319
Plant Molecular Biology 37: 319–335, 1998.
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 für Entwicklungs- und Molekularbiologie der Pflanzen, Heinrich-Heine-Universität, Universitätsstrasse 1,
40225 Düsseldorf, Germany ( author for correspondence); 2 Department of Soil and Crop Sciences, Texas A & M
University, College Station, TX 77843–2474, USA; 3 Xinjiang 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
C4 plants are characterized by high rates of photosynthesis as well as an efficient use of water and nitrogen
resources. For this reason, a number of C4 plants are
among the most productive crops in agriculture. The
high photosynthetic capacity of C4 plants is due to
their unique mode of carbon assimilation which concentrates CO2 at the site of Rubisco. As a consequence
of this CO2 pumping, the competitive inhibition of
Rubisco by oxygen is largely excluded and C4 plants
show drastically reduced rates of photorespiration. In
C3 plants, this process is responsible for the loss of up
to 30% of the net CO2 fixed in photosynthesis. The
CO2 pump provides high rates of photosynthesis even
when CO2 concentrations are low in the intercellular
air spaces of the leaf. Hence, C4 plants are able to limit the opening of their stomata and thereby minimize
water loss due to transpiration. As the CO2 pump results in saturating concentrations of CO2 at the site of
Rubisco, high photosynthetic rates can be maintained
with less enzyme than is required in C3 species and this
reflects in a higher nitrogen use efficiency (reviewed in
[13, 37, 38]).
The functioning of C4 photosynthesis is dependent
upon the strict compartmentation of the CO2 assimilatory enzymes into two distinct cell types, mesophyll
and bundle-sheath cells. The primary carboxylating
320
enzyme, phosphoenolpyruvate carboxylase (PEPC),
accumulates exclusively in the mesophyll cells while
the secondary carboxylase, Rubisco, as well as the
decarboxylating enzymes like NADP-dependent malic enzyme, are restricted to the bundle-sheath cells
(reviewed in [18]). A differential accumulation is
also observed for photorespiratory, nitrogen assimilating and carbohydrate synthesizing enzymes [2, 40,
54, 60]. C4 plants of the NADP-malic enzyme type
are even known to differ in the photosynthetic electron transport chains of mesophyll and bundle-sheath
chloroplasts. While thylakoid membranes of mesophyll chloroplasts possess a fully developed linear electron transport chain, those of the bundle-sheath chloroplasts are devoid of grana and are severely depleted in
photosystem II [11, 28, 46, 59].
This division of labour between mesophyll and
bundle-sheath cells is the result of differential gene
expression. In NADP-malic enzyme-type C4 species
transcripts for phosphoenolpyruvate carboxylase, pyruvate orthophosphate dikinase, NADP-malic enzyme
and the small subunit of Rubisco accumulate differentially in the two cell types. It was found that this differential accumulation is largely due to transcriptional
control (reviewed in [15, 23, 25]). A differential accumulation has also been reported for nuclear as well as
plastid-encoded transcripts for photosystem II proteins
[24, 48, 49, 56]. There is evidence that other genes may
be expressed differentially too, but the extent of this
differential gene expression in C4 photosynthesis has
to be elucidated.
A comprehensive knowledge of those genes which
are differentially expressed in mesophyll and bundlesheath cells could help in defining and understanding
the genetic basis of C4 photosynthesis. Therefore, we
have initiated a systematic search for these genes in the
NADP-malic enzyme type C4 grass Sorghum bicolor.
Sorghum has been selected for this purpose because
the gene expression patterns of mesophyll and bundlesheath cells differ drastically even at the very young
seedling stage [24, 28, 34]. This is of great advantage for gene cataloguing because cDNA libraries and
hybridization probes can be prepared from leaf tissue
which is still differentiating and may be expected to
express all the genes needed for the establishment and
maintenance of the C4 pathway of photosynthesis.
Sorghum is an agronomically important crop for
which detailed RFLP maps of intra- and interspecific
crosses are available [8, 39, 61]. The isolated cDNAs
can thus be mapped in the Sorghum genome. Due to the
extensive synteny in the grasses [1, 4, 31] orthologous
sequences may be easily identified in other C4 and C3
grasses. A comparative mapping of C4-photosynthesisassociated genes within the grasses is of prime interest
because the C4 photosynthetic pathway has evolved
several times independently within this family [20, 50]
and the grasses are an attractive model system in which
to study the evolution of this photosynthetic trait. An
identification of C4 -associated genes and their comparative analysis in the various evolutionary lines towards
C4 photosynthesis within the grasses may also be considered as the first step to elucidate the master gene(s)
of this evolutionary transition. Here we present a catalogue of genes which are differentially expressed in
the mesophyll and bundle-sheath cells of sorghum and
report on the location of these genes on the RFLP map
of this species.
Materials and methods
Plant material
Seeds of Sorghum bicolor cv. Tx430 (Pioneer HiBred, Plainview, TX) were germinated in soil. Plants
were grown in the greenhouse with supplementary
light (14 h per day from 07:00 to 21:00) provided
by a combination of sodium and mercury highpressure vapour lamps. Photon flux density was about
300 mol m,2 s,1 at plant height level and the growth
temperature varied between 20–26 C (day) and 18–
20 C (night). For the isolation of total leaf RNA
the entire second leaves of 8- to 10-day old seedlings
were used, while for the preparation of mesophyll and
bundle-sheath RNAs only the upper two thirds of the
second leaves were harvested. Root RNA was isolated
from seedlings grown for 14 days in the greenhouse.
For light/dark experiments seeds were germinated and
grown in the dark for 5 days and then illuminated for
24 h with white light.
Isolation of RNA from mesophyll protoplasts
Leaves (20–30 g) were cut into small pieces (5–10 mm)
with a sharp razor blade and stored in ice-cold medium
A (0.6 M sorbitol, 0.005 M MgCl2 , 0.5% w/v bovine
serum albumin, 0.050 M sodium ascorbate, 0.020 M
2-(N-morpholino)ethanesulfonic acid (MES)-KOH pH
5.5) until all the leaves had been processed. The leaf
pieces were resuspended in 200–300 ml fresh medium
A supplemented with 2% (w/v) Cellulase Onozuka R10 and 0.2% (w/v) Macerozyme Onozuka R-10 (both
321
from Yakult Honsha, Japan) followed by a 2 to 2.5 h
incubation in the dark at 25 C with gentle shaking.
After enzymatic digestion the suspension was poured
over a tea sieve and the remaining leaf pieces were
shaken in 100 ml fresh incubation medium for 5 min.
The filtrates were combined and re-filtered through a
80 m nylon mesh. Protoplasts in the filtrate were
sedimented by centrifugation at 430 g for 3 min
with a swinging bucket rotor (HS4 rotor, SorvallDupont). The pelleted protoplasts were microscopically examined for their intactness and then resuspended in 100 ml medium B (0.33 M sorbitol, 0.30 M
NaCl, 0.010 M EDTA, 0.010 M ethyleneglycol-bis(ßaminoethyl ether)-N,N,N0,N0 -tetraacetic acid (EGTA),
0.010 M dithiothreitol (DTT), 0.010 M diethyldithiocarbamic acid, 0.2 M Tris-HCl pH 9.0). Further
purification of the mesophyll RNA and isolation of
poly(A)+ RNA followed standard procedures [56].
Isolation of RNA from bundle-sheath strands
For the preparation of bundle-sheath strands 20–30 g
leaf material was divided into 2 to 3 portions and each
portion was homogenized in a Waring blender with
200 ml medium C (0.6 M sorbitol, 0.005 M MgCl2 ,
0.050 M sodium ascorbate, 0.001 M aurintricarboxylic acid, 0.005 M diethyldithiocarbamic acid, 0.020 M
MES/KOH, pH 5.5). The combined homogenates were
filtered through a 80 m mesh and the crude bundlesheath residue in the filter was resuspended in 200
ml medium A containing 2% Cellulase Onozuka R-10
and 0.2% Macerozyme Onozuka R-10. After 30 min
incubation at 25 C bundle-sheath strands were collected in a 80 m mesh and homogenized twice in a
Waring blender for 30 s each in 200 ml medium C.
The pure bundle-sheath strands were then recovered,
immediately frozen in liquid nitrogen and ground to a
fine powder with a aid of a mortar and a pestle. Further
isolation of bundle-sheath RNA was as described [56].
Synthesis and cloning of cDNA
Poly(A)+ RNA was isolated from total leaves of 8day old sorghum seedlings and converted into doublestranded cDNA using the cDNA cloning kit from
Stratagene Cloning Systems (San Diego, CA). The
double-stranded cDNAs were size-fractionated on a
Sepharose-4B column [43] and fragments larger than
600 bp were ligated into Lambda Uni ZAP XR (Stratagene Cloning Systems). Ligated DNAs were packaged
into phage using Gigapack II Gold Packaging extracts
(Stratagene) resulting in 9 105 individual phages.
The library was amplified following standard procedures [43].
Differential screening of the cDNA library
About 5000 phage were plated on a 20 cm 35 cm
rectangular plastic dish with Escherichia coli strain
XL1-Blue (Stratagene Cloning Systems, San Diego,
CA) as the recipient. Plaques were successively blotted in series to a total of four nitrocellulose filters (BA
85; Schleicher & Schüll, Dassel, Germany). The phage
DNAs were allowed to adsorb to the membrane for 0.5,
1, 2 and 4 min, respectively. Upon completion of transfer, the filters were incubated for 4 min in 0.5 M NaOH,
1.5 M NaCl, neutralized by 7 min incubation in 0.5 M
Tris-HCl, 3 M NaCl pH 7.4 and finally equilibrated
for 7 min in 250 mM Na2 HPO4 pH 7.2. DNA was
immobilized on the filters by baking for 2 h at 84 C.
Radioactively labelled cDNA probes were prepared from mesophyll and bundle-sheath poly(A)+
RNA essentially as described by Sambrook et al.
[43]. The poly(A)+ RNAs (1 g each in 5 l doubledistilled water) were heated for 5 min at 70 C, cooled
on ice and then added to a 22 l reaction mixture
containing 50 mM Tris-HCl pH 8.3, 10 mM DTT,
3 mM MgCl2 , 75 mM KCl, 800 M dGTP, dTTP and
dCTP, 4.8 M dATP, 100 Ci [32 P]-dATP (Amersham
Buchler, Braunschweig, Germany), 12.5 g random
hexamer primers (Boehringer, Mannheim, Germany),
20 U RNasin (Boehringer, Mannheim) and 400 U MMLV reverse transcriptase (Gibco–BRL, Eggenstein,
Germany). The reaction mixtures were incubated for
1 h at 37 C, and terminated by adding 12 l 1 M
NaOH, 2 l 0.25 M EDTA and 1 l 10% (w/v) SDS.
After 30 min incubation at room temperature the mixtures were neutralized by the addition of 8 l 2 M acetic
acid and 10 l 1 m Tris-HCl pH 7.6. The radioactively
labelled cDNAs were recovered by phenol/chloroform
extraction and precipitation with 3 volumes ethanol in
the presence of 0.3 M sodium acetate and 20 g of
glycogen (Boehringer, Mannheim). The precipitated
cDNAs were resuspended in sterile bidistilled water
and the incorporated radioactivity was determined by
spotting aliquots on DE 81 paper followed by liquid
scintillation counting.
Filters were prehybridized in phosphate/SDS medium [9] at 65 C for 2 h. A maximum of 8 filters
(17 cm 19 cm) were incubated in a glass tube with
2.6 ml hybridization medium for 14–18 h. The first
and second filters from each plate were hybridized
322
with 3 107 cpm of labelled mesophyll or bundlesheath cDNA probe, respectively. The third and fourth
filter were hybridized with a mixture of known cDNAs
for genes differentially expressed in mesophyll and
bundle-sheath cells. These cDNAs included sequences
for PEPC, pyruvate orthophosphate dikinase, NADPmalic enzyme, NADP malate dehydrogenase and the
small subunit of Rubisco. The hybridizations with the
known cDNAs were carried out to prevent re-isolation
of these abundant cDNAs. After hybridization, the
filters were washed once in 0:5 SSC, 0.5% (w/v)
SDS and four times in 0:1 SSC, 0.5% (w/v) SDS
at 65 C for 45 min each. Filters were exposed over
night on CEA RP X-ray films using Dupont intensifying screens.
Phage hybridizing differentially with the mesophyll and bundle-sheath cDNA probes, but not with
the mixture of known cDNAs were isolated, re-plated
at low density and re-hybridized with the mesophyll
and bundle-sheath cDNA probes. Only phage which
gave a clear differential signal were purified by a third
round of hybridization, and the inserted fragments of
the Lambda phage were excised in vivo with the f1
helper phage R408 [41] according to the manufacturer’s protocol.
Classification and identification of cDNA clones
The cDNAs isolated by differential screening were
grouped into classes of sequence identity/similarity
by cross-hybridization. For this purpose, the cDNAcontaining plasmids were digested with EcoRI/XhoI
(or BamHI/ApaI, if the EcoRI and XhoI cloning sites
were not intact), and the restricted DNAs were subjected 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
of the isolated cDNAs was successively hybridized
to the Southern-blotted DNAs with the exception of
those which had been identified in a previous round of
hybridization.
The largest cDNA clone of each identity/similarity
class was partially sequenced from the putative 50
end of the cDNA insert using a T7 DNA polymerase
kit (Pharmacia, Freiburg, Germany). If BLASTX
searches [45] did not reveal any significant matches
with sequences deposited in the non-redundant database at NCBI, additional sequence information was
gathered from the putative 30 end of the insert. In
addition, cDNA-specific oligonucleotide primers were
used to collect further sequence data. The cDNAs
were considered as being homologous to identified
sequences, if the BLASTX similarity scores were
greater than 200.
RNA analysis
Sizing and quantification of RNAs by northern analysis
and dot blot hybridization were carried out according
to standard procedures [56]. Hybridizing bands or dots
were visualized by fluorography with Kodak XAR-5
films and, if necessary, the bound radioactivity was
determined by liquid scintillation counting. To be able
to compare hybridization signals obtained with probes
of different sizes, all data were standardized to a probe
length of 1 kb. Based on these normalized hybridization signals the transcripts were arbitrarily assigned
to three abundance classes: I (>5000 cpm g leaf
poly(A)+ RNA), II (500–5000 cpm/g leaf poly(A)+
RNA) or III (<500 cpm/g leaf poly(A)+ RNA).
RFLP mapping
The mapping population, the molecular biological
techniques for RFLP analyses, the linkage analysis
and the nomenclature for loci and ‘linkage groups’
(chromosomes) are as described by Chittenden et al.
[8].
Miscellaneous
Isolation of recombinant plasmids, Southern analysis
of plasmid and genomic DNA and DNA sequence analysis was carried out according to standard protocols
[43].
Results
Differential screening of a leaf cDNA library and
sequence analysis of the isolated clones
The differential screening of a cDNA library was the
first step towards the identification of genes which
are differentially expressed in mesophyll and bundlesheath cells. For this technique pure mesophyll and
bundle-sheath RNA preparations were a prerequisite.
With the well established combination of mechanical and enzymatic treatments [28] cell preparations
of a sufficient purity were obtained. By assaying for
mesophyll and bundle-sheath marker RNAs, i.e. for
PEPC and NADP-malic enzyme transcripts, the cross-
323
contamination of both RNA fractions was estimated to
be less than 5%. RNAs which accumulate exclusively
in one of the two cell-types should, therefore, give
at least a 20-fold difference in hybridization intensity in the differential screening of the cDNA library.
To prevent re-isolation of cDNAs whose corresponding RNAs were known to accumulate differentially
in mesophyll and bundle-sheath cells, i.e. the transcripts encoding the C4 assimilatory enzymes, probes
for these genes were co-hybridized on separate filters
(see Materials and methods).
About 15 000 phage of the amplified cDNA library prepared from total leaf poly(A)+ RNA of
young sorghum leaves were screened differentially.
By visual comparison of the fluorographs 238 clones
were identified as candidates for mesophyll-specific
cDNAs and 116 for bundle-sheath-specific genes.
Cross-hybridization assays under stringent conditions
allowed grouping of the clones into 59 different classes
which contained from one to 46 (HHU17) individual
clones (see Table 1).
The largest clone of each class was partially
sequenced and the sequences obtained were subjected to data base searches using the BLASTX subroutine [42]. Three of the cDNA classes were found
to encode plastid genes (rbcL, psaA and atpH) and
were excluded from further analysis. Two cDNA
classes, as defined by cross-hybridization analysis had
to be merged since sequencing revealed that they were
derived from the same gene. Three cDNAs (HHU14,
HHU29 and HHU52) were found to be chimerical.
The analysis of HHU52 was not continued because
it contained sequences from two different members
of chlorophyll a/b-binding proteins already present
in the clone collection. In the case of HHU12 and
HHU29, the chimerical fragments were separated and
only those fragments re-cloned which gave rise to the
differential hybridization signals with mesophyll and
bundle-sheath RNA (HHU12
HHU71; HHU29
HHU72). Six additional sorghum cDNA clones coding
for phosphoenolpyruvate carboxylase, pyruvate orthophosphate dikinase, NADP-malic enzyme, the delta
subunit of the chloroplast ATP synthase, subunit D
of photosystem I reaction centre and a 70 kDa heat
shock protein had been isolated in previous studies
([21, 34] and unpublished data), thus a total of 58 different cDNAs were available for further analysis. 46 of
these cDNAs encoded known genes while 12 did not
reveal any significant matches to sequences deposited
in the data bases (Table 1).
!
!
Transcript size and abundance
To verify that the isolated cDNAs were derived from
expressed genes and to determine the size and the
abundance of the corresponding transcripts, RNA gel
blot analyses were carried out with total leaf RNA. All
clones with the exception of HHU22 were found to
hybridize to single RNAs (data not shown; Table 1).
This indicated that the genes represented by these
cDNAs are expressed in sorghum leaves and that these
genes are transcribed into RNAs of identical sizes, despite the fact that some occur in multiple copies in the
genome. HHU22 which encodes carbonic anhydrase
sequences, hybridized to two abundant leaf RNAs of
1700 and 2100 nucleotides. In addition, a faint hybridization signal to a transcript with a size of 1200 nucleotides was detected. This finding suggested that the
Sorghum genome contains several different carbonic
anhydrase genes and that the matter required further
analyses (see below).
Transcript abundance was roughly assessed by
measuring the radioactivity of the hybridized probe in
a liquid scintillation counter. The hybridization signals were normalized with respect to probe length
and the transcripts were arbitrarily assigned to three
abundance classes (see Materials and methods) consisting of highly (I), moderately (II) and less abundant (III) transcripts. The majority of the cDNAs isolated, encoded transcripts which accumulated to high
or moderate levels in sorghum leaves (Table 1). These
class I and II transcripts included, for instance, RNAs
encoding the C4 and Calvin cycle enzymes and components of the photosynthetic electron transport chain
(Table 1). Seven cDNAs (HHU21, HHU24, HHU25,
HHU40, HHU42, HHU49 and HHU56) were found to
encode less abundant transcripts. Two of the cDNAs
contained known sequences, i.e. for the chloroplast
adenine nucleotide translocator (HHU42) and Rubisco
activase (HHU56). The remaining five cDNAs did not
show any significant similarities to sequences in the
data bases. This suggested that they may encode novel
genes.
Mesophyll/bundle-sheath specificity of expression
The cDNAs were isolated due to their differential
hybridization to mesophyll and bundle-sheath probes.
To verify that these cDNAs encode transcripts that
accumulate differentially in mesophyll and bundlesheath 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
HHU2
–
phosphoenolpyruvate carboxylase (Ppc1)
1500
3600
I
> 20
HHU3
–
NADP-malic enzyme (Mod1)
1350
2500
I
< 0.05
HHU4
32
photosystem II, 33 kDa subunit (PsbO)
1400
1400
I
10–20
HHU5
13
photosystem II, 23 kDa subunit (PsbP)
1000
1300
I
10–20
HHU6
11
photosystem II, 16 kDa subunit (PsbQ)
800
1100
I
10–20
HHU7
HHU8
7
1
photosystem II, 10 kDa subunit (PsbR)
plastocyanin (PetE)
580
750
900
1000
I
I
10–20
1–10
HHU9
–
photosystem I, subunit D (PsaD)
775
1100
II
1–10
HHU10
–
heat shock protein HSP70
780
2600
II
1–10
HHU11
14
1250
1400
II
10–20
HHU12
12
1050
1200
I
10–20
HHU13
4
1230
1800
II
10–20
HHU15
6
chlorophyll a/b-binding protein CP24
(Lhcb6)
chlorophyll a/b-binding protein CP26
(Lhcb5)
(NADP+ )-glyceraldehyde-3-phosphate
dehydrogenase, chloroplast (GapB1)
ferredoxin-NADP-oxidoreductase (PetH)
920
1700
II
10–20
HHU16
10
1178
1400
II
10–20
HHU17
46
1150
1200
II
1–10
HHU18
HHU19
1
1
chlorophyll a/b-binding protein CP29
(Lhcb4)
chlorophyll a/b-binding protein type II
LHCII (Lhcb2)
photosystem II, 22 kDa subunit (PsbS)
proline-rich protein
529
1680
1400
1700
II
II
10–20
10–20
HHU20
4
ferredoxin (PetF)
890
1000
I
> 20
HHU21
1
?
1150
1100
III
> 20
HHU22
26
carbonic anhydrase (CAH)a
1730
II
> 20
HHU23
1
I
1–10
HHU24
960
1
chlorophyll a/b-binding protein type III
LHCI (Lhca3)
?
2100,
1700,
1200
1200
1880
3000
III
10–20
HHU25
HHU26
HHU27
1
1
1
?
?
S-adenosylmethionine decarboxylase
584
867
1880
2900
1300
2200
III
II
II
>20
HHU28
2
glutamyl tRNA reductase
1660
2100
II
1–10
1–10
1–10
356368;
356367
356370;
356369
356372;
366371
356374;
356373
356375;
356384
356377;
356376
356378
356380;
356379
356382;
356381
356363;
356365
356294;
–
356295;
1393256
356296;
–
356298;
–
356299
356300;
–
356301
356302;
356303
356304;
356305
356306;
356307
356308;
–
356309;
356310
356311;
356312
356313
356314
356315;
356316
356317;
356318
325
Table 1 continued.
Clone
design.
Class
size
(bp)
Putative identification
Insert
size
RNA
size (nt)
ratio
Expression
level
number
Mesophyll/
bundle-sheath
980
1100
I
1–10
588
1410
900
1400
II
II
1–10
1–10
221
940
1800
1100
II
I
1–10
1–10
HHU30
4
HHU31
HHU32
1
1
cytochrome b6 f-complex, Rieske Fe-S
subunit (PetC)
?
adenylate kinase, chloroplast
HHU33
HHU34
1
1
triosephosphate translocator, chloroplast
photosystem I, subunit F (PsaF)
HHU35
HHU36
1
1
?
?
863
1390
2100
1800
II
II
10–20
1–10
HHU37
HHU38
HHU39
2
2
1
vacuolar H+ -translocating pyrophosphatase
?
glutamine synthetase, chloroplast (Gln2)
481
1026
1740
3100
1600
1900
II
II
II
10–20
>20
1–10
HHU40
HHU42
HHU43
1
1
1
780
929
860
1800
2500
1500
III
III
II
10–20
1–10
>20
1100
1100
II
1–10
680
800
II
1–0.1
874
1100
II
1–0.1
<0.05
HHU44
HHU45
10
HHU46
1
HHU48
15
HHU49
?
adenine nucleotide translocator, chloroplast
triosephosphate isomerase, chloroplast
(TPIC1)
ATP synthetase, chloroplast, subunit
(AtpD)
metallothionein-like protein
?
,
1800
1900
I
1
fructose-1.6-bisphosphatase aldolase,
chloroplast (FBAC1)
?
1300
1700
III
1–0.1
HHU50
1
photosystem I, subunit G (PsaG)
1200
800
I
1–0.1
HHU51
HHU53
1
1
903
600
2500
900
II
I
HHU55
HHU56
1
2
2-oxoglutarate/malate translocator
water stress-induced protein (WSI729),
rice
?
rubisco activase (RCA1)
1000
800
1200
1900
II
III
<0.05
HHU57
3
HHU58
<0.05
<0.05
1–0.1
1480
1800
II
1–0.1
3
root nodule protein (MtN3), Medicago
trunculata
transketolase, chloroplast (TKLC1)
1910
3100
II
<0.05
HHU60
4
ribulose-5-phosphate 3-epimerase (RPE1)
1000
1500
II
<0.05
HHU61
5
metallothionein-like protein
760
900
I
1–0.1
HHU62
7
phosphoribulokinase (PRK1)
1500
1700
I
<0.05
dbEST
accession
356320;
356321
356322
356323;
–
356324
356325;
356326
356327;
–
356328
356329
356330;
–
35631
356332
356333;
–
X66004
356337;
–
356338;
–
356339;
356340
356341;
356342
356343;
–
356344
356347;
–
356350
356351;
–
356352;
356353
356354;
356355
356356;
–
356357;
–
356358;
–
326
Table 1 continued.
Clone
design.
HHU63
Class
size
(bp)
1
Putative identification
Insert
size
RNA
size (nt)
ratio
Expression
level
number
!-6 fatty acid desaturase
1200
1900
II
Mesophyll/
bundle-sheath
1–0.1
HHU68
–
carbonic anhydrase (CAH1)
1100
1200
–
–
HHU69
–
carbonic anhydrase (CAH2)
1100
II
>20
II
HHU71
4
photosystem II, 5 kDa subunit (PsbT)b
650
1700,
2100
900
HHU72
15
photosystem II, 7 kDa subunit (PsbW)
642
800
II
>20
1393258
10–20
dbEST
accession
356359;
–
–;
356334
356336;
356335
–;
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
by quantitative dot blot analysis; (8) dbEST accession numbers of the putative 50 and 30 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
each was spotted side by side onto a nylon membrane
and hybridized with the cDNA to be analysed. Hybridization signals were first visualized by fluorography
and the hybridized radioactivity was then quantitated
by liquid scintillation counting.
These values were used to group the transcripts
into three classes with the mesophyll/bundle-sheath
ratios differing more than 20-fold, between 10- and
20-fold, and less than 10-fold. A more than 20-fold difference indicates that the respective transcripts accumulate similarly as PEPC (Ppc1) and NADP-malic
enzyme (Mod1). Since these marker RNAs are known
to accumulate exclusively in mesophyll or bundlesheath cells, respectively, transcripts of this group are
considered to be mesophyll or bundle-sheath-specific
sensu strictu. Ratios between 10 to 20 indicate that the
transcripts do not accumulate in a strict cell-specific
manner, but accumulate preferentially in one or the
other cell type. Mesophyll/bundle-sheath differences
less than tenfold may still indicate a cell-preferential
accumulation. However, for the sake of experimental
accuracy a less than tenfold difference in steady-state
levels between mesophyll and bundle-sheath cells was
not considered to be diagnostic of a differential accumulation pattern.
With these criteria 25 cDNAs were found to encode
RNAs which accumulate specifically or preferentially
in mesophyll cells and 8 cDNAs were identified as
encoding bundle-sheath-specific or -preferential transcripts (Figure 1 and Tables 1 and 2). All cDNAs
which encode bundle-sheath-specific or -preferential
transcripts were identified by their significant sequence
similarities to known proteins. However, 6 of the 25
mesophyll-specific or -preferential transcripts encoded
proteins of hitherto unknown functions.
Expression patterns of the mesophyll-specific
transcripts of unknown identity
To characterize the mesophyll cDNA clones which
encoded unidentified sequences (HHU21, HHU24,
HHU26, HHU35, HHU38 and HHU40), the expression profiles of their corresponding RNAs were
linked to organ-type and light/dark growth. Three
more cDNAs, HHU19, HHU37 and HHU53 were
included in this analysis because the sequence similarities obtained for these clones (see Table 1) did
not reveal any clue about their possible function in C4
photosynthesis. HHU37 encodes a vacuolar protontranslocating pyrophosphatase [57] and HHU19 a
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)
pyruvate orthophosphate dikinase (Ppdk1; HHU1)
carbonic anhydrase (CAH2; HHU69)
Calvin cycle
NADPH glyceraldehyde-3-phosphate
dehydrogenase (GAPB1; HHU13)
triosephosphate isomerase, chloroplast (TPIC1; HHU43)
NADP-malic enzyme (Mod1; HHU3)
rubisco activase (RCA1; HHU56)
fructose-1.6-bisphosphate aldolase (FBAC1; HHU48)
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
bundle-sheath cells and are highly similar to a rice
mRNA which is up-regulated by water stress [53].
Figure 2 illustrates that HHU19, HHU35 and
HHU53 transcripts do not only accumulate in leaves
but also in roots, i.e. their expression is not restricted to the mesophyll (HHU19, HHU35) or bundlesheath-cells (HHU53) and these genes cannot be called
mesophyll- or bundle-sheath-specific in the strict sense
of the word. Light does not affect the accumulation
of these RNAs in the leaves of young seedlings. The
HHU35 transcript levels are even down-regulated during the greening of etiolated seedlings.
HHU21, HHU24, HHU26, HHU37, HHU38 and
HHU40 transcripts cannot be detected in root RNA
(Figure 2). This indicates that the expression of the
corresponding genes is confined to the leaves, i.e. to
the mesophyll cells. The levels of the HHU21, HHU26,
HHU38 and HHU40 transcripts increase strongly after
illumination of the etiolated seedlings and demonstrates that these genes are under the control of light. In
contrast, HHU24 and HHU37 RNA levels are similar
in etiolated and greening seedlings. This indicates that
these genes are not regulated by light.
Carbonic anhydrase sequences in sorghum: two
distinct genes, CAH1 and CAH2, with different
expression profiles
The northern blot analysis of RNA from leaves of
greenhouse-grown plants with HHU22 as a probe identified two abundant carbonic anhydrase transcripts of
2100 and 1700 nucleotides in size and trace amounts
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
with 1 g (HHU19, 26, 35, 37, 38 and 53) or 4 g poly(A)+ RNA
each (HHU21, 24 and 40). The filters were hybridized at 65 C in
SDS/phosphate medium. Filters were exposed for one to three days
depending on the signals obtained.
Figure 1. RNA dot blot analysis of cDNA sequences which are differentially expressed in mesophyll and bundle-sheath cells. Panel A.
Analysis of known-function cDNAs. Panel B. Analysis of unidentified cDNAs. Poly(A)+ RNA was prepared from mesophyll and
bundle-sheath preparations as described in Materials and methods.
Dilution series of the two RNA fractions (see figure) were spotted
onto nylon membranes and the membranes were hybridized with the
radiolabelled inserts of the HHU clones in SDS/phosphate buffer [9]
at 65 C. After washing, the filters were exposed to X-ray films for
one to two days depending on the signal strength. Dots were excised
from the filters and the radioactivity was determined in a liquid scintillation counter. The ratios of expression levels in mesophyll and
bundle-sheath cells are tabulated in Table 1. For gene designations,
see Table 1.
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
type or light, was analysed by RNA gel blot hybridization.
As might be predicted for a gene which is involved
in C4 photosynthesis carbonic anhydrase RNA levels
were found to be controlled by light. The 2100 and
1700 nucleotide transcripts were undetectable in the
leaves of etiolated seedlings and accumulated only
after illumination (Figure 3). A light-induced increase
in steady-state levels was also observed for the 1200
329
Figure 3. Organ specificity and light dependence of carbonic anhydrase RNA abundance in leaves (E, G) and roots (R) of sorghum seedlings 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
old plants grown in the greenhouse. One g of each RNA fraction
was analysed by northern blotting using the inserted fragment of
HHU22 as hybridization probe.
nucleotide RNA but traces of this RNA were already
present in the leaves of etiolated seedlings (Figure 3).
Traces of the 1200 nucleotide transcript were also
detectable in RNA which was isolated from roots of
greenhouse-grown seedlings, where the 2100 and 1700
nucleotide RNAs were absent (Figure 3). Collectively
these data suggested that sorghum possesses two types
of carbonic anhydrase transcripts with different expression profiles, i.e. the 2100 and 1700 nucleotide RNAs
on one hand and the 1200 nucleotide on the other.
This complexity of carbonic anhydrase transcripts contrasts with the C3 grass rice (Figure 3) where only a
single carbonic anhydrase transcript of about 1.4 kb
was detected.
To substantiate the existence of different carbonic anhydrase genes in sorghum the available carbonic anhydrase cDNA clones (see Table 1) were reanalyzed. Ten clones were randomly selected and
sequenced from the putative 30 end of the cDNA insert.
The sequences revealed two different types of cDNA
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
in sorghum. The most prominent and distinguishing
feature of the two types of carbonic anhydrase cDNA
clones is the presence of a 16 AT dinucleotide long
microsatellite in the 30 -untranslated segment of the
HHU68-type cDNAs (Figure 4).
The differences in the 30 -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 CAH1and CAH2-specific probes (HHU69-S and HHU68-S,
respectively) recognize different sets of DNA fragments (Figure 6) which appear as component and complementary fragments in the hybridization pattern of
the HHU68-L probe (Figure 6). The latter probe contains carbonic anhydrase coding sequences (Figure 4)
and recognizes both the CAH1 and CAH2 sequences
as well as other related carbonic anhydrase sequences,
when present. Hybridization of southern-blotted rice
DNA with this probe revealed a single-banded pattern
after digestion with various enzymes. This suggests
that rice contains only one carbonic anhydrase gene
and is in line with the northern hybridization analysis
of leaf RNA of this species (Figure 3). As only partial cDNA sequences were available for the CAH1 and
CAH2 carbonic anhydrase genes of sorghum their relationship with the rice gene [52] needs to be determined.
Genetic mapping of the cDNA sequences in Sorghum
For information about the chromosomal location in the
Sorghum genome those cDNAs which detected restriction fragment length polymorphisms and encoded lowcopy sequences were placed on the molecular map of
an interspecific cross [8]. A total of 43 cDNAs were
mapped and six of them yielded more than one locus
(Figure 7). Unfortunately, the mapping with HHU22
did not allow to resolve the location of the two carbonic anhydrase genes (CAH1 and CAH2) in the genome
since only one fragment showed length polymorphism.
Whether the second locus is located near the first one
or somewhere else in the genome needs to be investigated.
Discussion
While detailed information is available about the
physiological and biochemical context of C4 photosyn-
330
Figure 4. Analysis of CAH1 (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 molecular and genetic basis of this photosynthetic pathway is
still rather limited. It is well documented that the C4
cycle genes are differentially expressed in mesophyll
and bundle-sheath cells (reviewed in [15, 25, 25]).
There is also increasing information as to how this differential expression may be achieved by specific cisacting promoter elements [27, 47, 51]. However, the
trans-regulatory factors which interact with these promoter elements and the components of signal transduction pathways that are pertinent for the differentiation
of the two photosynthetic cell-types of a C4 leaf are
completely unknown.
To obtain additional information about the gene
expression patterns which are specific for the differentiation of mesophyll and bundle-sheath cells, differential screening of cDNA libraries was used to catalogue
genes which are differentially expressed in the two
cell-types of the monocotyledonous C4 plant sorghum.
A differential screening approach should pick up only
those cDNAs whose corresponding transcripts accumulate at high or possibly medium levels [44], i.e. rare
transcripts cannot be detected with this method. The
outcome of our analyses confirms this expectation. All
the cDNA clones which were identified as encoding
mesophyll- or bundle-sheath-specific transcripts could
easily be investigated by standard RNA gel and dot
blot hybridization techniques. It follows that these transcripts do not belong to the class of rare RNAs [33].
Most of the isolated mesophyll- and bundle-sheathspecific cDNA clones encode proteins whose putative
function could be deduced from database searches.
Component subunits of photosystem II reaction
centre and its light-harvesting antenna system were
the most prominent mesophyll-specific gene products
that were detected. Previous reports stated that the 33,
23 and 16 kDa proteins of the oxygen-evolving complex 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 g 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
a nylon membrane and hybridized with the labelled HHU68-S or
HHU69-S probes. Panel B. Northern hybridization with the CAH1and CAH2-specific probes. 1 g poly(A)+ RNA from seedlings
illuminated for 24 h after a 5-day period of etiolation was analysed.
The hybridizations were carried out in SDS/phosphate medium at
70 C.
mesophyll cells [34, 49]. This study adds four more
photosystem II mRNAs to this list, i.e. those from
the psbR, psbS, psbT and psbW genes [35]. In addition it was shown that the mRNAs which encode the
minor chlorophyll a/b-binding proteins CP24, CP26,
and CP29 of photosystem II [17] accumulate differentially in mesophyll and bundle-sheath cells. It has
been stated above that none of these photosystem II
mRNAs accumulate exclusively in the mesophyll cells
and that small amounts of these transcripts are also
detectable in bundle-sheath cells. This finding reflects
the known fact that bundle-sheath cells of NADP-malic
enzyme-type C4 species are not necessarily completely
devoid of any photosystem II but that the differences in
the levels of photosystem II proteins in mesophyll and
bundle-sheath cells are rather quantitative [16, 28].
The differential screening procedure resulted in the
isolation of almost the full complement of nuclearencoded mRNAs for photosystem II subunits. This
finding raises the hope that with a more sensitive
technique, i.e. subtractive hybridization [10] regulatory components of photosystem II will be detected.
Such an approach would nicely complement genetic
strategies which aim to identify regulatory genes of
photosystem II biogenesis by mutational analysis [3,
14, 29]. With gene probes available for both the constituent subunits of photosystem II as well as for the
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
biogenesis in the various C4 photosynthetic lineages of
the grasses [20].
Biochemical analyses of Calvin cycle activity
in the mesophyll and bundle-sheath chloroplasts of
NADP-malic enzyme type C4 plants have shown
that Rubisco and the enzymes of the regenerative phase of this cycle are confined to the bundlesheath cells. On the other hand, the reducing
phase of the Calvin cycle operates preferentially,
if not exclusively, in the mesophyll chloroplasts
[26]. Work described here supports this view at the
gene expression level. Transcripts encoding NADPHglyceraldehyde-3-phosphate dehydrogenase and the
chloroplast isoform of triosephosphate isomerase
accumulate only in mesophyll cells. RNAs which
encode fructose-1.6-bisphosphate aldolase, phosphoribulokinase, transketolase, ribulose-5-phosphate3-epimerase and Rubisco activase are bundle-sheath
specific transcripts. One may expect that also the
other enzymes of the regenerative phase of the
Calvin cycle, i.e. the fructose-1,6-bisphosphate and
sedoheptulose-1.7-bisphosphate phosphatases, and
ribulose-5-phosphate isomerase, are among the
bundle-sheath-specific transcripts. However, cDNAs
for these enzymes were not identified in the differential screening experiment.
It is established that the transcripts for the C4 cycle
enzymes PEPC, pyruvate orthophosphate dikinase
and NADP-malic enzyme accumulate differentially in
mesophyll and bundle-sheath cells. In contrast, the
intercellular distribution of carbonic anhydrase, another well-known C4 cycle enzyme has not been reported
yet. Carbonic anhydrase generates the rapid supply
of bicarbonate which is the CO2 substrate of PEPC
[19]. Biochemical and physiological analyses predict
that carbonic anhydrase should be exclusively found in
mesophyll cells [5, 6, 55]. It is demonstrated here that
in sorghum leaves carbonic anhydrase transcripts are
indeed confined to the mesophyll cells and are absent
in the bundle-sheath cells.
Two complications have been observed and need
to be considered. Firstly, the Sorghum genome contains two different carbonic anhydrase genes, CAH1
and CAH2, both of which are expressed in the leaves,
and secondly, the CAH2 gene is transcribed into two
different transcripts. Although both CAH1 and CAH2
are expressed in leaves, the steady-state levels of the
333
CAH2 transcripts are far more abundant than those
of the CAH1 transcripts. In fact, leaves of 10-day
old sorghum plants contain only traces of this RNA
suggesting that the CAH1 carbonic anhydrase are not
needed for C4 photosynthesis. The high levels of the
two CAH2 transcripts in the mesophyll cells of the
leaves, on the other hand, indicate that carbonic anhydrase proteins encoded by these latter transcripts are the
ones that supply the substrate bicarbonate to PEPC in
the mesophyll cells and hence are involved in the C4
photosynthetic pathway.
Northern and southern hybridization experiments
demonstrated that the two CAH2 transcripts are derived
from one single gene. To date, no full-size cDNA
clones for these two transcripts have been isolated from
sorghum and therefore one cannot say anything about
the differences in primary structure of the encoded
proteins and their putative functions. It is very likely,
however, that the sorghum CAH2 carbonic anhydrase transcripts correspond to two carbonic anhydrase
sequences from maize which are indentical in their
30 -untranslated regions and the carboxy-terminal parts
of the carbonic anhydrase reading frames but differ in
the aminoterminal halves of the proteins [7]. It will
be interesting to see whether the two carbonic anhydrase proteins of maize differ in function and cellular
location.
The comparison of the 30 -untranslated sequences
of the CAH1 and CAH2 cDNAs shows a high degree
of similarity. This suggests that the two genes diverged
quite recently from one another. Unfortunately the genomic locations of the CAH1 and CAH2 genes could
not be determined by RFLP analysis. It remains an
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
the same chromosome. One possible basis for duplication of CAH genes may be the finding that sorghum,
like maize, contains ancient duplications of some (perhaps all) chromosomal segments [8, 36]. Further mapping efforts which use the microsatellite sequence in
the 30 -untranslated region of CAH1 may help to solve
this problem.
Six cDNAs (HHU21, HHU24, HHU26, HHU35,
HHU38, HHU40) were isolated during the course
of this investigation whose corresponding transcripts
accumulated specifically in the mesophyll cells and
whose coding sequences did not match any knownfunction gene. The HHU35 gene is expressed also in
the roots suggesting that the HHU35 protein does not
function in C4 photosynthesis. Transcripts of all other
cDNAs were detected in leaves only which suggests
that the corresponding proteins are specific for leaf
functions. HHU21, HHU26, HHU38 and HHU40 transcript levels, moreover, are controlled by light. This
may indicate that these genes are involved in C4 photosynthesis. However, a full characterization of the
cDNAs has to be awaited to permit reliable conclusions about the putative functions of these genes.
It is known that the differential screening method is
not suitable for the detection of differentially expressed
genes with a low transcript abundance. Therefore,
more unknown genes with relevance to the C4 photosynthetic pathway are to be expected, when more
sensitive methods [10] are exploited for cDNA identification. The present study has initiated the cataloguing
of new genes which may be involved in the establishment and functioning of C4 photosynthesis. This has
to be followed by further studies until a more complete
view of the genetic basis of this fascinating pathway of
photosynthesis is available.
Acknowledgements
This work has been supported by grants from the Biotechnology Programme of the European Union (contract BIO2-CT93-0400) and from the German-Israeli
Foundation to P.W. A.H.P. acknowledges financial support from the USDA Plant Genome Program, and Pioneer Hibred International. D.Q. acknowledges a fellowship from the Chinese Academy of Sciences. Thanks
are due to Dr U.J. Santore for critically reading the
manuscript.
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