SummaryIt is thought that Na + and K + homeostasis is crucial for salt-tolerance in plants. To better understand the Na + and K + homeostasis in important crop rice (Oryza sativa L.), a cDNA homologous to the wheat HKT1 encoding K + -Na + symporter was isolated from japonica rice, cv Nipponbare (Ni-OsHKT1). We also isolated two cDNAs homologous to Ni-OsHKT1 from salt-tolerant indica rice, cv Pokkali (Po-OsHKT1, Po-OsHKT2). The predicted amino acid sequence of Ni-OsHKT1 shares 100% identity with Po-OsHKT1 and 91% identity with Po-OsHKT2, and they are 66±67% identical to wheat HKT1. Low-K + conditions (less than 3 mM) induced the expression of all three OsHKT genes in roots, but mRNA accumulation was inhibited by the presence of 30 mM Na + . We further characterized the ion-transport properties of OsHKT1 and OsHKT2 using an expression system in the heterologous cells, yeast and Xenopus oocytes. OsHKT2 was capable of completely rescuing a K + -uptake de®ciency mutation in yeast, whereas OsHKT1 was not under K + -limiting conditions. When OsHKTs were expressed in Na + -sensitive yeast, OsHKT1 rendered the cells more Na + -sensitive than did OsHKT2 in high NaCl conditions. The electrophysiological experiments for OsHKT1 expressed in Xenopus oocytes revealed that external Na + , but not K + , shifted the reversal potential toward depolarization. In contrast, for OsHKT2 either Na + or K + in the external solution shifted the reversal potential toward depolarization under the mixed Na + and K + containing solutions. These results suggest that two isoforms of HKT transporters, a Na + transporter (OsHKT1) and a Na + -and K + -coupled transporter (OsHKT2), may act harmoniously in the salt tolerant indica rice.
Plant HKT proteins comprise a family of cation transporters together with prokaryotic KtrB, TrkH, and KdpA transporter subunits and fungal Trk proteins. These transporters contain four loop domains in one polypeptide with a proposed distant homology to K ؉ channel selectivity filters. Functional expression in yeast and Xenopus oocytes revealed that wheat HKT1 mediates Na ؉ -coupled K ؉ transport. Arabidopsis AtHKT1, however, transports only Na ؉ in eukaryotic expression systems. To understand the molecular basis of this difference we constructed a series of AtHKT1͞HKT1 chimeras and introduced point mutations to AtHKT1 and wheat HKT1 at positions predicted to be critical for K ؉ selectivity. A single-point mutation, Ser-68 to glycine, was sufficient to restore K ؉ permeability to AtHKT1. The reverse mutation in HKT1, Gly-91 to serine, abrogated K ؉ permeability. This glycine in P-loop A of AtHKT1 and HKT1 can be modeled as the first glycine of the K ؉ channel selectivity filter GYG motif. The importance of such filter glycines for K ؉ selectivity was confirmed by interconversion of Ser-88 and Gly-88 in the rice paralogues OsHKT1 and OsHKT2. Surprisingly, all HKT homologues known from dicots have a serine at the filter position in P-loop A, suggesting that these proteins function mainly as Na ؉ transporters in plants and that Na ؉ ͞K ؉ symport in HKT proteins is associated with a glycine in the filter residue. These data provide experimental evidence that the glycine residues in selectivity filters of HKT proteins are structurally related to those of K ؉ channels.
SummaryEubacterial-type multi-subunit plastid RNA polymerase (PEP) is responsible for the principal transcription activity in chloroplasts. PEP is composed of plastid-encoded core subunits and one of multiple nuclearencoded sigma factors that confer promoter specificity on PEP. Thus, the replacement of sigma factors associated with PEP has been assumed to be a major mechanism for the switching of transcription patterns during chloroplast development. The null mutant (sig6-1) of plastid sigma factor gene AtSIG6 exhibited a cotyledon-specific pale green phenotype. Light-dependent chloroplast development was significantly delayed in the sig6-1 mutant. Genetic complementation of the mutant phenotype by the AtSIG6 cDNA demonstrated that AtSIG6 plays a key role in light-dependent chloroplast development. Northern and array-based global analyses for plastid transcripts revealed that the transcript levels of most PEP-dependent genes were greatly reduced in the sig6-1 mutant, but that the accumulation of nuclear-encoded RNA polymerase (NEP)-dependent transcripts generally increased. As the PEP a subunit and PEP-dependent trnV accumulated at normal levels in the sig6-1 mutant, the AtSIG6 knockout mutant probably retained functional PEP, and the transcriptional defects are likely to have been directly caused by AtSIG6 deficiency. Most of the AtSIG6-dependent genes are preceded by r 70 -type promoters comprised of conserved )35/)10 elements. Thus, AtSIG6 may act as a major general sigma factor in chloroplasts during early plant development. On the other hand, the mutant phenotype was restored in older seedlings. Arabidopsis probably contains another late general sigma factor, the promoter specificity of which widely overlaps with that of AtSIG6.
To express a foreign gene in plants effectively, a good expression system is required. Here we describe the identification of a transcriptional terminator that supports increased levels of expression. The terminators of several Arabidopsis genes were examined in transfected Arabidopsis T87 protoplasts. The heat shock protein 18.2 (HSP) terminator was the most effective in supporting increased levels of expression. The HSP terminator increases mRNA levels of both transiently and stably expressed transgenes approximately 2-fold more than the NOS (nopaline synthase) terminator. When combined with the HSP terminator, a translational enhancer increased gene expression levels approximately 60- to 100-fold in transgenic plants.
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