9 Springer-Verlag1992
Expression of the chlorophyll-a/b-protein multigene family in pea (Pisum sativum L.) Evidence for distinct developmental responses Michael J. White 1., Brian W. Fristensky 2, Denis Falconet 3, Lisa C. Childs 4, John C. Watson 5, Laura Alexander 6, Bruce A. Roe 6, and William F. Thompson 1'4 Departments of Botany 1 and Genetics4, North Carolina State University, Raleigh, NC 27695, USA 2 Department of Plant Science, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada 3 Laboratoire de Biologie Mol6culaire V6g6tale, Universit6 de Paris Sud, 91405 Orsay Cedex, France Department of Botany and Center for Agricultural Biotechnology, University of Maryland, College Park, MD 20742, USA 6 Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019, USA Received 9 March; accepted 20 April 1992
Abstract. To measure transcript levels for individual members of the Cab (chlorophyll a/b protein) multigene family in pea under a range of developmental situations, we developed a system using c D N A synthesis, the polymerase chain reaction (PCR), and chemiluminescence detection. In order to design gene-specific PCR primers for all genes, a partial genomic clone for a fifth, Type I L H C I I (light-harvesting complex of photosystem II) gene, Cab-91, was isolated and sequenced. All seven known Cab genes in pea are expressed in light-grown buds and leaves, including several genes previously known only from genomic clones. There appear to be at least two groups of Cab genes in pea which differ in their response to light and development. The first group (consisting o f Cab-8, AB96, Cab-215 and Cab-315) includes Type I, Type II and Type III genes, shows a relatively strong response to red light, and has bud transcript levels similar to or slightly higher than leaves. The second group, consisting of the Type I genes Cab-9, ABSO and AB66, shows little or no transcript accumulation 24 h after a red light pulse, and has higher transcript levels in leaves than in buds. Transcript levels for genes in this second group appear to be lower than those of the first group in all developmental situations examined. These data indicate that there has been an evolutionary divergence of the responses to light and development among the Type I L H C I I genes. Key words: Chlorophyll a/b protein - Gene expression (Cab) - Light and gene expression Light-harvesting * To whom correspondence should be addressed; FAX 1 (919) 515 3436 Abbreviations." Cab=gene for chlorophyll a/b binding protein; LHCI, LHCII = light-harvesting complexes of photosystems I and II; PCR = polymerase chain reaction 1 The Cab-9 sequence appears in the Genbank/EMBL databases under the accession number M86906
complex (photosystem II) - Multigene family (Cab) Pisum (Cab gene expression)
Introduction The Cab genes of higher plants encode proteins which bind chlorophylls a and b and carotenoids, and are major constituents of the light-harvesting antennae for the photosynthetic apparatus (Buetow et al. 1988; Green 1988; Green et al. 1991; Peter and Thornber 1991). Many studies of light-regulated gene expression have used the Cab genes as models, and these genes have been instrumental in advancing our knowledge of phytochrome-regulated gene expression, circadian rhythms, and promoter function (e.g. see Buetow et al. 1988; Gilmartin et al. 1990; Kellmann et al. 1990; Thompson and White 1991). Extended multigene families encode the polypeptides of L H C I I and LHCI, the light-harvesting antennae ofphotosystems II and I. In addition, a number of minor chlorophyll a/b proteins, CP29, CP27, and CP24 are also encoded by Cab genes (reviewed in Green 1988; Green et al. 1991). By far the most abundant of these complexes is LHCII, which binds about 50% of the total chlorophyll and most of the chlorophyll b in higher plants (Chitnis and Thornber 1988; Green 1988). The L H C I I genes can be classified into three types on the basis of gene structure. Type I genes do not contain introns and share a high degree of sequence similarity with each other. The Type II and Type III L H C I I genes are diverged from the Type I genes and from each other, contain introns, and encode more diverged polypeptides (Pichersky et al., 1987; Falconet et al. 1991 ; Schwartz et al. 1991). It is not known if the different types of L H C I I genes differ in their function or mode of expression.
M.J. White et al. : Expression of the chlorophyll-a/b-protein multigene family in pea Table 1. Known chlorophyll a/b protein (LHCII) genes in pea Gene
Type Introns Clones sequenced References (s)
AB96 AB80
I I
0 0
partial cDNA genomic
AB66 Cab-8 Cab-9 Cab-215 Cab-315
I I I II III
0 0 0 1 2
genomic genomic partial genomic genomic partial cDNA, genomic
Coruzzi et al. 1983 Cashmore 1984 Timko et al. 1985 Timko et al. 1985 Alexander et al. 1991 this paper Falconet et al. 1991a Falconet et al. 1991b
T o a d d r e s s these a n d o t h e r questions, we a r e c h a r a c terizing the c h l o r o p h y l l - a / b - p r o t e i n (Cab) m u l t i g e n e f a m i l y in pea. A l l the Cab genes in p e a identified to d a t e encode LHCII polypeptides. Complete or partial DNA sequences are presently available for seven genes (Table 1). F i v e o f these genes are T y p e I genes, a n d e n c o d e the m a j o r p o l y p e p t i d e s o f L H C I I . T w o o f these genes (AB80 a n d AB66) e n c o d e i d e n t i c a l p o l y p e p t i d e s , a n d their c o d i n g r e g i o n s differ o n l y b y t w o silent n u c l e o t i d e subs t i t u t i o n s ( T i m k o et al. 1985). W e h a v e r e c e n t l y seq u e n c e d a f o u r t h T y p e I L H C I I gene in pea, a c o m p l e t e g e n o m i c c l o n e for Cab-8 ( A l e x a n d e r et al. 1991). Cab-8 is m o r e d i v e r g e n t in sequence t h a n the o t h e r T y p e I Cab genes, AB96, AB80 a n d AB66 ( A l e x a n d e r et al. 1991). In this p a p e r we d e s c r i b e a g e n o m i c clone i n c l u d i n g m o s t o f the c o d i n g r e g i o n for a fifth T y p e I L H C I I gene, Cab-9. T h e r e m a i n i n g genes, Cab-215 ( F a l c o n e t et al. 1991 a) a n d Cab-315 ( F a l c o n e t et al. 1991b) a r e T y p e I I a n d T y p e I I I L H C I I genes, respectively. O u r p r e v i o u s studies o n Cab gene e x p r e s s i o n in p e a ( K a u f m a n et al. 1984, 1985, 1986; H o r w i t z et al. 1988) have used p r o b e s d e r i v e d f r o m the c D N A AB96, w h i c h h y b r i d i z e s to eight b a n d s o n S o u t h e r n b l o t s ( C o r u z z i et al. 1983), s h o w i n g t h a t it c r o s s - r e a c t s extensively w i t h o t h e r Cab genes. C o n s e q u e n t l y , it is n o t k n o w n a t w h a t level the i n d i v i d u a l m e m b e r s o f the Cab g e n e - f a m i l y are expressed, o r w h e t h e r different g e n e - f a m i l y m e m b e r s are r e g u l a t e d b y different d e v e l o p m e n t a l p r o g r a m s . Differential e x p r e s s i o n o f m e m b e r s o f the Cab gene f a m i l y o c c u r s b e t w e e n m a i z e b u n d l e - s h e a t h a n d m e s o p h y l l cells following i l l u m i n a t i o n o f d a r k - g r o w n seedlings (Sheen a n d B o g o r a d 1986). H o w e v e r , since m a i z e is a C4 species a n d p e a is a C3 species, different f a c t o r s m a y c o n t r o l the r e g u l a t i o n o r differential e x p r e s s i o n o f Cab g e n e - f a m i l y m e m b e r s in pea. S t u d y i n g i n d i v i d u a l m e m b e r s o f the p e a Cab genef a m i l y r e q u i r e s m e t h o d s w h i c h can d i s t i n g u i s h very closely r e l a t e d genes. These m e t h o d s m u s t also be v e r y sensitive, since s o m e g e n e - f a m i l y m e m b e r s are likely expressed at low levels in s o m e s i t u a t i o n s . I n this p a p e r we p r e s e n t a n e x t r e m e l y sensitive a s s a y system using c D N A synthesis a n d the p o l y m e r a s e c h a i n r e a c t i o n ( P C R ) w h i c h c a n d i s t i n g u i s h t r a n s c r i p t s o f i n d i v i d u a l Cab genef a m i l y m e m b e r s in pea. T h e system p r o v i d e s specificity a n d sensitivity n o t a v a i l a b l e w i t h c o n v e n t i o n a l m e t h o d s , a n d s h o w s a q u a n t i t a t i v e r e l a t i o n s h i p b e t w e e n i n p u t template a n d the final signal o b t a i n e d . W e h a v e i n c o r p o r a t e d
191
a sensitive c h e m i l u m i n e s c e n c e - d e t e c t i o n m e t h o d into this p r o t o c o l for m a x i m u m s p e e d a n d ease o f use. U s i n g these m e t h o d s we s h o w t h a t all seven k n o w n L H C I I Cab genes in p e a are e x p r e s s e d u n d e r s o m e c o n d i t i o n s . T h e s e genes c a n be classified i n t o at least t w o d i s t i n c t g r o u p s b a s e d o n the t i m i n g o f their e x p r e s s i o n d u r i n g seedling d e v e l o p m e n t a n d their r e s p o n s e s to r e d light.
Material and methods
Isolation and characterization of a partial 9enomic clonefor Cab-9. A genomic library of total cellular DNA from pea (Pisum sativum L., cv. Alaska) in the vector Charon 32 (Loenen and Blattner 1983) was provided by Mike Murray and Jerry Slightom (Department of Agrigenetics, University of Wisconsin, Madison, USA) and was screened using the isolated cDNA insert of pAB96 (Coruzzi et al. 1983). Positive clones were purified by three rounds of plaque hybridization prior to making plate stocks of the phage. Phage DNA digests were probed with the pAB96 insert to identify a 1.9-kilobase (kb) EcoRI fragment corresponding to a similar fragment in digests of pea genomic DNA. This fragment was cloned into the EcoRI site ofpUC8. Overlapping restriction fragments of this clone (pAB9) were subcloned into M13mpl8 or M13mpl9 and sequenced by a modification (Sanger et al. 1980) of the dideoxynucleotide method. Cab-9 would be named lhble according to the nomenclature proposed by Jansson and Gustafsson 1991 as modified by Jansson et al. 1992. Plant 9rowth conditions. Pea seedlings for experiments with red light were grown and irradiated as described by Horwitz et al. (1988). Briefly, plants were grown in complete darkness for 5.5 d, given a single 10-s red light pulse of photon flux 150 ~tmol 9m - z . s-1, and returned to darkness for 24 h. Dark-grown and light-grown seedlings were grown at room temperature (approx. 22 ~ C) in Kimpak (6-d seedlings) or vermiculite (9-d seedlings). Continuous white light was provided by a mixture of fluorescent and incandescent bulbs and had a photosynthetic photon flux density between 400 nm and 700 nm of 60 ~tmol. m - z . s-1 for the experiment with 6-d-old plants and 350-400 Ilmol - m -2 - s -1 for the experiment with 9-d plants. Quantitation o f relative Cab m R N A levels i) Isolation of RNA and cDNA synthesis. Total RNA was purified as described previously (Horwitz et al. 1988; Elliott et al. 1989). Copy-DNA was synthesized from an oligo dT18 primer (Sigma Chemical Co., St. Louis, Mo., USA) using MMLV H - reverse transcriptase (Gibco BRL, Gaithersburg, Md., USA) according to the manufacturer's protocol and then heated at 95~ C for 5 min. It is important to use a reverse transcriptase lacking an RNase H domain, since the absence of RNase H activity favors production of full-length cDNAs. Copy-DNAs were precipitated with ethanol and ammonium acetate (Sambrook et al. 1989), and pellets were resuspended in TE (10 mM Tris-HC1, 1 mM EDTA pH 8.0), heated at 65~ C for 5 min, and immediately chilled on ice. Aliquots were diluted 10 fold into sterile distilled water and stored at - 7 0 ~ C. ii) Construction of DNA standardsfor use in PCR. For all genes except Cab-215, a DNA standard sharing the same primer sites as the target to be amplified, but containing an internal deletion, was added to each PCR reaction to generate a product slightly smaller than that derived from the cDNA. For Cab-215, a genomic clone containing a 355-base-pair intron (Falconet et al. 1991a) between the primer sites was used, producing a larger PCR standard. Including such standards permitted correction for variables involved in PCR amplification and subsequent chemiluminescence detection. Since the amount of standard added to each PCR tube was known,
192
M.J. White et al.: Expression of the chlorophyll-a/b-protein multigene family in pea
the amount of co-amplified c D N A initially present in each PCR tube could be calculated (e.g. Gilliland et al. 1990). Internal deletion standards were constructed by cutting the full-length PCR products with restriction enzymes (specified below), ligating cohesive termini, and amplifying with the same primers. (This protocol produced at most four different-size products : a long product amplified by one primer, the original PCR product containing both primer sites, the desired internal deletion standard also containing both primer sites, and a short product amplified by the remaining primer.) Enzymes used to generate standards from full-length PCR products were NdeI for ABSO and AB66, AvaII for Cab-9, SspI and ScaI for Cab-315, and both NdeI and BglI for AB96 and Cab-& Overhangs produced by NdeI and BglI digestion were removed with the Klenow fragment of D N A polymerase I (Gibco BRL) (Sambrook et al. 1989) prior to ligation. In most cases, regeneration of a full-length PCR product was prevented by cutting at a restriction site unique to the region being deleted. The enzymes used were HinclI for AB80 and AB66, SmaI for Cab-9, and EcoRI for Cab-315. Standards were purified by successive cycles of PCR amplification and electrophoresis on gels containing differing agarose concentrations. All the resulting standards could be amplified only when both primers of a pair were present. These standards were quantitated as described in White et al. (1991).
iii) Amplification using PCR. The mixtures for PCR contained final concentrations of 200 nM primers and 100 nM each of dATP, dCTP, dGTP, 75 nM dTTP (Pharmacia Fine Chemicals, Piscataway, N.J. USA) and 25 nM biotin-11-dUTP (sodium salt; Sigma) in a common master mixture with a D N A standard (see preceding section) sharing the same primer sites as the target to be amplified. Taq D N A polymerase (Promega Corp., Madison, Wis., USA; 2.5 units per reaction) was added and 96 lal of the resulting master mixture was added to 4 lal of 0.1 • TE containing 0.1 or 1 ng cDNA. The PCR parameters were determined experimentally for each set of primers on a Perkin-Elmer (Norwalk, Conn. USA) thermal cycler. Cycle conditions were 1 min at 94 ~ C, 1 min at 42 ~ C, and 2 min at 72 ~ C for all genes except AB66, for which an annealing temperature of 40 ~ C was used. The number of cycles used to amplify 0.1 to 1 ng of c D N A was 16 for Cab-8 and AB96, 18 for Cab-215 and Cab-315, and 20 for Cab-9, AB80 and AB66. The amount of internal standard D N A added to each reaction was 0.2 pg for Cab-8, 0.02 pg for AB96 and AB66, 0.0005 pg for Cab-9 and AB80, 0.01 pg for Cab-215 and 0.1 pg for Cab-315. The products of PCR were electrophoresed on agarose gels in 1• E D T A (TAE; Sambrook et al. 1989) such that amplification products from the c D N A were well separated from those of the internal standard. Gels were denatured in 0.2 M NaOH, 0.6 M NaC1, neutralized in 25 mM NaPO4 pH 6.5, and blotted onto Tropilon membranes (Tropix Inc., Bedford, Mass., USA) using a pressure blotter (PosiBlot; Stratagene, La Jolla, Calif., USA) according to the manufacturer's instructions. Membranes were treated with 120 mJ of UV light (Lmax=254 rim) in a UV cross-linker (Stratagene), dried between sheets of Whatman (Maidstone, Kent., UK) 3MM paper, wrapped in plastic wrap and stored at 4 ~ C. iv) Quantitation of PCR products. Biotinylated PCR products were detected with streptavidin-alkaline phosphatase and the substrate AMPPD [3-(2'-spiroadamantane)-4-methoxy-4-(Y'-phosphoryloxy)-phenyt-l,2-dioxetane disodium salt]. Tropix reagents were used following the manufacturer's protocol except that filtered Gibco BRL streptavidin-alkaline phosphatase was used instead of Tropix avidin-alkaline phosphatase, since this substantially increased the sensitivity of detection. Blocking, staining and wash steps were carried out on a rocking shaker. Briefly, membranes were wetted in blocking solution for 10 min, then transferred to fresh blocking solution for 1 h. Streptavidin-alkaline phosphatase was diluted to 1/1600 in conjugate buffer, the mixture passed through a 0.45-tam Acrodisc (Gelman Sciences, Ann Arbor, Mich., USA), and then diluted a further tenfold in conjugate buffer (final dilution
of 1/16 000). Membranes were incubated in this solution for exactly 1 h, then given five washes (5-10 min each) of phosphate-buffered saline containing 3% Tween-20, and two washes of (fresh, sterilefiltered) substrate buffer (50 mM sodium carbonate-bicarbonate, 1 mM MgCI2, pH 9.5) prior to incubation in substrate buffer containing AMPPD. Moist membranes were sealed in plastic and exposed to Kodak XAR 5 X-ray film. For quantitation, a 1- to 3-min exposure 18 h after incubation in A M P P D was used. Images on film were quantitated on a soft laser densitometer (Model S L R - 2 D / 1 D ; Biomed Instruments Inc., Fullerton, Calif., USA) using two-dimensional scanning. For each PCR reaction, peak volumes were calculated for both the c D N A and internal standard. The ratio of c D N A signal/standard signal was multiplied by the starting amount of internal standard, to estimate the amount of a given Cab sequence present in the initial cDNA.
Results
Partial 9enomic sequence f o r Cab-9. G e n o m i c c l o n e s o r c D N A s f o r five g e n e s in p e a (AB80, AB66, A B 9 6 , Cab-8 a n d Cab-215) h a v e b e e n s e q u e n c e d t o d a t e as o u t l i n e d in t h e Introduction. S i n c e w e w i s h e d to p u r s u e a c o m p r e h e n sive s t u d y o f Cab g e n e e x p r e s s i o n in p e a , it was n e c e s s a r y to o b t a i n s e q u e n c e d a t a o n t h e r e m a i n i n g k n o w n genes, Cab-9 a n d Cab-315. T h i s d a t a c o u l d t h e n be u s e d to d e s i g n P C R p r i m e r s f o r e a c h g e n e , t h u s a l l o w i n g us to 1
2
5
2 1
-
_
_
2
UI
c~b-8
6 ~
Cab-9
7
-IE
I1 I
2
~,
As66
8
I 2
ABao
9 ~ - -
3
AB96
10 Cab-215
4
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J
1 O0 bp
Fig. l. Location of PCR primers for each of the seven known Cab genes in pea. The upstream sense primers indicated are 1, CAATC T T T T C A A T T T C A T T G C A A T A ; 2, G G T G A C T A C G G T T G G G A C A C T G C ; 3, Cab-215: T A C T G T G A A G A G T G C T C C T G ; 4, Cab-315: C A C C A T G G G A A A T G A T T T G . The downstream antisense primers indicated are 5, ABSO: A A A A A A G C A TCCATCATC; 6, A B66: A A G A A A A C A T C C A T G A T C ; 7, AB96 : A C A A T A A C A T A C T T C A C C ; 8, Cab-8: ACACTAACATTCAGTTGC; 9, Cab-9: TCATTCACATTCATTTCA; 10, Cab-215: TCAGCCACATACAGGTTC; 11, Cab-315: TTGTGC A T G A A C A A G G T T A G . For AB80, AB66 and Cab-8 where two primer combinations are possible, primer No. 1 was used as the 5' primer. The Cab-215 and Cab-315 introns are indicated with a triangle. Tentative assignment of locations of the two Cab-315 introns are based on those of the analogous Type III LHCII gene in tomato (Schwartz et al. 1991), and the observation that the Cab-315 genomic clone yields a PCR product approx. 200 bp larger than the PCR product from the Cab-315 c D N A (Falconer et al. 1991b)
M.J. White et al. : Expression of the chlorophyll-a/b-protein multigene family in pea
193
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quantitate m R N A levels using c D N A synthesis and PCR. A partial genomic clone was obtained for Cab-9 (see Material and methods for details) which contains approximately two-thirds o f the coding sequence and a b o u t 1100 nucleotides o f 3' flanking sequence. This sequence (1924 nucleotides) appears in the G e n b a n k / E M B L databases under the accession n u m b e r M86906. Based on the available amino-acid sequence, Cab-9 appears to be a Type I L H C I I protein. The Cab-9 gene is diverged f r o m the other Type I L H C I I genes of pea in its 3' untranslated sequence, allowing the design o f a gene-specific primer at the 3' end (Fig. 1). The Cab-315 primers (Fig. 1) are based on a partial c D N A sequence (data not shown). Sequencing o f a full-length genomic clone for Cab-315 is in progress.
A combination o f P C R amplification and subsequent detection o f chemiluminescence is quantitative. Since specificity is required to distinguish closely related genes and sensitivity is required to detect individual Cab-transcript levels in etiolated plants, we chose to use a procedure involving c D N A synthesis followed by P C R amplification. The primers used are shown in Fig. 1. Gene-specific antisense primers were used at the 3' end of each gene, while 5' sense primers were typically c o m m o n to several genes. To achieve a linear relationship between c D N A template and P C R product, it is necessary that all reagents
-
m
Fig. 2. Quantitation of RNA levels via cDNA synthesis. A twofold serial dilution of a cDNA was amplified with Cab-8 or Cab-215 primers in the presence of a constant amount of the appropriate DNA standards (denoted by S~ at left). The PCR reactions were set up by adding a master mixture already containing the DNA standard and PCR reagents to tubes containing 0.5, 0.25, 0.125, 0.0625 or 0.0313 ng of cDNA (see Material and methods for details). The far-right-hand lane of the cDNA dilution is a no-template control. M, biotinylated ~/HindlII molecular-weight markers (BRL); S on x-axis, sample to be quantitated (another cDNA)
be in m o l a r excess over P C R product throughout the amplification process. I f the n u m b e r of moles of product exceeds the n u m b e r o f moles o f T a q D N A polymerase, p r o d u c t amplification will no longer be exponential (Erlich et al. 1991; Robinson and Simon 1991). A linear relationship between template input and the output signal can be obtained by limiting the a m o u n t o f template and keeping the n u m b e r o f P C R cycles to a m i n i m u m
Fig. 3. Specificity of PCR amplification using the AB66 primers9 A PCR master mixture (see Material and methods) containing the AB66 primers was added to 0.1 pg of DNA standard for each of the seven Cab genes (indicated at bottom of figure). M, biotinylated L/HindIII molecular weight markers
194
M.J. White et al. : Expression of the chlorophyll-a/b-protein multigene family in pea
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Expression of the chlorophyll-a/b-protein multigene family in pea (Pisum sativum L.) Evidence for distinct developmental responses Michael J. White 1., Brian W. Fristensky 2, Denis Falconet 3, Lisa C. Childs 4, John C. Watson 5, Laura Alexander 6, Bruce A. Roe 6, and William F. Thompson 1'4 Departments of Botany 1 and Genetics4, North Carolina State University, Raleigh, NC 27695, USA 2 Department of Plant Science, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada 3 Laboratoire de Biologie Mol6culaire V6g6tale, Universit6 de Paris Sud, 91405 Orsay Cedex, France Department of Botany and Center for Agricultural Biotechnology, University of Maryland, College Park, MD 20742, USA 6 Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019, USA Received 9 March; accepted 20 April 1992
Abstract. To measure transcript levels for individual members of the Cab (chlorophyll a/b protein) multigene family in pea under a range of developmental situations, we developed a system using c D N A synthesis, the polymerase chain reaction (PCR), and chemiluminescence detection. In order to design gene-specific PCR primers for all genes, a partial genomic clone for a fifth, Type I L H C I I (light-harvesting complex of photosystem II) gene, Cab-91, was isolated and sequenced. All seven known Cab genes in pea are expressed in light-grown buds and leaves, including several genes previously known only from genomic clones. There appear to be at least two groups of Cab genes in pea which differ in their response to light and development. The first group (consisting o f Cab-8, AB96, Cab-215 and Cab-315) includes Type I, Type II and Type III genes, shows a relatively strong response to red light, and has bud transcript levels similar to or slightly higher than leaves. The second group, consisting of the Type I genes Cab-9, ABSO and AB66, shows little or no transcript accumulation 24 h after a red light pulse, and has higher transcript levels in leaves than in buds. Transcript levels for genes in this second group appear to be lower than those of the first group in all developmental situations examined. These data indicate that there has been an evolutionary divergence of the responses to light and development among the Type I L H C I I genes. Key words: Chlorophyll a/b protein - Gene expression (Cab) - Light and gene expression Light-harvesting * To whom correspondence should be addressed; FAX 1 (919) 515 3436 Abbreviations." Cab=gene for chlorophyll a/b binding protein; LHCI, LHCII = light-harvesting complexes of photosystems I and II; PCR = polymerase chain reaction 1 The Cab-9 sequence appears in the Genbank/EMBL databases under the accession number M86906
complex (photosystem II) - Multigene family (Cab) Pisum (Cab gene expression)
Introduction The Cab genes of higher plants encode proteins which bind chlorophylls a and b and carotenoids, and are major constituents of the light-harvesting antennae for the photosynthetic apparatus (Buetow et al. 1988; Green 1988; Green et al. 1991; Peter and Thornber 1991). Many studies of light-regulated gene expression have used the Cab genes as models, and these genes have been instrumental in advancing our knowledge of phytochrome-regulated gene expression, circadian rhythms, and promoter function (e.g. see Buetow et al. 1988; Gilmartin et al. 1990; Kellmann et al. 1990; Thompson and White 1991). Extended multigene families encode the polypeptides of L H C I I and LHCI, the light-harvesting antennae ofphotosystems II and I. In addition, a number of minor chlorophyll a/b proteins, CP29, CP27, and CP24 are also encoded by Cab genes (reviewed in Green 1988; Green et al. 1991). By far the most abundant of these complexes is LHCII, which binds about 50% of the total chlorophyll and most of the chlorophyll b in higher plants (Chitnis and Thornber 1988; Green 1988). The L H C I I genes can be classified into three types on the basis of gene structure. Type I genes do not contain introns and share a high degree of sequence similarity with each other. The Type II and Type III L H C I I genes are diverged from the Type I genes and from each other, contain introns, and encode more diverged polypeptides (Pichersky et al., 1987; Falconet et al. 1991 ; Schwartz et al. 1991). It is not known if the different types of L H C I I genes differ in their function or mode of expression.
M.J. White et al. : Expression of the chlorophyll-a/b-protein multigene family in pea Table 1. Known chlorophyll a/b protein (LHCII) genes in pea Gene
Type Introns Clones sequenced References (s)
AB96 AB80
I I
0 0
partial cDNA genomic
AB66 Cab-8 Cab-9 Cab-215 Cab-315
I I I II III
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genomic genomic partial genomic genomic partial cDNA, genomic
Coruzzi et al. 1983 Cashmore 1984 Timko et al. 1985 Timko et al. 1985 Alexander et al. 1991 this paper Falconet et al. 1991a Falconet et al. 1991b
T o a d d r e s s these a n d o t h e r questions, we a r e c h a r a c terizing the c h l o r o p h y l l - a / b - p r o t e i n (Cab) m u l t i g e n e f a m i l y in pea. A l l the Cab genes in p e a identified to d a t e encode LHCII polypeptides. Complete or partial DNA sequences are presently available for seven genes (Table 1). F i v e o f these genes are T y p e I genes, a n d e n c o d e the m a j o r p o l y p e p t i d e s o f L H C I I . T w o o f these genes (AB80 a n d AB66) e n c o d e i d e n t i c a l p o l y p e p t i d e s , a n d their c o d i n g r e g i o n s differ o n l y b y t w o silent n u c l e o t i d e subs t i t u t i o n s ( T i m k o et al. 1985). W e h a v e r e c e n t l y seq u e n c e d a f o u r t h T y p e I L H C I I gene in pea, a c o m p l e t e g e n o m i c c l o n e for Cab-8 ( A l e x a n d e r et al. 1991). Cab-8 is m o r e d i v e r g e n t in sequence t h a n the o t h e r T y p e I Cab genes, AB96, AB80 a n d AB66 ( A l e x a n d e r et al. 1991). In this p a p e r we d e s c r i b e a g e n o m i c clone i n c l u d i n g m o s t o f the c o d i n g r e g i o n for a fifth T y p e I L H C I I gene, Cab-9. T h e r e m a i n i n g genes, Cab-215 ( F a l c o n e t et al. 1991 a) a n d Cab-315 ( F a l c o n e t et al. 1991b) a r e T y p e I I a n d T y p e I I I L H C I I genes, respectively. O u r p r e v i o u s studies o n Cab gene e x p r e s s i o n in p e a ( K a u f m a n et al. 1984, 1985, 1986; H o r w i t z et al. 1988) have used p r o b e s d e r i v e d f r o m the c D N A AB96, w h i c h h y b r i d i z e s to eight b a n d s o n S o u t h e r n b l o t s ( C o r u z z i et al. 1983), s h o w i n g t h a t it c r o s s - r e a c t s extensively w i t h o t h e r Cab genes. C o n s e q u e n t l y , it is n o t k n o w n a t w h a t level the i n d i v i d u a l m e m b e r s o f the Cab g e n e - f a m i l y are expressed, o r w h e t h e r different g e n e - f a m i l y m e m b e r s are r e g u l a t e d b y different d e v e l o p m e n t a l p r o g r a m s . Differential e x p r e s s i o n o f m e m b e r s o f the Cab gene f a m i l y o c c u r s b e t w e e n m a i z e b u n d l e - s h e a t h a n d m e s o p h y l l cells following i l l u m i n a t i o n o f d a r k - g r o w n seedlings (Sheen a n d B o g o r a d 1986). H o w e v e r , since m a i z e is a C4 species a n d p e a is a C3 species, different f a c t o r s m a y c o n t r o l the r e g u l a t i o n o r differential e x p r e s s i o n o f Cab g e n e - f a m i l y m e m b e r s in pea. S t u d y i n g i n d i v i d u a l m e m b e r s o f the p e a Cab genef a m i l y r e q u i r e s m e t h o d s w h i c h can d i s t i n g u i s h very closely r e l a t e d genes. These m e t h o d s m u s t also be v e r y sensitive, since s o m e g e n e - f a m i l y m e m b e r s are likely expressed at low levels in s o m e s i t u a t i o n s . I n this p a p e r we p r e s e n t a n e x t r e m e l y sensitive a s s a y system using c D N A synthesis a n d the p o l y m e r a s e c h a i n r e a c t i o n ( P C R ) w h i c h c a n d i s t i n g u i s h t r a n s c r i p t s o f i n d i v i d u a l Cab genef a m i l y m e m b e r s in pea. T h e system p r o v i d e s specificity a n d sensitivity n o t a v a i l a b l e w i t h c o n v e n t i o n a l m e t h o d s , a n d s h o w s a q u a n t i t a t i v e r e l a t i o n s h i p b e t w e e n i n p u t template a n d the final signal o b t a i n e d . W e h a v e i n c o r p o r a t e d
191
a sensitive c h e m i l u m i n e s c e n c e - d e t e c t i o n m e t h o d into this p r o t o c o l for m a x i m u m s p e e d a n d ease o f use. U s i n g these m e t h o d s we s h o w t h a t all seven k n o w n L H C I I Cab genes in p e a are e x p r e s s e d u n d e r s o m e c o n d i t i o n s . T h e s e genes c a n be classified i n t o at least t w o d i s t i n c t g r o u p s b a s e d o n the t i m i n g o f their e x p r e s s i o n d u r i n g seedling d e v e l o p m e n t a n d their r e s p o n s e s to r e d light.
Material and methods
Isolation and characterization of a partial 9enomic clonefor Cab-9. A genomic library of total cellular DNA from pea (Pisum sativum L., cv. Alaska) in the vector Charon 32 (Loenen and Blattner 1983) was provided by Mike Murray and Jerry Slightom (Department of Agrigenetics, University of Wisconsin, Madison, USA) and was screened using the isolated cDNA insert of pAB96 (Coruzzi et al. 1983). Positive clones were purified by three rounds of plaque hybridization prior to making plate stocks of the phage. Phage DNA digests were probed with the pAB96 insert to identify a 1.9-kilobase (kb) EcoRI fragment corresponding to a similar fragment in digests of pea genomic DNA. This fragment was cloned into the EcoRI site ofpUC8. Overlapping restriction fragments of this clone (pAB9) were subcloned into M13mpl8 or M13mpl9 and sequenced by a modification (Sanger et al. 1980) of the dideoxynucleotide method. Cab-9 would be named lhble according to the nomenclature proposed by Jansson and Gustafsson 1991 as modified by Jansson et al. 1992. Plant 9rowth conditions. Pea seedlings for experiments with red light were grown and irradiated as described by Horwitz et al. (1988). Briefly, plants were grown in complete darkness for 5.5 d, given a single 10-s red light pulse of photon flux 150 ~tmol 9m - z . s-1, and returned to darkness for 24 h. Dark-grown and light-grown seedlings were grown at room temperature (approx. 22 ~ C) in Kimpak (6-d seedlings) or vermiculite (9-d seedlings). Continuous white light was provided by a mixture of fluorescent and incandescent bulbs and had a photosynthetic photon flux density between 400 nm and 700 nm of 60 ~tmol. m - z . s-1 for the experiment with 6-d-old plants and 350-400 Ilmol - m -2 - s -1 for the experiment with 9-d plants. Quantitation o f relative Cab m R N A levels i) Isolation of RNA and cDNA synthesis. Total RNA was purified as described previously (Horwitz et al. 1988; Elliott et al. 1989). Copy-DNA was synthesized from an oligo dT18 primer (Sigma Chemical Co., St. Louis, Mo., USA) using MMLV H - reverse transcriptase (Gibco BRL, Gaithersburg, Md., USA) according to the manufacturer's protocol and then heated at 95~ C for 5 min. It is important to use a reverse transcriptase lacking an RNase H domain, since the absence of RNase H activity favors production of full-length cDNAs. Copy-DNAs were precipitated with ethanol and ammonium acetate (Sambrook et al. 1989), and pellets were resuspended in TE (10 mM Tris-HC1, 1 mM EDTA pH 8.0), heated at 65~ C for 5 min, and immediately chilled on ice. Aliquots were diluted 10 fold into sterile distilled water and stored at - 7 0 ~ C. ii) Construction of DNA standardsfor use in PCR. For all genes except Cab-215, a DNA standard sharing the same primer sites as the target to be amplified, but containing an internal deletion, was added to each PCR reaction to generate a product slightly smaller than that derived from the cDNA. For Cab-215, a genomic clone containing a 355-base-pair intron (Falconet et al. 1991a) between the primer sites was used, producing a larger PCR standard. Including such standards permitted correction for variables involved in PCR amplification and subsequent chemiluminescence detection. Since the amount of standard added to each PCR tube was known,
192
M.J. White et al.: Expression of the chlorophyll-a/b-protein multigene family in pea
the amount of co-amplified c D N A initially present in each PCR tube could be calculated (e.g. Gilliland et al. 1990). Internal deletion standards were constructed by cutting the full-length PCR products with restriction enzymes (specified below), ligating cohesive termini, and amplifying with the same primers. (This protocol produced at most four different-size products : a long product amplified by one primer, the original PCR product containing both primer sites, the desired internal deletion standard also containing both primer sites, and a short product amplified by the remaining primer.) Enzymes used to generate standards from full-length PCR products were NdeI for ABSO and AB66, AvaII for Cab-9, SspI and ScaI for Cab-315, and both NdeI and BglI for AB96 and Cab-& Overhangs produced by NdeI and BglI digestion were removed with the Klenow fragment of D N A polymerase I (Gibco BRL) (Sambrook et al. 1989) prior to ligation. In most cases, regeneration of a full-length PCR product was prevented by cutting at a restriction site unique to the region being deleted. The enzymes used were HinclI for AB80 and AB66, SmaI for Cab-9, and EcoRI for Cab-315. Standards were purified by successive cycles of PCR amplification and electrophoresis on gels containing differing agarose concentrations. All the resulting standards could be amplified only when both primers of a pair were present. These standards were quantitated as described in White et al. (1991).
iii) Amplification using PCR. The mixtures for PCR contained final concentrations of 200 nM primers and 100 nM each of dATP, dCTP, dGTP, 75 nM dTTP (Pharmacia Fine Chemicals, Piscataway, N.J. USA) and 25 nM biotin-11-dUTP (sodium salt; Sigma) in a common master mixture with a D N A standard (see preceding section) sharing the same primer sites as the target to be amplified. Taq D N A polymerase (Promega Corp., Madison, Wis., USA; 2.5 units per reaction) was added and 96 lal of the resulting master mixture was added to 4 lal of 0.1 • TE containing 0.1 or 1 ng cDNA. The PCR parameters were determined experimentally for each set of primers on a Perkin-Elmer (Norwalk, Conn. USA) thermal cycler. Cycle conditions were 1 min at 94 ~ C, 1 min at 42 ~ C, and 2 min at 72 ~ C for all genes except AB66, for which an annealing temperature of 40 ~ C was used. The number of cycles used to amplify 0.1 to 1 ng of c D N A was 16 for Cab-8 and AB96, 18 for Cab-215 and Cab-315, and 20 for Cab-9, AB80 and AB66. The amount of internal standard D N A added to each reaction was 0.2 pg for Cab-8, 0.02 pg for AB96 and AB66, 0.0005 pg for Cab-9 and AB80, 0.01 pg for Cab-215 and 0.1 pg for Cab-315. The products of PCR were electrophoresed on agarose gels in 1• E D T A (TAE; Sambrook et al. 1989) such that amplification products from the c D N A were well separated from those of the internal standard. Gels were denatured in 0.2 M NaOH, 0.6 M NaC1, neutralized in 25 mM NaPO4 pH 6.5, and blotted onto Tropilon membranes (Tropix Inc., Bedford, Mass., USA) using a pressure blotter (PosiBlot; Stratagene, La Jolla, Calif., USA) according to the manufacturer's instructions. Membranes were treated with 120 mJ of UV light (Lmax=254 rim) in a UV cross-linker (Stratagene), dried between sheets of Whatman (Maidstone, Kent., UK) 3MM paper, wrapped in plastic wrap and stored at 4 ~ C. iv) Quantitation of PCR products. Biotinylated PCR products were detected with streptavidin-alkaline phosphatase and the substrate AMPPD [3-(2'-spiroadamantane)-4-methoxy-4-(Y'-phosphoryloxy)-phenyt-l,2-dioxetane disodium salt]. Tropix reagents were used following the manufacturer's protocol except that filtered Gibco BRL streptavidin-alkaline phosphatase was used instead of Tropix avidin-alkaline phosphatase, since this substantially increased the sensitivity of detection. Blocking, staining and wash steps were carried out on a rocking shaker. Briefly, membranes were wetted in blocking solution for 10 min, then transferred to fresh blocking solution for 1 h. Streptavidin-alkaline phosphatase was diluted to 1/1600 in conjugate buffer, the mixture passed through a 0.45-tam Acrodisc (Gelman Sciences, Ann Arbor, Mich., USA), and then diluted a further tenfold in conjugate buffer (final dilution
of 1/16 000). Membranes were incubated in this solution for exactly 1 h, then given five washes (5-10 min each) of phosphate-buffered saline containing 3% Tween-20, and two washes of (fresh, sterilefiltered) substrate buffer (50 mM sodium carbonate-bicarbonate, 1 mM MgCI2, pH 9.5) prior to incubation in substrate buffer containing AMPPD. Moist membranes were sealed in plastic and exposed to Kodak XAR 5 X-ray film. For quantitation, a 1- to 3-min exposure 18 h after incubation in A M P P D was used. Images on film were quantitated on a soft laser densitometer (Model S L R - 2 D / 1 D ; Biomed Instruments Inc., Fullerton, Calif., USA) using two-dimensional scanning. For each PCR reaction, peak volumes were calculated for both the c D N A and internal standard. The ratio of c D N A signal/standard signal was multiplied by the starting amount of internal standard, to estimate the amount of a given Cab sequence present in the initial cDNA.
Results
Partial 9enomic sequence f o r Cab-9. G e n o m i c c l o n e s o r c D N A s f o r five g e n e s in p e a (AB80, AB66, A B 9 6 , Cab-8 a n d Cab-215) h a v e b e e n s e q u e n c e d t o d a t e as o u t l i n e d in t h e Introduction. S i n c e w e w i s h e d to p u r s u e a c o m p r e h e n sive s t u d y o f Cab g e n e e x p r e s s i o n in p e a , it was n e c e s s a r y to o b t a i n s e q u e n c e d a t a o n t h e r e m a i n i n g k n o w n genes, Cab-9 a n d Cab-315. T h i s d a t a c o u l d t h e n be u s e d to d e s i g n P C R p r i m e r s f o r e a c h g e n e , t h u s a l l o w i n g us to 1
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Fig. l. Location of PCR primers for each of the seven known Cab genes in pea. The upstream sense primers indicated are 1, CAATC T T T T C A A T T T C A T T G C A A T A ; 2, G G T G A C T A C G G T T G G G A C A C T G C ; 3, Cab-215: T A C T G T G A A G A G T G C T C C T G ; 4, Cab-315: C A C C A T G G G A A A T G A T T T G . The downstream antisense primers indicated are 5, ABSO: A A A A A A G C A TCCATCATC; 6, A B66: A A G A A A A C A T C C A T G A T C ; 7, AB96 : A C A A T A A C A T A C T T C A C C ; 8, Cab-8: ACACTAACATTCAGTTGC; 9, Cab-9: TCATTCACATTCATTTCA; 10, Cab-215: TCAGCCACATACAGGTTC; 11, Cab-315: TTGTGC A T G A A C A A G G T T A G . For AB80, AB66 and Cab-8 where two primer combinations are possible, primer No. 1 was used as the 5' primer. The Cab-215 and Cab-315 introns are indicated with a triangle. Tentative assignment of locations of the two Cab-315 introns are based on those of the analogous Type III LHCII gene in tomato (Schwartz et al. 1991), and the observation that the Cab-315 genomic clone yields a PCR product approx. 200 bp larger than the PCR product from the Cab-315 c D N A (Falconer et al. 1991b)
M.J. White et al. : Expression of the chlorophyll-a/b-protein multigene family in pea
193
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quantitate m R N A levels using c D N A synthesis and PCR. A partial genomic clone was obtained for Cab-9 (see Material and methods for details) which contains approximately two-thirds o f the coding sequence and a b o u t 1100 nucleotides o f 3' flanking sequence. This sequence (1924 nucleotides) appears in the G e n b a n k / E M B L databases under the accession n u m b e r M86906. Based on the available amino-acid sequence, Cab-9 appears to be a Type I L H C I I protein. The Cab-9 gene is diverged f r o m the other Type I L H C I I genes of pea in its 3' untranslated sequence, allowing the design o f a gene-specific primer at the 3' end (Fig. 1). The Cab-315 primers (Fig. 1) are based on a partial c D N A sequence (data not shown). Sequencing o f a full-length genomic clone for Cab-315 is in progress.
A combination o f P C R amplification and subsequent detection o f chemiluminescence is quantitative. Since specificity is required to distinguish closely related genes and sensitivity is required to detect individual Cab-transcript levels in etiolated plants, we chose to use a procedure involving c D N A synthesis followed by P C R amplification. The primers used are shown in Fig. 1. Gene-specific antisense primers were used at the 3' end of each gene, while 5' sense primers were typically c o m m o n to several genes. To achieve a linear relationship between c D N A template and P C R product, it is necessary that all reagents
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Fig. 2. Quantitation of RNA levels via cDNA synthesis. A twofold serial dilution of a cDNA was amplified with Cab-8 or Cab-215 primers in the presence of a constant amount of the appropriate DNA standards (denoted by S~ at left). The PCR reactions were set up by adding a master mixture already containing the DNA standard and PCR reagents to tubes containing 0.5, 0.25, 0.125, 0.0625 or 0.0313 ng of cDNA (see Material and methods for details). The far-right-hand lane of the cDNA dilution is a no-template control. M, biotinylated ~/HindlII molecular-weight markers (BRL); S on x-axis, sample to be quantitated (another cDNA)
be in m o l a r excess over P C R product throughout the amplification process. I f the n u m b e r of moles of product exceeds the n u m b e r o f moles o f T a q D N A polymerase, p r o d u c t amplification will no longer be exponential (Erlich et al. 1991; Robinson and Simon 1991). A linear relationship between template input and the output signal can be obtained by limiting the a m o u n t o f template and keeping the n u m b e r o f P C R cycles to a m i n i m u m
Fig. 3. Specificity of PCR amplification using the AB66 primers9 A PCR master mixture (see Material and methods) containing the AB66 primers was added to 0.1 pg of DNA standard for each of the seven Cab genes (indicated at bottom of figure). M, biotinylated L/HindIII molecular weight markers
194
M.J. White et al. : Expression of the chlorophyll-a/b-protein multigene family in pea
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