P,ant.

197

Plmata 9 Springer-Verlag1992

Abscisic acid biosynthesis in roots II. The effects o f water-stress in wild-type and abscisic-acid-deficient mutant o f L ycopersicon esculentum M i l l .

(notabilis) plants

Andrew D. Parry 1, Allen Griffithsz, and Roger Horgan 1 1 Department of Biological Sciences, The UniversityCollege of Wales, Aberystwyth,Dyfed SY23 3DA, UK 2 Department of Biochemistry,The UniversityCollege of North Wales, Bangor, GwyneddLL57 2UW, UK Received 25 September; accepted 7 November 1991

Abstract. The ubiquity of the apo-carotenoid abscisic acid (ABA) biosynthetic pathway elucidated in waterstressed, etiolated leaves of Phaseolus vulgaris (see A.D. Parry and R. Horgan, 1991, Physiol. Plant. 82, 320-326), has been difficult to establish. Light-grown leaves contain very high carotenoid:ABA ratios, preventing correlative studies, and no etiolated leaves so far studied, other than those of Phaseolus, have been found capable of synthesising significant amounts of ABA in response to stress. Roots are known to synthesise ABA and contain low carotenoid levels; therefore A B A biosynthesis was investigated in soil- and hydroponically grown roots of Lycopersicon esculentum Mill. Hydroponically grown roots were stressed by immersion in 100 mM mannitol and soil-grown roots by withholding water. In both cases stress led to an increase in ABA levels and a decrease in the levels of specific xanthophylls, namely all-trans- and 9"-cis-neoxanthin and all-trans-violaxanthin. In hydroponically grown roots, and soil-grown roots stressed after removal of the shoot, ratios of xanthophyll cleaved: ABA synthesised of approx. 1 : 1 were obtained. These findings are consistent with the operation of an apo-carotenoid pathway in roots, involving the conversion of all-trans-violaxanthin via all-trans-neoxanthin, to 9"-cis-neoxanthin, and the specific cleavage of 9"-cisneoxanthin to yield the Cls ABA precursor xanthosin. Similar experiments with roots of the "leaky", ABA-deficient mutant of Lycopersicon, notabilis, indicate that the mutation does not affect the perception or transduction of stress, or the ability of the plant to cleave carotenoids. Rather, it appears that notabilis possesses an enzyme with reduced substrate specificity which cleaves more aU-trans- than 9"-cis-neoxanthin.

Key words: Abscisic acid biosynthesis - Carotenoid Lycopersicon (mutant) - Mutant (ABA-deficient) - Root (abscisic acid) - Water stress Abbreviations: ABA= abscisic acid; DPA = dihydrophaseic acid; not = notabilis; PA = phaseic acid; t = trans; XaA = xanthoxinacid; Xan = xanthoxin

Introduction Using etiolated leaves of Phaseolus vuloaris, which have reduced carotenoid levels but retain the ability to synthesise abscisic acid (ABA) in response to water stress, it has been shown that stress-induced ABA biosynthesis utilises an apo-carotenoid, or indirect, pathway (Li and Walton 1990; Parry et al. 1990a). Subsequent experiments with inhibitors of carotenogenesis and deuterium oxide (Parry and Horgan 1991a) have implicated 9"-cisneoxanthin as the immediate pre-cleavage ABA precursor, and all-trans-violaxanthin as a precursor of 9"-cisneoxanthin. The key regulatory step of ABA biosynthesis appears to be that involving the cleavage of 9"-cisneoxanthin (see Parry 1991 ; Parry and Horgan 1991b). Observations made using the etiolated Phaseolus system indicate that this cleavage is performed by an enzyme with high specificity, both for substrate (9"-cis-neoxanthin) and cleavage site (across the 11', 12' double bond) (Li and Walton 1990; Parry et al. 1990a; Parry and Horgan 1991a). The ubiquity of this pathway has been difficult to demonstrate, as light-grown leaves contain levels of xanthophylls up to 100-fold greater than those of ABA, and to date the only investigated etiolated leaves with the capacity to synthesise significant amounts of ABA in response to stress are those of Phaseolus (Li and Walton 1990; Parry et al. 1990a). Roots possess the ability to synthesise ABA (see Davies and Zhang 1991), by what is thought to be the same pathway as that operating in light-grown leaves (Creelman et al. 1987; Sindhu and Walton 1987). The aims of the present study were twofold. Firstly to investigate whether the apo-carotenoid pathway of ABA biosynthesis operates in roots, and if so to identify the intermediate between aU-trans-violaxanthin and 9"-cisneoxanthin and examine further the specificity of the cleavage reaction. Secondly to further characterise notabilis (not), an ABA-deficient mutant of Lycopersicon esculentum, which may be impaired in its ability to cleave 9"-cis-neoxanthin (Taylor 1987; Parry et al. 1988).

A.D. Parry et al.: Abscisic acid biosynthesis in roots. II Material and methods Plant material. Soil-grown wild-type and mutant plants of Lyeopersicon esculentum Mill. were grown as described in the preceding paper (Parry and Horgan 1992). Hydroponically grown Lycopersicon seedlings were imbided (with aeration) overnight and grown

in trays in Hoaglands solution (Hoagland and Arnon 1950) for 18 d, the Hoaglands solution being replaced after approx. 7, 10 and 16d. Water-stress. For soil-grown plants water-stress was imposed by withholding water for a period of 3 (notabilis) or 4 (wild-type) days. Shoots of certain plants were removed approx. 1 crn above soil level and the stumps covered with lanolin. Hydroponically grown roots were stressed by immersion in 50-200 nM mannitol for 148 h. Determination of cell turgor pressure was through use of a pressure probe as described in Husken et al. (1978). Determination of earotenoids, ABA, phaseic acid ( PA ) and dihydrophaseic acid (DPA). To enable appropriate comparisons to be

made, the levels of carotenoids, ABA, PA, and DPA were measured in the same extract, as described in the preceding paper (Parry and Horgan 1992) and Parry et al. 1990a. Results The identification, in root extracts, of those xanthophylls implicated in A B A biosynthesis, i.e. the all-trans- and 9 (9")-cis- isomers o f neoxanthin and violaxanthin, is described in the preceding paper (Parry and H o r g a n 1992). Abscisic acid and its two principle short-term metabolites PA and DPA, were identified, after two purification steps by high-performance liquid chromatography, from gasc h r o m a t o g r a p h y retention times and the presence o f characteristic fragment ions following analysis by gas chromatography-mass spectrometry. Experiments were carried out using both soil-grown and hydroponically grown roots of L y c o p e r s i c o n esculenturn. D a t a are presented on a fresh-weight basis as freezedrying root tissue was found to cause isomerisation o f neoxanthin (frozen roots - all-trans :9"-cis isomers 4.7:1 ; freeze-dried roots - all-trans :9'-cis isomers 0.7:1), formation o f furanoid artefacts, such as luteoxanthin, and the overall loss o f epoxy-xanthophylls.

193 Table 1. Effects of osmotic stress (100 mM mannitol) on the levels (nmol - (g FW)- 1) of xanthophylls and ABA, PA and DPA in roots of hydroponically grown wild-type seedlings of Lycopersicon Metabolitea

t-neo c-neo t-viola c-viola anthera lutein Decrease after stress ABA PA DPA Increase after stress

Expt. 1b

Expt. 2

non-str,

str.

non-str,

str.

0.188 0.043 0.157 0.013 0.010 0.040

0.120 0.022 0.093 0.007 0.007 0.046

0.168 0.037 0.130 0.010 0.009 0.032

0.150 0.028 0.102 0.007 0.007 0.033

0.t56 0.004 0.001 0.008

0.059 0.096 0.001 0.033

0.009 0.002 0.006

0.118

0.042 0.003 0.025 0.058

at = all-trans ; c = 9 /9'-eis ; neo = neoxanthin; viola = violaxanthin;

anthera = antheraxanthin bnon-str. =non-stressed; str. = stressed Table 2. Effects of osmotic stress (100 mM mannitol) on the levels (nmol - (g FW)-1) ofxanthophylls and ABA, PA and DPA in roots of hydroponically grown not seedlings of Lycopersicon Metabolite a

t-neo c-neo t-viola c-viola anthera lutein Decrease after stress ABA PA DPA Increase after stress

Expt. I b

Expt. 2

non-str,

str.

non-str,

str.

0.430 0.063 0.199 0.016 0.013 0.031

0.345 0.058 0.190 0.014 0.014 0.036

0.237 0.069 0.165 0.012 0.013 0.024

0.169 0.053 0.140 0.008 0.014 0.035

0.095 0.003 0.0001 n.d.

0.096 0.005 0.003 0.0003 n.d. n.d. n.d.

0.002

0.008 n.d. n.d. 0.005

at = all-trans ; c = 9/9'-cis ; neo = neoxanthin; viola = violaxanthin;

anthera = antheraxanthin bnon-str. = non-stressed; str. = stressed; n.d. = not detected

H y d r o p o n i c a l l y 9 r o w n roots. Wild-type 18-d-old seedlings

were subjected to osmotic stress by immersing their root systems in 100 m M mannitol for 3 h. Determinations of epidermal and cortical cell turgor pressure, by direct measurement using the pressure probe, confirmed that such treatments resulted in reduced cell turgor (data not shown). Detailed observations on the effects of such osmotic stresses on the water relations of roots of wildtype and notabilis (not.) plants will be reported separately. N o wilting o f wild-type leaves was observed during these experiments. The levels of xanthophyUs and ABA, P A and D P A extracted from such wild-type roots are given in Table 1. The results from two separate experiments are shown. In response to stress the levels o f A B A increased by between 5 to 24-fold, while the levels o f P A remained unchanged and those o f D P A rose by approx. 4-fold. The levels of certain xanthophylls decreased in response to stress, most o f the decrease being attributable

to a l l - t r a n s - n e o x a n t h i n and violaxanthin (30-45 % of the total decrease each), and 9 " - c i s - n e o x a n t h i n (15%). The average ratio, on a molar basis, o f xanthophyll "lost": A B A synthesised (measured as increases in the levels o f A B A + PA + D P A ) was 1.2:1. The magnitude o f the reductions in xanthophyll levels during this 3-h stress period varied from 15-34% o f the total root xanthophylls. A 23 % reduction in xanthophyll levels, together with equivalent increases in ABA, occurred within 1 h o f a 100 m M mannitol stress (data not shown). Periods o f stress up to 12 h led to decreases o f between 20-40% in total xanthophyll levels, while longer periods (up to 48 h) led to progressively smaller decreases (8-12%; data not shown). Similar experiments were carried out with hydroponically grown not plants (Table 2). In this case, wilting of

194

A.D. Parry et al.: Abscisic acid biosynthesis in roots. II

Table 3. Effects of soil-drying on the levels of xanthophylls, ABA, PA and DPA in roots of 13-week-old wild-type plants of L y c o p e r -

Table 4. Effects of soil-drying on the levels of xanthophylls, ABA, PA and DPA in roots of 13-week-old n o t plants of L y c o p e r s i c o n a

sicon a

Metabolite Metabolite

Control (watered)

Stressed (non-watered) - shoots

t-neo 0.483 4-0.007 0.2774-0.046 c-neo 0.028 4-0.008 0.0274-0.001 t-viola 0.092 4-0.009 0.0844-0.001 c-viola 0.008 4-0.001 0.009:60.001 anthera 0.004 4-0.001 0.0094-0.001 lutein 0.017 4-0.003 0.029:60.004 Decrease after stress 0.200 ABA 0.031 4-0.010 0.169:60.061 PA 0.0003 4- 0.0001 0.0064- 0.001 DPA 0.00024-0.00010.017:60.001 Increase after stress 0.161

Control (watered)

Stressed (non-watered) - shoots

+ shoots

1.3684-0.200 0.1424-0.043 0.315:50.063 0.0264- 0.004 0.0084- 0.002 0.0694-0.029 n.s. 0.0284-0.005 0.015 0.002 0.043

0.5324- 0.011 0.0594- 0.001 0.2544- 0.018 0.0254- 0.002 0.0134- 0.001 0.0454- 0.009 0.778 0.0014-< 0.001 n.d. n.d. n.s.

+ shoots 0.197:60.082 0.016:60.011 0.088:60.035 0.007:60.003 0.004:60.001 0.018:60.001 0.302 0.6804-0.172 0.225:60.087 0.149:60.023 1.022

t-neo 1.1834-0.023 c-neo 0.1104-0.024 t-viola 0.3164-0.042 c-viola 0.023 + 0.004 anthera 0.012 4-0.003 lutein 0.065 4-0.055 Decrease after stress ABA 0.0024-0.001 PA n.d. DPA n.d. Increase after stress

an.s. = not significant; for other abbreviations, see Table 2

a For abbreviations, see Table 2

the leaves was visible within 15 min of transfer to the mannitol solution. Basal levels o f ABA were 30-75 % of those of the wild-type, and PA and D P A were present at very low levels or were non-detectable. Stress produced increases in the levels of A B A of only 1.7-to 3.0-fold, leading to stress A B A levels only 5-19% of those in wild-type roots. However, decreases in the levels of total xanthophylls were, on average, very similar to the wild-type (0.11 n m o l - (g F W ) - I wild-type; 0.09 n m o l " (g FW) -1 not). Total xanthophyll levels in non-stressed roots of not were approx. 50% higher than those in the wild-type, which m e a n t that a 3-h stress period led to reductions of between 12-15% in the levels of total xanthophylls. The decrease in xanthophylls in not was due predominantly to decreases in the levels of alltrans-neoxanthin (71-89%). The average ratio, on a molar basis, o f xanthophyll "lost" : A B A synthesised in roots o f not was 33.3:1. The severity o f the osmotic stress was altered by using different concentrations o f mannitol (data not shown). F o r wild-type seedlings increasing osmotic stress, by using 200 m M mannitol, led to a 75% greater decrease in xanthophylls and a 71% greater increase in ABA, PA and D P A production c o m p a r e d with those measured in response to 100 m M mannitol treatments. Very little A B A was produced by not seedlings under any stress conditions, but the mildest stress (50 m M ) led to a greater decrease in xanthophylls (52% o f the total). Soil-grown roots. Thirteen-week-old soil-grown plants of Lyeopersicon were used for these experiments. Stress was imposed by withholding water for 3 (not) or 4 (wildtype) d. The contribution of shoot-synthesised ABA to the total accumulating in the roots was investigated by removing the shoots of certain plants prior to the imposition o f stress. The results are shown in Table 3. Basal wild-type A B A levels are approx, fourfold greater than in the younger hydroponically grown roots, but increased by only fivefold following stress in the detached roots, to give comparable stress levels in both tissues. In

the stressed whole plants, root ABA levels were 22-fold higher than in the non-stressed roots. Levels of PA and D P A were very low in non-stressed roots and although accumulating 20-fold in the detached roots they were still a small percentage (11%) o f ABA levels. In stressed whole plants, PA and D P A levels were 35% of those of ABA, approx. 750-fold higher than in non-stressed roots. Total xanthophyll levels were 40-60% higher in soilgrown roots compared with hydroponically grown ones, mainly as the result of increased levels of all-transneoxanthin, making up almost 80% of the total xanthophyll in soil-grown roots (approx. 40% in h y d r o p o n i cally grown roots). As with the hydroponically grown, roots there were specific decreases in the xanthophylls following stress, but in contrast the vast majority of the decrease was due to all-trans-neoxanthin (95-96%). In detached roots the ratio, on a molar basis, o f x a n t h o p h y l l "lost": ABA synthesised was 1.2:1, while in the roots o f whole plants the ratio was 0.3:1. As expected soil-grown not plants contained reduced ABA levels, only 6.5% of wild-type levels. Detached roots showed 14-fold higher ABA levels, but were still only 16% of the equivalent in wild-type roots (Table 4). Stressed roots of whole plants showed no increases in ABA levels. The relative carotenoid content in roots of not was very similar to the wild-type, but total levels were threefold higher. There were no significant differences in carotenoid levels between non-stressed roots and those of detached roots. In contrast, but similar to stressed hydroponically grown not roots, roots of stressed whole not plants "lost" approx. 45 % of their total xanthophylls, compared with 48 % in wild-type roots. The actual reduction in xanthophylls in roots of not was 2.5-fold greater than in wild-type roots. In a separate experiment, detached soil-grown wildtype roots were stressed by exposing them to a stream of w a r m air, as described by Cornish and Zeevaart (1985). In that report a loss of 50-60% fresh weight led to an optimal ABA accumulation of four- to fivefold. Unfortunately this could not be repeated in the present study,

A.D. Parry et al.: Abscisic acid biosynthesisin roots. II 40-60 % decreases in root flesh weight, followed by 6 h incubations in the dark, did not affect the levels of ABA, or those of the xanthophyUs, perhaps as a result of the rapid imposition of such a severe stress. This does, however, provide further evidence that the reduction in xanthophyll levels is directly linked with the increase in ABA, and is not a separate response to stress.

195 OH

HO O

~

V

d~~ + V ~

all-trans-violaxanthin

H0

-

-

Discussion It has proved possible to stimulate ABA biosynthesis, as measured by increases in the levels of ABA and its major metabolites, in both hydroponically grown and soilgrown roots of Lycopersion esculentum. There are many reports in the literature of stress-induced increases in the levels of ABA in roots, for example in Zea mays (Lachno and Baker 1986; Ribaut and Pilet 1991), Lycopersicon and Xanthium strurnarium (Cornish and Zeevaart 1985), Helianthus annuus (Neales et al. 1989), Commelina communis and Pisum sativum (Zhang and Davies 1987; Zhang et al. 1987), and many more measuring increased ABA levels in xylem exudates (see Davies and Zhang 1991). The levels of ABA measured in roots of Lycopersicon in the present study, and the magnitudes of the stress-induced increases, are in good agreement with the values of Cornish and Zeevaart (1985) for similar roots. The hydroponically grown seedlings were subjected to a relatively mild stress, which led to reduced turgor in root cells but not to visible leaf wilting. In contrast the soil-grown roots were subjected to a much more severe stress. Shoots of intact plants stressed by withholding water were severely wilted after 4 d. It is likely that the root systems of the intact plants suffered greater stress than the decapitated ones, due to withdrawal of water via the transpiration stream. However in roots of both soil- and hydroponically grown plants, in addition to stress causing increased ABA levels, there also occurred decreases in the levels of specific xanthophylls, all-trans- and 9"-cis-neoxanthin and all-trans-violaxanthin. The stoichiometry between decreases in the levels of these xanthophylls and the increases in the levels of ABA and its metabolites, on a molar basis, is very similar for both stressed hydroponically grown roots and detached soil-grown ones (1.2:1). Such ratios, approx. 1:1, have been reported for stressed etiolated Phaseolus leaves (Li and Walton 1990; Parry et al. 1990a), and are consistent with the operation of an apo-carotenoid ABA biosynthetic pathway in roots, and the existence of a specific cleavage enzyme. The fact that such stoichiometry has been observed indicates that rates of alternative, unaccounted for catabolism of ABA (e.g. conjugation), and de-novo xanthophyll synthesis are not significant under these conditions. In stressed etiolated leaves of Phaseolus, increases in the rate of ABA synthesis are correlated with decreases in the levels of 9'-cis-neoxanthin, all-trans-violaxanthin and to a much lesser extent 9-cis-violaxanthin (Li and Walton 1990; Parry et al. 1990a). All-trans-violaxanthin has been shown to be a precursor of 9'-cis-neoxanthin but the intermediate has not been identified (Parry and

"

violaxanfhin

V . HO

-

"

9 Ho~CHO

OH

Xan

9'-cis-ne

o~L~CHO ABA-ald HO

O,,C~'~CO 0H

ABA

Fig. 1. Proposed pathway of stress-induced ABA biosynthesisin roots of Lycopersicon esculentum. The ability of 9"-cis-violaxanthin to serve as a precursor of 9"-cis-neoxanthin and-or Xan is theoretically possible but unproven. ABA-ald = ABA-aldehyde

Horgan 1991a). In Lycopersicon roots a major portion of the reduction in xanthophylls following stress can be attributed to all-trans-neoxanthin, indicating that this xanthophyll is the intermediate between all-trans-violaxanthin and 9"-cis-neoxanthin (Fig. 1). Whether or not 9-cis-violaxanthin can serve as a substrate for the cleavage enzyme will probably not be known until the purification of this enzyme is achieved. In the stressed intact soil-grown plants the amount of ABA, PA and DPA present in the roots is about threefold higher than the observed decrease in xanthophyll levels. It seems likely that this "extra" ABA, PA and DPA is derived from the shoot, as the leaves were severly wilted and recirculation of ABA and its metabolites between shoots and roots is known to occur under such circumstances (Zeevaart and Boyer 1984; Wolf et al. 1990). The three ABA-deficient mutants of Lycopersicon, not, flacca (tic) and sitiens (sit), have been intensively studied, as vehicles to investigate the role of ABA in plant growth and development and also to investigate ABA biosynthesis (Parry 1991). Both tic and sit were shown to be impaired in their ability to convert xanthoxin (Xan) to ABA, specifically at the stage of ABAaldehyde oxidation (Parry et al. 1988; Sindhu and Walton 1988; Taylor et al. 1988) In contrast not converted both Xan and ABA-aldehyde to ABA, in detached leaves and cell-free systems, as efficiently as the wild-type (Parry et al. 1988; Sindhu and Walton 1988; Taylor et al. 1988). This led to suggestions that not could be affected either in the production of Xan, possibly at the stage of xanthophyll cleavage, or perhaps in the perception or transduction of the water-stress signal (Parry et al. 1988). One of the difficulties in attempting to characterise not is its "leakiness". Unlike tic and sit it can respond to

A.D. Parry et al.: Abscisic acid biosynthesis in roots. II

196

stress by synthesising some ABA (80% increase compared with 215% increase in the wild-type; Parry et al. 1988). The carotenoid content of not leaves (light-grown and etiolated) and roots is higher than in the wild-type, but this is not thought to be significant as the relative composition is the same in both genotypes (Parry et al. 1990a; Parry and Horgan 1991a). The conversion of all-trans-violaxanthin to 9"-cis-neoxanthin also takes place normally in greening not seedlings (Parry and Horgan 1991a). The fact that stress-induced xanthophyll cleavage could be observed in wild-type Lycopersicon roots allowed the hypothesis that this process was impaired in not to be investigated. The results from the present study show that the changes in xanthophyll levels observed in wild-type roots, which correlate with increases in ABA levels, also occur in roots of not, but without the concommitant synthesis of ABA. The first conclusion from this must be that the perception/transduction pathways in not are functional, and that the mutation must somehow affect the cleavage reaction. One possible explanation could be that cleavage of 9"-cis-neoxanthin is occurring as normal, but that the Xan generated is either being sequestered from the enzymes that convert it to ABA, or is being competitively metabolised via an alternative pathway. However previous work has shown that Xan does not accumulate in leaves of not and that rates of conversion of Xan to ABA are similar between not and wild-type (Parry et al. 1988; Sindhu and Walton 1988). A geometric isomer of Xan, 2-trans-xanthoxin (tXan), is present in plant tissues at much higher levels than Xan (Parry et al. 1990b). There is no evidence for a "Xan/t-Xan isomerase" and so it appears that the two isomers arise independently, through cleavage of cis and trans xanthophylls (Parry et al. 1988; Sindhu and Walton 1988). Levels of t-Xan in wild-type Lycopersicon leaves approximately double in response to stress (Parry et al. 1988), indicating that increased cleavage of all-transneoxanthin or violaxanthin occurs in addition to the increased cleavage of 9"-cis-neoxanthin. It may be that in not the ABA deficiency is due to an altered specificity of the cleavage enzyme, with more all-trans-than 9"-cisneoxanthin being cleaved. The basal levels of t-Xan in not leaves are approximately double those in the wild-type, but do not increase further in response to stress (Parry et al. 1988). However leaves of not were found to be able to metabolise externally-supplied t-Xan to t-xanthoxin acid (XaA) at much higher rates than either wild-type, fie or sit leaves, and this t-XaA was rapidly conjugated (Parry 1989). Endogenous levels of t-XaA were 6-to 17-fold higher than in the other genotypes, but still only 5-15% of the levels of the "missing" ABA. Any t-Xan formed by the cleavage of all-trans-neoxanthin could be metabolised through t-XaA and its ester, and-or via other routes such as the conversion to 2-trans-ABA-alcohol (Parry 1991; Fig. 2). Some t-Xan could be converted to t-ABA which is rapidy conjugated (Taylor and Burden 1973; Parry et al. 1988), but neither t-ABA nor alkaline hydrolyzable t-ABA conjugates were found to accumulate in leaves of the not mutant (Parry et al. 1988; Parry 1989). In stressed

~

OH 0

HO

all-trans-violaxanlhin

;xy~ Ho%

all-frans-neoxanthin

CaO

HO~r 4~r ]

f-Xan

,

t-XoA

...... ~

~%,,

-.

......

9 conjugate -""

...--"

9'-cis-neoxanfhin H~

~ < ~

O*~OH

9.~ ,,.

CH2OH

t-ABA-alc

9.

HO~'~CHO ~ Xan

o.d c,o I o.~:~'cooH

ABA-o,d

ABA

Fig. 2. A possible explanation for the ABA deficiency of notabilis. Cleavage of both all-trans- and 9'-cis-neoxanthin could result from the presence of an enzyme of lowered substrate specificity. A B A a M = ABA-aldehyde; t - A B A - a l c = t r a n s - A B A - a l c o h o l

light-grown not leaves there was a general decrease of 13-14% in the levels of the major xanthophylls, but a 30% decrease in the levels of all-trans-neoxanthin (Parry et al. 1990a). In contrast, no significant decreases in the levels of all-trans-neoxanthin were observed in stressed leaves of the wild-type, tic or sit. The purification of the cleavage enzyme should enable this theory to be properly tested. Roots of wild-type hydroponically grown plants show reductions in the levels of both all-trans- and 9"-cisneoxanthin and all-trans-violaxanthin following stress. In not the magnitude of the xanthophyll decrease is similar to that in the wild-type, but 80% of the decrease is due to all-trans-neoxanthin compared with only 37% in the wild-type. The conversion of all-trans-violaxanthin to all-trans-neoxanthin does not appear to have been triggered in these not roots, perhaps due to the much higher pre-stress levels of all-trans-neoxanthin. In soil-grown roots of both the wild-type and not stress led to a decrease in xanthophyll levels, mainly those of all-trans-neoxanthin. Unfortunately, from these data, it is not possible to distinguish between decreases resulting from direct cleavage, as might be happening in not, and those caused by isomerisation to 9'-cisneoxanthin prior to cleavage. The reason for the lack of an effect of soil-drying on xanthophyll levels in detached soil-grown not roots is unclear, but may be that the 3-d absence of irrigation did not cause sufficient stress to induce the cleavage enzyme. To conclude, it has been demonstrated that the ABA biosynthetic pathway that operates in roots appears to be the same as that in leaves, involving the conversion of all-trans-violaxanthin, via all-trans-neoxanthin, to 9"-cisneoxanthin, which is then cleaved to generate Xan. The ABA-deficient mutant of Lycopersicon, notabilis, may be

A.D. Parry et al. : Abscisic acid biosynthesis in roots. II

affected at the cleavage step, possessing an enzyme with altered substrate specificity which cleaves more all-tramthan 9"-cis-neoxanthin. The authors would like to thank Drs. A.D. Tomos (Department of Biochemistry, The University College of North Wales, Bangor) and H.G. Jones (AFRC Institute of Horticultural Research, Wellesbourne, Warwick, UK) for useful discussions, and Mr. J.K. Heald for his expert operation of the mass spectrometer. A.D.P. was supported by a grant from the Agricultural and Food Research Council (AFRC), from whom funds were also obtained to purchase the HPLC-photodiode-array detector. A.G. was supported by Science and Engineering Research Council-Cooperative Awards in Science and Engineering award to Drs. H.G. Jones and A.D. Tomos.

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