Improving palm oil quality through identification and mapping of the lipase gene causing oil deterioration.

The oil palm fruit mesocarp contains high lipase activity that increases free fatty acids and necessitates post-harvest inactivation by heat treatment of fruit bunches. Even before heat treatment the mesocarp lipase activity causes consequential oil losses and requires costly measures to limit free fatty acids quantities. Here we demonstrate that elite low-lipase lines yield oil with substantially less free fatty acids than standard genotypes, allowing more flexibility for post-harvest fruit processing and extended ripening for increased yields. We identify the lipase and its gene cosegregates with the low-/high-lipase trait, providing breeders a marker to rapidly identify potent elite genitors and introgress the trait into major cultivars. Overall, economic gains brought by wide adoption of this material could represent up to one billion dollars per year. Expected benefits concern all planters but are likely to be highest for African smallholders who would be more able to produce oil that meets international quality standards.

Characterization of proteinase activity in stratified Douglas-fir seeds.

We investigated the effect of stratification on the proteinase activity involved in mobilization of the major soluble approximately 45 kDa storage protein during germination of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seeds. Complete hydrolysis of the approximately 45 kDa protein was observed approximately 7 days after exposure of stratified seeds to germination conditions. Coincident with the onset of mobilization, proteinase activity was detected primarily in microsomal extracts from stratified seeds. Microsomal-associated proteinase activity was most active at pH 8.0 and had a molecular mass > 175 kDa as determined by gelatin SDS-PAGE gels. In vitro digestion of soluble protein extracts indicated that, following stratification, there was a significant increase in proteinase activity and hydrolysis of the approximately 45 kDa storage protein. Whether this increase was a result of activation of preexisting proteinase(s) or de novo synthesis remains unknown. In vitro digestion of soluble protein extracts in the presence of various proteinase inhibitors showed that digestion of the approximately 45 kDa storage protein is mediated primarily by a metalloproteinase and to a lesser degree by a serine proteinase. The accumulation of approximately 25 kDa protein products following in vitro digestion suggests that mobilization of the approximately 45 kDa soluble storage protein is mediated by a multi-step process involving the action of different classes of proteinases.

Regulation of NADPH-cytochrome P450 reductase expressed during Douglas-fir germination and seedling development.

NADH-cytochrome P450 is a key enzyme that transfers electrons from NADPH to the cytochrome P450 family of enzymes. To begin to determine the regulation of CPR gene expression and enzyme activity in Douglas-fir a full-length cDNA was isolated from a seedling lambda ZAP cDNA library and the ORF was used to develop a synthetic CPR-peptide-based antiserum. Northern blot analysis indicated CPR expression was regulated both developmentally prior to seed maturation and during germination, and differentially in the cotyledons, radicle and megagametophyte of seed and seedling tissues. The CPR-peptide antiserum detected a single CPR in seed and seedling microsomes analyzed by western blot of two-dimensional SDS-polyacrylamide gels. In microsomal extracts from whole seeds and seedlings, the amount of CPR protein remained constant while NADPH:cytochrome c reductase activity increased during stratification, germination and early seedling development. In contrast to cotyledons and megagametophyte, the level of CPR protein detected in radicles was higher than expected when compared to the amount of CPR transcript.

The isolation of a novel metallothionein-related cDNA expressed in somatic and zygotic embryos of Douglas-fir: regulation by ABA, osmoticum, and metal ions.

To isolate genes which are expressed preferentially during embryogenesis, a Douglas-fir embryogenesis cDNA library was constructed and differentially screened with cDNA probes made with mRNA from developing and mature embryos, respectively. The cDNA clone PM 2.1 was isolated based on its abundance in developing seeds and absence in mature seeds, and its predicted amino acid sequence was shown to have structural features characteristic of plant MT-like proteins. Alignment of the PM 2.1 predicted amino acid sequence with other plant MT-like protein sequences revealed a general paucity of Cys and Cys-Xaa-Cys sequences and the presence of novel serine residues within the conserved Cys-Xaa-Cys motifs in the C-terminal domain. The consensus sequence following the Cys-poor spacer in type 2 MT-like proteins, CXCXXXCXCXXCXCX, was modified in PM 2.1 to CXSXXXSXYXX-XCX. Phylogenetic analysis supported PM 2.1 was distinct from other MT and grouped with MT-like proteins from Arabidopsis (OEST), rice (AEST) and kiwifruit (AD1), which do not belong to type 1 or 2. The PM 2.1 gene was expressed in somatic and zygotic embryos, in haploid maternal tissue, as well as in hormone- and metal-treated seeds and seedlings. The PM 2.1 transcripts were detected in the needles of 14-week-old seedlings, but not the root tissue or mature pollen. The expression of the PM 2.1 gene in embryos was dependent upon ABA and osmoticum and in seedlings was differentially modulated by metals, suggesting a role of the PM 2.1 gene product in the control of microelement availability during Douglas-fir seed development and germination. The novel structural features, and the developmental, hormonal and metal modulation of PM 2.1 expression, are evidence for a new type of MT-related protein in plants.

Structure and expression of a developmentally regulated cDNA encoding a cysteine protease (pseudotzain) from Douglas fir.

We report the complete sequence and expression of a cDNA clone (Pm3-3) encoding a cysteine protease (CysP) from Pseudotsuga menziesii [Mirb] (Pm) Franco (Douglas fir). The sequence consists of a 5' untranslated region (UTR) of 153-bp followed by an open reading frame (ORF) of 1362 bp encoding a putative mature CysP flanked by N- and C-terminal propeptides. A 364-bp 3' UTR contains multiple putative AU-rich elements (ARE) that may be involved in the destabilization of transcripts. The deduced primary structure of the Pm CysP (designated pseudotzain) contains the same invariant amino acid (aa) residues that are involved in the catalytic reaction and make up the catalytic center of CysP from plants and animals. Northern blot analysis showed that cysP transcripts were most abundant in the megagametophyte (MGP) after germination and not detected in the MGP or embryo during embryogenesis. Various osmotic stresses slightly enhanced cysP transcript levels during early seedling development, whereas abscisic acid (ABA), gibberellin (GA) and other plant growth regulators and environmental conditions had little or no effect. The cysP transcripts were present in different amounts in the cotyledons, root and seed coat of 10-day-old seedlings, but were most abundant in the MGP, suggesting a role for this protease in storage protein mobilization. Phylogenetic analysis of mature CysP groups pseudotzain with other angiosperm CysP having both N- and C-terminal propeptides, suggesting a conserved function and/or targeting of this subgroup of enzymes.

Post-termination-induced and hormonally dependent expression of low-molecular-weight heat shock protein genes in Douglas fir.

We have isolated and sequenced two cDNA clones (PM 18.2A; PM 18.2B) from Douglas fir (Pseudotsuga menziesii (Mirb.) Franco) which encode for the low-molecular-weight heat shock proteins (LMW HSPs) of 18.2 kDa. The predicted amino acid sequences of the two Douglas fir proteins are 97.5% identical. A phylogenetic tree of class I LMW HSPs showed that the PM LMW HSPs are found within a subgroup consisting exclusively of dicot species indicating that class I LMW HSPs evolved from a common ancestor predating the divergence of gymnosperms and angiosperms. Northern blots of RNA from dry, imbibed, stratified and germinated seeds revealed a notable induction of LMW HSP transcripts during post-germination and early seedling growth. Unlike previous reports, the expression of these HSPs appears to be primarily restricted to seedlings as mRNA transcripts were detected at very low levels during seed development and desiccation. Maximum induction of LMW HSPs in seedlings occurred during heat shock treatment at 38-40 degrees C, whereas cold shock or wounding failed to induce HSP transcripts. The transcription of HSP genes is up regulated by GA, MeJA and auxin and is down regulated by ABA. Methyl jasmonate treatment induced expression of these genes in dormant seeds of Douglas fir. The expression of class I cytoplasmic LMW HSPs in seedlings and their regulation by plant growth regulators suggests specific roles in plant development other than desiccation tolerance.

A new family of lipolytic plant enzymes with members in rice, arabidopsis and maize.

We have noted a striking similarity between the sequences of proteins in a novel family of lipases we recently reported [Upton, C. and Buckley, J. T. (1995) Trends Biol. Sci. 20, 178-9] and more than 120 sequences from the database of Expressed Sequence Tags (dbEST) which correspond to at least 30 unique genes from arabidopsis, rice and maize. A cDNA (Arab-1) corresponding to one of these sequences was isolated, sequenced and translated. There was significant similarity to sequences in the new lipase family over the entire open reading frame of Arab-1 and when expressed in E. coli, the gene product was lipolytic. Arab-1 and genes for some of the other plant proteins appear to be differentially expressed. They may play a role in the regulation of lipid metabolism during plant development.

Expression and Accumulation Patterns of Nitrogen-Responsive Lipoxygenase in Soybeans.

Gene expression and protein accumulation patterns of nitrogen-responsive lipoxygenase (LOX-NR), as a representative vegetative storage protein, were investigated in nonnodulated soybeans (Glycine max [L.] Merr. cv Wye). The form of available nitrogen (supplied as NH4NO3, NH4+, NO3-, or urea) influenced the mRNA level and the amount of LOX protein, indicating that preferential accumulation of LOX may occur. Soybeans were grown with 0, 2, 5, and 16 mM total nitrogen to determine the extent to which LOX accumulation responded to soil nitrogen levels. Analysis of both mRNA and protein levels was conducted in shoot tips, stems, pod walls, and leaves over the entire life cycle of the plant. A general correlation between increasing available nitrogen level and LOX level was seen in the shoot tip and other organs throughout the soybean life cycle. However, appreciable amounts of LOX-NR mRNA and protein accumulated even when plants were grown under conditions of nitrogen deficiency. The results indicate that LOX may play an important role as a temporary storage site for amino acids in the developing shoot tip. The expression patterns of LOX-NR in plants grown under nitrogen deficiency suggest that these proteins, although responsive to nitrogen status, may not function solely as temporary storage pools for amino acids.

The soybean 94-kilodalton vegetative storage protein is a lipoxygenase that is localized in paraveinal mesophyll cell vacuoles.

Soybean leaves contain three proteins (the vegetative storage proteins or VSPs) that respond to nitrogen status and are believed to be involved in the temporary storage of nitrogen. One of these proteins, with a molecular mass of 94 kD and termed vsp94, was microsequenced. Partial amino acid sequence indicated that vsp94 was highly homologous to the lipoxygenase protein family. Further evidence that vsp94 is a lipoxygenase was obtained by demonstrating that vsp94 cross-reacted with a lipoxygenase antibody. Also, a lipoxygenase cDNA coding region was able to detect changes in an mRNA that closely parallel changes in vsp94 protein levels resulting from alteration of nitrogen sinks. Extensive immunocytochemical data indicate that this vsp94/lipoxygenase is primarily expressed in the paraveinal mesophyll cells and is subcellularly localized in the vacuole. These observations are significant in that they suggest that plant lipoxygenases may be bifunctional proteins able to function enzymatically in the hydroperoxidation of lipids and also to serve a role in the temporary storage of nitrogen during vegetative growth.