A two-staged model of Na+ exclusion in rice explained by 3D modeling of HKT transporters and alternative splicing

The HKT family of Na+ and Na+/K+ transporters is implicated in plant salinity tolerance. Amongst these transporters, the cereal HKT1;4 and HKT1;5 are responsible for Na+ exclusion from photosynthetic tissues, a key mechanism for plant salinity tolerance. It has been suggested that Na+ is retrieved from the xylem transpiration stream either in the root or the leaf sheath, protecting the leaf blades from excessive Na+ accumulation. However, direct evidence for this scenario is scarce. Comparative modeling and evaluation of rice (Oryza sativa) HKT-transporters based on the recent crystal structure of the bacterial TrkH K+ transporter allowed to reconcile transcriptomic and physiological data. For OsHKT1;5, both transcript abundance and protein structural features within the selectivity filter could control shoot Na+ accumulation in a range of rice varieties. For OsHKT1;4, alternative splicing of transcript and the anatomical complexity of the sheath needed to be taken into account. Thus, Na+ accumulation in a specific leaf blade seems to be regulated by abundance of a correctly spliced OsHKT1;4 transcript in a corresponding sheath. Overall, allelic variation of leaf blade Na+ accumulation can be explained by a complex interplay of gene transcription, alternative splicing and protein structure.Olivier Cotsaftis, Darren Plett, Neil Shirley, Mark Tester and Maria Hrmov

Recombinaison homologué chez le riz (Oryza sativa L.) : Développement d'un modèle d'étude de ciblage génique et caractérisation moléculaire du gène rad50

MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Enhancing gene targeting efficiency in higher plants: rice is on the move

UMR DAPInternational audienceMeeting the challenge of routine gene targeting (GT) in higher plants is of crucial interest to researchers and plant breeders who are currently in need of a powerful tool to specifically modify a given locus in a genome. Higher plants have long been considered the last lineage resistant to targeting technology. However, a recent report described an eficient method of T-DNA-mediated targeted disruption of a nonselectable locus in rice [Terada et al., Nat Biotechnol 20: 1030–1034 (2002)]. Though this study was an obvious breakthrough, further improvement of GT frequencies may derive from a better understanding of the natural mechanisms that control homologous recombination (HR) processes. In this review, we will focus on what is known about HR and the factors which may hamper the development of routine GT by HR in higher plants. We will also present the current strategies envisaged to overcome these limitations, such as expression of recombination proteins and refinements in the design of the transformation vector

Targeting Vector

The present invention relates generally to nucleic acid constructs, which are useful for inserting a nucleotide sequence of interest into a target nucleic acid molecule via homologous recombination. The present invention also relates to methods for producing such constructs

Proceedings of EPNADS'05, Emergent Properties in Natural and Artificial Dynamical Systems, a satellite workshop within ECCS'05

International audienc

Crystal structure of a TrkH (3PJZ) and molecular model of the rice OsHKT1;5 transporter.

<p>(<b>A–B</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. (<b>C–D</b>) 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.</p

Alternative splicing of the OsHKT1;4 rice gene.

<p>(<b>A</b>) 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>B</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. (<b>C</b>) 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039865#pone.0039865-Campbell1" target="_blank">[19]</a>. 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.</p