Photosynthesis Research 75: 183–192, 2003.
© 2003 Kluwer Academic Publishers. Printed in the Netherlands.
183
Emerging techniques
Quantification of photosynthetic gene expression in maize C3 and C4
tissues by real-time PCR
Silke Hahnen, Thorsten Joeris, Fritz Kreuzaler & Christoph Peterhänsel∗
Aachen University, Institute for Biology I, 52056 Aachen, Germany ∗ Author for correspondence
(e-mail:[email protected]; fax: +49-241-8022637)
Received 14 May 2002; accepted in revised form 24 September 2002
Key words: C3 , C4 , husk leaf, maize, real-time PCR, transcription
Abstract
Carbon assimilation in maize follows the C4 mechanism. This requires the tissue-specific and light-induced expression of a set of different genes involved in CO2 fixation as well as adaptations in the leaf anatomy including
a reduced distance between vascular bundles compared to C3 plants. However, several maize tissues exist with
larger bundle distances and there is significant evidence that CO2 fixation follows the C3 mechanism in these
tissues. We isolated maize C3 and C4 tissues and quantified the accumulation of mRNAs encoding PEPC, ME,
the small subunit of Rubisco, and PPDK. For this, primer systems for the specific and sensitive detection by realtime PCR were established. The observed patterns show the expected distribution for foliar leaf tissues. Also in
total husk leaves, all transcripts under investigation were detected, albeit at a lower level. When mesophyll cells
which are located distant from bundles were isolated from husk leaves, only accumulation of RbcS was observed.
Comparing the expression of two genes encoding for isoenzymes of the small subunit of RbcS in the different
tissues differential patterns of relative transcript abundance were observed. Transcripts for the DOF1 transcription
factor involved in the activation of photosynthetic genes in maize were found in leaf tissues performing both C4
and C3 photosynthesis with highest accumulation levels in C4 mesophyll cells, whereas the homologous DOF2
gene was not expressed in any of the investigated samples. The results provide novel insights into the regulation of
C3 and C4 carbon fixation pathways in maize.
Abbreviations: DMSO – dimethylsulfoxide; DOF1 – transcription factor DOF1; DOF2 – transcription factor
DOF2; GAPDH – glyceraldehyde-3-phosphate dehydrogenase; ME – malic enzyme; PEPC – phosphoenolpyruvate carboxylase; PPDK – pyruvate-Pi -dikinase; RbcS – small subunit of ribulose-1,5-bisphosphate carboxylase
oxygenase; RT – reverse transcription
Introduction
Maize performs CO2 fixation according to the C4
mechanism. This mechanism requires the separation
of primary and secondary CO2 fixation in two different
leaf tissues, the mesophyll and the chlorenchymatous
bundle sheath. An efficient exchange of metabolites
in between these two tissues is ensured by the high
density of vascular bundles in the blade of maize foliar
leaves with only two mesophyll cells separating two
bundles (for review see Dengler and Nelson 1999).
However, in the sheath of foliar leaves and in several leaf-like organs, e.g., the husk leaves surrounding
the female inflorescence, the distance between bundles
is greatly enhanced (Antonielli and Venanzi 1979).
There is significant evidence that mesophyll cells more
distant from bundles perform photosynthesis according to the C3 mechanism in these tissues (Nelson and
Langdale 1992b). This is reflected in the accumulation pattern of typical proteins (Langdale et al. 1988;
184
Hall et al. 1998), the oxygen sensitivity of photosynthesis (Langdale et al. 1988), and the carbon isotope
discrimination of the husk leaf (Yakir et al. 1991).
The biochemistry of C4 photosynthesis requires
the cell-type specific and light- induced expression
of numerous genes (reviewed in Sheen 1999). In the
mesophyll, CO2 is converted to HCO3 − and this is
fixed by PEPC to form oxaloacetate. In maize, oxaloacetate is converted to malate and this diffuses into
the bundle sheath and is decarboxylated by ME to
liberate CO2 that is again refixed by Rubisco. The
mechanism enhances the local CO2 concentration and
by this suppresses the oxygenase activity of Rubisco.
The remaining monocarbonic acid pyruvate serves to
regenerate phosphoenolpyruvate in the chloroplasts of
mesophyll cells under catalysis of PPDK.
The genes for the C4 -specific isoforms of these enzymes evolved on the basis of genes already existing
in C3 plants, but acquired new regulatory sequences to
ensure a high level of expression in a cell-type specific
and light-induced manner and to target the corresponding proteins to different subcellular compartments (Ku
et al. 1996). The situation is similarly complex for
Rubisco, the only enzyme involved in both C3 and C4
carbon assimilation. At least two different genes for
the small subunit of Rubisco have been described in
maize, which differ in expression patterns dependent
on light induction. Moreover, the RbcS1 gene is preferentially expressed in maize foliar leaves compared
to the RbcS2 gene and this effect is exacerbated further
in husk leaves (Ewing et al. 1998). Even more RbcS
genes have been differentiated by distinctions in their
positional and light-induced regulation of transcription
in an earlier study (Sheen and Bogorad 1986).
There is strong evidence that the expression of photosynthetic genes in maize is at least in part controlled
by the two transcription factors DOF1 and DOF2.
Transient overexpression in protoplasts together with
Northern analyses revealed that DOF1 might serve
as an ubiquitously expressed activator of transcription
whereas DOF2 may act as a tissue-specific repressor
(Yanagisawa and Sheen 1998).
In this study, we apply real-time PCR to quantify
the accumulation of genes related to carbon assimilation in maize C4 and C3 tissues. The developed
primer systems allow the specific detection of the
photosynthetic isoforms of these genes. The results
indicate different mechanisms for the light-induced
and tissue-specific control of photosynthesis-related
transcripts.
Materials and methods
Plant growth
Maize (Zea mays cv. Helix) was cultivated in growth
chambers at a temperature of 25 ◦ C in the light for
16 h and 20 ◦ C in the dark for 8 h. The plants were
illuminated with Osram Superstar HQI-T 400W/DH
lamps. The photon flux density was between 200 and
300 µmol m−2 s−1 .
Husk leaves were prepared from maize plants that
were grown in the greenhouse. Foliar leaves and the
prophyll shading the cob were removed 48 h before
tissue preparation to ensure optimal illumination of the
outer husk leaf.
Tissue preparation
All tissues were prepared 6 h after onset of illumination. Total foliar leaves and husk leaves were
frozen in liquid nitrogen immediately after harvest.
Bundle sheath and mesophyll preparations from foliar
leaves were done essentially as described by Sheen
and Bogorad (1987) with some adaptations.
For mesophyll preparation, cut primary leaves
were incubated in SMC buffer (0.5 M sorbitol, 5 mM
MES, 10 mM CaCl2 , pH 5.8) containing 3% w/v
Rohalase 7069, 2% w/v Rohament PL (Röhm, Darmstadt, Germany), and 0.12% w/v maceroenzyme R-10
(Serva, Heidelberg, Germany) for 1.5 h at 30 ◦ C
under illumination. Subsequently, cell debris was removed by filtering through a 100 µm sieve, the protoplast suspension was diluted with 0.5 Vol LinsmeierSkoog solution (0.36% w/v Murashige-Skoog medium, 0.27 M NaCl, pH 5.8), and protoplasts were
pelleted by centrifugation at 300 × g for 5 min.
The supernatant was aspirated and the pellet was immediately suspended in TRIZOL buffer (see RNA
preparation).
For bundle sheath preparations, leaves were treated
with diethylether for 5 s to remove cuticular waxes
and facilitate tissue disruption. Leaves were washed
extensively in ice-cold water and homogenised in a
cold Waring Blendor for 6 × 4 s. The mixture was
sieved through a household sieve and the homogenisation step was repeated with the filter residue. The
suspension was then filtered through a 100 µm sieve
and the residue was washed extensively with ice-cold
water. The remaining water was aspirated and the
bundle sheath strands were frozen in liquid nitrogen.
The more elaborate protocol minimised the contam-
185
ination of bundle sheath preparations with epidermal
tissue.
Mesophyll from husk leaves was prepared by
manually cutting sections under a binocular. Only
those regions of the outer husk leaves were used that
were subjected to illumination and were green. Care
was taken that a distance of at least three mesophyll
cells from bundles was kept and stripes of approximately 0.5 mm in width were cut from the leaves. The
sections were immediately frozen in liquid nitrogen.
All tissue samples were always mixed from at least
three individual plants and preparations were done
three times from plants grown independently from
each other.
for 20 s, 61 ◦ C for 10 s and 72 ◦ C for 20 s. A 1:500
dilution of the first reaction was subjected to a second
PCR with the same temperature profile but 45 cycles.
The amplification mix contained 2 mM MgCl2 and
was supplemented with 5% DMSO and 0.5 M betaine. A dilution series of a cDNA derived from C4
mesophyll was additionally subjected to the two-step
PCR protocol to prove that the relative abundances of
the transcripts are not changed during the preamplification. For the amplification of DOF2 transcripts the
same temperature profile as for DOF1 was used with
2 mM MgCl2 and 5% DMSO in the amplification mix.
Oligonucleotides were purchased from Sigma ARK
(Darmstadt, Germany).
RNA preparation and reverse transcription
Results
RNA was prepared from tissues following the
TRIZOL protocol (Chomczynski 1993) and finally
suspended in an appropriate volume of water. The
integrity of the preparation and the absence of genomic DNA were tested by gel electrophoresis. If
appropriate, 10 U of DNaseI (Roche Applied Science,
Mannheim, Germany) and 2 mM MgCl2 were added
to remove traces of contaminating DNA and reactions
were incubated for 15 min at 37 ◦ C followed by a
denaturation step of 15 min at 70 ◦ C.
Approximately 1 µg of the preparation were mixed
with 10 pmol oligo-dT primer or a gene-specific
primer, heated for 5 min to 68 ◦ C, and cooled down
on ice before adding 200 U of MMLV-RT (Promega,
Mannheim, Germany) and 1 mM dNTPs in reaction
buffer supplied by the manufacturer.
Real-time PCR
Real-time PCR was performed on a LightCycler using the FastStart DNA Master SYBR Green I kit
(Roche Applied Science, Mannheim, Germany). For
the amplifications of PEPC, ME, RbcS, and GAPDH
transcripts the final MgCl2 concentration was 3 mM
and amplification conditions were 94 ◦ C for 10 min
and 45 cycles, each cycle at 95 ◦ C for 10 s, 58 ◦ C for
10 s and 72 ◦ C for 20 s. The annealing temperature
was elevated to 60 ◦ C in amplification reactions with
primers specific for the RbcS1 and RbcS2 genes, respectively, and for RbcS2 reactions were additionally
supplemented with 0.5 M betain.
For the quantification of DOF1 cDNA a seminested RT-PCR protocol was applied with an initial
preamplification step of 25 cycles, each cycle at 95 ◦ C
Establishment of real-time PCR systems
Primer sequences were selected to detect specific isoforms of the investigated transcripts based on the
sequence information available in the databases and
are listed in Table 1.
For PEPC, primer sequences were derived from the
genomic sequence gi22396 encoding the C4 -specific
isoform of PEPC. The homologous region of a rootspecific isoform (gi3132309, Dong et al. 1998) is not
amplified.
For ME, the primer sequences were deduced from
sequence gi168527. This sequence has been published
before to encode the C4 -specific isoform of malic enzyme in maize (Rothermel and Nelson 1989). The
forward primer is specific for this coding sequence and
does not amplify other published isoforms (gi4096785
and gi18460984). The published genomic sequence
(gi2950394) did not show significant homology in the
respective region to the sequence chosen. However, we
were able to amplify an approximately 300 bp fragment with these primers from genomic DNA (data not
shown).
Primers amplifying both cDNAs under investigation that encode for the small subunit of Rubisco
(RbcS) were deduced from gi22464, but also fit
to the second isolated sequence (gi1673455). Additionally, primers specific for the respective isoforms
were designed and the specificity of amplification was
tested by direct sequencing of PCR products (data not
shown).
For PPDK, the primer sequences were deduced
from the mRNA sequence gi168579 that has been
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Table 1. Primer systems applied in this study
Name
Sequence
Derived
from gi
Product (bp)
DNA
cDNA
Tm cDNA
product (◦ C)
aga act caa gcc ctt tgg gaa gc
gtc ggc gaa ctc ctt gga cag c
22 396
327
248
88.4
gat ctc tgc gca cat cgc tgc
gca gca cta ccg gta gtt gcg g
168 527
∼ 300
145
89.2
RbcS
RUB 1
RUB 2
gct ccg ttc cag ggg ctc aag t
tct cgc ggt aca cga agc cga cct t
22 464
418
254
91.4
RbcS1
RUB 1
RUB 2
acg gac gac ctg ctg aag cag gtg g
ggt gga agg cgt ccg ggt agg att tg
22 464
226
226
90.3
RbcS2
RUB 1
RUB 2
ggt gta caa gga gct gca gga ggc cat
ggc aga ggc atg gcc atg ggt cg
1673 455
168
168
90.2
PPDK
PDK 1
PDK 2
PDK 3
ccg tcg acg atc tcg gcc cag
gtc gtt gac gcc gcg ccg ata cag
cgc cca tgt act cct cca ccc a
168 579
> 5000
244
93.6
GAPDH
GAP 1
GAP 2
ctg gtt tct acc gac ttc ctt g
cgg cat aca caa gca gca ac
22 302
327
204
88.7
DOF1
DOF1 1
DOF1 2
DOF1 3
aag aag cgc cgc gtc gtg gcg ccg
gag ggg aag tcg gag agg ccg agg
gcg cca ggg agt cgg agt cct cc
517 257
137
137
87.4
DOF2
DOF2 1
DOF2 2
gca gcg acg gtg gct gcc tcg gag
cct aac gcc gcc gct ccc agc atc
106 1305
153
153
90.6
PEPC
PEP 1
PEP 2
ME
ME 1
ME 2
published to encode the C4 -specific isoform of PPDK
(Sheen 1991). The forward primer is located in the
first exon and does not detect C3 -specific isoforms.
Because C4 - and C3 -specific isoforms only differ in
the 5 part of the coding sequence, reverse transcription was performed with a gene-specific primer. Due
to the large intron 1, no products are amplified from
DNA.
Primers amplifying DOF transcripts were deduced
from the published cDNA sequences (gi517257 for
DOF1 and gi1061305 for DOF2). As DOF coding sequences are extremely GC-rich, PCR reactions were
supplemented with DMSO and betaine to reduce the
melting temperature of primers and products. In order
to further optimise specificity, DOF1 amplifications
were performed in two semi-nested reactions, where
primer DOF1 1 was used in both reactions but primer
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DOF1 2 was exchanged for DOF1 3 in the second
reaction (see Table 1).
The genomic sequence encoding subunit C of
cytosolic GAPDH (gi22302) was used to select
GAPDH primers. The primers do not fit to any other
of the tested sequences encoding GAPDH mRNAs
from maize (gi293888, gi293886, gi22294, gi312178,
gi312180).
PCR reaction conditions were established with
cDNA derived from green maize leaves or with genomic DNA (for DOF genes), respectively. Amplifications were performed from a single cDNA preparation
started from an oligo-dT primer with the exception
of PPDK, where a gene-specific primer was used for
reverse transcription. Furthermore, whenever possible
primers were selected to amplify products of different
size from genomic DNA and cDNA in order to allow the discrimination of amplification products from
DNA that might contaminate the RNA preparations
(Table 1). In case such an arrangement of primers was
not possible, RNA samples were additionally digested
with DNAse and a second reverse transcription reaction without enzyme was performed in parallel to test
for the absence of amplification products that are not
dependent on reverse transcription.
Figure 1 shows the melting curves of the PCR
products. All curves show single product peaks in the
expected temperature range. None of the PCR systems produces peaks with lower melting points that
could be derived from primer dimers or additional
products. These results were confirmed by gel electrophoresis where only single products and no primer
dimers were visible (data not shown). Thus, the PCR
conditions are suitable for the accurate quantification of photosynthesis-related transcripts from RNA
preparations.
Quantification of transcripts encoding photosynthetic
enzymes
The abundance of the transcripts was determined in
the different tissues and results were standardised for
the expression of GAPDH. Standard curves for the
quantification were performed with a dilution series
of a cDNA preparation derived from green leaves and
units relative to the standard were defined arbitrarily.
As shown in Figure 2, the accumulation of transcripts
for RbcS, ME, and PEPC was comparable in foliar
leaves at the investigated conditions. All three transcripts accumulated to levels slightly lower than half
of the amount of GAPDH mRNA. The standard devi-
Figure 1. Melting curves of the amplification products derived from
cDNA. After amplification, PCR products were slowly heated and
SYBR Green mediated fluorescence was monitored. Shown is the
first derivative of the fluorescence. (A) Melting curves for PEPC,
ME, RbcS, and GAPDH. (B) Melting curves for PPDK, DOF1,
DOF2, RbcS1, and RbcS2.
ations of up to 30% of the measured value indicate the
biological variation because errors of repeated measurements from a single sample were always lower
than 10%. In husk leaves, also all three transcripts
accumulate, albeit to significantly lower levels presumably due to the lower photosynthetic activity of
this tissue. When comparing the abundance of each
transcript in husk leaves to the amount in foliar leaves,
a tendency for a higher relative accumulation of RbcS
transcripts is observable. In C3 mesophyll cells derived from husk leaves, only RbcS accumulated, but
not C4 -specific transcripts (PEPC or ME). Relative to
total husk leaves, the preparation was enriched for
RbcS transcript. Therefore, the isolated sections represent true C3 tissues that have been purified from the
tissues performing C4 photosynthesis. C4 mesophyll
cells from foliar leaves only showed significant accumulation of PEPC, whereas a complementary accumulation of RbcS and ME transcripts in bundle sheath
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Figure 2. Quantification of the mRNA accumulation of RbcS,
PEPC, and ME in different maize leaf tissues. The amount of RNA
transcribed from the listed genes was measured by real-time PCR
and calculated in arbitrary units by comparison to a standard dilution series. Each value is the relative accumulation of the respective
RNA compared to GAPDH levels measured in the preparation. Each
data point is based on three independent RNA preparations and for
each preparation the quantification was repeated at least three times.
Vertical bars show standard deviations.
cells could be observed. Again, an enrichment compared to total foliar leaves could be observed for PEPC
and ME. However, the amount of RbcS transcript in
bundle sheath cells was even slightly lower than the
amount detected in total leaves. This difference might
be due to the different preparation techniques applied
and differences in the stability of the RNAs during
preparation.
We additionally determined transcripts for the two
genes encoding RbcS separately. Figure 3 shows the
relative abundance of RbcS1 compared to RbcS2 transcripts. The ratio is highest in C3 mesophyll with a
factor of more than 10 and clearly lower in foliar
leaves performing C4 photosynthesis. For husk leaves
containing both kinds of photosynthetic tissues, the
ratio is intermediate. For the prepared bundle sheath
strands, again an unexpected relation with slightly
lower amounts of RbcS2 compared to RbcS1 is observed. As the bundle sheath is the only tissue in foliar
leaves containing RbcS transcripts, this is again due to
differences in the preparation techniques. The relative
abundances indicate that C3 tissues of maize show a
higher preference for RbcS1 expression compared to
C4 tissues.
We also attempted to quantify PPDK transcripts
(Figure 4). A high accumulation of PPDK transcripts
was found in foliar leaves and C4 mesophyll cells as
expected. For total husk leaves, values were again
on a similar level as described for the other photo-
Figure 3. Relative abundance of RbcS1 and RbcS2 transcripts in
different maize leaf tissues. The amount of RNA derived from the
respective genes was measured by real-time PCR and calculated in
arbitrary units by comparison to a standard dilution series. Each
value is the ratio of RbcS1 compared to RbcS2 transcripts measured
in the preparation. Each data point is based on three independent RNA preparations and for each preparation the quantification
was repeated at least three times. Horizontal bars show standard
deviations. n.a. = not applicable.
Figure 4. Quantification of the mRNA accumulation of PPDK in
different maize leaf tissues. The amount of RNA transcribed from
the PPDK gene was measured by real-time PCR and calculated in
arbitrary units by comparison to a standard dilution series. Each
value is the relative accumulation of the respective RNA compared
to GAPDH levels measured in the preparation. Each data point is
based on three independent RNA preparations and for each preparation the quantification was repeated at least three times. Vertical
bars show standard deviations.
synthetic transcripts. In bundle sheaths preparations
almost no PPDK transcripts could be detected and
the amounts in RNA preparations from mesophyll
cells distant from bundles was very low. However,
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DOF2 transcripts were neither detected in maize
tissues performing C3 photosynthesis nor in C4 tissues. However, clear accumulation was observed in
roots as described before (Yanagisawa and Sheen
1998). If any DOF2 transcript is present in leaf tissues,
its accumulation level is less than 1% of the amount
observed in roots as calculated from the detection limit
(data not shown).
In conclusion, the data suggest that the differential
expression of DOF genes does not control the photosynthetic mechanism adopted by maize C3 and C4
tissues.
Figure 5. Accumulation of DOF1 transcripts in different maize
leaf tissues. The amount of RNA transcribed from the listed genes
was measured by real-time PCR and calculated in arbitrary units
by comparison to a standard dilution series. Each value is the
relative accumulation of the respective RNA compared to the abundance measured in foliar leaves. Each data point is based on three
independent RNA preparations and for each preparation the quantification was repeated at least three times. Horizontal bars show
standard deviations.
standard deviations were clearly higher compared to
the other transcripts. As described above, a genespecific reverse transcription reaction was used for
PPDK and the values obtained were standardised for
the abundance of GAPDH as determined from a cDNA
preparation with oligo-dT primers. Thus, differences
in the efficiency of individual RT reactions might have
complicated quantification.
Quantification of transcripts encoding DOF
transcription factors
As DOF transcription factors are proposed to be main
regulators of photosynthetic gene expression (see ‘Introduction’) we investigated whether these factors
are differentially expressed in maize C3 and C4 tissues. Because RNA encoding these factors is very
low in abundance compared to other photosynthetic
transcripts, values were standardised for the level of
expression found in foliar leaf tissue. DOF1 transcripts
were found in all investigated tissues, albeit at clearly
different levels (Figure 5). Whereas the transcript
accumulation is highest in C4 mesophyll cells approximately three-fold lower levels are found in bundle
sheath tissues. Surprisingly, transcript amounts higher
than in foliar leaves were detected in C3 mesophyll
and total husk leaves.
Discussion
The cell-type specific and light-dependent expression
of genes related to carbon assimilation in maize has
been extensively studied in the past (for review see
Sheen 1999). These studies have been often performed
with RNA from separated foliar leaf tissues and Northern analyses requiring high amounts of template available for detection. Alternatively, in situ hybridisation
with RNA probes and/or antibodies has been used
to localise the expression of specific photosynthesisrelated genes (Langdale et al. 1988; Furumoto et al.
2000). Both techniques usually do not allow the discrimination of different isoforms of genes differing
only in a few nucleotide positions and are complicated to perform quantitatively. The importance of such
isoform-specific analyses has recently been emphasized by quantitative analyses on the expression of
different malic enzyme genes in Flaveria C3 and C4
species (Lai et al. 2002a, b).
RT-PCR is a very sensitive and specific alternative to these techniques and has also been applied in
maize for the detection of transcripts encoding PPDK
(Sheen 1991) or PEPC (Schäffner and Sheen 1992). In
these studies, the amplification products were detected
by gel electrophoresis at the endpoint of the reaction.
This does not allow a direct correlation between the
amounts of product and the amount of template as
the increase in product is unpredictable in the plateau phase of the reaction (Raeymaekers 2000). The
recent development of real-time PCR systems (Heid
et al. 1996; Wittwer et al. 1997) facilitates the detection of products during the exponential phase of
the reaction and a calculation of the input of template
from the kinetics of the amplification process. By this,
the exact quantification even of low amounts of template molecules is possible enabling the analysis of
190
less abundant tissues or even of single cells (Al-Taher
et al. 2000).
We applied this system for the detection of
photosynthesis-related transcripts in cuttings from
husk leaves. The amount of material that could be prepared from the light-exposed part of a single leaf was
only in the mg range but still enough to isolate RNA
for multiple PCR analyses. Dilution series with RNA
preparations from green foliar leaves showed that total
RNA in the pg range is sufficient for the specific detection of the transcripts under investigation in real-time
PCR (data not shown). This is clearly superior to the
amounts required for the detection of PEPC or PPDK
transcripts by conventional RT-PCR (see above). Thus,
the novel systems allow the quantitative detection of
RNAs in isolated tissues like the C3 mesophyll that
could not be anaylsed separately before.
With the exception of PPDK, the primer systems
were designed in a manner that all amplifications
could be done from a single reverse transcription
avoiding tube-to-tube variation in the efficiency of
cDNA synthesis that might interfere with the standardisation of transcript levels to the amount of GAPDH
transcript. This problem is reflected in the higher
standard deviations that were obtained when PPDK
levels were detected because here different reverse
transcription reactions had to be used. It is advisable
for future applications to design exclusively primer
systems that can amplify from a single cDNA preparation.
SYBR Green was used to detect the accumulation
of amplification products in the reactions. The advantage of this technique is that it is in principle applicable
to any primer system and does not require the complicated and cost-intensive design of specific probes.
However, this dye detects any double-stranded DNA
including primer dimers and thus care has to be taken
that PCR conditions ensure a specific and efficient
amplification (Ririe et al. 1997). This was tested by
determining the melting curves of the amplification
products at the end of the reaction. Each primer pair
showed one specific peak indicative for the melting
temperature of the respective PCR product. By this,
also products derived from genomic DNA or RNA
could be discriminated.
The results from our analyses show that the transcripts of genes involved in C4 metabolism all accumulate to a similar level in foliar leaves of maize under
the chosen conditions and the expected transcripts
were found in the two different photosynthetic tissues
of foliar leaves, the mesophyll and the bundle sheath,
respectively (Kanai and Edwards 1999). Samples were
taken 6 h after onset of light to detect highest possible
accumulation of these transcripts. It will be interesting to analyse the exact kinetics of light response
or the developmental profile of expression (Cribb et
al. 2001) of the different genes with the provided
system. The accumulation of the transcripts in husk
leaves was similar to foliar leaves ensuring that husk
leaves also perform C4 photosynthesis or at least that
they express the required enzymes. However, the isolated mesophyll cells distant from bundles in husk
leaves predominantly accumulated RbcS transcripts
and only very low levels of PEPC, PPDK, or ME.
The remaining amounts might have been derived from
gene expression in epidermal guard cells that contaminate the mesophyll preparations (Langdale et al.
1988) or from small transverse bundles interconnecting the bigger longitudinal bundles (although the latter
were excluded from the preparations as far as possible). Photosynthetic gene expression in maize is
controlled by positional information and by illumination and both factors might be overlapping (Nelson
and Langdale 1992a). Husk leaves are often shaded
by foliar leaves or by covering each other. It has
been previously shown that RbcS is also expressed
in etiolated leaf tissues (Langdale et al. 1988) or in
young leaves that did not develop full C4 anatomy
(Crespo et al. 1979; Cribb et al. 2001). We therefore
took care that only sufficiently illuminated husk leaves
were used for our analyses. This is reflected by the
green chloroplasts and the developed Kranz anatomy
of these tissues (data not shown) as well as the expression of C4 -specific enzymes that are not, or only
weakly, expressed in dark-grown plants (Sheen 1999).
Thus, the expression patterns observed are very likely
to be controlled by positional information, i.e., the
distance of the mesophyll cells to bundles.
We applied the developed systems to quantify the
relative contribution of two different RbcS genes to the
accumulation of RbcS transcripts. Ewing et al. (1998)
have shown before that RbcS1 transcripts are more
abundant than RbcS2 transcripts in foliar leaves and
that this effect is enhanced in husk leaves. They suggest that this is due to differences in the sensitivity of
the two promoters to blue and red light, respectively,
assuming that the different wavelengths permeate with
different efficiencies to the photosynthetic cells in partially shaded husk leaves. The data presented in this
study support a scenario where positional information additionally contributes to the differences in gene
activity. First, we avoided any impact of shading by
191
using only green outer parts of husk leaves as described above. Second, we found clearly highest ratios
of RbcS1 to RbcS2 expression in C3 tissue isolated
from husk leaves. The quotient was intermediate for
total husk leaves that constitute a mixture of C3 and C4
tissues and lowest for foliar leaves representing pure
C4 tissues. As light conditions are identical for both C3
mesophyll cells and total husk leaves, this effect can‘t
be contributed to this parameter. We conclude that the
position of a cell relative to a bundle influences the
relative contribution of both RbcS genes to the total
RbcS transcript levels.
DOF transcription factors are candidates for control elements of such quantitative differences in photosynthetic gene expression. Whereas DOF1 was
found in any tissue tested so far, DOF2 is not expressed in leaves and was therefore suspected to
restrict DOF1 activity to leaf tissues by competing
for the common binding site in non-photosynthetic
tissues (Yanagisawa and Sheen 1998). Our results
confirm that DOF2 transcripts are hardly detectable
in leaf tissues. However, we found no qualitative
differences when comparing expression patterns in
mesophyll cells performing C3 or C4 photosynthesis,
respectively. The data indicate that the photosynthetic
subtype executed by a specific leaf tissue is not controlled by the same mechanism that might control
specificity for photosynthetic and non-photosynthetic
tissues, respectively.
A quantitative assessment revealed that DOF1 is
expressed to unexpectedly high levels in husk leaves
compared to foliar leaves. We cannot exclude that
these differences are due to different developmental
stages of the tissues, however, the amounts of DOF1
detected in a specific tissue again do not correlate
with the amount of photosynthesis-related transcripts.
Within foliar leaf tissues, an approximately threefold difference was found when comparing mesophyll
to bundle sheath cells. Values for total foliar leaves
are intermediate making it unprobable that this effect
is due to selective degradation of DOF1 transcripts
in bundle sheath preparations. It is unclear whether
DOF proteins exert any effect on bundle sheath
specific gene expression as exclusively mesophyllspecific promoters were tested so far for interactions
with DOF1 (Yanagisawa 2000). We have recently sequenced the promoter of the C4 specific NADP-malic
enzyme gene from maize and found several potential binding sites for DOF proteins in this region (C.
Peterhänsel, unpublished results). The presence of
DOF1 transcripts in bundle sheath cells points to a
functional role of these binding sites.
Taken together, the available data as discussed
above portend specific mechanisms of transcriptional
regulation of photosynthesis-related genes by light induction, tissue specificity and positional information.
Acknowledgements
The authors are indebted to Barbara Rüger and Brigitte Miedl from Roche Applied Science for excellent
support during the establishment of real-time PCR
systems.
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