▪ Abstract Among the heavy metal-binding ligands in plant cells the phytochelatins (PCs) and metallothioneins (MTs) are the best characterized. PCs and MTs are different classes of cysteine-rich, heavy metal-binding protein molecules. PCs are enzymatically synthesized peptides, whereas MTs are gene-encoded polypeptides. Recently, genes encoding the enzyme PC synthase have been identified in plants and other species while the completion of the Arabidopsis genome sequence has allowed the identification of the entire suite of MT genes in a higher plant. Recent advances in understanding the regulation of PC biosynthesis and MT gene expression and the possible roles of PCs and MTs in heavy metal detoxification and homeostasis are reviewed.

PHYTOCHELATINS AND METALLOTHIONEINS: Roles in Heavy Metal Detoxification and Homeostasis

Stressful environments such as salinity, drought, and high temperature (heat) cause alterations in a wide range of physiological, biochemical, and molecular processes in plants. Photosynthesis, the most fundamental and intricate physiological process in all green plants, is also severely affected in all its phases by such stresses. Since the mechanism of photosynthesis involves various components, including photosynthetic pigments and photosystems, the electron transport system, and CO2 reduction pathways, any damage at any level caused by a stress may reduce the overall photosynthetic capacity of a green plant. Details of the stress-induced damage and adverse effects on different types of pigments, photosystems, components of electron transport system, alterations in the activities of enzymes involved in the mechanism of photosynthesis, and changes in various gas exchange characteristics, particularly of agricultural plants, are considered in this review. In addition, we discussed also progress made during the last two decades in producing transgenic lines of different C3 crops with enhanced photosynthetic performance, which was reached by either the overexpression of C3 enzymes or transcription factors or the incorporation of genes encoding C4 enzymes into C3 plants. We also discussed critically a current, worldwide effort to identify signaling components, such as transcription factors and protein kinases, particularly mitogen-activated protein kinases (MAPKs) involved in stress adaptation in agricultural plants.

Photosynthesis under stressful environments: An overview

The rhizosphere is the zone of soil immediately surrounding plant roots that is modified by root activity In this critical zone, plants perceive and respond to their environment As a consequence of normal growth and development, a large range of organic and inorganic substances are exchanged between the root and soil, which inevitably leads to changes in the biochemical and physical properties of the rhizosphere Plants also modify their rhizosphere in response to certain environmental signals and stresses Organic anions are commonly detected in this region, and their exudation from plant roots has now been associated with nutrient deficiencies and inorganic ion stresses This review summarizes recent developments in the understanding of the function, mechanism, and regulation of organic anion exudation from roots The benefits that plants derive from the presence of organic anions in the rhizosphere are described and the potential for biotechnology to increase organic anion exudation is highlighted

Function and mechanism of organic anion exudation from plant roots

Genome sequences reveal that a deluge of DNA from organelles has constantly been bombarding the nucleus since the origin of organelles. Recent experiments have shown that DNA is transferred from organelles to the nucleus at frequencies that were previously unimaginable. Endosymbiotic gene transfer is a ubiquitous, continuing and natural process that pervades nuclear DNA dynamics. This relentless influx of organelle DNA has abolished organelle autonomy and increased nuclear complexity.

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Endosymbiotic Gene Transfer: Organelle Genomes Forge Eukaryotic Chromosomes

This essay reviews comparative studies of animal mitochondrial DNA (mtDNA), with emphasis on findings made and ideas developed at Berkeley. It argues that such studies are bringing together two previous paths of progress in evolutionary biology. One path is that of those who worked far above the species level and were concerned with genealogical trees, time scales and the accumulation of new mutations on surviving molecular lineages. The other path is that of those who worked at and below the species level and were concerned mainly with population structure, migration and the frequencies of alleles that existed in an ancestral population. This fusion of paths is made possible by the high rate at which mutations accumulate on mtDNA lineages and by this molecule's uniparental and apparently haploid mode of inheritance. These properties make mtDNA a superb tool for building trees and time scales relating molecular lineages at and below the species level. In addition, owing to its mode of inheritance, mtDNA is more sensitive to bottlenecks in population size and to population subdivision than are nuclear genes. Joint comparative studies of both mtDNA and nuclear DNA variability give us valuable insights into how effective population size has varied through time. Such studies also give insight into the conditions under which mtDNA from one species can colonize another species.

Mitochondrial DNA and two perspectives on evolutionary genetics

We have determined the structure of the maize (Zea mays L. subsp.mays line B73) nuclear gene encoding the phosphoenolpyruvate (PEP) carboxylase isozyme involved in C4 photosynthesis. The gene is 5.3 kb long and has ten exons that range in size from 85 to 999 bp. The nine introns vary from 97 to 872 bp. The sequence of 663 bp of 5′-flanking and 205 bp of 3′-flanking DNA is reported along with the entire gene sequence. Several short repetitive sequences were found in the 5′-flanking DNA that have characteristics similar to elements important in the light regulation of pea genes encoding the small subunit of ribulose 1,5-bisphosphate carboxylase. In addition, some 5′-flanking sequence similarities were found in a comparison with other light-regulated genes from maize and wheat.

Structure and expression of the maize gene encoding the phosphoenolpyruvate carboxylase isozyme involved in C4 photosynthesis

Herbicide-resistant transgenic cotton (Gossypium hirsutum L.) plants carrying mutant forms of a native aceto- hydroxyacid synthase (AHAS) gene have been obtained by Agrobacterium and biolistic transformation. The native gene, A19, was mutated in vitro to create amino acid substitutions at residue 563 or residue 642 of the precursor polypeptide. Transformation with the mutated forms of the Al9 gene produced resistance to imidazolinone and sulfonylurea herbicides (563 substitution), or imidazolinones only (642 substitution). The herbicide-resistant phen- otype of transformants was also manifested in their in vitro AHAS activity. Seedling explants of both Coker and Acala cotton varieties were transformed with the mutated forms of the Al9 gene using Agrobacterium. In these experiments, hundreds of transformation events were obtained with the Coker varieties, while the Acala varieties were transformed with an efficiency about one-tenth that of Coker. Herbicide-resistant Coker and Acala plants were regenerated from a subset of transformation events. Embryonic cell suspension cultures of both Coker and Acala varieties were biolistically transformed at high frequencies using cloned cotton DNA fragments carrying the mutated forms of the Al9 gene. In these transformation experiments the mutated Al9 gene served as the selectable marker, and the efficiency of selection was comparable to that obtained with the NPT II gene marker of vector Bin 19. Using this method, transgenic Acala plants resistant to imidazolinone herbicides were obtained. Southern blot analyses indicated the presence of two copies of the mutated A 19 transgene in one of the biolistically transformed Ra plants, and a single copy in one of the Ro plants transformed with Agrobacterium. As expected, progeny seedlings derived from outcrosses involving the Ro plant transformed with Agrobacterium segregated in a 1: 1 ratio with respect to herbicide resistance. The resistant progeny grew normally after irrigation with 175 pg/l of the imidazolinone herbicide imazaquin, which is five times the field application rate. In contrast, untransformed sibling plants were severely stunted.

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Herbicide-resistant Acala and Coker cottons transformed with a native gene encoding mutant forms of acetohydroxyacid synthase

The expression of maize (Zea mays) phophoenolpyruvate carboxylase (PPC) gene constructions was studied in transgenic tobacco plants (Nicotiana tabacum). Where transcription was under the control of a maize PPC gene promoter, a low level of aberrantly large PPC transcript was detected. Analysis of this PPC transcript indicated that transcription initiation occurs upstream of the normal site. Despite the aberrant transcription initiation, expression of the PPC transcript was still light-regulated. Higher levels of maize PPC transcript of the correct size were obtained with a chimeric gene construction containing a tobacco (Nicotiana plumbaginifolia) chlorophyll a/b binding protein gene promoter. The PPC activities in the leaves of these transgenic plants were up to twofold higher than those of nontransformed plants. Two forms of PPC with different kinetic properties were identified in leaf extracts of the transgenic plants: one form with a high apparent Km for phosphoenolpyruvate (maize isozyme), and a second form exhibiting a low apparent Km (tobacco isozyme). Biochemical analyses of these plants indicated that the transgenic plants had significantly elevated levels of titratable acidity and malic acid. These biochemical differences did not produce any significant physiological changes with respect to photosynthetic rate or CO2 compensation point.

Expression of Maize Phosphoenolpyruvate Carboxylase in Transgenic Tobacco : Effects on Biochemistry and Physiology

Mitochondrial DNA sequences in the nuclear genome of Strongylocentrotus purpuratus.

New methods have been applied to the determination of single copy DNA sequence differences between the sea urchin species Strongylocentrotus purpuratus, S. franciscanus, S. drobachiensis, and Lytechinus pictus. The thermal stability of interspecies DNA duplexes was measured in a solvent (2.4 M tetraethylammonium chloride) that suppresses the effect of base composition on melting temperature. The lengths of duplexes were measured after digestion with S1 nuclease and correction made for the effect of length on thermal stability. The degree of base substitution that has occurred in the single copy DNA during sea urchin evolution is significantly larger than indicated by earlier measurements. We estimate that 19% of the nucleotides of the single copy DNA are different in the genomes of the two sea urchin congeners, S. purpuratus, and S. franciscanus, which apparently diverged only 15 to 20 million years ago.

John W. Grula

Papers

Structure and expression of the maize gene encoding the phosphoenolpyruvate carboxylase isozyme involved in C4 photosynthesis

Herbicide-resistant Acala and Coker cottons transformed with a native gene encoding mutant forms of acetohydroxyacid synthase

Expression of Maize Phosphoenolpyruvate Carboxylase in Transgenic Tobacco : Effects on Biochemistry and Physiology

Mitochondrial DNA sequences in the nuclear genome of Strongylocentrotus purpuratus.

Evolution of sea urchin non-repetitive DNA.