The Molecular Basis of C4 Photosynthesis in Sorghum

Plant Molecular Biology 37: 319–335, 1998. 319

c 1998 Kluwer Academic Publishers. Printed in Belgium.

The molecular basis of C4 photosynthesis in sorghum: isolation, characterization and RFLP mapping of mesophyll- and bundle-sheath-speciﬁc cDNAs obtained by differential screening

Ralf Wyrich1, Uta Dressen1, Stephan Brockmann1, Monika Streubel1, Charlene Chang2,

;ࣿ Dou Qiang2;3, Andrew H. Paterson2 and Peter Westhoff1 Institut fur¨ Entwicklungs- und Molekularbiologie der Pﬂanzen, Heinrich-Heine-Universitat,¨ Universitatsstrasse¨ 1,

40225 Dusseldorf,¨ Germany (ࣿ author for correspondence); 2Department of Soil and Crop Sciences, Texas A & M University, College Station, TX 77843–2474, USA; 3Xinjiang Institute of Chemistry, Chinese Academy of Sciences, Xinjiang, China 830011

Received 11 September 1997; accepted in revised form 11 January 1998

Key words: C4 photosynthesis, differential gene expression, Sorghum

Abstract

C4 photosynthesis depends upon the strict compartmentalization of the CO2-assimilatory enzymes of the C4 and Calvin cycle in two different cell types, mesophyll and bundle-sheath cells. A differential accumulation is also observed for enzymes of other metabolic pathways, and mesophyll and bundle-sheath chloroplasts of NADP-malic enzyme type C4 plants differ even in their photosynthetic electron transport chains. A large number of studies indicate that this division of labour between mesophyll and bundle-sheath cells is the result of differential gene expression. To investigate the extent of this differential gene expression and thus gain insight into the genetic basis of C4 photosynthesis, genes that are differentially expressed in the mesophyll and bundle-sheath cells were catalogued in the NADP-malic enzyme type C4 grass Sorghum bicolor. A total of 58 cDNAs were isolated by differential screening. Using a tenfold difference in transcript abundance between mesophyll and bundle-sheath cells as a criterion, 25 cDNAs were confirmed to encode mesophyll-specific gene sequences and 8 were found to encode bundle-sheath-specific sequences. Eight mesophyll-specific cDNAs showed no significant similarities within GenBank and may therefore represent candidates for the elucidation of hitherto unknown functions in the differentiation of mesophyll and bundle-sheath cells. The chromosomal location of 50 isolated cDNAs was determined by RFLP mapping using an interspecific sorghum cross.

Introduction CO2 pump provides high rates of photosynthesis even when CO2 concentrations are low in the intercellular C4 plants are characterized by high rates of photosyn- air spaces of the leaf. Hence, C4 plants are able to lim- thesis as well as an efficient use of water and nitrogen it the opening of their stomata and thereby minimize resources. For this reason, a number of C4 plants are water loss due to transpiration. As the CO2 pump res- among the most productive crops in agriculture. The ults in saturating concentrations of CO2 at the site of high photosynthetic capacity of C4 plants is due to Rubisco, high photosynthetic rates can be maintained their unique mode of carbon assimilation which con- with less enzyme than is required in C3 species and this centrates CO2 at the site of Rubisco. As a consequence reflects in a higher nitrogen use efficiency (reviewed in of this CO2 pumping, the competitive inhibition of [13, 37, 38]). Rubisco by oxygen is largely excluded and C4 plants The functioning of C4 photosynthesis is dependent show drastically reduced rates of photorespiration. In upon the strict compartmentation of the CO2 assimil- C3 plants, this process is responsible for the loss of up atory enzymes into two distinct cell types, mesophyll to 30% of the net CO2 fixed in photosynthesis. The and bundle-sheath cells. The primary carboxylating 320 enzyme, phosphoenolpyruvate carboxylase (PEPC), sequences may be easily identified in other C4 and C3 accumulates exclusively in the mesophyll cells while grasses. A comparative mapping of C4-photosynthesis- the secondary carboxylase, Rubisco, as well as the associated genes within the grasses is of prime interest decarboxylating enzymes like NADP-dependent mal- because the C4 photosynthetic pathway has evolved ic enzyme, are restricted to the bundle-sheath cells several times independently within this family [20, 50] (reviewed in [18]). A differential accumulation is and the grasses are an attractive model system in which also observed for photorespiratory, nitrogen assimil- to study the evolution of this photosynthetic trait. An ating and carbohydrate synthesizing enzymes [2, 40, identification of C4-associated genes and their compar- 54, 60]. C4 plants of the NADP-malic enzyme type ative analysis in the various evolutionary lines towards are even known to differ in the photosynthetic elec- C4 photosynthesis within the grasses may also be con- tron transport chains of mesophyll and bundle-sheath sidered as the first step to elucidate the master gene(s) chloroplasts. While thylakoid membranes of meso- of this evolutionary transition. Here we present a cata- phyll chloroplasts possess a fully developed linear elec- logue of genes which are differentially expressed in tron transport chain, those of the bundle-sheath chloro- the mesophyll and bundle-sheath cells of sorghum and plasts are devoid of grana and are severely depleted in report on the location of these genes on the RFLP map photosystem II [11, 28, 46, 59]. of this species. This division of labour between mesophyll and bundle-sheath cells is the result of differential gene expression. In NADP-malic enzyme-type C4 species Materials and methods transcripts for phosphoenolpyruvate carboxylase, pyruvate orthophosphate dikinase, NADP-malic enzyme Plant material and the small subunit of Rubisco accumulate differentially in the two cell types. It was found that this dif- Seeds of Sorghum bicolor cv. Tx430 (Pioneer Hi- ferential accumulation is largely due to transcriptional Bred, Plainview, TX) were germinated in soil. Plants control (reviewed in [15, 23, 25]). A differential accu- were grown in the greenhouse with supplementary mulation has also been reported for nuclear as well as light (14 h per day from 07:00 to 21:00) provided plastid-encoded transcripts for photosystem II proteins by a combination of sodium and mercury high-

[24, 48, 49, 56]. There is evidence that other genes may pressure vapour lamps. Photon flux density was about 2 1 be expressed differentially too, but the extent of this 300 ऌmol m s at plant height level and the growth differential gene expression in C4 photosynthesis has temperature varied between 20–26 C (day) and 18– to be elucidated. 20 C (night). For the isolation of total leaf RNA A comprehensive knowledge of those genes which the entire second leaves of 8- to 10-day old seedlings are differentially expressed in mesophyll and bundle- were used, while for the preparation of mesophyll and sheath cells could help in defining and understanding bundle-sheath RNAs only the upper two thirds of the the genetic basis of C4 photosynthesis. Therefore, we second leaves were harvested. Root RNA was isolated have initiated a systematic search for these genes in the from seedlings grown for 14 days in the greenhouse. NADP-malic enzyme type C4 grass Sorghum bicolor. For light/dark experiments seeds were germinated and Sorghum has been selected for this purpose because grown in the dark for 5 days and then illuminated for the gene expression patterns of mesophyll and bundle- 24 h with white light. sheath cells differ drastically even at the very young seedling stage [24, 28, 34]. This is of great advant- Isolation of RNA from mesophyll protoplasts age for gene cataloguing because cDNA libraries and hybridization probes can be prepared from leaf tissue Leaves (20–30 g) were cut into small pieces (5–10 mm) which is still differentiating and may be expected to with a sharp razor blade and stored in ice-cold medium express all the genes needed for the establishment and A (0.6 M sorbitol, 0.005 M MgCl2, 0.5% w/v bovine maintenance of the C4 pathway of photosynthesis. serum albumin, 0.050 M sodium ascorbate, 0.020 M Sorghum is an agronomically important crop for 2-(N-morpholino)ethanesulfonic acid (MES)-KOH pH which detailed RFLP maps of intra- and interspecific 5.5) until all the leaves had been processed. The leaf crosses are available [8, 39, 61]. The isolated cDNAs pieces were resuspended in 200–300 ml fresh medium can thus be mapped in the Sorghum genome. Due to the A supplemented with 2% (w/v) Cellulase Onozuka R- extensive synteny in the grasses [1, 4, 31] orthologous 10 and 0.2% (w/v) Macerozyme Onozuka R-10 (both 321

from Yakult Honsha, Japan) followed by a 2 to 2.5 h (Stratagene) resulting in 9 ࣾ 10 individual phages.

incubation in the dark at 25 C with gentle shaking. The library was ampliﬁed following standard proced- After enzymatic digestion the suspension was poured ures [43]. over a tea sieve and the remaining leaf pieces were shaken in 100 ml fresh incubation medium for 5 min. Differential screening of the cDNA library

The ﬁltrates were combined and re-ﬁltered through a ࣾ

80 ऌm nylon mesh. Protoplasts in the ﬁltrate were About 5000 phage were plated on a 20 cm 35 cm g sedimented by centrifugation at 430 ࣾ for 3 min rectangular plastic dish with Escherichia coli strain with a swinging bucket rotor (HS4 rotor, Sorvall- XL1-Blue (Stratagene Cloning Systems, San Diego, Dupont). The pelleted protoplasts were microscopic- CA) as the recipient. Plaques were successively blot- ally examined for their intactness and then resuspen- ted in series to a total of four nitrocellulose ﬁlters (BA ded in 100 ml medium B (0.33 M sorbitol, 0.30 M 85; Schleicher & Schull,¨ Dassel, Germany). The phage

NaCl, 0.010 M EDTA, 0.010 M ethyleneglycol-bis(ß- DNAs were allowed to adsorb to the membrane for 0.5, 0 aminoethyl ether)-N,N,N 0 ,N -tetraacetic acid (EGTA), 1, 2 and 4 min, respectively. Upon completion of trans- 0.010 M dithiothreitol (DTT), 0.010 M diethyldith- fer, the filters were incubated for 4 min in 0.5 M NaOH, iocarbamic acid, 0.2 M Tris-HCl pH 9.0). Further 1.5 M NaCl, neutralized by 7 min incubation in 0.5 M purification of the mesophyll RNA and isolation of Tris-HCl, 3 M NaCl pH 7.4 and finally equilibrated poly(A)+ RNA followed standard procedures [56]. for 7 min in 250 mM Na2HPO4 pH 7.2. DNA was immobilized on the filters by baking for 2 h at 84 C. Isolation of RNA from bundle-sheath strands Radioactively labelled cDNA probes were pre-

pared from mesophyll and bundle-sheath poly(A) +

For the preparation of bundle-sheath strands 20–30 g RNA essentially as described by Sambrook et al.

+ ऌ leaf material was divided into 2 to 3 portions and each [43]. The poly(A) RNAs (1 ऌg each in 5 l double-

portion was homogenized in a Waring blender with distilled water) were heated for 5 min at 70 C, cooled

200 ml medium C (0.6 M sorbitol, 0.005 M MgCl2, on ice and then added to a 22 ऌl reaction mixture 0.050 M sodium ascorbate, 0.001 M aurintricarboxyl- containing 50 mM Tris-HCl pH 8.3, 10 mM DTT,

ic acid, 0.005 M diethyldithiocarbamic acid, 0.020 M 3mMMgCl2,75mMKCl,800ऌM dGTP, dTTP and

32 ऌ

MES/KOH, pH 5.5). The combined homogenates were dCTP, 4.8 ऌM dATP, 100 Ci [ P]-dATP (Amersham ऌ ﬁltered through a 80 ऌm mesh and the crude bundle- Buchler, Braunschweig, Germany), 12.5 g random sheath residue in the ﬁlter was resuspended in 200 hexamer primers (Boehringer, Mannheim, Germany), ml medium A containing 2% Cellulase Onozuka R-10 20 U RNasin (Boehringer, Mannheim) and 400 U M- and 0.2% Macerozyme Onozuka R-10. After 30 min MLV reverse transcriptase (Gibco–BRL, Eggenstein,

incubation at 25 C bundle-sheath strands were col- Germany). The reaction mixtures were incubated for

ऌ

lected in a 80 ऌm mesh and homogenized twice in a 1hat37 C, and terminated by adding 12 l1M ऌ Waring blender for 30 s each in 200 ml medium C. NaOH, 2 ऌl0.25MEDTAand1 l 10% (w/v) SDS. The pure bundle-sheath strands were then recovered, After 30 min incubation at room temperature the mix-

immediately frozen in liquid nitrogen and ground to a tures were neutralized by the addition of 8 ऌl 2 M acetic

ﬁne powder with a aid of a mortar and a pestle. Further acid and 10 ऌl 1 m Tris-HCl pH 7.6. The radioactively isolation of bundle-sheath RNA was as described [56]. labelled cDNAs were recovered by phenol/chloroform extraction and precipitation with 3 volumes ethanol in

Synthesis and cloning of cDNA the presence of 0.3 M sodium acetate and 20 ऌgof glycogen (Boehringer, Mannheim). The precipitated

Poly(A) + RNA was isolated from total leaves of 8- cDNAs were resuspended in sterile bidistilled water day old sorghum seedlings and converted into double- and the incorporated radioactivity was determined by stranded cDNA using the cDNA cloning kit from spotting aliquots on DE 81 paper followed by liquid Stratagene Cloning Systems (San Diego, CA). The scintillation counting. double-stranded cDNAs were size-fractionated on a Filters were prehybridizedin phosphate/SDS medi-

Sepharose-4B column [43] and fragments larger than um [9] at 65 C for 2 h. A maximum of 8 ﬁlters

600 bp were ligated into Lambda Uni ZAP XR (Strata- (17 cm ࣾ 19 cm) were incubated in a glass tube with gene Cloning Systems). Ligated DNAs were packaged 2.6 ml hybridization medium for 14–18 h. The ﬁrst into phage using Gigapack II Gold Packaging extracts and second ﬁlters from each plate were hybridized 322

with 3 ࣾ 10 cpm of labelled mesophyll or bundle- were considered as being homologous to identified sheath cDNA probe, respectively. The third and fourth sequences, if the BLASTX similarity scores were filter were hybridized with a mixture of known cDNAs greater than 200. for genes differentially expressed in mesophyll and bundle-sheath cells. These cDNAs included sequences RNA analysis for PEPC, pyruvate orthophosphate dikinase, NADP- malic enzyme, NADP malate dehydrogenase and the Sizing and quantification of RNAs by northern analysis small subunit of Rubisco. The hybridizations with the and dot blot hybridization were carried out according known cDNAs were carried out to prevent re-isolation to standard procedures [56]. Hybridizing bands or dots

of these abundant cDNAs. After hybridization, the were visualized by ﬂuorography with Kodak XAR-5 ࣾ

ﬁlters were washed once in 0 :5 SSC, 0.5% (w/v) ﬁlms and, if necessary, the bound radioactivity was ࣾ SDS and four times in 0 :1 SSC, 0.5% (w/v) SDS determined by liquid scintillation counting. To be able

at 65 C for 45 min each. Filters were exposed over to compare hybridization signals obtained with probes night on CEA RP X-ray ﬁlms using Dupont intensify- of different sizes, all data were standardized to a probe ing screens. length of 1 kb. Based on these normalized hybridiza-

Phage hybridizing differentially with the meso- tion signals the transcripts were arbitrarily assigned ऌ

phyll and bundle-sheath cDNA probes, but not with to three abundance classes: I ( >5000 cpm g leaf

+ +

the mixture of known cDNAs were isolated, re-plated poly(A) RNA), II (500–5000 cpm/ऌg leaf poly(A)

+ ऌ at low density and re-hybridized with the mesophyll RNA) or III (<500 cpm/ g leaf poly(A) RNA). and bundle-sheath cDNA probes. Only phage which gave a clear differential signal were purified by a third RFLP mapping round of hybridization, and the inserted fragments of the Lambda phage were excised in vivo with the f1 The mapping population, the molecular biological helper phage R408 [41] according to the manufacturer- techniques for RFLP analyses, the linkage analysis ’s protocol. and the nomenclature for loci and ‘linkage groups’ (chromosomes) are as described by Chittenden et al. Classification and identification of cDNA clones [8].

The cDNAs isolated by differential screening were Miscellaneous grouped into classes of sequence identity/similarity by cross-hybridization. For this purpose, the cDNA- Isolation of recombinant plasmids, Southern analysis containing plasmids were digested with EcoRI/XhoI of plasmid and genomic DNA and DNA sequence ana- (or BamHI/ApaI, if the EcoRI and XhoI cloning sites lysis was carried out according to standard protocols were not intact), and the restricted DNAs were sub- [43]. jected to Southern analysis using stringent conditions

of hybridization (70 C; phosphate/SDS medium) and

ࣾ washings (70 C; 0 :1 SSC, 0.5% (w/v) SDS). Each Results of the isolated cDNAs was successively hybridized to the Southern-blotted DNAs with the exception of Differential screening of a leaf cDNA library and those which had been identiﬁed in a previous round of sequence analysis of the isolated clones hybridization. The largest cDNA clone of each identity/similarity The differential screening of a cDNA library was the

class was partially sequenced from the putative 50 first step towards the identification of genes which end of the cDNA insert using a T7 DNA polymerase are differentially expressed in mesophyll and bundle- kit (Pharmacia, Freiburg, Germany). If BLASTX sheath cells. For this technique pure mesophyll and searches [45] did not reveal any significant matches bundle-sheath RNA preparations were a prerequisite. with sequences deposited in the non-redundant data- With the well established combination of mechan- base at NCBI, additional sequence information was ical and enzymatic treatments [28] cell preparations

gathered from the putative 3 0 end of the insert. In of a sufficient purity were obtained. By assaying for addition, cDNA-specific oligonucleotide primers were mesophyll and bundle-sheath marker RNAs, i.e. for used to collect further sequence data. The cDNAs PEPC and NADP-malic enzyme transcripts, the cross- 323 contamination of both RNA fractions was estimated to Transcript size and abundance be less than 5%. RNAs which accumulate exclusively in one of the two cell-types should, therefore, give To verify that the isolated cDNAs were derived from at least a 20-fold difference in hybridization intens- expressed genes and to determine the size and the ity in the differential screening of the cDNA library. abundance of the corresponding transcripts, RNA gel To prevent re-isolation of cDNAs whose correspond- blot analyses were carried out with total leaf RNA. All ing RNAs were known to accumulate differentially clones with the exception of HHU22 were found to in mesophyll and bundle-sheath cells, i.e. the tran- hybridize to single RNAs (data not shown; Table 1). scripts encoding the C4 assimilatory enzymes, probes This indicated that the genes represented by these for these genes were co-hybridized on separate filters cDNAs are expressed in sorghum leaves and that these (see Materials and methods). genes are transcribed into RNAs of identical sizes, des- About 15 000 phage of the amplified cDNA lib- pite the fact that some occur in multiple copies in the rary prepared from total leaf poly(A) + RNA of genome. HHU22 which encodes carbonic anhydrase young sorghum leaves were screened differentially. sequences, hybridized to two abundant leaf RNAs of By visual comparison of the fluorographs 238 clones 1700 and 2100 nucleotides. In addition, a faint hybrid- were identified as candidates for mesophyll-specific ization signal to a transcript with a size of 1200 nuc- cDNAs and 116 for bundle-sheath-specific genes. leotides was detected. This finding suggested that the Cross-hybridization assays under stringent conditions Sorghum genome contains several different carbonic allowed grouping of the clones into 59 different classes anhydrase genes and that the matter required further which contained from one to 46 (HHU17) individual analyses (see below). clones (see Table 1). Transcript abundance was roughly assessed by The largest clone of each class was partially measuring the radioactivity of the hybridized probe in sequenced and the sequences obtained were subjec- a liquid scintillation counter. The hybridization sig- ted to data base searches using the BLASTX sub- nals were normalized with respect to probe length routine [42]. Three of the cDNA classes were found and the transcripts were arbitrarily assigned to three to encode plastid genes (rbcL, psaA and atpH)and abundance classes (see Materials and methods) con- were excluded from further analysis. Two cDNA sisting of highly (I), moderately (II) and less abund- classes, as defined by cross-hybridization analysis had ant (III) transcripts. The majority of the cDNAs isol- to be merged since sequencing revealed that they were ated, encoded transcripts which accumulated to high derived from the same gene. Three cDNAs (HHU14, or moderate levels in sorghum leaves (Table 1). These HHU29 and HHU52) were found to be chimerical. class I and II transcripts included, for instance, RNAs The analysis of HHU52 was not continued because encoding the C4 and Calvin cycle enzymes and com- it contained sequences from two different members ponents of the photosynthetic electron transport chain of chlorophyll a/b-binding proteins already present (Table 1). Seven cDNAs (HHU21, HHU24, HHU25, in the clone collection. In the case of HHU12 and HHU40, HHU42, HHU49 and HHU56) were found to HHU29, the chimerical fragments were separated and encode less abundant transcripts. Two of the cDNAs only those fragments re-cloned which gave rise to the contained known sequences, i.e. for the chloroplast

differential hybridization signals with mesophyll and adenine nucleotide translocator (HHU42) and Rubisco ! bundle-sheath RNA (HHU12 ! HHU71; HHU29 activase (HHU56). The remaining five cDNAs did not HHU72). Six additional sorghum cDNA clones coding show any significant similarities to sequences in the for phosphoenolpyruvate carboxylase, pyruvate ortho- data bases. This suggested that they may encode novel phosphate dikinase, NADP-malic enzyme, the delta genes. subunit of the chloroplast ATP synthase, subunit D of photosystem I reaction centre and a 70 kDa heat Mesophyll/bundle-sheath specificity of expression shock protein had been isolated in previous studies ([21, 34] and unpublished data), thus a total of 58 dif- The cDNAs were isolated due to their differential ferent cDNAs were available for further analysis. 46 of hybridization to mesophyll and bundle-sheath probes. these cDNAs encoded known genes while 12 did not To verify that these cDNAs encode transcripts that reveal any significant matches to sequences deposited accumulate differentially in mesophyll and bundle- in the data bases (Table 1). sheath cells their cell-specific accumulation pattern was analysed by quantitative dot blot hybridization. A 324

Table 1. List of isolated cDNA clones.

HHU1 – pyruvate orthophosphate dikinase (Ppdk1) 1900 3600 I 10–20 356368; 356367

HHU2 – phosphoenolpyruvate carboxylase (Ppc1) 1500 3600 I > 20 356370; 356369

HHU3 – NADP-malic enzyme (Mod1) 1350 2500 I < 0.05 356372; 366371 HHU4 32 photosystem II, 33 kDa subunit (PsbO) 1400 1400 I 10–20 356374; 356373 HHU5 13 photosystem II, 23 kDa subunit (PsbP) 1000 1300 I 10–20 356375; 356384 HHU6 11 photosystem II, 16 kDa subunit (PsbQ) 800 1100 I 10–20 356377; 356376

HHU7 7 photosystem II, 10 kDa subunit (PsbR) 580 900 I 10–20 356378ࣿ HHU8 1 plastocyanin (PetE) 750 1000 I 1–10 356380; 356379 HHU9 – photosystem I, subunit D (PsaD) 775 1100 II 1–10 356382; 356381 HHU10 – heat shock protein HSP70 780 2600 II 1–10 356363; 356365 HHU11 14 chlorophyll a/b-binding protein CP24 1250 1400 II 10–20 356294; (Lhcb6)– HHU12 12 chlorophyll a/b-binding protein CP26 1050 1200 I 10–20 356295; (Lhcb5) 1393256

HHU13 4 (NADP + )-glyceraldehyde-3-phosphate 1230 1800 II 10–20 356296; dehydrogenase, chloroplast (GapB1)– HHU15 6 ferredoxin-NADP-oxidoreductase (PetH) 920 1700 II 10–20 356298; –

HHU16 10 chlorophyll a/b-binding protein CP29 1178 1400 II 10–20 356299ࣿ (Lhcb4) HHU17 46 chlorophyll a/b-binding protein type II 1150 1200 II 1–10 356300; LHCII (Lhcb2) –

HHU18 1 photosystem II, 22 kDa subunit (PsbS) 529 1400 II 10–20 356301 ࣿ HHU19 1 proline-rich protein 1680 1700 II 10–20 356302; 356303

HHU20 4 ferredoxin (PetF) 890 1000 I > 20 356304; 356305

HHU21 1 ? 1150 1100 III > 20 356306; 356307 a HHU22 26 carbonic anhydrase (CAH) 1730 2100, II > 20 356308; 1700, – 1200 HHU23 1 chlorophyll a/b-binding protein type III 960 1200 I 1–10 356309; LHCI (Lhca3) 356310 HHU24 1 ? 1880 3000 III 10–20 356311; 356312

HHU25 1 ? 584 2900 III 1–10 356313ࣿ ࣿ

HHU26 1 ? 867 1300 II >20 356314 HHU27 1 S-adenosylmethionine decarboxylase 1880 2200 II 1–10 356315; 356316 HHU28 2 glutamyl tRNA reductase 1660 2100 II 1–10 356317; 356318 325

Table 1 continued.

Clone Class Putative identiﬁcation Insert RNA Expression Mesophyll/ dbEST design. size size size (nt) level bundle-sheath accession (bp) ratio number

HHU30 4 cytochrome b6f-complex, Rieske Fe-S 980 1100 I 1–10 356320; subunit (PetC) 356321

HHU31 1 ? 588 900 II 1–10 356322ࣿ HHU32 1 adenylate kinase, chloroplast 1410 1400 II 1–10 356323; –

HHU33 1 triosephosphate translocator, chloroplast 221 1800 II 1–10 356324ࣿ

HHU34 1 photosystem I, subunit F (PsaF) 940 1100 I 1–10 356325;

HHU35 1 ? 863 2100 II 10–20 356326ࣿ HHU36 1 ? 1390 1800 II 1–10 356327;

– ࣿ

HHU37 2 vacuolar H + -translocating pyrophosphatase 481 3100 II 10–20 356328 ࣿ

HHU38 2 ? 1026 1600 II >20 356329 HHU39 1 glutamine synthetase, chloroplast (Gln2) 1740 1900 II 1–10 356330; –

HHU40 1 ? 780 1800 III 10–20 35631ࣿ

HHU42 1 adenine nucleotide translocator, chloroplast 929 2500 III 1–10 356332 ࣿ

HHU43 1 triosephosphate isomerase, chloroplast 860 1500 II >20 356333;

(TPIC1) – ࣿ

HHU44 ATP synthetase, chloroplast, subunit 1100 1100 II 1–10 X66004 (AtpD) HHU45 10 metallothionein-like protein 680 800 II 1–0.1 356337; – HHU46 1 ? 874 1100 II 1–0.1 356338; –

HHU48 15 fructose-1.6-bisphosphatase aldolase, 1800 1900 I <0.05 356339; chloroplast (FBAC1) 356340 HHU49 1 ? 1300 1700 III 1–0.1 356341; 356342 HHU50 1 photosystem I, subunit G (PsaG) 1200 800 I 1–0.1 356343;

– ࣿ

HHU51 1 2-oxoglutarate/malate translocator 903 2500 II <0.05 356344

HHU53 1 water stress-induced protein (WSI729), 600 900 I <0.05 356347; rice –

HHU55 1 ? 1000 1200 II 1–0.1 356350ࣿ

HHU56 2 rubisco activase (RCA1) 800 1900 III <0.05 356351; – HHU57 3 root nodule protein (MtN3), Medicago 1480 1800 II 1–0.1 356352; trunculata 356353

HHU58 3 transketolase, chloroplast (TKLC1) 1910 3100 II <0.05 356354; 356355

HHU60 4 ribulose-5-phosphate 3-epimerase (RPE1) 1000 1500 II <0.05 356356; – HHU61 5 metallothionein-like protein 760 900 I 1–0.1 356357; –

HHU62 7 phosphoribulokinase (PRK1) 1500 1700 I <0.05 356358; – 326

Table 1 continued.

Clone Class Putative identiﬁcation Insert RNA Expression Mesophyll/ dbEST design. size size size (nt) level bundle-sheath accession (bp) ratio number

HHU63 1 ! -6 fatty acid desaturase 1200 1900 II 1–0.1 356359; – HHU68 – carbonic anhydrase (CAH1) 1100 1200 – – –; 356334

HHU69 – carbonic anhydrase (CAH2) 1100 1700, II >20 356336; 2100 356335 b

HHU71 4 photosystem II, 5 kDa subunit (PsbT) 650 900 II >20 –; 1393258 HHU72 15 photosystem II, 7 kDa subunit (PsbW) 642 800 II 10–20 1393259; –

The columns refer respectively to: (1) the designation of the locus and the corresponding cDNA; (2) the number of clones of this cDNA family as isolated in the primary screening; (3) putative identiﬁcation as deﬁned by BLASTX searches (if available gene symbols are given in parenthesis); (4) insert size of the cDNA clone designated with the HHU number; (5) size of the RNA(s); (6)

expression level as estimated by quantitative RNA gel blot analysis; (7) mesophyll/bundle-sheath ratio of RNA levels as estimated 0 by quantitative dot blot analysis; (8) dbEST accession numbers of the putative 50 and 3 ends of the cDNAs (fully sequenced clones are labelled by asterisk). a The identity of HHU22, i.e. whether the cDNA encodes CAH1 or CAH2 carbonic anhydrase sequences,

could not be determined, since the sequencing from the putative 30 end was not possible. b The naming of psbT is ambiguous because this designation has been given to a plastid-encoded subunit of photosystem II [30] as well as to a nuclear-encoded subunit of this photosystem [22]. dilution series of mesophyll and bundle-sheath RNAs With these criteria 25 cDNAs were found to encode each was spotted side by side onto a nylon membrane RNAs which accumulate specifically or preferentially and hybridized with the cDNA to be analysed. Hybrid- in mesophyll cells and 8 cDNAs were identified as ization signals were first visualized by fluorography encoding bundle-sheath-specific or -preferential tran- and the hybridized radioactivity was then quantitated scripts (Figure 1 and Tables 1 and 2). All cDNAs by liquid scintillation counting. which encode bundle-sheath-specific or -preferential These values were used to group the transcripts transcripts were identified by their significant sequence into three classes with the mesophyll/bundle-sheath similarities to known proteins. However, 6 of the 25 ratios differing more than 20-fold, between 10- and mesophyll-specific or -preferential transcripts encoded 20-fold, and less than 10-fold. A more than 20-fold dif- proteins of hitherto unknown functions. ference indicates that the respective transcripts accumulate similarly as PEPC (Ppc1) and NADP-malic Expression patterns of the mesophyll-specific enzyme (Mod1). Since these marker RNAs are known transcripts of unknown identity to accumulate exclusively in mesophyll or bundle- sheath cells, respectively, transcripts of this group are To characterize the mesophyll cDNA clones which considered to be mesophyll or bundle-sheath-specific encoded unidentified sequences (HHU21, HHU24, sensu strictu. Ratios between 10 to 20 indicate that the HHU26, HHU35, HHU38 and HHU40), the expres- transcripts do not accumulate in a strict cell-specific sion profiles of their corresponding RNAs were manner, but accumulate preferentially in one or the linked to organ-type and light/dark growth. Three other cell type. Mesophyll/bundle-sheath differences more cDNAs, HHU19, HHU37 and HHU53 were less than tenfold may still indicate a cell-preferential included in this analysis because the sequence sim- accumulation. However, for the sake of experimental ilarities obtained for these clones (see Table 1) did accuracy a less than tenfold difference in steady-state not reveal any clue about their possible function in C4 levels between mesophyll and bundle-sheath cells was photosynthesis. HHU37 encodes a vacuolar proton- not considered to be diagnostic of a differential accu- translocating pyrophosphatase [57] and HHU19 a mulation pattern. proline-rich protein [58] both of which accumulate preferentially in the mesophyll cells (Figure 1). 327

Table 2. Compilation of mesophyll- and bundle-sheath-speciﬁc cDNA sequences.

Mesophyll cells Bundle-sheath cells

Photosystem II

light-harvesting complex: CP29 (Lhcb4; HHU16), CP26 (Lhcb5; HHU12), CP24 (Lhcb6; HHU11)

oxygen-evolving complex: PSII-33K (PsbO; HHU4), PS II-23K (PsbP; HHU5), PSII-16K (PsbQ; HHU6)

other subunits: PSII-22K (PsbS; HHU18), PSII-10K (PsbR; HHU7), PSII-7K (PsbW; HHU72), PSII-5K (PsbT, HHU71)

NADP reduction

ferredoxin (PetF; HHU20)

NADP-ferredoxin oxidoreductase (PetH; HHU15)

C4 cycle phosphoenolpyruvate carboxylase(Ppc1; HHU2) NADP-malic enzyme (Mod1; HHU3)

pyruvate orthophosphate dikinase (Ppdk1; HHU1)

carbonic anhydrase (CAH2; HHU69)

Calvin cycle NADPH glyceraldehyde-3-phosphate rubisco activase (RCA1; HHU56)

dehydrogenase (GAPB1; HHU13) fructose-1.6-bisphosphate aldolase (FBAC1; HHU48) triosephosphate isomerase, chloroplast (TPIC1; HHU43) phosphoribulokinase (PRK1; HHU62)

transketolase (TKLC1; HHU58)

ribulose-5-phosphate-3-epimerase (RPE1; HHU60)

Translocators

+ vacuolar H -translocating pyrophosphatase (HHU37) 2-oxoglutarate/malate translocator (HHU51)

Other identiﬁed sequences proline-rich protein (HHU19) water-stress-induced protein (HHU53)

unidentiﬁed sequences

HHU21, HHU24, HHU26, HHU35, HHU38, HHU40

HHU53 transcripts accumulate preferentially in the the mesophyllcells. The levels of the HHU21, HHU26, bundle-sheath cells and are highly similar to a rice HHU38 and HHU40 transcripts increase strongly after mRNA which is up-regulated by water stress [53]. illumination of the etiolated seedlings and demon- Figure 2 illustrates that HHU19, HHU35 and strates that these genes are under the control of light. In HHU53 transcripts do not only accumulate in leaves contrast, HHU24 and HHU37 RNA levels are similar but also in roots, i.e. their expression is not restric- in etiolated and greening seedlings. This indicates that ted to the mesophyll (HHU19, HHU35) or bundle- these genes are not regulated by light. sheath-cells (HHU53) and these genes cannot be called mesophyll- or bundle-sheath-specific in the strict sense Carbonic anhydrase sequences in sorghum: two of the word. Light does not affect the accumulation distinct genes, CAH1 and CAH2, with different of these RNAs in the leaves of young seedlings. The expression profiles HHU35 transcript levels are even down-regulated during the greening of etiolated seedlings. The northern blot analysis of RNA from leaves of HHU21, HHU24, HHU26, HHU37, HHU38 and greenhouse-grownplants with HHU22 as a probe iden- HHU40 transcripts cannot be detected in root RNA tified two abundant carbonic anhydrase transcripts of (Figure 2). This indicates that the expression of the 2100 and 1700 nucleotides in size and trace amounts corresponding genes is confined to the leaves, i.e. to of a third, 1200 nucleotide RNA (see above). Evidence 328

Figure 2. Organ-speciﬁcity and light dependence of the expression of the unidentiﬁed cDNA sequences that are differentially expressed in mesophyll and bundle-sheath cells of sorghum. RNA was isolated from roots (R) of 14-day old light-grown seedlings, from the leaves of seedlings which had been grown for 5 days in the dark (E) and from seedlings which had been illuminated for 24 h after a 5-day

period of etiolation (G). RNA gel blot analyses were carried out

+ ऌ with 1 ऌg (HHU19, 26, 35, 37, 38 and 53) or 4 g poly(A) RNA

each (HHU21, 24 and 40). The ﬁlters were hybridized at 65 Cin SDS/phosphate medium. Filters were exposed for one to three days depending on the signals obtained.

from dot blot (Figure 1) and RNA gel blot analyses (data not shown) proves that at least the 2100 and 1700 transcripts accumulate in mesophyll cells only. The accumulation preference of the 1200 nucleotide RNA was not resolved because the amounts were too small. To dissect this complex pattern of carbonic anhydrase transcripts, their accumulation, as affected by organ Figure 1. RNA dot blot analysis of cDNA sequences which are dif- type or light, was analysed by RNA gel blot hybridiz- ferentially expressed in mesophyll and bundle-sheath cells. Panel A. Analysis of known-function cDNAs. Panel B. Analysis of uniden- ation. tified cDNAs. Poly(A) + RNA was prepared from mesophyll and As might be predicted for a gene which is involved bundle-sheath preparations as described in Materials and methods. in C4 photosynthesis carbonic anhydrase RNA levels Dilution series of the two RNA fractions (see figure) were spotted were found to be controlled by light. The 2100 and onto nylon membranes and the membranes were hybridized with the radiolabelled inserts of the HHU clones in SDS/phosphate buffer [9] 1700 nucleotide transcripts were undetectable in the at 65 C. After washing, the filters were exposed to X-ray films for leaves of etiolated seedlings and accumulated only one to two days depending on the signal strength. Dots were excised after illumination (Figure 3). A light-induced increase from the filters and the radioactivity was determined in a liquid scin- in steady-state levels was also observed for the 1200 tillation counter. The ratios of expression levels in mesophyll and bundle-sheath cells are tabulated in Table 1. For gene designations, see Table 1. 329

microsatellite in the 3 0 -untranslated segment of the HHU68-type cDNAs (Figure 4).

The differences in the 3 0 -untranslated sequences of the HHU68 and HHU69 carbonic anhydrase sequences were used to construct gene-specific hybridization probes (Figure 5A) and to correlate transcripts and genes (Figure 5B). The HHU68-S probe (gene designation CAH1) hybridized only to the 1200 nucleotide RNA, while HHU69-S (gene designation CAH2) labelled both the 2100 and 1700 nucleotide transcripts. The existence of these two genes was confirmed at the genomic level by southern analyses. The CAH1- and CAH2-specific probes (HHU69-S and HHU68-S, respectively) recognize different sets of DNA fragments (Figure 6) which appear as component and com- plementary fragments in the hybridization pattern of the HHU68-L probe (Figure 6). The latter probe con- Figure 3. Organ specificity and light dependence of carbonic anhyd- tains carbonic anhydrase coding sequences (Figure 4) rase RNA abundance in leaves (E, G) and roots (R) of sorghum seed- and recognizes both the CAH1 and CAH2 sequences lings and in rice leaves (L). Sorghum RNAs used are as described in the legend to Figure 2. The rice leaf RNA was isolated from 13-day as well as other related carbonic anhydrase sequences,

old plants grown in the greenhouse. One ऌg of each RNA fraction when present. Hybridization of southern-blotted rice was analysed by northern blotting using the inserted fragment of DNA with this probe revealed a single-banded pattern HHU22 as hybridization probe. after digestion with various enzymes. This suggests that rice contains only one carbonic anhydrase gene and is in line with the northern hybridization analysis nucleotide RNA but traces of this RNA were already of leaf RNA of this species (Figure 3). As only par- present in the leaves of etiolated seedlings (Figure 3). tial cDNA sequences were available for the CAH1 and Traces of the 1200 nucleotide transcript were also CAH2 carbonic anhydrase genes of sorghum their rela- detectable in RNA which was isolated from roots of tionship with the rice gene [52] needs to be determined. greenhouse-grownseedlings, where the 2100 and 1700 nucleotide RNAs were absent (Figure 3). Collectively Genetic mapping of the cDNA sequences in Sorghum these data suggested that sorghum possesses two types of carbonic anhydrase transcripts with different expres- For information about the chromosomal location in the sion profiles, i.e. the 2100 and 1700 nucleotide RNAs Sorghum genome those cDNAs which detected restric- on one hand and the 1200 nucleotide on the other. tion fragment length polymorphisms and encoded low- This complexity of carbonic anhydrase transcripts con- copy sequences were placed on the molecular map of trasts with the C3 grass rice (Figure 3) where only a an interspecific cross [8]. A total of 43 cDNAs were single carbonic anhydrase transcript of about 1.4 kb mapped and six of them yielded more than one locus was detected. (Figure 7). Unfortunately, the mapping with HHU22 To substantiate the existence of different carbon- did not allow to resolve the location of the two carbonic anhydrase genes in sorghum the available carbonic anhydrase genes (CAH1 and CAH2) in the genome ic anhydrase cDNA clones (see Table 1) were re- since only one fragment showed length polymorphism. analyzed. Ten clones were randomly selected and Whether the second locus is located near the first one sequenced from the putative 3 0 end of the cDNA insert. or somewhere else in the genome needs to be investig- The sequences revealed two different types of cDNA ated. clones for which HHU68 and 69 are presented as examples (Figure 4). This finding reinforces the existence of at least two different carbonic anhydrase genes Discussion in sorghum. The most prominent and distinguishing feature of the two types of carbonic anhydrase cDNA While detailed information is available about the clones is the presence of a 16 AT dinucleotide long physiological and biochemical context of C4 photosyn- 330

Figure 4.AnalysisofCAH1 (HHU68) and CAH2 (HHU69) carbonic anhydrase sequences of sorghum. Panel A. Sequence alignment of the carboxy-terminal part of the CAH1/CAH2 reading frames and of the 30 -untranslated regions. The translational stop codon is marked in bold, the microsatellite sequence of HHU68 by a shaded box. Panel B. Schematic representation of the structure of HHU68 and HHU69 and location of hybridization probes for southern and northern analysis (see Figures 5 and 6.). thesis [12, 18, 32, 38] our understanding of the molecu- A differential screening approach should pick up only lar and genetic basis of this photosynthetic pathway is those cDNAs whose corresponding transcripts accu- still rather limited. It is well documented that the C4 mulate at high or possibly medium levels [44], i.e. rare cycle genes are differentially expressed in mesophyll transcripts cannot be detected with this method. The and bundle-sheath cells (reviewed in [15, 25, 25]). outcome of our analyses confirms this expectation. All There is also increasing information as to how this dif- the cDNA clones which were identified as encoding ferential expression may be achieved by specific cis- mesophyll- or bundle-sheath-specific transcripts could acting promoter elements [27, 47, 51]. However, the easily be investigated by standard RNA gel and dot trans-regulatory factors which interact with these pro- blot hybridization techniques.It follows that these tran- moter elements and the components of signal transduc- scripts do not belong to the class of rare RNAs [33]. tion pathways that are pertinent for the differentiation Most of the isolated mesophyll- and bundle-sheath- of the two photosynthetic cell-types of a C4 leaf are specific cDNA clones encode proteins whose putative completely unknown. function could be deduced from database searches. To obtain additional information about the gene Component subunits of photosystem II reaction expression patterns which are specific for the differen- centre and its light-harvesting antenna system were tiation of mesophyll and bundle-sheath cells, differen- the most prominent mesophyll-specific gene products tial screening of cDNA libraries was used to catalogue that were detected. Previous reports stated that the 33, genes which are differentially expressed in the two 23 and 16 kDa proteins of the oxygen-evolving com- cell-types of the monocotyledonous C4 plant sorghum. plex of photosystem II accumulate preferentially in 331

Figure 6. Genomic southern blot analysis of carbonic anhydrase sequences in sorghum and rice. Genomic RNA from sorghum and

rice (10 ऌg each) was digested with the indicated restriction enzyme and the southern-blotted DNA fragments were hybridized with the HHU68 and HHU69 probes as depicted at the top of the ﬁgure.

Hybridization was carried out at 65 C. EcoRI/HindIII-digested Lambda DNA was used as a size marker (in kb). Filters were exposed for 5 days. Figure 5. Identification of CAH1 and CAH2 transcripts in leaves of greening sorghum seedlings Panel A. Cross-hybridization analysis of the CAH1 (HHU68-S) and CAH2 (HHU69-S) gene probes. 1, 10 and 100 pg of each double-stranded fragment were immobilized on detectable in bundle-sheath cells. This finding reflects a nylon membrane and hybridized with the labelled HHU68-S or the known fact that bundle-sheath cells of NADP-malic HHU69-S probes. Panel B. Northern hybridization with the CAH1-

enzyme-type C4 species are not necessarily completely + and CAH2-speciﬁc probes. 1 ऌg poly(A) RNA from seedlings illuminated for 24 h after a 5-day period of etiolation was analysed. devoid of any photosystem II but that the differences in The hybridizations were carried out in SDS/phosphate medium at the levels of photosystem II proteins in mesophyll and 70 C. bundle-sheath cells are rather quantitative [16, 28]. The differential screening procedure resulted in the isolation of almost the full complement of nuclear- mesophyll cells [34, 49]. This study adds four more encoded mRNAs for photosystem II subunits. This photosystem II mRNAs to this list, i.e. those from ﬁnding raises the hope that with a more sensitive the psbR, psbS, psbT and psbW genes [35]. In addi- technique, i.e. subtractive hybridization [10] regulat- tion it was shown that the mRNAs which encode the ory components of photosystem II will be detected. minor chlorophyll a/b-binding proteins CP24, CP26, Such an approach would nicely complement genetic and CP29 of photosystem II [17] accumulate differ- strategies which aim to identify regulatory genes of entially in mesophyll and bundle-sheath cells. It has photosystem II biogenesis by mutational analysis [3, been stated above that none of these photosystem II 14, 29]. With gene probes available for both the con- mRNAs accumulate exclusively in the mesophyll cells stituent subunits of photosystem II as well as for the and that small amounts of these transcripts are also regulatory factors of its biogenesis, one will be able to 332

Figure 7. Genetic mapping of cDNA sequences on Sorghum, maize and rice. The mapping population (F2) was derived from a cross between S. bicolor cv. BTx623 and S. propinquum. The chromosomal locations in maize, rice and wheat were inferred for as many HHU loci as possible, based on comparative mapping [36]. Where two maize chromosomes are indicated, the two chromosomes are homologous. Most cDNA clones detected duplicated loci on each of the chromosomes. In two adjacent regions of sorghum linkage group C, the corresponding rice chromosome could not be determined with certainty, but the two most likely candidates have been marked. investigate the evolution of differential photosystem II bundle-sheath-specific transcripts. However, cDNAs biogenesis in the various C4 photosynthetic lineages of for these enzymes were not identified in the differen- the grasses [20]. tial screening experiment. Biochemical analyses of Calvin cycle activity It is established that the transcripts for the C4 cycle in the mesophyll and bundle-sheath chloroplasts of enzymes PEPC, pyruvate orthophosphate dikinase NADP-malic enzyme type C4 plants have shown and NADP-malic enzyme accumulate differentially in that Rubisco and the enzymes of the regenerat- mesophyll and bundle-sheath cells. In contrast, the ive phase of this cycle are confined to the bundle- intercellular distribution of carbonic anhydrase, anoth- sheath cells. On the other hand, the reducing er well-known C4 cycle enzyme has not been reported phase of the Calvin cycle operates preferentially, yet. Carbonic anhydrase generates the rapid supply if not exclusively, in the mesophyll chloroplasts of bicarbonate which is the CO2 substrate of PEPC [26]. Work described here supports this view at the [19]. Biochemical and physiological analyses predict gene expression level. Transcripts encoding NADPH- that carbonic anhydrase should be exclusively found in glyceraldehyde-3-phosphate dehydrogenase and the mesophyll cells [5, 6, 55]. It is demonstrated here that chloroplast isoform of triosephosphate isomerase in sorghum leaves carbonic anhydrase transcripts are accumulate only in mesophyll cells. RNAs which indeed confined to the mesophyll cells and are absent encode fructose-1.6-bisphosphate aldolase, phos- in the bundle-sheath cells. phoribulokinase, transketolase, ribulose-5-phosphate- Two complications have been observed and need 3-epimerase and Rubisco activase are bundle-sheath to be considered. Firstly, the Sorghum genome con- specific transcripts. One may expect that also the tains two different carbonic anhydrase genes, CAH1 other enzymes of the regenerative phase of the and CAH2, both of which are expressed in the leaves, Calvin cycle, i.e. the fructose-1,6-bisphosphate and and secondly, the CAH2 gene is transcribed into two sedoheptulose-1.7-bisphosphate phosphatases, and different transcripts. Although both CAH1 and CAH2 ribulose-5-phosphate isomerase, are among the are expressed in leaves, the steady-state levels of the 333

CAH2 transcripts are far more abundant than those cDNAs were detected in leaves only which suggests of the CAH1 transcripts. In fact, leaves of 10-day that the corresponding proteins are speciﬁc for leaf old sorghum plants contain only traces of this RNA functions. HHU21, HHU26, HHU38 and HHU40 tran- suggesting that the CAH1 carbonic anhydrase are not script levels, moreover, are controlled by light. This needed for C4 photosynthesis. The high levels of the may indicate that these genes are involved in C4 pho- two CAH2 transcripts in the mesophyll cells of the tosynthesis. However, a full characterization of the leaves, on the other hand, indicate that carbonic anhyd- cDNAs has to be awaited to permit reliable conclu- rase proteins encoded by these latter transcripts are the sions about the putative functions of these genes. ones that supply the substrate bicarbonate to PEPC in It is known that the differential screening method is the mesophyll cells and hence are involved in the C4 not suitable for the detection of differentially expressed photosynthetic pathway. genes with a low transcript abundance. Therefore, Northern and southern hybridization experiments more unknown genes with relevance to the C4 pho- demonstrated that the two CAH2 transcripts are derived tosynthetic pathway are to be expected, when more from one single gene. To date, no full-size cDNA sensitive methods [10] are exploited for cDNA identi- clones for these two transcripts have been isolated from ﬁcation. The present study has initiated the cataloguing sorghum and therefore one cannot say anything about of new genes which may be involved in the establish- the differences in primary structure of the encoded ment and functioning of C4 photosynthesis. This has proteins and their putative functions. It is very likely, to be followed by further studies until a more complete however, that the sorghum CAH2 carbonic anhyd- view of the genetic basis of this fascinating pathway of rase transcripts correspond to two carbonic anhydrase photosynthesis is available. sequences from maize which are indentical in their

3 0 -untranslated regions and the carboxy-terminal parts of the carbonic anhydrase reading frames but differ in Acknowledgements the aminoterminal halves of the proteins [7]. It will be interesting to see whether the two carbonic anhyd- This work has been supported by grants from the Bio- rase proteins of maize differ in function and cellular technology Programme of the European Union (con- location. tract BIO2-CT93-0400) and from the German-Israeli

The comparison of the 3 0 -untranslated sequences Foundation to P.W.A.H.P.acknowledgesfinancial sup- of the CAH1 and CAH2 cDNAs shows a high degree port from the USDA Plant Genome Program, and Pion- of similarity. This suggests that the two genes diverged eer Hibred International. D.Q. acknowledges a fellow- quite recently from one another.Unfortunately the gen- ship from the Chinese Academy of Sciences. Thanks omic locations of the CAH1 and CAH2 genes could are due to Dr U.J. Santore for critically reading the not be determined by RFLP analysis. It remains an manuscript. open question therefore whether the two CAH genes are located at different places in the Sorghum genome or whether they are arranged adjacent to each other on References the same chromosome. One possible basis for duplic- ation of CAH genes may be the finding that sorghum, 1. Ahn S, Tanksley SD: Comparative linkage maps of the rice like maize, contains ancient duplications of some (per- and maize genomes. Proc Natl Acad Sci USA 90: 7980–7984 (1993). haps all) chromosomal segments [8, 36]. Further map- 2. Baldy P, Cavalie´ G: Compartmentation of photorespiratory ping efforts which use the microsatellite sequence in enzymes in a C4 photosynthesis plant, Zea mays. Z Pflan- the 3 0 -untranslated region of CAH1 mayhelptosolve zenphysiol 114: 255–259 (1984). 3. Barkan A: Nuclear mutants of maize with defects in chloroplast this problem. polysome assembly have altered chloroplast RNA metabolism. Six cDNAs (HHU21, HHU24, HHU26, HHU35, Plant Cell 5: 389–402 (1993). HHU38, HHU40) were isolated during the course 4. Bennetzen JL, Freeling M: Grasses as a single genetic system: of this investigation whose corresponding transcripts genome composition, collinearity and compatibility. Trends Genet 9: 259–261 (1993). accumulated specifically in the mesophyll cells and 5. Burnell JN: Immunological study of carbonic anhydrase in C3 whose coding sequences did not match any known- and C4 plants using antibodies to maize cytosolic and spinach function gene. The HHU35 gene is expressed also in chloroplastic carbonic anhydrase. Plant Cell Physiol 31: 423– the roots suggesting that the HHU35 protein does not 427 (1990). function in C4 photosynthesis. Transcripts of all other 334

6. Burnell JN, Hatch MD: Low bundle sheath carbonic anhydrase processing in isolated chloroplasts. J Biol Chem 270: 12197– is apparently essential for effective C4 pathway operation. Plant 12202 (1995). Physiol 86: 1252–1256 (1988). 23. Ku MSB, Kano-Murakami Y, Matsuoka M: Evolution and 7. Burnell JN, Ludwig M: Characterization of two cDNAs encod- expression of C4 photosynthesis genes. Plant Physiol 111: ing carbonic anhydrase in maize leaves. Aust J Plant Physiol 949–957 (1996). 24: 451–458 (1997). 24. Kubicki A, Steinmuller¨ K, Westhoff P: Differential transcrip- 8. Chittenden LM, Schertz KF, Lin Y-R, Wing RA, Paterson AH: tion of plastome-encoded genes in the mesophyll and bundle-

A detailed RFLP map of Sorghum bicolor ࣾ S. propinquum, sheath chloroplasts of the monocotyledonous NADP-malic suitable for high-density mapping, suggests ancestral duplic- enzyme type C4 plants maize and Sorghum. Plant Mol Biol ation of Sorghum chromosomes or chromosomal segments. 25: 669–679 (1994). Theor Appl Genet 87: 925–933 (1994). 25. Langdale JA, Nelson T: Spatial regulation of photosynthetic 9. Church GM, Gilbert W: Genomic sequencing. Proc Natl Acad development in C4 plants. Trends Genet 7: 191–196 (1991). Sci USA 81: 1991–1995 (1984). 26. Leegood RC: The regulation of C4 photosynthesis. Adv Bot 10. Diatchenko L, Lau YFC, Campbell AP, Chenchik A, Moqadam Res 26: 251–316 (1997). F, Huang B, Lukyanov S, Lukyanov K, Gurskaya N, Sverd- 27. Matsuoka M, Kyozuka J, Shimamoto K, Kano-Murakami Y: lov ED, Siebert PD: Suppression subtractive hybridization: A The promoters of two carboxylases in a C4 plant (maize) direct method for generating differentially regulated or tissue-specific cell-specific, light-regulated expression in a C3 plant (rice). cDNA probes and libraries. Proc Natl Acad Sci USA 93: 6025– Plant J 6: 311–319 (1994). 6030 (1996). 28. Meierhoff K, Westhoff P: Differential biogenesis of pho- 11. Downton WJS, Berry JA, Tregunna EB: C4-photosynthesis: tosystem II in mesophyll and bundle-sheath cells of mono- non-cyclic electron flow and grana development in bundle cotyledonous NADP-malic enzyme-type C4 plants: The non- sheath chloroplasts. Z Pflanzenphysiol 63: 194–198 (1970). stoichiometric abundance of the subunits of photosystem II in 12. Edwards GE, Ku MSB, Monson RK. C4 photosynthesis and the bundle-sheath chloroplasts and the translational activity of its regulation. In: Barber J, Baker NR (eds) Photosynthet- the plastome-encoded genes. Planta 191: 23–33 (1993). ic Mechanisms and the Environment, pp. 287–327. Elsevier 29. Meurer J, Meierhoff K, Westhoff P: Isolation of high- Science Publishers (Biomedical Division), Amsterdam/New chlorophyll-fluorescence mutants of Arabidopsis thaliana and York/Oxford (1985). their characterisation by spectroscopy, immunoblotting and 13. Ehleringer JR, Monson RK: Evolutionary and ecological Northern hybridisation. Planta 198: 385–396 (1996). aspects of photosynthetic pathway variation. Annu Rev Ecol 30. Monod C, Takahashi Y, Goldschmidt-Clermont M, Rochaix Syst 24: 411–439 (1993). J-D: The chloroplast ycf8 open reading frame encodes a pho- 14. Erickson JM, Rochaix JD. The molecular biology of photosys- tosystem II polypeptide which maintains photosynthetic activ- tem II. In: Barber J (ed) Topics in Photosynthesis, vol. 11. The ity under adverse growth conditions. EMBO J 13: 2747–2754 Photosystems: Structure, Function and Molecular Biology, pp. (1994). 101–177. Elsevier Science Publishers, Amsterdam (1992). 31. Moore G: Cereal genome evolution: Pastoral pursuits with 15. Furbank RT, Taylor WC: Regulation of photosynthesis in C3 ‘Lego’ genomes. Curr Opin Genet Devel 5: 717–724 (1995). and C4 plants: a molecular approach. Plant Cell 7: 797–807 32. Nester EW, Kosuge T: Plasmids specifying plant hyperplasias. (1995). Annu Rev Microbiol 35: 531–565 (1981). 16. Golbeck JH, Martin IF, Velthuys BR, Radmer R. A critical 33. Okamuro JK, Goldberg RB. Regulation of plant gene expres- reassessment of the photosystem II content in bundle sheath sion: general principles. In: Marcus A (ed) The Biochemistry chloroplasts of young leaves of Zea mays. In: Akoyunoglou of Plants, vol. 15. Molecular Biology, pp. 1–82. Academic J (ed) Proceedings of the Vth International Congress of Pho- Press, San Diego, FL (1989). tosynthesis, vol. 5. Chloroplast Development, pp. 533–546. 34. Oswald A, Streubel M, Ljungberg U, Hermans J, Eskins K, Balaban International Science Services, Philadelphia (1981). Westhoff P: Differential biogenesis of photosystem II in meso- 17. Green BR, Durnford DG: The chlorophyll-carotenoid proteins phyll and bundle sheath cells of NADP malic enzyme-type of oxygenic photosynthesis. Annu Rev Plant Physiol Plant Mol C4 plants. A comparative protein and RNA analysis. Eur J Biol 47: 685–714 (1996). Biochem 190: 185–194 (1990). 18. Hatch MD: C4 photosynthesis: a unique blend of modified 35. Pakrasi HB: Genetic analysis of the form and function of pho- biochemistry, anatomy and ultrastructure. Biochim Biophys tosystem I and photosystem II. Annu Rev Genet 29: 755–776 Acta 895: 81–106 (1987). (1995). 19. Hatch MD, Burnell JN: Carbonic anhydrase activity in leaves 36. Paterson AH, Lin Y-R, Li Z, Schertz KF, Doebley JF, Pinson and its role in the first step of C4 photosynthesis. Plant Physiol SRM, Liu S-C, Stansel JW, Irvine JE: Convergent domestica- 93: 825–828 (1990). tion of cereal crops by independent mutations at corresponding 20. Hattersley PW, Watson L. Diversification of photosynthesis. genetic loci. Science 269: 1714–1718 (1995). In: Chapman GP (ed) Grass evolution and domestication, pp. 37. Pearcy RW, Bjorkman¨ O, Caldwell MM, Keely JE, Monson 38–116. Cambridge University Press, Cambridge, UK (1992). RK, Strain BR: Carbon gain by plants in natural environments. 21. Hoesche JA, Berzborn RJ: Primary structure, deduced from Carbon assimilation analysis provides an understanding of how cDNA, secondary structure analysis and conclusions concern- plants function in diverse environments. BioScience 37: 21–29 ing interaction surfaces of the d subunit of the photosynthetic (1987). ATP-synthase (E.C. 3.6.1.34) from millet (Sorghum bicolor) 38. Pearcy RW, Ehleringer J: Comparative ecophysiology of C3 and maize (Zea mays). Biochim Biophys Acta 1142: 293–305 and C4 plants. Plant Cell Environ 7: 1–13 (1984). (1993). 39. Pereira MG, Lee M, Bramel-Cox P, Woodman W, Doebley 22. Kapazoglou A, Sagliocco F, Dure L,III: PSII-T, a new nuclear J, Whitkus R: Construction of an RFLP map in sorghum and encoded lumenal protein from photosystem II. Targeting and comparative mapping in maize. Genome 37: 236–243 (1994). 335

40. Rathnam CKM, Edwards GE: Distribution of nitrate- phosphoenolpyruvate carboxylase directs mesophyll speciﬁc assimilating enzymes between mesophyll protoplasts and expression in transgenic C4 Flaveria spp. Plant Cell 9: 479– bundle sheath cells in leaves of three groups of C4 plants. 489 (1997). Plant Physiol 57: 881–885 (1976). 52. Suzuki S, Burnell JN: Nucleotide sequence of a cDNA encod- 41. Russel M, Kidd S, Kelley MR: An improved ﬁlamentous helper ing rice chloroplastic carbonic anhydrase. Plant Physiol 107: phage for generating single-stranded plasmid DNA. Gene 45: 299–300 (1995). 333–338 (1986). 53. Takahashi R, Joshee N, Kitagawa Y: Induction of chilling 42. Sakakibara Y, Kobayashi H, Kasamo K: Isolation and charac- resistance by water stress, and cDNA sequence analysis and

terization of cDNAs encoding vacuolar H(+)-pyrophosphatase expression of water stress-regulated genes in rice. Plant Mol isoforms from rice (Oryza sativa L.). Plant Mol Biol 31: 1029– Biol 26: 339–352 (1994). 1038 (1996). 54. Usuda H, Edwards GE: Localization of glycerate kinase and 43. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A some enzymes for sucrose synthesis in C3 and C4 plants. Plant Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Physiol 65: 1017–1022 (1980). Press, Cold Spring Harbor, NY (1989). 55. Utsunomiya E, Muto S: Carbonic anhydrase in the plasma 44. Sargent TD: Isolation of differentially expressed genes. Meth membranes from leaves of C3 and C4 plants. Physiol Plant 88: Enzymol 152: 423–432 (1987). 413–419 (1993). 45. Schuler GD, Boguski MS, Gish W. Sequence similarity search- 56. Westhoff P, Offermann-Steinhard K, Hofer¨ M, Eskins K, ing using the BLAST family of programs. In: Dracopoli NC, Oswald A, Streubel M: Differential accumulation of plastid Haines JL, Korf BR, Moir DT, Morton CC, Seidman CE, transcripts encoding photosystem II components in the meso- Seidman JG, Smith DR (eds) Current Protocols in Human phyll and bundle-sheath cells of monocotyledonous NADP- Genetics, vol. 2, pp. 11.3.1–11.3.44. John Wiley, New York malic enzyme-type C4 plants. Planta 184: 377–388 (1991). (1995). 57. Westhoff P, Svensson P, Ernst K, Blasing¨ O, Burscheidt J, 46. Schuster G, Ohad I, Martineau B, Taylor WC: Differentiation Stockhaus J: Molecular evolution of C4 phosphoenolpyruvate and development of bundle sheath and mesophyll thylakoids in carboxylase in the genus Flaveria. Aust J Plant Physiol 24: maize. Thylakoid polypeptide composition, phosphorylation, 429–436 (1997). and organization of photosystem II. J Biol Chem 260: 11866– 58. Wilson RC, Long F, Maruoka EM, Cooper JB: A new 11873 (1985). proline-rich early nodulin from Medicago truncatula is highly 47. Schaffner¨ AR, Sheen J: Maize C4 photosynthesis involves expressed in nodule meristematic cells. Plant Cell 6: 1265– differential regulation of phosphoenolpyruvate carboxylase 1275 (1994). genes. Plant J 2: 221–232 (1992). 59. Woo KC, Anderson JM, Boardman NK, Downton WJS, 48. Sheen JY, Bogorad L: Differential expression in bundle sheath Osmond CB, Thorne SW: Deﬁcient photosystem II in agranal and mesophyll cells of maize of genes for photosystem II bundle sheath chloroplasts of C4 plants. Proc Natl Acad Sci components encoded by the plastid genome. Plant Physiol 86: USA 67: 18–25 (1970). 1020–1026 (1988). 60. Wurtele ES, Nikolau BJ: Enzymes of glucose oxidation in leaf 49. Sheen JY, Sayre RT, Bogorad L: Differential expression of tissues. The distribution of the enzymes of glycolysis and the oxygen-evolving polypeptide genes in maize leaf cell types. oxidative pentose phosphate pathway between epidermal and Plant Mol Biol 9: 217–226 (1987). mesophyll tissues of C3-plants and epidermal, mesophyll, and 50. Sinha NR, Kellogg EA: Parallelism and diversity in multiple bundle sheath tissues of C4-plants. Plant Physiol 82: 503–510 origins of C4 photosynthesis in the grass family. Am J Bot 83: (1986). 1458–1470 (1996). 61. Xu G-W, Magill CW, Schertz KF, Hart GE: A RFLP linkage 51. Stockhaus J, Schlue U, Koczor M, Chitty JA, Taylor WC, West- map of Sorghum bicolor (L.) Moench. Theor Appl Genet 89: hoff P: The promoter of the gene encoding the C4 form of 139–145 (1994).