Tomato SP-interacting proteins define a conserved signaling system that regulates shoot architecture and flowering.

Divergent architecture of shoot models in flowering plants reflects the pattern of production of vegetative and reproductive organs from the apical meristem. The SELF-PRUNING (SP) gene of tomato is a member of a novel CETS family of regulatory genes (CEN, TFL1, and FT) that controls this process. We have identified and describe here several proteins that interact with SP (SIPs) and with its homologs from other species: a NIMA-like kinase (SPAK), a bZIP factor, a novel 10-kD protein, and 14-3-3 isoforms. SPAK, by analogy with Raf1, has two potential binding sites for 14-3-3 proteins, one of which is shared with SP. Surprisingly, overexpression of 14-3-3 proteins partially ameliorates the effect of the sp mutation. Analysis of the binding potential of chosen mutant SP variants, in relation to conformational features known to be conserved in this new family of regulatory proteins, suggests that associations with other proteins are required for the biological function of SP and that ligand binding and protein-protein association domains of SP may be separated. We suggest that CETS genes encode a family of modulator proteins with the potential to interact with a variety of signaling proteins in a manner analogous to that of 14-3-3 proteins.

The SELF-PRUNING gene of tomato regulates vegetative to reproductive switching of sympodial meristems and is the ortholog of CEN and TFL1.

Vegetative and reproductive phases alternate regularly during sympodial growth in tomato. In wild-type 'indeterminate' plants, inflorescences are separated by three vegetative nodes. In 'determinate' plants homozygous for the recessive allele of the SELF-PRUNING (SP) gene, sympodial segments develop progressively fewer nodes until the shoot is terminated by two consecutive inflorescences. We show here that the SP gene is the tomato ortholog of CENTRORADIALIS and TERMINAL FLOWER1, genes which maintain the indeterminate state of inflorescence meristems in Antirrhinum and Arabidopsis respectively. The sp mutation results in a single amino acid change (P76L), and the mutant phenotype is mimicked by overexpressing the SP antisense RNA. Ectopic and overexpression of the SP and CEN transgenes in tomato rescues the 'indeterminate' phenotype, conditions the replacement of flowers by leaves in the inflorescence and suppresses the transition of the vegetative apex to a reproductive shoot. The SELF-PRUNING gene is expressed in shoot apices and leaves from very early stages, and later in inflorescence and floral primordia as well. This expression pattern is similar to that displayed by the tomato ortholog LEAFY and FLORICAULA. Comparison of the sympodial, day-neutral shoot system of tomato and the monopodial, photoperiod-sensitive systems of Arabidopsis and Antirrhinum suggests that flowering genes that are required for the processing of floral induction signals in Arabidopsis and Antirrhinum are required in tomato to regulate the alternation between vegetative and reproductive cycles in sympodial meristems.

The dominant developmental mutants of tomato, Mouse-ear and Curl, are associated with distinct modes of abnormal transcriptional regulation of a Knotted gene.

The Curl (Cu) and Mouse-ear (Me) mutations of tomato cause two seemingly unrelated developmental syndromes with a wide range of pleiotropic phenotypes. Yet, the distinct morphogenic alterations in shoots, leaves, and inflorescences conferred by the two mutations appear to be caused by unchecked meristematic activity that characterizes dominant mutations in Knotted1 (Kn1)-like genes of monocot plants. We have been unable to separate the two closely linked Cu and Me mutations, and they may lie in the same gene. A homeobox-containing class I Kn1-like gene, TKn2, also maps to the same location. Significantly, the dominant mutations are associated with two aberrant modes of TKn2 transcription. Overexpression of the two in-frame wild-type transcripts of TKn2 is associated with the Cu mutation, whereas misexpression of an abundant and oversized fusion mRNA is associated with the Me mutation. Available molecular evidence strongly suggests that the defective Me-TKn2 transcript is generated via a novel splicing event that merges transcripts of two closely linked genes. The translated fusion product is comprised of most of the 5' end of the adjacent PPi-dependent fructose 6-phosphate phosphotransferase (PFP) transcript spliced in-frame to coding position 64 of the TKn2 transcript, leaving the TKn2 homeobox intact. We suggest that class I Kn1-like genes were selected early during evolution to regulate basic programs of aerial meristems and that subtle alterations in their function may be the basis for the wide diversity in growth parameters of shoot systems, leaves, and inflorescences among plant species.

The making of a compound leaf: genetic manipulation of leaf architecture in tomato.

The most distinctive morphogenetic feature of leaves is their being either simple or compound. To study the basis for this dichotomy, we have exploited the maize homeobox-containing Knotted-1 (Kn1) gene in conjunction with mutations that alter the tomato compound leaf. We show that misexpression of Kn1 confers different phenotypes on simple and compound leaves. Up to 2000 leaflets, organized in compound reiterated units, are formed in tomato leaves expressing Kn1. In contrast, Kn1 induces leaf malformations but fails to elicit leaf ramification in plants with inherent simple leaves such as Arabidopsis or in tomato mutant plants with simple leaves. Moreover, the tomato Kn1 ortholog, unlike that of Arabidopsis, is expressed in the leaf primordia. Presumably, the two alternative leaf forms are conditioned by different developmental programs in the primary appendage that is common to all types of leaves.

The tomato 66.3-kD polyphenoloxidase gene: molecular identification and developmental expression.

A gene coding for a polypeptide abundant in tomato floral meristems was isolated and shown to represent a tomato 66.3-kD polyphenoloxidase. Analysis of cDNA clones and a corresponding intronless genomic clone indicated that the plastid-bound 587-residue-long polypeptide, designated P2, contains two conserved copper-binding domains, similar to those found in fungal and mammalian tyrosinases. P2 transcripts and polypeptides are accumulated in the arrested floral primordia of the anantha mutant inflorescences and are equally abundant in primordia of wild-type flowers; the gene continues to be expressed at high levels in developing floral organs. In young expanding leaves, P2 protein is concentrated in palisade cells and in epidermal trichomes. Expression patterns of P2 in plant meristems permit molecular distinction between floral and vegetative primordia, and, in a companion study, comparison with dUTPase suggests that the two genes mark two alternative complementary developmental programs in the floral and vegetative meristems of the tomato plants.

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

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 greater than 40% overall similarity. The Td gene is unique in the tomato genome and its coding region is interrupted by eight introns. Its expression is greater than 50-fold higher in sepals and greater than 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.

cDNA sequence of adrenodoxin reductase. Identification of NADP-binding sites in oxidoreductases.

Adrenodoxin reductase is an NADP dependent flavoenzyme which functions as the reductase of mitochondrial P 450 systems. We sequenced two adrenodoxin reductase cDNAs isolated from a bovine adrenal cortex cDNA library. The deduced amino acid sequence shows no similarity to the sequence of the microsomal P 450 systems or other known protein sequences. Nonetheless, by sequence analysis and c comparisons with known sequences of dinucleotide-binding folds of two NADP-binding flavoenzymes, two regions of adrenodoxin reductase sequence were identified as the FAD- and NADP-binding sites. These analyses revealed a consensus sequence for the NADP-binding dinucleotide fold (GXGXXAXXXAXXXXXXG, in one-letter amino acid code) that differs from FAD and NAD-binding dinucleotide-fold sequences. In the data base of protein sequences, the NADP-binding-site sequence appears solely in NADP-dependent enzymes, the binding sites of which were not known to date. Thus, this sequence may be used for identification of a certain type of NADP-binding site of enzymes that show no significant sequence similarity.

Isolation of a cDNA for adrenodoxin reductase (ferredoxin-NADP+ reductase). Implications for mitochondrial cytochrome P-450 systems.

Using specific antibodies against adrenodoxin reductase (AR), we screened lambda gt11 cDNA expression libraries constructed from bovine adrenal cortex mRNA, and isolated several putative clones coding for this enzyme. Concurrently we determined the amino acid sequences of fragments from it. A deoxyinosine-containing oligonucleotide probe, generated for one of the sequences, reacted specifically with one of the cloned cDNAs of about 1600 base pairs. The codon sequence of this cDNA matched the peptide sequences, further confirming its identity as a copy of AR mRNA. RNA blot analysis indicates that in the adrenal cortex and corpus luteum there is only one major mRNA (approximately 2000 bp) for AR. The levels of this mRNA are at least 40-fold lower in the liver and kidney which are also known to contain in homologue of AR. As compared to adrenodoxin and cytochrome P-450scc mRNAs, AR mRNA levels in the adrenal cortex appear to be about 10-fold lower. Southern blot analysis of bovine and human genomic DNAs reveals that in both of these species there is only one gene for AR. These results indicate that only a single reductase serves the different mitochondrial P-450 systems in steroidogenic tissues.