Ilga Winicov

Post-transcriptional regulation of a salt-inducible alfalfa gene encoding a putative chimeric proline-rich cell wall protein

A cDNA previously shown to identify a salt-inducible root-specific transcript in Medicago sativa was used to screen an alfalfa library for the corresponding genomic sequence. One positive clone was recovered. The nucleotide sequence of a subclone contained a 329 bp 5′ region upstream of the first ATG codon, a 1143 bp coding segment, and a 447 bp 3′-untranslated region interrupted by a single 475 bp intron. Translation of the coding segment, which was designated MsPRP2, suggested it encodes a chimeric 40 569 Da cell wall protein with an amino-terminal signal sequence, a repetitive proline-rich sequence, and a cysteine-rich carboxyl-terminal sequence homologous to nonspecific lipid transfer proteins. The 3′-untranslated region of MsPRP2 contained a sequence similar to one found to destabilize mRNAs transcribed from the elicitor-regulated proline-rich protein gene PvPRP1. Transcription run-on experiments using nuclei from salt-sensitive and salt-tolerant alfalfa callus suggested that the accumulation of MsPRP2 transcripts in salt-tolerant alfalfa cells grown in the presence of salt is due primarly to increased mRNA stability. The MsPRP2 gene thus may be a useful model for studying post-transcriptional salt-regulated expression of cell wall proteins.

Characterization of salt tolerant alfalfa (Medicago sativa L.) plants regenerated from salt tolerant cell lines.

Salt tolerant cell lines have been selected from Medicago sativa, by a single step selection process on tissue culture medium containing 1% NaCl. Plants regenerated from these lines show improved salt tolerance compared to parent plants. The regenerated plants are vigorous, have flowered and are self fertile. The cellular salt tolerance characteristic can be passaged through the regenerated plants, since callus cultures initiated from immature ovaries of the salt tolerant regenerated plants are salt tolerant without additional selection on 1% NaCl. Several of these “second generation” callus cultures have been regenerated to produce vigorous plants which maintain the salt tolerance characteristic. The tolerance phenotype appears dominant in seeds obtained from self fertilization of the tolerant plants. The regenerated salt tolerant plants are therefore a valuable source as genotypes in plant breeding for salt tolerance and isolation, identification and manipulation of genes which confer salt tolerance in alfalfa.

Salt tolerance in crop plants: new approaches through tissue culture and gene regulation

Recent approaches to study of salinity tolerance in crop plants have ranged from genetic mapping to molecular characterization of gene products induced by salt/drought stress. Transgenic plant design has allowed to test the effects of overexpression of specific prokaryotic or plant genes that are known to be up-regulated by salt/drought stress. This review summarizes current progress in the field in the context of adaptive metabolic and physiological responses to salt stress and their potential role in long term tolerance. Specifically considered are gene activation by salt, in view of proposed avenues for improved salt tolerance and the need to ascertain the additional influences of developmental regulation of such genes. Discussion includes the alternate genetic strategy we have pursued for improving salinity tolerance in alfalfa (Medicago sativa L.) and rice (Oryza sativa L.). This strategy combines single-step selection of salt-tolerant cells in culture, followed by regeneration of salt-tolerant plants and identification of genes important in conferring salt tolerance. We have postulated that activation or improved expression of a subset of genes encoding functions that are particularly vulnerable under conditions of salt-stress could counteract the molecular effects of such stress and could provide incremental improvements in tolerance. We have proceeded to identify the acquired specific changes in gene regulation for our salt-tolerant mutant cells and plants. One particularly interesting and novel gene isolate from the salt-tolerant cells is Alfin1, which encodes a putative zinc-finger regulatory protein, expressed predominantly in roots. We have demonstrated that this protein binds DNA in a sequence specific manner and may be potentially important in gene regulation in roots in response to salt and an important marker for salt tolerance in crop plants.

Characterization of rice (Oryza sativa L.) plants regenerated from salt-tolerant cell lines

Cell lines were established from the seed of two elite US rice lines (L-202 and M-202) as well as Indica rice varieties Pokkali, IR 28 and IR 42. Salt-tolerant cell lines were selected from these callus cultures by a single step selection process on tissue culture medium containing 1% (0.171 M) NaCl. Plants were regenerated from the salt-tolerant lines. The salt selection process and regeneration of the embryogenic callus had minimal adverse effect on subsequent plant growth, since the regenerated plants were vigorous, have flowered and in most cases were fertile. Seed was collected from the regenerated plants and the germinated seedlings were tested for salt tolerance. The R2 seedling salt tolerance was compared for plants regenerated from salt selected cell lines of L-202 that had been maintained on 1% NaCl for 1, 4 and 5 months. Heritable improvement in salt tolerance was obtained in R2 seedlings from one plant that had been regenerated after 5 months of selection on salt-containing medium. These results indicate that short-term salt selected rice callus contains many embryogenic calli that do not carry heritable changes in the salt tolerance phenotype and therefore necessitates screening for the tolerance phenotype in the progeny of the regenerated plants. Thus, as shown in the case of L-202 (R4), prolonging the selection process in vitro for rice improves the likelihood of regenerating plants with improved salt tolerance.

New concepts in protein-DNA recognition: sequence-directed DNA bending and flexibility.

Publisher Summary This chapter discusses the unusual DNA structures with known sequence dependencies such as bends, and the relatively new concept of sequence-directed structural softness or flexibility, and correlates these with protein structural motifs wherever possible. It shows that analyses of consensus binding sequences in DNA can provide important clues both for identifying possible roles of localized DNA structures (or microstructures) in protein-DNA interactions and for interpreting these roles in structure-function terms. An important consequence is that most biological processes are modulated at the molecular level by the interactions of regulatory proteins with themselves or other proteins, and with their characteristic operator DNA. A corollary is that the trajectory of the DNA is precisely defined, particularly in large, multi-subunit nucleoprotein complexes. Because both of these factors are highly site specific, they can confer a corresponding level of site specificity to the processes they control, which include transcriptional regulation, the action of hormone receptors, and certain types of site-specific recombination, including the precise insertion of viral DNA into host genomes.

Accumulation of photosynthesis gene transcripts in response to sodium chloride by salt-tolerant alfalfa cells

A cell line from diploid alfalfa (Medicago sativa L.), HG2-N1, which is stably tolerant to 1% (0.171 M) NaCl in the medium and noticeably more green than the parent line (HG2), was investigated to determine if increased chlorophyll accumulation was related to chloroplast development toward greater photosynthetic activity and increases in chloroplast-gene expression in this cell line. The ctDNA (chloroplast-DNA) copy number in HG2-N1 was found to be increased by 50% from that of the salt-sensitive parent HG2 line. RNA accumulation, as detected by hybridization to a nuclear actin gene, appeared comparable in HG2 and HG2-N1. In contrast, mRNA levels of the plastid-encoded psbA gene were increased in the salt-tolerant HG2-N1 grown on normal medium. The mRNA levels of HG2-N1 were further increased in response to added NaCl in the medium. The mRNA levels from other chloroplast genes necessary for photosynthesis (psbD, psaB, atpB, rbcL), as well as from several nuclear genes (pCab4, pCab1, rbcS) encoding polypeptides participating in photosynthesis, also increased in the salt-tolerant HG2-N1 when it was grown in the presence of NaCl whereas actin-mRNA and chloroplast-rRNA levels remained comparable under growth conditions ± NaCl. Several independently isolated salt-tolerant cell lines showed a similar response to salt, as monitored by accumulation of rbcL mRNA, indicating that transcript accumulation for photosynthesis genes is correlated with the salt response in these salt-tolerant alfalfa cell lines.

Messenger RNA induction in cellular salt tolerance of Alfalfa (Medicago sativa)

A salt tolerant alfalfaMedicago sativa L. cell line (HG2-N1) has been selected for growth in 171 mM NaCl. The salt tolerance characteristic is stable and is retained after growth in absence of salt selection for two months.In vitro translation was used to compare mRNA composition from the salt tolerant HG2-N1 and parent salt sensitive HG2 cell lines grown in the presence and absence of 171 mM NaCl. The results suggest that the mRNA composition differs between HG2-N1 and HG2 in a number of RNA species. The salt tolerant HG2-N1 shows both increases and decreases in specific polypeptides as compared to HG2. Many of the enhanced polypeptide bands from mRNA in the salt tolerant HG2-N1 variant appear to be constitutively expressed, since they can be detected from HG2-N1 cells grown in presence and absence of NaCl, but the expression of a few bands may depend on the presence of added NaCl. Most enhanced polypeptides, which are detected from mRNA in the salt tolerant variant HG2-N1 (grown on NaCl) are different from polypeptide bands enhanced in the salt sensitive HG2 line as a result of 24 hour salt stress. Similar results were obtained from two dimensional analysis ofin vivo labeled polypeptides. At least one isolated cDNA clone shows selective expression of mRNA in salt tolerant cells grown in NaCl. These results indicate that adaptive mechanisms for salt tolerance may differ in some aspects from acute stress mechanisms.

Histone variants and acetylated species from the alfalfa plant Medicago sativa

The histones from the alfalfa plant Medicago sativa have been characterized in terms of type variants and levels of acetylation. Histones were isolated directly from total plant tissue (callus), eliminating the need to develop methods for nuclear isolation. An acid-urea-polyacrylamide gel with a transverse Triton X-100 gradient resolved and identified in a single gel at least one type of histone H4, two variant forms of histone H2B, two variant forms of histone H3, and four variant forms of histone H2A from a crude histone preparation. Histone H4 was present 25% in an unmodified state and 75% as monomodified, presumably as monoacetylated histone. Both histone H3 variants displayed five bands, consistent with up to four internal sites of acetylation. The two H3 variants differed in their steady-state level of acetylation, suggesting that they may reside in different chromatin environments. Several histone H1 species were identified by solubility and cross-reactivity with antiserum raised against the globular part of bovine H1(0), indicating conservation of epitopes between histone H1 of mammals and higher plants.

Characterization of a cDNA Clone from Salt-Tolerant Alfalfa Cells that Identifies Salt-Inducible Root-Specific Transcripts

Summary A novel cDNA clone has been isolated by differential screening of a cDNA library from salt-tolerant alfalfa ( Medicago sativa ) cells using cDNA probes derived from poly-A + RNAs from salt-sensitive and salt-tolerant callus. This clone, which is designated pA9, contains a 600 bp insert that hybridizes to a 1.6 kb RNA transcript. The concentration of this transcript increases in salt-tolerant callus cells in response to either NaCl or abscisic acid (ABA). By contrast, the concentration of transcripts hydridizing to pA9 decreases in salt-sensitive callus cells in response to salt or ABA. In whole plants, the transcripts detected by clone pA9 occur specifically in roots and cannot be found in leaves. The concentration of these transcripts increases in both salt-sensitive and salt-tolerant plant roots in response to NaCl. There are only a small number of copies of the gene corresponding to pA9 in the alfalfa genome. The sequence of the pA9 insert suggests that it represents a gene encoding a protein related to proline-rich cel1 wall proteins previously found in tomato, maize, and carrot. Clone pA9 thus identifies a potentially important gene that is regulated in a tissue-specific manner by NaCl.

Transcriptional and post-transcriptional activation of genes in salt-tolerant alfalfa cells

Salt-tolerant cell lines of alfalfa (Medicago sativa L.) selected in this laboratory showed increased mRNA accumulation for both nuclear- and chloroplastencoded genes involved in photosynthesis as well as in several non-photosynthetic related functions. The basis for this constitutive and salt-dependent gene activation was investigated by measuring both nuclear and plastid run-on transcription from the salt-sensitive parent line and from selected salt-tolerant lines. Plastids from tolerant cells showed a 2.5-fold increase in transcription rate over those from sensitive cells and a 4.5-fold increase if isolated from tolerant cells grown in salt. Nuclei isolated from salt-tolerant cells grown on normal medium showed higher transcription of the photosynthesis-related genes rbcS, cab1 and cab4 than those from salt-sensitive cells, confirming that the salt-tolerant cells had acquired altered transcriptional regulation of these genes. However, the major salt-induced increase in steady-state mRNA accumulation, from photosynthesis-related and other genes (alfin1, pA18 and histone H3cI and H3cII genes), was not reflected in run-on assays from these same cells. These results indicated that salt-dependent post-transcriptional mRNA stabilization led to the steady-state mRNA accumulation. The mRNA stabilization appears to be transcript specific, since transcripts of a constitutively-expressed gene (Msc27) remained unaffected by growth of the tolerant cells in 171 mM NaCl.