Abscisic acid (ABA) is an essential hormone that not location and metabolism of organic substances in plants (Wayne and John 1996). Numerous reports show that ABA enhances assimilate unloading in economic sink organs such as crop grains and fleshy fruits, stimulates active uptake of organic nutrients by cells and regulates the metabolism of assimilates in cells (Opaskornkul et al. 1999, Rock and Quatrano 1995, Wayne and John 1996, Yamaki and Asakura 1991). The mechanism of ABA action on the translocation and metabolism of organic substances, however, remains essentially unknown. Acid invertase (b-fructosidase; EC 3.2.1.26), an enzyme http://www.cusabio.com/catalog-26-1.html catalyzing the irreversible cleavage of sucrose into glucose and fructose, is one of the key enzymes in sucrose metabolism and sugar transportation in grape berry (Davies et al. 1997, Davies and Robinson 1996, Patrick 1997, Quick and Schaffer 1996) as also in sink organs of other plants (Patrick 1997, Quick and Schaffer 1996). Acid invertase can be grouped into the cell wall-bound acid invertase (CWI) located in the apoplasmic space and the soluble acid invertase (SAI) compartmented in the vacuole (Patrick, 1997, Quick and Schaffer, 1996). It has been suggested that the cell wall-bound invertase catalyzes sucrose cleavage in apoplasmic space and decreases the apoplasmic sucrose concentration, thus creating the driving force for sucrose transport from the source to the sink cells. The enzyme is, therefore, considered highly relevant to the modulation of sink strength in crops (Patrick 1997, Quick and Schaffer 1996). On the other hand, the vacuole is the largest subcellular storage sink for fleshy fruits such as grape berry and other fleshy harvest organs, and the vacuole-located acid invertase has an important role in sucrose metabolism and storage (Famiani et al. 2000, Quick and Schaffer 1996). Sugar unloading in grape berry is considered to occur through the apoplasmic route (Lang and During 1991, Patrick 1997, Xia and Zhang 2000) in which the apoplasmically unloaded sucrose from the phloem may be hydrolyzed by the functional cell wall-located acid invertase before it is loaded into sink cells. Thus, acid invertase should play a key role in grape berry development.
Regarding the possible role of ABA in the regulation of acid invertases, some reports indicate that ABA can enhance the activity of acid invertases or upregulate their gene expression in soybean seeds (Ackerson 1985), pea seedling (Zhang et al. 1996), avocado fruit (Richings et al. 2000) and maize leaves and roots (Trouverie et al. 2004), whereas other reports show that gene expression of acid invertases and their activities cannot be upregulated by ABA (Bussis et al. 1997, Davies et al. 1997, Goupil et al. 1998, Kobashi et al. 1999). In these previous experiments, however, the assessment of the regulation of acid invertases by ABA has been limited to one of their two isoforms, i.e. either SAI or CWI. Furthermore, this assessment has been based in most cases on either analysis of the enzyme catalyzing activity or measurement of the mRNA level. A study focused on the effects of ABA on both the SAI and CWI, based on the assessment of both the activity and expressed amount of the enzyme, is lacking. Also, it remains unknown whether the induced effects by ABA [the previously used form being only cis, trans-(±)ABA] is specific to the physiologically active cis-(+)ABA and by what mechanism ABA acts on acid invertases. The answers to these questions are essential to assert the role of ABA in the regulation of the enzyme. This is highly significant in grape berry where accumulation of soluble sugars is believed to be modulated mainly by ABA (Coombe 1989, Davies et al. 1997), but where it remains obscure whether ABA can regulate acid invertases. We report here that ABA specifically activates both the SAI and CWI in grape berry during its development and that this activation is dependent upon the pH of the medium, treatment time, ABA dose and in vivo tissue. A complex reversible protein phosphorylation appears to be involved in the ABA-induced activation of acid invertases, independently of the expression of the enzymes. The data suggest that ABA acts on acid invertases at all levels of mRNA transcription, translation and post-translation to activate the enzymes and may be involved in fruit development by activating acid invertases.
The present experiments, done with in vivo pre-incubation of the grape berry tissue in the ABA-containing medium and in vivo infiltration of ABA into intact berries, show that ABA activates both the SAI and the CWI. The effects of ABA were dependent on the pH of the medium, time of treatments and ABA dose (Figs 1–3) and also on the living state of the tissues, because ABA could not induce any effect when applied directly on crude extracts of non-treated tissues (data not shown). The uptake rate of exogenous ABA by the berry tissues was dependent on the pH of the medium as shown by the pre-incubation of in vivo tissue where the uptake rate increased with decreasing pH (Fig. 1), probably due to the unionized ABA being able to easily cross the plasma membrane at pH values lower than its pKa of 4.8 (Parry and Horgan 1991). However, the pH dependence of the ABA-induced activation of acid invertases cannot be attributed to these differences in ABA concentrations within the treated tissues, because an effective level of ABA did accumulate in these tissues (Fig. 1). This pH dependence of ABA-induced effects is probably associated with the role of protons in ABA signal transduction in living cells (Leung and Giraudat 1998), where an appropriate acid environment may be necessary at the perceiving site of the ABA signal. As a matter of fact, the optimum pH for binding of ABA to its binding protein (putative receptor) is between 5.5 and 6.0 in grape berry (Zhang et al. 1999), which may partly explain the pH dependence of the ABA-induced activation of invertases in this fruit.
The ABA-induced activation of acid invertases is ABA dose dependent. ABA showed an increasing effect of activating acid invertases in levels from the physiological concentration (applied 2 mM with 1.5 or 1.6 mM in the treated tissues, Figs 1–3) to a moderately higher level (applied 20 mM with 6–17 mM in the treated tissues, Figs 1–3). The unstable effects of the application of 100 mM ABA with about 30 mM or more in the treated tissues (Figs 1–3) are difficult to explain but may be due to a general negative effect of excessive concentrations of the hormone. The time dependence of the ABA activation of acid invertases may be either associated with the time course of ABA accumulation in the acting sites of the treated tissues as shown in the pre-incubation of tissue in vivo (Fig. 2) or, more probably, with the time required for the enzyme-encoding gene expression involved in these ABA-induced effects.
As a matter of fact, immunoblotting and ELISA revealed that upregulation of enzyme expression by ABA enhances the activities of acid invertase (Figs 1,3,4 and 7). This suggests that expression of genes encoding for acid invertases is involved in the ABA-induced activation of acid invertases, which is a cell-signalling process depending on the living state of the berry cells for signal transduction. The decreased response of ABA by longer incubation (5 h, Fig. 2) may be due to a decline in the binding of ABA to its binding protein (putative perceiving site) after a prolonged stimulation by exogenous ABA (Zhang et al. 1999).
Whether the effect of ABA on acid invertases is specific to the physiologically active form of ABA is fundamental in establishing the function of ABA. The two ABA analogues (–)ABA and trans-ABA, used in this study to test the specificity, are structurally similar to physiologically active cis-(+)ABA but are functionally inactive (Balsevich et al. 1994, Walton 1983, Walker-Simmons et al. 1997). We have previously shown that the two ABA analogues are unable to bind to ABA-specific binding proteins with receptor nature (Zhang et al. 1999, Zhang et al. 2002). The finding that the two ABA analogues are unable to activate the acid invertases in grape berry (Fig. 4) shows the specificity of the effect of ABA. It also provides further evidence for the effect of ABA on acid invertases and the significance of this effect in berry development, where only living cells can perceive the physiologically active ABA signal (Leung and Giraudat 1998, Zhang et al. 2002).
Reversible phosphorylation of proteins has been believed to be involved in ABA signal transduction (Leung and Giraudat 1998). May such a phosphorylation be involved also in the ABA-induced activation of acid invertase? The pharmaceutical assays conducted in the present study gave a positive answer. Inhibiting (S/T-PK) or promoting protein dephosphorylation (by a nonspecific AP) increased the ABA-induced activation of acid invertase (Figs 5 and 6). Conversely, okadaic acid, a protein phosphatase inhibitor, tended to reduce the ABA-induced effects (data not shown). Inhibiting TY-PKs reduced or even completely suppressed the ABA-induced effects (Fig. 5). The reducing effects by the TY-PK inhibitor were much more intense, and the enhancing effects by S/T-PK inhibitors were more apparent when the inhibitors were applied to the living tissues before the treatment by ABA in vivo (Fig. 5A,B). This suggests that the triggering of the protein kinases–phosphatases network related to the ABA-induced effects depends on a signalling process in living cells. The involvement of both S/T-PK and TY-PK with different effects in the ABA-induced acid invertase activation suggests the involvement of a highly complex phosphorylation–dephosphorylation cascade rather than a single step in the transduction of the ABA signal towards acid invertases. In the present study, however, it could not be determined whether acid invertases are the direct substrates of protein kinases or phosphatases.
Neither the improvement nor suppression of the ABA-induced effects by the protein kinase inhibitors was dependent on the alteration of the acid invertase expression (Fig. 5A). This suggests that reversible phosphorylation of proteins may act on post-translational modification of acid invertases. We did not directly evaluate the level of mRNA of the enzymes, but a pharmaceutical assay with an inhibitor of mRNA transcription, Actidione D, showed that the inhibitor cancelled the ABA-induced effects (data not shown), indicating that a process related to mRNA transcription is also involved in the ABA-induced activation of acid invertases. This is consistent with previously reported data showing that ABA enhances the gene transcription level of acid invertases (Trouverie et al. 2004, Zhang et al. 1996).
The developmental changes in the ABA-induced effects and their related parameters were pursued to provide further information on their biological significance. The contents of soluble sugars and the activities and amounts of acid invertases were shown to be different in their patterns during berry development (Fig. 7). Similar results have been reported previously (Davies and Robinson 1996). The present study shows also that the amounts of the enzyme do not correspond to their activities in late fruit development (Fig. 7), suggesting a post-translational regulation that may involve a reversible protein http://www.cusabio.com/ phosphorylation according to the findings described above. Nevertheless, ABA steadily induced increases in both the activities and amounts of the two acid invertases during fruit development (Fig. 7). May ABA be involved in phloem unloading by promoting apoplasmic sucrose cleavage as well as in the storage of soluble sugars by stimulating sucrose metabolism in the vacuole during fruit development? It will be of interest to further study the physiological significance of the complex action of ABA on acid invertases.
It is, finally, noteworthy that the previous conflicting conclusions regarding the effect of ABA on acid invertase, based mostly on single measurements, are probably due to the complexity of the ABA-induced effects, involving as they do the pH of the medium, treatment time and ABA dose. The present characterization of the action of ABA on acid invertases suggests that ABA can function in different ways under different environmental and physiological conditions.
