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PHYSIOLOGIA PLANTARUM 97: 3 11-320, 1996 Primed in Deimmrk - all lighls reserved

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Partial blocks in the early steps of the chlorophyll synthesis pathway: A common feature of chlorophyll Z>-deficient mutants Tanya G, Falbel and L. Andrew Staehelin

Falbel, T. G. and Staehelin, L. A. 1996. Partial blocks in the early steps of the chlorophyll synthesis pathway: A common feature of chlorophyll 6-deficient mutants. — Physiol. Plant. 97: 311-320. We have analyzed precursor pools in the chlorophyll (Chl) synthesis pathway for a set of eighteen well studied Chi fi-deficient mutants in monocotyledonous (barley, maize and wheat) and dicotyledonous plants (Antirrhinum, Arahidopsis, soybean, tobacco and tomato) thai form abnomial thylakoid membrane systems. All of these mutants have a partial block in Chl synthesis and nearly all of them accumulate protoporphyrin IX (Proto), the lasl porphyrin compound common to both heme and Chl synthesis. The lai-ge number of mutants al several genetic loci affecting this critical branchpoint in telrapjTrole biosynthesis suggests that the Mg-chelatase enzyme, catalyzing the first committed step of Chl biosynthesis, is a multimeric complex composed of the products of some of these genetic loci, and perhaps regulated by others. We hypothesize that these mutants are Chl 6-deficient and have reduced amounts of light-han'esting antenna complexes (LHCs) and deveiop' abnormal thylakoid membranes as a direct result of limited Chl synthesis. The observed bottleneck in Chl synthesis can also explain the light-intensity-dependent and temperature-dependent expression of the mutant phenot>'pe. This hypothesis offers a simple explanation for the wide vanety of phenotypes that have been reported for the many Chl-deficient mutants in the literature. Our findings are alsO' consistent with the notion that Chl b is made from *'lefl over" Chl a molecules and suggest that the Ch! /^-deficient mutants should be considered more appropriately as leaky Chl-deficient mutants. Key words - Chlorophyll biosynthesis, chlorophyll-deficient mutants, photosynthesis. T. G. Falhei (corresponding author, e-maif falbei@nmcc.wisc.edu: present address: Dept of Horticulture, 1575 Linden Drive, Univ. of Wisconsin, Madison. WI 53706, USA) and L. A. Staeheiin, Dept of Moiecuiar. Cellular and Developmental Biology', Univ. of Colorado. Boulder. CO 80309, USA.

introduction In the 1970s many genetic loci were identified that affect the chlorophyll (Chl) synthesis pathway of higher plants io ways that resulted in ultrastructural changes in mutant chloroplasts (reviewed by von Wettstein etal, 1971, Henningsen et al. 1993), More recently, Klein et al, (1988) and Kim et al, (1994) used a Chl-less barley mutant and performed experiments to demonstrate that the accumulation of chloroplast-encoded Chl binding proteins requires the presence of Chl a, an important link between the biosynthetic pathways for Chl and its binding proteins. This litik is a tight one, since in plants one finds littie or no free Chl or Chl-apoproteins, It is largely un-

knovt-n how a plant determines the appropriate amounts ^^ j ^ ^ ^ , ^^^ ^^^ ^^ ^.^ o/b-binding antenna proteins per reaction center (the photosynthetic unit size), which are strongly dependent upon the plant's light environment, What are the controls involved in partitioning the biosynthesis of Chl a and Chl b, and what determines the choice between the synthesis of reaction centers aod antenna molecules under different growth conditions? The maize mutant, oil yello-rv-yellow green.. (OY-YG), also known as Oy-700, contains significantly reduced amounts of Chl b and reduced numbers of antenna complexes and membrane stacks when grown under high light conditions (Greene et al, 1988a), suggesting that it might be over-responding to changes in light intensity.

Received 31 August, 1995; revised 5 Febniao', 1996 Physiol. Plant. 97, 1996

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Since another mutant at the same oil yellow locus had been shown to have a partial block in Chi biosynthesis (Mascia 1978), we began a general study of the consequences of mutants with partial bottlenecks in the Chi synthesis pathway to investigate how the Chi synthesis pathway might be involved in determining a plant's photosynthetic unit size. Our first report on this work demonstrated that a set of similar wheat and barley Chi bdeficieot mutants also had pattial blocks in the Chi synthesis pathway (Falbel and Staehelin 1994), To test the generality of this finding further, we compiled from the literature a list of eighteen Chi i)-deficient mutants that exhibit typical features of the nnultifaceted structural and light intensity-dependent pheootypes of Chi i>-deficient maize, wheat and barley mutants already studied in otir laboratory (Alien et al, 1988, Greene et al, 1988a,b, Falbel and Staehelin 1994), In addition, we included mutants that were yellowgreen in color and showed genetic behavior similar to either the semidominant OY-YG maize mutant, the recessive and viable CD3 wheat mutant, or the recessive and seedling lethal viridis k23 barley mutant (demonstrated also to have a partial block in the pathway, see below). The collection was not intended to be an exhaustive list of mutants, A series of similar sweetclover mutants have also been studied (e,g. Markwell et al, 1985), Not included in this study are a number of yellow-green mutants in barley (Simpson and von Wettstein 1980, Simpson et al, 1985 ) and mutants in other species (described in King 1974) such as tomato (Xa-3. Jau) (Clayberg et al. 1966), Antirrhinum (Aur, Aurahnlich) (King 1974), cotton (yellow-green) (Rhyne 1954), seven mutants in Brassica eampestris (Stringam 1973, 1978) and many similar mutants in pea (Blixt 1972), We consider the Chi f>-deficient and Chi b-]ess mutants to belong to different subclasses of Chl-deficient mutants. The Chi b-]ess mutants of barley (cblorina f2) and Arabidopsis (chl) are not included in this report, because in general they have nearly normal amounts of Chl a and they synthesize no Chl b regardless of the light condition (SimpsoD et al, 1985, Murray and Kohom 1991), They are thought to possess a lesion in the Chl b biosynthetic machinery itself. Some reported alleles of these Chl i)-less mutants both in barley and Arabidopsis are leaky in their block in Chl b synthesis and have pigment contents resembling the Chi bdeficient mutants to some extent. However, these mutants have not been characterized well enough to be included in this work. If leaky alleles of a Chl i-less mutant can indeed become Chl i>-deficient mutants (also deficient in Chl a), that would imply a direct regtiiatory link between a step of the main Chi synthesis pathway and the biosynthetic machinery for Chl b. The existence of such a relationship is not clear from our present data, so we have restricted the mutants in this report to those Chl ii-deficient mutants that have been demonstrated to express the light intensity-dependent multi-faceted phenotype described above.

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In the present paper, we demonstrate that a partial block in Chl biosynthesis is a new common feature of all eighteen of the Chl ii-deficient mutants that we have assembled for this study. For many of the mutants, we do not know if this block in the pathway is the result of direct or indirect effects of mutations on Ch! synthesis. Surprisingly, all but one are blocked at the same point in the pathway, at protoporphyrin IX — the branch point between heme and Chl synthesis. Although it has been known for some time that reaction centers bind Chl a with higher priority than antenna proteins (e.g, Akoyunoglou and Akoyunoglou 1985), a block in the pathway had not been demonstrated for the set of mutants as a whole. Therefore, we hypothesize that the limited Chl synthesis could be directly responsible for the reduced amounts of LHCs and abnormal thylakoid membrane development io these Chi 6-deficient mutants.

Dcseription of the Chl /i-deficient mutants analyzed The series of mutants assembled for this study have a variety of origins: some were naturally occurring mutations, others were generated through the use of a variety of mutagens. We chose lo use such a varied collection in an effort to eliminate bias and be as complete as possible without doing an exhaustive study of all available mutants. The light intensity-dependent CD3 mutant of hexaploid wheat and four related mutant alleles (Driscoll's chlorina, chlorina-1, chlorina-214 and CDd-1) have been the main focus of much of our recent work (Allen et al, 1988, Falbel and Staehelin 1994, Falbel et al, 1994), These mutants all survive to maturity as homozygotes, Driscoll's chlorina was discovered in a field, and the remaining four mutants were generated by ethylmethane sulfonate (EMS) mutagenesis. In our previous work we compared the wheat mutants to the recessive, light- and temperature-sensitive Chlorina-104 mutant of barley because of phenotypic similarities to OY-YG and CD3 mutants (Knoetzel and Simpson 1991), Chlorina-104 was generated by Na-azide mutagenesis (Simpson et al, 1985), The barley mutaot viridis-k^^ (vir-lc^) was included for comparison because of its similarities to the wheat mutants above, having both unusual thylakoid ultrastructure (Nielsen et al, 1979, Simpson and von Wettstein 1980) and a strong but incomplete block in the Chl synthesis pathway. The block is in a later step in the pathway than the block in wheat mutants; in the conversion of Mgprotoporphyrin IX (Mg-Proto) to protochlorophylhde (Pchlide) (Simpson and von Wettstein 1980, Henoingsen et al, 1993), This seedling lethal mutant makes some Chl and has limited photosynthetic capacity, Vir-tr^ is a recessive mutation generated by X-ray mutagenesis. In addition to the incompletely dominant OY-YG mutant of maize (Hopkins et al, 1980a,b), four other incompletely dominant Chl-deficient mutants have been dePhysiol. Plant, 97, 1996


scribed in the literature: the Sulfur mutant of tobacco (Burk and Menser 1964), the YH mutant of soybean (Palmer and Kilen 1987), and the Xantha and Xantha-2 mutants of tomato (Young and MacArthur 1947, Butler and Chang 1958, Grober 1963), These mutants have unstacked thylakoid mennbranes and their phenotypes are light-intensity dependent {e,g, Schmid et al, 1966, Schindler et al, 1994), All of these mutants segregate 1:2:1 (green:yellow-green:yellow) when heterozygous lines are self-fertilized, Honnozygous mutants are lethal, and the heterozygous yellow-green individuals show a light-inteosity-dependent phenotype. The Oy700 and Xantha-2 mutants were generated by mutagenesis, and the Xantha, Sulfur and Yll mutants arose spontaneously. The chlorina-} 6! mutant of barley was also included in this study because of its incomplete domioance, but has not been well characterized previously. The allelic yellow-green cs and cb42 mutants in Arabidopsis thaliana (Fischerova 1975, Koncz et al, 1990) and the olive mutant of Antirrhinum (Hudson et al, 1993) also showed a phenotype similar to that of the rest of the mutants in the study and were included because their DNA sequences were known and were homologous to Rhodobacter genes required for bacteriochlorophyli biosynthesis (Armstrong et ai, 1993, Hudson et al, 1993), The X-ray-induced ch42 mutant is a lethal allele and can only survive on medium that contains sucrose, while the cs and olive mutants were generated by transposon tagging and survive to maturity on soil as homozygotes. Other mutants that were included were the the recessive Jg mutant (Nolla 1934) of tobacco because of its similarity to Su, and the recessive y9 mutant of soybean (Eskins et al, 1981, Droppa et al, 1988, Ghirardi and Melis 1988) because of its similarity to Yll. Both of these mutants arose spontaneously and were discovered in a field. The recessive ch5 mutant of Arabidopsis thaliana (Koornneef et al. 1983) is a commonly used genetic marker line originating from EMS mutagenesis that expresses a light-intensity dependent Chl-deficient phenotype. Abbreviations - ALA, 5-aminolevulinale; Chl, chlorophyU; EMS, ethylmethane sulfonate; LHC, lighl-hai-vesting antenna complex; Mg-Proto, Mg-protoporphyrin IX; Pchlide, protochloropJiyllide; Proto, protoporphyrin IX; RC, reaction center.

Materials and methods Plant and seed sources Dicotyledonous plants Antirrhinum. - Wild type Antirrhinum, majus plants and the olive mutant plants were obtained from Dr Andrew Hudson, University of Edinburgh, Scotland, Arabidopsis. — Wild type Arabidopsis thaliana ecotypes Columbia and Dijon, and mutants cs (parent: Columbia) and ch42 (parent: Dijon) were obtained from Dr Csaba Koncz, Max Planck Institiit fiir Zuchtungsforschung, Cologne, Gennany, The ch5 mutant was obtained from Physiol. Plant. 97, 1996

both the Ohio State Arabidopsis Biological Resource Center aad the Nottingham Arabidopsis Stock Center, Soybean, - Wild type soybean Glycine max, and mutants )'9 and yll were obtained from Dr Kenneth Eskins, University of Missouri, Tobacco, - Nicotiana tabacum mutants Su and yg were obtained from Dr Verne Sisson, Tobacco Stock Center, USDA Crops Research Eaboratory, Oxford, NC, Wild type seed was produced by selfing wild type progeny from the selfed Su heterozygotes. Tomato, -Lycopersicon escuientum mutants XG, andXa2 were obtained from Dr Charles Rick, University of California at Davis, Wild type seed was produced by selfing wild type progeny froro the selfed Xa-2 heterozygotes, Monoeotyledonous plants Barley, - Wild type barley Hordeum vulgare strain "Svalov's Bonus" and mutant barley chlorina-104. chlorina-161. and viridis k23 were obtained from Dr David Simpson, Carlsberg Research Laboratories, Copenhagen, Denmark. Wheat, — Wild type hexaploid wheat Triticum aestivum strains ND496-25 (CD3 parent strain), and "Waldron", and tetraploid wheat Triticum turgidum strain "Langdon16'" (CDd-1 parent straiti) and all mutant wheat strains (CD3; chlorina-l, chlorina-2i4, Driscoll's chlorina, CDd-1) were obtained from Dr Murray Duysen, North Dakota State University, Triticum aestivum strain "Alex" a close relative of ND496-25 was obtained from AgriPro Seeds in Berthoud, Colorado, Growth conditions

Plants were grown in moist vermictilite (monocotyledons), or a veniiiculite:potting soil (1:1, v/v) mix (dicotyledons), Arabidopsis plants were germinated on 0,5 MSAR medium (Koncz et al, 1990 and C, Koncz, personal communication: 10 g T' sucrose for mutants, 5 g T' sucrose for wiid type) on Petri plates, Monocotyledonous plants and soybean and tobacco plants were grown in a growth chamber at 24°C under a 14-h light/10-h dark cycle under cool white fluorescent light (~ 100 ^lmoi m"" s"'. Philips F30T12/CW/RS, Somerset, NJ, USA) for approximately 14 days (3 weeks or more for tobacco). Antirrhinum and tomato plants were grown for 3 weeks or more under greenhouse conditions. Wild lype and cs mutant Arabidopsis plants were grown under the same growth chamber conditions (light intensity was ca 30 jimol m^" s^^ for the ch42 mutant) for 3 ^ weeks until the plants had at least 6 leaves but had not yet begun to flower.

Pigment determination

Potential bottlenecks in the Chl synthesis pathway were determined by incubating the plants with 5-aminolevulinic acid (ALA) in darkness. For Arabidopsis plants 313


this was done by adding 3 ml of a solution of ALA (10 mM ALA, 5 mM MgClo, 10 m^^ phosphate, pH 7) to the agar on which the piants were growing (on 85-mm diam, Petri dishes), and incubating the plants in darkness for 12 h. For all other piants, this was done by adding 1 ml of the same solution of ALA to a 13 x 100 mm culture tube, immersing (for monocotyledons) plants cut at soil level, and immersing (for dicotyledons) the petioles of individually cut leaves, and incubating in darkness for 12 h. The parts of the leaves that were submerged were not used. Leaf material (approximately 0,5 g) was ground in 7 ml of ice-cold acetone:0. IM NH4OH (9; 1) in a "Tissumizer" (Tekmar) homogenizer for 15 s under a green safelight. Steady-state pools of Chl precursors were determined for plants untreated with ALA, Daytime pools were extracted from plants homogenized within 10 s of being removed from the growth chamber, and nighttime pools were extracted from plants homogenized under a green safelight, as controls for the ALA treated plants (data not shown). Absolute amounts of Chl precursors were not determined, because they mean very little in ALA feeding experiments. In our experience ALA uptake is not reproducible between experiments. Because chlorophyll synthesis has a light-dependent step, increasing amounts of Pchlide will accumulate in the dark during an ALA incubation over time. In this 12-h experiment, the relative proportion of the three prectirsors was reproducible regardless of uptake and therefore the results are expressed as mol percent of total porphyrin. Additionally, the method of ALA treatment varied among plants. Most plants were cut and dipped into an ALA solution for 12 h in the dark. For Arabidopsis plants, similar results were obtained when either the agar on which the plants were growing was soaked with ALA or when the plants were cut and dipped in a large welled dissection slide filled with a puddle of ALA, This was not the case for tobacco seedlings grown on agar plates. When ALA was added to the agar, very little was taken up through tobacco roots, and no blocks were observed (not shown). Thus, tobacco seedlings were treated by dipping cut leaves into a solution of ALA. Painting or spraying the leaves with ALA resulted in very irreproducible ALA uptake. For all extracts, insoluble material was pelleted at 4째C for 10 min at 12000 g. The supematant acetone extract was stored at -20째C until analysis, and was stable for several weeks at this step. After removal of the mature Chls by two hexane extractions, Chl precursors protoporphyrin IX (Proto), Mg-protoporphyrin IX (MgProto), and protochlorophyilide (Pchlide) were quantitated from these isolates by spectrofluorimetry, Spectroscopic quantitation of Chl precursors in the hexatie-extracted acetone residue was done on an SLM 48000 (Champaign, IL, USA) or Perkin Elmer LS50 (Norwalk, CT, USA) spectrofluorimeter as described by Tripathy and Rebeiz (1985), Quantitation was based upon standard curves generated for each Chl precursor (detailed in T. O. Falbel 1994, The:sis, University of Colorado, Boul314

der, CO, USA), using the millimolar extinction coefficients in 80% acetone reported by Gough (1972), Nearly all pigment determinations were repeated at least twice, on separate occasions, and the error between determinations was less than 5%. The Arabidopsis ch5 mutant was only analyzed once because of problems with seed germination that could not be resolved within the time available. Results Bottlenecks in the Chl synthesis pathway can be determined by two methods, 5-aminolevulinic acid (ALA) feeding (Fig. 1) and direct fluorescence spectroscopy (Fig, 2). The ALA feeding experiments involve overloading the pathway with the precursor ALA and monitoring the accumulated precursors by absorption spectroscopy (Gough 1972) or fluorescence spectroscopy (e.g, Falbel and Staehelin 1994), The direct fluorescence spectroscopy approach can be used to detennine the natural levels of the precursor pools during the light cycle in vivo without first perturbing the pathway with ALA. However, because the pool levels are very low, they can only be detected with very sensitive fluorescence techniques which limit their use in plants with certain types of autofluorescence. We have also found that because the precursors are in constant flux through the pathway, it is also critical that the plants be homogenized immediately (< 10 s) after being removed from their light environment (T, G, Falbei 1994. Thesis, University of Colorado, Boulder CO, USA), Ideally one would like to confirm the bottleneck for each mutant by both methods. However, in this study technical problems (autofluorescence of samples, poor germination and limited availability of mutant seeds) prevented us from analyzing some of the mutants using both types of measurements. Nevertheless, the general agreement in the results obtained by the two techniques would seem to validate both approaches. Determination of bottlenecks in the pathway by ALA feeding Figure 1 illustrates the relative pools of three porphyrin precursors (Proto, Mg-Proto, and Pchlide) of the eighteen mutants quantitated by fluorescence spectroscopy after treatment with ALA, With the exception of the yg tobacco and the vir-lt.23 barley, these mutants all accumulate relatively more Proto and less Pchlide than wild type. Because all of the mutants being considered have only partial blocks in Chl synthesis, all mutant and wild type plants accumulate only Pchlide in the dark without ALA treatment (not shown). The wheat mutant Driscoll's chlorina has been described previously (Newell and Rienits 1975) to accumulate Proto, and the barley vir k23 mutant has been described previously (Simpson and von Wettstein 1980) to accumulate Mg-Proto after treatment with ALA, Physiol. Plant. 97, 1996


Monocotyl ed'on s

Dicotyledons Antirrhinum

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S Mg-Proto D Pchlide

Fig 1 Porphwin pools after ALA incubation of wild type and mutant planls quantifred by spectrofluorimetrj' and expressed in mo) percent of the three compounds measured: protoporphyrin IX (Proto), Mg-protoporphyrin IX (Mg-Proto), and protochlorophyllrde (Pchlide) Presentation of the data shown m Figs 1 and 2 as raol percent of eaeh precursor is possible in these studies because the total preeursor poo] in each mutant was about the same as the total precursor pool in wild type for the ALA feeding expenments (see Fie 2 legend See also the note in the Materials and methods section regarding the reproducibilitj' of the absolute amounts ot each precurso? accumulated in the ALA feeding experiments). Without ALA U'eatment, wild type and all mutants accumulate only Pchlide (not shown) With ALA treatment, the wild type contains significantly more Pchlide than do the mutants. A relative increase in Proto or Mg-Proto accumulation along with a relative decrease in Pchlide accumulation for each mutant compared to wild type is indicative of a block in the pathway. For wheat samples, wt 4 designates the tetraploid wheat parent strain of ftie tetraploid tU-dy mutant wt 6 designates the hexaploid wheat parent strain of the CDS mutant used as a control sample for all of the hexaploid wheat mutants The data generated for the monocots (barley and wheat) and l\e Arabidopsis cs and eh42 mutants are parts of more detailed characterizations, of these mutants (reported elsewhere) bot are included in this figure and in Fig^ 2 for con^ipanson porposes the wheat and barky mutant characterization has been published previously ( F a t e l and Staehelin 1994), and the report on the Arabidopsis mutant characteriza.ion is currently m preparation (P Putnoky, T. Falbel and C. Koncz unpublished data). The as^terisb bring attention to the fact * a t the barley viridis 123 and Arabidopsis eh5 mutants accumulate Mg-Proto rather than Proto like the rest of the mutants The point of the block for the Driscoil's chlorina mutant and the barley viridis k23 mutant had both been established previously (see text) We have confirmed the presence of the block for each of these two mutants as a part ot this study.

Physiol. Plant. 97, 1996

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Monocotyledons

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Fig. 2. In vivo poi"phyrin poois measured in wiid type and mutant planls quantified by spectroiHuorimetry and expressed as mo] percent of each precursor as in Fig. 1. All samples were homogenized within 10 s of being removed from the growth chamber. With the exception of the barley viridis k-23 mutant, noted by an asterisk, all mutants show bottlenecks at PrO'to. The barley mutant shows a bottleneck at MgProto. The # sign denotes the fact that the size of the precursor pool for the homozygous lethal barley and soybean mutants was significantly smaller than the other pools, bul was 100% Proto for these two muLants. Homozygous mutants for the tobacco Su and tomato Xa and Xa-2 strains generated insufficient material to measure the accumulation of any precursors.

mol percent total porphyrins

• • Proto

S Mg-Proto G Pchlide

Determination of bottlenecks from in vivo levels of Ch! precursors As illustrated in Fig, 2, a block in the pathway at Proto is clearly evident for all of the Arabidopsis, Antirrhinum, soybean, tobacco, tomato, barley and wheat mutants except the Arabidopsis ch5 mutant, and the vir k23 of barley, but including the yg tobacco mutant which showed no block in the ALA experiment (Fig, 1). The barley c]61 homozygote and the soybean Yll homozygote show total blocks at Proto; heterozygotes show partial blocks. In general, the daytime levels of porphyrin precursors (Fig. 2) are consistent with the results of the ALA experiment (Fig, 1), Discussion Complexity of the first committed step of Chl synthesis Unexpectedly, among the eighteen Ch! 6-deficient mutants in eight species selected for this study all but two accumulate the same Chl precursor, protoporphyrin IX.

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Only the Arabidopsis ch5 and the barley vir-k23 mutants are blocked at a later step. Figure 3 shows a compilation of all characterized Chi synthesis mutants of plants, algae and photosynthetic bacteria that accumulate Proto and Mg-Proto (Gough 1972, Wang et al, 1975, 1977, Mascia 1978 and reviews by von Wettstein et al, 1971, 1995, Henningsen and Stummann 1982, Baoer et al, 1993, Henningsen et al, 1993), Clearly, mutants that accumulate Proto exist commonly among all photosynthetic organisms. Of the higher plant mutants listed in Fig, 3, the loci identified by the Arabidopsis cslch42 mutant and the olive mutant of Antirrhinum majus, as well as their homologous loci in barley {xan-flolive) and (xan-hlch42) have been cloned and sequenced (Koncz et al, 1990, Hudson et al, 1993, von Wettstein et al. 1995), Ch42 and olive are the plant homologs of Rhodobacter genes bchi and bchH, respectively, which are involved in the Mg-insertion step of bacteriochlorophyll synthesis (Bauer et al, 1993, Gibson et al. 1995), The plant proteins are also directly involved in Mg-chelatase activity (T. Falbel, J, Physiol. Plant. 97, 1996


(1986) showed that the suifur locus in tobacco had at least five antimorphic alleles of various strengths. Recent cloning and sequencing of the sulfur gene has demProtoporphyriti IX onstrated that it identifies a locus homologous to the m^j Rhodobacter bchI and Arabidopsis ch42 components of the Mg-chelatase (L. Nguyen, North Carolina State UniCttlemytSomonas bi-i, brc Mg-Protoporphyrin IX ChlotiellB W5B versity, personal communication). We showed in our â&#x20AC;˘ bcM,, bc-hH, b previous study (Falbe! and .Staehelin 1994) that five dosArabSttopsis cR^ Mg-Protoporphyrin \, age-dependent, antimorphic alleles of wheat accumulate M on omet hyl ester Proto, but the genes involved have not yet been cloned. The simplest model to explain the dominant behavior Protochlorophyliide of these mutants is a multi-enzyme complex for the MgFig. 3. A portion of the chlorophyll and heme biosynthelic path- chelatase which would be consistent with the results of ways illustrating the mutants known that accumulate Proto or Walker and Weinstein (1991) and Gibson et al. (1995). It Mg-Proto. Numbers in parentheses indicate Ehe number of alle- is thought to be composed of the products of three genes le.s'identified at that locus. The lower box points to both MgProto and Mg-I^rolo monomethyl ester because our spectro- in Rhodobacler (bchI, bchD and bchlT) and at least two scopic method used to reveal the point of the block can not dis- genes in plants (homologs to bchl and bchH-, homologs tineuish between these two compounds, and also probably both to bchD have not yet been found). In addition, a working of these compounds accumulate in certain of these mutants. model for the enzyme's multi-subunit architecture is Compiled from the results of this study and from the references based on the homoiogy of the Antirrhinum olive gene listed in the text. product to the large subunit of the Fseudomonas denitrificans colbaltochelatase noted by several groups (HudRoper and A. Hudson, unpublished data: von Wettstein son et al. 1993, Walker and Weinstein 1994, Gibson et al. 1995). Within any enzyme that is a hetero-oligomeric etal, 1995). A bottleneck in the Chl synthesis pathway is easy to multi-subunit complex, dominant mutations can be exaccount for when it is the result of a direct lesion in one pected to be found (Herskowitz 1987), and conversely of the biosynthetic enzymes. However, since the inser- damage to any number of loci might have a dominant eftion of Mg"* into Proto is the first committed step of Chi fect in a multi-enzyme complex, tlirough the introducsynthesis one might expect that it is a highly regulated tion of "poison products"' (discussed in T. G. Falbel process. The existence of the large number of both domi- 1994, Thesis, University of Colorado, Boulder, CO, nant and recessive mutants at a number of genetic loci, USA). that all accumulate Proto attests to the complex nature of the enzymatic and regulatory machinery involved in this Origin ofthe speetrum of Chl S-defieient thylakoid critical step of Cht synthesis. AdditiortalJy, the Proto-ac- phenotypes cumulating Oy-700 mutant io maize has beeti crossed into eight different inbred lines, and its mutant phenotype The set of mutants investigated in tlie present sttidy share is expressed to a different extent in each of these lines both a pleiotropic, Chl-deficient and Chl i>-deficient phe(Polacco and Walden 1987). This implies that additional notype and a common bottleneck in the Chl synthesis patliway at protoporphyrin IX. Similarities have already undefined genetic loci also participate in the expression been noted in the literature for CDS, OY-YG, chlorina of the mutant phenotype. Clearly some of the mutants 104 and viridis k23 (Allen et al. 1988, Greene et al. listed in Fig, 3 may acciimttiate their prectirsor and result 1988a,b, Knoetzel and Simpson 1991), Our data expand in a Chl fc-deficiency through indirect effects of their tiiuthis list to include all of the mutants that have been retations. Indeed, Plumley and Schmidt (1995) recently ported as Ch! iÂť-deficient mutants in the photosynthesis found an example of a Clil 6-deficiency associated with a literature. Together this set of mutants forms a spectrum defect in thylakoid insertion of LHCIl, adding further of Chl deficiencies, depending upon the severity of their support to a tight interrelationship between the accumula- blocks at Proto. For the alielic series of wheat mutants tion of Chl b and its binding proteins, and Arabidopsis mutants, the severity of the bottleneck in Protoporphyrinogen

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j AnUrrtilnum .olive ; Anbittopsla cs/ch-IS ' barlay jcan -1(3}. -^(5), -hi'>). vif-y, vk-s, do-104, c, ; malie o>'f>I'f), I'-Bianay4,1 -s.oytmany9, yfi I totMlcct) Su(>6},. yg(2)

Protoporphyrin IX accumulating mutants are frequently dominant The genes leading to Proto accumtilation commonly give rise to dominant negative mutant alleles. For example, Neuffer has commented (cited in Walbot 1991) on Ihe unusually high frequency with which dotninant Oy mutant alleles are reported in maize and the existence of a wide number of semidominant mutants with similar yellow-green phenotypes In many other species, Dulieu Physiol. Ptant. 97, 1996

Chl synthesis is coiTelated to the reduction in Chl content of the leaves (Falbel and Staehelin 1994, and unpublished data) and other facets of the phenotype (T. Falbel, I, Meehl and L, A, Staehelin, unpublished data). All of the mutants we included in our study are Chl Z)-deficient, LHC antetina deficient, and grana deficient but the degree to which these deficiencies ate manifested under a given growth regimen varies with the severity of the Chl synthesis block (T, Falbel, J, Meeltl and L, A, Staehelin, uttpublished data). Because the viridis k23 barley mutant. 317


blocked at the following step (Mg-Proto), also shows similarities to these mutants (Nielsen et al. 1979, HoyerHansen et al. 1988a,b), as well as a mutant of sweetclover which has a block at an earlier step (coproporphyrinogen ffl) (Markwell et al, 1985, 1986, Markwell and Chelgren 1988, Bevins et al. 1992), as do plants grown under limiting light conditions (limiting the rate of protochlorophyllide photoreduction) (DeGreef et al. 1971, Akoyunoglou ef al, 1978, Akoyunoglou and Akoyunoglou 1985), we propose that any partial block in Chl synthesis will give a mutant phenotype which will fall into this spectrum of deficiencies. In a previous study (Falbel and Staehelin 1994), we proposed that Chl b is made from a set of "left over" Chl a molecules, meaning that nascent RCs being synthesized on chloroplast ribosomes are able to "win" the putative competition for Chl a with the unidentified Chl b biosynthetic machinery. If this theory is correct, then the synthetic mechanism that regulates the ratio between Chl a and Chl b must be governed by the total supply of Chl a and the demand for Chl by the nascent polypeptides of the RC core. As long as the supply of Chl is limiting, by either the light environment or by a block in any step of the pathway, fhis mechanism will lead to a Chl 6-deficiency, LHC-antenna deficiency and grana-deficiency. The phenotypes are most notable early in chioroplast development and also likely in high light environments when the demands on the Chl synthesis pathway are high due to enhanced rates of turnover of chioroplast components.

provide a starting point for further research. Whether the regulation of Chl synthesis at the ftrst committed step is an important mechanism by which a plant determines its photosynthetic unit size and Chl alb ratio will have to await a tnore detailed molecular characterization of the Mg-chelatase complex in plants. The recent identification of some Mg-chelatase subunits should allow that characterization in the near future. Acknowledgments - We thank all of the suppliers of mutant seed listed above, Dr M. G. Neuffer for pointing out the existence of a large number of similar mutants, Dr Joseph Falke in the Department of Chemistry and Biochemistry, University of Colorado, for the use ofthe fluorimeter, and Dr Long Ngyuen, North Carolina State University, for eommunicating his unpublished data. Thanks also to Dr Alison G. Smith and the Department of Plant Sciences, University of Cambridge. UK, for hosting T. G. F. during the summer of 1994, while several of the atialyses included here were performed. Supported by NIH grant GM22912 to L. A. S. and a Howard Hughes Medical Institite Predoetoral Fellowship and International Human Frontiers in Science ShortTerm Fellowship to T. G. F.

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1996 Physiol Plant