Overexpression of Arabidopsis thaliana farnesyldiphosphate synthase (FPS1S) in transgenic Arabidopsisinduces a cell death/senescence-like response and reducedcytokinin levels

Angela Masferrer1, Montserrat Arro 1, David Manzano1, Hubert Schaller2, Xavier FernaÂndez-Busquets1,

Paloma MoncaleaÂn4, BeleÂn FernaÂndez4, Nu ria Cunillera1, Albert Boronat3 and Albert Ferrer1,*1Departament de BioquõÂmica i Biologia Molecular, Facultat de FarmaÁcia, Universitat de Barcelona, Avinguda, Diagonal

643, 08028-Barcelona, Spain.2Institut de Biologie MoleÂculaire des Plantes, DeÂpartement d'Enzymologie Cellulaire et MoleÂculaire, UPR 406 du CNRS,

28 rue Goethe, 67083 Strasbourg CeÂdex, France.3Departament de BioquõÂmica i Biologia Molecular, Facultat de QuõÂmica, Universitat de Barcelona, c. MartõÂ i FranqueÁs 1,

08028-Barcelona, Spain.4Departamento de BiologõÂa de Organismos y Sistemas, Facultad de BiologõÂa, Universidad de Oviedo, c/CatedraÂtico

Rodrigo UrõÂa s/n, 33071-Oviedo, Spain.

Received 5 November 2001; revised 21 December 2001; accepted 11 January 2002.*For correspondence (fax +34 93 4021896; e-mail aferrer@farmacia.far.ub.es).

Summary

To investigate the contribution of farnesyl diphosphate synthase (FPS) to the overall control of the

mevalonic acid pathway in plants, we have generated transgenic Arabidopsis thaliana overexpressing

the Arabidopsis FPS1S isoform. Despite high levels of FPS activity in transgenic plants (8- to 12-fold as

compared to wild-type plants), the content of sterols and the levels of 3-hydroxy-3-methylglutaryl-CoA

reductase activity in leaves were similar to those in control plants. Plants overexpressing FPS1S showed

a cell death/senescence-like phenotype and grew less vigorously than wild-type plants. The onset and

the severity of these phenotypes directly correlated with the levels of FPS activity. In leaves of plants

with increased FPS activity, the expression of the senescence activated gene SAG12 was prematurely

induced. Transgenic plants grown in the presence of either mevalonic acid (MVA) or the cytokinin 2-

isopentenyladenine (2-iP) recovered the wild-type phenotype. Quanti®cation of endogenous cytokinins

demonstrated that FPS1S overexpression speci®cally reduces the levels of endogenous zeatin-type

cytokinins in leaves. Altogether these results support the notion that increasing FPS activity without a

concomitant increase of MVA production leads to a reduction of IPP and DMAPP available for cytokinin

biosynthesis. The reduced cytokinin levels would be, at least in part, responsible for the phenotypic

alterations observed in the transgenic plants. The ®nding that wild-type and transgenic plants

accumulated similar increased amounts of sterols when grown in the presence of exogenous MVA

suggests that FPS1S is not limiting for sterol biosynthesis.

Keywords: Arabidopsis thaliana, cytokinin, farnesyl diphosphate synthase, isoprenoid, mevalonic acid,

senescence

Introduction

All plant isoprenoids derive from a common precursor,

isopentenyl diphosphate (IPP), which is synthesized by

two different pathways. The mevalonic acid (MVA) path-

way, which operates in the cytosol/endoplasmic reticulum

(ER) to supply IPP for the synthesis of cytosolic and

mitochondrial isoprenoids (Disch et al., 1998; Newman and

Chappell, 1997), and the 2-C-methyl-D-erythritol 4-phos-

phate (MEP) pathway, which is localized in the plastids

The Plant Journal (2002) 30(2), 123±132

ã 2002 Blackwell Science Ltd 123

(Lichtenthaler, 1999; Rohmer, 1999). In all three compart-

ments, IPP is utilized by prenyltransferases as a substrate

in condensation reactions with different allylic prenyl

diphosphates, to produce a variety of linear prenyl dipho-

sphates of increasing size. These compounds serve in turn

as donors or intermediates in the synthesis of the wide

range of isoprenoid end products (Bach, 1995; McGarvey

and Croteau, 1995).

The evidence available indicates that 3-hydroxy-3-

methylglutaryl coenzyme A reductase (HMGR), plays a

key regulatory role in the overall control of the MVA

pathway (Bach et al., 1999; Schaller et al., 1995; Stermer

et al., 1994). Nevertheless, it is also accepted that other

enzymes are involved in the control of the pathway.

Among these is farnesyl diphosphate synthase (FPS; EC

2.5.1.1/EC 2.5.1.10), the prenyltransferase that catalyses

the two sequential 1¢-4 condensation reactions of IPP with

the allylic diphosphates, dimethylallyl diphosphate

(DMAPP) and the resulting geranyl diphosphate, to pro-

duce farnesyl diphosphate (FPP). The proposal of a regu-

latory role for FPS is mainly based on the fact that FPP is

the starting point of different branches of the pathway

leading to the synthesis of a large variety of essential

isoprenoid end products (Figure 1), so that the concentra-

tion of this prenyl diphosphate should be closely con-

trolled. Previous reports suggesting that FPS plays a

regulatory role in plant sesquiterpenoid phytoalexin syn-

thesis (Chen et al., 2000; Dudley et al., 1986; Hugueney

et al., 1996; Ramos-Valdivia et al., 1997) are in keeping with

this proposal. However, the real contribution of FPS to the

overall control of the mevalonic acid pathway in plants

remains to be demonstrated.

A. thaliana contains two genes, FPS1 and FPS2, encod-

ing isoforms FPS1S, FPS1L and FPS2 (Cunillera et al., 1996,

1997). FPS1S and FPS1L are both encoded by the FPS1

gene and differ only at their N-terminus. FPS1L has an N-

terminal extension of 41 amino acid residues with respect

to FPS1S, which targets the enzyme into mitochondria

(Cunillera et al., 1997). In contrast, FPS1S and FPS2 are

presumably located in the cytosol/ER compartment. From

the analysis of transgenic A. thaliana plants harboring

chimeric FPS:GUS genes, it has been postulated that each

cytosolic FPS isoform might have a specialized role in

directing the ¯ux of pathway intermediates into speci®c

isoprenoid end products. FPS1S might function in a

housekeeping capacity to provide FPP for conversion into

isoprenoids serving general plant cell functions, whereas

Figure 1. Simpli®ed scheme of the mevalonic acid pathway leading tothe synthesis of isoprenoids in the cytosol/endoplasmic reticulum (ER)and mitochondria.The position of the reactions catalysed by farnesyl diphosphate synthase(FPS) and 3-hydroxy-3-methylglutaryl coenzyme a reductase (HMGR) isshown.IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; GPP,geranyl diphosphate; FPP, farnesyl diphosphate.

Figure 2. Overexpression of FPS1S in transgenic A. thaliana plants.(a) Schematic representation (not to scale) of the CaMV35S:FPS1S geneused to generate transgenic A. thaliana plants. A grey box indicates theFPS1S coding sequence. Crosshatched boxes represent 5¢ and 3¢untranslated sequences. Restriction sites relevant for cloning are alsoindicated. 35S-P, cauli¯ower mosaic virus 35S promoter. Nos-T, nopalynesynthase gene terminator region.(b) Northern blot analysis of total RNA (20 mg) isolated from leaves of 4-week-old wild-type (wt) and transgenic (2.4, 4.3, and 9.1) plants. The blotwas hybridized with the 370-bp BglII-HindIII fragment from A. thalianaFPS1 gene (Cunillera et al., 1996). Ethidium bromide staining of the gelbefore transfer is shown below.(c) Western blot analysis of FPS protein in the 200 g supernatant fractionprepared from leaves of wild-type and transgenic plants. Samples (20 mgof protein) were fractionated by SDS-PAGE, transferred onto amembrane and blotted with polyclonal anti-FPS1S antibodies.(d) FPS activity measured in the same fractions used for Western blotanalysis. Enzyme activity is expressed in nanomols of IPP incorporatedinto acid-labile products per minute and mg of protein at 37°C. Valuesare means 6 SE of three experiments.

124 Angela Masferrer et al.

ã Blackwell Science Ltd, The Plant Journal, (2002), 30, 123±132

FPS2 would be involved in the synthesis of isoprenoids

serving more specialized functions (Cunillera et al., 2000).

To investigate the contribution of each FPS isoform to

the overall control of the mevalonic acid pathway in plants,

we have generated and characterized transgenic

A. thaliana plants expressing a chimeric CaMV35S:FPS1S

gene. In spite of the high levels of active FPS1S produced

in these plants, their content of sterols is similar to that in

wild-type plants. In addition, FPS1S overexpression trig-

gers a cell death/senescence-like response in leaves,

concomitant to a reduction of the levels of endogenous

zeatin-type cytokinins. Our results suggest that FPS1S is

not limiting for sterol biosynthesis in A. thaliana.

Results

Generation of transgenic A. thaliana plants

overexpressing isoform FPS1S

To overexpress FPS1S, a chimeric CaMV35S:FPS1S gene

(Figure 2a) was introduced into A. thaliana using

Agrobacterium-mediated transformation. Segregation

analysis of the kanamycin resistance trait through suc-

ceeding generations of 10 randomly chosen kanamycin-

resistant primary transformants (T1), indicated that trans-

genic lines 2.4, 4.3, and 9.1 (T2 generation) contained a

single T-DNA insertion and were homozygous with respect

to the transgene. Northern blot analysis revealed that plant

lines 2.4, 4.3, and 9.1 accumulated much higher levels of

the FPS1S transcript than wild-type plants (Figure 2b).

Consistent with this result, these lines also contained

increased levels of FPS protein (Figure 2c) and activity (8-

to 12-fold) (Figure 2d) as compared to wild-type plants.

Altogether, these results demonstrated that in the selected

transgenic lines the CaMV35S:FPS1S gene was intensely

expressed giving rise to high levels of active FPS1S.

Sterol content and composition in transgenic A. thaliana

plants

Sterols are the major isoprenoid end products synthesized

from FPP in the cytosol/ER compartment. The presence of

increased amounts of active FPS1S in transgenic plants

would be expected to enhance the ¯ux through the sterol

pathway, thus leading to increased levels of sterols.

However, sterol analysis in green rosette leaves from

plant lines 2.4, 4.3, 9.1, and wild-type plants revealed no

signi®cant differences neither in the amount (all plants

contained approximately 1.9 mg of total sterols g±1 DW)

nor in the pro®le of sterols between transgenic and wild-

type plants (results not shown). The levels of the two

major plastidic isoprenoids, namely chlorophylls and

carotenoids, in leaves of plants overexpressing FPS1S

were also similar to those measured in leaves of wild-type

plants.

Overexpression of FPS1S induces a cell death/

senescence-like response in leaves

During the early stages of growth, transgenic plants were

morphologically indistinguishable from wild-type plants.

However, by approximately 4 weeks of culture, the ®rst

produced rosette leaves started to show pale brown

lesions with the appearance of regions in which premature

cell death had occurred. Typically, these lesions were ®rst

observed in zones near the veins and then progressed

towards the leaf margins (Figure 3a,b). As plants con-

tinued developing, all rosette leaves and also cauline

leaves displayed this phenotype. In addition, plants over-

expressing FPS1S grew much less vigorously than their

non-transgenic counterparts. In comparison with wild-type

plants, transgenic plants showed a marked reduction in

the size (by approximately 40%) of the basal rosette

(Figure 3c) and a signi®cant reduction in height as well

as in the number of reproductive shoots, in¯orescences,

and siliques. Interestingly, the onset of premature cell

death and the severity of the phenotypes (Figure 3) directly

correlated with the levels of FPS activity (Figure 2d), thus

con®rming the causal relationship between FPS1S over-

expression and the altered growth of transgenic plants. No

signi®cant differences were observed between wild-type

and transgenic plants in terms of number of leaves, time of

¯owering, and rate of germination of seeds.

Given that leaf senescence is considered a form of

programmed cell death, we investigated whether FPS1S

overexpression affected the onset of leaf senescence. In

many plants detachment of leaves promotes their senes-

cence, and this feature has been frequently used to

characterize this process. Thus, fully expanded green

rosette leaves were excised from wild-type and transgenic

plants and incubated on wetted ®lter papers under the

same conditions used for growing whole plants.

Interestingly, detached leaves of plants overexpressing

FPS1S started to show typical symptoms of natural

senescence, in terms of patterning and pigmentation

(Quirino et al., 2000), much earlier than detached leaves

of wild-type plants (Figure 4). In the case of plant line 9.1,

leaves displayed evident symptoms of senescence 4 days

after detachment and turned almost completely yellow

after 6 days of detachment. On the contrary, leaves from

control plants were still green 6 days after detachment.

Detached leaves of plants from lines 2.4 and 4.3 started to

senesce slightly later than detached leaves of plants from

line 9.1 (results not shown). It is also remarkable that the

visible effects of FPS1S overexpression on attached or

detached leaves were clearly different. Thus, whereas

leaves on the transgenic plants showed symptoms of

Overexpression of FPP synthase induces cell death/senescence 125

ã Blackwell Science Ltd, The Plant Journal, (2002), 30, 123±132

Figure 3. Premature cell death/senescence-like phenotype and altered growth ofA. thaliana plants overexpressing FPS1S.Basal rosette (a)and close-up view ofdetached rosette leaves (b)of 5-week-oldwild-type (wt) and transgenic (2.4, 4.3, and9.1) plants. Basal rosette (c)of wild-type (wt)and transgenic (2.4, 4.3, and 9.1) plantsgrown for 6 weeks on soil.

Figure 4.

Figure 7.

126 Angela Masferrer et al.

ã Blackwell Science Ltd, The Plant Journal, (2002), 30, 123±132

premature cell death (Figures 3a,b), the appearance of

detached leaves closely resembled that of naturally±

senescing leaves (Figure 4). Consequently, the phenotypic

alterations observed in leaves of plants overexpressing

FPS1S will be further referred to as a cell death/senes-

cence-like phenotype.

Overexpression of FPS1S activates the expression of the

SAG12 gene.

The A. thaliana SAG12 gene is expressed late in natural

senescence and it has been postulated that the encoded

cysteine protease may have a senescence speci®c cell

death function (Lohman et al., 1994; Weaver et al., 1998).

To investigate whether the expression of SAG12 was

induced in plants overexpressing FPS1S, total RNA was

extracted from rosette leaves of two different ages har-

vested from 5-week-old plants from lines 2.4, 4.3 and 9.1.

At this stage of development the ®fth and sixth rosette

leaves were completely green, whereas the third and

fourth rosette leaves showed evident symptoms of cell

death/senescence. Total RNA was also extracted from the

third and fourth rosette leaves of wild-type plants grown

under the same conditions, as well as from naturally

senescing leaves of older wild-type plants. As shown in

Figure 5, high levels of SAG12 mRNA were detected both

in leaves of transgenic plants showing cell death/senes-

cence-like symptoms and in naturally senescing leaves,

whereas as expected, the SAG12 transcript was not

detected in green leaves of wild-type plants. In addition,

the levels of the SAG12 transcript in leaves of transgenic

plants directly correlated with the levels of FPS activity. It is

also remarkable that SAG12 mRNA was detected in leaves

of transgenic plants even before they showed any symp-

tom of cell death/senescence (Figure 5). This is in contrast

to the pattern of expression of SAG12 in naturally

senescing leaves, in which the expression of this gene is

not detectable until leaves show evident symptoms of

senescence (Lohman et al., 1994; Noh and Amasino, 1999).

Mevalonic acid reverse the cell death/senescence-like

phenotype

The phenotypic alterations displayed by plants overex-

pressing FPS1S raised the question of how increased

levels of FPS1S could induce the cell death/senescence-like

Figure 5. Expression of the SAG12 mRNA associated to cell death/senescence induced by FPS1S overexpression.Total RNA was isolated from leaves showing the cell death/senescence-like phenotype (third and fourth rosette leaves) and completely greenleaves (®fth and sixth rosette leaves) of 5-week-old plants from lines 2.4,4.3, 9.1. Total RNA was also extracted from completely green (wt) andnaturally senescing (wt-s) rosette leaves of wild-type plants. RNAsamples (10 mg) were subjected to Northern blot analysis using the1.1 kb EcoRI-EcoRI fragment from the A. thaliana SAG12 cDNA (Lohmanet al., 1994) as a probe. Ethidium bromide staining of the gel beforetransfer is shown below.

Figure 6. HMGR activity in A. thaliana plants overexpressing FPS1S.HMGR activity was measured in the 200 g supernatant fraction preparedfrom leaves of wild-type (wt) and transgenic (2.4, 4.3, and 9.1) plants.HMGR activity in transgenic lines is expressed relative to enzyme activityin control plants (4.95 6 0.6 units mg±1 of protein) which was consideredas 100%. Values are means 6 SE of three separate experiments.

Figure 7. Reversion of the premature cell death/senescence-like phenotype by exogenously added mevalonic acid (MVA) and 2-isopentenyladenine (iP).Wild-type and transgenic (line 9.1) plants were grown axenically in Magenta vessels containing germination medium (GS) or germination mediumsupplemented with either 5 mM MVA or 0.1 mM iP, under a 16-h light/8-h dark illumination regime for 5 weeks.

Figure 4. Rapid senescence of leaves detached of A. thaliana plants overexpressing FPS1S. Leaves detached of wild-type (wt) and transgenic (line 9.1)plants were placed on wetted ®lter papers and incubated under a 16-h light/8-h dark illumination regime for 2, 4, and 6 days.

Overexpression of FPP synthase induces cell death/senescence 127

ã Blackwell Science Ltd, The Plant Journal, (2002), 30, 123±132

phenotype. Given that the reaction catalysed by HMGR is

considered a key rate-limiting step for MVA biosynthesis,

and subsequently for IPP and DMAPP production, we

investigated the effect of FPS1S overexpression on the

levels of HMGR activity. As shown in Figure 6, HMGR

activity in leaves of transgenic plants was similar to that

found in leaves of wild-type plants, thus indicating that a

drastic increase in FPS activity does not substantially

modify the rate of MVA production. Thus, a plausible

hypothesis was that increasing FPS1S activity without a

concomitant increase of the metabolic ¯ux through pre-

ceding steps in the pathway, could alter the partitioning of

IPP and DMAPP among the different branches of the

pathway arising from these C5 prenyl diphosphates

(Figure 1). This in turn might lead to a reduction of the

synthesis of isoprenoids other than sterols that would then

cause the observed phenotypes. According to this hypoth-

esis, feeding transgenic plants with MVA would be

expected to restore the wild-type phenotype. As shown

in Figure 7, plants from line 9.1 grown in the presence of

5 mM MVA were indistinguishable from control plants

grown under the same conditions, thus indicating that the

addition of MVA fully reversed the cell death/senescence-

like phenotype induced by FPS1S overexpression.

Interestingly, sterol analysis in these plants revealed that

leaves of both transgenic and wild-type plants accumu-

lated similar levels of sterols which were, in addition, 6- to

7-fold higher than those found in leaves of plants grown in

the absence of MVA.

Overexpression of FPS1S causes a reduction in

endogenous cytokinin levels

Cytokinins are a class of hormones synthesized from

cytosolic IPP and DMAPP (Figure 1) that are known to be

strong antagonists of senescence (Gan and Amasino,

1995; NoodeÂn, 1988; Ori et al., 1999). As a ®rst approach

to investigate the effects of FPS1S overexpression on

cytokinin biosynthesis, we examined the effect of exogen-

ously supplied 2-isopentenyladenine (iP) on plant line 9.1.

As shown in Figure 7, iP completely reversed the pheno-

typic alterations of transgenic plants. This, together with

the results obtained in the MVA feeding experiments,

strongly supported the notion that increased levels of

FPS1S activity cause a reduction of the cellular IPP and

DMAPP available for cytokinin biosynthesis, thus leading

to reduced levels of these hormones. To con®rm this

hypothesis, the contents of endogenous cytokinins were

measured in fully green leaves from plant line 9.1 and

wild-type plants. As shown in Table 1, zeatin (Z) levels

were drastically reduced (about 80%) in leaves of plants

from line 9.1 relative to wild-type plants; dihydrozeatin

(DHZ) levels were also reduced, although to a lesser extent

(about 35%). No signi®cant changes were detected in the

levels of zeatin riboside (ZR), dihydrozeatin riboside

(DHZR), iP and 2-isopentenyladenosine (iPR). These results

conclusively demonstrated that FPS1S overexpression

causes a speci®c reduction of the levels of zeatin-type

cytokinins.

Discussion

A. thaliana plants containing increased levels of FPS1S

activity displayed a premature cell death/senescence-like

phenotype, which directly correlated with the levels of FPS

activity. In leaves of transgenic plants the contents of the

three major isoprenoid end products (sterols, carotenoids

and chlorophylls) were similar to those found in leaves of

wild-type plants. The observation that carotenoid and

chlorophyll contents remained unchanged is consistent

with the fact that these compounds derive from geranyl-

geranyl diphosphate synthesized in the plastids through

the MEP pathway (Licktentnaler, 1999; Rohmer, 1999). On

the contrary, the ®nding that a drastic raise in FPS1S

activity did not result in a detectable increase of total

sterols was unexpected, since they are the major iso-

prenoids synthesized from cytosolic FPP. These results,

together with the analysis of the sterol pro®les, ruled out

the possibility that the phenotypes caused by FPS1S

overexpression were due to changes in the levels of any

of these isoprenoids or to a novel balance of speci®c

sterols. All these results contrast with those previously

obtained when the Saccharomyces cerevisiae FPS gene

was overexpressed in tobacco. A 12-fold increase of FPS

activity relative to wild-type plants, was correlated with a

4-fold increase of sterol and carotenoid biosynthesis, and

did not cause any signi®cant phenotypic alteration

(Daudonnet et al., 1997). The reason for these con¯icting

Table 1. Endogenous cytokinin levels (pmol g±1 DW) in leavesa of wild type and line 9.1 plants. Cytokinin levels were measured bytriplicate in three independently harvested samples of plant material. Each value represents the mean 6 SE

Plant Z DHZ ZR DHZR iP iPR

Wt 174.09 6 18.17 106.19 616.56 156.16 620.02 114.40 6 10.43 28.96 6 5.72 40.48 6 6.499.1 34.80 6 7.23 71.40 6 15.03 176.13 6 10.75 112.50 6 13.47 27.78 6 1.78 43.76 6 8.29

aLeaves showing no symptoms of cell death/senescence were harvested from plants grown on soil for 4 weeks. Z, zeatin; DHZ,dihydrozeatin; ZR zeatin riboside; DHZR, dihydrozeatin riboside; iP, N6-isopentenyladenine; iPR, N6-isopentenyladenosine.

128 Angela Masferrer et al.

ã Blackwell Science Ltd, The Plant Journal, (2002), 30, 123±132

results is not obvious, although it might arise from the fact

that the enzyme overproduced in tobacco was a hetero-

logous FPS, while in the case of A. thaliana the enzyme

overexpressed was an authentic Arabidopsis FPS isoform.

It is also conceivable that these discrepancies might re¯ect

subtle differences in the organization and/or the regulatory

mechanisms between the MVA pathway in A. thaliana and

tobacco.

Overexpression of FPS1S in A. thaliana triggers a

phenotype of rapid cell death in intact leaves, whereas it

induces premature senescence rather than rapid cell death

in detached leaves. During natural senescence, which is

considered the last stage of leaf development and one type

of programmed cell death, the expression of certain genes

potentially involved in the catabolism and mobilization of

nutrients is activated. This is the case of the A. thaliana

SAG12 gene, which encodes a cysteine protease that may

have a senescence speci®c cell death function (Lohman

et al., 1994; Weaver et al., 1998). Interestingly, in plants

overexpressing FPS1S the expression of SAG12 was

activated, not only in leaves showing cell death/senes-

cence-like symptoms, but also in completely green leaves.

This is in sharp contrast with the normal pattern of

expression of SAG12, which is induced late in natural

senescence (Weaver et al., 1998) but not when other cell

death pathways are activated (Quirino et al., 2000). It has

been postulated that this is due to the deleterious effect

that the encoded protease may have to the cells, so that its

expression needs to be restricted to late senescence

(Weaver et al., 1998). Altogether these observations sug-

gest that the premature expression of SAG12, and possibly

of other SAG-encoded proteins potentially involved in the

breakdown of cellular constituents, might cause the

phenotype of premature cell death observed in intact

leaves. The reason for the different appearance of

detached leaves is currently unknown, although it seems

clear that different molecular events are occurring under

these conditions. This is consistent with previous obser-

vations showing that different molecular programmes of

senescence are activated depending on the senescence-

inducing factors, including the detachment of leaves

(Becker and Apfel, 1993; Park et al., 1998; Weaver et al.,

1998).

The activation of SAG12 has been correlated with the

decline of endogenous cytokinins at the onset of natural

senescence (Gan and Amasino, 1995). There is also

compelling evidence supporting the view that cytokinin

biosynthesis is preferentially inhibited when the levels of

MVA, and subsequently of IPP and DMAPP, are decreased

(Astot et al., 2000; Cowan et al., 1997; Crowel and Salaz,

1992; Laureys et al., 1998). Furthermore, reduced levels of

these intermediates have been correlated with premature

cell death and senescence. The phenotypically small fruits

produced by the `Hass' avocado cultivar have a drastic

reduction in HMGR activity and display early seed coat

senescence and/or death (Cowan et al., 1997). In this

context, we hypothesized that increased levels of FPS1S

activity might cause a signi®cant decrease of the cellular

contents of IPP and DMAPP available for cytokinin

biosynthesis. This, in turn, would lead to a premature

drop of cytokinin content below a certain threshold at

which the expression of SAG12 is activated. The ®nding

that transgenic plants grown in the presence of MVA or the

cytokinin iP recovered a wild-type phenotype was consist-

ent with this hypothesis. The analysis of endogenous

cytokinin levels in transgenic plants con®rmed that FPS1S

overexpression caused a speci®c reduction in the content

of the free bases (active forms) of Z-type cytokinins. An

80% decrease of Z levels and a 35% decrease of DHZ levels

was detected in transgenic plants relative to control plants,

whereas the levels of the corresponding ribosides (inactive

forms) and iP-type cytokinins remained virtually

unchanged. These results unequivocally demonstrated

the detrimental effect of FPS1S overexpression on cytoki-

nin biosynthesis. It is also worth mentioning that the

speci®c effect on Z-type cytokinins is in agreement with

previous reports showing that HMGR inhibition speci®c-

ally reduces the levels of Z-type cytokinins (Astot et al.,

2000; Laureys et al., 1998).

The results of this study indicate that FPS1S over-

expression mimics the effects of a reduction in HMGR

activity. However, the ®nding that HMGR activity in

transgenic plants was similar to that found in wild-type

plants ruled out the possibility that increased levels of

FPS1S activity might reduce indirectly the levels of MVA,

IPP and DMPP by downregulation of HMGR activity

through an unknown regulatory mechanism. Therefore,

the reduction of substrate available for cytokinin biosynth-

esis is most likely due to the metabolic imbalance caused

by the competition for a limited pool of IPP and DMAPP

between overexpressed FPS1S and endogenous dimethy-

lallyl PPi:AMP-dimethylallyl transferase (IPT), the ®rst

committed enzyme of the cytokinin pathway. Neverthe-

less, our results do not exclude the possibility that FPS1S

overexpression might also detrimentally affect the ¯ux of

other pathways starting from cytosolic IPP/DMAPP, as for

instance the synthesis of the prenyl-side chain of ubiqui-

none (Disch et al., 1998). Ubiquinone is known to play a key

role in preventing cell death by providing mitochondrial

antioxidant protection (Alleva et al., 2001), so that reduced

levels of this compound would be expected to increase the

risk of necrosis by oxidative stress. If this were the case,

the phenotypes of plants overexpressing FPS1S might

result not only from the reduced endogenous cytokinin

levels but also from a reduced rate of mitochondrial

isoprenoid biosynthesis. It is also tempting to speculate

that the relative contribution of each of these two pro-

cesses to the observed phenotypes might vary depending

Overexpression of FPP synthase induces cell death/senescence 129

ã Blackwell Science Ltd, The Plant Journal, (2002), 30, 123±132

on whether leaves are attached or detached, thus explain-

ing the different phenotypes displayed under these two

conditions.

The results of this study strongly support the notion that

plants accumulate high levels of sterols only when the

limitation imposed by the HMGR bottleneck is overcome.

This may be accomplished either by increasing the levels

of HMGR activity (Chappell et al., 1995; Gondet et al., 1992;

Schaller et al., 1995) or by supplying exogenous MVA, as

shown in cell suspension cultures (Wilkinson et al., 1994)

and in whole plants (this study). Moreover, the ®nding that

A. thaliana plants grown in the presence of exogenous

MVA accumulated similar high amounts of sterols irre-

spective of the levels of FPS activity, suggests that

endogenous levels of FPS1S are not limiting for sterol

biosynthesis. In this context, an increase of FPS1S activity

might have a negligible effect on the amount of total

sterols despite the fact that it might reduce the concentra-

tions of IPP and DMAPP below a certain threshold at which

cytokinin biosynthesis is reduced. In fact, IPT has been

suggested to have a low af®nity for DMAPP (Crowell and

Salaz, 1992), so that it would be highly sensitive even to a

slight decrease in the concentration of this substrate.

These effects would be ampli®ed by the fact that the

synthesis of a single sterol molecule requires the conden-

sation of 6 units of C5 prenyl diphosphates, whereas only

one C5 unit is required to produce a single cytokinin

molecule. Finally, our results demonstrate that strategies

for bioengineering the MVA pathway must take into

account that speci®c intermediates of this pathway serve

as precursors in the synthesis of isoprenoid end products

essential for normal plant growth and development.

Experimental procedures

Construction of plasmid pBIFPS1S and generation of

transgenic plants

The A. thaliana FPS1S cDNA in plasmid pDD7 (Delourme et al.,1994) was cloned in the reverse orientation into the same vectoryielding plasmid pDD7R. To incorporate the polyadenylationsignals of FPS1 gene into the CaMV35S:FPS1S gene, the BglII-SacI fragment including the last 148 bp of the FPS1S cDNA wasexcised from plasmid pDD7R and replaced by a genomicfragment containing the sequence of the excised cDNA fragmentand 231 additional bp of the 3¢ region of the FPS1 gene. Thisfragment was ampli®ed by PCR using a forward primer (5¢-GCAGTGCTAAAATCCTTCTTGGC-3¢) extending from positions3480±3502 in the FPS1 gene, a reverse primer (5¢-CCGAGCTC-AAGCTTTATTTTCTTGCCTTTGG-3¢) complementary to thesequence 3861±3883 in the FPS1 gene, and DNA from clonepgNC242 (Cunillera et al., 1996) as a template. To facilitate thecloning, a SacI restriction site (underlined) was added to the 5¢end of the reverse primer. The ampli®ed fragment was digestedwith SacI and BglII (internal to the fragment; positions 3505±3510in the FPS1 gene) and cloned into pDD7R. The PCR fragment wassequenced to exclude artifacts. The chimeric cDNA was excised as

a XbaI-SacI fragment and cloned into the corresponding sites ofthe binary vector pBI121 (Clontech, San Diego, CA, USA), yieldingpBIFPS1S.

A. thaliana plants (ecotype Columbia) were transformed usingthe in planta vacuum in®ltration method (Bechtold et al., 1993)and Agrobacterium tumefaciens, strain C58C1 (pGV2260), har-boring pBIFPS1S. Seeds from in®ltrated plants were surface-sterilized, sown in Petri dishes containing solid (0,8% w/v agar)germination medium (GM; Murashige and Skoog medium sup-plemented with 0,5 g l±1 MES pH 5.7, 10 g l±1 sucrose, and 50 mgml±1 kanamycin), and incubated at 22 6 2°C for 10±12 days undera 16-h light/8-h dark illumination regime with 130 mmol m±2 sec±1

`daylight' ¯uorescent illumination. Kanamycin-resistant seedlings(T1) were transplanted into soil and grown to maturity under thesame conditions.

RNA extraction and gel blot analysis

Total RNA was isolated according to Dean et al. (1985). RNAsamples were subjected to denaturing 1% formaldehyde-agarosegel electrophoresis and transferred onto a positively chargednylon membrane (Roche, Mannheim, Germany) by capillaryblotting. Membranes were pre-hybridized in DIGEasyHyb hybri-dization solution (Roche, Mannheim, Germany) for 1 h at 68°C.Hybridization was performed overnight at 68°C using the samesolution supplemented with the corresponding digoxigenin (DIG)-labelled RNA probes, which were synthesized using the DIG RNAlabelling kit (Roche, Mannheim, Germany). The blots werewashed twice at room temperature in 2 3 SSC, 0.1% SDS andtwice at 68°C in 0.1 3 SSC, 0.1% SDS. Detection of the probe wasperformed according to the manufacturer's recommendations.

Production of antibodies against FPS1S and Western blot

analysis

A. thaliana FPS1S expressed in E. coli. was solubilized frominclusion bodies and puri®ed according to Pujol et al. (1998).Antibodies were raised in New Zealand white rabbits as previ-ously described (Pujol et al., 1998). Blood was collected 14 daysafter the last injection and serum was recovered by centrifugationat 5000 g for 10 min. The antibodies were concentrated byprecipitation with a 50% of saturation of ammonium sulfate,and dialysed against phosphate-buffered saline (pH 7.5).

For Western blot analysis, about 250 mg of leaves was frozen inliquid nitrogen, ground to a ®ne powder, and mixed with 2 ml ofpre-chilled extraction buffer containing 50 mM Tris±HCl, pH 7.5,5 mM DTT, 6% (w/v) polivinylpolypirrolidone (PVP-40), 40 mM

ascorbic acid, 10 mg/ml aprotinin, 1 mg/ml E64, 0.5 mg/ml leupep-tin, 1 mg/ml pepstatin, and 0.1 mM PMSF at 4°C. The slurry wascentrifuged at 200 g for 10 min at 4°C to remove cell debris. Theresulting supernatant was collected and its protein concentrationwas determined by the method of Bradford (1976) using bovineserum albumin as a standard. The supernatant was used directlyor stored frozen at ±80°C. Aliquots of protein extracts werefractionated by 12.5% SDS-PAGE (Laemmli, 1970) and electro-transferred onto a Hybond-P polyvinylidene di¯uoride membrane(Amersham, Buckinghamshire, UK), which was blocked in100 mM Tris±ClH, pH 7.4, 100 mM MgCl2, 0.5% (v/v) Tween 20,1% (v/v) Triton X-100, 1% (w/v) bovine serum albumin, and 5% (v/v) fetal calf serum, for 1 h at 4°C. The membrane was incubatedwith anti-FPS1S antibodies (1 : 2000 dilution in blocking solution)for 16 h at 4°C. The FPS1S-antibody complex was visualizedusing the ECL PlusTM.. Western blotting system (Amersham,

130 Angela Masferrer et al.

ã Blackwell Science Ltd, The Plant Journal, (2002), 30, 123±132

Buckinghamshire, UK) according to the manufacturer's instruc-tions. The secondary antibody was goat antirabbit IgG peroxidaseconjugate diluted 1 : 2000 with blocking solution.

FPS and HMGR enzyme activity assays

FPS activity was measured essentially as described by Chambonet al. (1990) in freshly prepared protein extracts obtained asdescribed above. Brie¯y, 30±60 mg of protein in 50 ml of extractionbuffer were mixed with 50 ml of assay solution containing 100 mM

sodium phosphate, pH 7.5, 120 mM [4-C14] IPP (3.75 mCi mmol±1),400 mM GPP, 2 mM MgCl2, 10 mg ml±1 aprotinin, 1 mg ml±1 E64,0.5 mg ml±1 leupeptin, 1 mg ml±1 pepstatin, and 0.1 mM PMSF.After incubation for 15 min at 37°C, the reaction was terminatedby the addition of 500 ml of distilled water pre-chilled at 0°C. Themixture was ice-chilled and acidi®ed by the addition of 200 ml of 1M HCl. Solid NaCl was added to saturation and the reactionproducts were acid hydrolysed for 10 min at 37°C. The mixturewas extracted with 1 ml of n-hexane and the radioactivity wasquanti®ed in an aliquot of the hexanic phase by scintillationcounting. FPS speci®c activity is reported in nanomols of IPPincorporated into acid-labile products per minute and mg ofprotein at 37°C.

HMGR activity was assayed as described in Dale et al. (1995), infreshly prepared protein extracts obtained as described forWestern blot analysis, but using as extraction buffer a solutioncontaining 100 mM sucrose, 40 mM sodium phosphate, pH 7.5,30 mM EDTA, 50 mM sodium chloride, 10 mM DTT, 10 mg ml±1

aprotinin, 1 mg ml±1 E64, 0.5 mg ml±1 leupeptin, 1 mg ml±1

pepstatin, and 0.5 mM PMSF, and 0,25% (w/v) Triton X-100. Oneunit of HMGR activity is de®ned as the amount of enzyme thatconverts one picomol of 3-hydroxy-3-methylglutaryl coenzyme Ainto MVA per min and mg of protein at 37°C.

Measurements of sterols, chlorophylls and carotenoids

Quanti®cation of total sterol content and determination of sterolpro®les were performed as previously reported by Schaller et al.(1998). Chlorophyll and carotenoid contents were determined byacetone extraction as described by Chory (1992).

Measurements of cytokinins

Leaves were frozen in liquid nitrogen, ground to a ®ne powder,and lyophilised. Cytokinins were extracted with 80% (v/v)methanol containing 10 mg l±1 of butylated hydroxytoluene,isolated on Sep±Pak C18 cartridges (Waters Associates, Milford,MA, USA), and puri®ed by immunoaf®nity chromatography(FernaÂndez et al., 1995). Cytokinins were fractionated by reverse-phase HPLC and quanti®ed by enzyme immunoassay (ELISA)using three different polyclonal rabbit antibodies (FernaÂndezet al., 1995): anti-ZR antibodies to measure Z and ZR, anti-DHZRantibodies to measure DHZ and DHZR, and anti-iPR antibodies tomeasure iP and iPR. Standard curves were prepared for eachcytokinin and its ribosilated form. The alkaline phosphatase-citokinin conjugates and the ELISA assays were carried out aspreviously described by Centeno et al. (1996). Triplicate measure-ments of each HPLC-puri®ed cytokinin were performed, andcytokinins were quanti®ed in three independent replicate sam-ples.

Acknowledgements

This work was supported by Grants PB96-0176 and BIO2000-0334from the Direccio n General de Investigacio n del Ministerio deCiencia y TecnologõÂa, and 1999SGR-00032 from the ComissioÂInterdepartamental de Recerca i Innovacio TecnoloÁ gica de laGeneralitat de Catalunya. A. M. was the recipient of a predoctoralfellowship from the Comissio Interdepartamental de Recerca iInnovacio TecnoloÁ gica de la Generalitat de Catalunya and D. M. isthe recipient of a predoctoral fellowship from the SpanishMinisterio de Ciencia y TecnologõÂa. X. F. is supported by apostdoctoral contract from the Spanish Ministerio de Ciencia yTecnologia. We are indebted to Richard G. Amasino (University ofWisconsin) for providing us the A. thaliana SAG12 cDNA clone.

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