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.

Metabolism of Indole-3-Acetic Acid in Arabidopsis1

The metabolism of indole-3-acetic acid (IAA) was investigated in 14-d-old Arabidopsis plants grown in liquid culture. After ruling out metabolites formed as an effect of nonsterile conditions, high-level feeding, and spontaneous interconversions, a simple metabolic pattern emerged. Oxindole-3-acetic acid (OxIAA), OxIAA conjugated to a hexose moiety via the carboxyl group, and the conjugates indole-3-acetyl aspartic acid (IAAsp) and indole-3-acetyl glutamate (IAGlu) were identified by mass spectrometry as primary products of IAA fed to the plants. Refeeding experiments demonstrated that none of these conjugates could be hydrolyzed back to IAA to any measurable extent at this developmental stage. IAAsp was further oxidized, especially when high levels of IAA were fed into the system, yielding OxIAAsp and OH-IAAsp. This contrasted with the metabolic fate of IAGlu, since that conjugate was not further metabolized. At IAA concentrations below 0.5 μm, most of the supplied IAA was metabolized via the OxIAA pathway, whereas only a minor portion was conjugated. However, increasing the IAA concentrations to 5 μm drastically altered the metabolic pattern, with marked induction of conjugation to IAAsp and IAGlu. This investigation used concentrations for feeding experiments that were near endogenous levels, showing that the metabolic pathways controlling the IAA pool size in Arabidopsis are limited and, therefore, make good targets for mutant screens provided that precautions are taken to avoid inducing artificial metabolism.

Hexose Transport in Growing Petunia Pollen Tubes and Characterization of a Pollen-Specific, Putative Monosaccharide Transporter1

We investigated the molecular and physiological processes of sugar uptake and metabolism during pollen tube growth and plant fertilization. In vitro germination assays showed that petunia (Petunia hybrida) pollen can germinate and grow not only in medium containing sucrose (Suc) as a carbon source, but also in medium containing the monosaccharides glucose (Glc) or fructose (Fru). Furthermore, high-performance liquid chromatography analysis demonstrated a rapid and complete conversion of Suc into equimolar amounts of Glc and Fru when pollen was cultured in a medium containing 2% Suc. This indicates the presence of wall-bound invertase activity and uptake of sugars in the form of monosaccharides by the growing pollen tube. A cDNA designated pmt1 (petunia monosaccharide transporter 1), which is highly homologous to plant monosaccharide transporters, was isolated from petunia. Pmt1 belongs to a small gene family and is expressed specifically in the male gametophyte, but not in any other vegetative or floral tissues. Pmt1 is activated after the first pollen mitosis, and high levels of mRNA accumulate in mature and germinating pollen. A model describing the transport of sugars to the style, the conversion of Suc into Glc and Fru, and the active uptake by a monosaccharide transporter into the pollen tube is presented.

A Model for Signal Transduction during Gamete Release in the Fucoid Alga Pelvetia compressa1

Fucoid algae release gametes into seawater following an inductive light period (potentiation), and gamete expulsion from potentiated receptacles of Pelvetia compressa began about 2 min after a light-to-dark transition. Agitation of the medium reversed potentiation, with an exponential time course completed in about 3 h. Light regulated two signaling pathways during potentiation and gamete expulsion: a photosynthetic pathway and a photosynthesis-independent pathway in which red light was active but blue light was not. Uptake of K+ appears to have an important role in potentiation, because a 50% inhibition of potentiation occurred in the presence of the tetraethylammonium ion, a K+-channel blocker. A central role of anion channels in the maintenance of potentiation is suggested by the premature release of gametes in the light when receptacles were incubated with inhibitors of slow-type anion channels. An inhibitor of tyrosine kinases, tyrphostin A63, also inhibited potentiation. A model for gamete release from P. compressa is presented that proposes that illumination results in the accumulation of ions (e.g. K+) throughout the cells of the receptacle during potentiation, which then move into the extracellular matrix during gamete expulsion to generate osmomechanical force, resulting in gamete release.

Distinct Biochemical and Topological Properties of the 31- and 27-Kilodalton Plasma Membrane Intrinsic Protein Subgroups from Red Beet1

Plasma membrane vesicles from red beet (Beta vulgaris L.) storage tissue contain two prominent major intrinsic protein species of 31 and 27 kD (X. Qi, C.Y Tai, B.P. Wasserman [1995] Plant Physiol 108: 387–392). In this study affinity-purified antibodies were used to investigate their localization and biochemical properties. Both plasma membrane intrinsic protein (PMIP) subgroups partitioned identically in sucrose gradients; however, each exhibited distinct properties when probed for multimer formation, and by limited proteolysis. The tendency of each PMIP species to form disulfide-linked aggregates was studied by inclusion of various sulfhydryl agents during tissue homogenization and vesicle isolation. In the absence of dithiothreitol and sulfhydryl reagents, PMIP27 yielded a mixture of monomeric and aggregated species. In contrast, generation of a monomeric species of PMIP31 required the addition of dithiothreitol, iodoacetic acid, or N-ethylmaleimide. Mixed disulfide-linked heterodimers between the PMIP31 and PMIP27 subgroups were not detected. Based on vectorial proteolysis of right-side-out vesicles with trypsin and hydropathy analysis of the predicted amino acid sequence derived from the gene encoding PMIP27, a topological model for a PMIP27 was established. Two exposed tryptic cleavage sites were identified from proteolysis of PMIP27, and each was distinct from the single exposed site previously identified in surface loop C of a PMIP31. Although the PMIP31 and PMIP27 species both contain integral proteins that appear to occur within a single vesicle population, these results demonstrate that each PMIP subgroup responds differently to perturbations of the membrane.

The Two Genes Encoding Starch-Branching Enzymes IIa and IIb Are Differentially Expressed in Barley1

The sbeIIa and sbeIIb genes, encoding starch-branching enzyme (SBE) IIa and SBEIIb in barley (Hordeum vulgare L.), have been isolated. The 5′ portions of the two genes are strongly divergent, primarily due to the 2064-nucleotide-long intron 2 in sbeIIb. The sequence of this intron shows that it contains a retro-transposon-like element. Expression of sbeIIb but not sbeIIa was found to be endosperm specific. The temporal expression patterns for sbeIIa and sbeIIb were similar and peaked around 12 d after pollination. DNA gel-blot analysis demonstrated that sbeIIa and sbeIIb are both single-copy genes in the barley genome. By fluorescence in situ hybridization, the sbeIIa and sbeIIb genes were mapped to chromosomes 2 and 5, respectively. The cDNA clones for SBEIIa and SBEIIb were isolated and sequenced. The amino acid sequences of SBEIIa and SBEIIb were almost 80% identical. The major structural difference between the two enzymes was the presence of a 94-amino acid N-terminal extension in the SBEIIb precursor. The (β/α)8-barrel topology of the α-amylase superfamily and the catalytic residues implicated in branching enzymes are conserved in both barley enzymes.

Expression of the Yeast FRE Genes in Transgenic Tobacco

Two yeast genes, FRE1 and FRE2 (encoding Fe(III) reductases) were placed under the control of the cauliflower mosaic virus 35S promoter and introduced into tobacco (Nicotiana tabacum L.) via Agrobacterium tumefaciens-mediated transformation. Homozygous lines containing FRE1, FRE2, or FRE1 plus FRE2 were generated. Northern-blot analyses revealed mRNA of two different sizes in FRE1 lines, whereas all FRE2 lines had mRNA only of the expected length. Fe(III) reduction, chlorophyll contents, and Fe levels were determined in transgenic and control plants under Fe-sufficient and Fe-deficient conditions. In a normal growth environment, the highest root Fe(III) reduction, 4-fold higher than in controls, occurred in the double transformant (FRE1 + FRE2). Elevated Fe(III) reduction was also observed in all FRE2 and some FRE1 lines. The increased Fe(III) reduction occurred along the entire length of the roots and on shoot sections. FRE2 and double transformants were more tolerant to Fe deficiency in hydroponic culture, as shown by higher chlorophyll and Fe concentrations in younger leaves, whereas FRE1 transformants did not differ from the controls. Overall, the beneficial effects of FRE2 were consistent, suggesting that FRE2 may be used to improve Fe efficiency in crop plants.

The H+-Sucrose Cotransporter NtSUT1 Is Essential for Sugar Export from Tobacco Leaves1

In many species translocation of sucrose from the mesophyll to the phloem is carrier mediated. A sucrose/H+-symporter cDNA, NtSUT1, was isolated from tobacco (Nicotiana tabacum) and shown to be highly expressed in mature leaves and at low levels in other tissues, including floral organs. To study the in vivo function of NtSUT1, tobacco plants were transformed with a SUT1 antisense construct under control of the cauliflower mosaic virus 35S promoter. Upon maturation, leaves of transformants expressing reduced amounts of SUT1 mRNA curled downward, and strongly affected plants developed chloroses and necroses that led to death. The leaves exhibited impaired ability to export recently fixed 14CO2 and were unable to export transient starch during extended periods of darkness. As a consequence, soluble carbohydrates accumulated and photosynthesis was reduced. Autoradiographs of leaves show a heterogenous pattern of CO2 fixation even after a 24-h chase. The 14C pattern does not change with time, suggesting that movement of photosynthate between mesophyll cells may also be impaired. The affected lines show a reduction in the development of the root system and delayed or impaired flowering. Taken together, the effects observed in a seed plant (tobacco) demonstrate the importance of SUT1 for sucrose loading into the phloem via an apoplastic route and possibly for intermesophyll transport as well.

Expression Patterns Conferred by Tyrosine/Dihydroxyphenylalanine Decarboxylase Promoters from Opium Poppy Are Conserved in Transgenic Tobacco1

Opium poppy (Papaver somniferum) contains a large family of tyrosine/dihydroxyphenylalanine decarboxylase (tydc) genes involved in the biosynthesis of benzylisoquinoline alkaloids and cell wall-bound hydroxycinnamic acid amides. Eight members from two distinct gene subfamilies have been isolated, tydc1, tydc4, tydc6, tydc8, and tydc9 in one group and tydc2, tydc3, and tydc7 in the other. The tydc8 and tydc9 genes were located 3.2 kb apart on one genomic clone, suggesting that the family is clustered. Transcripts for most tydc genes were detected only in roots. Only tydc2 and tydc7 revealed expression in both roots and shoots, and TYDC3 mRNAs were the only specific transcripts detected in seedlings. TYDC1, TYDC8, and TYDC9 mRNAs, which occurred in roots, were not detected in elicitor-treated opium poppy cultures. Expression of tydc4, which contains a premature termination codon, was not detected under any conditions. Five tydc promoters were fused to the β-glucuronidase (GUS) reporter gene in a binary vector. All constructs produced transient GUS activity in microprojectile-bombarded opium poppy and tobacco (Nicotiana tabacum) cell cultures. The organ- and tissue-specific expression pattern of tydc promoter-GUS fusions in transgenic tobacco was generally parallel to that of corresponding tydc genes in opium poppy. GUS expression was most abundant in the internal phloem of shoot organs and in the stele of roots. Select tydc promoter-GUS fusions were also wound induced in transgenic tobacco, suggesting that the basic mechanisms of developmental and inducible tydc regulation are conserved across plant species.

Callose Deposition Is Responsible for Apoplastic Semipermeability of the Endosperm Envelope of Muskmelon Seeds1

Semipermeable cell walls or apoplastic “membranes” have been hypothesized to be present in various plant tissues. Although often associated with suberized or lignified walls, the wall component that confers osmotic semipermeability is not known. In muskmelon (Cucumis melo L.) seeds, a thin, membranous endosperm completely encloses the embryo, creating a semipermeable apoplastic envelope. When dead muskmelon seeds are allowed to imbibe, solutes leaking from the embryo are retained within the envelope, resulting in osmotic water uptake and swelling called osmotic distention (OD). The endosperm envelope of muskmelon seeds stained with aniline blue, which is specific for callose (β-1,3-glucan). Outside of the aniline-blue-stained layer was a Sudan III- and IV-staining (lipid-containing) layer. In young developing seeds 25 d after anthesis (DAA) that did not exhibit OD, the lipid layer was already present but callose had not been deposited. At 35 DAA, callose was detected as distinct vesicles or globules in the endosperm envelope. A thick callose layer was evident at 40 DAA, coinciding with development of the capacity for OD. Removal of the outer lipid layer by brief chloroform treatment resulted in more rapid water uptake by both viable and nonviable (boiled) seeds, but did not affect semipermeability of the endosperm envelope. The aniline-blue-staining layer was digested by β-1,3-glucanase, and these envelopes lost OD. Thus, apoplastic semipermeability of the muskmelon endosperm envelope is dependent on the deposition of a thick callose-containing layer outside of the endosperm cell walls.

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.

Characterization of a Maize Tonoplast Aquaporin Expressed in Zones of Cell Division and Elongation1

We studied aquaporins in maize (Zea mays), an important crop in which numerous studies on plant water relations have been carried out. A maize cDNA, ZmTIP1, was isolated by reverse transcription-coupled PCR using conserved motifs from plant aquaporins. The derived amino acid sequence of ZmTIP1 shows 76% sequence identity with the tonoplast aquaporin γ-TIP (tonoplast intrinsic protein) from Arabidopsis. Expression of ZmTIP1 in Xenopus laevis oocytes showed that it increased the osmotic water permeability of oocytes 5-fold; this water transport was inhibited by mercuric chloride. A cross-reacting antiserum made against bean α-TIP was used for immunocytochemical localization of ZmTIP1. These results indicate that this and/or other aquaporins is abundantly present in the small vacuoles of meristematic cells. Northern analysis demonstrated that ZmTIP1 is expressed in all plant organs. In situ hybridization showed a high ZmTIP1 expression in meristems and zones of cell enlargement: tips of primary and lateral roots, leaf primordia, and male and female inflorescence meristems. The high ZmTIP1 expression in meristems and expanding cells suggests that ZmTIP1 is needed (a) for vacuole biogenesis and (b) to support the rapid influx of water into vacuoles during cell expansion.

High Expression of the Tonoplast Aquaporin ZmTIP1 in Epidermal and Conducting Tissues of Maize1

Aquaporins are integral membrane proteins of the tonoplast and the plasma membrane that facilitate the passage of water through these membranes. Because of their potentially important role in regulating water flow in plants, studies documenting aquaporin gene expression in specialized tissues involved in water and solute transport are important. We used in situ hybridization to examine the expression pattern of the tonoplast aquaporin ZmTIP1 in different organs of maize (Zea mays L.). This tonoplast water channel is highly expressed in the root epidermis, the root endodermis, the small parenchyma cells surrounding mature xylem vessels in the root and the stem, phloem companion cells and a ring of cells around the phloem strand in the stem and the leaf sheath, and the basal endosperm transfer cells in developing kernels. We postulate that the high level of expression of ZmTIP1 in these tissues facilitates rapid flow of water through the tonoplast to permit osmotic equilibration between the cytosol and the vacuolar content, and to permit rapid transcellular water flow through living cells when required.

RNA Polymerase Subunits Encoded by the Plastid rpo Genes Are Not Shared with the Nucleus-Encoded Plastid Enzyme1

Plastid genes in photosynthetic higher plants are transcribed by at least two RNA polymerases. The plastid rpoA, rpoB, rpoC1, and rpoC2 genes encode subunits of the plastid-encoded plastid RNA polymerase (PEP), an Escherichia coli-like core enzyme. The second enzyme is referred to as the nucleus-encoded plastid RNA polymerase (NEP), since its subunits are assumed to be encoded in the nucleus. Promoters for NEP have been previously characterized in tobacco plants lacking PEP due to targeted deletion of rpoB (encoding the β-subunit) from the plastid genome. To determine if NEP and PEP share any essential subunits, the rpoA, rpoC1, and rpoC2 genes encoding the PEP α-, β′-, and β"-subunits were removed by targeted gene deletion from the plastid genome. We report here that deletion of each of these genes yielded photosynthetically defective plants that lack PEP activity while maintaining transcription specificity from NEP promoters. Therefore, rpoA, rpoB, rpoC1, and rpoC2 encode PEP subunits that are not essential components of the NEP transcription machinery. Furthermore, our data indicate that no functional copy of rpoA, rpoB, rpoC1, or rpoC2 that could complement the deleted plastid rpo genes exists outside the plastids.

Chemotropic and Contact Responses of Phytophthora sojae Hyphae to Soybean Isoflavonoids and Artificial Substrates1

We have investigated the role of the isoflavones daidzein and genistein on the chemotropic behavior of germinating cysts of Phytophthora sojae. Hyphal germlings were shown to respond chemotropically to daidzein and genistein, suggesting that hyphal tips from zoospores that have encysted adjacent to the root may use specific host isoflavones to locate their host. Observations of the contact response of hyphal germlings were made on several different substrates in the presence and absence of isoflavones. Hyphal tips of germlings detected and penetrated pores in membranes and produced multiple appressoria on smooth, impenetrable surfaces. Hyphae that successfully penetrated the synthetic membrane were observed to grow away from the membrane surface. The presence of isoflavones in the medium surrounding the hyphal germlings did not appear to alter any of those habits. Daidzein and genistein did not inhibit germination or initial hyphal growth at concentrations up to 20 μm.