John C. Cushman

Genomic approaches to plant stress tolerance.

Past efforts to improve plant tolerance to drought, high salinity and low-temperature through breeding and genetic engineering have had limited success owing to the genetic complexity of stress responses. Progress is now anticipated through comparative genomics studies of an evolutionarily diverse set of model organisms, and through the use of techniques such as high-throughput analysis of expressed sequence tags, large-scale parallel analysis of gene expression, targeted or random mutagenesis, and gain-of-function or mutant complementation. The discovery of novel genes, determination of their expression patterns in response to abiotic stress, and an improved understanding of their roles in stress adaptation (obtained by the use of functional genomics) will provide the basis of effective engineering strategies leading to greater stress tolerance.

Salt stress leads to differential expression of two isogenes of phosphoenolpyruvate carboxylase during Crassulacean acid metabolism induction in the common ice plant.

The common ice plant is a facultative halophyte in which Crassulacean acid metabolism, a metabolic adaptation to arid environments, can be induced by irrigating plants with high levels of NaCl or by drought. This stress-induced metabolic transition is accompanied by up to a 50-fold increase in the activity of phosphoenolpyruvate carboxylase (PEPCase). To analyze the molecular basis of this plant response to water stress, we have isolated and characterized two members of the PEPCase gene family from the common ice plant. The PEPCase isogenes, designated Ppc1 and Ppc2, have conserved intron-exon organizations, are 76.4% identical at the nucleotide sequence level within exons, and encode predicted polypeptides with 83% amino acid identity. Steady-state levels of mRNAs from the two genes differ dramatically when plants are salt-stressed. Transcripts of Ppc1 increase about 30-fold in leaves within 5 days of salt stress. In contrast, steady-state levels of Ppc2 transcripts decrease slightly in leaf tissue over the same stress period. Steady-state levels of transcripts of both genes decrease in roots over 5 days of salt stress. We have used in vitro transcription assays with nuclei isolated from leaves to demonstrate that the increased expression of Ppc1 caused by water stress occurs in part at the transcriptional level.

Mesembryanthemum crystallinum, a higher plant model for the study of environmentally induced changes in gene expression

Plants have evolved several strategies to deal with water stress brought about by changes in soil water potential or solute concentration (Hanson & Hitz, 1982; Morgan, 1984). One solution to long-term periods of water stress is avoidance. This often involves rapid completion of ontogeny or prolonged periods of dormancy. Many halophytes adapt to changes in soil salinity by accumulating inorganic ions in the vacuole. The osmotic potential of the cytoplasm is balanced by the synthesis and accumulation of biologically compatible solutes such as proline, betaine, polyamines, sugars or sugar alcohols (Hanson & Hitz, 1982; Flores et al., 1985). A few species actively secrete or sequester salt via salt glands or salt hairs (Hill & Hill, 1976). Some facultative halophytes such as Mesembryanthemum crystallinum switch to crassulacean acid metabolism (CAM),

Molecular cloning and expression of chloroplast NADP-malate dehydrogenase during Crassulacean acid metabolism induction by salt stress

A full-length cDNA clone for NADP+-dependent malate dehydrogenase (NADP-MDH; EC 1.1.1.82) from the facultative CAM plant,Mesembryanthemum crystallinum has been isolated and characterized. NADP-MDH is responsible for the reduction of oxaloacetate to malate in the chloroplasts of higher plants. The cDNA clone is 1747 bp in size and contains a single open reading frame encoding a 441 amino acid long precursor polypeptide with a predicted molecular weight of 47 949. The predicted, mature NADP-MDH polypeptide sequence fromM. crystallinum shares 82.7% to 84% amino acid identity with other known higher plant sequences. Genomic Southern blot analysis ofM. crystallinum DNA indicates that MDH is encoded by a small gene family. Steady-state transcript levels for chloroplast NADP-MDH decrease transiently in the leaves after salt stress and then increase to levels greater than two-fold higher than in unstressed plants. Transcript levels in roots are extremely low and are unaffected by salt-stress treatment. In vitro transcription run-on experiments using isolated nuclei from leaf tissue confirm that the accumulation of NADP-MDH transcripts is, at least in part, the result of increased transcription of this gene during salt stress. The salt-stress-induced expression pattern of this enzyme suggests that it may participate in the CO2 fixation pathway during CAM.

Salt stress alters A/T-rich DNA-binding factor interactions within the phosphoenolpyruvate carboxylase promoter from Mesembryanthemum crystallinum

The common ice plant, Mesembryanthemum crystallinum, shifts from C3 to crassulacean acid metabolism (CAM) photosynthesis in response to osmotic stress. The expression of a number of genes encoding enzymes involved in the CAM pathway increases as a result of increased transcription rates. To begin to investigate the mechanisms responsible for the transcriptional activation, we have characterized the 5′ control region of a specific isoform of phosphoenolpyruvate carboxylase gene (Ppc1) that plays a key role in CAM. We have determined the nucleotide sequence of the 5′ flanking region of this gene. Ppc1 contains a long 5′-leader sequence with the transcriptional start site located 332/333 nucleotides 5′ of the translational initiation codon. Multiple DNA interactions with nuclear factors are detectable within the 5′-flanking region of Ppc1. We have used copper orthophenanthroline footprinting to demonstrate that one particularly abundant factor (designated PCAT-1) binds the Ppc1 promoter at two distinct A/T-rich sites located −128 to −158 and −187 to −205 bp upstream of the transcriptional start site. These binding sites share a loose consensus motif having the sequence AARTAAC(T/A)A(G/T)TTTY. Gel retardation competition experiments with oligonucleotides containing these A/T-rich binding sites suggest that both sites bind the same factor, but with different affinities. Fractionation of crude nuclear extracts by heparin-agarose chromatography indicates that PCAT-1 is more prevalent in extracts prepared from salt-stressed leaf tissue. Additional binding activities that interact with the PCAT-1 binding sites have been detected that either increase or decrease in abundance or binding affinity in response to salt stress.

Organization of ribosomal protein genes rp123, rp12, rps19, rp122 and rps3 on the Euglena gracilis chloroplast genome

SummaryThe nucleotide sequence (4,814 bp) was determined for a cluster of five ribosomal protein genes and their DNA flanking regions from the chloroplast genome of Euglena gracilis. The genes are organized as rp123 — 150 by spacer — rpl2 — 59 by spacer —rps19 — 110 by spacer — rp122 — 630 by spacer — rps3. The genes are all of the same polarity and reside 148 bp downstream from an operon for two genes of photosystem I and four genes of photosystem II. The Euglena ribosomal protein gene cluster resembles the S-10 ribosomal protein operon of Escherichia coli in gene organization and follows the exact linear order of the analogous genes in the tobacco and liverwort chloroplast genomes. The number and positions of introns in the Euglena ribosomal protein loci are different from their higher plant counterparts. The Euglena rp123, rps19 and rps3 loci are unique in that they contain three, two and two introns, respectively, whereas rp12 and rp122 lack introns. The introns found in rpl23 (106, 99 and 103 bp), rps19 (103 and 97 bp) and rps3 intron 2 (102 bp) appear to represent either a new class of chloroplast intron found only in constitutively expressed genes, or possibly a degenerate version of Euglena chloroplast group II introns. They are deficient in bases C and G and extremely rich in base T, with a base composition of 53–76% T, 25–34% A, 3–10% G and 2–7% C in the mRNA-like strand. These six introns show minimal resemblance to group IT chloroplast introns. They have a degenerate version of the group II intron conserved boundary sequences at their 5′ and 3′ ends. No conserved internal secondary structures are apparent. By contrast, rps3 intron 1 (409 bp) has a potential group II core secondary structure. The five genes, rpl23 (101 codons), rpl2 (278 codons), rpsl9 (95 codons), rpl22 (114 codons) and rps3 (220 codons) encode lysine-rich polypeptides with predicted molecular weights of 12,152, 31,029, 10,880, 12,819, and 25,238, respectively. The Euglena gene products are 18–50%, and 29–58% identical in primary structure to their E. coli and higher plant counterparts, respectively. Oligonucleotide sequences corresponding to Euglena chloroplast ribosome binding sites are not apparent in the intergenic regions. Inverted repeat sequences are found in the upstream flanking region of rp123 and downstream from rps3.

Identification of enhancer and silencer regions involved in salt-responsive expression of Crassulacean acid metabolism (CAM) genes in the facultative halophyte Mesembryanthemum crystallinum

In response to salinity or drought stress, the facultative halophyte Mesembryanthemum crystallinum will switch from C3 photosynthesis to Crassulacean acid metabolism (CAM). During this switch, the transcription rates of many genes encoding glycolytic, gluconeogenic, and malate metabolism enzymes are increased. In particular, transcription of the Ppcl and Gapl genes encoding a CAM-specific isozyme of phosphoe nolpyruvate carboxylase and NAD-dependent glyceraldehyde-3-phosphate dehydrogenase, respectively, is increased by salinity stress. To investigate the molecular basis of salt-induced gene regulation, we examined the Ppcl and Gapl promoters for cis-elements and trans-acting factors that may participate in their expression. Ppcl or Gapl promoter-β-glucuronidase chimeric gene constructs containing various deletions were introduced into intact, detached M. crystallinum leaves by microprojectile bombardment. The Ppcl 5′-flanking region contains several salt-responsive enhancer regions and one silencer region reflecting the complex regulation patterns exhibited by this promoter in vivo. A region localized between nucleotides -977 and -487 relative to the transcriptional start site appears to regulate the magnitude of salt-inducibility. In contrast, the Gapl promoter contains a single region from -735 to -549 that confers salt-responsive gene expression. Alignment of these 5′-flanking regions reveals several common sequence motifs that resemble consensus binding sites for the Myb class of transcription factors. Electrophoretic gel mobility shift assays indicate that both the -877 to -679 region of Ppcl and the -735 to -549 region of Gapl form a DNA-protein complex unique to nuclear extracts from salt-stressed plants. The appearance of this DNA-protein complex upon salt stress suggests that it may participate in salt-induced transcriptional activation of Ppcl and Gapl.

Induction of a cysteine protease cDNA from Mesembryanthemum crystallinum leaves by environmental stress and plant growth regulators

Abstract A cDNA clone encoding a cysteine protease was isolated from salinity-stressed Mesembryanthemum crystallinum. The deduced sequence of this full-length clone of 1483 bp encodes a pre-proprotein of 367 amino acids with a predicted molecular mass of 41 kDa. Southern blot analysis of genomic DNA indicated that this cDNA is a member of a small multigenic family. Northern and western blot analysis showed that both mRNA and protein expression are strongly induced in leaves in response to salinity stress. mRNA abundance is also enhanced by drought stress and exogenous application of cytokinin and methyl jasmonate, but not ABA. Sequence comparisons showed the encoded gene product shows highest sequence homology (47.7–57.2% identity) to a group of cysteine proteases proposed to have diverse proteolytic functions in higher plants.

A salinity-induced gene from the halophyte M. crystallinum encodes a glycolytic enzyme, cofactor-independent phosphoglyceromutase

In the facultative halophyte Mesembryanthemum crystallinum (ice plant), salinity stress triggers significant changes in gene expression, including increased expression of mRNAs encoding enzymes involved with osmotic adaptation to water stress and the crassulacean acid metabolism (CAM) photosynthetic pathway. To investigate adaptive stress responses in the ice plant at the molecular level, we generated a subtracted cDNA library from stressed plants and identified mRNAs that increase in expression upon salt stress. One full-length cDNA clone was found to encode cofactor-independent phosphoglyceromutase (PGM), an enzyme involved in glycolysis and gluconeogenesis. Pgm1 expression increased in leaves of plants exposed to either saline or drought conditions, whereas levels of the mRNA remained unchanged in roots of hydroponically grown plants. Pgm1 mRNA was also induced in response to treatment with either abscisic acid or cytokinin. Transcription run-on experiments confirmed that Pgm1 mRNA accumulation in leaves was due primarily to increased transcription rates. Immunoblot analysis indicated that Pgm1 mRNA accumulation was accompanied by a modest but reproducible increase in the level of PGM protein. The isolation of a salinity-induced gene encoding a basic enzyme of glycolysis and gluconeogenesis indicates that adaptation to salt stress in the ice plant involves adjustments in fundamental pathways of carbon metabolism and that these adjustments are controlled at the level of gene expression. We propose that the leaf-specific expression of Pgm1 contributes to the maintenance of efficient carbon flux through glycolysis/gluconeogenesis in conjunction with the stress-induced shift to CAM photosynthesis.

Expression of the CAM-form of phospho(enol)pyruvate carboxylase and nucleotide sequence of a full length cDNA from Mesembryanthemum crystallinum

SummaryWe have determined the complete nucleotide sequence of a full length cDNA encoding the Crassulacean acid metabolism (CAM) isogene of phospho(enol)pyruvate carboxylase (PEPCase). The cDNA clone, 3348 bp in length, was obtained from mRNA isolated from Mesembryanthemum crystallinum (common ice plant) which had undergone salt stress and subsequent induction of CAM. The long open reading frame encodes PEPCase (EC 4.1.1.31) with a predicted molecular mass of 110533 daltons. The deduced amino acid sequence of the ice plant PEPCase is most similar to that from maize having an amino acid identity of 74.9%. Sequence identity in corresponding regions of the PEPCase proteins from Escherichia coli and the cyanobacterium Anacystis nidulans are 41.4% and 33.5%, respectively. A compilation of the four amino acid sequences permitted the identification of phylogenetically conserved regions within the proteins which may play a role in the function of this important enzyme in plant metabolism. Gene specific probes from 3′ coding and noncoding regions of the cDNA clone used to probe genomic Southern blots established that this PEPCase gene is present in one copy in the nuclear genome of M. crystallinum. Transcripts arising from this gene increase dramatically when M. crystallinum is irrigated with 0.5 M NaCl, a stress which induces this plant to switch the primary fixation of CO2 from C3 (Calvin cycle) to CAM mode. The salt-induced mRNA encodes a PEPCase isoform which is undetectable in plants in the C3 mode as demonstrated by Northern hybridization.