Riccardo Caccia

Tomato fruit set driven by pollination or by the parthenocarpic fruit allele are mediated by transcriptionally regulated gibberellin biosynthesis

We investigated the role of gibberellins (GAs) in the phenotype of parthenocarpic fruit (pat), a recessive mutation conferring parthenocarpy in tomato (Solanum lycopersicum L.). Novel phenotypes that parallel those reported in plants repeatedly treated with gibberellic acid or having a GA-constitutive response indicate that the pat mutant probably expresses high levels of GA. The retained sensitivity to the GA-biosynthesis inhibitor paclobutrazol reveals that this condition is dependent on GA biosynthesis. Expression analysis of genes encoding key enzymes involved in GA biosynthesis shows that in normal tomato ovaries, the GA20ox1 transcript is in low copy number before anthesis and only pollination and fertilization increase its transcription levels and, thus, GA biosynthesis. In the unpollinated ovaries of the pat mutant, this mechanism is de-regulated and GA20ox1 is constitutively expressed, indicating that a high GA concentration could play a part in the parthenocarpic phenotype. The levels of endogenous GAs measured in the floral organs of the pat mutant support such a hypothesis. Collectively, the data indicate that transcriptional regulation of GA20ox1 mediates pollination-induced fruit set in tomato and that parthenocarpy in pat results from the mis-regulation of this mechanism. As genes involved in the control of GA synthesis (LeT6, LeT12 and LeCUC2) and response (SPY) are also altered in the pat ovary, it is suggested that the pat mutation affects a regulatory gene located upstream of the control of fruit set exerted by GAs.

Constitutive co-suppression of the GA 20-oxidase1 gene in tomato leads to severe defects in vegetative and reproductive development.

To dissect the role of gibberellins in tomato development, we have constitutively down-regulated the gene GA 20-oxidase1 (GA20ox1). Plants co-suppressed for GA20ox1 (referred to as CO-6 plants) showed vegetative defects typical of GA deficiency such as darker and mis-shaped leaves and dwarfism. CO-6 plants flowered as the controls, although their flowers had subtle defects in the pedicel and in organ insertion. Analysis of male development revealed defects before, during and after meiosis, and a final pollen viability of 22%. The development of female organs and gametes appeared normal. Pollination experiments indicated that the pollen produced by CO-6 plants was able to fertilize control ovaries, but the analysis of the progeny showed that the construct was not transmitted. Ovaries of CO-6 plants showed high fruit set and normal fruit development when pollinated with control pollen. However these fruits were completely seedless due to a stenospermocarpic behaviour that was evidenced by callose layering in the endothelium between 7 and 15 days after pollination. We conclude that GA20ox1 in tomato exerts specific developmental roles that are not redundantly shared with other members of this gene family. For reproductive male development, silencing of this gene is detrimental for pollen production and either gametophytically lethal or severely hampering seed germination. In the pistil, the co-suppression construct does not affect the progamic phase, nor fruit set and growth, but it interferes with seed development after fertilization leading to seed abortion.

Redundancy modulation of nuclear DNA sequences in Dasypyrum villosum

In order to assess fluid domains in the genome of Dasypyrum villosum, Feulgen/DNA cytophotometric determinations and molecular and cytological DNA-DNA hybridization experiments were carried out in resting embryos and developing seedlings from yellow and brown caryopses belonging to different populations. The cytophotometric data showed that the basic amount of nuclear DNA is, on average, 12% higher in 2-day-old seedlings from yellow caryopses as compared to those from brown caryopses. It increases in each individual during seed germination, to a higher extent in seedlings from yellow caryopses than in those from brown caryopses. DNA content also differs up to 13% between plants within a caryopsis-colour group and up to 40% between populations. Dot-blot hybridization of a 396-bp D. villosum-specific DNA repeat to genomic DNA extracted from embryos in dry seeds, or from seedlings belonging to single progenies of plants from different populations, confirmed the cytophotometric results. The redundancy in the genome of sequences hybridizing to the 396-bp element differs significantly both between populations and between plant progenies within a population. During seed germination these sequences are the more amplified the less they are redundant in the genome of resting embryos, and amplification occurs to a significantly-greater extent in seedlings from yellow caryopses than in those from brown caryopses. 3H-labelled 396-bp sequences hybridize at or near the telomeres of most chromsome pairs though only to the shorter of the two subtelocentric pairs. The hybridization level is higher in seedlings from yellow caryopses that in those from brown caryopses, and a linear correlation exists between the number of silver grains counted over the labelled regions of each chromosome pair in the two groups of seedlings. Possible control mechanisms of the observed changes in the nuclear genome, and the role of these changes in developmental pregulation and environmental adaptation, are discussed.

A H2O2-forming peroxidase rather than a NAD(P)H-dependent O2.- synthase may be the major player in cell death responses controlled by the Pto-Fen complex following fenthion treatment

Four tomato (Lycopersicon esculentum Mill.) near-isogenic lines were treated by foliar spraying with the insecticide fenthion. Two, Riogrande and Rimone, differed from each other only for the presence in the latter of the Fen gene, conferring propensity to develop foliar symptoms upon exposure to fenthion. The other two, namely RC332 and RC131, were the transgenic versions of Riogrande and Rimone, respectively, harbouring the Gox gene encoding for glucose oxidase of Aspergillus niger. The production of H2O2 as well as the activities of H+-ATPase, NAD(P)H-dependent superoxide synthase, catalase, peroxidase, and Cu,Zn-superoxide dismutase were evaluated in the foliar tissues up to 24 h after exposure to fenthion. The Fen gene conferred sensitivity to fenthion, regardless of the expression of a Gox transgene. A prolonged accumulation of H2O2 was observed in the leaves of Rimone and of RC131, which was instead transient in Riogrande and in RC332. In all the tomato lines, exposure to fenthion induced rapid but transient changes in the activities of most enzymes. The only exception was peroxidase activity in the leaves of Rimone and of RC131, which steadily increased until the end of the sampling period. It is suggested that the sensitivity of Rimone to fenthion might be due to the sustained activity of a H2O2-forming peroxidase.

Tomato transgenic lines and Tetranychus urticae: changes in plant suitability and susceptibility.

A critical aspect dealing with the use of transgenic plants is the global evaluation of their environmental impact. The polyphagous mite Tetranychus urticae can be considered a suitable species to investigate unpredictable and undesirable effects on phytophagous arthropods. Three tomato near isogenic lines, that is, the cv. Riogrande (RIG), the transgenic lines RC332 (containing the Gox gene and showing high glucose oxidase activity), and MS498 (containing the KTI3 gene and exhibiting a high trypsin inhibition) were used in laboratory and greenhouse trials. Trichomes and contents of C and N of the leaves, differences in development and oviposition of T. urticae and damage caused were evaluated for each line. The laboratory trials evidenced that (1) the intrinsic rate of increase of two strains of T. urticae (T from tomato, B from bindweed), reared on the lower surface of tomato leaflets, was significantly lower in RIG than in transgenic lines and doubling time ranged between 6.9 and 11.6 days in the first and between 3.9 and 5.3 days in the latter; (2) the glandular four-lobed trichomes were always higher in RIG than in other genotypes; (3) the N leaf content was from 1.3 to 1.9 fold lower and the C/N ratio from 1.3 to 1.9 fold higher in RIG than in other lines. The greenhouse experiment, that lasted over a month and was performed by inducing an initially equal infestation of strain T, evidenced: (1) no significant difference between plant lines in the final mite infestation (motile stages per plant), nevertheless an almost double number of spider mites was counted in RC332; (2) a significantly higher percentage of damaged leaves and a significant higher average damage index on RC332 than on RIG (79% and 2.3 in the former, and 62% and 2.1 in the latter, respectively), even if in both transgenics a higher level of the most severe damages and a shorter time to approach them were observed; (4) a comparable number of mites causing the same damage level in all genotypes and a strong linear relation between the first four levels of damage and mite infestation. Although in the laboratory studies both transgenic lines enhanced the T. urticae population increase, the glasshouse studies were not as conclusive and they only suggest the possibility of any real difference between the transgenic and non-transgenic genotypes.

The Cf-2 / Rcr3esc gene interaction in tomato (Lycopersicon esculentum) induces autonecrosis and triggers biochemical markers of oxidative burst at cellular level

A tomato plant (Lycopersicon esculentum Mill.) with necrotic leaf spots mimicking disease lesions was singled out in progeny under selection in Moscow (breeding material of Ignatova Svetlana). The progeny from spontaneous selfing of such a plant (V20368), in the presence of increasing temperature and high light intensity, exhibited spontaneous necrotic lesions on the leaves, with acropetal progression (autonecrosis). A similar phenotype, described in 1948 by Langford, appeared to be associated with the Cf-2 resistance gene, introgressed from L. pimpinellifolium. Recently, Kruger et al. (2002) demonstrated that the Cf-2 effect depends on a second gene (Rcr3pim) encoding a cysteine protease, and that autonecrosis is activated by the contemporary presence of Cf-2 and the L. esculentum allele Rcr3esc. In this work we characterised the V20368 mutant and verified that autonecrosis is caused by the presence of an interaction between Cf-2 and Rcr3esc. When the environmental conditions are favourable, this interaction triggers an oxidative burst, as evidenced by a strong increase in H2O2 production and activities of catalase (EC 1.11.1.6), peroxidase (EC 1.11.1.7) and ATPase (EC 3.6.1.3). In addition, by grafting the necrotic mutant on the cv. Riogrande and vice versa, we proved that the necrotic phenotype is not associated with the movement of a signal molecule, since the autonecrosis was not transmitted across the grafting point. Finally, the interaction between Cf-2 and Rcr3esc appeared to lower the threshold of stress perception, as evidenced by an increased sensitivity to the insecticide Fenthion.

Apoptosis-like DNA fragmentation in leaves and floral organs precedes their developmental senescence

ABSTRACT We investigated the senescence of flag leaves of durum wheat (Triticum durum) during grain-filling, and of petal-like ray flowers of Jerusalem artichoke (Helianthus tuberosus) at anthesis. In both systems, we observe cleavage of DNA to high molecular weight fragments, followed by further degradation to nucleosomal fragments (laddering), a classical hallmark of apoptosis. We show that DNA fragmentation in such specialised leaves is triggered early in organ development, before the appearance of visual symptoms of senescence. Our observations support the hypothesis that senescence and cell death are part of the plant developmental program, activated by developmental cues.