Figure 1.
Root-to-shoot Na+ transfer control in rice upon salt stress.
(A) Radioactive 22Na+ fluxes measurement of root Na+ uptake (+SEM; n = 8) (uptake: 2 min). (B) Flame photometry measurement of plant total Na+ concentration (+SEM; n = 5). (C) Flame photometry measurement of tissue Na+ concentrations (+SEM; n = 5). (D) Radioactive 22Na+ fluxes measurement of the shoot-to-plant ratio of Na+ amount (+SEM; n = 8). (E) Correlation between root OsHKT1;5 expression level measured by qRT-PCR (three biological replicates per line +/− SEM; three technical replicates per biological sample) and shoot Na+ concentration measured by flame photometry (+/− SEM; n = 5) in salt treated rice lines with either a Leu or a Val residue in position 395 of the OsHKT1;5 gene. The abbreviations Po = Pokkali; NB = Nona Bokra; Ni = Nipponbare; KV = Kallurundai Vellai; Ka = Kalurundai; NSI = NSICRC106; and SAL = SAL208 stand for individual varieties, variously represented through the panels A–E. IR29 and FL478 are full names of rice varieties.
Figure 2.
Crystal structure of a TrkH (3PJZ) and molecular model of the rice OsHKT1;5 transporter.
(A–B) Cartoon representations of a bacterial TrkH K+ transporter and a model of Ni-OsHKT1;5 illustrating the overall folds of transporters (Ni = Nipponbare). Both structures are coloured in rainbow, except helix 0 in TrkH that is in grey and that is absent in Ni-OsHKT1;5. Four variations between Ni-OsHKT1;5 and Po-OsHKT1;5 (Po = Pokkali) are indicated in green cpk (A140P, H184R, D332H and V395L). Thr328 in TrkH (A) corresponds to Leu395 in Ni-OsKHT1;5 (B). Black lines indicate the Gly tetrad in TrkH, while the Ser-Gly-Gly-Gly signature is present in Ni-OsHKT1;5. Purple spheres indicate K+ (TrkH) and Na+ (Ni-OsHKT1;5) ions. The entry into the pores of transporters is indicated by black arrows. (C–D) The view in A–B is rotated by 90 degrees along the x-axis and shows TrkH and Ni-HKT1;5 viewed from a cytoplasmic side.
Figure 3.
Selectivity filters of TrkH and the rice Ni-OsHKT1;5 and Po-OsHKT1;5 transporters.
(A) Views of the pore regions with K+ (TrkH) and Na+ (Ni-OsHKT1;5 and Po-OsHKT1;5) ions coloured in purple spheres (Ni = Nipponbare; and Po = Pokkali). The Gly-Gly-Gly-Gly (TrkH) and Ser-Gly-Gly-Gly (Ni-OsHKT1;5 and Po-OsHKT1;5) signatures are coloured in black. The residue (stick) at the entrance of the pore in TrkH is Thr326 (orange), while Leu395 (dark magenta) is present in Ni-OsHKT1;5, and Val395 (light magenta) occurs in Po-OsHKT1;5. These residues are likely to affect pore rigidity and dispositions of residues controlling the rates of Na+ transport. (B) A stereo superposition of TrkH (yellow) and Ni-OsHKT1;5 (green). (C) A stereo superposition of Ni-OsHKT1;5 (green) and Po-OsHKT1;5 (wheat).
Figure 4.
Alternative splicing of the OsHKT1;4 rice gene.
(A) Flame photometry measurement of the blade-to-shoot ratio of Na+ concentrations (+SEM; n = 5) in nine rice lines (The abbreviations Po = Pokkali; NB = Nona Bokra; SAL = SAL208; Ka = Kalurundai; NSI = NSICRC106; KV = Kallurundai Vellai; and Ni = Nipponbare stand for individual varieties; IR29 and FL478 are full names of rice varieties). (B) Schematic representation of the OsHKT1;4 cDNA with its three exons (Ex) and the Val residue in position 344. This residue has been identified through the sequencing of the OsHKT1;4 locus genomic DNA in nine rice lines and a 522 bp fragment spanning the three exons in Nipponbare and Pokkali. (C) The sequencing of these products shows the presence of two splicing variants of OsHKT1;4: OsHKT1;4-SV1 (+104 bp, i.e. 626 bp total) and OsHKT1;4-SV2 (+186 bp, i.e. 708 bp total). Whereas the first intron (In) is always spliced correctly between a 5′-GT donor site and 3′-AG donor site, the presence of a 5′-GC donor site in the second intron causes alternative splicing [19]. Another intron of a shorter size is spliced in SV1 but not in SV2. Both spliced variants are translated into a truncated OsHKT1;4 protein due to the presence of an in-frame STOP codon at the beginning of the second intron.
Figure 5.
Sheath-to-blade Na+ transfer control in rice upon salt stress.
(A) Total OsHKT1;4 transcript level measured by qRT-PCR (+SEM; n = 3 to 4 replicates per sheath; three technical replicates per biological sample) in individual leaf sheath of Pokkali (Po) and Nipponbare (Ni). Three leaf sheaths were assessed, S2 to S4, from the oldest one to the youngest. (B) HPLC quantification of functional OsHKT1;4 transcripts and non-functional spliced variants SV1 and SV2 (+/− SEM; n = 3 to 4 replicates per sheath) in individual leaf sheath of Po and Ni, as a percentage of the total amount of transcripts reported in panel A. Leafs 2 and 3 were fully mature whereas leaf 4, although bigger than leaf 3, was still expending. (C) Correlation between functional OsHKT1;4 expression levels in individual sheath (Total OsHKT1;4 transcript level from panel A multiplied by the percentage of functional OsHKT1;4 transcript from panel B; +/− SEM; n = 3 to 4 replicates per sheath) and Na+ concentrations in the corresponding blades, measured by flame photometry (+/− SEM; n = 5) in three independent leaves (L2 to L4, from the oldest to the youngest) in Po and Ni.
Figure 6.
A two-staged Na+ exclusion model in rice.
Na+ ions from the external medium penetrate the root and are transported throughout the plant via xylem vessels. OsHKT1;5 proteins present in xylem parenchymal cells pump Na+ ions back into the root to minimize the amount of Na+ reaching the shoot, where it is harmful to the plant. This root-to shoot Na+ transfer mechanism represents the first stage of a Na+ exclusion model in rice, which is controlled by both OsHKT1;5 transcript levels and structural determinants of the OsHKT1;5 protein. High excluding lines carry a Val instead of a Leu in position 395, and this protein variation mediates a faster Na+ transport rate. The remaining Na+ ions that arrive into the shoot are diverted into different leaves. There, OsHKT1;4 proteins load the sheath tissues with Na+ ions before they can reach the photosynthetic part of the shoot, i.e. the blades. This sheath-to-blade Na+ transfer mechanism represents the second stage of the Na+ exclusion model in rice. Na+ excluding lines maximize this second dimension by firstly, having higher OsHKT1;4 expression levels in younger sheaths to protect the more energy-producing young blades and secondly, by controlling the ratio of spliced transcripts in favor of transcripts translated into functional proteins. Older leaves, with lower levels of the OsHKT1;4 proteins in the sheath, let Na+ go through to the senescing leaf blades, where Na+ can safely be stored and does no harm the plant.