Genes for the enzymes that make plant cell wall hemicellulosic polysaccharides remain to be identified. We report here the isolation of a complementary DNA (cDNA) clone encoding one such enzyme, mannan synthase (ManS), that makes the beta-1, 4-mannan backbone of galactomannan, a hemicellulosic storage polysaccharide in guar seed endosperm walls. The soybean somatic embryos expressing ManS cDNA contained high levels of ManS activities that localized to Golgi. Phylogenetically, ManS is closest to group A of the cellulose synthase-like (Csl) sequences from Arabidopsis and rice. Our results provide the biochemical proof for the involvement of the Csl genes in beta-glycan formation in plants.

Reduced phytic acid content in seeds is a desired goal for genetic improvement in several crops. Low-phytic acid mutants have been used in genetic breeding, but it is not known what genes are responsible for the low-phytic acid phenotype. Using a reverse genetics approach, we found that the maize (Zea mays) low-phytic acid lpa2 mutant is caused by mutation in an inositol phosphate kinase gene. The maize inositol phosphate kinase (ZmIpk) gene was identified through sequence comparison with human and Arabidopsis Ins(1,3,4)P 3 5/6-kinase genes. The purified recombinant ZmIpk protein has kinase activity on several inositol polyphosphates, including Ins(1,3,4)P 3 , Ins(3,5,6)P 3 , Ins(3,4,5,6)P 4 , and Ins(1,2,5,6)P 4 . The ZmIpk mRNA is expressed in the embryo, the organ where phytic acid accumulates in maize seeds. The ZmIpk Mutator insertion mutants were identified from a Mutator F 2 family. In the ZmIpk Mu insertion mutants, seed phytic acid content is reduced approximately 30%, and inorganic phosphate is increased about 3-fold. The mutants also accumulate myo-inositol and inositol phosphates as in the lpa2 mutant. Allelic tests showed that the ZmIpk Mu insertion mutants are allelic to the lpa2. Southern-blot analysis, cloning, and sequencing of the ZmIpk gene from lpa2 revealed that the lpa2-1 allele is caused by the genomic sequence rearrangement in the ZmIpk locus and the lpa2-2 allele has a nucleotide mutation that generated a stop codon in the N-terminal region of the ZmIpk open reading frame. These results provide evidence that ZmIpk is one of the kinases responsible for phytic acid biosynthesis in developing maize seeds.Phytic acid, myo-inositol 1,2,3,4,5,6-hexakisphosphate, is an abundant component of plant seeds and is deposited in protein bodies as a mixed salt of mineral cations, such as K ϩ , Mg 2ϩ , Ca 2ϩ , Zn 2ϩ , and Fe 3ϩ . Typically, 50% to 80% of the phosphorus in seeds is found in this compound. Phytic acid serves as a major storage form for myo-inositol, phosphorus, and mineral cations for use during seedling growth. The other known role of phytic acid is the control of inorganic phosphate (Pi) levels in both developing seeds and seedlings (Strother, 1980). In maize (Zea mays) kernels, nearly 90% of the phytic acid is accumulated in embryo and about 10% in aleurone layers. Maize endosperm contains only trace amount of phytic acid (O'Dell et al., 1972). In rice (Oryza sativa), barley (Hordeum vulgare), and wheat (Triticum aestivum), most of the phytic acid (approximately 90%) is found in the aleurone layers and only about 10% in embryo. Reduced phytic acid content in seeds is a desired goal for genetic improvement in several crops, including maize, rice, barley, wheat, and soybean (Glycine max). Because monogastric animals digest phytic acid poorly, animal feed is supplemented with Pi to meet the phosphorus requirement for animal growth. Undigested phytic acid is eliminated and is a leading phosphorus pollution source (Cromwell and Coffey, 1991). Although phytic acid as an antioxidant is sugges...

SummaryPhytic acid, myo-inositol-1,2,3,4,5,6-hexakis phosphate or Ins P 6 , is the most abundant myo-inositol phosphate in plant cells, but its biosynthesis is poorly understood. Also uncertain is the role of myo-inositol as a precursor of phytic acid biosynthesis. We identified a low-phytic acid mutant, lpa3, in maize. The Mu-insertion mutant has a phenotype of reduced phytic acid, increased myo-inositol and lacks significant amounts of myo-inositol phosphate intermediates in seeds. The gene responsible for the mutation encodes a myo-inositol kinase (MIK). Maize MIK protein contains conserved amino acid residues found in pfkB carbohydrate kinases. The maize lpa3 gene is expressed in developing embryos, where phytic acid is actively synthesized and accumulates to a large amount. Characterization of the lpa3 mutant provides direct evidence for the role of myo-inositol and MIK in phytic acid biosynthesis in developing seeds. Recombinant maize MIK phosphorylates myo-inositol to produce multiple myo-inositol monophosphates, Ins(1/3)P, Ins(4/6)P and possibly Ins(5)P. The characteristics of the lpa3 mutant and MIK suggest that MIK is not a salvage enzyme for myo-inositol recycling and that there are multiple phosphorylation routes to phytic acid in developing seeds. Analysis of the lpa2/lpa3 double mutant implies interactions between the phosphorylation routes.

As the use of tocopherols as natural antioxidants increases, it is economically and agronomically important to determine the range, composition, and factors that affect their levels in oilseed crops, a major commercial source. In this study, tocopherols were quantified from seeds of wheat, sunflower, canola, and soybean. The breeding lines analyzed possessed a broad range of economically important phenotypic traits such as disease or herbicide resistance, improved yield and agronomic characteristics, and altered storage oil fatty acid composition. Complete separation of all four native tocopherols was achieved using normal-phase high-performance liquid chromatography with ultraviolet detection. Total tocopherol concentration among wheat germ oil samples ranged from 1947 to 4082 µg g −1 . Total tocopherol concentration ranges varied from 534 to 1858 µg g −1 in sunflower, 504 to 687 µg g −1 in canola, and 1205 to 2195 µg g −1 among the soybean oils surveyed. Although the composition of tocopherols varied substantially among crops, composition was stable within each crop. Total tocopherol concentration and the percentage linolenic acid were correlated positively in soybean oils with modified and unmodified fatty acid compositions. Tocopherol concentration and degree of unsaturation were not correlated in sunflower or canola seeds with genetically altered fatty acid composition. These findings suggest that breeding for altered storage oil fatty acid composition did not negatively impact tocopherol concentrations in sunflower and canola as they apparently did in soybeans. When 12 soybean breeding lines were grown at each of five locations, significant correlations were observed among planting location, breeding line, tocopherol concentration, and fatty acid composition. Analysis of seeds that matured under three different controlled temperature regimes suggests that the relationship between tocopherol concentration level and unsaturated fatty acids in commodity (not genetically modified for fatty acid composition) oil types is due to temperature effects on the biosynthesis of both compounds.Paper no. J8930 in JAOCS 76, 349-355 (March 1999).

To assess the potential of traditional selection breeding to develop varieties with increased phytosterol content, we determined concentrations of those sterols in canola, sunflower, and soybean seed oils produced from breeding lines of diverse genetic backgrounds. Seed oils were extracted and saponified, and the nonsaponifiable fractions were subjected to silylation. The major phytosterols, brassicasterol, campesterol, stigmasterol and β-sitosterol, were quantified by capillary gas chromatography with flame-ionization detection. Canola contained approximately twice the amount of total phytosterols (4590-8070 µg g −1 ) as sunflower (2100-4540 µg g −1 ) or soybean (2340-4660 µg g −1 ) oils. Phytosterol composition varied among crops as expected, as well as within a crop. Both genetic background and planting location significantly affected total phytosterol concentrations. Soybean plants were maintained from flower initiation to seed maturity under three temperature regimes in growth chambers to determine the effect of temperature during this period on seed oil phytosterol levels. A 2.5-fold variability in total phytosterol content was measured in these oils (3120-7920 µg g −1 ). Total phytosterol levels increased with higher temperatures. Composition also changed, with greater percent campesterol and lower percent stigmasterol and β-sitosterol at higher temperatures. In these soybean oils, total phytosterol accumulation was correlated inversely with total tocopherol levels. Owing to the relatively limited variability in phytosterol levels in seed oils produced under field conditions, it is unlikely that a traditional breeding approach would lead to a dramatic increase in phytosterol content or modified phytosterol composition.