The transcriptional activator - Springer Link

M o l G e n G e n e t (1996) 2 5 0 : 6 4 % 6 5 4

© Springer-Verlag 1996

Massimo Maddaloni • Giovanna Donini C a r l o t t a B a l c o n i • Eduardo R i z z i • Philippe G a l l u s c i F a b i o F o r l a n i • Stephan L o h m e r • Richard T h o m p s o n Francesco Salamini • Mario Motto

The transcriptional activator Opaque.2controls the expression of a cytosolic form of pyruvate orthophosphate dikinase-1 in maize endosperms Received: 30 August 1995/Accepted: 26 October 1995

The maize Opaque-2 (02) protein is a tran- Key words Endosperm • Opaque-2 locus • scription factor of the basic/leucine-zipper class, General control • Amino acid biosynthesis • Carbon involved in the regulation of endosperm proteins partitioning including the 22 kDa e-zein storage proteins and b32 protein. In this study we have focussed our attention on the relationship between 02 and the cyPPDK1 Introduction gene, which encodes a cytoplasmic pyruvate orthophosphate dikinase (PPDK) isoform. The results of this Zeins, the major storage proteins in maize endosperm, study showed that P P D K activity is detectable in provide a convenient model for the study of mechanisms wild-type maize endosperms, while in 02 mutant endo- that coordinate gene expression in higher plants. Zeins sperms, the levels of P P D K protein, mRNA, and represent a group of alcohol-soluble proteins, which are enzymatic activity are reduced, indicating that 02 is encoded by distinct classes of structural genes belonging involved in the regulation of cyPPDK1 in this tissue. By to a multigene family (Heidecker and Messing 1986). employing transient expression experiments in tobacco Their expression is coordinately regulated and several mesophyll protoplasts, we have demonstrated that the independent loci positively control the rate of zein de02 protein can activate expression of a chloram- position during kernel development (reviewed by Motto phenicol acetyl transferase reporter gene placed under et al. 1989); the best characterized of these loci is Opaquethe control of the cyPPDK1 promoter. An in vitro 2 (02). Maize homozygous for the o2 mutation, in addibinding assay and DNaseI footprint analysis demon- tion to showing modification of endosperm appearance strated that a specific sequence in the cyPPDK1 pro- and increased lysine content (Mertz et al. 1964), has moter can be recognized and protected by maize 02 reduced levels of 22 kDa e-zeins (Jones et al. 1977). The protein. The regulation by the 02 locus of cyPPDK1 02 mutants also lack a number of non-zein polypeptides reported here, and control of e-zein synthesis by 02 present in the wild-type endosperm, including b32 and suggest that the 02 protein may play a more general b70 (Soave et al. 1981; Marocco et al. 1991). role in maize endosperm development than previously The gene corresponding to the 02 locus was cloned thought. by transposon tagging (Schmidt et al. 1987; Motto et al. 1988). It encodes a protein belonging to the basic domain/leucine zipper (bZIP) class of eukaryotic transcriptional activators (Hartings et al. 1989; Schmidt et al. 1990). The 02 protein binds to the b32 and 22 kDa c~-zeingene promoters and regulates their tranCommunicated by H. Saedler scription (Lohmer et al. 1991; Schmidt et al. 1992). M. Maddaloni • G. Donini • C. Balconi Furthermore, studies in yeast have shown that the 02 E. Rizzi • F. Forlani • M. Motto ( [ ] ) protein can substitute for GCN4 protein (Mauri et al. Istituto Sperimentale per la Cerealicoltura, Via Stezzano 24, 1993), a transcriptional activator of amino acid biosyn24126 Bergamo, Italy thetic genes (Hinnebusch 1990). Similarly to GCN4, the S. Lohmer - R. Thompson - F. Salamini presence of three short upstream open reading frames Max-Planck-Institut ffir Ziichtungsforschung, D-50829 K61n, Germany (uORFs) in the leader of the 02 mRNA mediates a mechanism of control at the translational level P. Gallusci (Lohmer et al. 1993). LPCV Universit6e de Bordeaux I, Talence, France Abstract

648 Storage protein synthesis in the developing maize kernel begins a r o u n d 10 days after pollination and creates a large d e m a n d for a m i n o acids ( M o t t o et al. 1989). M o s t of the nitrogen arriving via the translocation stream is in the f o r m of aspartic acid, a n d the amides asparagine a n d glutamine (Arruda a n d D a Silva 1979; Sodek a n d D a Silva 1977). In order to meet the d e m a n d s of storage protein synthesis for the various a m i n o acids, a p o r t i o n of the nitrogen in these t r a n s p o r t c o m p o u n d s m u s t be i n c o r p o r a t e d into other a m i n o acids within the developing kernels (Misra and O a k s 1985; Pernollet et al. 1986). Previous observations in different plant species have suggested that the enzyme p y r u v a t e o r t h o p h o s p h a t e dikinase ( P P D K ; E C 2.7.9.1), k n o w n primarily for the key role it plays in C4 photosynthesis (Hatch and Slack 1968), might also be involved in a m i n o acid interconversion during seed development, b y providing p h o s p h o e n o l p y r u v a t e (PEP) to be c o n s u m e d for a m i n o acid synthesis (Davies 1979; M e y e r et al. 1982; Aoyagi et al. 1984; Aoyagi and B a s s h a m 1984a, b). In maize P P D K transcripts h a v e been identified. T h e y are differentially expressed in plant tissues ( H u d s p e t h et al. 1986; G l a c k i n and G r u l a 1990; Sheen 1991). O n e encodes the C4 chloroplast P P D K (C4PPDK), and two others (cyPPDK1, cyPPDK2) encode cytosolic P P D K activities. It has been found that the C 4 P P D K transcript overlaps the c y P P D K 1 transcript and b o t h are derived from the same gene using different transcription startpoints (Sheen 1990). In the present study we report t h a t the 0 2 gene p r o d u c t regulates P P D K a c c u m u l a t i o n in the endos p e r m and is c a p a b l e of transactivating the cyPPDK1 gene b y direct interaction with the cyPPDK1 p r o m o t e r region. A role in a m i n o acid interconversion a n d / o r c a r b o n partitioning for this regulatory relationship is discussed.

Materials and methods Plant materials and sampling Plants of the inbred lines W22 + and A69Y + , carrying wild-type alleles or their mutant counterparts were grown in the field in Bergamo, Italy, in 1992. The mutant alleles o2R, o2m(r) and o7 were described elsewhere (Di Fonzo et al. 1979; Hartings et al. 1995). Plants were hand-pollinated and ears harvested at different stages of development. The endosperms from the cultured kernels were sampled as described in Balconi et aI. (1993).

PPDK activity assay PPDK activity was assayed as previously described (Aoyagi et al. 1984) with minor modifications. The supernatant was incubated at 25°C for 30 rain to activate the enzyme, before the equilibration with (NH~)2SO4, as indicated in Ashton et al. (1990). One unit of activity was defined as the amount of enzyme required for the conversion of 1 nmol/min substrate into product at 30° C. Protein

concentrations in the enzyme extracts were determined by the method of Lowry et al. (1951).

In vitro endosperm culture Basic experimental procedures and growth conditions for endosperm culture were described previously (Balconi et al. 1993).

Transient expression in protoplasts Tobacco protoplasts for transient gene expression assays and stable transformation experiments were isolated from young expanded leaves of axenic cuttings of Nicotiana tabacum Petit Havana SR1 and transformed as described in Negrutiu et al. (1987), with minor modification. Protoplasts were resuspended in K3 medium, incubated in the dark for 17 h at 26° C, and analyzed for chloramphenicol acetyltransferase (CAT) activity as described in Seed and Sheen (1988).

RNA isolation and Northern analysis RNA was isolated from endosperms grown in vitro, using the procedure of Kloppstech and Schweiger (1976). Poly(A)+ RNA was selected by passage over oligo(dT)-cellulose. Northern blots of poly(A)+ RNA were prepared according to Sambrook et al. (1989). Hybridization and washing conditions were as described (Motto et al. 1988). RNA hybridization signals were quantified by densitometric scanning of autoradiograms to determine the relative intensity of each band.

Nucleic acid manipulations Recombinant DNA techniques were carried out following standard procedures as described by Sambrook et al. (1989), with enz~anes supplied by New England Biolabs and Bethesda Research Laboratory. Escherichia coli HB101 was used as host strain for all plasmids except cyPPDK1CAT, which was cloned into JM110 (dam- dcrn-; a gift of Dr. J. Messing, State University of New Jersey, Piscataway, N J) to allow the use of BclI. The PPDK probe used was a HindIII-KpnI subclone of the pH2 23 clone described in Glackin and Grula (1990). A 2.2 kb PstI cDNA fragment, isolated from a maize seedling library, was used as well with identical results. The plasmids cyPPDK1CAT and C4PPDKCAT were previously described by Sheen (1990).

DNA-protein interactions Gel shift assays were performed basically as described in Mauri et al. (1993) with minor differences.To obtain the fragment used in the gel shift assay, the plasmid cyPPDK1CAT described in Sheen (1991) was cloned into E. coil JMll0. The 363 bp BclI-SmaI fragment was then subcloned into pGEM7 cut with BamHI and Sinai, and rescued as an XbaI-SstI fragment. Probes were labelled by filling in recessed termini with labelled dNTPs using the Klenow fragment of DNA polymerase I. Conditions for footprinting were as in Lohmer et al. (1991). DNA fragment and labelling conditions used for footprinting were the same as those used for bandshifting, based on the assumption that the 5' recessed SstI end would not be labelled. The same end-labeled DNA was treated with G + A chemical cleavage reaction, according

649 to the Maxam and Gilbert (1980) method, and run in lanes adjacent to the footprinting reactions.

Results

a

02 regulates the accumulation of two PPDK isoforms in maize endosperm

Total protein extracts from endosperms harvested at 15 days after pollination (DAP), were fractionated by SDS gel electrophoresis, electroblotted, and incubated with an antibody against maize P P D K (Fig. 1). o2R and o2m(r) mutant extracts show reduced signal relative to wild-type samples and extracts from other strains mutant in zein synthesis. The apparent increase in P P D K levels in theft2, DeB30 and o7 mutants over wild-type levels has not been consistently observed. One of two cross-reacting bands is absent, and the second species, of slightly higher mobility, is reduced in intensity. Therefore, 02 positively regulates P P D K protein accumulation. Neither of the two bands seems to correspond to C4PPDK, which is slightly larger.

1

2

3

4

5

6

4501 400035003000250015001000- i 0

PPOK

b

1800-1) 1600-{I

~

Ni

1000 8O0 20

PPDK transcript analysis in wild-type and o2 endosperms To assess the degree to which changes in P P D K protein synthesis are reflected at the mRNA level, the relative abundance of P P D K transcripts was examined by Northern blot analysis. Poly(A) ÷ mRNA was isolated from wild-type and o2 endosperms, grown in vitro, with varying concentrations of ammonium nitrate and asparagine (the exact composition of media is

+

f12

DeB30

o7

o2R

o2m(r)

+

M

106

80

Fig. 1 Analysis of pyruvate orthophosphate dikinase (PPDK) proteins in the endosperms of different maize mutants. Aliquots (80 to 100 ~g) of total protein were loaded in each lane and probed with anti-PPDK antibodies. Lane +, A69Y wild-type;fl2, A69Y floury-2; DeB30, Defective endosperm B30; 07, W22 opaque-7; o2R, A69Y opaque-2 (spontaneous mutant); o2m(r), A69Y opaque-2 (insertion allele)

0

1

2

3

4

2?kD 7-zein

5

6

Fig. 2 a RNA gel blot analysis showing the relative levels of PPDK transcript in wild type (lanes 1, 2, 3) and o2t? mutant (lanes 4, 5, 6) endosperms grown in vitro on medium 1, 2, 3, respectively. Poly(A) + mRNA, was extracted as described in Materials and methods, and 5 gg samples were assayed in each lane. Densitometric scan of autoradiograph obtained after hybridization with a PPDK probe (h) and with a probe for 27 kDa 7-zein genes (e). Values are the average of three independent extractions and measurements, and the standard deviations are less than 5%

described in Balconi et al. 1993), size fractionated and immobilized onto membrane filters. The filters were hybridized and washed under stringent conditions with cDNA probes for the cT~PPDK1 gene. The results, shown in Fig. 2, clearly demonstrated that P P D K mRNA is more abundant in cultured wild-type endosperm than in o2 endosperm. This difference was also seen in kernels grown in vivo (our unpublished data). The 0 2 protein is, therefore, indeed involved in the regulation of PPDK gene expression in maize endosperm. The same filter was checked for uniformity of loading by rehybridizing with a specific probe for the 27 kDa 7-zein, which, in our hands, is not affected by the o2 mutation. PPDK activity in endosperms of wild-type plants and in plants mutant for zein synthesis Crude extracts, prepared in parallel from normal, o2R, and o7 endosperms of the A69Y or W22 inbreds,

650 Table 1 Pyruvate orthophosphate dikinase (PPDK) activity at 24 DAP in wild-type and opaque mutant endosperms of the inbred iines A69Y and W22 Genotype

Units/mg soluble protein ~

Units/g fresh weight

A69Y wt A69Y o2m(r) W22 wt W22 07

1.328 (0.058) 0.301 (0.094) 2.59 (0.060) 3.73 (0.341)

3.224 (0.030) 1.016 (0.309) 6.78 (0.239) 9.05 (0,679)

a One unit of enzyme activity catalyzes conversion of I gtmol substrate into product per min. The values are means of three independent assays. Standard error is given in parentheses, wt, wild type

sampled at 24 DAP were assayed for P P D K activity. P P D K activity of o2 endosperms was 3-4 times lower than that of normal endosperms (Table 1), while the P P D K activity in the o7 endosperm mutant, which also negatively affects the synthesis and/or accumulation of zein protein, was comparable to that of normal endosperm. This results suggest that the lower enzymatic activity of P P D K in the 02 mutant is specifically under the control of 02 and not caused by reduced zein accumulation alone.

The Opaque-2 protein, when transiently expressed in tobacco protoplasts, transactivates the cyPPDK1 promoter An assay for transient gene expression in tobacco mesophyll protoplasts has been employed to investigate a possible direct activation of the cyPPDK1 gene by the 02 gene product. The assay is based on cotransfection of mesophyll protoplasts with an effector plasmid and a reporter construct (Lohmer et al. 1991). The effector plasmid (pCaMVO2) consisted of the full-length 0 2 cDNA placed, as a transcriptional fusion, under the control of the strong promoter of the 35S gene from cauliflower mosaic virus. The reporter plasmid, cyPPDK1CAT (Sheen 1991), consists of a cyPPDK1 promoter region extending from - 1 kb up to + 96 bp relative to the translation start, fused to the chloramphenicol acetyltransferase (CA T) gene. The specificity of transactivation was tested with a construct bearing a promoter region of the C4PPDK gene spanning from - 3 1 0 up to q-158bp coupled to the CAT gene as a negative control. Such a construct contains all the essential control elements needed for the expression of C4PPDK (J. Sheen, personal communication). The plasmids were used separately or as a mixture to transfect tobacco mesophyll protoplasts and CAT activity was measured after 17 h. Transfection of reporter or activating plasmids alone resulted in a weak, basal level of CAT activity. Cotransfection of cyPPDKICAT together with pCaMVO2 stimulated CAT activity up to 19-fold over the background. In

Table 2 Chloramphenicol acetyltransferase (CAT) activity in transiently transformed mesophyll protoplasts Transfected plasmids

CAT activity ~

Activation ratio

No DNA C4PPDKCAT cyPPDK1CAT C4PPDKCAT + CaMVO2 cyPPDK1CAT + CaMVO2

66 468 1460 452 27966

1 19.1

(20) (30). (142) (55) (2567)

a The CAT activity is expressed in counts per minute (cpm) per gg of total protein. CAT assays were performed with cell extracts prepared from 5 x 104 protoplasts for 90 min as described (Seed and Sheen 1988). Aliquots (10 gg) containing equal molar concentrations of the various P P D K promoter/CAT constructs were used. CaMVO2 contains the CaMV 35S promoter fused to the 0 2 coding region. The values shown are the means of four independent assays performed for each construct using the same batch of protoptasts, to show the consistency among different samples. The experiment was repeated twice with similar results. Standard error is given in parentheses

contrast, the C4PPDK promoter was only slightly stimulated by the 0 2 protein. The results of the transient expression experiments are reported in Table 2. It must be noted, however, that the cyPPDK1 promoter showed a residual level of CAT activity even in absence of the 0 2 protein; this result is in agreement with results reported by Sheen (1990), who assigned a housekeeping role to the cyPPDK1 gene, and with the mRNA analysis reported above.

In vitro interactions between 02 protein and the cyPPDKt promoter To establish whether the transactivation of cyPPDK1 by 02 was through the binding of 0 2 protein to a specific target, a band-shift assay was employed. A 363 bp fragment of the cyPPDK1 promoter containing two putative 0 2 binding sites, very similar to those bound by the 0 2 protein in the b32 gene promoter (Table 3), was isolated and labelled as described in Materials and methods. Incubation of purified GST-O2 protein with this cyPPDK1 promoter fragment gave two proteinDNA complexes of reduced electrophoretic mobility compared to free DNA (Fig. 3). We have assumed (i) that the two complexes arise from the binding of 0 2 to two binding sites, one of high affinity and one of low affinity, in the target sequence, and (ii) that the binding is specific, since a 1000-fold molar excess of an unspecific competitor does not prevent binding. The binding can be completed, however, by a 100-fold molar excess of unlabelled cyPPDK1 promoter fragment. The site of interaction of 0 2 protein on the cyPPDK1 promoter fragment was mapped using DNase I footprinting. For this purpose, the same protein extract and end-labelled promoter fragment as used for the band shift assay were employed (Fig. 4). This promoter

651 Table 3 Comparison of the 02 binding sites in distinct promoters

Gene

DNA sequence

Re~rence

b32

G G G G G

A A A A T

T T T T T

G G G G G

A A A A A

C T T T C

A A G A G

T T T T T

G G G G G

G G G A A

Lohmer et al. (1991) Lohmer et al. (1991) Lohmer et al. (1991) Lohmer et al. (1991) Lohmer et al. (1991)

02

G

T

T

G

A

C

G

T

T

G

L0hmer et aL (1991)

cyPPDK1

G C

T A

T T

G G

A A

T C

C G

T T

G G

A T

This study This study

G 88

A/T 100

T 100

G 100

A 100

Py 100

Pu 88

T 100

G 88

Pu 88

Consensus a

The values below the consensus sequence indicate the frequency (in %) of the gene bases at that position

ng hot ¢yppaklprom ng G S T - 0 2 fusion protein ng c o l d cyppakl

1

1

1

1

C

100 100 100 -

-

50

100 -134

-125

Fig, 3 Gel retardation assay of GST-O2 fusion protein binding to the cyPPDK1 promoter. A 363 bp promoter region extending from position - 371 to - 8 relative to the translation start point was used as the probe fragment as described in Materials and methods f r a g m e n t c o n t a i n s t w o regions w h i c h are p u t a t i v e l y c a p a b l e o f b i n d i n g the 0 2 protein. T h e t w o regions are l o c a t e d b e t w e e n - 125 to - 134 a n d b e t w e e n - 257 to - 266, relative to the t r a n s l a t i o n start; they are very similar to e a c h o t h e r (see T a b l e 3) a n d t h e y fit the general c o n s e n s u s sequence 5 ' - G A / T T G A P y P u T G T 3', closely resembling 0 2 b i n d i n g sites in the b32 gene ( L o h m e r et al. 1991). T h e D N A s e I p r o t e c t i o n experim e n t revealed t h a t the - 125 to - 133 site was p r o tected b y 0 2 protein. T h e - 2 5 7 t o - 2 6 6 region, being t o o far a w a y f r o m the labelled end, c o u l d n o t be

Fig. 4 Footprinting analysis of GST-O2 protein binding sites on the cyPPDK1 promoter. The same cyPPDK1 promoter fragment was

used as in the gel retardation assay. Lanes 1 and 4, DNaseI digestion without protein (control); lanes 2 and 3, DNasei digestion in the presence of GST-O2 fusion protein. G + A lanes show a sequencing reaction obtained with the promoter fragment resolved b u t it is r e a s o n a b l e to s u p p o s e it t o o is b o u n d , o n the basis o f its close similarity to the consensus.

Discussion E u k a r y o t i c genes e n c o d i n g functionally c o o p e r a t i n g e n z y m e s m a y be subject to a c o m m o n genetic c o n t r o l

652 mediated by similar cis and trans-acting elements. In yeast, regulatory systems such as the GAL4-dependent induction of galactose-metabolizing enzymes (Johnston 1987) and the general control of amino acid biosynthesis (reviewed by Hinnebusch 1.990) fit these criteria. However, little is known in higher plants about the molecular mechanisms that regulate different metabolic pathways that are connected by common regulators (Kuhlemeier 1992). There are indications that the maize 02 locus represents such a connecting regulator gene. This gene is known from classical genetic studies to activate 22 kDa a-zein genes and the gene b32 in trans, and encodes a member of the bZIP class of transcription factors (Hartings et al. 1989; Schmidt et al. 1990; Lohmer et al. 1991). A role for its product in connecting different aspects of endosperm metabolism would be consistent with the numerous pleiotropic effects previously reported in 02 mutant endosperm (reviewed in Motto et al. 1989). In addition to effects on zein synthesis, major differences exist between the metabolism of normal and o2 endosperms. For example, it has been shown that 02 maize varieties, similarly to high lysine varieties of barley, have an altered amino acid metabolism (Sodek and Wilson 1971; Murphy and Dalby 1971; Brandt 1975; Dalby and Tsai 1975; Da Silva and Arruda 1979) and lower levels of carbohydrate at maturity (Murphy and Dalby 1971). These observations suggest that 02 may, like the yeast transcription factor GCN4 (Hinnebusch 1990), coordinate the expression of a number of amino acid biosynthetic genes and/or genes involved in carbon partitioning between protein and starch. In this report, we have followed up the observation that one of the cytosolic PPDK gene promoters contained two putative 0 2 binding sites. The cytoplasmic form of the P P D K enzyme has been reported to be present in developing seeds and has been suggested to play a role in providing PEP for amino acid interconversion during seed development, in order to sustain storage protein synthesis (Aoyagi and Bassham 1984a). PEP is a key compound at the biochemical interface between carbon and nitrogen metabolism, being the first compound common to the pathways for the biosynthesis of aromatic amino acids; moreover, it can be used by PEP carboxylase as a substrate for recapturing respiratory CO2, thus generating oxolacetate which, in turn, can be transaminated to generate aspartate (reviewed in Davies 1979). The data reported in this study confirm that P P D K activity is detectable in normal maize endosperm, as previously reported (Aoyagi and Bassham 1984b). Two polypeptides differing by < 5 kDa in apparent molecular weight were detected with antiserum raised against C4PPDK from maize. One band disappears in o2 mutants, whereas the second, slightly smaller, species is decreased in abundance. As we could detect CyPPDK1 and CyPPDK2 transcripts but not C4PPDK mRNA

(Gallusci et al., in preparation), it is assumed that the polypeptides are derived from cyPPDK1 and cyPPDK2. Only cyPPDK1 mRNA showed o2-dependent expression, and therefore is probably responsible for the o2-regulated polypeptide component. Enzyme measurements are consistent with both polypeptides corresponding to active PPDK. In addition, the decreased PPDK activity and reduced mRNA level in the o2 mutant are consistent with 02 being involved in the regulation ofcyPPDK1 gene expression in maize endosperm. The results showing that 0 2 protein transactivates the cyPPDK1 promoter in vivo and binds to it in vitro, indicate that the interaction between 0 2 and cyPPDK1 is likely to occur at the transcriptional level. However, the detection of P P D K transcript in o2 mutant cells suggests that O2-independent expression of PPDK can also occur in the endosperm. The accumulation of zeins in o2 mutant endosperm grown under high-nitrogen regimes also points to the existence of an O2-independent metabolic regulation mechanism in the grain (Balconi et al. 1993). The 0 2 binding sites in the cyPPDK1 promoter established by DNase I footprinting correspond to a conserved decamer, with the consensus sequence 5'GA/TTGAPyPuTGPu-3', which is highly homologous to already published 0 2 binding sites in the b32 gene (Table 4) (Lohmer et al. 1991). In addition, the TGAPy core is found in many promoters of plant genes encoding seed proteins and it was demonstrated that such cores mediate important functions in gene expression (Yunes et al. 1994; Miiller and Knudsen 1993; De Pater et al. 1993; Ottoboni et al. 1993; Cord Neto et al. 1995). Finally, this consensus sequence shares a striking homology with the binding sites for a class of plant b-ZIP proteins, collectively referred to as TGACGT/C binding proteins (Izawa et al. 1993), all of which interact with the TGACGTAC sequence or close variants. In conclusion, the data presented indicate a transcriptional control by 02 of cyPPDK1 expression during endosperm development. The notion of a direct transactivation by 02 of cyPPDKI is supported by the observation that other mutants producing an opaquetype phenotype, such as o7, DeB30 andfl2, leave P P D K unaffected. Thus, it appears that 02 plays a rote in coordinating the expression not only of certain a-zein genes, and genes such as b32 and bTO, implicated in zein deposition, but also genes involved in carbon partitioning between protein and starch. Changes in the supply of nitrogen compounds to endosperm are known to affect mainly the synthesis of the zein fraction of storage proteins (Tsai et al. 1978; Balconi et al. 1993), which is accumulated coordinately with carbohydrate in the kernel (Tsai et al. 1978). In barley, the high-lysine trait is associated with a block in starch synthesis, and an accumulation of soluble sugars and free amino acids (Kreis et al. 1980). Direct evidence for an effect on starch synthesis has not yet been demonstrated in high

653

lysine maize, although a lower content of dry matter in mature kernels compared with wild type is characteristic of o2 andfl2 seeds (Murphy and Dalby 1971; Tsai 1979). Acknowledgements This work was funded by the European Communities BIOTECH Programme, as part of the Project of Technological Priority 1993-1996. We are grateful to Dr. Jane Sheen (Massachussetts General Hospital) for providing the constructs C4PPDKCAT and cyPPDK1CAT, to Dr. J. W. Grula (Phytogen) for supplyings the clone pH2 231.0, and to Dr. Matenola (NIAR, Tsukuba, Japan) for PPDK antibody.

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