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.

Expression of an amino acid biosynthesis gene in tomato flowers: developmental upregulation and MeJa response are parenchyma-specific and mutually compatible.

The gene coding for threonine deaminase (TD), the enzyme which catalyzes the first committed step in the biosynthesis of isoleucine, was isolated from tomato as a consequence of its unusual 500-fold upregulation in floral organs. It was subsequently shown that TD is induced in potato leaves in response to wounding, abscisic acid and methyl jasmonate (MeJa). Detailed analysis presented here, reveals an intricate developmental regulation pattern of gene expression in flowers that is operating solely in parenchyma territories. Yet, despite its high pre-existing expression level, TD in flowers can be further induced by MeJa. Induction of TD in flowers as well as in leaves is effective only in the parenchyma domains, irrespective of the prior expression levels. TD is neither expressed nor induced in epidermal, vascular or sporogenous tissues. Promoter analysis in transgenic tomato plants indicates that induction of TD follows identical kinetics in flowers and leaves. Furthermore, the 'conditioning' of developmental upregulation in flowers, the response to MeJa in flowers and leaves, and the parenchyma-specific expression are all mediated by the cis-elements within the proximal 192 bp of the promoter. Promoter elements regulating the correct organ-specific expression are located, however, further upstream. The promoter constructs used in this study can serve as useful tools for expressing extremely high levels of transgenes in specific cells. A scheme explaining tissue-specific response to MeJa, in conjunction with developmental control, is discussed.

Isolation of the tomato AGAMOUS gene TAG1 and analysis of its homeotic role in transgenic plants.

To understand the details of the homeotic systems that govern flower development in tomato and to establish the ground rules for the judicious manipulation of this floral system, we have isolated the tomato AGAMOUS gene, designated TAG1, and examined its developmental role in antisense and sense transgenic plants. The AGAMOUS gene of Arabidopsis is necessary for the proper development of stamens and carpels and the prevention of indeterminate growth of the floral meristem. Early in flower development, TAG1 RNA accumulates uniformly in the cells fated to differentiate into stamens and carpels and later becomes restricted to specific cell types within these organs. Transgenic plants that express TAG1 antisense RNA display homeotic conversion of third whorl stamens into petaloid organs and the replacement of fourth whorl carpels with pseudocarpels bearing indeterminate floral meristems with nested perianth flowers. A complementary phenotype was observed in transgenic plants expressing the TAG1 sense RNA in that first whorl sepals were converted into mature pericarpic leaves and sterile stamens replaced the second whorl petals.

The TM5 MADS Box Gene Mediates Organ Differentiation in the Three Inner Whorls of Tomato Flowers.

The tomato MADS box gene no. 5 (TM5) is shown here to be expressed in meristematic domains fated to form the three inner whorls-petals, stamens, and gynoecia-of the tomato flower. TM5 is also expressed during organogenesis and in the respective mature organs of these three whorls. This is unlike the major organ identity genes of the MADS box family from Antirrhinum and Arabidopsis, which function in overlapping primordial territories consisting of only two floral whorls each. The developmental relevance of the unique expression pattern of this putative homeotic gene was examined in transgenic plants. In agreement with the expression patterns, antisense RNA of the TM5 gene conferred both early and late alterations of morphogenetic markers. Early defects consist of additional whorls or of a wrong number of organs per whorl. Late, organ-specific changes include evergreen, cauline, and unabscised petals; green, dialytic, and sterile anthers; and sterile carpels and defective styles on which glandular trichomes characteristic of sepals and petals are ectopically formed. However, a complete homeotic transformation of either organ was not observed. The early and late floral phenotypes of TM5 antisense plants suggest that TM5 mediates two unrelated secondary regulatory systems. One system is the early function of the floral meristem identity genes, and the other system is the function of the major floral organ identity genes.

A meristem-related gene from tomato encodes a dUTPase: analysis of expression in vegetative and floral meristems.

A meristem-specific gene coding for deoxyuridine triphosphatase (EC 3.6.1.23) (dUTPase) in tomato was isolated, and its developmental expression in vegetative and floral apices was monitored. An 18-kD polypeptide, P18, was isolated as a consequence of its accumulation in arrested floral meristems of anantha mutant plants. The corresponding cDNA isolated from an expression library exhibited a 40 to 60% similarity with the pseudoprotease sequences of poxviruses, genes that have been suggested to encode dUTPases. Enzymatic tests and conservation of peptide motifs common to bacterial and viral genes verified that the P18 cDNA clone indeed represents a eukaryotic dUTPase. Immunogold localization and in situ hybridization experiments showed that polypeptides and transcripts of dUTPase are maintained at high levels in apical meristematic cells of vegetative and floral meristems. dUTPase gene activity is also high in the potentially meristematic cells of the provascular and vascular system. Its expression is lower in the immediate parenchymal derivatives of the apical meristematic cells, and this downregulation marks, perhaps, the transition from totipotency to the first differentiated state.

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.

The MADS box gene family in tomato: temporal expression during floral development, conserved secondary structures and homology with homeotic genes from Antirrhinum and Arabidopsis.

Five genes with homology to the floral homeotic genes deficiens of Antirrhinum and agamous of Arabidopsis were isolated from tomato. Each of the five genes is unique in the genome and could be localized to a different chromosome by RFLP mapping. Four of the tomato genes (hereafter TM) are flower-specific with distinguishable temporal expression. TM4 and TM8 are 'early', while TM5 and TM6 are 'late' genes. TM4 is homologous to squamous and TM6 is similar to deficiens, which are, respectively, 'early' and 'late' bona fide homeotic genes in Antirrhinum. The proteins encoded by the five tomato genes, like several known homeotic genes from other plants, contain within their N-terminus a highly conserved DNA-binding domain, the MADS box. All known plant MADS box genes also share, however, other properties. They all contain a central, moderately conserved, and rather basic domain, and a highly divergent or even missing C-terminal domain. Furthermore, molecular modelling predicts the presence of a conserved amphipatic alpha helix, at a constant distance from the MADS box in each of these proteins. The common properties of eight MADS box proteins from three plant families indicate that all their domains were coded for by the same ancestor gene. The sequence homology between pairs of MADS genes from different species indicates that the MADS ancestor gene multiplied and diverged in an ancestor plant common to several dicotyledon families.

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.