Joshua C. Stein

Engineering Vitamin E Content: From Arabidopsis Mutant to Soy Oil

We report the identification and biotechnological utility of a plant gene encoding the tocopherol (vitamin E) biosynthetic enzyme 2-methyl-6-phytylbenzoquinol methyltransferase. This gene was identified by map-based cloning of the Arabidopsis mutation vitamin E pathway gene3-1 (vte3-1), which causes increased accumulation of δ-tocopherol and decreased γ-tocopherol in the seed. Enzyme assays of recombinant protein supported the hypothesis that At-VTE3 encodes a 2-methyl-6-phytylbenzoquinol methyltransferase. Seed-specific expression of At-VTE3 in transgenic soybean reduced seed δ-tocopherol from 20 to 2%. These results confirm that At-VTE3 protein catalyzes the methylation of 2-methyl-6-phytylbenzoquinol in planta and show the utility of this gene in altering soybean tocopherol composition. When At-VTE3 was coexpressed with At-VTE4 (γ-tocopherol methyltransferase) in soybean, the seed accumulated to >95% α-tocopherol, a dramatic change from the normal 10%, resulting in a greater than eightfold increase of α-tocopherol and an up to fivefold increase in seed vitamin E activity. These findings demonstrate the utility of a gene identified in Arabidopsis to alter the tocopherol composition of commercial seed oils, a result with both nutritional and food quality implications.

SRK, the stigma-specific S locus receptor kinase of Brassica, is targeted to the plasma membrane in transgenic tobacco.

The S locus receptor kinase (SRK) gene is one of two S locus genes required for the self-incompatibility response in Brassica. We have identified the product of the SRK6 gene in B. oleracea stigmas and have shown that it has characteristics of an integral membrane protein. When expressed in transgenic tobacco, SRK6 is glycosylated and targeted to the plasma membrane. These results provide definitive biochemical evidence for the existence in plants of a plasma membrane-localized transmembrane protein kinase with a known cell-cell recognition function. The timing of SRK expression in stigmas follows a time course similar to that previously described for another S locus-linked gene, the S locus glycoprotein (SLG) gene, and correlates with the ability of stigmas to mount a self-incompatibility response. Based on SRK6 promoter studies, the site of gene expression overlaps with that of SLG and exhibits predominant expression in the stigmatic papillar cells. Although reporter gene studies indicated that the SRK promoter was active in pollen, SRK protein was not detected in pollen, suggesting that SRK functions as a cell surface receptor exclusively in the papillar cells of the stigma.

Transformation of Brassica oleracea with an S-locus gene from B. campestris changes the self-incompatibility phenotype

SummaryAn SLG gene derived from the S-locus and encoding and S-locus-specific glycoprotein of Brassica campestris L. was introduced via Agrobacterium-mediated transformation into B. oleracea L. A self-incompatible hybrid and another with partial self-compatibility were used as recipients. The transgenic plants were altered in their pollen-stigma interaction and were fully compatible upon self-pollination. Reciprocal crosses between the transgenic plants and untransformed control plants indicated that the stigma reaction was changed in one recipient strain while the pollen reaction was altered in the other. Due to interspecific incompatibility, we could not demonstrate whether or not the introduced SLG gene confers a new allelic specificity in the transgenic plants. Our results show that the introduced SLG gene perturbs the self-incompatibility phenotype of stigma and pollen.

An alternative transcript of the S locus glycoprotein gene in a class II pollen-recessive self-incompatibility haplotype of Brassica oleracea encodes a membrane-anchored protein.

Recent reports have shown that SLG, one of two genes linked to the S locus of Brassica, encodes a secreted glycoprotein. We have used RNA gel blot analysis, genomic and cDNA clone analysis, expression in transgenic plants, and immunodetection to characterize SLG2, the SLG gene derived from the S2 haplotype. This haplotype belongs to the class II group of S haplotypes that exhibit a weak incompatibility phenotype and are pollen recessive. We showed that SLG2 produces two transcript forms: the expected 1.6-kb transcript that predicts a secreted glycoprotein and an alternative 1.8-kb transcript that predicts a membrane-anchored protein. Stigmas of the S2 haplotype and pistils of transgenic tobacco plants transformed with the SLG2 gene produce a membrane-associated 62-kD protein as well as soluble 57- and 58-kD glycoforms. Because of the sequence similarity between SLG2 and the extracellular domain of the S Locus Receptor Kinase (SRK2) gene, the membrane-anchored form of SLG2 may be viewed as a naturally occurring truncated form of the receptor that lacks the kinase catalytic domain. The occurrence of this protein has potential implications for the activity of the full-length receptor. Furthermore, the underlying structure of the SLG2 gene suggests the evolution of SLG from an ancestral SRK-like gene.

A Plant Receptor-Like Gene, the S-Locus Receptor Kinase of Brassica oleracea L., Encodes a Functional Serine/Threonine Kinase

To investigate the catalytic properties of the Brassica oleracea S-locus receptor kinase (SRK), we have expressed the domain that is homologous to protein kinases as a fusion protein in Escherichia coli. Following in vivo labeling of cultures with 32P-labeled inorganic phosphate, we observed phosphorylation of the fusion protein on serine and threonine, but not on tyrosine. In contrast, labeling was not observed when lysine-524, a residue conserved among all protein kinases, was mutated to arginine, thus confirming that SRK phosphorylation was the result of intrinsic serine/threonine kinase activity.

Signaling the Arrest of Pollen Tube Development in Self-Incompatible Plants

Self-incompatibility (SI), the cellular recognition system that limits inbreeding, has served as a paradigm for the study of cell-to-cell communication in plants since the phenomenon was first described by Darwin. Recent studies indicate that SI is achieved by diverse molecular mechanisms in different plant species. In the mustard family, the mechanism of SI shows parallels to the signaling systems found in animals that are mediated by cell-surface receptors with signal-transducing protein kinase activity.

Avian adipose lipoprotein lipase: cDNA sequence and reciprocal regulation of mRNA levels in adipose and heart

cDNA clones for chicken adipose lipoprotein lipase were isolated from an expression library in lambda gt11 by antibody screening and characterized by hybridization selection and nucleotide sequencing. Based on the cDNA sequence and on N-terminal sequence analysis of the purified enzyme, chicken adipose lipoprotein lipase is a mature protein of 465 amino acids with a signal peptide of 19 or 25 amino acids, depending on which of two methionine residues is used for translation initiation. The predicted amino-acid sequence was found to be 73-77% identical to the four known mammalian adipose lipoprotein lipase sequences, with conservation of position of cysteine residues and putative functional domains, and number of potential N-glycosylation sites. Chicken lipoprotein lipase differs from mammalian lipoprotein lipases with respect to the position of one N-glycosylation site and the presence of an additional 15-17 C-terminal amino acids. 32P-labeled cDNA clones hybridized to mRNA species of 3.7 and 4.0 kb in Northern blots of heart and adipose, but not of liver RNA. In chickens that were fasted for 48 h and then refed, lipoprotein lipase mRNA levels in adipose increased to a maximal level of 350% that of controls at 10 h, whereas heart lipoprotein lipase mRNA levels fell to 40% of controls at 14 h. Concomitantly, no changes in total RNA were observed. Thus, avian lipoprotein lipase is subject to reciprocal pretranslational regulation in adipose and heart.

Srk: S-locus receptor PK (Brassica spp.)

The chapter discusses the S-locus receptor PK (Srk) that has a receptor-like structure, possesses intrinsic serine/threonine PK activity, and is capable of autophosphorylation. The pattern of expression of SRK gene and its high degree of sequence polymorphism among different self-incompatibility haplotypes suggest a role for Srk as a receptor in pollen/stigma recognition. Plants bearing null alleles of SRK exhibit loss of the self-incompatibility response. Potential ligands and substrates remain to be identified. Each S-locus haplotype (>50 are known) is believed to encode a distinct Srk allele. Srk6, Srk2, and Srk910 have been characterized. These show as much as 32% sequence divergence at the amino acid level. The S-locus glycoprotein (Slg) is highly similar to the extracellular domain of Srk and is also encoded at the S locus. When derived from the same S-locus haplotype, Srk and S1g have as high as 90% amino acid identity. SRK transcript variants potentially encode N-terminally truncated and C-terminally truncated forms. SRK6 and SRK2 were sequenced from B. oleracea. SRK910 was sequenced from a B. campestris haplotype that was introgressed into B. napus.