Isolation and Characterization of a Histidine Biosynthetic Gene in Arabidopsis Encoding a Polypeptide with Two Separate Domains for Phosphoribosyl-ATP Pyrophosphohydrolase and Phosphoribosyl-AMP Cyclohydrolase

Phosphoribosyl-ATP pyrophosphohydrolase (PRA-PH) and phosphoribosyl-AMP cyclohydrolase (PRA-CH) are encoded by HIS4 in yeast and by hisIE in bacteria and catalyze the second and the third step, respectively, in the histidine biosynthetic pathway. By complementing a hisI mutation of Escherichia coli with an Arabidopsis cDNA library, we isolated an Arabidopsis cDNA (At-IE) that possesses these two enzyme activities. The At-IE cDNA encodes a bifunctional protein of 281 amino acids with a calculated molecular mass of 31,666 D. Genomic DNA-blot analysis with the At-IE cDNA as a probe revealed a single-copy gene in Arabidopsis, and RNA-blot analysis showed that the At-IE gene was expressed ubiquitously throughout development. Sequence comparison suggested that the At-IE protein has an N-terminal extension of about 50 amino acids with the properties of a chloroplast transit peptide. We demonstrated through heterologous expression studies in E. coli that the functional domains for the PRA-CH (hisI) and PRA-PH (hisE) resided in the N-terminal and the C-terminal halves, respectively, of the At-IE protein.

wrinkled1: A Novel, Low-Seed-Oil Mutant of Arabidopsis with a Deficiency in the Seed-Specific Regulation of Carbohydrate Metabolism1

During oil deposition in developing seeds of Arabidopsis, photosynthate is imported in the form of carbohydrates into the embryo and converted to triacylglycerols. To identify genes essential for this process and to investigate the molecular basis for the developmental regulation of oil accumulation, mutants producing wrinkled, incompletely filled seeds were isolated. A novel mutant locus, wrinkled1 (wri1), which maps to the bottom of chromosome 3 and causes an 80% reduction in seed oil content, was identified. Wild-type and homozygous wri1 mutant plantlets or mature plants were indistinguishable. However, developing homozygous wri1 seeds were impaired in the incorporation of sucrose and glucose into triacylglycerols, but incorporated pyruvate and acetate at an increased rate. Because the activities of several glycolytic enzymes, in particular hexokinase and pyrophosphate-dependent phosphofructokinase, are reduced in developing homozygous wri1 seeds, it is suggested that WRI1 is involved in the developmental regulation of carbohydrate metabolism during seed filling.

Interaction of Cryptochrome 1, Phytochrome, and Ion Fluxes in Blue-Light-Induced Shrinking of Arabidopsis Hypocotyl Protoplasts1

Protoplasts isolated from red-light-adapted Arabidopsis hypocotyls and incubated under red light exhibited rapid and transient shrinking within a period of 20 min in response to a blue-light pulse and following the onset of continuous blue light. Long-persisting shrinkage was also observed during continuous stimulation. Protoplasts from a hy4 mutant and the phytochrome-deficient phyA/phyB double mutant of Arabidopsis showed little response, whereas those from phyA and phyB mutants showed a partial response. It is concluded that the shrinking response itself is mediated by the HY4 gene product, cryptochrome 1, whereas the blue-light responsiveness is strictly controlled by phytochromes A and B, with a greater contribution by phytochrome B. It is shown further that the far-red-absorbing form of phytochrome (Pfr) was not required during or after, but was required before blue-light perception. Furthermore, a component that directly determines the blue-light responsiveness was generated by Pfr after a lag of 15 min over a 15-min period and decayed with similar kinetics after removal of Pfr by far-red light. The anion-channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid prevented the shrinking response. This result, together with those in the literature and the kinetic features of shrinking, suggests that anion channels are activated first, and outward-rectifying cation channels are subsequently activated, resulting in continued net effluxes of Cl− and K+. The postshrinking volume recovery is achieved by K+ and Cl− influxes, with contribution by the proton motive force. External Ca2+ has no role in shrinking and the recovery. The gradual swelling of protoplasts that prevails under background red light is shown to be a phytochrome-mediated response in which phytochrome A contributes more than phytochrome B.

Complementation of the Arabidopsis pds1 Mutation with the Gene Encoding p-Hydroxyphenylpyruvate Dioxygenase

Plastoquinone and tocopherols are the two major quinone compounds in higher plant chloroplasts and are synthesized by a common pathway. In previous studies we characterized two loci in Arabidopsis defining key steps of this biosynthetic pathway. Mutation of the PDS1 locus disrupts the activity of p-hydroxyphenylpyruvate dioxygenase (HPPDase), the first committed step in the synthesis of both plastoquinone and tocopherols in plants. Although plants homozygous for the pds1 mutation could be rescued by growth in the presence of homogentisic acid, the product of HPPDase, we were unable to determine if the mutation directly or indirectly disrupted HPPDase activity. This paper reports the isolation of a cDNA, pHPPD, encoding Arabidopsis HPPDase and its functional characterization by expression in both plants and Escherichia coli. pHPPD encodes a 50-kD polypeptide with homology to previously identified HPPDases, including 37 highly conserved amino acid residues clustered in the carboxyl region of the protein. Expression of pHPPD in E. coli catalyzes the accumulation of homogentisic acid, indicating that it encodes a functional HPPDase enzyme. Mapping of pHPPD and co-segregation analysis of the pds1 mutation and the HPPD gene indicate tight linkage. Constitutive expression of pHPPD in a pds1 mutant background complements this mutation. Finally, comparison of the HPPD genomic sequences from wild type and pds1 identified a 17-bp deletion in the pds1 allele that results in deletion of the carboxyterminal 26 amino acids of the HPPDase protein. Together, these data conclusively demonstrate that pds1 is a mutation in the HPPDase structural gene.

Molecular Characterization of an Arabidopsis Gene Encoding Hydroperoxide Lyase, a Cytochrome P-450 That Is Wound Inducible1

Hydroperoxide lyase (HPL) cleaves lipid hydroperoxides to produce volatile flavor molecules and also potential signal molecules. We have characterized a gene from Arabidopsis that is homologous to a recently cloned HPL from green pepper (Capsicum annuum). The deduced protein sequence indicates that this gene encodes a cytochrome P-450 with a structure similar to that of allene oxide synthase. The gene was cloned into an expression vector and expressed in Escherichia coli to demonstrate HPL activity. Significant HPL activity was evident when 13S-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid was used as the substrate, whereas activity with 13S-hydroperoxy-9(Z),11(E)-octadecadienoic acid was approximately 10-fold lower. Analysis of headspace volatiles by gas chromatography-mass spectrometry, after addition of the substrate to E. coli extracts expressing the protein, confirmed enzyme-activity data, since cis-3-hexenal was produced by the enzymatic activity of the encoded protein, whereas hexanal production was limited. Molecular characterization of this gene indicates that it is expressed at high levels in floral tissue and is wound inducible but, unlike allene oxide synthase, it is not induced by treatment with methyl jasmonate.

Characterization of Two Glutamate Decarboxylase cDNA Clones from Arabidopsis

Two distinct cDNA clones encoding for the glutamate decarboxylase (GAD) isoenzymes GAD1 and GAD2 from Arabidopsis (L.) Heynh. were characterized. The open reading frames for GAD1 and GAD2 were expressed in Escherichia coli and the recombinant proteins were purified by affinity chromatography. Analysis of the recombinant proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis suggest that GAD1 and GAD2 encode for 58- and 56-kD peptides, respectively. The enzymatic activities of the pure recombinant GAD1 and GAD2 proteins were stimulated 35- and 13-fold, respectively, by Ca2+/calmodulin but not by Ca2+ or calmodulin alone. Southern-blot analysis of genomic DNA suggests that there is only one copy of each gene in Arabidopsis. The GAD1 transcript and a corresponding 58-kD peptide were detected in roots only. Conversely, the GAD2 transcript and a corresponding 56-kD peptide were detected in all organs tested. The specific activity, GAD2 transcript, and 56-kD peptide increased in leaves of plants treated with 10 mm NH4Cl, 5 mm NH4NO3, 5 mm glutamic acid, or 5 mm glutamine as the sole nitrogen source compared with samples from plants treated with 10 mm KNO3. The results from these experiments suggest that in leaves GAD activity is partially controlled by gene expression or RNA stability. Results from preliminary analyses of different tissues imply that these tendencies were not the same in flower stalks and flowers, suggesting that other factors may control GAD activity in these organs. The results from this investigation demonstrate that GAD activity in leaves is altered by different nitrogen treatments, suggesting that GAD2 may play a unique role in nitrogen metabolism.

Multiple Genes Encoding the Conserved CCAAT-Box Transcription Factor Complex Are Expressed in Arabidopsis

The CCAAT motif is found in the promoters of many eukaryotic genes. In yeast a single complex of three proteins, termed HAP2, HAP3, and HAP5, binds to this sequence, and in mammals the three components of the equivalent complex (called variously NF-Y, CBF, or CP1) are also represented by single genes. Here we report the presence of multiple genes for each of the components of the CCAAT-binding complex, HAP2,3,5, from Arabidopsis. Three independent Arabidopsis HAP subunit 2 (AtHAP2) cDNAs were cloned by functional complementation of a yeast hap2 mutant, and two independent forms each of AtHAP3 and AtHAP5 cDNAs were detected in the expressed sequence tag database. Additional homologs (two of AtHAP3 and one of AtHAP5) have been identified from available Arabidopsis genomic sequences. Northern-blot analysis indicated ubiquitous expression for each AtHAP2 and AtHAP5 cDNA in a range of tissues, whereas expression of each AtHAP3 cDNA was under developmental and/or environmental regulation. The unexpected presence of multiple forms of each HAP homolog in Arabidopsis, compared with the single genes in yeast and vertebrates, suggests that the HAP2,3,5 complex may play diverse roles in gene transcription in higher plants.

A New Class of Arabidopsis Mutants with Reduced Hexadecatrienoic Acid Fatty Acid Levels1

Chloroplast glycerolipids in a number of higher-plant species, including Arabidopsis thaliana, are synthesized by two distinct pathways termed the prokaryotic and eukaryotic pathways. The molecules of galactolipids produced by the prokaryotic pathway contain substantial amounts of hexadecatrienoic acid fatty acid. Here we describe a new class of mutants, designated gly1, with reduced levels of hexadecatrienoic acid. Lipid fatty acid profiles indicated that gly1 mutants exhibited a reduced carbon flux through the prokaryotic pathway that was compensated for by an increased carbon flux through the eukaryotic pathway. Genetic and biochemical approaches revealed that the gly1 phenotype could not be explained by a deficiency in the enzymes of the prokaryotic pathway. The flux of fatty acids into the prokaryotic pathway is sensitive to changes in glycerol-3-phosphate (G3P) availability, and the chloroplast G3P pool can be increased by exogenous application of glycerol to leaves. Exogenous glycerol treatment of gly1 plants allowed chemical complementation of the mutant phenotype. These results are consistent with a mutant lesion affecting the G3P supply within the chloroplast. The gly1 mutants may therefore help in determining the pathway for synthesis of chloroplast G3P.

An Arabidopsis VPS45p Homolog Implicated in Protein Transport to the Vacuole1

The Sec1p family of proteins is required for vesicle-mediated protein trafficking between various organelles of the endomembrane system. This family includes Vps45p, which is required for transport to the vacuole in yeast (Saccharomyces cerevisiae). We have isolated a cDNA encoding a VPS45 homolog from Arabidopsis thaliana (AtVPS45). The cDNA is able to complement both the temperature-sensitive growth defect and the vacuolar-targeting defect of a yeast vps45 mutant, indicating that the two proteins are functionally related. AtVPS45p is a peripheral membrane protein that associates with microsomal membranes. Sucrose-density gradient fractionation demonstrated that AtVPS45p co-fractionates with AtELP, a potential vacuolar protein sorting receptor, implying that they may reside on the same membrane populations. These results indicate that AtVPS45p is likely to function in the transport of proteins to the vacuole in plants.

Function and Substrate Specificity of the Gibberellin 3β-Hydroxylase Encoded by the Arabidopsis GA4 Gene1

cDNA corresponding to the GA4 gene of Arabidopsis thaliana L. (Heynh.) was expressed in Escherichia coli, from which cell lysates converted [14C]gibberellin (GA)9 and [14C]GA20 to radiolabeled GA4 and GA1, respectively, thereby confirming that GA4 encodes a GA 3β-hydroxylase. GA9 was the preferred substrate, with a Michaelis value of 1 μm compared with 15 μm for GA20. Hydroxylation of these GAs was regiospecific, with no indication of 2β-hydroxylation or 2,3-desaturation. The capacity of the recombinant enzyme to hydroxylate a range of other GA substrates was investigated. In general, the preferred substrates contained a polar bridge between C-4 and C-10, and 13-deoxy GAs were preferred to their 13-hydroxylated analogs. Therefore, no activity was detected using GA12-aldehyde, GA12, GA19, GA25, GA53, or GA44 as the open lactone (20-hydroxy-GA53), whereas GA15, GA24, and GA44 were hydroxylated to GA37, GA36, and GA38, respectively. The open lactone of GA15 (20-hydroxy-GA12) was hydroxylated but less efficiently than GA15. In contrast to the free acid, GA25 19,20-anhydride was 3β-hydroxylated to give GA13. 2,3-Didehydro-GA9 and GA5 were converted by recombinant GA4 to the corresponding epoxides 2,3-oxido-GA9 and GA6.

Aluminum Resistance in the Arabidopsis Mutant alr-104 Is Caused by an Aluminum-Induced Increase in Rhizosphere pH1

A mechanism that confers increased Al resistance in the Arabidopsis thaliana mutant alr-104 was investigated. A modified vibrating microelectrode system was used to measure H+ fluxes generated along the surface of small Arabidopsis roots. In the absence of Al, no differences in root H+ fluxes between wild type and alr-104 were detected. However, Al exposure induced a 2-fold increase in net H+ influx in alr-104 localized to the root tip. The increased flux raised the root surface pH of alr-104 by 0.15 unit. A root growth assay was used to assess the Al resistance of alr-104 and wild type in a strongly pH-buffered nutrient solution. Increasing the nutrient solution pH from 4.4 to 4.5 significantly increased Al resistance in wild type, which is consistent with the idea that the increased net H+ influx can account for greater Al resistance in alr-104. Differences in Al resistance between wild type and alr-104 disappeared when roots were grown in pH-buffered medium, suggesting that Al resistance in alr-104 is mediated only by pH changes in the rhizosphere. This mutant provides the first evidence, to our knowledge, for an Al-resistance mechanism based on an Al-induced increase in root surface pH.

Cloning, Expression in Escherichia coli, and Characterization of Arabidopsis thaliana UMP/CMP Kinase1

A cDNA encoding the Arabidopsis thaliana uridine 5′-monophosphate (UMP)/cytidine 5′-monophosphate (CMP) kinase was isolated by complementation of a Saccharomyces cerevisiae ura6 mutant. The deduced amino acid sequence of the plant UMP/CMP kinase has 50% identity with other eukaryotic UMP/CMP kinase proteins. The cDNA was subcloned into pGEX-4T-3 and expressed as a glutathione S-transferase fusion protein in Escherichia coli. Following proteolytic digestion, the plant UMP/CMP kinase was purified and analyzed for its structural and kinetic properties. The mass, N-terminal sequence, and total amino acid composition agreed with the sequence and composition predicted from the cDNA sequence. Kinetic analysis revealed that the UMP/CMP kinase preferentially uses ATP (Michaelis constant [Km] = 29 μm when UMP is the other substrate and Km = 292 μm when CMP is the other substrate) as a phosphate donor. However, both UMP (Km = 153 μm) and CMP (Km = 266 μm) were equally acceptable as the phosphate acceptor. The optimal pH for the enzyme is 6.5. P1, P5-di(adenosine-5′) pentaphosphate was found to be a competitive inhibitor of both ATP and UMP.

Isolation of the Ornithine-δ-Aminotransferase cDNA and Effect of Salt Stress on Its Expression in Arabidopsis thaliana1

To evaluate the relative importance of ornithine (Orn) as a precursor in proline (Pro) synthesis, we isolated and sequenced a cDNA encoding the Orn-δ-aminotransferase (δ-OAT) from Arabidopsis thaliana. The deduced amino acid sequence showed high homology with bacterial, yeast, mammalian, and plant sequences, and the N-terminal residues exhibited several common features with a mitochondrial transit peptide. Our results show that under both salt stress and normal conditions, δ-OAT activity and mRNA in young plantlets are slightly higher than in older plants. This appears to be related to the necessity to dispose of an easy recycling product, glutamate. Analysis of the expression of the gene revealed a close association with salt stress and Pro production. In young plantlets, free Pro content, Δ1-pyrroline-5-carboxylate synthase mRNA, δ-OAT activity, and δ-OAT mRNA were all increased by salt-stress treatment. These results suggest that for A. thaliana, the Orn pathway, together with the glutamate pathway, plays an important role in Pro accumulation during osmotic stress. Conversely, in 4-week-old A. thaliana plants, although free Pro level also increased under salt-stress conditions, the δ-OAT activity appeared to be unchanged and δ-OAT mRNA was not detectable. Δ1-pyrroline-5-carboxylate synthase mRNA was still induced at a similar level. Therefore, for the adult plants the free Pro increase seemed to be due to the activity of the enzymes of the glutamate pathway.

Aluminum-Resistant Arabidopsis Mutants That Exhibit Altered Patterns of Aluminum Accumulation and Organic Acid Release from Roots1

Al-resistant (alr) mutants of Arabidopsis thaliana were isolated and characterized to gain a better understanding of the genetic and physiological mechanisms of Al resistance. alr mutants were identified on the basis of enhanced root growth in the presence of levels of Al that strongly inhibited root growth in wild-type seedlings. Genetic analysis of the alr mutants showed that Al resistance was semidominant, and chromosome mapping of the mutants with microsatellite and random amplified polymorphic DNA markers indicated that the mutants mapped to two sites in the Arabidopsis genome: one locus on chromosome 1 (alr-108, alr-128, alr-131, and alr-139) and another on chromosome 4 (alr-104). Al accumulation in roots of mutant seedlings was studied by staining with the fluorescent Al-indicator dye morin and quantified via inductively coupled argon plasma mass spectrometry. It was found that the alr mutants accumulated lower levels of Al in the root tips compared with wild type. The possibility that the mutants released Al-chelating organic acids was examined. The mutants that mapped together on chromosome 1 released greater amounts of citrate or malate (as well as pyruvate) compared with wild type, suggesting that Al exclusion from roots of these alr mutants results from enhanced organic acid exudation. Roots of alr-104, on the other hand, did not exhibit increased release of malate or citrate, but did alkalinize the rhizosphere to a greater extent than wild-type roots. A detailed examination of Al resistance in this mutant is described in an accompanying paper (J. Degenhardt, P.B. Larsen, S.H. Howell, L.V. Kochian [1998] Plant Physiol 117: 19–27).

The GA2 Locus of Arabidopsis thaliana Encodes ent-Kaurene Synthase of Gibberellin Biosynthesis

The ga2 mutant of Arabidopsis thaliana is a gibberellin-deficient dwarf. Previous biochemical studies have suggested that the ga2 mutant is impaired in the conversion of copalyl diphosphate to ent-kaurene, which is catalyzed by ent-kaurene synthase (KS). Overexpression of the previously isolated KS cDNA from pumpkin (Cucurbita maxima) (CmKS) in the ga2 mutant was able to complement the mutant phenotype. A genomic clone coding for KS, AtKS, was isolated from A. thaliana using CmKS cDNA as a heterologous probe. The corresponding A. thaliana cDNA was isolated and expressed in Escherichia coli as a fusion protein. The fusion protein showed enzymatic activity that converted [3H]copalyl diphosphate to [3H]ent-kaurene. The recombinant AtKS protein derived from the ga2–1 mutant is truncated by 14 kD at the C-terminal end and does not contain significant KS activity in vitro. Sequence analysis revealed that a C-2099 to T base substitution, which converts Gln-678 codon to a stop codon, is present in the AtKS cDNA from the ga2–1 mutant. Taken together, our results show that the GA2 locus encodes KS.