The GA5 locus of Arabidopsis thaliana encodes a multifunctional gibberellin 20-oxidase: molecular cloning and functional expression.

The biosynthesis of gibberellins (GAs) after GA12-aldehyde involves a series of oxidative steps that lead to the formation of bioactive GAs. Previously, a cDNA clone encoding a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing), EC 1.14.11.-] was isolated by immunoscreening a cDNA library from liquid endosperm of pumpkin (Cucurbita maxima L.) with antibodies against partially purified GA 20-oxidase. Here, we report isolation of a genomic clone for GA 20-oxidase from a genomic library of the long-day species Arabidopsis thaliana Heynh., strain Columbia, by using the pumpkin cDNA clone as a heterologous probe. This genomic clone contains a GA 20-oxidase gene that consists of three exons and two introns. The three exons are 1131-bp long and encode 377 amino acid residues. A cDNA clone corresponding to the putative GA 20-oxidase genomic sequence was constructed with the reverse transcription-PCR method, and the identity of the cDNA clone was confirmed by analyzing the capability of the fusion protein expressed in Escherichia coli to convert GA53 to GA44 and GA19 to GA20. The Arabidopsis GA 20-oxidase shares 55% identity and > 80% similarity with the pumpkin GA 20-oxidase at the derived amino acid level. Both GA 20-oxidases share high homology with other 2-oxoglutarate-dependent dioxygenases (2-ODDs), but the highest homology was found between the two GA 20-oxidases. Mapping results indicated tight linkage between the cloned GA 20-oxidase and the GA5 locus of Arabidopsis. The ga5 semidwarf mutant contains a G-->A point mutation that inserts a translational stop codon in the protein-coding sequence, thus confirming that the GA5 locus encodes GA 20-oxidase. Expression of the GA5 gene in Ara-bidopsis leaves was enhanced after plants were transferred from short to long days; it was reduced by GA4 treatment, suggesting end-product repression in the GA biosynthetic pathway.

Evidence for five divergent thioredoxin h sequences in Arabidopsis thaliana.

Five different clones encoding thioredoxin homologues were isolated from Arabidopsis thaliana cDNA libraries. On the basis of the sequences they encode divergent proteins, but all belong to the cytoplasmic thioredoxins h previously described in higher plants. The five proteins obtained by overexpressing the coding sequences in Escherichia coli present typical thioredoxin activities (NADP(+)-malate dehydrogenase activation and reduction by Arabidopsis thioredoxin reductase) despite the presence of a variant active site, Trp-Cys-Pro-Pro-Cys, in three proteins in place of the canonical Trp-Cys-Gly-Pro-Cys sequence described for thioredoxins in prokaryotes and eukaryotes. Southern blots show that each cDNA is encoded by a single gene but suggest the presence of additional related sequences in the Arabidopsis genome. This very complex diversity of thioredoxins h is probably common to all higher plants, since the Arabidopsis sequences appear to have diverged very early, at the beginning of plant speciation. This diversity allows the transduction of a redox signal into multiple pathways.

Use of Arabidopsis thaliana defense-related mutants to dissect the plant response to pathogens.

The plant defense response to microbial pathogens had been studied primarily by using biochemical and physiological techniques. Recently, several laboratories have developed a variety of pathosystems utilizing Arabidopsis thaliana as a model host so that genetic analysis could also be used to study plant defense responses. Utilizing a pathosystem that involves the infection of Arabidopsis with pathogenic pseudomonads, we have cloned the Arabidopsis disease-resistance gene RPS2, which corresponds to the avirulence gene avrRpt2 in a gene-for-gene relationship. RPS2 encodes a 105-kDa protein containing a leucine zipper, a nucleotide binding site, and 14 imperfect leucine-rich repeats. The RPS2 protein is remarkably similar to the product of the tobacco N gene, which confers resistance to tobacco mosaic virus. We have also isolated a series of Arabidopsis mutants that synthesize decreased levels of an Arabidopsis phytoalexin called camalexin. Analysis of these mutants indicated that camalexin does not play a significant role in limiting growth of avirulent Pseudomonas syringae strains during the hypersensitive defense response but that it may play a role in limiting the growth of virulent strains. More generally, we have shown that we can utilize Arabidopsis to systematically dissect the defense response by isolation and characterization of appropriate defense-related mutants.

A gene encoding a phosphatidylinositol-specific phospholipase C is induced by dehydration and salt stress in Arabidopsis thaliana.

A cDNA corresponding to a putative phosphatidylinositol-specific phospholipase C (PI-PLC) in the higher plant Arabidopsis thaliana was cloned by use of the polymerase chain reaction. The cDNA, designated cAtPLC1, encodes a putative polypeptide of 561 aa with a calculated molecular mass of 64 kDa. The putative product includes so-called X and Y domains found in all PI-PLCs identified to date. In mammalian cells, there are three types of PI-PLC, PLC-beta, -gamma, and -delta. The overall structure of the putative AtPLC1 protein is most similar to that of PLC-delta, although the AtPLC1 protein is much smaller than PLCs from other organisms. The recombinant AtPLC1 protein synthesized in Escherichia coli was able to hydrolyze phosphatidylinositol 4,5-bisphosphate and this activity was completely dependent on Ca2+, as observed also for mammalian PI-PLCs. These results suggest that the AtPLC1 gene encodes a genuine PI-PLC of a higher plant. Northern blot analysis showed that the AtPLC1 gene is expressed at very low levels in the plant under normal conditions but is induced to a significant extent under various environmental stresses, such as dehydration, salinity, and low temperature. These observations suggest that AtPLC1 might be involved in the signal-transduction pathways of environmental stresses and that an increase in the level of AtPLC1 might amplify the signal, in a manner that contributes to the adaptation of the plant to these stresses.

Gene-for-gene disease resistance without the hypersensitive response in Arabidopsis dnd1 mutant

The cell death response known as the hypersensitive response (HR) is a central feature of gene-for-gene plant disease resistance. A mutant line of Arabidopsis thaliana was identified in which effective gene-for-gene resistance occurs despite the virtual absence of HR cell death. Plants mutated at the DND1 locus are defective in HR cell death but retain characteristic responses to avirulent Pseudomonas syringae such as induction of pathogenesis-related gene expression and strong restriction of pathogen growth. Mutant dnd1 plants also exhibit enhanced resistance against a broad spectrum of virulent fungal, bacterial, and viral pathogens. The resistance against virulent pathogens in dnd1 plants is quantitatively less strong and is differentiable from the gene-for-gene resistance mediated by resistance genes RPS2 and RPM1. Levels of salicylic acid compounds and mRNAs for pathogenesis-related genes are elevated constitutively in dnd1 plants. This constitutive induction of systemic acquired resistance may substitute for HR cell death in potentiating the stronger gene-for-gene defense response. Although cell death may contribute to defense signal transduction in wild-type plants, the dnd1 mutant demonstrates that strong restriction of pathogen growth can occur in the absence of extensive HR cell death in the gene-for-gene resistance response of Arabidopsis against P. syringae.

High temperature promotes auxin-mediated hypocotyl elongation in Arabidopsis

Physiological studies with excised stem segments have implicated the plant hormone indole-3-acetic acid (IAA or auxin) in the regulation of cell elongation. Supporting evidence from intact plants has been somewhat more difficult to obtain, however. Here, we report the identification and characterization of an auxin-mediated cell elongation growth response in Arabidopsis thaliana. When grown in the light at high temperature (29°C), Arabidopsis seedlings exhibit dramatic hypocotyl elongation compared with seedlings grown at 20°C. This temperature-dependent growth response is sharply reduced by mutations in the auxin response or transport pathways and in seedlings containing reduced levels of free IAA. In contrast, mutants deficient in gibberellin and abscisic acid biosynthesis or in ethylene response are unaffected. Furthermore, we detect a corresponding increase in the level of free IAA in seedlings grown at high temperature, suggesting that temperature regulates auxin synthesis or catabolism to mediate this growth response. Consistent with this possibility, high temperature also stimulates other auxin-mediated processes including auxin-inducible gene expression. Based on these results, we propose that growth at high temperature promotes an increase in auxin levels resulting in increased hypocotyl elongation. These results strongly support the contention that endogenous auxin promotes cell elongation in intact plants.

Identification of a family of zinc transporter genes from Arabidopsis that respond to zinc deficiency

Millions of people worldwide suffer from nutritional imbalances of essential metals like zinc. These same metals, along with pollutants like cadmium and lead, contaminate soils at many sites around the world. In addition to posing a threat to human health, these metals can poison plants, livestock, and wildlife. Deciphering how metals are absorbed, transported, and incorporated as protein cofactors may help solve both of these problems. For example, edible plants could be engineered to serve as better dietary sources of metal nutrients, and other plant species could be tailored to remove metal ions from contaminated soils. We report here the cloning of the first zinc transporter genes from plants, the ZIP1, ZIP2, and ZIP3 genes of Arabidopsis thaliana. Expression in yeast of these closely related genes confers zinc uptake activities. In the plant, ZIP1 and ZIP3 are expressed in roots in response to zinc deficiency, suggesting that they transport zinc from the soil into the plant. Although expression of ZIP2 has not been detected, a fourth related Arabidopsis gene identified by genome sequencing, ZIP4, is induced in both shoots and roots of zinc-limited plants. Thus, ZIP4 may transport zinc intracellularly or between plant tissues. These ZIP proteins define a family of metal ion transporters that are found in plants, protozoa, fungi, invertebrates, and vertebrates, making it now possible to address questions of metal ion accumulation and homeostasis in diverse organisms.

The protein kinase CK2 is involved in regulation of circadian rhythms in Arabidopsis

A wide range of processes in plants, including expression of certain genes, is regulated by endogenous circadian rhythms. The circadian clock-associated 1 (CCA1) and the late elongated hypocotyl (LHY) proteins have been shown to be closely associated with clock function in Arabidopsis thaliana. The protein kinase CK2 can interact with and phosphorylate CCA1, but its role in the regulation of the circadian clock remains unknown. Here we show that plants overexpressing CKB3, a regulatory subunit of CK2, display increased CK2 activity and shorter periods of rhythmic expression of CCA1 and LHY. CK2 is also able to interact with and phosphorylate LHY in vitro. Additionally, overexpression of CKB3 shortened the periods of four known circadian clock-controlled genes with different phase angles, demonstrating that many clock outputs are affected. This overexpression also reduced phytochrome induction of an Lhcb gene. Finally, we found that the photoperiodic flowering response, which is influenced by circadian rhythms, was diminished in the transgenic lines, and that the plants flowered earlier on both long-day and short-day photoperiods. These data demonstrate that CK2 is involved in regulation of the circadian clock in Arabidopsis.

Photorepair mutants of Arabidopsis

UV radiation induces two major DNA damage products, the cyclobutane pyrimidine dimer (CPD) and, at a lower frequency, the pyrimidine (6–4) pyrimidinone dimer (6–4 product). Although Escherichia coli and Saccharomyces cerevisiae produce a CPD-specific photolyase that eliminates only this class of dimer, Arabidopsis thaliana, Drosophila melanogaster, Crotalus atrox, and Xenopus laevis have recently been shown to photoreactivate both CPDs and 6–4 products. We describe the isolation and characterization of two new classes of mutants of Arabidopsis, termed uvr2 and uvr3, that are defective in the photoreactivation of CPDs and 6–4 products, respectively. We demonstrate that the CPD photolyase mutation is genetically linked to a DNA sequence encoding a type II (metazoan) CPD photolyase. In addition, we are able to generate plants in which only CPDs or 6–4 products are photoreactivated in the nuclear genome by exposing these mutants to UV light and then allowing them to repair one or the other class of dimers. This provides us with a unique opportunity to study the biological consequences of each of these two major UV-induced photoproducts in an intact living system.

Gene dosage and stochastic effects determine the severity and direction of uniparental ribosomal RNA gene silencing (nucleolar dominance) in Arabidopsis allopolyploids

Nucleolar dominance is an epigenetic phenomenon in which one parental set of ribosomal RNA (rRNA) genes is silenced in an interspecific hybrid. In natural Arabidopsis suecica, an allotetraploid (amphidiploid) hybrid of Arabidopsis thaliana and Cardaminopsis arenosa, the A. thaliana rRNA genes are repressed. Interestingly, A. thaliana rRNA gene silencing is variable in synthetic Arabidopsis suecica F1 hybrids. Two generations are needed for A. thaliana rRNA genes to be silenced in all lines, revealing a species-biased direction but stochastic onset to nucleolar dominance. Backcrossing synthetic A. suecica to tetraploid A. thaliana yielded progeny with active A. thaliana rRNA genes and, in some cases, silenced C. arenosa rRNA genes, showing that the direction of dominance can be switched. The hypothesis that naturally dominant rRNA genes have a superior binding affinity for a limiting transcription factor is inconsistent with dominance switching. Inactivation of a species-specific transcription factor is argued against by showing that A. thaliana and C. arenosa rRNA genes can be expressed transiently in the other species. Transfected A. thaliana genes are also active in A. suecica protoplasts in which chromosomal A. thaliana genes are repressed. Collectively, these data suggest that nucleolar dominance is a chromosomal phenomenon that results in coordinate or cooperative silencing of rRNA genes.

Separate jasmonate-dependent and salicylate-dependent defense-response pathways in Arabidopsis are essential for resistance to distinct microbial pathogens

The endogenous plant hormones salicylic acid (SA) and jasmonic acid (JA), whose levels increase on pathogen infection, activate separate sets of genes encoding antimicrobial proteins in Arabidopsis thaliana. The pathogen-inducible genes PR-1, PR-2, and PR-5 require SA signaling for activation, whereas the plant defensin gene PDF1.2, along with a PR-3 and PR-4 gene, are induced by pathogens via an SA-independent and JA-dependent pathway. An Arabidopsis mutant, coi1, that is affected in the JA-response pathway shows enhanced susceptibility to infection by the fungal pathogens Alternaria brassicicola and Botrytis cinerea but not to Peronospora parasitica, and vice versa for two Arabidopsis genotypes (npr1 and NahG) with a defect in their SA response. Resistance to P. parasitica was boosted by external application of the SA-mimicking compound 2,6-dichloroisonicotinic acid [Delaney, T., et al. (1994) Science 266, 1247–1250] but not by methyl jasmonate (MeJA), whereas treatment with MeJA but not 2,6-dichloroisonicotinic acid elevated resistance to Alternaria brassicicola. The protective effect of MeJA against A. brassicicola was the result of an endogenous defense response activated in planta and not a direct effect of MeJA on the pathogen, as no protection to A. brassicicola was observed in the coi1 mutant treated with MeJA. These data point to the existence of at least two separate hormone-dependent defense pathways in Arabidopsis that contribute to resistance against distinct microbial pathogens.

RNA editing in Arabidopsis mitochondria effects 441 C to U changes in ORFs

On the basis of the sequence of the mitochondrial genome in the flowering plant Arabidopsis thaliana, RNA editing events were systematically investigated in the respective RNA population. A total of 456 C to U, but no U to C, conversions were identified exclusively in mRNAs, 441 in ORFs, 8 in introns, and 7 in leader and trailer sequences. No RNA editing was seen in any of the rRNAs or in several tRNAs investigated for potential mismatch corrections. RNA editing affects individual coding regions with frequencies varying between 0 and 18.9% of the codons. The predominance of RNA editing events in the first two codon positions is not related to translational decoding, because it is not correlated with codon usage. As a general effect, RNA editing increases the hydrophobicity of the coded mitochondrial proteins. Concerning the selection of RNA editing sites, little significant nucleotide preference is observed in their vicinity in comparison to unedited C residues. This sequence bias is, per se, not sufficient to specify individual C nucleotides in the total RNA population in Arabidopsis mitochondria.

The Arabidopsis cullin AtCUL1 is modified by the ubiquitin-related protein RUB1

The ubiquitin-like protein RUB1 is conjugated to target proteins by a mechanism similar to that of ubiquitin conjugation. Genetic studies in Arabidopsis thaliana have implicated the RUB-conjugation pathway in auxin response. The first step in the pathway is RUB activation by a bipartite enzyme composed of the AXR1 and ECR1 proteins. Ubiquitin activation is an ATP-dependent process that involves the formation of an AMP-ubiquitin intermediate. Here we show that RUB activation by AXR1-ECR1 also involves formation of an AMP-RUB intermediate and that this reaction is catalyzed by the ECR1 subunit alone. In addition, we identified an Arabidopsis protein called RCE1 that is a likely RUB-conjugating enzyme. RCE1 works together with AXR1-ECR1 to promote formation of a stable RUB conjugate with the Arabidopsis cullin AtCUL1 in vitro. Using a tagged version of RUB1, we show that this modification occurs in vivo. Because AtCUL1 is a component of the ubiquitin protein ligase SCFTIR1, a complex that also functions in auxin response, we propose that RUB modification of AtCUL1 is important for auxin response.

A PII-like protein in Arabidopsis: Putative role in nitrogen sensing

PII is a protein allosteric effector in Escherichia coli and other bacteria that indirectly regulates glutamine synthetase at the transcriptional and post-translational levels in response to nitrogen availability. Data supporting the notion that plants have a nitrogen regulatory system(s) includes previous studies showing that the levels of mRNA for plant nitrogen assimilatory genes such as glutamine synthetase (GLN) and asparagine synthetase (ASN) are modulated by carbon and organic nitrogen metabolites. Here, we have characterized a PII homolog (GLB1) in two higher plants, Arabidopsis thaliana and Ricinus communis (Castor bean). Each plant PII-like protein has high overall identity to E. coli PII (50%). Western blot analyses reveal that the plant PII-like protein is a nuclear-encoded chloroplast protein. The PII-like protein of plants appears to be regulated at the transcriptional level in that levels of GLB1 mRNA are affected by light and metabolites. To initiate studies of the in vivo function of the Arabidopsis PII-like protein, we have constructed transgenic lines in which PII expression is uncoupled from its native regulation. Analyses of these transgenic plants support the notion that the plant PII-like protein may serve as part of a complex signal transduction network involved in perceiving the status of carbon and organic nitrogen. Thus, the PII protein found in archaea, bacteria, and now in higher eukaryotes (plants) is one of the most widespread regulatory proteins known, providing evidence for an ancestral metabolic regulatory mechanism that may have existed before the divergence of these three domains of life.

An Arabidopsis mutant that requires increased calcium for potassium nutrition and salt tolerance

Potassium (K+) nutrition and salt tolerance are key factors controlling plant productivity. However, the mechanisms by which plants regulate K+ nutrition and salt tolerance are poorly understood. We report here the identification of an Arabidopsis thaliana mutant, sos3 (salt-overly-sensitive 3), which is hypersensitive to Na+ and Li+ stresses. The mutation is recessive and is in a nuclear gene that maps to chromosome V. The sos3 mutation also renders the plant unable to grow on low K+. Surprisingly, increased extracellular Ca2+ suppresses the growth defect of sos3 plants on low K+ or 50 mM NaCl. In contrast, high concentrations of external Ca2+ do not rescue the growth of the salt-hypersensitive sos1 mutant on low K+ or 50 mM NaCl. Under NaCl stress, sos3 seedlings accumulated more Na+ and less K+ than the wild type. Increased external Ca2+ improved K+/Na+ selectivity of both sos3 and wild-type plants. However, this Ca2+ effect in sos3 is more than twice as much as that in the wild type. In addition to defining the first plant mutant with an altered calcium response, these results demonstrate that the SOS3 locus is essential for K+ nutrition, K+/Na+ selectivity, and salt tolerance in higher plants.