Suicidal tomato cells: programmed cell death in suspension-cultured tomato cells and ripening fruit

 Tomato fruit ripening involves a series of highly organised biochemical, physiological and structural changes that are under strict genetic control. The plant hormone ethylene (C 2 H 4 ), in synergy with certain developmental cues, regulates fruit ripening by initiating and co-ordinating the expression of genes responsible for different aspects of the ripening process, such as the respiratory rise, chlorophyll degradation, carotenoid biosynthesis, conversion of starch to sugars, and cell wall degradation. The economical importance of tomato as a crop has resulted in substantial research efforts aimed at the improvement of fruit quality traits, such as shelf life. Inhibition of ethylene action or perception, either by the use of inhibitors or by transgenic approaches, has been proved to be a powerful means to control ripening.In this thesis, research aimed at providing new tools to further improve tomato fruit quality is described. It was hypothesised that fruit quality could be improved if (further) ripening was inhibited only after the fruit had reached a certain degree of maturity, including the associated stages of coloration and taste development. Genes that encode for proteins involved in ethylene biosynthesis or perception are potential targets for a transgenic approach within this strategy. It was believed that ethylene acts as a rheostat rather than a trigger during tomato fruit ripening, which means that ethylene signalling is required throughout the complete course of ripening. Indeed, treatment of tomato fruit with 1-MCP, a potent inhibitor of ethylene action, delayed colour development, softening, and ethylene production not only in tomato fruit harvested at the mature green stage, but also in the breaker, and orange stages. 1-MCP treatment decreased the mRNA abundance of PSY1 , EXP1 , and ACO1 , three ripening-related tomato genes, in mature green as well as in breaker, orange, and red ripe fruit. These results demonstrate that the ripening process can be inhibited both on a physiological and molecular level, even at very advanced stages of ripening.In plant transgenic approaches, the most widely used promoter is the CaMV 35S promoter. It expresses the corresponding transgene constitutively and at high levels in all plant cells. A number of endogenous, fruit-specific tomato promoters, such as the 2A11 , ACO1 , E8 , E4 , and polygalacturonase (PG) promoter, have also been successfully used to obtain transgenic fruit. These promoters are generally active throughout ripening. To obtain DNA sequences that potentially could direct expression of a transgene towards the later stages of ripening, two new promoters were cloned. CEL2 (encoding an endo-ß-1,4-glucanase) and EXP1 (an expansin) are specifically and highly expressed during the advanced stages of ripening. The 1341 bp long EXP1 promoter fragment contains a 19 bp stretch also present in the promoter of fruit-specific polygalacturonase (PG) gene, and two putative ethylene-responsive elements (EREs). The 660 bp long CEL2 promoter fragment does not contain any putative EREs. Several promoter deletion constructs were transiently expressed in fruit tissue by particle bombardment, using firefly luciferase as a reporter gene. Some positively and negatively regulating regions could be identified, but it was concluded that this method is not very suitable to determine promoter activity in tomato fruit tissue.In addition to disrupting ethylene biosynthesis or perception, new possibilities to inhibit tomato fruit ripening were searched for. It was postulated that programmed cell death (PCD) might play a role during the advanced stages of ripening and postharvest senescence. PCD is a process aimed at eliminating unnecessary or harmful cells during growth and development of multicellular organisms. It is indispensable for normal development and survival of plants. To study a possible role for PCD in tomato fruit ripening and postharvest senescence, first, a model system of suspension-cultured tomato cells was established. These cells can be induced to undergo PCD by treatment with chemicals that are known to induce a specific form of PCD in animal cells, named apoptosis. This chemical-induced cell death in tomato is accompanied by characteristic features of animal apoptosis, such as typical changes in nuclear morphology, the fragmentation of the nucleus and genomic DNA degradation. Moreover, inhibitor studies suggest that, like in animal systems, caspase-like proteases are involved in this apoptotic-like cell death pathway.To identify genes potentially involved in plant PCD, changes in gene expression during chemical-induced PCD were studied. Tomato homologues of DAD1 and HSR203 , two genes that had been implicated in PCD previously, were isolated. LeDAD1 mRNA levels are reduced by approximately 50%, whereas LeHSR203 mRNA levels increase 5-fold during chemical-induced PCD tomato. A differential display approach, used to identify novel genes, resulted in isolation of two up-regulated (CTU1 and CTU1 shows high homology to various gluthatione S-transferases, whereas CTU2 shows homology to human PIRIN . CTD1 is highly similar to early-auxin-responsive Aux/IAA genes. CTD2 corresponds to the tomato RSI-1 gene, CTD4 is an unknown clone, and CTD5 shows limited homology with a proline-rich protein from maize. A tomato metacaspase gene, designated LeMCA1 , that encodes a protein homologous to mammalian caspases was also cloned. Caspases are cysteinyl aspartate-specific proteases that constitute the core component of animal apoptosis. Southern analysis indicates that there is at least one more metacaspase present in the tomato genome. Unexpectedly, LeMCA1 gene expression is constitutive in suspension-cultured tomato cells. However, LeMCA1 is rapidly induced upon infection of tomato leaves with the fungal pathogen Botrytis cinerea , suggesting a possible role in disease-related cell death.Taken together, the data derived from the model system of tomato suspension cells suggest that plants make use of cell death pathways that share homology with animal apoptotic pathways. These conserved mechanisms, however, can be activated and regulated by plant-specific factors such as plant hormonesThe occurrence of PCD during fruit ripening and postharvest senescence was investigated using DNA laddering and the expression of several genes as markers. No DNA laddering could be detected in tomato fruit tissue. The tomato homologue of a putative negative regulator of apoptosis, LeDAD1 , is constitutively expressed during ripening of wildtype, rin , and Nr tomato fruit. LePIRIN(a tomato homologue of a human gene encoding a putative nuclear factor that is believed to be involved in NF-κB/IκB signalling) mRNA could not be detected in fruit tissue.LeHSR203 (correlated with HR-like cell death) transcripts were clearly detectable in tomato fruit and significantly increased during postharvest senescence of MG, BR, OR, and RR fruit. CTD1 (IAA/Aux factor) mRNA was detected at very low levels in ripening tomato fruit, but, unlike during chemical-induced PCD, transcript levels did not decrease during the course of ripening.In conclusion, these data do not support the hypothesis that PCD occurs during postharvest senescence of tomato fruit. Although LeHSR203 gene expression patterns during postharvest senescence of tomato fruit were similar to those observed during various forms of plant PCD, those of the other genes under investigation were not. In addition, the degradation of genomic DNA (DNA laddering) could not be detected. The results up to now did not lead to novel opportunities for the improvement of tomato fruit quality by targeting plant PCD signalling pathways. However, improvement of tomato fruit quality through inhibition of ethylene biosynthesis or perception at the later stages of ripening seems feasible.