Michel Hernould

Regulatory Features Underlying Pollination-Dependent and -Independent Tomato Fruit Set Revealed by Transcript and Primary Metabolite Profiling

Indole Acetic Acid 9 (IAA9) is a negative auxin response regulator belonging to the Aux/IAA transcription factor gene family whose downregulation triggers fruit set before pollination, thus giving rise to parthenocarpy. In situ hybridization experiments revealed that a tissue-specific gradient of IAA9 expression is established during flower development, the release of which upon pollination triggers the initiation of fruit development. Comparative transcriptome and targeted metabolome analysis uncovered important features of the molecular events underlying pollination-induced and pollination-independent fruit set. Comprehensive transcriptomic profiling identified a high number of genes common to both types of fruit set, among which only a small subset are dependent on IAA9 regulation. The fine-tuning of Aux/IAA and ARF genes and the downregulation of TAG1 and TAGL6 MADS box genes are instrumental in triggering the fruit set program. Auxin and ethylene emerged as the most active signaling hormones involved in the flower-to-fruit transition. However, while these hormones affected only a small number of transcriptional events, dramatic shifts were observed at the metabolic and developmental levels. The activation of photosynthesis and sucrose metabolism-related genes is an integral regulatory component of fruit set process. The combined results allow a far greater comprehension of the regulatory and metabolic events controlling early fruit development both in the presence and absence of pollination/fertilization.

SlY1, the first active gene cloned from a plant Y chromosome, encodes a WD‐repeat protein

Unlike the majority of flowering plants, which possess hermaphrodite flowers, white campion (Silene latifolia) is dioecious and has flowers of two different sexes. The sex is determined by the combination of heteromorphic sex chromosomes: XX in females and XY in males. The Y chromosome of S.latifolia was microdissected to generate a Y‐specific probe which was used to screen a young male flower cDNA library. We identified five genes which represent the first active genes to be cloned from a plant Y chromosome. Here we report a detailed analysis of one of these genes, SlY1 (S.latifolia Y‐gene 1). SlY1 is expressed predominantly in male flowers. A closely related gene, SlX1, is predicted to be located on the X chromosome and is strongly expressed in both male and female flowers. SlY1 and SlX1 encode almost identical proteins containing WD repeats. Immunolocalization experiments showed that these proteins are localized in the nucleus, and that they are most abundant in cells that are actively dividing or beginning to differentiate. Interestingly, they do not accumulate in arrested sexual organs and represent potential targets for sex determination genes. These genes will permit investigation of the origin and evolution of sex chromosomes in plants.

Flower development schedule in tomato Lycopersicon esculentum cv. sweet cherry

The ontogeny of tomato (Lycopersicon esculentum cv. sweet cherry) flowers was subdivided into 20 stages using a series of landmark events. Stamen primordia emergence and carpel initiation occur at stage 4; archesporial and parietal tissue differentiate at stage 6 and meiosis in anthers begins at stage 9. Subepidermal meristematic ovule primordia are formed on the placenta at stage 9; megasporogenesis begins at stage 11–12 and embryo sac differentiation and ovule curvature take place at stage 14, once the pollen is maturing. We established a correlation between the characteristic cellular events in carpels and stamens and morphological markers of the perianth. The model of tomato flower development schedule was then used to analyse the spatial, temporal and tissue-specific expression of gene(s) involved in the regulation of floral organ development. As an example, the expression pattern of ORFX, a gene controlling cell size in tomato fruits, shows that expression starts very early during the ontogeny of reproductive organs.

Elucidating the functional role of endoreduplication in tomato fruit development.

BACKGROUND Endoreduplication is the major source of endopolyploidy in higher plants. The process of endoreduplication results from the ability of cells to modify their classical cell cycle into a partial cell cycle where DNA synthesis occurs independently from mitosis. Despite the ubiquitous occurrence of the phenomenon in eukaryotic cells, the physiological meaning of endoreduplication remains vague, although several roles during plant development have been proposed, mostly related to cell differentiation and cell size determination. SCOPE Here recent advances in the knowledge of endoreduplication and fruit organogenesis are reviewed, focusing on tomato (Solanum lycopersicum) as a model, and the functional analyses of endoreduplication-associated regulatory genes in tomato fruit are described. CONCLUSIONS The cyclin-dependent kinase inhibitory kinase WEE1 and the anaphase promoting complex activator CCS52A both participate in the control of cell size and the endoreduplication process driving cell expansion during early fruit development in tomato. Moreover the fruit-specific functional analysis of the tomato CDK inhibitor KRP1 reveals that cell size and fruit size determination can be uncoupled from DNA ploidy levels, indicating that endoreduplication acts rather as a limiting factor for cell growth. The overall functional data contribute to unravelling the physiological role of endoreduplication in growth induction of fleshy fruits.

Impairment of tapetum and mitochondria in engineered male-sterile tobacco plants

Flowers of tobacco transformed with an unedited copy of the mitochondrial atp9 gene sequence fused to the yeast coxIV mitochondrial targeting presequence, showed several anther abnormalities leading to pollen abortion. The gene was expressed in vegetative and reproductive tissues of the plant. Cytological analysis revealed that tapetum development was impaired. Mitochondria of the tapetum cells were severely affected showing characteristic signs of degeneration: loss of cristae and swelling. These mitochondrial modifications were correlated with the presence of the transcript and translated product of the ‘unedited’ atp9 and a significant decrease in oxygen consumption in non-photosynthetic tissues. The main effect of the unedited atp9 expression in transgenic plants was male sterility.

Cyclin-dependent Kinase (CDK) Inhibitors Regulate the CDK-Cyclin Complex Activities in Endoreduplicating Cells of Developing Tomato Fruit

The jelly-like locular (gel) tissue of tomato fruit is made up of large thin-walled and highly vacuolized cells. The development of the gel tissue is characterized by the arrest of mitotic activities, the inhibition of cyclin-dependent kinase A (CDKA) activity, and numerous rounds of nuclear DNA endoreduplication. To decipher the molecular determinants controlling these developmental events, we investigated the putative involvement of CDK inhibitors (p27Kip-related proteins, or KRPs) during the endoreduplication process. Two cDNAs, LeKRP1 and LeKRP2, encoding tomato CDK inhibitors were isolated. The LeKRP1 and LeKRP2 transcript expression was shown to be enhanced in the differentiating cells of the gel undergoing endoreduplication. At the translational level, LeKRP1 was shown to accumulate in the gel tissue and to participate in the inhibition of the CDK-cyclin kinase activities occurring in endoreduplicating cells of the gel tissue. We here propose that LeKRP1 participates in the control of both the cell cycle and the endoreduplication cycle.

Functional analysis of the anaphase promoting complex activator CCS52A highlights the crucial role of endo-reduplication for fruit growth in tomato.

Tomato fruit growth is characterized by the occurrence of numerous rounds of DNA endo-reduplication in connection with cell expansion and final fruit size determination. Endo-reduplication is an impairment of mitosis that originates from the selective degradation of M phase-specific cyclins via the ubiquitin-mediated proteolytic pathway, requiring the E3 ubiquitin ligase anaphase promoting complex/cyclosome (APC/C). Two types of APC/C activators, namely CCS52 and CDC20 proteins, exist in plants. We report here the molecular characterization of such APC/C activators during fruit development, and provide an in planta functional analysis of SlCCS52A, a gene that is specifically associated with endo-reduplication in tomato. Altering SlCCS52A expression in either a negative or positive manner had an impact on the extent of endo-reduplication in fruit, and fruit size was reduced in both cases. In SlCCS52A over-expressing fruits, endo-reduplication was initially delayed, accounting for the altered final fruit size, but resumed and was even enhanced at 15 days post anthesis (dpa), leading to fruit growth recovery. This induction of growth mediated by endo-reduplication had a considerable impact on nitrogen metabolism in developing fruits. Our data contribute to unravelling of the physiological role of endo-reduplication in growth induction during tomato fruit development.

Tomato Flower Abnormalities Induced by Stolbur Phytoplasma Infection Are Associated with Changes of Expression of Floral Development Genes

Tomato (Lycopersicon esculentum cv. Micro-Tom) plants infected by the stolbur phytoplasma (isolate PO) display floral abnormalities, including sepal hypertrophy, virescence, phyllody, and aborted reproductive organs, which are reminiscent of those observed in Arabidopsis thaliana mutants affected in flower development genes. Semiquantitative reverse transcription-polymerase chain reaction and in situ RNA hybridization were used to compare expressions of meristem and flower development genes in healthy and stolbur phytoplasma-infected tomatoes. In infected plants, FALSIFLORA (FA), controlling the identity of the inflorescence meristem, was up-regulated, whereas LeWUSCHEL (LeWUS) and LeCLAVATA1 (LeCLV1), regulating the meristem development, and LeDEFICIENS (LeDEF), responsible for the organ (petals and stamens) identity within the flower, were down-regulated regardless of the development stage of the flower bud. In contrast, expression of TAG1, which regulates stamen and carpel identities and negatively controls LeWUS, was up-regulated at the early stages and down-regulated at the late stages. In situ RNA hybridization analyses revealed that TAG1 transcripts were restricted to the same floral meristem territories in healthy and infected tomatoes, indicating that tissue-specific expression of TAG1 was not affected by the stolbur phytoplasma infection. Taken together, these data indicate that flower malformations of stolbur phytoplasma-infected tomatoes are associated with early changes in the expression of key flower development genes. The possible mechanisms by which the multiplication of stolbur phytoplasma in tomato sieve tubes deregulates floral development are discussed.

The synthesis of the rhamnogalacturonan II component 3-deoxy-D-manno-2-octulosonic acid (Kdo) is required for pollen tube growth and elongation

Despite a very complex structure, the sugar composition of the rhamnogalacturonan II (RG-II) pectic fraction is extremely conserved. Among its constituting monosaccharides is the seldom-observed eight-carbon sugar 3-deoxy-D-manno-octulosonic acid (Kdo), whose phosphorylated precursor is synthesized by Kdo-8-P synthase. As an attempt to alter specifically the RG-II structure in its sugar composition and assess the consequences on the function of RG-II in cell wall and its relationship with growth, Arabidopsis null mutants were sought in the genes encoding Kdo-8-P synthase. Here, the isolation and characterization of one null mutant for the isoform 1 (AtkdsA1-S) and two distinct null mutants for the isoform 2 of Arabidopsis Kdo-8-P synthase (AtkdsA2-V and AtkdsA2-S) are described. Evidence is provided that AtkdsA2 gene expression is preferentially associated with plantlet organs displaying a meristematic activity, and that it accounts for 75% of the mRNAs to be translated into Kdo-8-P synthase. Furthermore, this predominant expression of AtKDSA2 over AtKDSA1 was confirmed by quantification of the cytosolic Kdo content in the mutants, in a variety of ecotypes. The inability to identify a double knockout mutant originated from pollen abortions, due to the inability of haploid pollen of the AtkdsA1- AtkdsA2- genotype to form an elongated pollen tube properly and perform fertilization.

Molecular characterization of a WEE1 gene homologue in tomato (Lycopersicon esculentum Mill.)

Early fruit development in tomato (Lycopersicon esculentum Mill.) proceeds in two distinct phases of growth that comprise cell division and cell expansion, respectively. In pericarp and the jelly like locular tissue of tomato fruit, the transition between cell division to cell expansion is characterized by the arrest of mitotic activity, numerous rounds of nuclear DNA endoreduplication and the inhibition of Cyclin-Dependent Kinase A (CDKA) activity. To investigate whether the WEE1 kinase may play a role during the endoreduplication process, we isolated and characterized the tomato homologue for WEE1. The LeWEE1 gene consisted of 10 exons with a predicted 510 amino acid-long protein. The accumulation of the corresponding transcripts was associated with mitotically active organs: developing fruits, seeds and roots. Interestingly, LeWEE1was expressed in the jelly like locular tissue concomitant with endoreduplication during fruit development. Using tobacco BY-2 synchronized cells, we showed that the WEE1 gene expression is cell-cycle regulated with a maximum transcript accumulation at S phase. Our data indicate the putative dual contribution of LeWEE1 in the classical cell cycle and the endocycle.