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BMC Plant Biol
2019 Jun 20;191:268. doi: 10.1186/s12870-019-1885-9.
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Disruption of an amino acid transporter LHT1 leads to growth inhibition and low yields in rice.
Wang X
,
Yang G
,
Shi M
,
Hao D
,
Wei Q
,
Wang Z
,
Fu S
,
Su Y
,
Xia J
.
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BACKGROUND: Research on plant amino acid transporters was mainly performed in Arabidopsis, while our understanding of them is generally scant in rice. OsLHT1 (Lysine/Histidine transporter) has been previously reported as a histidine transporter in yeast, but its substrate profile and function in planta are unclear. The aims of this study are to analyze the substrate selectivity of OsLHT1 and influence of its disruption on rice growth and fecundity.
RESULTS: Substrate selectivity of OsLHT1 was analyzed in Xenopus oocytes using the two-electrode voltage clamp technique. The results showed that OsLHT1 could transport a broad spectrum of amino acids, including basic, neutral and acidic amino acids, and exhibited a preference for neutral and acidic amino acids. Two oslht1 mutants were generated using CRISPR/Cas9 genome-editing technology, and the loss-of-function of OsLHT1 inhibited rice root and shoot growth, thereby markedly reducing grain yields. QRT-PCR analysis indicated that OsLHT1 was expressed in various rice organs, including root, stem, flag leaf, flag leaf sheath and young panicle. Transient expression in rice protoplast suggested OsLHT1 was localized to the plasma membrane, which is consistent with its function as an amino acid transporter.
CONCLUSIONS: Our results indicated that OsLHT1 is an amino acid transporter with wide substrate specificity and with preference for neutral and acidic amino acids, and disruption of OsLHT1 function markedly inhibited rice growth and fecundity.
Fig. 1. Substrate specificity of OsLHT1. a Representative inward currents induced by amino acids. Oocytes injected with water (upper trace, control) and OsLHT1 cRNA (lower trace) were voltage-clamped at − 70 mV and superfused with different amino acids (10 mM, pH 5.4). b Amino acid transport activity of OsLHT1. Oocytes expressing OsLHT1 were voltage-clamped at − 70 mV and superfused with different amino acids (10 mM, pH 5.4). Substrate-induced currents (background subtracted) were normalized to the Asn-induced current. Data are shown as means ± SD of six independent cells. c Kinetic analysis of Asn transport by OsLHT1. Asn-induced currents were recorded at − 140 mV (holding potential of − 40 mV) in different Asn concentrations. Net Asn-induced currents (background subtracted) were normalized to that obtained in 10 mM Asn, and then fitted to the Michaelis-Menten equation, as shown by the solid line. Data are shown as means ± SD of six independent cells
Fig. 2. Targeted mutagenesis of OsLHT1 by CRISPR/Cas9 led to rice growth inhibition. a Gene structure of OsLHT1 and the two targeted sites. Black boxes indicate exons. Red letters indicate the PAM of the recognition sequence. b Sequencing chromatography of wild type, mutants oslht1–1 and oslht1–2. Red arrows indicate mutation sites. c Phenotype of 5-day-old WT, oslht1–1 and oslht1–2 grown on 0.5 mM CaCl2 solution. Bar =1 cm. d Time-dependent root elongation. Germinated seedlings were exposed to a 0.5 mM CaCl2 solution and the root length was measured at different days. Error bars represent ± SD (n = 10). e-f Gross morphological phenotypes of WT, oslht1–1and oslht1–2 mutants grown in the field. Bar = 30 cm (e), 20 cm (f). g Symptoms of early senescence were present in oslht1 plants at reproductive growth stage. Bar = 20 cm. h-j Comparison of plant height (h), stem length (i) and tiller number (j) of the wild type (WT) and oslht1 plants at harvest. Values (h to j) are the mean ± SD (n = 15). Asterisks indicate significant differences from the wild type (*P < 0.05; **P < 0.01 by Student’s t-test)
Fig. 3. Loss of OsLHT1 function reduced rice grain yield. a Total grains per plant of WT and oslht1 plants grown in field. Scale bars, 1 cm. b-f Comparison of grain yield per plant (b), panicle number per plant (c), grain number per panicle (d), seed setting rate (e) and 1000-grain weight (f) of the WT and oslht1 plants. Values (b to f) are the mean ± SD (n = 15). Asterisks indicate significant differences from the wild type (*P < 0.05; **P < 0.01 by Student’s t-test)
Fig. 4. Organ-specific expression and subcellular localization of OsLHT1. a Expression of OsLHT1 in various organs of the wild-type plants, analyzed by quantitative real-time PCR. The data are shown as the mean ± SD (n = 3). b Subcellular localization of the OsLHT1. GFP:OsLHT1 or GFP was transiently introduced into rice protoplast together with mCherry-OsRac3 by PEG-mediated transformation. Fluorescence signals from GFP, mCherry-OsRac3, and the merged images are shown. Free GFP was used as a control. Bars, 10 μm
Fig. 5. Phylogenetic relationship and protein motifs of OsLHT1 and its homologs. a Phylogenetic tree of OsLHT1 and its homologs from maize, sorghum, Brachypodium and Arabidopsis. With the exception of OsLHT1–6 and AtLHT1–10, the first two letters of each protein label represent the abbreviated species name, followed by GenBank accession number. Zm, Zea mays; Sb, Sorghum bicolor; Bd, Brachypodium distachyon. The phylogenetic tree was constructed by Mega 6.0 software using ClustalW for the alignment and the neighbour-joining method for the construction of the phylogeny [28]. The bootstrap values, shown at the nodes, are percentages for 1000 replications. The red triangle marks the OsLHT1. b Schematic representation of conserved motifs in OsLHT1–6 and its closest homologs in maize, sorghum, Arabidopsis and Brachypodium. Each motif is represented by different color boxes. The order of the motifs corresponds to their position within individual protein sequences
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