Molecular genetics of lipid synthesis in barley

transcript

Barley Genetics VI (1992)

MOLECULAR GENETICS OF LIPID SYNTHESIS IN BARLEY

P. von Wettstein-Knowles

Department of Physiology Carlsberg Laboratory

Gamle Carlsberg Vej 10 DK-2500 Copenhagen Valby, Denmark

and Genetics Institute

University of Copenhagen

INTRODUCTION

Lipids can be broadly defined as water insoluble, organic solvent soluble compounds. Included among them are the acyl lipids, derivatives of long chain fatty acids, which occur in a wide range of forms allowing them to carry out an equally diverse range of functions . In barley, for example, they are the major structural components of the membranes, contribute to the energy reserves stored in the caryopses, as well as being prominent constituents of the cell walls, especially the outermost cuticular and epicuticular layers. Synthesis of the long carbon skeletons of these lipids takes place by repetitive joining of short activated carbon chains, most frequently acetate (C2) donated by malonyl-ACP (Fig. 1). Each condensation introduces a ~-keto group into the elongating carbon chain. This keto group is normally removed by a series of three reactions: a ~-keto

reduction, a dehydration and an enoyl reduction (Fig. 1 left) . The complexes of proteins carrying out these reactions yielding saturated acyl chains are known as fatty acid synthetase (FAS) and elongases (eg C16 elongase). Various fates await the synthesized fatty acids. Many are transferred to glycerol or one of its phosphate containing derivatives before and/or after being desaturated to various extents. For example, in the caryopses addition of three acyl chains to glycerol gives the triacylglycerols. In leaves addition of two acyl chains to phosphatidylcholine or phosphatidylethanolamine leads to formation of two of the prominent complex lipids of the plasmalemma and nuclear membranes, or when added to monogalactosyldiglyceride results in a major complex lipid of chloroplast membranes.

Variations of this basic condensation elongation synthetic mechanism occur giving rise to compounds classified as polyketides. Their modified acyl chains can be recognized by the presence of keto groups, hydroxy groups or double bonds that were not removed before the next condensation reaction took place (Fig. 1 right). In barley 6-methylsalicylic acid synthetase joins four C2-units leaving keto groups after the first and third rounds of elongation and a double bond after the second. The most extreme example is chalcone synthase which condenses three C2-units to p-coumaric acid with no intervening reactions to give the flavonoid presursor naringenin chalcone. The molecular organization of the functional units

Vol. ll (ed. L. Munck) Munksgaard International Publishers Ltd., Copenhagen, Denmark

754 Barley Genetics VI (1992)

0 0 II II

HO-C-CH 2-C-S-ACP .. .. +

0 " ••• CH2-C-S-ACP

0 0 II II

• • • CH2 -C-CH2-C- S- ACP / ... .. ',,,

/ Protected

/ 0 0 0 " II II

• • • CH 2-CH2-CH2-C- S-ACP • • • CH2-C- CH 2-C- S- ACP

Fig. 1. Simplified scheme to illustrate the reiterated cyclic nature of acyl chain synthesis. In the first cycle a C2-unit (.t.-.t.) from malonyi-ACP is condensed to the C2-primer acetyi-ACP to produce acetoacetyl-ACP with four carbons. Removal of the ~-keto group in three steps (arrows left side) of each cycle yields saturated fatty acyl chains while retention of the ~-keto group (right side) or one of its derivatives (see text) in one or more cycles yields modified fatty acyl chains.

participating in the FAS, elongase and polyketide synthetase pathways range from multifunctional proteins coded for by single genes to complexes of proteins each coded for by a different gene. The limited, presently available primary sequences in data bases support the notion that the genes encoding all proteins and domains with a similar function have evolved from the same ancestral gene. The only apparent exception is chalcone synthase vs all the other condensing enzymes and domains (Lanz et al 1991).

In barley the elongases synthesizing the very long carbon chains of the epicuticular waxes are an interesting mixture of the different types of enzyme complexes participating in the just described condensation elongation pathways. The two designated acyl and ~-ketoacyl are the best known. The former produces saturated acyl chains. By comparison the latter during specified steps of elongation condenses a C2-unit to a chain that retains keto groups from either one or two of the previous cycles of elongation (Fig. 2). Before the majority of the resulting very long carbon chains synthesized by these two elongases arrive on the surface of the plant, they are modified by associated enzyme systems. Those from the acyl elongase can serve as substrates for a reductive system yielding aldehydes, alkan-1-ols and alkan-1-ol esters or as precursors for a decarboxylation system producing very long chain alkanes and alkan-2-ols. As diagrammed in figure 2 products from the ~-ketoacyl elongase are channeled into ~-diketones

Barley Genetics VI (1992) 755

and alkan-2-ol esters plus their derivatives. Herein, I shall summarize what is known with respect to the genetics of acyl

chain synthesis in barley which is essentially the only facet of lipid synthesis thus far investigated. Small exceptions can be noted such as the assignment of cer-j to the reductive associated enzyme system of epicuticular wax synthesis (Avato et a/1982). Emphasis is laid upon the types of mutants which can be expected, and the necessity of characterizing those obtained both genetically and biochemically. Well developed experimental systems are available in this plant. The accumulated basic information has opened avenues which are being exploited to isolate the desired structural genes participating in fatty acid biosynthesis. This in turn has and will lead to the other members of the same multigene families. Once these are characterized the techniques of molecular biology will enable exploration of the waiting myriad of fascinating questions in lipid metabolism.

MUTANTS IN PLANT LIPID SYNTHESIS

The nature of the non-lethal mutants which can be expected in lipid metabolism are dependent on the function of the end product of the given pathway. Natural variation within the plant kingdom is probably a good indicator of the range of mutant types which can be expected. The composition of the storage lipids of the seeds and of the epicuticular waxes on the aerial surfaces of plants are known to diverge widely, apparently without detracting from the viability of the plant. By comparison membrane lipid composition is much more circumscribed. The number of identified mutants is in part a direct result of the potential use by man of the end products or of their visibility. Plant breeders have been successful in exploiting natural and induced mutations to modify the composition of many oil seeds even though the mutants can not be distinguished by eye (see Downey 1987). No such mutants have been described in barley with its starchy endosperm. On the other hand, since many wax mutants markedly alter the color and/or appearance of the cuticle surface, they have been collected and used as markers by breeders and geneticists. Those in barley, referred to as eceriferum (cer), glossy (gl) etc, are described below. Recently, non-lethal mutants in Arabidopsis with blocks in leaf membrane lipid biosynthesis have been successfully sought among M2 progenies by assaying the total fatty acyl profile of individual plants in the laboratory (see Browse et a/1990). None were potential mutants in the C16 elongase complex, rather most altered the extent of desaturation reflecting the range of natural variation. In any given plant an important factor contributing to the potential isolation of mutants in a given biosynthetic step is the question of gene copy number. For example, chalcone synthase Chs mutants are known in a number of plants, but have not been identified among the extensive flavonoid mutant collection in barley (jende-Strid 1991 ). One possible explanation is the existence of more than one functional gene, a hypothesis which has recently been supported by the mapping of two hybridizing sites on chromosome 2 (Kleinhofs and Nilan personal communication) .

c ----- - ~ - 5 - CoA • c 2 8cer~cqu polypeptide

A IS · kacel cond 7q·s • q1440 t

VJ 4q ·s

0 0 11 II

C--- - - C - CH 2- C - S - CoA

, OH c ------ c

m 0 " CoA - 5 - C ---- - C

' ' I I I I I I I I I

Be 's J:... I I I I I

sc ·s~

I I I I I \

{},\r', .) \ 0 0

I II C---- - - C- CH2- C- S - CoA

,,~ a' 0 0

I II II C----- - C- CH 2- C- CH2- C- S - CoA

0 0 0 I I II

C------ C- CH 2- C - CH2- C - S - CoA

o c ---- c 11 % c ------ c '

yC2l C02 . /'-.

0 0 \CH

3 11 II

24 % c ---- -- c-CH2-c----- - c alkan-2-ol ester

1S- d iketone

{;) 1ou·s t [OH [

OH 0 0 I 11 II

" ,o -c -- c o.2<t c - - c - 26 % c -- c --- c- cH2- c------ c -c

'cH3 hydroxy - /1- diketone 7 - oxoalkan -2 - ol ester

Fig. 2. The multifunctional cer--cqu polypeptide plays at least three roles in synthesis of barley epicuticular waxes. Its condensing activity (~-kacel cond) produces ~-ketoacyl key intermediates. When these are C14 and C16 components (x = 12 and 14) they are converted primarily into alkan-2-ol esters (left side) by the coordinated action of a decarboxylase (1), reductase (II) and ester synthase (Ill) . Thereafter, an oxo group may be inserted onto carbon 7 of the C15 alkan-2-ol moieties. In ~-diketone synthesis (right side) the key intermediate is predominantly a C18 compound (x = 16)_ Retention of keto groups in two successive rounds of elongation are shown by I and A _ Generally y = 6, if not then 5. An apparent decarboxylation yields ~-diketones into which an hydroxyl group can be inserted on carbon 25. Whether CoA or ACP is involved in this pathway is unknown. Since CoA is implicated in synthesis of C20-C24 acyl chains

THE cer MUTANTS IN BARLEY

The more than 1560 studied cer mutants in barley are distributed among 85 complementation groups which are expressed at specified developmental stages (see von Wettstein-Knowles 1989). That is, they affect the phenotypes of the leaf blades, the spikes (essentially the lemmas) and/or the uppermost leaf sheaths plus culms. In part this organ specificity can be correlated with the differences in composition of the wax on the various surfaces. The presence in the wild type of the highly lobed plates on the leaf blades are attributable to large amounts of alkan-1-ols (primarily C26) and the long thin tubes on the other surfaces to ~diketones (primarily C31). As described above these two lipid classes represent products of different elongases. An intriguing set of complementation groups are those in which the just noted correlation does not hold. Twenty three cer groups affect only the waxes of the spikes or only those of the uppermost leaf sheaths and culms (Lundqvist and Lundqvist 1988). The best characterized is Cer-yy to which more than 17 mutant alleles have been assigned, all of them dominantly inherited (Lundqvist and von Wettstein-Knowles 1982, von Wettstein-Knowles 1991). Compositional analyses of five mutant alleles led to the conclusion that the dominant mutations resulted in spike waxes very similar to that on leaf blades (Lundqvist and von Wettstein-Knowles 1982, von Wettstein-Knowles unpublished). Thus Cer-yy does not function in the wax biosynthetic pathways, but rather as a regulatory gene, conceivably one associated with the cascade of events specifying differentiation into leaf blades vs lemmas. Tools for isolating this gene are accumulating. These include i) its location between the Hor2 and Mlra(Reg7) loci (see von Wettstein-Knowles 1991) combined with isolation of YAC clones (Dunford 1991) and construction of physical maps in this region ( Siedler and Graner 1991, S0rensen 1989) plus ii) the creation of tissue specific libraries for use in differential screening (Wissenbach 1991).

The 21 complementation groups affecting surface waxes mapped to distinct sites in the barley genome (Fig. 3) must represent 21 genes. In the calculation cerv, -n and -s have been counted as one since definitive evidence is lacking to demonstrate they they represent two or three genes. An additional gene designated cer-cqu has been assigned to chromosome 4 unlinked to the other cer genes (see references in S0gaard and von Wettstein-Knowles 1987). Recent observations, however, infer a location in chromosome 2 (Schulze-Lefert and von Wettstein-Knowles unpublished observations). More than a third (522) of the induced mutations in the cer collection have been assigned to this gene. Of these 204, 157 and 148 belong to complementation groups cer-e, -q and -u. The other 13

in oil seeds CoA is used in this figure (Fehling et al 1990). The amounts of the four lipids present in Bonus spike wax are given as weight percentages. The sites of action of 28 blocked and 10 leaky mutants are denoted by single and double arrows, respectively. All cer-cqu mutants are recessive except for cer-q1440 which is dominant.

s(gs5)

(JI_5_

a(gs3)

zh(gl) zg······· ((~I~)

YY(Gie)

~ 10 map unit s

x(g s4)

Fig. 3. The cer loci affecting leaf waxes(. .. .... ) plus spikes and/or uppermost leaf sheath and culm waxes(--) are widely distributed in the barley genome. The symbols gl for the former and gs or Gle for the latter type are used in other mutant collections. Only letter designations for the individual cer loci are given in thi s figure. Brackets indicate allelism and a ? potential allelism between complementation groups in the different mutant collections. Centromere regions are not shown in this ideogram as their relationships to the cer loci are unknown, although all four loci in chromosome 3 are in the long arm . Distances between the cer loci are estimated despite a lack of knowledge on the genetic lengths of the chromosomes. Based on data summarized in 50gaard and von Wettstein-Knowles 1987, von Wettstein-Knowles 1991, and unpublished observations of B. S0gaard.

belong to more than one of these, that is, -cu, -cq, -qu or -cqu. Genetic testing with the latter initially demonstrated that the cer-e, -q and -u complementation g rou ps were very tightly linked, on the order of 0.0012 eM (von Wettstein-Knowles and

S0gaard, 1980). These results implied that they represented the same gene, a hypothesis substantiated by the recovery of partial and wild type revertants after NaN3 mutagenesis which would have been highly improbable if the multiple mutants had originated from concurrent mutational events.

Before carrying out the NaN3 treatment the multiple mutants were combined with cer-j, one of the comlementation groups affecting leaf waxes situated on chromosome 4 (Fig. 3). All13 apparent multiple mutants have been reverted and hence are· attributable to individual mutational events. The average reversion frequency of the cer-cqu mutants (5.8 X 10·5, ranging from 2.6-15.1 X 10·5) is very similar to that for cer-l9 (4.2 x w-5). The 39 cer-cqu revertants recovered from these experiments arose from mutational events within the same or other loci. To clarify this question homozygous revertant cer-cquR, -/9 strains were identified and crossed to the wild type Bonus. Subsequent genetic analyses divulge that the one partial revertant to cer-u and 37 to wild type stem from mutations within the cer-cqu locus. The other wild type revertant cer-cu947R:2 is due to mutation of a non-linked gene to a dominant suppressor of the cer-cu947 allele. Furthermore, the suppressor locus- another cer complementation group- is not linked to cer-j and does not affect the expression of the cer-l9 allele.

Combined with the original522 cer-cqu mutants, the revertants provide a large storehouse of tools for the molecular dissection of the cer-cqu locus when cloned sequences for it become available. Attaining the latter objective requires knowledge of the roles of the cer-cqu determined polypeptide in ~-diketone

synthesis. Briefly, this polypeptide has at least three functional domains corresponding to the complementation groups cer-e -q and -u (Fig. 2) . Biochemical studies have revealed that the ~-ketoacyl condensing enzyme activity is defective in cer-q mutants (Mikkelsen 1984) and that hydroxyl group insertion is impaired in cer-u mutants (von Wettstein-Knowles 1972). The activity specified by the cer-e domain is unknown although it must act in two succesive rounds of elongation. Earlier studies intimate that the cer-cqu determined protein is located in the plasmalemma and/or walls of the epidermal cells (Mikkelsen 1980) where it functions as a multimer with a minimum of two units (von Wettstein-Knowles 1989). The described characteristics combined with the lack of an in vitro assay system suggested the following approach. Namely, to capitalize on the deduction that the condensing activity region of the cer-cqu gene would be coded for by sequences in the same multigene family as those coding for the soluble condensing activities of FAS and C16 elongase localized in the chloroplasts. Progress towards this goal is summarized below. Differential screening is a supplementary approach which takes advantage of the tissue and organ specific expression of cercqu . Thus far, however, no positive clones have been obtained from a leaf sheath library by probing with leaf sheath and leaf mRNAs (Wissenbach 1991). Starting with epidermal tissue would have presumably increased the chances of success. Finally, initial anlyses of the DNA from the parents and F2s of crosses between cer-cqu mutants in the Scandinavian cultivar Bonus x Hs yh in the Japanese linkage stock Kimugi imply a tight linkage between the restriction fragment bBE54f and the cer-cqu locus (von Wettstein-Knowles and Schulze-Lefert unpublished observations) .

SYSTEMS TO STUDY SYNTHESIS OF ACYL CHAINS IN BARLEY

In many plants seeds have been selected for studies of acyl chain formation since elevated amounts of lipids are synthesized during their development, and the lipids are major components. In barley the total amount of lipids increases circa 15-fold from 5-37 days after anthesis when 42 days are required for maturation (de Man and Cauberghe 1988). Not only is the embryo rich in lipids, but much lipid is stored as triacylglycerols in the oleosomes which occur in large numbers in the aleurone cells and Jess extensively in the endosperm (CameronMills and von Wettstein 1980). Significant quantities of Iysophosphatidylcholine and phosphatidylcholine are present in the endosperm associated with starch granules (Stokes et aJ 1986, Morrison 1988). Lipids, however, are minor constituents of barley caryopses (starch:protein:lipid 30:5:1, Briggs et a/1981 ). This means that the lipid rich tissues would have to be dissected from caryopses at the same stage of development which would severely limit the starting material from which to try and isolate enzymes. This system is thus less than optimal for studying the genes and enzymes participating in acyl lipid synthesis.

Seedling leaves are another extensively exploited experimental system in barley. Large numbers of seeds can be germinated in the dark and the etiolated seedlings transferred into the light. While the focus of interest has been on photosynthesis and development of chloroplasts in the mesophyll cells, especially the pigment and proteins involved, studies of the lipid components have also been carried out. Initial total fatty acyl and lipid class analyses (eg Hawke and Stumpf 1965, Appelqvist et aJ 1968) have been supplemented with detailed characterizations of the lipids associated with specific membranes, such as the plasmalemma (eg Rochester et al 1987), as well as membrane fractions and individual proteins, such as those of the photosystem II reaction center from the chloroplast grana (eg Henry et al 1983, Hinz 1985). The comparison of lipids in proplastids of etiolated tissue with those in chloroplasts of green tissue revealed that the latter is unique in having major amounts of two complex lipid classes, namely monogalactosyldiglycerols and digalactosyldiglycerols. These complex lipids are characterized by a high content of linolenic acid C18:3 which is synthesized by the reiterated reactions of FAS and C16 elongase to give the 18 carbon skeleton followed by three desaturations. This happens concomitantly with the differentiation of the chloroplast membranes. An analyses of total fatty acyl chains in greening leaves is presented in Fig. 4. Both segments of light exposed leaves have 2-3 times as much C18:3 as the respective segments from etiolated leaves. Furthermore the top halves of both types of leaves are circa twice as rich in C18:3 as the bottom halves which include the basal meristematic region with its undifferentiated plastids. These results infer that the relatively high constitutive level of C18:3 synthesis in etioplasts is stimulated several fold by light. The relatively constant amounts of the other fatty acids in all the leaf samples assayed demonstrate the marked specificity of the light stimulation. The extent of C18:3 synthesis (compare amounts in lower half of etiolated leaf with those in upper half of illuminated leaf, Fig. 4) is roughly paralleled by the increase in number and size of plastids. According to Baumgartner et al (1989) during meso-

1- 6 .. ~ .. ::t: ..

· ~ (/) w

( c:: ,, u.. 4 a> ..... 0 E :::_

Dark Light

16:0 18•1 18•2 18 •3

NUMBER OF CARBONS: DOUBLE BONDS

Fig. 4. Spectrum of fatty acyl chains from the upper and lower halves of Bonus seedling leaves plus coleoptiles six days after planting. After five days in the dark at 22° half of the seedlings were transferred to the light for 24 hours before their lipids were extracted and quantified as described (Hinz 1985).

phyll cell elongation plastid number increases from 10-15 to 60 per cell and their diameter from circa 2.3 to 6.7flm. In leaves analogous to those studied here max cell length is attained 3-4 em above the leaf base while in leaves grown under constant light it is within 1-2 em. The fatty acyl composition of the chloroplast lacking coleoptiles resembles that characteristic of plasmalemma membranes, and light does not influence the low constitutive level of c18:3 formation .

Exposing etiolated leaves to light also influences formation of the epicuticular wax lipids which takes place in the plasmalemma and/or walls of the epidermal cells. This is shown in Fig. 5. Primary alcohols are the major wax class (83% by weight) on the surfaces of both etiolated and light grown, barley seedling leaves (Giese 1975). Their composition on the etiolated leaf surfaces is C22 = 734, C24 = 343 and C26 = 1896 ng · cm-1 whereas on the green leaf surfaces the values are 133, 374 and 7377 respectively (Giese 1976). Thus, exposure to light stimulates constitutive synthesis of fatty acyl chains several fold both in the mesophyll and epidermal cells of seedling leaves. In the experiments summarized below both protein and mRNA have been isolated from greening leaves 6-12 hours after transfer to light.

8 E ()

....... 6 ><

~ Cl 4 ~

---------0---------~ 0 0

.,----------.

• --------6 ~.~-6

Fig. 5. Total wax per em seedling leaf. Bonus seedlings were grown for the specified number of days with cycles of 15° for 16 hours I 10° for 8 hours in constant dark (.o.), constant light (o) or transferrred from dark to light (•). Data from Giese 1975.

THE Acl GENE FAMILY CODING FOR ACYL CARRIER PROTEINS INCLUDES HOUSEKEEPING AND TISSUE SPECIFIC MEMBERS

During their biosynthesis fatty acyl chains are bound via a thioester linkage to a small member of the fatty acid synthetase complex called acyl carrier protein (ACP). Why was this component of barley FAS selected for study? Firstly, ACP participates in other reactions of lipid synthesis in addition to those of elongation implying that it might occur in higher amounts. It had been extensively studied in other organisms and shown to have characteristics that could simplify its isolation. Finally, with the exception of chalcone synthase, all well characterized FAS and polyketide synthetase complexes have an ACP domain .

To date, three isoforms have been identified in barley seedling leaves. ACP I and ACP II, encoded by the genes Acl1 and Acl2, were initially isolated from chloroplasts and amino acid sequence data obtained (Hej and Svendsen 1983, 1984). Suitable oligonucleotides were then synthesized and used as probes to screen for clones in eDNA libraries constructed from RNA isolated from analogous greening seedling leaves (Hansen 1987, Hansen and Kauppinen 1991, Hansen and von Wettstein-Knowles 1989). The three groups of clones characterized are schematized in Fig. 6. Ac/1 and Ac/2 include 5' sequences coding for transit peptides, which despite a lack of homology, target preACP I and

Transit Mature

Act 1 C=Ef:::]:::::[:=::}:s9~a!!a~:::]:::::[:=:=:••ii]g~o~aa~··-========]IPolyA I s;r I

ATG TAA AeCC Ge

ATG ecce Ge

eeTe GC

GAATAA

AATAAG

AATATG

Fig. 6. Diagrams of eDNA sequences of Ac/1, Ac/2 and Ac/3 coding for ACP I, ACP II and ACP III, respectively. Included are the apparent initiation sequences encompassing A TG, stop codons, presumed polyadenylation signals, poly A tails and the regions coding for the transit peptides and mature proteins which contain the serine residue to which 4' phosphopantetheine is attached .

preACP II to the chloroplasts. The third group of clones, representing processed transcripts of the Ac/3 gene, encodes ACP III for which primary structure data is lacking. That the Ac/3 gene includes a sequence coding for a transit peptide directing this isoform also into chloroplasts is suggested by the identity of 33 of the 49 NH2-terminal residues deduced from the Acl2 and Ac/3 clones. Whether the deduced mature ACP Ill is identical to a nonsequenced isoform identified in seedling extracts (H0j and Svendsen 1984) remains to be determined. Differential processing of primary transcripts of Acl2 and Acl3 is revealed by the isolation of clones that used alternate polyadenylation signals. If this also occurs with the Acl1 primary transcript is unknown as only one of the characterized clones had a poly (A+) tail. The biological significance of this phenomenon for barley A CPs is unknown.

Southern analyses using the Chinese Spring wheat - Betzes barley addition lines localized the three barley genes to chromosomes 1 and 7 (von WettsteinKnowles 1989, Hansen and Kauppinen 1991, Hansen and von Wettstein-Knowles 1991). No cross hybridization was observed. Similar studies using the barley probes and DNA from nullisomic-tetrasomic and ditelosomic lines of Chinese Spring wheat identified which arms of the homoeologous wheat chromosomes contained hybridizing sequences (Devos et al 1991, Gale unpublished observations). Extrapolation from the latter data infers that in barley Ac/1 is on the short arm of chromosome 7 while Acl2 and Acl3 are on the long and short

arms, respectively, of chromosome 1. The number, intensity and sizes of the hybridizing bands resulting after digesting Bonus and Betzes barley DNA with different restriction enzymes suggests that these three genes are single copy members of a small multigene family . This is in marked contrast to the preliminary estimates of 35 members in the rape and a minimum of 8 members in the spinach ACP gene families (Schmid and Ohlrogge 1990, de Silva et al1990).

Size selected genomic libraries were constructed and screened for clones of Acl1 and Acl3. A summary of the results is presented in Fig. 7. Both genes are

A eft single copy on 7S

Xbal Bglll Sac I EcoRI Bglll Xbal

10 , 2 4 - 6 kb , , -, I n m N --I s· , -c ITl DG -, 3 .

' s· '

.g G o-o _. 3. ' ' I n m w ' ' ' ' ' --' ', 0 2 4 - 6 8 kb

Xbal Bglll EcoRI EcoRI Bglll Xbal

Ac/3 single copy on 1S

Fig. 7. The mosaic structures of the Acll and Acl3 genes are diagrammed below and above their respective restriction maps. The dotted regions of exons I and II code for the transit peptides and the open regions of exons II and/or Ill plus IV code for the mature ACP I and ACP Ill proteins. The serine binding 4' phosphopantetheine is shown. Thick black lines between the exons represent introns removed during processing of the mRNA as well as the non translated 5' and 3' ends of the cDNAs. Adapted from Hansen and von Wettstein-Knowles 1991.

a mosaic of four exons and three introns extending over more than 2.5 kb. If the requisite promoter regions are included, a minimum of 3 kb is required for expression of the small mature proteins. The latter are essentially coded for by exons Ill and IV which are the only homologous segments of these genes; > 70% at the DNA level. The prosthetic group binding serine is part of a block of 19 amino acids spanning the junction of these two exons which with the exception of one residue in barley ACP II is totally conserved in all plant ACPs thus far

described (Hansen 1991). The seven extra residues in ACP I vs ACP III are at its NHTterminal end . The most likely origin of these is a small, in frame duplication event including the last three bases of the original second intron plus the first five codons of exon III. Aligning the specified 18 bp of Acl1 with the following 18 reveals that 14 of them are identical (Hansen and von Wettstein-Knowles 1991). In both genes the first intron in the transit peptide and the second intron at or adjacent to the transit peptide cleavage site are fairly long (=1100 and 600 bp, respectively), and are the only structural feature distinguishing the barley genes from those in rape and Arabidopsis (Lamppa and Jacks 1991, Post-Beittenmiller et al1989, de Silva et al1990).

Primer extension analyses revealed three transcription start sites for both genes giving leader sequences ranging from 85-145 bases. In other aspects, however, the two promoters are quite different (Hansen and von WettsteinKnowles 1991). Firstly, the transcription start sites for Acl1 are accompanied by appropriately located TAT A boxes, but those for Acl3 are not. Secondly, a circa 275 bp 5' flanking region including the transcription start sites, leader sequences and first bases of the mature message is 51% GC in Ad1 but 66% in Acl3. Thirdly, the GC enriched region of the Acl3 promoter contains three GC elements (consensus GGGCGG) that do not occur in the Acl1 promoter. These GC elements serve as recognition (binding) sites for the Spl factor of RNA polymerase II (Mitchel and Tjian 1989). The just noted attributes of the Acl3 promoter are characteristics demarking promoters of mammalian housekeeping genes, eg goose FAS (Kameda and Goodridge 1991). That Acl3 may well be such a gene is in accord with the presence of its message in all tissues thus far analyzed, namely, seedling leaves grown under various photo and thermoperiods as well as developing caryopses (10 and 30 days after flowering) and germinating embryos (over night). By comparison Acl1 appears to be a tissue specifically expressed gene since its messages are relatively abundant in seedling leaves, absent in the developing caryopses and just detectable in the germinating embryos (Hansen 1987, 1991). An interesting question for the future will be to delimit the cis acting sequences determining leaf specific expression.

A FIRST INSIGHT INTO THE NATURE OF THE Kas MULTIGENE FAMILY CODING FOR CONDENSING ENZYMES

The condensing enzyme, P-ketoacyl (ACP) synthase I, of the soluble chloroplast localized FAS complex uses C2-C14 fatty acyl chains as primers. This component of FAS is the most difficult to assay enzymatically. Thus an alternative detection method based on its extreme sensitivity to the antibiotic cerulenin was developed which consists of two parts (Kauppinen et al 1988, Siggaard-Andersen 1988). Firstly, 3H-cerulenin bound covalently to the active site cysteine provides a radiolabelled tag that can be followed during purification and characterization. Secondly, addition of unlabelled cerulenin to a crude FAS preparation destroys its ability to incorporate 14C-acetate into C16 acyl chains. The FAS activity can be restored upon addition of a protein fraction contain ing the

appropriate condensing enzyme (complementation analysis). This technique was established during the course of isolating and characterizing the more readily obtainable E. coli condensing enzyme and its gene (jabB) .

When 3H-cerulenin was taken up by isolated barley chloroplasts three proteins were tagged . Structural characterization revealed that two were homodimers (~, ~) and one a heterodimer (a~) of the closely related a- and ~-polypeptides . Complementation analysis demonstrated a synthase I activity associated with all three proteins (Siggaard-Andersen et al 1991). Using oligonucleotides corresponding to the NH2-terminal end of the ~-polypeptide and internal amino acid sequences plus the polymerase chain reaction (PCR) on a eDNA library yielded a 311 bp clone. Screening eDNA libraries with the latter gave 15 full length clones (Siggaard-Andersen et al 1991, Kauppinen unpublished). As summarized in Fig. 8, Kas12 (~-1eto~cyl §_ynthase ! polypeptide ~ or ~) cON A codes for a mature protein of 427 residues with an NH2-terminal transit peptide of 35 residues. The latter has no homology to the ACP transit pcptides that also target to the chloroplast. By comparison the mature KAS 12 protein shares 35% identical residues with the fabB determined protein of E. coli, and an additional 19% are classified as conservative changes.

Kas 12 single copy on 2

Xba I BstE II

I Hin?mJ Sail I

Pvul I

EcoRV Hind III BamHI Xba I I I I I I I ······---4

0 2 3 4 5 -9 kb

I n m NV1ll "llii

Promoter 3.

Transit Mature 35aa 427aa

Fig. 8. Restriction map (top) of a tiny Xbal fragment of chromosome 2. Sequencing o:S.S kb at the 5' end of the Xbai fragment and comparison with the sequence of the Kas12 eDNA revealed the mosaic structure of this gene (center) coding for KAS12 (bottom). The active site cysteine residue is indicated . See Fig. 7 for explanation of symbols.

As illustrated in Fig. 8 the first exon encodes the transit peptide plus 109 residues of the mature protein which is in marked contrast to the structure of the Acl genes. Both the first two exons and the first intron are relatively long compared to the other segments of the gene. Results of Southern analyses (Kauppinen unpublished) with the exon II-VI probe and DNA from the Bonus,

Betzes and the Chinese Spring - Betzes addition lines revealed that Ktzs12 is a single copy gene (Fig. 9) on chromosome 2. A DNA fragment containing Ktzs12 was isolated from a size selected genomic library and partially characterized (Kauppinen unpublished). Sequencing and comparison with the eDNA sequence divulged that the gene consists of 7 exons and 6 introns.

Kas 12 Probe

exon I exon II-Yl

23.1 - = -9 .4- = == = - -= 6 .6- =

4 .4- - -3.7-

B E X B E X

Fig. 9. Schematic illustration of a Southern analysis of Bonus barley DNA digested with BamHI (B), EcoRI (E) and Xbal (X). Probes to detect sequences homologous to the NH2- and C02H-terminal ends of mature KAS12 (exon I vs exons II-VI, see Fig. 8) disclose a marked difference between the two ends of the Ktzs12 gene.

Although more primary sequence data was available for the a- than the Ppolypeptide (Siggaard-Andersen et al1991), the oligonucleotide-PeR approach did not lead to isolation of Ktzs11 clones which might have been expected on the basis of the homology of the available sequences for the KAS 11 and KAS 12 proteins. The region of Ktzs12 coding for the mature protein was fused to the 3' end of the glutathion-S-transferase gene and expressed in E. coli. Polyclonal antibodies to the resulting purified fusion protein react at low dilutions with a-polypeptide containing fractions (Wissenbach and Kauppinen unpublished). These antibodies may thus provide a tool for isolating condensing enzymes less sensitive to cerulenin than synthase I, such as that in C16 elongase or that determined by the Q domain of the polypeptide encoded by cer-cqu. In this connection the observation that using exons II-VI as a probe in Southerns lights up only a single band while exon I, excluding the transit peptide coding segment, lights up at least 3-4 additional bands (Fig. 9) is intriguing. Do any of these bands represent Ktzs11 or a C16 elongase condensing enzyme gene? Possibly, although this suggestion conflicts with (i) the just noted failure to isolate Ktzs11 clones using the exon I probe and (ii) preliminary comparisons among all available FAS and polyketide condensing sequences, excluding Chs, which indicate a greater homology in the C-terminal halves than in the NH2-terminal halves (Siggaard-Andersen unpublished). Once the other members of the condensing gene family have been

obtained, the domains of the enzymes contributing to the diversity of products arising from the condensation elongation mechanism can be ascertained.

ACKNOWLEDGEMENTS

The reported research from the author's laboratory was in part financially supported by the Danish Biotechnology Programme 1991-1995, the Academy of Finland and the Deutsche Forschungsgemeinschaft.

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Molecular genetics of lipid synthesis in barley

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