Arnd Sturm

cDNA cloning of carrot extracellular beta-fructosidase and its expression in response to wounding and bacterial infection.

We isolated a full-length cDNA for apoplastic (extracellular or cell wall-bound) beta-fructosidase (invertase), determined its nucleotide sequence, and used it as a probe to measure changes in mRNA as a result of wounding of carrot storage roots and infection of carrot plants with the bacterial pathogen Erwinia carotovora. The derived amino acid sequence of extracellular beta-fructosidase shows that it is a basic protein (pl 9.9) with a signal sequence for entry into the endoplasmic reticulum and a propeptide at the N terminus that is not present in the mature protein. Amino acid sequence comparison with yeast and bacterial invertases shows that the overall homology is only about 28%, but that there are short conserved motifs, one of which is at the active site. Maturing carrot storage roots contain barely detectable levels of mRNA for extracellular beta-fructosidase and these levels rise slowly but dramatically after wounding with maximal expression after 12 hours. Infection of roots and leaves of carrot plants with E. carotovora results in a very fast increase in the mRNA levels with maximal expression after 1 hour. These results indicate that apoplastic beta-fructosidase is probably a new and hitherto unrecognized pathogenesis-related protein [Van Loon, L.C. (1985). Plant Mol. Biol. 4, 111-116]. Suspension-cultured carrot cells contain high levels of mRNA for extracellular beta-fructosidase and these levels remain the same whether the cells are grown on sucrose, glucose, or fructose.

Antisense Repression of Vacuolar and Cell Wall Invertase in Transgenic Carrot Alters Early Plant Development and Sucrose Partitioning

To unravel the functions of cell wall and vacuolar invertases in carrot, we used an antisense technique to generate transgenic carrot plants with reduced enzyme activity. Phenotypic alterations appeared at very early stages of development; indeed, the morphology of cotyledon-stage embryos was markedly changed. At the stage at which control plantlets had two to three leaves and one primary root, shoots of transgenic plantlets did not separate into individual leaves but consisted of stunted, interconnected green structures. When transgenic plantlets were grown on media containing a mixture of sucrose, glucose, and fructose rather than sucrose alone, the malformation was alleviated, and plantlets looked normal. Plantlets from hexose-containing media produced mature plants when transferred to soil. Plants expressing antisense mRNA for cell wall invertase had a bushy appearance due to the development of extra leaves, which accumulated elevated levels of sucrose and starch. Simultaneously, tap root development was markedly reduced, and the resulting smaller organs contained lower levels of carbohydrates. Compared with control plants, the dry weight leaf-to-root ratio of cell wall invertase antisense plants was shifted from 1:3 to 17:1. Plants expressing antisense mRNA for vacuolar invertase also had more leaves than did control plants, but tap roots developed normally, although they were smaller, and the leaf-to-root ratio was 1.5:1. Again, the carbohydrate content of leaves was elevated, and that of roots was reduced. Our data suggest that acid invertases play an important role in early plant development, most likely via control of sugar composition and metabolic fluxes. Later in plant development, both isoenzymes seem to have important functions in sucrose partitioning.

Isolation of a Mutant Arabidopsis Plant That Lacks N-Acetyl Glucosaminyl Transferase I and Is Unable to Synthesize Golgi-Modified Complex N-Linked Glycans

The complex asparagine-linked glycans of plant glycoproteins, characterized by the presence of [beta]1->2 xylose and [alpha]1->3 fucose residues, are derived from typical mannose9(N-acetylglucosamine)2 (Man9GlcNAc2) N-linked glycans through the activity of a series of glycosidases and glycosyl transferases in the Golgi apparatus. By screening leaf extracts with an antiserum against complex glycans, we isolated a mutant of Arabidopsis thaliana that is blocked in the conversion of high-manne to complex glycans. In callus tissues derived from the mutant plants, all glycans bind to concanavalin A. These glycans can be released by treatment with endoglycosidase H, and the majority has the same size as Man5GlcNAc1 glycans. In the presence of deoxymannojirimycin, an inhibitor of mannosidase I, the mutant cells synthesize Man9GlcNAc2 and Man8GlcNAc2 glycans, suggesting that the bio-chemical lesion in the mutant is not in the biosynthesis of high-mannose glycans in the endoplasmic reticulum but in their modification in the Golgi. Direct enzyme assays of cell extracts show that the mutant cells lack N-acetyl glucosaminyl transferase I, the first enzyme in the pathway of complex glycan biosynthesis. The mutant plants are able to complete their development normally under several environmental conditions, suggesting that complex glycans are not essential for normal developmental processes. By crossing the complex-glycan-deficient strain of A. thaliana with a transgenic strain that expresses the glycoprotein phytohemagglutinin, we obtained a unique strain that synthesizes phytohemagglutinin with two high-mannose glycans, instead of one high-mannose and one complex glycan.

Development- and organ-specific expression of the genes for sucrose synthase and three isoenzymes of acid β-fructofuranosidase in carrot

The steady-state levels of transcripts for cellwall β-fructofuranosidase (cwβF), for isoenzymes I and II of soluble acid β-fructofuranosidase (sI, sII), and for sucrose synthase (ss) were determined in the sink and source organs of developing carrot (Daucus carota L.) plants. The expression patterns of the four genes clearly differed. The expression of the gene for cwβF was development-specific but not organ-specific; high transcript levels were only found in plants with primary roots, with about equal amounts in leaf lamina, petioles and roots. The genes for sI and sII were mainly expressed in roots, sI predominating in primary roots and sII in developing tap roots. Transcripts for ss were found at a low level in all developing plant organs and were markedly up-regulated during the development of young leaves and during the transition of primary roots to tap roots. Developing tap roots contained only transcripts for sII and for ss. Marked alterations in the expression of these two genes after manipulation of the source/sink balance of these plants indicates their importance in sucrose partitioning. We suggest that ss regulates sucrose utilization in developing tap roots, whereas sII located in the vacuole controls sucrose storage and sugar composition.

Purification and characterization of neutral and alkaline invertase from carrot

Neutral and alkaline invertase were identified in cells of a suspension culture of carrot (Daucus carota L.) and purified to electrophoretic homogeneity. Neutral invertase is an octamer with a molecular mass of 456 kD and subunits of 57 kD, whereas alkaline invertase is a tetramer with a molecular mass of 504 kD and subunits of 126 kD. Both enzymes had sharp pH profiles, with maximal activities at pH 6.8 for neutral invertase and pH 8.0 for alkaline invertase, and both hydrolyzed sucrose with typical hyperbolic kinetics and similar Km values of about 20 mM at pH 7.5. Neutral invertase also hydrolyzed raffinose and stachyose and, therefore, is a [beta]-fructofuranosidase. In contrast, alkaline invertase was highly specific for sucrose. Fructose acted as a competitive inhibitor of both enzymes, with Ki values of about 15 mM. Glucose was a noncompetitive inhibitor of both neutral and alkaline invertase, with a Ki of about 30 mM. Neither enzyme was inhibited by HgCl2. Alkaline invertase was markedly inhibited by CaCl2, MgCl2, and MnCl2, and neutral invertase was not. In contrast to alkaline invertase, neutral invertase was inhibited by the nucleotides ATP, CTP, GTP, and UTP.

cDNA Cloning of Carrot (Daucus carota) Soluble Acid [beta]-Fructofuranosidases and Comparison with the Cell Wall Isoenzyme

Carrot (Daucus carota), like most other plants, contains various isoenzymes of acid [beta]-fructofuranosidase ([beta]F) (invertase), which either accumulate as soluble polypeptides in the vacuole (isoenzymes I and II) or are ionically bound to the cell wall (extracellular [beta]F). Using antibodies against isoenzyme I of carrot soluble [beta]F, we isolated several cDNA clones encoding polypeptides with sequences characteristic of [beta]Fs, from bacteria, yeast, and plants. The cDNA-derived polypeptide of one of the clones contains all partial peptide sequences of the purified isoenzyme I and thus codes for soluble acid [beta]F isoenzyme I. A second clone codes for a related polypeptide (63% identity and 77% similarity) with characteristics of isoenzyme II. These two soluble [beta]Fs, have acidic isoelectric points (3.8 and 5.7, respectively) clearly different from the extracellular enzyme, which has a basic isoelectric point of 9.9. Marked differences among the three nucleotide sequences as well as different hybridization patterns on genomic DNA gel blots prove that these three isoenzymes of carrot acid [beta]F are encoded by different genes and do not originate from differential splicing of a common gene, as is the case in the yeast Saccharomyces cerevisiae. All three carrot acid [beta]Fs, are preproenzymes with signal peptides and N-terminal propeptides. A comparison of the sequences of the soluble enzymes with the sequence of the extracellular protein identified C-terminal extensions with short hydrophobic amino acid stretches that may contain the information for vacuolar targeting.

Cloning and Characterization of a Glutathione S-Transferase That Can Be Photolabeled with 5-Azido-indole-3-acetic Acid

Previously, we identified a soluble protein from Hyoscyamus muticus that was photolabeled by 5-azido-indole-3-acetic acid. This protein was determined to be a glutathione S-transferase (GST; J. Bilang, H. Macdonald, P.J. King, and A. Sturm [1993] Plant Physiol 102: 29–34). We have examined the effect of auxin on the activity of this H. muticus GST. Auxins reduced enzyme activity only at high concentrations, with 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid being more effective than indole-3-acetic acid (IAA) and naphthylacetic acid. IAA was a noncompetitive inhibitor, whereas inhibition by 2,4-D was competitive with respect to 1-chloro-2,4-dinitro-benzene. We also present the sequence of a full-length cDNA clone that codes for a GST and contains all partial amino acid sequences of the purified protein. The auxin-binding GST was found in high amounts in roots and stems and low amounts in leaves and flower buds. The steady-state mRNA level was not regulated by IAA or naphthylacetic acid, whereas 2,4-D and 2,3-dichlorophenoxyacetic acid increased mRNA levels. We propose a model in which 2,4-D is a substrate for GST, whereas IAA binds at a second site, known as a ligandinbinding site for the purpose of intracellular transport.

Correct glycosylation, Golgi-processing, and targeting to protein bodies of the vacuolar protein phytohemagglutinin in transgenic tobacco.

We used a heterologous system (transgenic Nicotiana tabacum L.) to investigate the processing, assembly and targeting of phytohemagglutinin (PHA), the lectin of the common bean, Phaseolus vulgaris L. In the bean, this glycoprotein accumulates in the protein bodies of the storage parenchyma cells in the cotyledons, and each polypeptide has a high-mannose glycan attached to Asn12 and a complex glycan on Asn60. The gene for PHA-L, dlec2, with 1200 basepairs (bp) 5′ upstream and 1600 bp 3′ downstream from the coding sequence was introduced into tobacco using Agrobacterium-mediated transformation (T. Voelker et al., 1987, EMBO J. 6, 3571–3577). Examination of thin sections of tobacco seeds by immunocytochemistry with antibodies against PHA showed that PHA-L accumulated in the amorphous matrix of the protein bodies in the embryo and endosperm. This localization was confirmed using a non-aqueous method to isolate the protein bodies from mature tobacco seeds. The biochemical analysis of tobacco PHA indicated that the signal peptide had been correctly removed, and that the polypeptides formed 6.4 S oligomers; tobacco PHA had a high-mannose glycan at Asn12 and a complex glycan at Asn60. The presence of the complex glycan shows that transport to the protein bodies was mediated by the Golgi complex. At seed maturity, a substantial portion of the PHA-L remained associated with the endoplasmic reticulum and the Golgi complex, as indicated by fractionation experiments using aqueous media and the presence of two high-mannose glycans on some of the polypeptides. Taken together, these data show that insertion of the nascent PHA into the endoplasmic reticulum, signal peptide processing, glycosylation, assembly into oligomers, glycan modification in the Golgi, and targeting of the protein occur faithfully in this heterologous system, although transport may not be as efficient as in bean cotyledons.

Differences in expression between two seed lectin alleles obtained from normal and lectin-deficient beans are maintained in transgenic tobacco

Using Agrobacterium‐mediated transformation, two genes for phytohemagglutinin‐L (PHA‐L), the lectin seed protein of the common bean Phaseolus vulgaris, were stably integrated into the tobacco genome. The two alleles for PHA‐L, dlec2 and pdlec2, were obtained from a normal cultivar (Greensleeves) and a lectin‐deficient cultivar (Pinto) respectively. In the bean embryos, the expression of dlec2 is 30 times greater than the expression of pdlec2. In the dlec2‐transformed tobacco, PHA‐L accumulated specifically in the seeds at the same stages as the tobacco seed storage proteins and was degraded after germination. PHA‐L was found in the embryo, and at a 5–7 times lower concentration in the endosperm tissue of the mature tobacco seeds. No PHA could be detected in other parts of the plants. We conclude that the signals for temporal and spatial regulation of the dlec2 gene are present in the DNA fragment used for transformation. Transformation with the second PHA‐L allele pdlec2 from the cultivar Pinto caused the accumulation of about 50 times less PHA‐L in tobacco seeds when compared to dlec2. We conclude from analyzing the 5′ sequences of dlec2 and Pdlec2 that the low expression phenotype of the Pdlec2 allele could be due to the absence or mutation of a cis‐acting element carried by the dlec2 fragment.

Characterization of β-fructosidase: an extracellular glycoprotein of carrot cells

Abstract Seedlings and suspension-cultured cells of carrot (Daucus carota) contain a cell wall associated as well as a soluble form of β-fructosidase (βF). These two forms have different pH optima: 4.6 for cell wall βF and 5.6 for soluble βF. Soluble βF is relatively more abundant in the seedlings and cell wall βF is relatively much more abundant in the cultured cells. Protoplasts of cultured cells have only the soluble form (pH optimum 5.6) indicating that the cell wall associated form is indeed extracellular in situ. Cell wall βF was purified to homogeneity and has an Mr = 63 000. Antibodies raised against the deglycosylated enzyme cross-reacted with two soluble enzyme forms: in cultured cells, the soluble enzyme has an Mr = 58 000 and, in seedlings, there are two forms of Mr = 58 000 and 52 000. Treatment of purified cell wall βF with endoglycosidase H and trifluoromethanesulfonic acid (complete deglycosylation) indicated that the enzyme probably has one high mannose and two complex glycans. This was confirmed by HPLC analysis of [3H]GlcNAc- and [3H]fucose-labeled glycopeptides obtained after trypsin digestion of radioactively-labeled βF. The amino acid composition shows that cell wall βF has 18.6% glycine.