Dana Hareven

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.

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.

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.

Gene expression and the control of spermatid morphogenesis in Drosophila melanogaster

Abstract The genetic control of spermatid morphogenesis was studied by light microscopy through the analysis of meiotic and premeiotic lesions. Sperm disfunction-type male-sterile mutations were screened for novel “early effect” mutations: (1) timing mutations, in which mitochondrial aggregation occurs before instead of after meiosis; (2) mutations which affect the spindle structure, e.g., a mutant with second-division monoastral spindle; (3) mutations which cause deformations in primary spermatocyte structures. It is shown, in addition to the examples cited above, that normal meiosis may often serve as an early marker for normal differentiation, and that approximately 20% of male-sterile mutations are meiotic mutants. The role of the Y chromosome was reexamined. The interaction between Y factors and X -linked male steriles is in many cases additive, indicating that Y gene products are essential for normal development of the primary spermatocytes. Furthermore, XO males are shown to be extreme meiotic mutants. It is argued that spermatid morphogenesis is totally dependent on developmental processes in the primary spermatocyte stage. The relations among developmental processes in early spermatogenesis are discussed in terms of gene activity.

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.

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.

Heterochromatin markers: arrangement of obligatory heterochromatin, histone genes and multisite gene families in the interphase nucleus of D. melanogaster.

Localization, as detected by in situ hybridization, of major heterochromatic blocks in interphase nuclei of larval brain and imaginal discs is reported. We conclude that the position of heterochromatic regions in interphase nuclei is correlated with their respective position in metaphase chromosomes and hence, independent of sequence recognition. Furthermore, chromocentral associations of X-, Y- or autosomal-based heterochromatin are not formed in these cells. Homologues do align in close proximity, but heterochromatin plays no role in this arrangement. Heterochromatin, and probably nucleoli, establish their membrane links in situ, and have no prefixed recognition sites. The most intimate association between homologous repetitive sequences was found in the histone locus, but no tendency for clustering was found among loci of multisite euchromatic gene families.

Heterochromatin Markers: A Search for Heterochromatin Specific Middle Repetitive Sequences in Drosophila

We sought for cloned sequences of middle repetitive (MR) complexity that mark obligatory heterochromatic regions. Total genome probes were employed in a differential screening procedure to recover X-specific, Y-specific and autosomal specific heterochromatic sequences. X- and Y-linked sequences were recovered in the same experiment. (Y-linked clones will be described elsewhere). All nine independent, non-identical X-specific clones were found to be partially homologous to one another and to type I rDNA insertion. No other X-specific Bam HI or HindIII clones were found. In situ hybridization to normal and inverted chromosomes revealed extensive homology in the heterochromatin spanning the nucleolus organizer (NOR) and the eu-heterochromatin junction. Eleven clones which are underrepresented in polytene chromosomes were selected in another differential screening. None was autosome-specific. Five were of nucleolar origin. Among them a presumptive type II 28SrDNA insertion sequence was clearly localized within the X-chromosome proximal heterochromatin in addition to the known localization of the X and Y nucleolar organizers. We mapped three clones to major sites on the Y chromosome and to secondary autosomal sites. The results are discussed with regard to the complexity of heterochromatin organization.

The floral system of tomato

The tomato floral system is distinguished from that of Arabidopsis and Antirrhinum in several ways. The shoot is a sympodium, the first flower of the inflorescence is apical and no bracts are formed. We discuss the possible function of genes affecting growth habit and inflorescence development and analyze in detail the unique developmental alterations caused by inhibition or ectopic expression of tomato-specific MADS-box genes. Preliminary analysis of transgenic tomato plants expressing an alien homeotic gene and several different chimeric genes of the MADS-box family is also reported.