NaCl-regulated gene expression in Distichlis spicata
NaCl-induced and -repressed cDNA clones had previously been isolated by differential screening of a cDNA library, prepared from poly(A(^+)) RNA isolated from Distichlis spicata (salt grass) cell cultures grown in the presence of 260 mM NaCl (Zhao, et al., 1989). Eight of these cDNA clones have now been subcloned and/or sequenced and the predicted polypeptides compared with owl sequence data base. Three clones pDZ6.2, pDZVIII 1.2.1 and pDZIX 3.1 encode proline rich proteins, containing an amino acid repeat [PPKKDH(H)Y(Y)]. They have similar amino acid usage to proline-rich cell wall proteins, being rich in P, K, H and Y. The first 20 amino acid residues encode a putative leader sequence, supporting the proposed extracellular role as a cell wall protein. This N-terminal sequence (MPLLVALLLVLAVVAAAGAD) shares some similarity with die leader sequence of a soyabean proline-rich cell wall protein precursor and other extracellular proteins (the conserved residues are underlined). There is an increase in abundance of transcripts hybridising to the inserts from pDZ6.2 and pDZVUI 1.2.1 in response to either 520 mM NaCl or 100 µM ABA, but a decrease in response to 5 mM exogenous proline. It is suggested that the corresponding gene(s) are regulated at the level of either transcription or transcript stability, in response to elevated NaCl, with ABA as a mediator of (or part of) tills response. pDZ6.2 and pDZXI 3.1 have identical nucleotide sequences, whilst pDZVni 1.2.1 differs in three base paks within the putative open reading frame, suggesting that there may be at least two members of a multi gene family. A 68 bp OA repeat has been found in the 5' untranslated region of pDZ6.2 and a corresponding transcript identified by northern analysis using this OA sequence as a probe. Such nucleotide repeats can form triplexes (DNA) or hakpin loops (RNA), which is dependent on pH and ionic conditions. Therefore this OA repeat may play a role in the regulation of the gene corresponding to pDZ6.2 at the level of transcription or translation, possibly by attenuation of these processes, either by the formation of triplexes or hah-pins, or the binding of a protein to this GA region, at low ionic strength. However initial in vitro ttanscription experiments, to compare the transcriptional activity of pDZ6.2 and pDZVin 5.1.1 at different ionic strengths, proved inconclusive. An attempt was also made to identify the corresponding genomic region from D. spicata by anchored PGR.A fourth clone pDZ2.8L encodes a histone 2B protein, having 97.9% similarity to a wheat histone 2B. Its transcript abundance decreased in response to either 520 mM NaCl, 5 mM proline or 100 µM ABA. The sequences of the remaining clones either revealed no significant similarity to any known sequences or were assigned as being cloning artefacts .D. spicata cells accumulate proline within eight hours of exposure to 260 mM NaCl (Heyser, et al., 1989b). An unsuccessful attempt was also made to isolate a pyrroline-5- carboxylate reductase gene homologue from D. spicata, by heterologous probing of Southern blots with a soyabean cDNA pProCl and PCR.