Structural features of the ABA molecule relevant to this study

The ABA analogues used in the present study were (-)-ABA, (±)-trans, trans- A B A, PBI-63 and PBI-51; in addition (+)-ABA and (±)-ABA were used (see Figure 1.4 and Section 1.7.4 for detailed descriptions of these molecules). Several studies have either directly examined the effect of these molecules on guard cell turgor or examined the effect o f the alterations to the structure of (+)-ABA apparent in these molecules (see below).

2.1.3(a) (-)-A B A

(-)-ABA (Figure 1.4) has very little, or no effect on stomatal closure (Walton, 1983) (see Section 1.7.4). (-)-ABA showed very little effect on stomatal conductance (measured by monitoring leaf temperature) o f excised leaves o f Hordeum vulgare (Cummins & Sondheimer, 1973). It was suggested that the small amount o f activity that was detected could be attributed to slight contamination o f the (-)-ABA with (+)- ABA (Cummins & Sondheimer, 1973). It was shown that (±)-ABA had approximately twice the activity of (+)-ABA in the closure of stomata (measured by monitoring leaf CO2 and water exchange) on leaves o f Xanthium pennsylvanicum thus suggesting that

(-)-A BA was inactive (Kriedman et al, 1972). In addition, (-)-ABA did not alter stomatal aperture in detached epidermis from the leaves o f C. communis, Trapaeolum majus (Milborrow, 1980) or V. faba (Hornberg & Weiler, 1984).

2.1.3(b) (:±)-trans, trans-ABA

Altering the side chain from C-2 cis, C-4 trans to C-2 trans, C-4 trans to form trans, trans-ABA (Figure 1.4) resulted in a loss of activity in the stomatal system (Kriedmann et al, 1972 - Xanthium pennsylvanicum leaves; Hornberg & Weiler, 1984 - V. faba, detached epidermis). The low level of biological activity reported for trans, trans-ABA in one experiment (Cummins et al, 1971 - barley seedlings) was later attributed to light-induced conversion of trans, trans-ABA to the active cis, trans- ABA (Milborrow, 1970; Walton, 1983).

2.1.3(c) PBI-63 and PBI-51

The effect o f PBI-63 and PBI-51 on guard cell turgor has not been investigated previously. However, the individual changes to the structure o f (+)-ABA apparent in

these molecules [see Figure 1.4 and Section 1.7.4(c)] have been investigated. The individual changes include:

(i) Saturation o f the ring double bond. The ABA analogue [(+)- or (+)-] dihydro ABA, formed by the saturation o f the ring double bond, was active (Oritani & Yamashita, 1982), had one tenth of the activity o f ABA (Yamamoto & Oritani, 1995 - C. communis, detached epidermis) or was inactive (Hornberg & Weiler, 1984 - V. faba, detached epidermis; Orton & Mansfield, 1974 - C. communis, detached epidermis) at promoting stomatal closure.

(ii) Changing the C-l group to an alcoholic group. Altering the C-l carboxylic group o f ABA to an alcoholic group resulted in no loss o f activity (Uehara et al., 1975 - B. napus leaves; Orton & Mansfield, 1974 - C. communis detached epidermis).

(iii) Changing the C4, C-5 double bond to an acetylenic linkage. To date, the effect of acetylenic ABA on stomatal closure has not been measured directly; changing the C4, C5 bond to a acetylenic linkage has always been accompanied by other changes. An ABA analogue with an acetylenic link in the side-chain and a C -l alcohol group was active as an antitranspirant (Blake et al., 1990a & b - three coniferous species, leaves).

2.1.4 Aims

This study aimed to investigate the effects o f several ABA analogues (see Figure 1.4 and Section 2.1.3) on stomatal aperture with a view to using the structure- activity data, in conjunction with similar data for ABA-induced changes in guard cell gene expression (Chapter 4), to probe ABA perception in stomatal guard cells (see Montgomery et al., 1996a & b). It was envisaged that if the “receptors” for the two ABA-induced responses (control of guard cell turgor and regulation o f gene expression in guard cells) differed, for example in either structure or location, then their

responsiveness to a range o f ABA analogues may vary (see Sections 1.7.2 and 1.8). In addition it was o f interest to determine whether one o f the ABA analogues, PBI-51 (Figure 1.4) would act as an ABA antagonist in the guard cell system as it does in other plant systems [see Section 1.7.4(c); Wilen etal., 1993, 1996].

The effects o f the ABA analogues described in Figure 1.4 on stomatal opening were tested in several plant species. For C. communis, the effects o f the ABA analogues on stomatal opening and on stomata which were already open were investigated bearing in mind that ABA inhibits stomatal opening and promotes closure (Assmann, 1993; and see Section 1.5). The mechanisms by which ABA inhibits stomatal opening differ from those by which it promotes stomatal closure (Assmann, 1993; Giraudat, 1995; Willmer & Fricker, 1996; Hey et al., 1997; see Sections 1.3 and 1.5). Therefore, a study o f the effects of the ABA analogues on these two processes may help to determine whether they have similar ABA perception mechanisms.

C. communis and V faba were chosen as the plant material in the investigations because they have easily detachable epidermis and a well-characterized and well- defined stomatal response to ABA (Weyers & Meidner, 1990), C. communis being the more sensitive to ABA o f the two species (Weyers & Meidner, 1990). Nicotiana tabacum (tobacco) was chosen because it is a species in which ABA regulated gene promoter activity in the guard cell has been reported (Taylor et al, 1995; see Section 1.7.2). It is thus a potential candidate for the investigation o f two types o f response to ABA (turgor changes and gene expression changes) in guard cells o f a single species. Other reasons for choosing tobacco include the fact that some characterization o f the effect o f ABA on guard cells o f tobacco has been carried out (Thomas, 1970; H. Clayton, personal communication). In addition, tobacco was the preferred choice o f the plants which were available for guard cell gene expression studies (Taylor et al., 1995).

This was because tobacco leaf epidermis was more suitable for detached epidermal work than that from A. thalicma (see Chapter 4). The reasons for this include: (i) the abaxial epidermis was easier to detach from the leaf; (ii) there was a higher percentage o f viable epidermal cells (other than guard cells) in the detached epidermis; and (iii) the stomata and stomatal apertures were larger and therefore easier to measure than those o f A. thaliana.