Biosynthetic threonine deaminase gene of tomato: isolation, structure, and upregulation in floral organs.

Abstract

The gene encoding the plant biosynthetic threonine deaminase (Td; EC 4.2.1.16) has been cloned as a result of its unusual upregulation in tomato flowers. The Td gene of tomato encodes a polypeptide of 595 residues, the first 80 of which comprise a putative two-domain transit peptide cleaved at position 51. Comparison of the amino acid sequence with the corresponding enzymes from yeast and bacteria reveals a near identity of the important catalytic regions and >40% overall similarity. The Td gene is unique in the tomato genome and its coding region is interrupted by eight introns. Its expression is >50-fold higher in sepals and >500-fold higher in the rest of the flower than in leaves or roots. Its overexpression, however, is strictly confined to the parenchymal cells of the floral organs. In young tomato leaves, the chloroplast-bound enzyme is found almost exclusively in the subepidermal spongy mesophyll cells.The biosynthetic and biodegradative threonine deaminase (Td) enzymes [L-threonine hydro-lyase (deaminating), EC 4.2.1.16] catalyze the conversion of L-threonine to a-ketobutyrate (1). The biosynthetic reaction is the first committed step of the pathway leading to the formation of the essential amino acid isoleucine. Regulation ofTd activity by isoleucine was the first example discovered of allosteric inhibition by the end product of a biosynthetic pathway. L-Threonine is also a feedback inhibitor of aspartate kinase and homoserine dehydrogenase, thus regulating the biosynthesis of threonine and other amino acids of the aspartate family (2, 3).The biosynthetic Td genes from Escherichia coli (ilvA) and yeast (ilvi) have been isolated and characterized (4-7). In plants, biosynthetic Td activity is associated with chloroplasts and the enzyme is feedback-regulated by isoleucine (8,9). Isoleucine-dependent Td-cell lines have been isolated from Nicotiana plumbaginifolia and shown to be complemented by ilvi from yeast (10, 11). The tomato Td reported here was cloned as a consequence of its unusual upregulation in floral organs. Flower development is initiated by a series of events that cause the irreversible conversion of a vegetative meristem to a floral meristem (12-14). As a working hypothesis it was proposed that upon induction, meristematic cells fated to form an inflorescence acquire a basic, cell-autonomous developmental program that distinguishes a floral cell from a vegetative cell (15). This distinction persists throughout floral development and is a prerequisite for the proper execution of homeotic transformations-i.e., organogenesis-as well as the ensuing organspecific gene activity. We have tried to approach the problem experimentally, by isolation of common regulatory elements of genes that are upregulated in all floral organs. Consequently, an extensive comparative study ofprotein profiles of leaves and flowers was undertaken. It was discovered that the gene encoding the most abundant protein of tomato flowers is, in fact, the structural gene for the tomato biosynthetic Td.t MATERIALS AND ME...

Molecular Interactions between the Specialist HerbivoreManduca sexta (Lepidoptera, Sphingidae) and Its Natural Host Nicotiana attenuata. I. Large-Scale Changes in the Accumulation of Growth- and Defense-Related Plant mRNAs

“…The ORF of the pTD13 insert encodes a polypeptide of 601 amino acids with a calculated molecular mass of 65.6 kD. Alignments of the amino acid sequence of N. attenuata with other sequences available in databases revealed similarities of 73% to tomato TD (Samach et al, 1991) (Kielland-Brandt et al, 1984), 58% to Escherichia coli TD (Cox et al, 1987), and 46% to human Ser deaminase (Ogawa et al, 1989). Catalytic domains, regulatory domains, and exon-intron boundaries are highly conserved, whereas the putative chloroplast transit peptide shows only moderate similarity between the three plant species (Fig.…”

“…A cytoskeletal compound is represented by pDH9.4, which is similar to ␤-tubulin of the fungus Glomus coronatum (M. Stommel and P. Franken, unpublished data). Carbon metabolism is represented by pDH14.2 encoding biosynthetic Thr deaminase (TD), which catalyzes the committed step in iso-Leu biosynthesis by converting Thr to 2-ketobutyrate (Samach et al, 1991). As indicated by the similarity to the Arabidopsis ferredoxin-dependent Glu synthase (Fd-GOGAT; Lin et al, 1999), the gene product encoded by pDH55.2 is involved in nitrogen metabolism.…”

“…9). The conservation of catalytic domains (Taillon et al, 1988), exon-intron boundaries, and putative plastidtargeting sequences (Samach et al, 1991) in combination with the similar developmental expression of TD in potato, tomato, and N. attenuata provide strong evidence that pTD13 indeed encodes biosynthetic TD and not biodegradative TD (Szamosi et al, 1993). In undamaged plants, TD transcripts (pDH14.2) accumulated mainly in the apex of the emerging axis, which consists of vegetative and flower buds (Fig.…”

“…The five catalytic domains C1 to C5 and the eight regulatory domains R1 to R7 as defined by Taillon et al (1988) are displayed as bars. Exon-intron boundaries determined for the tomato gene by Samach et al (1991) are indicated by triangles. Pro residues in a putative chloroplast transit peptide of N. attenuata and tomato are indicated by dots in the upper and lower row, respectively, above the alignment.…”

Identification and Characterization of a Biodegradative Form of Threonine Dehydratase in Senescing Tomato (Lycopersicon esculentum) Leaf

“…A gene encoding TD was recently isolated from tomato (Samach et al, 1991). Expression of this gene at the mRNA leve1 was >50-fold higher in sepals, >500-fold higher in the rest of flower, and two to five times higher in fruits and stems than in leaves or roots.…”