Distinct Evolutionary Origins of Intron Retention Splicing Events in NHX1 Antiporter Transcripts Relate to Sequence Specific Distinctions in Oryza Species.

The genome of Asian cultivated rice (Oryza sativa L.) shows the presence of six organelle-specific and one plasma membrane (OsNHX1-7) NHX-type cation proton antiporters. Of these, vacuolar-localized OsNHX1 is extensively characterized. The genus Oryza consists of 27 species and 11 genome-types, with cultivated rice, diploid O. sativa, having an AA-type genome. Oryza NHX1 orthologous regions (gene organization, 5' upstream cis elements, amino acid residues/motifs) from closely related Oryza AA genomes cluster distinctly from NHX1 regions from more ancestral Oryza BB, FF and KKLL genomes. These sequence-specific distinctions also extend to two separate intron retention (IR) events involving Oryza NHX1 transcripts that occur at the 5' and 3' ends of the NHX1 transcripts. We demonstrate that the IR event involving the 5' UTR is present only in more recently evolved Oryza AA genomes while the IR event governing retention of the 13th intron of Oryza NHX1 (terminal intron) is more ancient in origin, also occurring in halophytic wild rice, Oryza coarctata (KKLL). We also report presence of a retro-copy of the OcNHX1 cDNA in the genome of O. coarctata (rOcNHX1). Preferential species and tissue specific up- or down-regulation of the correctly spliced NHX1 transcript/5' UTR/13th intron-retaining splice variants under salinity was observed. The implications of IR on NHX1 mRNA stability and ORF diversity in Oryza spp. is discussed.

Expression of wild rice Porteresia coarctata PcNHX1 antiporter gene (PcNHX1) in tobacco controlled by PcNHX1 promoter (PcNHX1p) confers Na+-specific hypocotyl elongation and stem-specific Na+ accumulation in transgenic tobacco.

Soil salinization is a major abiotic stress condition that affects about half of global agricultural lands. Salinity leads to osmotic shock, ionic imbalance and/or toxicity and build-up of reactive oxygen species. Na⁺/H⁺ antiporters (NHXs) are integral membrane transporters that catalyze the electro-neutral exchange of K⁺/Na⁺ for H⁺ and are implicated in cell expansion, development, pH/ion homeostasis and salt tolerance. Porteresia coarctata is a salt secreting halophytic wild rice that thrives in the coastal-riverine interface. P. coarctata NHX1 (PcNHXI) expression is induced by salinity in P. coarctata roots and shows high sequence identity to Oryza sativa NHX1. PcNHX1 confers hygromycin and Li+ sensitivity and Na+ tolerance transport in a yeast strain lacking sodium transport systems. Additionally, transgenic PcNHX1 expressing tobacco seedlings (PcNHX1 promoter) show significant growth advantage under increasing concentrations of NaCl and MS salts. Etiolated PcNHX1 seedlings also exhibit significantly elongated hypocotyl lengths in 100 mM NaCl. PcNHX1 expression in transgenic tobacco roots increases under salinity, similar to expression in P. coarctata roots. Under incremental salinity, transgenic lines show reduction in leaf Na+, stem specific accumulation of Na+ and K+ (unaltered Na+/K+ ratios). PcNHX1 transgenic plants also show enhanced chlorophyll content and reduced malondialdehyde (MDA) production in leaves under salinity. The above data suggests that PcNHX1 overexpression (controlled by PcNHX1p) enhances stem specific accumulation of Na+, thereby protecting leaf tissues from salt induced injury.

T-homoeolog specific plasma membrane protein 3 [Nt(t)PMP3-2] in polyploid Nicotiana tabacum shows conserved alternative splicing, derived from extant Nicotiana tomentosiformis parent.

Abiotic stress induced plasma membrane protein 3 (PMP3) genes occur as multigene families in plants, coding for hydrophobic proteins. Group I PMP3s code for shorter ORFs while Group II PMP3s code for proteins with C-terminal extensions. Allotetraploid Nicotiana tabacum (SSTT; 2n = 48) derives its parentage from extant ancestors related to Nicotiana sylvestris (SS) and Nicotiana tomentosiformis (TT). Polyploidization triggers complex genetic and epigenetic changes, often leading to homoeolog-specific retention or loss of function, sub-functionalization or neo-functionalization. Genomic sequences of Nt(t)PMP3-1/Nt(t)PMP3-2 cloned from N. tabacum show near identity with N. tomentosiformis NtoPMP3-1/NtoPMP3-2 genomic sequences respectively (distinct from N. sylvestris NsPMP3-1/NsPMP3-2 genomic regions). RT-PCR with exon 1,2 primer pairs amplified only single fragments for Nt(t)PMP3-1 and Nt(t)PMP3-2. In contrast, for Nt(t)PMP3-2, three variants were detected using exon 2,3 primers by RT-PCR. Cloning revealed (i) a transcript coding for a Group I PMP3 [Nt(t)PMP3-2CS], (ii) a transcript with complete retention of the second intron [Nt(t)PMP3-2IR] and (iii) a transcript with an alternative (exon 2) 5' splice site [Nt(t)PMP3-2AS], coding for a longer protein, similar to ORFs of Group II PMP3 genes. All three Nt(t)PMP3-2 variants have conserved counterparts in the N. tomentosiformis transcriptome, suggesting the transcriptional machinery governing alternative splicing of Nt(t)PMP3-2 in N. tabacum has conserved origins, derived from a N. tomenosiformis lineage. The above data shows alternative splicing of PMP3 genes contributes to transcript and ORF diversity in plants. All three Nt(t)PMP3-2 splice variants show increased root-specific expression. Implications of Nt(t)PMP3-2 alternative splicing on transcript stability and ORF features are discussed.

A vacuolar antiporter is differentially regulated in leaves and roots of the halophytic wild rice Porteresia coarctata (Roxb.) Tateoka.

Vacuolar NHX-type antiporters play a role in Na(+)/K(+) uptake that contributes to growth, nutrition and development. Under salt/osmotic stress they mediate the vacuolar compartmentalization of K(+)/Na(+), thereby preventing toxic Na(+)K(+) ratios in the cytosol. Porteresia coarctata (Roxb.) Tateoka, a mangrove associate, is a distant wild relative of cultivated rice and is saline as well as submergence tolerant. A vacuolar NHX homolog isolated from a P. coarctata cDNA library (PcNHX1) shows 96 % identity (nucleotide level) to OsNHX1. Diurnal PcNHX1 expression in leaves was found to be largely unaltered, though damped by salinity. PcNHX1 promoter directed GUS expression is phloem-specific in leaves, stem and roots of transgenic plants in the absence of stress. Under NaCl stress, GUS expression was also seen in the epidermal and sub-epidermal layers (mesophyll, guard cells and trichomes) of leaves, root tip. The salinity in the rhizosphere of P. coarctata varies considerably due to diurnal/semi-diurnal tidal inundation. The diurnal expression of PcNHX1 in leaves and salinity induced expression in roots may have evolved in response to dynamic changes in salinity of in the P. coarctata rhizosphere. Despite high sequence conservation between OsNHX1 and PcNHX1, the distinctive expression pattern of PcNHX1 exemplifies how variation in expression is fine tuned to suit the halophytic growth habitat of a plant.