McGaughey SA et al. (2016), Roles of Aquaporins in Setaria viridis Stem Dev...

XB-ART-52880

Front Plant Sci 2016 Jan 01;7:1815. doi: 10.3389/fpls.2016.01815.

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Roles of Aquaporins in Setaria viridis Stem Development and Sugar Storage.

McGaughey SA , Osborn HL , Chen L , Pegler JL , Tyerman SD , Furbank RT , Byrt CS , Grof CP .

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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.

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Species referenced: Xenopus laevis
Genes referenced: app

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	FIGURE 1. Phylogenetic tree based on protein sequences of aquaporins from Setaria viridis and Zea mays. S. viridis aquaporins were identified in the genome via HMMER search using aquaporins sequences from Arabidopsis, barley, maize, and rice. Maize aquaporins were included in the phylogenetic tree for ease of interpretation. The addition of aquaporin sequences from other grasses did not change the groupings. Tree was generated by neighbor-joining method using the Geneious Tree Builder program, Geneious 9.0.2. The scale bar indicates the evolutionary distance, expressed as changes per amino acid residue. Aquaporins can be grouped into four subfamilies: PIPs (plasma membrane intrinsic proteins), TIPs (tonoplast intrinsic proteins), NIPs (nodulin-like intrinsic proteins), and SIPs (small basic intrinsic proteins). ∗Sevir.5G141800.1 protein sequence is truncated, 112 amino acids in length. ‡SvNIP5;3 (Sevir.6G06000.1) may have a related pseudogene Sevir.6G061300.1.
	FIGURE 2. Expression of putative aquaporins across the developmental zones of an elongating S. viridis internode. (A) Schematic of the developmental regions in an elongating internode of S. viridis as reported by Martin et al. (2016): meristematic zone, residing at the base of the internode, where cell division occurs; the cell expansion zone where cells undergo turgor driven expansion; transitional zone where cells begin to differentiate and synthesize secondary cell walls; and the maturation zone whereby expansion, differentiation and secondary cell wall synthesis cease and sugar is accumulated. (B) The expression profiles of putative S. viridis aquaporins, as identified by phylogeny to Z. mays aquaporins, were mined in the S. viridis elongating internode transcriptome (Martin et al., 2016). RNA-seq data is presented as mean FPKM ± SEM for four biological replicates from each developmental zone.
	FIGURE 3. Comparison of relative fold changes between RNA-seq and RT-qPCR of SvPIP2;1 and SvNIP2;2 in an elongating internode of S. viridis. (A) SvPIP2;1. (B) SvNIP2;2. Data is mean relative fold change in expression ± SEM. Data for RNA-seq and RT-qPCR was normalized relative to the cell expansion zone expression level.
	FIGURE 4. Co-expression network of putative S. viridis aquaporin and sugar transporter genes identified in an elongating internode. The co-expressed gene network was generated from the stem specific aquaporins (Figure 2) and sugar transporters identified in the S. viridis elongating internode transcriptome reported by Martin et al. (2016). Raw FPKM values were Log2 transformed and Pearson’s correlation coefficients (0.8–1.0) were calculated in the MetScape app in Cytoscape v3.4.0. Sugar transporters in the S. viridis elongating internode were identified by homology to rice sugar transporter genes (Supplementary Figures S2–S4). Sugar transport related genes are color filled with blue and aquaporin genes with orange. SvNIP2;2 and SvPIP2;1 are in bold font.
	FIGURE 5. Osmotic permeability (Pf) of Xenopus laevis oocytes injected with SvNIP2;2 and SvPIP2;1 cRNA. (A) Osmotic permeability (Pf) of water (H2O) injected and SvNIP2;2 and SvPIP2;1 cRNA (46 ng) injected oocytes. Oocytes were transferred into a hypo-osmotic solution, pH 7.4, and Pf was calculated by video monitoring of the rate of oocyte swelling. (B) Effect of lowering oocyte cytosolic pH on osmotic permeability (Pf) of H2O and SvNIP2;2 and SvPIP2;1 cRNA injected oocytes by bathing in hypo-osmotic solution supplemented with 50 mM Na-acetate, pH 5.6, n = 12–14; a = non-significant; b = p < 0.05; c = p < 0.005.

References [+] :

Azad, Genome-Wide Characterization of Major Intrinsic Proteins in Four Grass Plants and Their Non-Aqua Transport Selectivity Profiles with Comparative Perspective. 2016, Pubmed