Wound-healing process of tomato fruits
A fast and efficient wound closure stops excessive water loss and prevents pathogenic organisms from entering through wounded plant surfaces. Suberization is supposed to be a common plant response to wounding and plays an important role in the formation of a protective, impervious layer (Kolattukudy, 1981). Suberin is a specific lipophilic biopolymer characterized by the deposition of a polyaromatic domain associated with the cell wall, and a polyaliphatic domain thought to be deposited between the cell wall and plasma membrane (Bernards, 2002; Lulai et al., 2008). The aromatic domain primarily consists of p-coumaric and ferulic acids, which are presumably involved in covalently linking the aromatic domain to the cell wall (Bernards, 2002; Santos & Graça, 2006), whereas the aliphatic domain of suberin comprises long-chain aliphatic compounds, such as alkandioic acids, hydroxyalkanoic acids, alkanoic acids and alkanols (Kolattukudy et al., 1975; Holloway, 1983). It still remains hypothetical how these monomeric units are assembled at the macromolecular level (Bernards, 2002).
Tomato fruits are predominantly harvested by removal from their pedicel (Poole & McLeod, 1994). Consequently, a stem scar wound is generated, which causes significant water loss and makes the fruit amenable to post-harvest microbial infections. Hence, a rapid biological response is crucial for tomato fruits to reduce dehydration, as well as the efflux of nutrients, and to restrain sites of microbial invasion, aiming to ensure proper ripening and/or a prolonged shelf life. The compositional nature, time course, regulation and efficiency of these wound-healing processes of tomato fruits have so far not been investigated in detail.
Suberized stem scar membrane
Our analyses confirmed the presence of aromatic and aliphatic components characteristic of the biopolyester suberin in the newly formed stem scar membrane, with the exception of glycerol (Moire et al., 1999) which was not analyzed because of experimental limitations. The detected components could not have originated from the cuticular membrane because the cuticle surrounding the stem scar tissue was removed completely before chemical analysis. The abundance of ω-hydroxyalkanoic acids, α,ω-alkandioic acids and a significant proportion of C18 unsaturated aliphatic compounds is in agreement with the suberin monomer composition reported from tomato fruit and other plant sources (Dean & Kolattukudy, 1976; Zeier & Schreiber, 1998). Compositional changes at the stem scar during the time course of suberization remain to be determined.
Soluble wax-like substances integrated into the suberin matrix have been shown to be largely responsible for the formation of a water-loss barrier of native potato tuber periderm (Soliday et al., 1979; Schreiber et al., 2005). In potato wound periderm, the permeability was, on average, 100-fold higher than that of the native periderm, although the total amounts of wax-like substances and suberin biopolymer were c. 50–60% of the amounts of the native periderm without any pronounced qualitative differences in chemical composition. These results show that the water barrier properties of suberized tissue cannot be inferred from the occurrence of suberin with depositions of wax-like substances (Schreiber et al., 2005). From the suberized stem scar tissue of tomato fruits, only small amounts of chloroform-soluble aliphatic compounds, C20, C22, C24 alkanols and alkanoic acids – probably embedded in the suberin polymer matrix – were detected. These soluble compounds showed a completely different composition from waxes embedded in the tomato fruit cutin matrix. Previous studies have shown that the main difference between potato periderm and other suberized plant tissues is the significant fraction of aliphatic wax compounds sorbed to the suberin biopolymer of potato, whereas this fraction is basically missing in suberized tissues of other plants (Schreiber et al., 2005; Efetova et al., 2007). Accordingly, our results suggest that soluble wax-like compounds are presumably not the major determinants for the formation of an effective barrier at the stem scar tissue of tomato fruits, restricting water loss, which, however, remains to be elucidated.
Wound healing and water loss of tomato fruits
According to Cameron & Yang (1982), transpiration through the tomato fruit stem scar accounts for c. 67% of the whole-fruit water loss. This is in full agreement with the results provided in the present study. The stem scar tissue of tomato fruit is also known to account for c. 97% of the fruit gas exchange (Cameron & Yang, 1982). It has long been recognized that a gas-tight sealing of stem scar tissue causes reduced ripening rates and extends the storage life of tomato fruits (Brooks, 1937; Yang & Shewfelt, 1999). Thus, post-harvest suberization of the stem scar wound should at least reduce gas exchange to a level still sufficient to allow for a nonretarded ripening of tomato fruit.
As a result of their comparably small surface area and fragility, the enzymatically isolated stem scar tissues were not suitable for the direct determination of water permeability. Our results demonstrate an effective reduction in fruit water loss as a result of the efficient sealing of the stem scar wound by suberization, reflected by the permeance for water and the ion flows out of the fruit, approximating those of tomato fruits artificially sealed with paraffin wax immediately after harvest. The dependence on active suberin biosynthesis to attain efficient closure of the stem scar wound is demonstrated by the low-temperature storage experiment.
Function of the stem scar membrane as a microbial barrier
The greatly reduced intrusion of actively growing mycelium of the post-harvest tomato fruit pathogen F. solani (Amadioha & Uchendu, 2003) as a result of prolonged fruit storage indicates a role for suberized stem scar tissue simply as a mechanical barrier and/or for prevention of entry by accumulating antibiotic/antimicrobial compounds. Although the aliphatic domain of potato tuber suberin has been shown to specifically provide resistance against the fungal pathogen Fusarium sambucinum (Lulai & Corsini, 1998), suggesting a chemical mode of action, the toluidine blue infiltration experiment corroborated the effective wound closure. The newly formed stem scar barrier membrane prevents the seepage of water through the stem scar wound and, consequently, reduces the risk of infiltration with microbial contaminants. From the present data, it can be suggested that the pretreatment of traditionally harvested tomato fruits to accelerate the suberization process at the stem scar wound might reduce significantly microbial infection, improve the quality and extend the shelf life of tomato fruits. This pretreatment of harvested tomato fruits includes conditions of moderate temperature and reduced humidity for a 2-d period of storage before washing and further processing. Likewise, a prestorage period of kiwifruit (Actinidia deliciosa) has been shown to markedly reduce stem scar infection by the fungal pathogen Botrytis cinerea (Pennycook & Manning, 1992; Bautista-Baños et al., 1997).
Nevertheless, there are also indications that soluble compounds associated with the suberin biopolymer, such as phenolics or wax-like components, may themselves act as antifungal agents (Kolattukudy, 1984; Thomas et al., 2007). Similarly, H2O2-generating systems associated with suberization might additionally affect microbial growth, resulting in an overall reduced microbial amenability of the tomato fruit stem scar during the wound-healing process.
Impact of the CER6 β-ketoacyl-coenzyme A synthase
Fatty acid elongases catalyze the elongation of the carbon chain of C16 and C18 fatty acids to different lengths as found in cuticular wax biosynthesis (Domergue et al., 1998). MicroTom lecer6 is defective in a β-ketoacyl-coenzyme A synthase involved in very-long-chain fatty acid elongation, resulting in a largely increased cuticular permeance for water of tomato fruits (Vogg et al., 2004; Leide et al., 2007). Despite the overall elevated weights of the stem scar tissue of the MicroTom lecer6 mutant, probably as a result of a generally enlarged stem scar area, the time course and efficiency of wound healing were not modified in this mutant. An induction of tomato Cer6 gene expression was not found at the transcriptional level of the stem scar tissue during tomato fruit storage. Hence, our results indicate that the CER6 enzyme is not required for the biosynthesis of suberin and associated wax-like compounds in tomato fruits. These findings are not consistent with the expression of the KCS6 protein of the potato periderm, which shares a high homology with the CER6 protein of tomato (Serra et al., 2009). As stem scar wound healing was not modified in the MicroTom lecer6 mutant, our results indicate that enzymes other than CER6 might be substantially responsible for fatty acid elongation in tomato fruit suberin biosynthesis.
Relationship between gene expression and wound healing
So far, only a few genes involved in the biosynthesis of the suberin biopolymer have been identified (Ranathunge et al., 2011). Interestingly, genes found to be differentially expressed in the stem scar tissue during wound healing are related to desiccation and regulation by ABA.
Some genes encoding nonspecific lipid transfer proteins, such as the Tsw12 gene, are inducible by ABA, and thus mediate in plant responses to environmental stress conditions (Torres-Schumann et al., 1992; Yubero-Serrano et al., 2003). Lipid transfer proteins are known to bind a variety of hydrophobic fatty acids and lipids (Kader, 1996; Blein et al., 2002), potentially also providing monomers for suberin biosynthesis. Therefore, increased transcript levels of Tsw12 may indicate a post-harvest stimulation of transport processes of lipophilic compounds in the stem scar tissue. The formation of suberized cell layers, in turn, prevents excessive water loss from the detached tomato fruit, as generally suggested for plant organ abscission zones (Bleecker & Patterson, 1997; Roberts et al., 2002).
In the present study, we also expected to identify the dehydrin gene Tas14 as being induced in the early stage of fruit storage on wounding at the stem scar tissue. Dehydrins have the ability to bind to lipid vesicles that contain acidic phospholipids, scavenge hydroxyl radicals or display protective activity towards lipid membranes against peroxidation (Close, 1996; Richard et al., 2000). The Tas14 gene has also been described to accumulate in response to ABA or environmental stress (Godoy et al., 1990; del Mar Parra et al., 1996).
Strikingly, the expression pattern of the Asr4 gene was similar to that of the highly induced Tsw12 gene and the moderately up-regulated Tas14 gene. The ABA, water stress and ripening-inducible Asr gene family has been reported to be up-regulated under different environmental stress conditions in an ABA-dependent manner and during the process of tomato fruit ripening (Dóczi et al., 2005; Fischer et al., 2011). Accumulating functional evidence suggests that Asr genes have several functions in tomato plant adaptation to desiccation (Maskin et al., 2001; Frankel et al., 2006), probably effective in transcriptional regulation of post-harvest changes in tomato fruits.
The only tomato gene detected in the present study that showed a decrease in expression after fruit harvest was the Erd7 gene encoding an early-responsive to dehydration protein. In Arabidopsis, the Erd7 gene has been shown to be induced by ABA and dehydration, and repressed by rehydration (Seki et al., 2002; Oono et al., 2003). Hence, the down-regulation of the tomato Erd7 gene might be an early indication for desiccation stress recovery, probably because of the fairly efficient closure of the stem scar wound already 1 d after harvest. However, as the senescence-related Erd7 gene has also been reported to be up-regulated under high light stress in Arabidopsis (Kimura et al., 2003), post-harvest storage in darkness might have caused the subsequent decrease in gene expression.
These genes display a rapid, although to some extent, transient reaction in response to tomato fruit wounding as a consequence of the harvesting process, suggesting a significant role in the mediation of plant stress responses and/or suberization. The precise function of these enzymes has not been characterized to date. The effect of wounding on transcript accumulation in tomato fruits could be linked to a higher water loss rate through more open surfaces arising from the mechanical treatment and with subsequent closure caused by the wound-healing process.
Importance of endogenous ABA in wound healing
The gene expression analysis results of the present study support the idea that ABA could play a pivotal role in the stem scar wound-healing process of tomato fruits, although the induction of genes putatively involved in wound healing does not prove the direct involvement of ABA in stem scar suberization of tomato fruits. As documented previously for tomato leaves, ABA accumulation was also found in the stem scar tissue after harvest (Herde et al., 1999). Furthermore, during the wound-healing process, an induction at the transcriptional level was found for the Sit gene encoding an aldehyde oxidase, which is known to be the catalyst of the abscisic aldehyde oxidation to ABA – the final step of ABA biosynthesis in tomato plants (Taylor et al., 1988; Seo et al., 2000). The application of tomato mutants with reduced ABA levels and the assessment of ABA contents of the stem scar tissue during wound healing, in combination with the functional characterization of the wound-healing process, provided appropriate tools to investigate in vivo the role of ABA in the stem scar suberization of tomato fruits. The regulatory involvement of ABA in the wound-healing processes of tomato fruits has not been demonstrated unequivocally by reduced ABA concentrations and the determination of the resulting functional effects. A rapid and transient boost in ABA content has also been reported for potato tubers after wounding (Lulai et al., 2008; Kumar et al., 2010).
The decreased ABA contents in the stem scar tissues of RR flacca, RR sitiens and AC notabilis were accompanied by a retarded and attenuated stem scar wound-healing response in the ABA-defective tomato fruits. These results unambiguously indicate that ABA is involved as a mediating agent in the wound-healing processes of the stem scar tissue of tomato fruits. The presence of basal ABA levels in combination with readily synthesized ABA significantly accelerates the suberization response after stem scar wounding. Lulai et al. (2008) showed that ABA plays a similar role in wounded potato tubers. Nevertheless, the specific role of ABA in the regulation/promotion of wound-induced gene expression at the stem scar tissue of tomato fruits remains to be elucidated. Future experimental approaches need to address the functional involvement and interplay of other regulatory molecules, such as jasmonic acid, salicylic acid, auxin, ethylene and nitric oxide (León et al., 2001; París et al., 2007; Lulai et al., 2011), in the wound responses at the tomato fruit stem scar tissue.