Striking sequence similarity in inter- and intra-specific comparisons of class I SLG alleles from Brassica oleracea and Brassica campestris: Implications for the evolution and recognition mechanism

Self-incompatibility in Brassica is controlled by a single multi-allelic locus (S locus), which contains at least two highly polymorphic genes expressed in the stigma: an S glycoprotein gene (SLG) and an S receptor kinase gene (SRK). The putative ligand-binding domain of SRK exhibits high homology to the secretory protein SLG, and it is believed that SLG and SRK form an active receptor kinase complex with a self-pollen ligand, which leads to the rejection of self-pollen. Here, we report 31 novel SLG sequences of Brassica oleracea and Brassica campestris. Sequence comparisons of a large number of SLG alleles and SLG-related genes revealed the following points. (i) The striking sequence similarity observed in an inter-specific comparison (95.6% identity between SLG14 of B. oleracea and SLG25 of B. campestris in deduced amino acid sequence) suggests that SLG diversification predates speciation. (ii) A perfect match of the sequences in hypervariable regions, which are thought to determine S specificity in an intra-specific comparison (SLG8 and SLG46 of B. campestris) and the observation that the hypervariable regions of SLG and SRK of the same S haplotype were not necessarily highly similar suggests that SLG and SRK bind different sites of the pollen ligand and that they together determine S specificity. (iii) Comparison of the hypervariable regions of SLG alleles suggests that intragenic recombination, together with point mutations, has contributed to the generation of the high level of sequence variation in SLG alleles. Models for the evolution of SLG/SRK are presented.

Structure, Properties, and Tissue Localization of Apoplastic α-Glucosidase in Crucifers1

Apoplastic α-glucosidases occur widely in plants but their function is unknown because appropriate substrates in the apoplast have not been identified. Arabidopsis contains at least three α-glucosidase genes; Aglu-1 and Aglu-3 are sequenced and Aglu-2 is known from six expressed sequence tags. Antibodies raised to a portion of Aglu-1 expressed in Escherichia coli recognize two proteins of 96 and 81 kD, respectively, in vegetative tissues of Arabidopsis, broccoli (Brassica oleracea L.), and mustard (Brassica napus L.). The acidic α-glucosidase activity from broccoli flower buds was purified using concanavalin A and ion-exchange chromatography. Two active fractions were resolved and both contained a 96-kD immunoreactive polypeptide. The N-terminal sequence from the 96-kD broccoli α-glucosidase indicated that it corresponds to the Arabidopsis Aglu-2 gene and that approximately 15 kD of the predicted N terminus was cleaved. The 81-kD protein was more abundant than the 96-kD protein, but it was not active with 4-methylumbelliferyl-α-d-glucopyranoside as the substrate and it did not bind to concanavalin A. In situ activity staining using 5-bromo-4-chloro-3-indolyl-α-d-glucopyranoside revealed that the acidic α-glucosidase activity is predominantly located in the outer cortex of broccoli stems and in vascular tissue, especially in leaf traces.

The Biosynthesis of Erucic Acid in Developing Embryos of Brassica rapa1

The prevailing hypothesis on the biosynthesis of erucic acid in developing seeds is that oleic acid, produced in the plastid, is activated to oleoyl-coenzyme A (CoA) for malonyl-CoA-dependent elongation to erucic acid in the cytosol. Several in vivo-labeling experiments designed to probe and extend this hypothesis are reported here. To examine whether newly synthesized oleic acid is directly elongated to erucic acid in developing seeds of Brassica rapa L., embryos were labeled with [14C]acetate, and the ratio of radioactivity of carbon atoms C-5 to C-22 (de novo fatty acid synthesis portion) to carbon atoms C-1 to C-4 (elongated portion) of erucic acid was monitored with time. If newly synthesized 18:1 (oleate) immediately becomes a substrate for elongation to erucic acid, this ratio would be expected to remain constant with incubation time. However, if erucic acid is produced from a pool of preexisting oleic acid, the ratio of 14C in the 4 elongation carbons to 14C in the methyl-terminal 18 carbons would be expected to decrease with time. This labeling ratio decreased with time and, therefore, suggests the existence of an intermediate pool of 18:1, which contributes at least part of the oleoyl precursor for the production of erucic acid. The addition of 2-[{3-chloro-5-(trifluromethyl)-2-pyridinyl}oxyphenoxy] propanoic acid, which inhibits the homodimeric acetyl-CoA carboxylase, severely inhibited the synthesis of [14C]erucic acid, indicating that essentially all malonyl-CoA for elongation of 18:1 to erucate was produced by homodimeric acetyl-CoA carboxylase. Both light and 2-[{3-chloro-5-(trifluromethyl)-2-pyridinyl}oxyphenoxy]-propanoic acid increased the accumulation of [14C]18:1 and the parallel accumulation of [14C]phosphatidylcholine. Taken together, these results show an additional level of complexity in the biosynthesis of erucic acid.

Fatty Acid Elongation Is Independent of Acyl-Coenzyme A Synthetase Activities in Leek and Brassica napus1

In both animal and plant acyl elongation systems, it has been proposed that fatty acids are first activated to acyl-coenzyme A (CoA) before their elongation, and that the ATP dependence of fatty acid elongation is evidence of acyl-CoA synthetase involvement. However, because CoA is not supplied in standard fatty acid elongation assays, it is not clear if CoA-dependent acyl-CoA synthetase activity can provide levels of acyl-CoAs necessary to support typical rates of fatty acid elongation. Therefore, we examined the role of acyl-CoA synthetase in providing the primer for acyl elongation in leek (Allium porrum L.) epidermal microsomes and Brassica napus L. cv Reston oil bodies. As presented here, fatty acid elongation was independent of CoA and proceeded at maximum rates with CoA-free preparations of malonyl-CoA. We also showed that stearic acid ([1-14C]18:0)-CoA was synthesized from [1-14C]18:0 in the presence of CoA-free malonyl-CoA or acetyl-CoA, and that [1-14C]18:0-CoA synthesis under these conditions was ATP dependent. Furthermore, the appearance of [1-14C]18:0 in the acyl-CoA fraction was simultaneous with its appearance in phosphatidylcholine. These data, together with the s of a previous study (A. Hlousek-Radojcic, H. Imai, J.G. Jaworski [1995] Plant J 8: 803–809) showing that exogenous [14C]acyl-CoAs are diluted by a relatively large endogenous pool before they are elongated, strongly indicated that acyl-CoA synthetase did not play a direct role in fatty acid elongation, and that phosphatidylcholine or another glycerolipid was a more likely source of elongation primers than acyl-CoAs.

Extensive purification of a putative RNA polymerase I holoenzyme from plants that accurately initiates rRNA gene transcription in vitro

RNA polymerase I (pol I) is a nuclear enzyme whose function is to transcribe the duplicated genes encoding the precursor of the three largest ribosomal RNAs. We report a cell-free system from broccoli (Brassica oleracea) inflorescence that supports promoter-dependent RNA pol I transcription in vitro. The transcription system was purified extensively by DEAE-Sepharose, Biorex 70, Sephacryl S300, and Mono Q chromatography. Activities required for pre-rRNA transcription copurified with the polymerase on all four columns, suggesting their association as a complex. Purified fractions programmed transcription initiation from the in vivo start site and utilized the same core promoter sequences required in vivo. The complex was not dissociated in 800 mM KCl and had a molecular mass of nearly 2 MDa based on gel filtration chromatography. The most highly purified fractions contain ≈30 polypeptides, two of which were identified immunologically as RNA polymerase subunits. These data suggest that the occurrence of a holoenzyme complex is probably not unique to the pol II system but may be a general feature of eukaryotic nuclear polymerases.

Arabidopsis Sec21p and Sec23p Homologs. Probable Coat Proteins of Plant COP-Coated Vesicles1

Intracellular protein transport between the endoplasmic reticulum (ER) and the Golgi apparatus and within the Golgi apparatus is facilitated by COP (coat protein)-coated vesicles. Their existence in plant cells has not yet been demonstrated, although the GTP-binding proteins required for coat formation have been identified. We have generated antisera against glutathione-S-transferase-fusion proteins prepared with cDNAs encoding the Arabidopsis Sec21p and Sec23p homologs (AtSec21p and AtSec23p, respectively). The former is a constituent of the COPI vesicle coatomer, and the latter is part of the Sec23/24p dimeric complex of the COPII vesicle coat. Cauliflower (Brassica oleracea) inflorescence homogenates were probed with these antibodies and demonstrated the presence of AtSec21p and AtSec23p antigens in both the cytosol and membrane fractions of the cell. The membrane-associated forms of both antigens can be solubilized by treatments typical for extrinsic proteins. The amounts of the cytosolic antigens relative to the membrane-bound forms increase after cold treatment, and the two antigens belong to different protein complexes with molecular sizes comparable to the corresponding nonplant coat proteins. Sucrose-density-gradient centrifugation of microsomal cell membranes from cauliflower suggests that, although AtSec23p seems to be preferentially associated with ER membranes, AtSec21p appears to be bound to both the ER and the Golgi membranes. This could be in agreement with the notion that COPII vesicles are formed at the ER, whereas COPI vesicles can be made by both Golgi and ER membranes. Both AtSec21p and AtSec23p antigens were detected on membranes equilibrating at sucrose densities equivalent to those typical for in vitro-induced COP vesicles from animal and yeast systems. Therefore, a further purification of the putative plant COP vesicles was undertaken.

Restrictions to Carbon Dioxide Conductance and Photosynthesis in Spinach Leaves Recovering from Salt Stress

Salt accumulation in spinach (Spinacia oleracea L.) leaves first inhibits photosynthesis by decreasing stomatal and mesophyll conductances to CO2 diffusion and then impairs ribulose-1,5-bisphosphate carboxylase/oxygenase (S. Delfine, A. Alvino, M. Zacchini, F. Loreto [1998] Aust J Plant Physiol 25: 395–402). We measured gas exchange and fluorescence in spinach recovering from salt accumulation. When a 21-d salt accumulation was reversed by 2 weeks of salt-free irrigation (rewatering), stomatal and mesophyll conductances and photosynthesis partially recovered. For the first time, to our knowledge, it is shown that a reduction of mesophyll conductance can be reversed and that this may influence photosynthesis. Photosynthesis and conductances did not recover when salt drainage was restricted and Na content in the leaves was greater than 3% of the dry matter. Incomplete recovery of photosynthesis in rewatered and control leaves may be attributed to an age-related reduction of conductances. Biochemical properties were not affected by the 21-d salt accumulation. However, ribulose-1,5-bisphosphate carboxylase/oxygenase activity and content were reduced by a 36- to 50-d salt accumulation. Photochemical efficiency was reduced only in 50-d salt-stressed leaves because of a decrease in the fraction of open photosystem II centers. A reduction in chlorophyll content and an increase in the chlorophyll a/b ratio were observed in 43- and 50-d salt-stressed leaves. Low chlorophyll affects light absorptance but is unlikely to change light partitioning between photosystems.

Rhizosphere Bacteria Enhance Selenium Accumulation and Volatilization by Indian Mustard1

Indian mustard (Brassica juncea L.) accumulates high tissue Se concentrations and volatilizes Se in relatively nontoxic forms, such as dimethylselenide. This study showed that the presence of bacteria in the rhizosphere of Indian mustard was necessary to achieve the best rates of plant Se accumulation and volatilization of selenate. Experiments with the antibiotic ampicillin showed that bacteria facilitated 35% of plant Se volatilization and 70% of plant tissue accumulation. These results were confirmed by inoculating axenic plants with rhizosphere bacteria. Compared with axenic controls, plants inoculated with rhizosphere bacteria had 5-fold higher Se concentrations in roots (the site of volatilization) and 4-fold higher rates of Se volatilization. Plants with bacteria contained a heat-labile compound in their root exudate; when this compound was added to the rhizosphere of axenic plants, Se accumulation in plant tissues increased. Plants with bacteria had an increased root surface area compared with axenic plants; the increased area was unlikely to have caused their increased tissue Se accumulation because they did not accumulate more Se when supplied with selenite or selenomethionine. Rhizosphere bacteria also possibly increased plant Se volatilization because they enabled plants to overcome a rate-limiting step in the Se volatilization pathway, i.e. Se accumulation in plant tissues.

Distribution of Sulfur within Oilseed Rape Leaves in Response to Sulfur Deficiency during Vegetative Growth1

The distribution of S to sulfate, glucosinolates, glutathione, and the insoluble fraction within oilseed rape (Brassica napus L.) leaves of different ages was investigated during vegetative growth. The concentrations of glutathione and glucosinolates increased from the oldest to the youngest leaves, whereas the opposite was observed for SO42−. The concentration of insoluble S was similar among all of the leaves. At sufficient S supply and in the youngest leaves, 2% of total S was allocated to glutathione, 6% to glucosinolates, 50% to the insoluble fraction, and the remainder accumulated as SO42−. In the middle and oldest leaves, 70% to 90% of total S accumulated as SO42−, whereas glutathione and glucosinolates together accounted for less than 1% of S. When the S supply was withdrawn (minus S), the concentrations of all S-containing compounds, particularly SO42−, decreased in the youngest and middle leaves. Neither glucosinolates nor glutathione were major sources of S during S deficiency. Plants grown on nutrient solution containing minus S and low N were less deficient than plants grown on solution containing minus S and high N. The effect of N was explained by differences in growth rate. The different responses of leaves of different ages to S deficiency have to be taken into account for the development of field diagnostic tests to determine whether plants are S deficient.

Regulation of Apoplastic NH4+ Concentration in Leaves of Oilseed Rape1

Regulation of apoplastic NH4+ concentration in leaves of oilseed rape (Brassica napus L.) was studied using a vacuum-infiltration technique that allowed controlled manipulations of the apoplastic solution. In leaves infiltrated with NH4+-free solution, the apoplastic NH4+ concentration returned in less than 1.5 min to the preinfiltration level of 0.8 mm. Infiltrated 15NH4+ was rapidly diluted by 14NH4+/14NH3 effluxed from the cell. The exchange rate of 15N/14N over the apoplast due to combined 14N efflux from the symplast and 15N influx from the apoplastic solution was 29.4 μmol g−1 fresh weight h−1 between 0 and 5 min after infiltration. The net uptake of NH4+ into the leaf cells increased linearly with apoplastic NH4+ concentrations between 2 and 10 mm and could be partially inhibited by the channel inhibitors La3+ and tetraethylammonium and by Na+ and K+. When apoplastic pH increased from 5.0 to 8.0, the steady-state apoplastic NH4+ concentration decreased from 1.0 to 0.3 mm. Increasing temperature increased the rate of NH4+ net uptake and reduced the apoplastic steady-state NH4+ concentration. We conclude that the apoplastic solution in leaves of oilseed rape constitutes a highly dynamic NH4+ pool.

A Phosphothreonine Residue at the C-Terminal End of the Plasma Membrane H+-ATPase Is Protected by Fusicoccin-Induced 14–3–3 Binding1

We have isolated the plasma membrane H+−ATPase in a phosphorylated form from spinach (Spinacia oleracea L.) leaf tissue incubated with fusicoccin, a fungal toxin that induces irreversible binding of 14–3–3 protein to the C terminus of the H+-ATPase, thus activating H+ pumping. We have identified threonine-948, the second residue from the C-terminal end of the H+-ATPase, as the phosphorylated amino acid. Turnover of the phosphate group of phosphothreonine-948 was inhibited by 14–3–3 binding, suggesting that this residue may form part of a binding motif for 14–3–3. This is the first identification to our knowledge of an in vivo phosphorylation site in the plant plasma membrane H+-ATPase.

d-Ribulose-5-Phosphate 3-Epimerase: Cloning and Heterologous Expression of the Spinach Gene, and Purification and Characterization of the Recombinant Enzyme1

We have achieved, to our knowledge, the first high-level heterologous expression of the gene encoding d-ribulose-5-phosphate 3-epimerase from any source, thereby permitting isolation and characterization of the epimerase as found in photosynthetic organisms. The extremely labile recombinant spinach (Spinacia oleracea L.) enzyme was stabilized by dl-α-glycerophosphate or ethanol and destabilized by d-ribulose-5-phosphate or 2-mercaptoethanol. Despite this lability, the unprecedentedly high specific activity of the purified material indicates that the structural integrity of the enzyme is maintained throughout isolation. Ethylenediaminetetraacetate and divalent metal cations did not affect epimerase activity, thereby excluding a requirement for the latter in catalysis. As deduced from the sequence of the cloned spinach gene and the electrophoretic mobility under denaturing conditions of the purified recombinant enzyme, its 25-kD subunit size was about the same as that of the corresponding epimerases of yeast and mammals. However, in contrast to these other species, the recombinant spinach enzyme was octameric rather than dimeric, as assessed by gel filtration and polyacrylamide gel electrophoresis under nondenaturing conditions. Western-blot analyses with antibodies to the purified recombinant enzyme confirmed that the epimerase extracted from spinach leaves is also octameric.

Potent Inhibition of Ribulose-Bisphosphate Carboxylase by an Oxidized Impurity in Ribulose-1,5-Bisphosphate1

Oxidation of d-ribulose-1,5-bisphosphate (ribulose-P2) during synthesis and/or storage produces d-glycero-2,3-pentodiulose-1,5-bisphosphate (pentodiulose-P2), a potent slow, tight-binding inhibitor of spinach (Spinacia oleracea L.) ribulose-P2 carboxylase/oxygenase (Rubisco). Differing degrees of contamination with pentodiulose-P2 caused the decline in Rubisco activity seen during Rubisco assay time courses to vary between different preparations of ribulose-P2. With some ribulose-P2 preparations, this compound can be the dominant cause of the decline, far exceeding the significance of the catalytic by-product, d-xylulose-1,5-bisphosphate. Unlike xylulose-1,5-bisphosphate, pentodiulose-P2 did not appear to be a significant by-product of catalysis by wild-type Rubisco at saturating CO2 concentration. It was produced slowly during frozen storage of ribulose-P2, even at low pH, more rapidly in Rubisco assay buffers at room temperature, and particularly rapidly on deliberate oxidation of ribulose-P2 with Cu2+. Its formation was prevented by the exclusion of transition metals and O2. Pentodiulose-P2 was unstable and decayed to a variety of other less-inhibitory compounds, particularly in the presence of some buffers. However, it formed a tight, stable complex with carbamylated spinach Rubisco, which could be isolated by gel filtration, presumably because its structure mimics that of the enediol intermediate of Rubisco catalysis. Rubisco catalyzes the cleavage of pentodiulose-P2 by H2O2, producing P-glycolate.

Participation of Mitochondrial Metabolism in Photorespiration1 : Reconstituted System of Peroxisomes and Mitochondria from Spinach Leaves

In this study the interplay of mitochondria and peroxisomes in photorespiration was simulated in a reconstituted system of isolated mitochondria and peroxisomes from spinach (Spinacia oleracea L.) leaves. The mitochondria oxidizing glycine produced serine, which was reduced in the peroxisomes to glycerate. The required reducing equivalents were provided by the mitochondria via the malate-oxaloacetate (OAA) shuttle, in which OAA was reduced in the mitochondrial matrix by NADH generated during glycine oxidation. The rate of peroxisomal glycerate formation, as compared with peroxisomal protein, resembled the corresponding rate required during leaf photosynthesis under ambient conditions. When the reconstituted system produced glycerate at this rate, the malate-to-OAA ratio was in equilibrium with a ratio of NADH/NAD of 8.8 × 10−3. This low ratio is in the same range as the ratio of NADH/NAD in the cytosol of mesophyll cells of intact illuminated spinach leaves, as we had estimated earlier. This result demonstrates that in the photorespiratory cycle a transfer of redox equivalents from the mitochondria to peroxisomes, as postulated from separate experiments with isolated mitochondria and peroxisomes, can indeed operate under conditions of the very low reductive state of the NADH/NAD system prevailing in the cytosol of mesophyll cells in a leaf during photosynthesis.

Characterization of a Gene for Spinach CAP160 and Expression of Two Spinach Cold-Acclimation Proteins in Tobacco1

The cDNA sequence for CAP160, an acidic protein previously linked with cold acclimation in spinach (Spinacia oleracea L.), was characterized and found to encode a novel acidic protein of 780 amino acids having very limited homology to a pair of Arabidopsis thaliana stress-regulated proteins, rd29A and rd29B. The lack of similarity in the structural organization of the spinach and Arabidopsis genes highlights the absence of a high degree of conservation of this cold-stress gene across taxonomic boundaries. The protein has several unique motifs that may relate to its function during cold stress. Expression of the CAP160 mRNA was increased by low-temperature exposure and water stress in a manner consistent with a probable function during stresses that involve dehydration. The coding sequences for CAP160 and CAP85, another spinach cold-stress protein, were introduced into tobacco (Nicotiana tabacum) under the control of the 35S promoter using Agrobacterium tumefaciens-based transformation. Tobacco plants expressing the proteins individually or coexpressing both proteins were evaluated for relative freezing-stress tolerance. The killing temperature for 50% of the cells of the transgenic plants was not different from that of the wild-type plants. As determined by a more sensitive time/temperature kinetic study, plants expressing the spinach proteins had slightly lower levels of electrolyte leakage than wild-type plants, indicative of a small reduction of freezing-stress injury. Clearly, the heterologous expression of two cold-stress proteins had no profound influence on stress tolerance, a result that is consistent with the quantitative nature of cold-stress-tolerance traits.