Proc. Natl. Acad. Sci. USAVol. 91, pp. 12760-12764, December 1994Applied Biological Sciences

Targeting of the polyhydroxybutyrate biosynthetic pathway to theplastids of Arabidopsis thaliana results in high levels ofpolymer accumulationCHRISTIANE NAWRATH, YVES POIRIER*, AND CHRIS SOMERVILLEtDepartment of Plant Biology, Carnegie Institution of Washington, 290 Panama Street, Stanford, CA 94305

Communicated by W. L. Ogren, September 20, 1994 (received for review June 20, 1994)

ABSTRACT In the bacterium Akaligenes eutrophus, threegenes encode the enzymes necessary to catalyze the synthesis ofpoly[(R)-(-)-3-hydroxybutyrate] (PHB) from acetyl-CoA. Inorder to target these enzymes into the plastids of higher plants,the genes were modified by addition ofDNA fragments encod-ing a pea chloroplast transit peptide, a constitutive plantpromoter, and a poly(A) addition sequence. Each of themodified bacterial genes was introduced into Arabidopsisthaliana by Agrobacterium-mediated transformation, andplants containing all three genes were obtained by sexualcrosses. These plants accumulated PHB up to 14% of the dryweight as 0.2- to 0.7-,um granules within plastids. In contrastto earlier experiments in which expression of the PHB biosyn-thetic pathway in the cytoplasm led to a deleterious effect ongrowth, expression ofthe PHB biosynthetic pathway in plastidshad no obvious effect on the growth or fertility of the transgenicplants and resulted in a 100-fold increase in the amount ofPHBthat accumulated. We conclude that there does not appear tobe any biological barrier to high-level production of PHB inhigher plants. The high level of PHB accumulation also sug-gests that the synthesis of plastid acetyl-CoA is regulated by amechanism which responds to metabolic demand.

Poly[(R)-(-)-3-hydroxybutyrate] (PHB) and other relatedpolyhydroxyalkanoates are aliphatic polyesters with thermo-plastic properties. Alcaligenes eutrophus and many otherspecies of bacteria produce polyhydroxyalkanoates as acarbon reserve when grown in medium with an excess ofcarbon but limited in an essential nutrient, such as nitrogenor phosphorus. PHB is synthesized as granules of 0.2-1 Amand may accumulate up to 80% of the dry weight. In Alcali-genes eutrophus, PHB is synthesized from acetyl-CoA by theconsecutive action of three enzymes: 3-ketothiolase, ace-toacetyl-CoA reductase, and PHB synthase, which are en-coded by the phbA, phbB, and phbC genes, respectively (1).A wide variety of microorganisms can hydrolyze polyhy-

droxyalkanoate polyesters to monomers which are metabo-lized as a carbon source. Therefore, polyhydroxyalkanoateshave attracted interest as a potential source of renewablebiodegradable thermoplastics. The cost ofPHB produced bybacterial fermentation is significantly higher than the cost ofstarch or oil produced from agricultural plants. Therefore, thefeasibility of producing PHB in plants was initially exploredin a pilot experiment in which expression of the acetoacetyl-CoA reductase and PHB synthase of Alcaligenes eutrophusin the cytoplasm of transgenic Arabidopsis resulted in accu-mulation of granules ofPHB (2, 3). The polymer produced inplants was of high molecular weight and similar in structureand physical properties to bacterial PHB (4). However, theyield of PHB was low [20-100 ,g/g of fresh weight (g fwt)],and plants were severely stunted in growth. In addition, the

PHB granules were found in several subcellular compart-ments that included the nucleus, vacuole, and cytoplasm.The main metabolic role of cytoplasmic acetyl-CoA in

higher plants is thought to be as a precursor for mevalonatesynthesis. It has been estimated that the accumulation ofPHB at 100 ,ug/g fwt could account for as much as 50% ofthetotal flux through the mevalonate pathway. Thus, the lowyield of PHB and the growth inhibition observed in PHB-producing plants were hypothesized to be due to depletion ofthe cytoplasmic acetyl-CoA pool (2, 5). A possible strategyfor overcoming this problem is to change the subcellularlocation of PHB production (6). In plants, the plastid is thesite of fatty acid biosynthesis for membrane and storagelipids. Therefore, the flux through acetyl-CoA is high in theplastid. Furthermore, a high level of starch accumulation inplastids seems not to interfere with the function ofthe plastid,indicating that the organelle can accommodate the physicaldistortion associated with accumulation of insoluble storagecompounds.The feasibility of plastid-localized PHB production was

tested by targeting the three bacterial enzymes involved inPHB biosynthesis to the plastid. We report here that trans-genic Arabidopsis plants expressing all three enzymes in theplastid accumulated high levels ofPHB in the plastid withoutany readily apparent deleterious effects on growth and seedproduction. This observation indicates that there do notappear to be biological constraints which would preventhigh-level PHB production in plants and provides insight intothe mechanisms that regulate acetyl-CoA synthesis in plants.

MATERIALS AND METHODSPlant Material. Arabidopsis thaliana, race Rschew, was

transformed by the in planta method (7) with the binary Tiplasmid constructs pBI-TP-Thio, pBI-TP-Red, and pBI-TP-Syn, containing modified genes encoding for 3-ketothiolase,acetoacetyl-CoA reductase, and PHB synthase, respectively(6). Seeds developing from cross-pollinated flowers weregerminated on soil and hybrids were grown under naturallight at 16-280C.

Protein Analysis. For Western blot analysis and enzymeactivity measurements of 3-ketothiolase and acetoacetyl-CoA reductase, protein extracts of fresh leaf samples wereprepared as described (6) and assayed according to theprocedures of Senior and Dawes (8). For measurements ofPHB synthase accumulation, leafsamples were homogenizedin extraction buffer (100 mM Tris-HCl, pH 6.8/10 mMEDTA/4 mM 2-mercaptoethanol with phenylmethylsulfonylfluoride at 20 ,g/ml) and clarified by centrifugation for 5 min

Abbreviations: g fwt, g of fresh weight; PHB, poly[(R)-(-)-3-hydroxybutyrate].*Present address: Institut de Biologie et Physiologie Vegetales,Batiment de Biologie, Universite de Lausanne, CH-1015 Lausanne,Switzerland.tTo whom reprint requests should be addressed.

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at 5000 x g. The membranes and particulates were thensolubilized in extraction buffer containing 1% SDS and againclarified by centrifugation.

Analysis of PHB. For gas chromatographic analyses, 20-100 mg of leaf material was extracted three or four times for1 hr at 55°C with 50% ethanol and then with 100% methanol.Dry residues were extracted at 55°C with 0.5 ml ofchloroformfor 12 hr. The ethyl ester was produced by addition of 0.2 mlHCI and 1.7 ml ethanol and incubation for 4-6 hr at 100°C.Three milliliters of 0.9 M NaCl was added, the phases wereseparated, and the chloroform phase was analyzed on aHewlett Packard 5890 series II gas chromatograph (9). Bac-terial PHB (Sigma) was used as a standard. PHB granuleswere visualized by epifluorescence microscopy and trans-mission electron microscopy (2).

RESULTSTargeting of the PHB Biosynthetic Enzymes to the Plastid.

The three bacterial enzymes necessary for PHB production(i.e., 3-ketothiolase, acetoacetyl-CoA reductase, and PHBsynthase) were targeted to the plastid by fusing a DNAfragment encoding the transit peptide of the small subunit ofpea ribulose-bisphosphate carboxylase to the coding se-quence ofeachphb gene from Alcaligenes eutrophus (6) (Fig.1). To obtain constitutive expression of the genes in plants,the modified coding regions were fused to the cauliflowermosaic virus 35S promoter present in the binary Ti plasmidvector pBI121, resulting in the constructs pBI-TP-Thio,pBI-TP-Red, and pBI-TP-Syn. Three sets of transgenic Ara-bidopsis lines were established by transformation with thesevectors, each ofwhich expressed one of the three constructs.Transgenic lines which expressed the proteins were identifiedby using antibodies to the bacterial proteins (6) to probeWestern blots of SDS/polyacrylamide gels containing clari-fied leaf extracts from each of the transgenic lines. Theapparent molecular weights of the PHB enzymes on Westernblots indicated that they were proteolytically cleaved asexpected for enzymes which had been correctly targeted tothe plastid. Northern blot analysis of RNA from the trans-genic lines probed with the phb genes indicated that therelative amount of mRNA for each of the genes was corre-lated with the amount of the corresponding polypeptides asdetermined by Western blot analysis (data not shown).Heterozygous plants of the three transgenic lines harboring

the pBI-TP-Thio construct exhibited a 3-ketothiolase activityranging from 0.27 to 0.32 unit/mg of protein versus 0.005

ThiolaseTDV----ER G

small subunit of Rubisco-- .- ..-. .- .. ._ ..

[transit e tide mature protein1 linker reductaseM A S---V K C M Q V---T R D S R VYT Q R------H M G1 55 78

A T G------A K R

FIG. 1. Diagram showing the amino acid sequences of the threetransit peptide-Phb fusion proteins used to generate the transgenicPHB-producing plants (6). The constructs are composed of fourregions denoted by boxes: the 55-amino acid transit peptide from thepea ribulose-bisphosphate carboxylase (Rubisco) small subunit 3.6,the first 23 amino acids ofthe Rubisco small subunit, a short syntheticlinker sequence, and the complete coding sequences (except for theamino-terminal methionine) of the 3-ketothiolase (phbA), ace-

toacetyl-CoA reductase (phbB), and PHB synthase (phbC) genesfrom Alcaligenes eutrophus. The amino acid sequences between thejunctions, indicated by hyphens, have been described (10, 11).

unit/mg in wild type. Heterozygous plants of the seventransgenic lines harboring the pBI-TP-Red construct con-tained acetoacetyl-CoA reductase activities ranging from0.28 to 3.39 units/mg of protein, compared with 0.05 unit/mgin wild type (6). These levels of activity correlated with theamount of protein detected on Western blots and are similarto those seen in transgenic plants expressing the enzymes inthe cytoplasm (ref. 2 and unpublished results). Thus, additionof the transit peptide did not markedly reduce the catalyticactivity of these proteins. The amount of PHB synthaseactivity in the transgenic lines containing the PHB synthasegene was not assayed.

Construction and Analysis of Triple Hybrid Lines Accumu-lating High Levels ofPHB. A series of sexual crosses was usedto construct plant lines containing all three PHB enzymes.Three transgenic lines expressing high amounts of ace-toacetyl-CoA reductase (lines TP-Red-1, -2, and -3) werecross-pollinated with transgenic plants producing highamounts of PHB synthase (lines TP-Syn-1, -2, and -3). Theresulting hybrid TP-Red/TP-Syn plants expressing ace-toacetyl-CoA reductase and PHB synthase in the plastid didnot produce measurable amounts ofPHB in expanding leaves(<20 p,g of PHB per g fwt; data not shown). By comparison,transgenic plants producing the acetoacetyl-CoA reductaseand the PHB synthase to similar levels in the cytoplasmproduced PHB (2). A possible reason for this difference isthat plastids may not have sufficient 3-ketothiolase to supportPHB production. This is consistent with evidence that theearly steps of the mevalonate pathway leading from acetyl-CoA to isopentenyl pyrophosphate are absent in differenti-ated plastids (12, 13).

In order to establish a complete PHB biosynthetic pathwayin plastids, a transgenic Arabidopsis thaliana line having ahigh amount of thiolase (line TP-Thio-1) was cross-pollinatedwith the TP-Red/TP-Syn hybrids. Progeny from thesecrosses were tested for the presence of all three genes byprobing Western blots with antibodies against the threeproteins. From five combinations of crosses, a number ofhybrids were obtained having all three enzymes necessary forPHB production (TP-Thio-1/TP-Red-1/TP-Syn-1, TP-Thio-1/TP-Red-3/TP-Syn-1, TP-Thio-1/TP-Red-3/TP-Syn-2, TP-Thio-l/TP-Red-3/TP-Syn-3, and TP-Thio-1/TP-Red-2/TP-Syn-3). These hybrids produced PHB which was quantitatedby GC and identified by GC-MS analysis. The amount ofPHB in expanding leaves of 20- to 30-day-old plants rangedfrom 20 to 700 pg/g fwt (Fig. 2A). The triple hybridscontinued to accumulate PHB throughout the life of theplants, so that in the leaves of senescing plants levels ofPHBwere 8-13 times higher than the amount in expanding leaves(i.e., up to 7 mg of PHB per g fwt). This increase in PHBaccumulation with time was observed in plants with a lowinitial PHB content (100 pg/g fwt in expanding leaves,reaching 1.1 mg/g fwt in presenescing leaves) as well as inplants with a high initial PHB content (700 ,ug/g fwt inexpanding leaves, reaching 7 mg/g fwt in presenescingleaves). In a TP-Thio-1/TP-Red-1/TP-Syn-1 hybrid, themaximal amount ofPHB measured in the presenescing leaveswas 14% of their dry weight (10 mg/g fwt).

Plants producing PHB in the plastid showed wild-typegrowth and fertility even at the highest level of PHB accu-mulation (Fig. 3). No differences were observed betweenwild type and the transgenic PHB-producing plants in the rateof seed germination. However, plants that accumulated morethan 300-400 pg ofPHB per g fwt in expanding leaves couldbe distinguished from the wild type by visual inspectionduring the later stages of growth. In these plants, the leavesshowed slight chlorosis after 50-60 days of growth, suggest-ing that very high levels ofPHB accumulation (>3 mg/g fwt)are deleterious to normal chloroplast function (Fig. 4). Aquantitative analysis of the effects of PHB production on

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< 0.1 0.1-0.2 0.2-0.3 0.3-0.4 0.4-0.5 0.5-0.6 0.6-0.7

PHB [mg/g fresh weight]

< 1 1 -2 2-3 3-4 4-5 5-6 6-7

PHB [mg/g fresh weight]

FIG. 2. PHB accumulation in leaves of different hybrid TP-Thio/TP-Red/TP-Syn plants. (A) Measurements made on expandingleaves (20-30 days old). (B) Measurements made on mature leaves(50-60 days old).

growth and seed yield must await the development of linesthat are homozygous for all three transgenes.Appearance of PHB Granules. Transmission electron mi-

croscopy of leaf samples ofPHB-producing hybrids revealedthat PHB accumulated as agglomerations of electron-lucentgranules of 0.2-0.7 ,um, surrounded by a thin layer ofelectron-dense material (Fig. SA). PHB granules of similarappearance have been described for bacteria (14) and trans-genic plants (2). In plants expressing the plastid-targetedPHB enzymes, these granules were exclusively located in theplastids. This is in contrast to transgenic plants expressing thePHB biosynthetic pathway in the cytoplasm, which accumu-lated PHB in the nucleus, vacuole, and cytoplasm, but not inthe plastid (2). PHB granules were clearly distinguished fromstarch granules in form, appearance, and organization of thegranules as visualized by conventional electron microscopyfixation protocols. PHB granules are comparatively small,round, electron-lucent granules that form dense agglomera-tions (Fig. 5A). In comparison, starch appears as singular,electron-dense granules ofoval form, surrounded by a diffusearea (Fig. SB).

In triple hybrids producing only a low amount of PHB inexpanding leaves, it appeared that not all chloroplasts accu-mulated PHB. The reason for this is not known. There wereno indications of differential expression of the transgenes indifferent tissues. Conceivably it could reflect the action ofcosuppression, which may lead to a mosaic of differentialexpression, as observed in Petunia (ref. 15; see Discussion).In old leaves of triple hybrids accumulating high levels ofPHB, almost all chloroplasts contained PHB.

Levels of PHB Accumulation. To gain insight into themechanisms responsible for the different amounts of PHBproduced in the various hybrids, the level of the 3-ketothio-lase and acetoacetyl-CoA reductase activity present in PHB-producing plants was measured in clarified crude leaf protein

FIG. 3. Fully developed rosettes ofwild type (WT) and transgenicArabidopsis expressing the PHB biosynthetic enzymes in the plastid(A) and in the cytoplasm (B). The leaves of the hybrid TP-Thio-l/TP-Red-1/TP-Syn-1 producingPHB in the plastid contained -1.2 mgofPHB per g fwt (PHB+ in A). The Red/Syn hybrid expressing thePHB enzymes in the cytoplasm contained -100 ,ug ofPHB per g fwt(PHB+ in B). Wild-type and transgenic plants were grown underidentical conditions.

extracts. The amount ofPHB synthase protein was analyzedby Western blot analysis.The 3-ketothiolase activity in TP-Thio-1/TP-Red/TP-Syn

hybrid plants ranged from 0.001 to 0.8 unit/mg of protein. Incontrast, the 3-ketothiolase activity in the parental TP-Thio-1plants was 0.84 ± 0.15 unit/mg. Southern blot analysis of theTP-Thio-1 line revealed four bands which hybridized to the

FIG. 4. Effect ofhigh-level accumulation ofPHB in the plastid onleaf pigmentation. Shown are a leaf of a 50-day-old transgenicArabidopsis hybrid producing 700 pg ofPHB per g fwt in the plastidof expanding leaves (Right) and a wild-type leaf (Left).

A WT PHBRA14

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phbA gene, probably corresponding to several independentintegrations of the construct (data not shown). Therefore,segregation ofthe different pBI-TP-Thio constructs may havecaused the range of 3-ketothiolase activities in the F1 gener-ation.The acetoacetyl-CoA reductase activity in TP-Thio-1/TP-

Red-1/TP-Syn-1 hybrid plants ranged from 0.007 to 0.78unit/mg of protein. The acetoacetyl-CoA reductase activityin the TP-Red-1/TP-Syn-1 hybrid, which served as a parentin the cross with TP-Thio-1, was 2.09 ± 0.23 unit/mg.Southern blot analysis and segregation analysis of the select-able marker indicated that TP-Red-1 plants harbored theconstruct pBI-TP-Red at a single integration site. Westernblot analysis indicated that the decreased activity was cor-related with an -10-fold decrease in protein in the clarifiedextracts ofPHB-producing plants. Similarly, PHB-producinghybrid plants TP-Thio-1/TP-Red-3/TP-Syn-1 A, TP-Thio-1/TP-Red-3/TP-Syn-1 B, and TP-Thio-1/TP-Red-2/TP-Syn-3also had widely varying and unexpectedly low reductaseactivities in comparison to their parent plants and the de-crease in activity was correlated with low amounts of proteinin Western blot analysis.

FIG. 5. Transmission electronmicrograph of thin section from aPHB-producing triple hybrid ex-pressing the PHB enzymes in theplastid (TP-Thio-1/TP-Red-1/TP-Syn-1). (A) Agglomeration ofelec-tron-translucent granules in thechloroplast of a mesophyll cell isindicated by the arrows. The plantwas placed in darkness for 48 hrbefore sampling in order to re-move starch. (B) Transmission

s electron micrograph of a thin sec-tion of a chloroplast from a wild-type plant. (Bars = 1 ,um.)

The presence of PHB synthase was investigated by West-ern blot analysis of leaf protein extracts of PHB-producingplants. In TP-Thio/TP-Red/TP-Syn hybrid plants, PHB syn-thase was not detected in the soluble protein fraction. PHBsynthase was readily detectable on Western blots of SDS-solubilized membrane and particulate fractions of plant ex-tracts. In contrast, in plants transformed only with thepBI-TP-Syn, PHB synthase was detected in the solublefraction (Fig. 6). This finding suggests that the PHB synthaseis closely associated with the PHB granules formed in plants,as has been found for the PHB synthase in bacteria (16, 17).Attempts to establish a correlation between PHB accumu-

lation and the measured 3-ketothiolase and acetoacetyl-CoAreductase activities in developing leaves ofthe various hybridlines failed to provide a clear indication of what limits PHBaccumulation in these plants. This may be related to theunexplained variability in the amount of enzyme activity inthe various hybrids as noted above. In general, plants withthe highest levels ofboth 3-ketothiolase and acetoacetyl-CoAreductase activities had the highest levels ofPHB, and plantswith low levels of both activities had the lowest levels ofPHB. There was no obvious plateau in PHB production at thehighest levels of measurable enzyme activity. Thus, it ap-

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C 1 2 3 4

- 63 kDa

FIG. 6. Western blot analysis using the anti-PHB synthase anti-body to probe protein extracts of soluble proteins (lanes 1 and 2) andsolubilized proteins of the membrane and particulate fraction (lanes3 and 4) of two different transgenic TP-Thio-1/TP-Red-1/TP-Syn-1plants. Lane C contained an extract of soluble proteins ofa TP-Syn-1plant.

peared that PHB accumulation was limited by the amount ofenzyme activity rather than by the availability of acetyl-CoA.

DISCUSSIONArabidopsis plants engineered to express the PHB biosyn-thetic pathway in their plastids accumulate PHB to highamounts. By changing the location of PHB production froma cellular compartment with a low flux through acetyl-CoA(cytoplasm) to a compartment with a high flux throughacetyl-CoA (plastid), the maximal level of PHB productionwas increased 100-fold. In contrast to the previous generationof transgenic plants, in which the PHB enzymes were tar-geted to the cytoplasm (2), plants producing high levels ofPHB in the plastids appeared to have normal growth andvigor. Thus, it appears that depletion of metabolites fromessential metabolic pathways of the cytoplasm may havebeen the reason for the deleterious effect ofPHB productionin plants expressing the PHB enzymes in the cytoplasm (2).Alternatively, since PHB granules were also located in thenucleus, it might be that the interaction of the PHB granuleswith nuclear constituents was a cause of the deleterious effectof PHB production in these plants (2).

In plastids of leaves, a surplus of assimilated carbon istransiently deposited in the form of starch during the photo-synthetically active period. In Arabidopsis, transient leafstarch accumulates to 12 mg/g fwt at the end of a 12-hrphotoperiod and is largely degraded during the night (18). Asfar as we are aware, PHB cannot be reutilized by the plantand, therefore, acts as a terminal carbon sink. PHB accu-mulates up to 10 mg/g fwt (14% of the dry weight) in leavesduring an extended growth period ('60 days). Thus, theaverage daily rate of PHB synthesis is relatively low com-pared with starch biosynthesis.

In plastids of leaves, acetyl-CoA is mainly utilized asprecursor of fatty acid biosynthesis for membrane lipids. Thenormal vigor of the PHB-producing plants indicates that thesupply of acetyl-CoA for synthesis of fatty acids was suffi-cient in spite of the competition for acetyl-CoA for PHBsynthesis. Mature leaves of Arabidopsis contain -4.2 mg ofglycerolipid per g fwt (19). Thus, if we ignore the fact thatmembrane lipids are probably degraded and resynthesized tosome extent, the accumulation ofPHB appears to have beenassociated with a significant increase in the net flux throughthe plastid acetyl-CoA pool. The implication is that a mech-anism exists to enhance synthesis of acetyl-CoA in responseto demand.

Analysis of the activities and protein levels of the 3-ke-tothiolase and acetoacetyl-CoA reductase revealed that in thetriple hybrid lines the PHB enzyme activities varied widelyand were generally lower than the activities observed in theparental lines. For example, in a cross of a hybrid plantcarrying a single integration of pBI-TP-Red and a singleintegration of pBI-TP-Syn with a plant carrying severalintegrations of pBI-TP-Thio, all hybrid plants inheriting the

pBI-TP-Red construct are expected to produce the sameamount of reductase. Instead, the amount (measured asactivity of the reductase) varied from 1% to 30% of thereductase activity in the parent plant. The reason for the lowamount of reductase activity might be that all transcripts ofthese modified genes contain a common sequence of 243 bpencoding the transit peptide (6). Negative effects on geneexpression resulting from the presence ofhomology betweengenes in transgenic plants is caused by an unexplainedmechanism termed cosuppression (15, 20). Cosuppressionmay also provide an explanation of why, upon examinationby transmission electron microscopy, the plastids of somecells had large numbers ofPHB granules whereas plastids inadjoining cells showed no PHB granules. Whatever the basisfor these observations, we believe they are related to theparticular constructs used and do not bear directly on thebasic biological issue addressed by these experiments. Themain result is that there appear to be no biological bafflers toPHB production in plastids.

In addition to the fact that PHB production in plants maybe of applied significance, PHB represents a new sink forcarbon which may afford new opportunities to study carbonflux through major biosynthetic pathways and give insightinto the regulation of acetyl-CoA synthesis in plastids.

We thank Jacqueline Wood and Karen Klomparens for assistancewith microscopy. This work was supported in part by grants from theNational Science Foundation (MCB 9305269) and the U.S. Depart-ment of Energy (DE-FG02-94ER20133). C.N. was the recipient of afellowship from the German Scholarship Foundation (program sup-ported by BASF Aktiengesellschaft). This is Carnegie Institution ofWashington Department of Plant Biology publication no. 1232.

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