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This Update is concerned with the mechanism of synthesis of heavy metal-binding thiol peptides, phytochelatins (PCs), by the enzyme PC synthase (EC 2.3.2.15). The bulk of the considerations in this review centers on what has been learned recently of the fundamental mechanics of PC synthesis, the domain organization and phylogenetic distribution of PC synthases, and PC synthase-like enzymes, and what this tells us about the chemistry underlying and the enzyme residues necessary for PC synthesis. It was decided to prepare a review of this type rather than aim at a more comprehensive treatment of heavy metal homeostasis and detoxification in plants for two reasons. The first is that there are already several contemporary reviews dealing with the more global aspects of plant heavy metal physiology. Excellent examples are Cobbett (2000), Clemens (2001), and Cobbett and Goldsbrough (2002). Readers who have not already read these are encouraged to do so. The second reason is that some of the most fascinating and unexpected developments for our understanding in this area of late derive from investigations of the catalytic mechanism and distribution of PC synthases, facets of this field of research that have yet to be reviewed in detail.

Living systems are supported and sustained by the genome through the action of the transcriptome, proteome, metabolome, and ionome, the four basic biochemical pillars of functional genomics. These pillars represent the sum of all the expressed genes, proteins, metabolites, and elements within an

A nitrate reductase (NR)-null mutant of Arabidopsis was constructed that had a deletion of the major NR gene NIA2 and an insertion in the NIA1 NR gene. This mutant had no detectable NR activity and could not use nitrate as the sole nitrogen source. Starch mobilization was not induced by nitrate in this mutant but was induced by ammonium, indicating that nitrate was not the signal for this process. Microarray analysis of gene expression revealed that 595 genes responded to nitrate (5 mm nitrate for 2 h) in both wild-type and mutant plants. This group of genes was overrepresented most significantly in the functional categories of energy, metabolism, and glycolysis and gluconeogenesis. Because the nitrate response of these genes was NR independent, nitrate and not a downstream metabolite served as the signal. The microarray analysis also revealed that shoots can be as responsive to nitrate as roots, yet there was substantial organ specificity to the nitrate response.

Genetic and physiological data establish that Arabidopsis AtHKT1 facilitates Na(+) homeostasis in planta and by this function modulates K(+) nutrient status. Mutations that disrupt AtHKT1 function suppress NaCl sensitivity of sos1-1 and sos2-2, as well as of sos3-1 seedlings grown in vitro and plants grown in controlled environmental conditions. hkt1 suppression of sos3-1 NaCl sensitivity is linked to higher Na(+) content in the shoot and lower content of the ion in the root, reducing the Na(+) imbalance between these organs that is caused by sos3-1. AtHKT1 transgene expression, driven by its innate promoter, increases NaCl but not LiCl or KCl sensitivity of wild-type (Col-0 gl1) or of sos3-1 seedlings. NaCl sensitivity induced by AtHKT1 transgene expression is linked to a lower K(+) to Na(+) ratio in the root. However, hkt1 mutations increase NaCl sensitivity of both seedlings in vitro and plants grown in controlled environmental conditions, which is correlated with a lower K(+) to Na(+) ratio in the shoot. These results establish that AtHKT1 is a focal determinant of Na(+) homeostasis in planta, as either positive or negative modulation of its function disturbs ion status that is manifested as salt sensitivity. K(+)-deficient growth of sos1-1, sos2-2, and sos3-1 seedlings is suppressed completely by hkt1-1. AtHKT1 transgene expression exacerbates K(+) deficiency of sos3-1 or wild-type seedlings. Together, these results indicate that AtHKT1 controls Na(+) homeostasis in planta and through this function regulates K(+) nutrient status.

Physicochemical similarities between K(+) and Na(+) result in interactions between their homeostatic mechanisms. The physiological interactions between these two ions was investigated by examining aspects of K(+) nutrition in the Arabidopsis salt overly sensitive (sos) mutants, and salt sensitivity in the K(+) transport mutants akt1 (Arabidopsis K(+) transporter) and skor (shaker-like K(+) outward-rectifying channel). The K(+)-uptake ability (membrane permeability) of the sos mutant root cells measured electrophysiologically was normal in control conditions. Also, growth rates of these mutants in Na(+)-free media displayed wild-type K(+) dependence. However, mild salt stress (50 mm NaCl) strongly inhibited root-cell K(+) permeability and growth rate in K(+)-limiting conditions of sos1 but not wild-type plants. Increasing K(+) availability partially rescued the sos1 growth phenotype. Therefore, it appears that in the presence of Na(+), the SOS1 Na(+)-H(+) antiporter is necessary for protecting the K(+) permeability on which growth depends. The hypothesis that the elevated cytoplasmic Na(+) levels predicted to result from loss of SOS1 function impaired the K(+) permeability was tested by introducing 10 mm NaCl into the cytoplasm of a patch-clamped wild-type root cell. Complete loss of AKT1 K(+) channel activity ensued. AKT1 is apparently a target of salt stress in sos1 plants, resulting in poor growth due to impaired K(+) uptake. Complementary studies showed that akt1 seedlings were salt sensitive during early seedling development, but skor seedlings were normal. Thus, the effect of Na(+) on K(+) transport is probably more important at the uptake stage than at the xylem loading stage.

Transcriptome analysis, using Affymetrix ATH1 arrays and a real-time reverse transcription-PCR platform for >1,400 transcription factors, was performed to identify processes affected by long-term nitrogen-deprivation or short-term nitrate nutrition in Arabidopsis. Two days of nitrogen deprivation led to coordinate repression of the majority of the genes assigned to photosynthesis, chlorophyll synthesis, plastid protein synthesis, induction of many genes for secondary metabolism, and reprogramming of mitochondrial electron transport. Nitrate readdition led to rapid, widespread, and coordinated changes. Multiple genes for the uptake and reduction of nitrate, the generation of reducing equivalents, and organic acid skeletons were induced within 30 min, before primary metabolites changed significantly. By 3 h, most genes assigned to amino acid and nucleotide biosynthesis and scavenging were induced, while most genes assigned to amino acid and nucleotide breakdown were repressed. There was coordinate induction of many genes assigned to RNA synthesis and processing and most of the genes assigned to amino acid activation and protein synthesis. Although amino acids involved in central metabolism increased, minor amino acids decreased, providing independent evidence for the activation of protein synthesis. Specific genes encoding expansin and tonoplast intrinsic proteins were induced, indicating activation of cell expansion and growth in response to nitrate nutrition. There were rapid responses in the expression of many genes potentially involved in regulation, including genes for trehalose metabolism and hormone metabolism, protein kinases and phosphatases, receptor kinases, and transcription factors.

Full genome microarrays were used to assess transcriptional responses of Arabidopsis seedlings to changing external supply of the essential macronutrient potassium (K(+)). Rank product statistics and iterative group analysis were employed to identify differentially regulated genes and statistically significant coregulated sets of functionally related genes. The most prominent response was found for genes linked to the phytohormone jasmonic acid (JA). Transcript levels for the JA biosynthetic enzymes lipoxygenase, allene oxide synthase, and allene oxide cyclase were strongly increased during K(+) starvation and quickly decreased after K(+) resupply. A large number of well-known JA responsive genes showed the same expression profile, including genes involved in storage of amino acids (VSP), glucosinolate production (CYP79), polyamine biosynthesis (ADC2), and defense (PDF1.2). Our findings highlight a novel role of JA in nutrient signaling and stress management through a variety of physiological processes such as nutrient storage, recycling, and reallocation. Other highly significant K(+)-responsive genes discovered in our study encoded cell wall proteins (e.g. extensins and arabinogalactans) and ion transporters (e.g. the high-affinity K(+) transporter HAK5 and the nitrate transporter NRT2.1) as well as proteins with a putative role in Ca(2+) signaling (e.g. calmodulins). On the basis of our results, we propose candidate genes involved in K(+) perception and signaling as well as a network of molecular processes underlying plant adaptation to K(+) deficiency.

A combined bioinformatic and experimental approach is being used to uncover the functions of a novel family of cation/H(+) exchanger (CHX) genes in plants using Arabidopsis as a model. The predicted protein (85-95 kD) of 28 AtCHX genes after revision consists of an amino-terminal domain with 10 to 12 transmembrane spans (approximately 440 residues) and a hydrophilic domain of approximately 360 residues at the carboxyl end, which is proposed to have regulatory roles. The hydrophobic, but not the hydrophilic, domain of plant CHX is remarkably similar to monovalent cation/proton antiporter-2 (CPA2) proteins, especially yeast (Saccharomyces cerevisiae) KHA1 and Synechocystis NhaS4. Reports of characterized fungal and prokaryotic CPA2 indicate that they have various transport modes, including K(+)/H(+) (KHA1), Na(+)/H(+)-K(+) (GerN) antiport, and ligand-gated ion channel (KefC). The expression pattern of AtCHX genes was determined by reverse transcription PCR, promoter-driven beta-glucuronidase expression in transgenic plants, and Affymetrix ATH1 genome arrays. Results show that 18 genes are specifically or preferentially expressed in the male gametophyte, and six genes are highly expressed in sporophytic tissues. Microarray data revealed that several AtCHX genes were developmentally regulated during microgametogenesis. An exciting idea is that CHX proteins allow osmotic adjustment and K(+) homeostasis as mature pollen desiccates and then rehydrates at germination. The multiplicity of CHX-like genes is conserved in higher plants but is not found in animals. Only 17 genes, OsCHX01 to OsCHX17, were identified in rice (Oryza sativa) subsp. japonica, suggesting diversification of CHX in Arabidopsis. These results reveal a novel CHX gene family in flowering plants with potential functions in pollen development, germination, and tube growth.

The frd3 mutant of Arabidopsis exhibits constitutive expression of its iron uptake responses and is chlorotic. These phenotypes are consistent with defects either in iron deficiency signaling or in iron translocation and localization. Here we present several experiments demonstrating that a functional FRD3 gene is necessary for correct iron localization in both the root and shoot of Arabidopsis plants. Reciprocal grafting experiments with frd3 and wild-type Arabidopsis plants reveal that the phenotype of a grafted plant is determined by the genotype of the root, not by the genotype of the shoot. This indicates that FRD3 function is root-specific and points to a role for FRD3 in delivering iron to the shoot in a usable form. When grown under certain conditions, frd3 mutant plants overaccumulate iron in their shoot tissues. However, we demonstrate by direct measurement of iron levels in shoot protoplasts that intracellular iron levels in frd3 are only about one-half the levels in wild type. Histochemical staining for iron reveals that frd3 mutants accumulate high levels of ferric iron in their root vascular cylinder, the same tissues in which the FRD3 gene is expressed. Taken together, these results clearly indicate a role for FRD3 in iron localization in Arabidopsis. Specifically, FRD3 is likely to function in root xylem loading of an iron chelator or other factor necessary for efficient iron uptake out of the xylem or apoplastic space and into leaf cells.

Dennis R. Hoagland, who was Professor of Plant Nutrition at the University of California at Berkeley from 1927 until his death in 1949, is generally acknowledged as the father of modern plant nutrition research. In 1942 he was invited to deliver the prestigious series of John M. Prather Lectures at