Coskun D et al. (2019), Si permeability of a deficient Lsi1 aquaporin i...

XB-ART-56238

Plant Direct 2019 Aug 01;38:e00163. doi: 10.1002/pld3.163.

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Si permeability of a deficient Lsi1 aquaporin in tobacco can be enhanced through a conserved residue substitution.

Coskun D , Deshmukh R , Sonah H , Shivaraj SM , Frenette-Cotton R , Tremblay L , Isenring P , Bélanger RR .

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Silicon (Si) is a beneficial substrate for many plants, conferring heightened resilience to environmental stress. A plant's ability to absorb Si is primarily dependent on the presence of a Si-permeable Lsi1 (NIP2-1) aquaporin in its roots. Structure-function analyses of Lsi1 channels from higher plants have thus far revealed two key molecular determinants of Si permeability: (a) the amino acid motif GSGR in the aromatic/arginine selectivity filter and (b) 108 amino acids between two highly conserved NPA domains. Curiously, tobacco (Nicotiana sylvestris) stands as a rare exception as it possesses an Lsi1 (NsLsi1) with these molecular signatures but is reported as a low Si accumulator. The aim of this study was therefore to identify whether additional determinants influence Si permeability via Lsi1 channels, focusing on the role of residues that differ uniquely in NsLsi1 relative to functional Lsi1 homologs. We observed tobacco indeed absorbed Si poorly (0.1% dw), despite NsLsi1 being expressed constitutively in planta. Si influx measured in NsLsi1-expressing Xenopus oocytes was very low (<13% that of OsLsi1 from rice (Oryza sativa) over a 3-hr time course), which likely explains why tobacco is a low Si accumulator. Interestingly, NsLsi1P125F displayed a significant gain of function (threefold increase in Si influx relative to NsLsi1WT), which coincided with a threefold increase in plasma membrane localization in planta, as measured by transient expression of GFP constructs in Nicotiana benthamiana leaves. These findings thus reveal a novel molecular determinant of Si transport in plants and inform breeding, biotechnological, and agricultural practices to effectively utilize Si in the context of plant resilience to environmental stress.

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	Figure 1. Tobacco is a low Si accumulator despite possessing the known molecular determinants for Si permeability. (a) Leaf Si content (% dw) of one‐month‐old plants grown with 1.7 mM Si supplementation. Error bars denote SEM of five biological replicates. (b) Phylogenetic tree of Lsi1 (NIP2‐1) AQPs corresponding to the species from panel a. (c) Amino acid sequence alignment of Lsi1 AQPs corresponding to panel b. Residues highlighted in red denote the positions of the ar/R selectivity filter. Residues highlighted in yellow denote the positions of the NPA domains. 108 amino acids separate the NPA domains of all sequences except for SlNIP2‐1, which has an extra amino acid located at the position of the asterisk
	Figure 2. NsLsi1 expression in planta. qPCR results showing NsLsi1 expression in roots and shoots of tobacco plants grown for one month with (+Si) and without (−Si) silicon. Gene expression was normalized to NsActin and NsEF1A1. Error bars denote SEM of four biological and two technical replicates
	Figure 3. NsLsi1 possesses limited Si‐uptake capabilities. Si content of Xenopus laevis oocytes expressing OsLsi1 (filled symbols) or NsLsi1 (open symbols) as a function of exposure time to Si in the external medium ([Si]ext = 2 mM). Si‐content values were corrected for background Si and normalized to the highest time value (i.e., OsLsi1 at 180 min; see text for details). Data represent means of nine replicates (error bars were smaller than symbols) and fitted to a Michaelis–Menten regression
	Figure 4. Amino acid sequence alignment of Lsi1 homologs. Residues marked by a position (based on the sequence of NsNIP2‐1) are unique to this homolog and indicate potential candidates for mutagenesis (see text and Table 1 for details). Residues composing the ar/R selectivity filter are highlighted in red and those composing the NPA domains in yellow. The number of amino acids spanning the NPA domains for all proteins is 108. Green bars denote the position of the six transmembrane (TM) helices, interspersed by five interconnecting loops (A–E) denoted by black lines. NsNIP2‐1 from tobacco (Nicotiana sylvestris), OsNIP2‐1 from rice (Oryza sativa), ZmNIP2‐1 from maize (Zea mays), SbNIP2‐1 from sorghum (Sorghum bicolor), GmNIP2‐1 from soybean (Glycine max), LaNIP2‐1 from blue lupin (Lupinus angustifolius), MtNIP2‐1 from barrelclover (Medicago truncatula), AiNIP2‐1 and AdNIP2‐1 from peanut ancestors Arachis ipaensis and Arachis duranensis, respectively, CaNIP2‐1 from chickpea (Cicer arietinum), PsNIP2‐1 from pea (Pisum sativum), CcNIP2‐1 from pigeon pea (Cajanus cajan), and SiNIP2‐1 form foxtail millet (Setaria italica)
	Figure 5. Kinetic analysis of Si influx mediated by NsLsi1WT and NsLsi1P125F. Si influx in Xenopus laevis oocytes expressing NsLsi1WT (filled symbol) or NsLsi1P125F (open symbol) as a function of external Si concentration ([Si]ext). Exposure time = 3 hr. Si influx was corrected for background Si and normalized to the highest concentration value (i.e., NsLsi1P125F at 2 mM Si; see text for details). Error bars denote SEM of nine replicates. Data were fitted to a Michaelis–Menten regression
	Figure 6. NsLsi1P125F displays enhanced plasma membrane localization relative to wild‐type. (a) Confocal micrographs displaying cellular localization of GFP fused to the C terminus of NsLsi1 wild‐type (left) or P125F mutant (right) in an Nicotiana benthamiana leaf transient expression assay. Scale bar denotes 50 μm. (b) Quantitative analysis of fluorescence intensity at the plasma membrane (n = 6–17; ****p < .0001, Student's t test)

References [+] :

Bragg, The C-terminal region of the Barley stripe mosaic virusgammab protein participates in homologous interactions and is required for suppression of RNA silencing. 2004, Pubmed