Arabidopsis cytosolic alpha-glycan phosphorylase, PHS2, is important during carbohydrate imbalanced conditions.

Arabidopsis thaliana has two isoforms of alpha-glycan phosphorylase (EC 2.4.1.1), one residing in the plastid and the other in the cytosol. The cytosolic phosphorylase, PHS2, acts on soluble heteroglycans that constitute a part of the carbohydrate pool in a plant. This study aimed to define a physiological role for PHS2. Under standard growth conditions phs2 knock-out mutants do not show any clear growth phenotype, and we hypothesised that during low-light conditions where carbohydrate imbalance is perturbed, this enzyme is important. Soil-grown phs2 mutant plants developed leaf lesions when placed in very low light. Analysis of soluble heteroglycan (SHG) levels showed that the amount of glucose residues in SHG was higher in the phs2 mutant compared to wild-type plants. Furthermore, a standard senescence assay from soil-grown phs2 mutant plants showed that leaves senesced significantly faster in darkness than the wild-type leaves. We also found decreased hypocotyl extension in in vitro-grown phs2 mutant seedlings when grown for long time in darkness at 6 °C. We conclude that PHS2 activity is important in the adult stage during low-light conditions and senescence, as well as during prolonged seedling development when carbohydrate levels are unbalanced.

Phospholipid:diacylglycerol acyltransferase: an enzyme that catalyzes the acyl-CoA-independent formation of triacylglycerol in yeast and plants.

Triacylglycerol (TAG) is known to be synthesized in a reaction that uses acyl-CoA as acyl donor and diacylglycerol (DAG) as acceptor, and which is catalyzed by the enzyme acyl-CoA:diacylglycerol acyltransferase. We have found that some plants and yeast also have an acyl-CoA-independent mechanism for TAG synthesis, which uses phospholipids as acyl donors and DAG as acceptor. This reaction is catalyzed by an enzyme that we call phospholipid:diacylglycerol acyltransferase, or PDAT. PDAT was characterized in microsomal preparations from three different oil seeds: sunflower, castor bean, and Crepis palaestina. We found that the specificity of the enzyme for the acyl group in the phospholipid varies between these species. Thus, C. palaestina PDAT preferentially incorporates vernoloyl groups into TAG, whereas PDAT from castor bean incorporates both ricinoleoyl and vernoloyl groups. We further found that PDAT activity also is present in yeast microsomes. The substrate specificity of this PDAT depends on the head group of the acyl donor, the acyl group transferred, and the acyl chains of the acceptor DAG. The gene encoding the enzyme was identified. The encoded PDAT protein is related to lecithin:cholesterol acyltransferase, which catalyzes the acyl-CoA-independent synthesis of cholesterol esters. However, budding yeast PDAT and its relatives in fission yeast and Arabidopsis form a distinct branch within this protein superfamily, indicating that a separate PDAT enzyme arose at an early point in evolution.

Characterisation of acyl-ACP desaturases from Macadamia integrifolia Maiden & Betche and Nerium oleander L.

The seed oil in Macadamia integrifolia contains about 30% palmitoleic acid (16:1(Delta9)) and Nerium oleander about 12% isoricinoleic acid (Delta9-hydroxy-18:1(Delta12)). It has been shown that palmitoleic acid can be produced by acyl-acyl carrier protein (ACP) desaturases and it has also been shown that fatty acid hydroxylation can occur via direct substitution of a hydrogen atom. Therefore it seemed possible that the enzymes responsible for the making of these unusual fatty acids in M. integrifolia and N. oleander were of acyl-ACP desaturase type. Extracts from developing M. integrifolia developing seeds showed a relative ratio of 16:0-ACP to 18:0-ACP desaturation that was about 13 times higher than in sunflower seeds. N. oleander seed extracts catalysed conversion of 18:0-ACP to 18:1(Delta9) but only trace amounts of Delta9-hydroxy fatty acids were formed. A total of four cDNAs were isolated from developing seeds, of both species, using a fragment isolated with PCR amplification. The M. integrifolia acyl-ACP desaturase cDNA was expressed in Escherichia coli. A partly purified fraction of the enzyme showed a 16:0-ACP to 18:0-ACP desaturation ratio about 90-fold less than that in the Macadamia extracts. Expressed N. oleander acyl-ACP desaturase cDNAs showed predominantly 18:0-ACP desaturase activity and no hydroxylase activity. Thus it is not likely that any of the four acyl-ACP desaturases cloned from M. integrifolia or N. oleander is involved in the production of unusual fatty acids.

The involvement of phospholipid:diacylglycerol acyltransferases in triacylglycerol production.

We have characterized three CoA-independent types of enzyme, phospholipases, phospholipid:diacylglycerol acyltransferases (PDATs) and cholinephosphotransferases, responsible for the removal of unusual fatty acids from phosphatidylcholine (PC) in microsomal preparations from developing oil seeds. The metabolism of sn-2-[(14)C]acyl-PC was monitored in microsomal preparations from various oilseeds having either medium-chain, acetylenic, epoxy or hydroxy fatty acids as their major fatty acids in the oil. The results indicate that PDAT plays a major role in removing ricinoleic acid and vernolic acid from phospholipids in Ricinus communis and Crepis palaestina seeds, respectively. However, vernolic, crepenynic and capric acids are primarily removed from phospholipids by phospholipases in Euphorbia lagascae, Crepis rubra and elm seeds, respectively. Further, we show that significant PDAT activity is also present in vegetative tissues of Arabidopsis thaliana.

An acyl-CoA:cholesterol acyltransferase (ACAT)-related gene is involved in the accumulation of triacylglycerols in Saccharomyces cerevisiae.

The major route for the synthesis of triacylglycerol (TAG) in yeast as well as in all TAG-accumulating organisms has been suggested to occur via the acylation of diacylglycerol (DAG) by acyl-CoA:diacylglycerol acyltransferase (DAGAT). Genes encoding DAGAT have been identified in both plant and animal tissues. These genes show strong sequence similarities to genes encoding acyl-CoA:cholesterol acyltransferase (ACAT). So far no Saccharomyces cerevisiae DAGAT gene has been published; however, two ACAT-like genes, ARE1 and ARE2, are present in the yeast genome. Both these genes have been suggested to be involved in the synthesis of sterol esters. We have now shown that the ARE1 gene in yeast also is involved in the synthesis of TAG, whereas the ARE2 gene is more specifically involved in the synthesis of sterol esters.

Identification of non-heme diiron proteins that catalyze triple bond and epoxy group formation.

Acetylenic bonds are present in more than 600 naturally occurring compounds. Plant enzymes that catalyze the formation of the Delta12 acetylenic bond in 9-octadecen-12-ynoic acid and the Delta12 epoxy group in 12,13-epoxy-9-octadecenoic acid were characterized, and two genes, similar in sequence, were cloned. When these complementary DNAs were expressed in Arabidopsis thaliana, the content of acetylenic or epoxidated fatty acids in the seeds increased from 0 to 25 or 15 percent, respectively. Both enzymes have characteristics similar to the membrane proteins containing non-heme iron that have histidine-rich motifs.

Characterization of rapeseed myrosinase-binding protein.

Myrosinase-binding proteins (MBPs) were purified from seeds of Brassica napus L. (oilseed rape). The proteins were characterized with respect to amino-acid composition, peptide sequence and isoelectric points. Gel electrophoresis and Western blotting of protein extracts from mature seeds showed the existence of at least ten proteins reacting with a monoclonal anti-MBP antibody and ranging in molecular size from 110 to 30 kDa. Proteins other than MBP reacting with the anti-MBP antibody were assigned as myrosinase-binding protein-related proteins (MBPRPs). Two MBPRPs were purified by immunoaffinity chromatography and characterized with respect to partial amino-acid sequence. Sequence identities were found between MBP and MBPRP. Western blot analysis of protein extracts from different tissues of B. napus showed that MBPRP is present in the whole plant, whereas MBP mostly occurs in the mature seed. A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was used to investigate the occurrence of MBP and MBPRP in developing seeds of some species in the Brassicaceae family.

Cruciferin gene families are expressed coordinately but with tissue-specific differences during Brassica napus seed development.

The major storage protein in seeds of Brassica napus, the 12S globulin cruciferin, is composed of three different groups of subunits; cru1, cru2/3 and cru4. By using gene family-specific probes, we have investigated the accumulation, rate of synthesis and spatial distribution of transcripts corresponding to the different groups of cruciferin subunits in developing seeds. Cruciferin transcripts derived from different gene families accumulate coordinately to comparable amounts during seed development. The corresponding gene families are, however, transcribed at different rates. Investigation of the spatial distribution of transcripts corresponding to each group of cruciferin subunits in the developing seed by in situ hybridization, revealed that mRNAs of all three types accumulate in both axis and cotyledons. Transcripts derived from cru1 and cru4 gene families show a similar cell specificity and accumulate in a similar spatial manner during seed development. In contrast, mRNAs corresponding to the cru2/3 gene family are expressed with a partly different cell specificity and show a slightly different pattern of accumulation in the axis and cotyledons, with a delayed accumulation in epidermal cells. In the cotyledons, the initial accumulation of this type of cruciferin mRNAs is also distinguished from the two other types. The differences in cell specificity are seen in the root cap and in provascular cells, where mRNAs belonging to the cru2/3 family are absent.

Differential expression of myrosinase gene families.

In mature seeds of Brassica napus three major and three minor myrosinase isoenzymes were identified earlier. These myrosinases are known to be encoded by at least two different families of myrosinase genes, denoted MA and MB. In the work described in this paper the presence of different myrosinase isoenzymes in embryos, seedlings, and vegetative mature tissues of B. napus was studied and related to the expression of myrosinase MA and MB genes in the same tissues to facilitate future functional studies of these enzymes. In developing seeds, myrosinases of 75, 73, 70, 68, 66, and 65 kD were present. During seedling development there was a turnover of the myrosinase pool such that in 5-d-old seedlings the 75-, 70-, 66-, and 65-kD myrosinases were present, with the 70- and 75-kD myrosinases predominating. In 21-d-old seedlings the same myrosinases were present, but the 66- and 65-kD myrosinase species were most abundant. At flowering the mature organs of the plant contained only a 72-kD myrosinase. MA genes were expressed only in developing seeds, whereas MB genes were most highly expressed in seeds, seedling cotyledons, young leaves, and to a lesser extent other organs of the mature plant. During embryogenesis of B. napus, myrosinase MA and MB gene transcripts started to accumulate approximately 20 d after pollination and reached their highest level approximately 15 d later. MB transcripts accumulated to about 3 times the amount of MA transcripts. In situ hybridization analysis of B. napus embryos showed that MA transcripts were present predominatly in myrosin cells in the axis, whereas MB genes were expressed in myrosin cells of the entire embryo. The embryo axiz contained 75-, 70-, and 65-kD myrosinases, whereas the cotyledons contained mainly 70- and 65-kD myrosinases. Amino acid sequencing revealed the 75-kD myrosinase to be encoded by the MA gene family. The high degree of cell and tissue specificity of the expression of myrosinase genes suggests that studies of their transcription should provide interesting information concerning a complex type of gene regulation.

Characterization of a Brassica napus myrosinase pseudogene: myrosinases are members of the BGA family of beta-glycosidases.

Myrosinase isoenzymes are known to be encoded by two different families of genes denoted MA and MB. Nucleotide sequence analysis of a Brassica napus genomic clone containing a gene for myrosinase revealed it to be a pseudogene of the MA family. The gene spans more than 5 kb and contains at least 12 exons. The exon sequence of the gene is highly similar to myrosinase cDNA sequences. However, the gene displays three potential or actual pseudogene characters. Southern blot analysis using probes from the 3' portions of the genomic and B. napus MA and MB cDNA clones showed that MA type myrosinases are encoded by approximately 4 genes, while MB type myrosinases are encoded by more than 10 genes in B. napus. Northern blots with mRNA from seeds and young leaves probed with the MA- and MB-specific probes showed that the MA and MB myrosinase gene families are differentially expressed. Myrosinases are highly similar to proteins of a beta-glycosidase enzyme family comprising both beta-glycosidases and phospho-beta-glycosidases of as diverged species as archaebacteria, bacteria, mammals and plants. By homology to these beta-glycosidases, putative active site residues in myrosinase are discussed on the basis of the similarity between beta-glycosidases and cellulases.

The glucosinolate-degrading enzyme myrosinase in Brassicaceae is encoded by a gene family.

A full-length cDNA clone (MB3) and three partial clones (MA1, MB1 and MB2) which encode myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1) were isolated from a Sinapis alba (white mustard) cDNA library. Nucleotide sequence analysis of these clones revealed that they are encoded by a gene family. Southern blot analysis with gene-specific probes showed that the gene family consists of a least two subfamilies (MA and MB) each with several members both in S. alba and in Brassica napus (oilseed rape). In Arabidopsis thaliana (wall cress) only three myrosinase genes seem to be present. Northern blot analysis indicated that all the myrosinase mRNA species have the same size, approximately 1.95 kb.

Distribution of myrosinase in rapeseed tissues.

Immunocytochemical studies on Brassica napus (rapeseed) tissues using a monoclonal antibody against myrosinase (thioglucoside glucohydrolase) showed that the enzyme was only present in a small number of cells. In the developing embryo, scattered myrosinase-containing cells were present in both cotyledons and axis. The enzyme accumulated in these cells during the later stages of seed development, approximately from day 20 until day 40 after pollination. Parallel staining with the immunocytochemical technique and a histochemical method identified these cells as myrosin cells. Myrosinase appeared to be located outside the myrosin grains, although the occasional association with the membrane of the grains also was noted. In leaves, petals, and siliques, scattered parenchyma cells were stained in the mesophyll as well as in the vascular tissue. In young leaves, guard cells also contained myrosinase. The enzyme was also present in xylem cells of the stem.

Immunological characterization of rapeseed myrosinase.

A purified 75-kDa myrosinase and a crude rapeseed myrosinase fraction were used as antigens to produce mouse anti-myrosinase monoclonal antibodies. The 75-kDa myrosinase was also used to produce a polyclonal rabbit antiserum. The antiserum and one monoclonal antibody reacted with three distinct rapeseed polypeptides of 75, 70 and 65 kDa (M75, M70 and M65, respectively). A second set of monoclonal antibodies reacted exclusively with the 75-kDa form of myrosinase, and a third set showed specificity towards two components of 52 and 50 kDa (myrosinase-binding proteins, MBP52 and MBP50, respectively). MBP52 and MBP50 lack inherent myrosinase activity, but are nevertheless capable of mediating immunoprecipitation of myrosinase due to their interaction with myrosinase. Gel chromatography and glycerol gradient centrifugation experiments resolved two myrosinase-containing fractions. One of these had an approximate molecular mass of 140 kDa and consisted of disulfide-linked dimers of the 75-kDa myrosinase. The other fraction was heterogeneous in size with molecular masses ranging from 250 kDa to approximately 1 MDa. The high-molecular-mass fractions contained complexes consisting of disulfide-linked 70-kDa and 65-kDa myrosinases and non-covalently bound 52-kDa and 50-kDa myrosinase-binding proteins.

Structure of a gene encoding the 1.7 S storage protein, napin, from Brassica napus.

A rapeseed chromosomal region containing a gene (napA), which encodes the 1.7 S seed storage protein (napin), was isolated in several overlapping recombinant clones from a phage lambda genomic library. Following restriction enzyme mapping of the genomic region, a subclone containing the napA coding region as well as some 1.1 and 1.4 kilobases of DNA from the 5' and 3' regions, respectively, was mapped and sequenced. The gene turned out to lack introns. Southern blotting analyses utilizing a napin cDNA clone as a probe revealed the presence of on the order of 10 napin genes in the rapeseed genome. The major polyadenylated transcript encoded by these genes was shown to be an 850-nucleotide species, the initiation site of which was mapped onto the napA gene. The major initiation site for transcription is located some 33 nucleotides downstream from a sequence perfectly conforming to the consensus sequence of a TATA box. Further analyses of the sequence revealed several features that may be of relevance for the expression of the napin genes.

Structure of the rapeseed 1.7 S storage protein, napin, and its precursor.

Napin (1.7 S protein) is a basic, low molecular weight storage protein synthesized in rapeseed (Brassica napus) embryos during seed development. Napin is composed of two polypeptide chains with molecular weights of 9000 and 4000 that are held together by disulfide bonds. Comparison of the deduced amino acid sequence of a napin cDNA clone with that of napin peptide fragments established that napin is initially synthesized as a precursor of 178 residues. This polypeptide is subsequently processed through several proteolytic events, which ultimately generate the two mature napin chains, of 86 and 29 residues, respectively. Protein biosynthesis in vitro showed that the initial translation product (Mr 20,000) contains a signal sequence which is removed during transfer of the protein into the endoplasmic reticulum. Two additional peptides, of 22 and 19 residues, as well as the COOH-terminal residue, are also removed during maturation of napin, as deduced from the sequence comparison. Comparisons of the napin sequence with other known protein sequences established that there is a significant homology between napin and two other small seed proteins, the castor bean storage protein and a trypsin inhibitor from barley.