|
Plant Physiol, January 2000, Vol. 122, pp. 49-56
Antisense HEMA1 RNA Expression Inhibits Heme and
Chlorophyll Biosynthesis in Arabidopsis1
A. Madan
Kumar and
Dieter
Söll*
Department of Molecular Biophysics and Biochemistry (A.M.K., D.S.)
and Department of Molecular, Cellular and Developmental Biology (D.S.),
Yale University, New Haven, Connecticut 06520-8114.
 |
ABSTRACT |
5-Aminolevulinic acid (ALA) is a
precursor in the biosynthesis of tetrapyrroles including chlorophylls
and heme. The formation of ALA involves two enzymatic steps which take
place in the chloroplast in plants. The first enzyme, glutamyl-tRNA
reductase, and the second enzyme,
glutamate-1-semialdehyde-2,1-aminomutase, are encoded by the nuclear
HEMA and GSA genes, respectively. To
assess the significance of the HEMA gene for chlorophyll
and heme synthesis, transgenic Arabidopsis plants that expressed
antisense HEMA1 mRNA from the constitutive cauliflower
mosaic virus 35S promoter were generated. These plants exhibited
varying degrees of chlorophyll deficiency, ranging from patchy yellow
to total yellow. Analysis indicated that these plants had decreased
levels of chlorophyll, non-covalently bound hemes, and ALA; their
levels were proportional to the level of glutamyl-tRNA reductase
expression and were inversely related to the levels of antisense
HEMA transcripts. Plants that lacked chlorophyll failed
to survive under normal growth conditions, indicating that
HEMA gene expression is essential for growth.
| |
INTRODUCTION |
Porphyrin compounds such as chlorophyll and heme play vital roles
in plant metabolism. The porphyrin ring system is derived from
5-aminolevulinic acid (ALA). In plants ALA is formed from the
five-carbon skeleton of Glu in two steps known as the
C5 pathway (Beale, 1978 ; von Wettstein et al.,
1995; Kumar et al., 1996b). All of the components required for such a
conversion are located in the chloroplasts. The initial metabolite of
the C5 pathway is chloroplast-specific
glutamyl-tRNAGlu (Schön et al., 1986). In
the first step, glutamyl-tRNA reductase (GluTR) reduces the glutamyl
moiety of Glu-tRNA to Glu-1-semialdehyde (GSA). In the subsequent
reaction, GSA is converted to ALA by GSA-2,1-aminomutase (GSA-AM).
GluTR and GSA-AM are encoded by the nuclear HEMA and
GSA genes. In Arabidopsis and other plants two
HEMA genes exist (Ilag et al., 1994; Kumar et al., 1996a; Tanaka et al., 1996; Grimm, 1998; Sangwan and O'Brian, 1999). The
HEMA1 gene is regulated by light and expressed in all parts of the plant, while the expression of the HEMA2 gene was
only found in roots and flowers in a light-independent fashion. The HEMA1 and HEMA2 genes are very similar (79% at
the nucleotide level in the coding region and differ in the 5'- and
3'-untranslated regions), while the gene products also show high
similarity (83%).
Although it is widely accepted that all ALA in plants is formed in the
C5 pathway, the presence of the
C4 pathway in plants has been implied (Beale,
1978). In this biosynthetic route, which operates in animal
mitochondria and in yeast, ALA is generated by ALA synthase from Gly
and succinyl coenzyme A (May et al., 1990). ALA synthase-like activity
has been monitored in greening potato skin (Ramaswamy and Nair, 1973)
and soybean callus (Meller and Gassman, 1982); however, the enzyme was
never characterized from plants. Other lines of evidence, e.g.
photodestruction of chloroplasts (Thomsen et al., 1993), mutations
affecting the tRNAGlu (Hess et al., 1992), and
inhibition of GSA-AT activity by gabaculin (Flint, 1984), also support
the C4 pathway of ALA biosynthesis in plants. We
describe studies of antisense HEMA1 Arabidopsis plants that
unequivocally demonstrate the involvement of GluTR in ALA formation.
| |
MATERIALS AND METHODS |
Bacterial Strains
Escherichia coli strain DH5
was used for routine recombinant DNA work. Agrobacterium
tumefaciens strains EHA101 (for hypocotyl infection) and GV 3101 (for vacuum infiltration) were used in the generation of Arabidopsis
transgenic lines.
Construction of the HEMA Antisense Vector
A NcoI site was created by site-directed mutagenesis at
the translation start site in HEMA1 cDNA (Sambrook et al.,
1989). After confirming the sequence, the entire coding region of the HEMA1 cDNA was released by digestion with NcoI
and SphI restriction endonucleases. This fragment was
blunt-ended with Klenow DNA polymerase and ligated to
NcoI/NruI-digested, blunt-ended pRTL2-GUS
plasmid. Transformants were analyzed for the constructs containing the HEMA nucleotide sequence in the antisense orientation
downstream of the cauliflower mosaic virus 35S promoter. From the
confirmed construct, a DNA fragment containing the cauliflower mosaic
virus 35S promoter, HEMA cDNA (in antisense orientation),
and the terminator was isolated as a cassette and ligated to
SalI-digested, blunt-ended binary vector pCIT20. Plasmids
pRTL2-GUS and pCIT20 were from Dr. X.-W. Deng's laboratory (Yale
University). The resultant construct was transformed into the A. tumefaciens strains EHA101 and GV3101.
Plant Growth and Transformation
Hypocotyl Infection
Arabidopsis (ecotype Columbia) plants were grown from
surface-sterilized seeds on sterile Murashige and Skoog agar medium containing 0.1% (w/v) Suc and 0.1 g/L myo-inositol under
standard conditions (22°C, 60% relative humidity with a regimen of
16 h of 90 µE m 2
s1 white light and 8-h dark day cycle).
Hypocotyls were isolated from 10-d-old seedlings and
hand-prepared for A. tumefaciens infection as previously
described (Akama et al., 1992). Growth conditions involved in
generating well-differentiated plants from the infected hypocotyls
using root- and growth-inducing media were as previously described
(Akama et al., 1992).
Vacuum Infiltration
Arabidopsis seeds were grown in vermiculite under the standard
growth conditions. When primary inflorescence shoots attained a
reasonable height (2-3 cm), they were cut off to generate multiple secondary inflorescences (which took about 7-10 d). At this stage, plants were used for A. tumefaciens infection by vacuum
infiltration as described previously (Berthold et al., 1993).
DNA Isolation and Analysis
Total DNA (5 µg) isolated from the 2-week-old transgenic and
control Arabidopsis plants was digested with BamHI, and the
restricted fragments were separated on a 0.8% (w/v) agarose
gel. Transfer of restriction fragments to a nitrocellulose membrane,
prehybridization, hybridization, and washing of the membrane were
performed under stringent conditions (Sambrook et al., 1989). The probe
(a 600-bp NheI fragment from the HEMA1 cDNA) was
labeled with [-32P]dATP as described
previously (Sambrook et al., 1989).
RNA Extractions and Northern-Blot Analysis
Total RNA was isolated (Ilag et al., 1994) from 2-week-old
transgenic and control Arabidopsis plants. RNA (20 µg) was
heat-denatured (85°C for 2 min) in the presence of formamide (50%,
w/v) and separated on a denaturing 1.2% (w/v) agarose
gel as described previously (Sambrook et al., 1989). Equal loading of
RNA in northern-blot analysis was confirmed by hybridization with a pea
18S rDNA fragment. Prehybridization, hybridization, and washing
conditions of the membranes were described above. To monitor the
expression levels of endogenous genes, respective DNA fragments were
labeled with [-32P]dATP using random
hexanucleotide priming (Sambrook et al., 1989). We used strand-specific
RNA probes prepared from HEMA cDNA clone using T3 and T7 RNA
polymerase for the estimation of sense and antisense expression of the
HEMA gene in these plants (Sambrook et al., 1989).
Protein Extractions and Western-Blot Analysis
Plants were ground in liquid nitrogen and suspended
(100 µL/100 mg) in extraction buffer containing 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 50 mM  -mercaptoethanol, 0.5% (w/v) Triton X-100, 0.2 mM phenylmethylsulfonylfluoride, 1 µg/mL leupeptin, and 1 µg/mL apoprotein. After a brief vortexing, the suspension was centrifuged at 13,000g for 10 min and the supernatant
collected. The protein in the supernatant was measured with the
dye-binding assay (Bradford, 1976). Separation of proteins by SDS-PAGE
and blotting was performed as described previously (Sambrook et al., 1989). Blots were incubated for 1 h with respective antibodies (1:1,000 dilution) in a solution containing 50 mM
Tris-HCl (pH 10.2), 150 mM NaCl, 1% (w/v) bovine
serum albumin, and 0.1% (w/v) Triton X-100. The blots were washed
three times (for 5 min each) with the same solution without bovine
serum albumin. The western-blot analysis of protochlorophyllide
oxidoreductase (Por) proteins was performed as previously described
(Armstrong et al., 1995).
Biochemical Analysis
Chlorophyll Estimation
Chlorophyll pigment was extracted into 80% (w/v) aqueous
acetone and the total chlorophyll (chlorophyll
a+b) was estimated as described previously
(Lichtenthaler, 1987).
Quantitation of Heme
Non-covalently bound hemes (protoheme and heme a) were
extracted by acid acetone and purified on a DEAE-Sepharose column as reported previously (Weinstein and Beale, 1984). Pyridine (final concentration, 25%) and NaOH (final concentration, 0.1 N) were added to the processed eluate (to convert
the protoheme and heme a in the eluate to pyridine
homochrome), followed by few crystals of sodium dithionite (to reduce
the pyridine homochromes), as described previously (Stillman and
Gassman, 1978). The absorption spectrum of the reduced pyridine
homochrome was recorded between wavelengths of 400 to 600 nm with a
UV/VIS spectrometer (Lambda 2, Perkin-Elmer, Foster City, CA).
ALA Determination
ALA was extracted from Arabidopsis plants, purified, and measured
colorimetrically, as described previously (Weinstein and Beale, 1985).
However, prior to the extraction, plants were incubated for 6 h in
Murashige and Skoog medium containing levulinic acid (100 mM).
| |
RESULTS |
HEMA1 and HEMA2 Map to Chromosome I
Several restriction enzymes were used to reveal the polymorphisms
between pure Arabidopsis ecotypes Columbia and Landsberg erecta for HEMA1 and HEMA2 genes
(Lister and Dean, 1993). The 3'-untranslated DNA fragments of
HEMA1 and HEMA2 genes were used as gene-specific
probes. After identifying the RFLP pattern for HEMA1 and
HEMA2 genes, the genomic DNA isolated from 29 inbred lines
derived from a cross of Columbia and Landsberg erecta
ecotypes was digested with EcoRV (for HEMA1) and
BglII (for HEMA2) and Southern-blot analysis was
carried out with gene-specific probes as described above. The data on
segregation of polymorphisms in these lines were analyzed with the
Mapmaker program (Lander et al., 1987), which placed the
HEMA1 and HEMA2 genes to chromosome I in
Arabidopsis (Fig. 1).
View larger version (5K):
[in this window]
[in a new window]
|
Figure 1.
Schematic representation of the location of
HEMA1 (hma1) and HEMA2 (hma2) genes on
the Arabidopsis chromosome I. The distance (in centiMorgans [cM]) and
the markers nearest to these genes are shown. Numbers in parentheses
indicate the marker identification.
|
|
Generation and Characterization of Antisense Plants
Infection of Arabidopsis hypocotyls with the A. tumefaciens strain EHA101 harboring the binary
pCIT20-HEMA antisense construct resulted in the growth of
several hygromycin-resistant shoots. Plantlets from these shoots were
generated by transferring onto root-inducing medium followed by
growth-inducing medium, as described previously (Akama et al., 1992).
Similarly, seeds obtained from Arabidopsis plants vacuum infiltrated
with transformed A. tumefaciens were plated on Murashige and
Skoog medium containing Suc (3%, w/v) and hygromycin (20 µg/mL) to select hygromycin-resistant growth. The transformation
efficiency by this protocol was found to be less than 0.1%. However,
all the transgenic plants characterized in this study are derived from
hypocotyl infection. From a total of 25 transformants that were
established for hygromycin-resistant growth, four lines (2-1B2,
2-10PG1, 2-10PG2, and 17-10) exhibiting varying degrees of chlorophyll
deficiency were selected for analysis. The phenotypes of these plants
are shown in Figure 2.
View larger version (45K):
[in this window]
[in a new window]
|
Figure 2.
Phenotypes of the selected 3-week-old transgenic
Arabidopsis plants. A control plant and four transgenic plants (2-1B2,
2-10PG1, 2-10PG2, and 17-10) representing four different lines
exhibiting varying degrees of chlorophyll deficiency are shown. The
2-B12 line represents the severe phenotype and the 17-10 line shows
negligible loss of chlorophyll. The other lines (2-10PG1 and 2-10PG2)
show intermediary effects of antisense HEMA RNA expression.
|
|
Correlation of Chlorophyll and Heme Content
As a first step to demonstrate the effect of the expression of
antisense HEMA1 RNA, the levels of chlorophyll and
non-covalently bound hemes were measured in the selected four
transformants. All of the transgenic plants showed reduced levels of
total chlorophyll (23%-82%) compared with the control plants (Fig.
3). Similarly, protoheme content was also
lowered (22%-60%) in these plants (Fig. 3). We observed that the
levels of chlorophyll and protoheme were proportionally decreased.
View larger version (19K):
[in this window]
[in a new window]
|
Figure 3.
Chlorophyll (top), protoheme (middle), and ALA
(bottom) levels in 2-week-old Arabidopsis transgenic lines. The amounts
are expressed relative to the amounts present in control plants (taken
as 100%). Data are the means of three replicates; SEs are
shown.
|
|
The dithionite-reduced pyridine homochrome spectra showed two
absorption peaks (data not shown) around 418 and 557 nm. The authentic
protoheme when processed similarly also gave two peaks at 418 and 557 nm. Furthermore, it was previously reported that the protoheme purified
from the maize seedlings showed two peaks at 418 and 557 nm (Schneegurt
and Beale, 1986). Therefore, it was concluded that the protocol used in
the present study measures mostly protoheme. However, heme a
could not be measured satisfactorily as we did not detect peaks at 429 nm and 588 nm as shown previously (Schneegurt and Beale, 1986).
Reduced ALA Levels in the Transformants
To determine if the decreased concentrations of chlorophyll and
non-covalently bound hemes in the transgenic plants reflect the amount
of ALA synthesis, we measured the level of ALA. Plants treated with
levulinic acid showed detectable levels of ALA. As in the case of
chlorophyll and hemes, plant line 2-1B2 accumulated the least amount of
ALA. The levels of ALA in the tested plants ranged from 21% to 56%
compared with the control (Fig. 3).
Nucleic Acid Analysis of Transgenic Plants
DNA Analysis
Based on the restriction map of the pCIT20-HEMA
antisense construct it was expected that digestion with
BamHI would release a 600-bp fragment from the transgenic
plant DNA and not from the control (Fig.
4). However, such an analysis with the
transgenic plant DNA yielded not only the 600-bp fragment but also
multiple fragments, suggesting that one or multiple copies of the
antisense HEMA1 gene were integrated into the different
locations of the chromosome. The Southern-blot analysis also showed
hybridization signals in all the transgenic lines that coincided with
the BamHI restriction pattern in the control plant (Fig. 4).
This result implied that the native HEMA gene in the
transgenic lines is intact and the phenotype in these plants did not
arise due to the disruption of the wild-type gene.
View larger version (144K):
[in this window]
[in a new window]
|
Figure 4.
DNA analysis of the transgenic Arabidopsis lines.
The BamHI-digested restriction fragments from the
pCIT20-HEMA antisense construct (lane A) and from the
Arabidopsis plants (lanes 1, 2-1B2; 2, 2-10PG1; 3, 2-10PG2; 4, 17-10;
and 5, control plant) were size-fractionated by agarose electrophoresis
and probed with the [-32P]dATP labeled 600-bp
NheI HEMA fragment. The white arrow shows
the fragment in the plasmid digest and in the transgenic lines,
indicating the integration of the fragment from the construct into the
genome of the transgenic plants. The white ovals show the positions of
the hybridization fragments in the control plant and in the transgenic
plants, suggesting that the native HEMA gene in these
transgenic plants is not disrupted by the transformation.
|
|
RNA Analysis
To examine if the observed ALA, chlorophyll, and heme levels of
the selected transgenic Arabidopsis plants were due to the inhibition
of HEMA mRNA expression by antisense mRNA, the steady-state HEMA mRNA, and the antisense HEMA1 mRNA levels
were analyzed. All transgenic lines showed an equal amount of the
endogenous HEMA mRNA levels. However, these lines expressed
different levels of antisense mRNA (Fig.
5). The transgenic line expressing the severe phenotype (2-1B2) contained an almost 4-fold elevated level of
antisense transcript. Interestingly, the transgenic line (17-10) that
did not exhibit phenotypic variation showed a 2-fold increase of
antisense transcript compared with the control plant. Thus, there is no
strict correlation of the antisense transcript level (as measured by
northern blot) with the observed phenotype. The GSA gene
transcript levels were similar in all the transgenic lines.
View larger version (126K):
[in this window]
[in a new window]
|
Figure 5.
RNA analysis of the transgenic Arabidopsis lines
expressing antisense HEMA1 RNA. Total RNA (20 µg) was
isolated from 2-week-old Arabidopsis plants and blotted onto a
nitrocellulose membrane as described in "Materials and Methods."
Lanes indicate the selected lines: 1, 2-1B2; 2, 2-10PG1; 3, 2-10PG2; 4, 17-10; and 5, control plant. Probes are HEMA1 cDNA sense
strand (A), HEMA1 cDNA antisense strand (B),
GSA cDNA (C), small subunit of Rubisco (D), and 18S rDNA
(E).
|
|
Antisense HEMA1 RNA Expression Lowers GluTR Levels
The GluTR protein level in the extracts was monitored by
western-blot analysis using polyclonal anti-GluTR antibodies. These antibodies recognized a 53-kD protein; in the transgenic plants the
amount GluTR protein was reduced (Fig.
6). The transgenic line with the most
severe phenotype (2-1B2) contained only 1% of the amount found in
control plants. To determine if this reduction is specific to the
HEMA gene product GluTR, we analyzed the expression of
Glu-1-semialdehyde-2,1-aminomutase and protochlorophyllide oxidoreductase by western blots using respective antibodies. We observed that all transgenic lines expressed GSA-AM and Por proteins as
much as in they were expressed in control plants. This indicates that
the HEMA antisense RNA specifically suppresses the GluTR expression.
View larger version (48K):
[in this window]
[in a new window]
|
Figure 6.
Western-blot analysis of the transgenic
Arabidopsis lines expressing antisense HEMA1 RNA. Lane
1, 2-1B2; lane 2, 2-10PG1; lane 3, 2-10PG2; lane 4, 17-10; and lane 5, control plant.
|
|
Results obtained by this analysis establish that the expression of
antisense HEMA1 RNA suppresses the formation of ALA,
resulting in the reduction of chlorophyll and heme levels in
Arabidopsis. This is the first direct demonstration of the vital role
of GluTR expression in higher plants.
| |
DISCUSSION |
The C5-Pathway Is Indispensable for Plant Survival
Transgenic Arabidopsis plants expressing antisense
HEMA1 mRNA were generated to monitor the significance of the
HEMA gene in the formation of ALA. The coding region of the
HEMA1 gene was used to construct the antisense mRNA
expression vector. As the HEMA1 and HEMA2 genes
have extensive similarity at the nucleotide level in the coding region,
an antisense construct made from either HEMA1 cDNA was
expected to block the translation of both HEMA1 and
HEMA2 mRNAs. Some of the transgenics showed chlorophyll
deficiency, ranging from patchy yellow to total yellow. We also
observed that plantlets that completely lacked chlorophyll failed to
survive under the growth conditions described. Attempts to rescue these plants by growing them in either low light or supplementing the medium
with varying concentrations of ALA failed. An earlier study carried out
with transgenic tobacco plants expressing antisense GSA mRNA
also showed that plants with reduced chlorophyll levels failed to
survive (Höfgen et al., 1994). These observations suggest that
suppression of the enzymes of the C5 pathway
affect the growth of the plant. In addition, an analysis of
chlorophyll-deficient Arabidopsis mutants revealed plants with lesions
in steps of chlorophyll synthesis beyond the point of ALA formation
(Runge et al., 1995); the fact that none were found in the
C5 pathway underscores the vital nature of ALA
biosynthesis in plants.
The Antisense HEMA1 Phenotype Is Variable
All of the selected transgenic plants expressed different levels
of antisense HEMA1 mRNA. The antisense RNA is thought to form a duplex with the endogenous HEMA mRNA and thus to
prevent gene expression. As these duplexes are substrates for
double-strand-specific RNases, the detection of either sense or
antisense mRNAs in transgenic plants is difficult (e.g. Zrenner et al.,
1993; Höfgen et al., 1994). However, we detected both antisense
and sense mRNAs in the transgenic plants, suggesting that this was
either due to sluggish double-stranded RNA-specific RNase activity or a
rapid production of the sense and antisense mRNAs that surpasses the RNase activity. The transgenic plant with the weakest phenotype (17-10)
was found to contain several copies of the antisense gene (Fig. 4).
Nevertheless, the amount of antisense transcript present in this line
was not proportional. This phenomenon has been observed in several
instances and could be due to silencing phenomena or position effects
(Tabler, 1993). On the other hand, although line 2-1B2 showed fewer
copies of antisense HEMA1 (as judged by Southern blots), the
severe phenotype observed was due to the expression of significant
amounts of HEMA antisense RNA reducing the GluTR level (Fig.
6).
While probing the antisense transcripts with the sense RNA probe, we
also noticed a hybridization signal in the control plants. This
hybridization is either from the minute quantities of antisense probes
that are made by aberrant priming process during the probe preparation
(Sambrook et al., 1989) or, more interestingly, it may due to the
naturally occurring antisense RNA in the wild-type plants. Such
complementary RNA species, although not widely reported to be involved
in gene regulation in plants, have been described; e.g. there is
evidence for naturally occurring antisense RNA of an -amylase gene
in barley (Rogers, 1988).
While there is a slight difference between RNA and protein expression
values, it is most satisfying to see that the protein levels correlate
with the severity of the antisense plant phenotype.
Does Heme Regulate the ALA Synthesis at the Level of GluTR?
Based on the biochemical analysis it was postulated that plants
contain two pools of ALA with distinct regulation, phytochrome or
feedback regulated (Huang et al., 1990). In all plants studied so far,
expression of at least one of the HEMA and GSA
genes is known to respond to light, whereas the other members of these gene families are not responsive to light (Kumar et al., 1996b). It is
not clear if expression of this set of HEMA and
GSA genes is regulated by a feedback mechanism. The end
product, heme, exhibited such a feedback inhibition on ALA biosynthesis
(Beale, 1978). Lowering heme levels by iron chelators increased ALA
accumulation (Duggan and Gassman, 1974). Of the two enzymes of the
C5 pathway, we believe that heme may not have a
regulatory role on GSA-AT protein as our results in the present study
show that decreased content of non-covalently bound hemes failed
elevate the GSA-AT levels (Figs. 5 and 6). While this observation
suggests that GluTR probably is the site of heme action, there is no
conclusive evidence except an in vitro study in which GluTR activity
was shown to be affected by heme (Huang and Wang, 1986). A direct proof
would be the examination of the GluTR levels in antisense
GSA transgenics or in mutant plants that synthesize
suboptimal levels of heme.
Chlorophyll Deficiency in Antisense HEMA1
Transgenics Is Not Due to Lowered
Protochlorophyllide Oxidoreductase Levels
NADPH-dependent protochlorophyllide oxidoreductase catalyzes the
reduction of protochlorophyllide to chlorophyllide in a light-dependent manner. Similar to HEMA and GSA genes, a set of
POR genes (PORA and PORB) is involved
in chlorophyll biosynthesis (Armstrong et al., 1995; Holtorf et al.,
1995). Transfer of light-grown plants to the dark causes accumulation
of Pchlide and decline of ALA synthesis. In light, however, Pchlide is
reduced and ALA synthesis increases. The inverse levels of ALA and
Pchlide in light and dark suggested the possible existence of a
regulatory circuit between ALA and Pchlide synthesis (Beale, 1978). In
the present study we observed that the anti-Por antibodies recognized a
36-kD protein in all the Arabidopsis lines including the control
plants. While PORA mRNA disappears within a few hours of
illumination (Armstrong et al., 1995), the expression of
PORB proceeds throughout the greening process; thus, the
detected signal was assumed to be the PorB protein. The constant levels
of PorB suggested that the chlorophyll deficiency in the transgenic
plants was primarily due to insufficient levels of Pchlide that had
resulted from decreased ALA synthesis.
Is the C5-Pathway the Sole Source for ALA in Plants?
While the existence of the C5 pathway is
widely accepted, other pathways of ALA formation in plants have been
reported (Ramaswamy and Nair, 1973; Meller and Gassman, 1982). Efforts
to thoroughly characterize these "alternative ALA-forming pathways"
have been unsuccessful. More convincing yet indirect evidence for an
extraplastidic ALA synthesis was obtained with a barley
albina mutant (Hess et al., 1991, 1992). The lack of
detectable levels of chloroplastic tRNAGlu, and
the presence of minute amounts of Pchlide, chlorophyll, and heme in
these mutants indicated an additional pathway for tetrapyrrole biosynthesis.
We show that the levels of protoheme are decreased in the transgenic
plants expressing antisense HEMA mRNA. On the other hand, a decrease
(more than 50%) in absorption units at 588 nm was noticed in the
severe phenotype (compared with the control); however, heme
a was not detected as no heme a specific peaks
were noticed in the absorption spectra. Nevertheless, radiotracer
studies conducted with maize seedlings demonstrated that all of the
cellular hemes including protoheme and the heme a (the
mitochondrial heme, a marker for alternative pathway for tetrapyrrole
biosynthesis) were made from ALA that is derived from
C5 pathway (Schneegurt and Beale, 1986).
Furthermore, lethal effect produced in some lines by antisense
HEMA1 (present study) and antisense GSA
(Höfgen et al., 1994) favors the C5 pathway
as a sole source for ALA biosynthesis.
| |
ACKNOWLEDGMENTS |
We thank Larry Ilag for help, Carl Simmons for suggestions and
critical comments regarding the manuscript, Xing-Wang Deng (Yale
University) for various plasmids, Gregory Armstrong and Klaus Apel
(Eidgenossiche Technische Hochschule, Zurich) for
PORA and PORB probes and anti-Por antibodies, and
Michael Timko (University of Virginia, Charlottesville) for
anti-Por antibodies. We are indebted to Clare Lister and Caroline Dean
(John Innes Centre, Norwich, UK) for mapping the HEMA1 and
HEMA2 genes.
| |
FOOTNOTES |
Received July 7, 1999; accepted September 23, 1999.
1
This work was supported by the Department of
Energy (grant no. DE-FG02-87ER13734).
*
Corresponding author; e-mail soll{at}trna.chem.yale.edu; fax
203-432-6202.
| |
LITERATURE CITED |
-
Akama K, Shiraishi H, Ohta S, Nakamura K, Okada K, Shimura Y
(1992)
Efficient transformation of Arabidopsis thaliana: comparison of efficiencies with various organs, plant ecotypes and Agrobacterium strains.
Plant Cell Rep
12: 7-11
-
Armstrong GA, Runge S, Frick G, Sperling U, Apel K
(1995)
Identification of NADPH:protochlorophyllide oxidoreductases A and B: a branched pathway for light-dependent chlorophyll biosynthesis in Arabidopsis thaliana.
Plant Physiol
108: 1505-1517
[Abstract]
-
Beale SI
(1978)
 -Aminolevulinic acid in plants: its biosynthesis, regulation, and role in plastid development.
Annu Rev Plant Physiol
29: 95-120
[CrossRef] -
Berthold N, Ellis J, Pelletier G
(1993)
In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants.
C R Acad Sci Paris
316: 1194-1199
-
Bradford M
(1976)
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal Biochem
72: 248-254
[CrossRef][Web of Science][Medline]
-
Duggan J, Gassman M
(1974)
Induction of porphyrin synthesis in etiolated bean leaves by chelators of iron.
Plant Physiol
53: 206-215
[Abstract/Free Full Text]
-
Flint DH
(1984)
Gabaculine inhibits -ALA synthesis in chloroplasts (abstract no. S-965).
Plant Physiol
75: 170
[Abstract/Free Full Text]
-
Grimm B
(1998)
Novel insights in the control of tetrapyrrole metabolism of higher plants.
Curr Opin Plant Biol
1: 245-250
[CrossRef][Web of Science][Medline]
-
Hess WR, Schendel R, Börner T, Rüdiger W
(1991)
Reduction of mRNA levels for two nuclear encoded light-regulated genes in barley mutant albostrians is not correlated with phytochrome content and activity.
J Plant Physiol
138: 292-298
-
Hess WR, Schendel R, Rüdiger W, Fieder B, Börner T
(1992)
Components of chlorophyll biosynthesis in a barley albina mutant unable to synthesize -aminolevulinic acid by utilizing the transfer RNA for glutamic acid.
Planta
188: 19-27
[CrossRef]
-
Höfgen R, Axelsen KB, Kannangara CG, Schüttke I, Pohlenz HD, Willmitzer L, Grimm B, von Wettstein D
(1994)
A visible marker for antisense mRNA expression in plants: inhibition of chlorophyll synthesis with glutamate-1-semialdehyde aminotransferase antisense gene.
Proc Natl Acad Sci USA
91: 1726-1730
[Abstract/Free Full Text]
-
Holtorf H, Reinbothe S, Reinbothe C, Bereza B, Apel K
(1995)
Two routes of chlorophyllide synthesis that are differentially regulated by light in barley (Hordeum vulgare L.).
Proc Natl Acad Sci USA
92: 3254-3258
[Abstract/Free Full Text]
-
Huang DD, Wang WY
(1986)
Chlorophyll biosynthesis in Chlamydomonas starts with the formation of glutamyl-tRNA.
J Biol Chem
261: 13451-13455
[Abstract/Free Full Text]
-
Huang L, Bonner BA, Castelfranco PA
(1990)
Regulation of 5-aminolevulinic acid (ALA) synthesis in developing chloroplasts.
Plant Physiol
92: 172-178
[Abstract/Free Full Text]
-
Ilag LL, Kumar AM, Söll D
(1994)
Light regulation of chlorophyll biosynthesis at the level of 5-aminolevulinate formation in Arabidopsis.
Plant Cell
6: 265-275
[Abstract]
-
Kumar AM, Csankovszki G, Söll D
(1996a)
A second and differentially expressed glutamyl-tRNA reductase gene from Arabidopsis thaliana.
Plant Mol Biol
30: 419-426
[CrossRef][Web of Science][Medline]
-
Kumar AM, Schaub U, Söll D, Ujwal M
(1996b)
Glutamyl-transfer RNA: at the crossroad of chlorophyll and protein biosynthesis.
Trends Plant Sci
1: 371-376
[CrossRef]
-
Lander ES, Green P, Abrahamson J, Barlow A, Daly MJ, Lincoln SE, Newburg L
(1987)
MAPMAKER: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations.
Genomics
1: 174-181
[CrossRef][Medline]
-
Lichtenthaler HK
(1987)
Chlorophylls and carotinoids: pigments of photosynthetic biomembranes.
Methods Enzymol
148: 350-382
[Web of Science]
-
Lister C, Dean C
(1993)
Recombinant inbred lines for mapping RFLP and phenotypic markers in Arabidopsis thaliana.
Plant J
4: 745-750
[CrossRef][Web of Science]
-
May BK, Bhasker CR, Bawden MJ, Cox TC
(1990)
Molecular regulation of 5-aminolevulinate synthase.
Mol Biol Med
7: 405-421
[Web of Science][Medline]
-
Meller E, Gassman ML
(1982)
Biosynthesis of 5-aminolevulinic acid: two pathways in higher plants.
Plant Sci Lett
26: 23-29
-
Ramaswamy NK, Nair PM
(1973)
-Aminolevulinate synthetase from cold-stored potatoes.
Biochim Biophys Acta
293: 269-277
[Medline]
-
Rogers JC
(1988)
RNA complementary to -amylase mRNA in barley.
Plant Mol Biol
11: 125-138
-
Runge S, van Cleve B, Lebedev N, Armstrong G, Apel K
(1995)
Isolation and classification of chlorophyll-deficient xantha mutants of Arabidopsis thaliana.
Planta
197: 490-500
[Medline]
-
Sambrook J, Fritsch EF, Maniatis T
(1989)
Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
-
Sangwan I, O'Brian MR
(1999)
Expression of a soybean gene encoding the tetrapyrrole-synthesis enzyme glutamyl-tRNA reductase in symbiotic root nodules.
Plant Physiol
119: 593-598
[Abstract/Free Full Text]
-
Schneegurt MA, Beale SI
(1986)
Biosynthesis of protoheme and Hema a from glutamate in maize.
Plant Physiol
81: 965-971
[Abstract/Free Full Text]
-
Schön A, Krupp G, Gough S, Berry-Lowe S, Kannangara CG, Söll D
(1986)
The RNA required in the first step of chlorophyll biosynthesis is a chloroplast glutamate tRNA.
Nature
322: 281-284
[CrossRef][Medline]
-
Stillman LC, Gassman ML
(1978)
Protoheme extraction from plant tissue.
Anal Biochem
91: 166-172
[CrossRef][Web of Science][Medline]
-
Tabler M
(1993)
Antisense RNA in plants: a tool for analysis and suppression of gene function.
In
KA Roubelakis-Angelakis, KTT Van, eds, Morphogenesis in Plants. Plenum Press, New York, pp 237-258
-
Tanaka R, Yoshida K, Nakayashiki T, Masuda T, Tsuji H, Inokuchi H, Tanaka A
(1996)
Differential expression of two HEMA mRNAs encoding glutamyl-tRNA reductase proteins in greening cucumber seedlings.
Plant Physiol
110: 1223-1230
[Abstract]
-
Thomsen B, Oelze-Karow H, Schuster C, Mohr H
(1993)
Stimulation of appearance of extraplastidic tetrapyrroles by photooxidative treatment of plastids.
Photochem Photobiol
58: 711-717
-
von Wettstein D, Gough S, Kannangara CG
(1995)
Chlorophyll biosynthesis.
Plant Cell
7: 1039-1057
[CrossRef][Web of Science][Medline]
-
Weinstein JD, Beale SI
(1984)
Biosynthesis of protoheme and heme a precursors solely from Glu in the unicellular red alga Cyanidium caldarium.
Plant Physiol
74: 146-151
[Abstract/Free Full Text]
-
Weinstein JD, Beale SI
(1985)
Enzymatic conversion of Glu to 5-aminolevulinate in soluble extracts of the unicellular alga Chlorella vulgaris.
Arch Biochem Biophys
237: 454-464
[CrossRef][Medline]
-
Zrenner R, Willmitzer L, Sonnewald U
(1993)
Analysis of the expression of potato uridine diphosphate-Glc pyrophosphorylase and its inhibition by antisense RNA.
Planta
190: 247-252
[Web of Science][Medline]
© 2000 American Society of Plant Physiologists
This article has been cited by other articles:
|
|
|
|
 
L. A. Nogaj, A. Srivastava, R. van Lis, and S. I. Beale
Cellular Levels of Glutamyl-tRNA Reductase and Glutamate-1-Semialdehyde Aminotransferase Do Not Control Chlorophyll Synthesis in Chlamydomonas reinhardtii
Plant Physiology,
September 1, 2005;
139(1):
389 - 396.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Srivastava and S. I. Beale
Glutamyl-tRNA Reductase of Chlorobium vibrioforme Is a Dissociable Homodimer That Contains One Tightly Bound Heme per Subunit
J. Bacteriol.,
July 1, 2005;
187(13):
4444 - 4450.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. C. McCormac and M. J. Terry
Loss of Nuclear Gene Expression during the Phytochrome A-Mediated Far-Red Block of Greening Response
Plant Physiology,
September 1, 2002;
130(1):
402 - 414.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Kanamaru, A. Nagashima, M. Fujiwara, H. Shimada, Y. Shirano, K. Nakabayashi, D. Shibata, K. Tanaka, and H. Takahashi
An Arabidopsis Sigma Factor (SIG2)-Dependent Expression of Plastid-Encoded tRNAs in Chloroplasts
Plant Cell Physiol.,
October 1, 2001;
42(10):
1034 - 1043.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
X. Chen, M. D. Smith, L. Fitzpatrick, and D. J. Schnell
In Vivo Analysis of the Role of atTic20 in Protein Import into Chloroplasts
PLANT CELL,
March 1, 2002;
14(3):
641 - 654.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION