporter by expression in yeast. The estimated K for sucrose of the ShSUT1 transporter was 2 mM at pH 5.5. ShSUT1 was expressed predominantly in mature leaves of sugarcane that were exporting sucrose and in stem internodes that were actively accumulating sucrose. Immunolocalization with a ShSUT1-specific antiserum identified the protein in cells at the periphery of the vascular bundles in the stem. These cells became lignified and suberized as stem development proceeded, forming a barrier to apoplasmic solute movement. However, the movement of the tracer dye, carboxyfluorescein from phloem to storage parenchyma cells suggested that symplasmic connections are present. ShSUT1 may have a role in partitioning of sucrose between the vascular tissue and sites of storage in the parenchyma cells of sugarcane stem internodes

Reference gene identification for reliable normalisation of quantitative RT-PCR data in Setaria viridis

Abstract Background Quantitative real-time polymerase chain reaction (RT-qPCR) is the key platform for the quantitative analysis of gene expression in a wide range of experimental systems and conditions. However, the accuracy and reproducibility of gene expression quantification via RT-qPCR is entirely dependent on the identification of reliable reference genes for data normalisation. Green foxtail (Setaria viridis) has recently been proposed as a potential experimental model for the study of C4 photosynthesis and is closely related to many economically important crop species of the Panicoideae subfamily of grasses, including Zea mays (maize), Sorghum bicolor (sorghum) and Sacchurum officinarum (sugarcane). Setaria viridis (Accession 10) possesses a number of key traits as an experimental model, namely; (i) a small sized, sequenced and well annotated genome; (ii) short stature and generation time; (iii) prolific seed production, and; (iv) is amendable to Agrobacterium tumefaciens-mediated transformation. There is currently however, a lack of reference gene expression information for Setaria viridis (S. viridis). We therefore aimed to identify a cohort of suitable S. viridis reference genes for accurate and reliable normalisation of S. viridis RT-qPCR expression data. Results Eleven putative candidate reference genes were identified and examined across thirteen different S. viridis tissues. Of these, the geNorm and NormFinder analysis software identified SERINE/THERONINE-PROTEIN PHOSPHATASE 2A (PP2A), 5′-ADENYLYLSULFATE REDUCTASE 6 (ASPR6) and DUAL SPECIFICITY PHOSPHATASE (DUSP) as the most suitable combination of reference genes for the accurate and reliable normalisation of S. viridis RT-qPCR expression data. To demonstrate the suitability of the three selected reference genes, PP2A, ASPR6 and DUSP, were used to normalise the expression of CINNAMYL ALCOHOL DEHYDROGENASE (CAD) genes across the same tissues. Conclusions This approach readily demonstrated the suitably of the three selected reference genes for the accurate and reliable normalisation of S. viridis RT-qPCR expression data. Further, the work reported here forms a highly useful platform for future gene expression quantification in S. viridis and can also be potentially directly translatable to other closely related and agronomically important C4 crop species