Plant Physiol. (1999) 120: 411-420
Symbiotic Root Nodules of the Actinorhizal Plant
Datisca glomerata Express Rubisco Activase
mRNA1
Patricia A. Okubara2,
Katharina Pawlowski3,
Terence M. Murphy, and
Alison M. Berry*
Department of Environmental Horticulture (P.A.O., A.M.B.), and
Plant Biology Section (T.M.M.), University of California, Davis,
California 95616; and University of California, Davis,
California 95616Department of Molecular Biology, Wageningen
Agricultural University, 6703HA Wageningen, The Netherlands (K.P.)
 |
ABSTRACT |
N2-fixing
symbiotic root nodules of the actinorhizal host Datisca
glomerata express Dgrca
(D.
glomerata Rubisco activase) mRNA, a
transcript usually associated with photosynthetic organs or tissues. In
northern blots a mature, 1700-nucleotide Dgrca mRNA was
detected in green plant organs (leaves, flowers, and developing fruits)
and in nodules but was not detected in roots. A second message of 3000 nucleotides was observed only in nodules. Both size classes of
transcripts were polyadenylated. The larger transcript was 2- to 5-fold
more abundant than the mature mRNA; it was hybridized to an intronic
probe, indicating that a stable, incompletely spliced transcript was
accumulating. Treatment with light on excised nodules did not alter the
relative abundance of the two species. In in situ hybridizations the
Dgrca message was expressed intensely in the nuclei of
infected cells. The Dgrca transcripts also accumulated
at lower levels in uninfected cortical cells adjacent to the periderm
and the vascular cylinder. mRNA encoding the large subunit of Rubisco
(DgrbcL) was abundant in mature infected cells and in
the amyloplast-rich sheath of uninfected cortical cells lying between
the infected cells and nodule periderm. The proteins Rubisco activase,
Rubisco, and the 33-kD O2-evolving complex subunit did not
accumulate to detectable levels, indicating that a functional
photosynthetic apparatus was not prevalent in nodule tissue. Signals or
factors required for the transcription of Dgrca appeared
to be present in nodules, but efficient splicing and translation of the
message were not observed in Frankia-infected tissue
where transcript accumulation was highest.
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INTRODUCTION |
Organogenesis of N2-fixing symbiotic root
nodules represents an intricate interplay between two separate
organisms: a plant and a microbial endosymbiont. Development of a
functionally specialized organ, such as the symbiotic root nodule,
depends on environmentally and developmentally induced cellular,
physiological, and molecular events. Actinorhizal root nodules are
induced by N2-fixing actinomycetes of the genus
Frankia on woody dicotyledonous plants belonging to eight
angiosperm families (Benson and Silvester, 1993
). Actinorhizal symbioses contribute substantially to the global nitrogen economy, with
particular impact on land reclamation, forestry, and management of
sustainable ecosystems worldwide.
Functional aspects of the establishment of the endosymbiont, e.g. the
infection process, N2 assimilation,
O2 regulation, and C reduction, are shared
between actinorhizal and legume nodules (for review, see Pawlowski and
Bisseling, 1996; Pawlowski et al., 1996). However, there is
considerable diversity in the morphological and organizational
characteristics of these two nodule types. The actinorhizal nodule is a
modified lateral root, having an organized meristem that arises from
the pericycle and a central vascular cylinder surrounded by cortical
cells, some of which contain Frankia. Legume nodules arise
from interior cortical cell layers of the parent root and have
peripheral vasculature (Hirsch and LaRue, 1997).
Molecular and physiological studies on a number of actinorhizal systems
are gradually providing information on patterns of gene expression and
biochemical processes relevant to nodule organogenesis. Homologs of
genes coding for enzymes in primary N2 and C
fixation, including Gln synthetase, Orn carbamoyl transferase, Suc
synthase, and enolase, have been cloned from Alnus
glutinosa by differential screening of cDNA libraries
(Pawlowski et al., 1993). The characterization of expression of genes
for leghemoglobin from Casuarina glauca (Jacobsen-Lyon et
al., 1995; Gherbi et al., 1997), for a subtilisin-like protease
(Ribeiro et al., 1995), for an enzyme involved in thiazole biosynthesis (Ribeiro
et al., 1996), both from A. glutinosa, and for an A. glutinosa Cys protease of the papain superfamily (Goetting-Minesky and Mullin, 1994) is helping to identify metabolic processes in young
and mature actinorhizal nodules. Recent reports of
glutamate-and-Pro-rich, putative cell wall protein cDNA cell wall
protein cDNA (Guan et al., 1997) and two Gly- and His-rich mRNAs
expressed in the early infection zone (Pawlowski et
al., 1997) contribute to an emerging picture of cell- and
tissue-specific gene expression and their corresponding
biochemical processes in nodule organogenesis.
To better understand actinorhizal nodule development, the relationship
between nodule organization and function, and the cytological and
biochemical diversity underlying different host-microbe
N2-fixing symbioses, we initiated studies with
Datisca glomerata. D. glomerata is an herbaceous
perennial that grows in sandy soils of riparian ecosystems in
California and northern Mexico (Davidson, 1973; Liston et al., 1989).
It is closely related to the Indo-European species Datisca
cannabina (Davidson, 1973); it is also related to begonias and
cucurbits based on molecular systematics of rbcL (Rubisco large subunit
genes (Swensen et al., 1994). The herbaceous nature of the foliage and
the low tissue levels of phenolics render D. glomerata
amenable to molecular biological studies. D. glomerata, with
Coriaria, is distinguished from other actinorhizal hosts in
that the symbiotic N2-fixing cells form a
distinct, dense sector within the nodule cortex, to one side of the
central vascular cylinder (Hafeez et al., 1984).
Because of our interest in gene expression in developing nodules of
D. glomerata, we isolated a nodule cDNA clone, designated Dgrca (D.
glomerata
Rubisco activase), encoding a
putative Rubisco activase. Rubisco activase normally accumulates in
greening or photosynthetic tissues expressing Rubisco. The molecular
mode of action of Rubisco activase has not been completely elucidated;
however, it is postulated to cause conformational changes in Rubisco
that promote the ordered binding of substrates required for optimal
enzymatic activity and stability (Portis, 1990; Lan and Mott, 1991).
Hence, it has a regulatory role in photosynthetic C reduction via the
action of Rubisco. The significance of an mRNA encoding Rubisco
activase in symbiotic root nodules of D. glomerata remains
unknown, but might be attributed to: (a) a role in photosynthesis as a
minor process that occurs in some nodules; (b) a role in
O2 partitioning, whereby it activates the oxygenase function of Rubisco; and (c) part of a general induction of
cellular processes during nodule organogenesis. In this paper we
characterize the pattern of expression of Dgrca and other
components of the photosynthetic apparatus to examine the possible role
of Dgrca in nodules.
The accession numbers for the sequences of Dgrca mRNA
and the 1.2-kb Dgrca gene segment reported in this
article are AF047352 and AF052424, respectively.
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MATERIALS AND METHODS |
Plant Material
Datisca glomerata (Presl) Baill seeds were obtained
from plants in Vaca Hills, California. Plants were grown either in
liquid culture medium consisting of one-quarter-strength Hoagland
solution or in a 2:1 (v/v) mixture of Perlite and sand:peat:fir bark
(1:1:1, v/v). Root nodules of D. glomerata were induced on
greenhouse-grown seedlings by inoculation with crushed nodules of
Ceanothus griseus var. horizontalis (Liu and
Berry, 1991). Inoculated plants were fertilized with
one-quarter-strength Hoagland medium. Nodules and root tips were
excised from intact root systems of plants grown in soil or in liquid
culture, and immediately transferred to liquid
N2. Nodules were harvested 4, 5, 7, and 11 weeks
after inoculation. Enhanced greening and vigorous growth of plants at 4 to 5 weeks after inoculation correlated with nodule lobe expansion and
suggested that the onset of N2 fixation occurred
at this time. Young leaves, flowers (sepals, anthers, stamens, and
styles), and fruits containing immature seed (developing fruits) were
collected from 5-month-old plants. To examine the effect of light on
nodule greening and Dgrca mRNA levels, washed root systems
on intact plants inoculated 17 weeks earlier were either wrapped in
clear plastic and placed under continuous white light at 20°C for 16 to 20 h or placed in darkness for 20 h before excision of
nodules. Nodules were also harvested from light-treated roots excised
from plants 10 weeks after inoculation. Plant materials for RNA and DNA
preparations were stored at 
80°C.
RNA and DNA Isolation and Blot Analysis
Total RNA was isolated from D. glomerata nodules as
previously described (Pawlowski et al., 1994). For blot analysis,
poly(A
) RNA was recovered after two passages
over oligo(dT25) magnetic beads (Dynal, Lake
Success, NY) to remove poly(A+) RNA. RNA was
partitioned on 1% agarose containing 6% formaldehyde (Sambrook et
al., 1989), transferred to a nylon membrane (Zeta Probe, Bio-Rad), and
hybridized as recommended by the manufacturer. Loading of equal amounts
of total RNA for northern blots was determined from
A260 titers and by visualization of
ethidium bromide-stained rRNA bands. For cDNA library construction
and blot analysis, poly(A+) RNA was obtained by
two passes through oligo(dT)-cellulose columns (Boehringer
Mannheim). Total DNA was obtained from young leaves of D. glomerata, essentially as described by Ribeiro et al. (1995), and
transferred to the Zeta Probe nylon membrane. Hybridization of DNA
blots was performed according to instructions (Bio-Rad). Autoradiography was carried out using preflashed Kodak XAR 5 film at
80°C with an intensifying screen (Lightning II Plus, DuPont).
Hybridization probes for RNA and DNA blots consisted of the 400-bp
partial Dgrca cDNA insert, the full-length 1.7-kb cDNA insert, or intron A (128 bp) and intron C (160 bp) amplified by PCR.
DNA probes were purified from agarose gels (JETSORB Gel Extraction kit,
Genomed, Research Triangle Park, NC) and radiolabeled using [
-32P]dCTP (Amersham) and the Multiprime DNA
Labeling System (Amersham) to specific activities of 3.5 to 7.0 × 104 Bq µg1
(2.0-4.0 × 106 cpm
ng1). The intron A probe used in Figure 3B was
labeled with both [-32P]dCTP and
[-32P]dATP (Amersham).
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| Figure 3.
Total DNA of D. glomerata
hybridized to the 400-bp partial cDNA insert (A) and intron-A probe
(B). DNA was treated with HindIII (lane H) or
EcoRI (lanes E) before transfer to nylon membrane. Sizes
(in kb) of phage Lambda HindIII fragments are shown in
the middle. Autoradiography was carried out at 80°C for 3 (A)
and 5 (B) d.
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32P radiolabel was quantified from freshly
hybridized nylon membranes using a phosphor imager (Storm, Molecular
Dynamics, Sunnyvale, CA) and imaging plate (BAS IIIs, Fuji) and
analyzed with ImageQuant software (version 4.0, Molecular Dynamics).
cDNA Library Construction and Screening
Two Lambda Zap cDNA libraries were made representing mRNAs from
D. glomerata nodules harvested 4, 5, and 7 weeks after
inoculation with Frankia. cDNA synthesis and ligation was
carried out according to the protocol for Lambda Zap Express cDNA
synthesis (Stratagene).
After mass excision, DNA was prepared from approximately 400 randomly
selected phagemids and differentially screened with either
radiolabeled-nodule or root cDNA. The average insert size of the first
library was 500 to 600 bp. The second cDNA library was constructed to
obtain a full-length clone. Double-stranded cDNA was fractionated on
Sepharose CL-4B (Pharmacia); the 1- to 2-kb fraction was ligated to the
Lambda Zap Express phage vector. The average size of cDNA inserts in
this library was approximately 1.6 kb.
Primers
The oligonucleotide primers (Operon Technologies, Alameda, CA) for
nucleotide sequencing of Dgrca cDNA and the Dgrca
gene segment and for PCR of the Dgrca gene segment, intron A
and intron C, are listed in Table I.
In Situ Localization
Whole nodules were harvested 6 to 8 weeks after inoculation with
Frankia and fixed in buffered 4% paraformaldehyde and
0.25% glutaraldehyde under a vacuum. Nodules were then dehydrated,
embedded in paraffin, and sectioned. Nodule lobe sections were
hybridized to 35S-radiolabeled RNA probes (see
"RNA and DNA Isolation and Blot Analysis"), as described previously
(van de Wiel et al., 1990; Ribeiro et al., 1995). Adjacent sections
were used for hybridization to antisense and sense RNA probes. Sections
were stained with ruthenium red and counterstained with toluidine blue.
A 500-bp HindIII fragment was obtained from the full-length
clone of Dgrca and ligated to the Bluescript pBKS+ vector
(Stratagene). The resulting subclone was either treated with
SalI and transcribed with T7 RNA polymerase (antisense
probe) or digested with EcoRI and transcribed with T3 RNA
polymerase (sense probe). To obtain an antisense probe of
DgrbcL, plasmid pDgrbcL, containing the rbcL
coding sequence cloned in pBKS+, was linearized with SalI and transcribed with T7 RNA polymerase. The DgrbcL sense
probe was generated with BamHI and T3 RNA polymerase. Probes
for the nitrogenase subunit H gene (nifH) of
Frankia were described by Ribeiro et al. (1995). Antisense
and sense RNA probes were radiolabeled with
[35S]UTP, as described by van de Wiel et al.
(1990), without a cold chase. Maximum lengths of the probes were
therefore 400 to 500 nucleotides.
Western Blots
Protein extracts were obtained from D. glomerata roots,
root-free nodules and leaves, and leaves of soybean and tobacco by the
following procedure: Tissue samples were frozen at 70°C, then
homogenized with a mortar and pestle in 50 mM
Tris-Cl, pH 7.4, 200 mM NaCl, and 10 mM MgCl2 (4 mL
g1 fresh weight tissue), and centrifuged for 15 min at 27,000g at 4°C. Equal amounts (by fresh weight) of
clarified supernatants were used directly for SDS-PAGE (Laemmli, 1970).
Protein determinations were performed according to the method of
Bradford (1976). Immunodetection was performed according to the method
of Sambrook et al. (1989) using rabbit polyclonal antibodies made to
cotton Rubisco activase (provided by M. Salvucci, U.S. Department of
Agriculture-Agricultural Research Service, Western Cotton Research
Laboratory, Phoenix, AZ), to the soybean large subunit protein (Murphy,
1978), and to the OEC33 of pea (provided by S. Theg, University of
California, Davis). Secondary anti-rabbit IgG conjugated to alkaline
phosphatase was obtained from Sigma. Protein bands were visualized by
staining with 5-bromo-4-chloro-3-indolyl phosphate (Sigma) and
nitroblue tetrazolium (Eastman Kodak). At least two western-blot
analyses using independent protein extractions were conducted with each primary antiserum.
| |
RESULTS |
We isolated an unusually expressed full-length (1634-bp) cDNA from
symbiotic root nodules of D. glomerata. In BLASTX alignments (Altschul et al., 1990), the deduced amino acid sequence of the cDNA
shared up to 89% similarity with Rubisco activases from many higher
plants and cyanobacteria. We therefore designated our clone Dgrca. For clarity, only the three highest-scoring matches
to sequences of apple, Arabidopsis, and tobacco are shown (Fig.
1).
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| Figure 1.
The deduced amino acid sequence of
Dgrca (Dg) is conserved with respect to Rubisco activase
coding sequences from other photosynthetic species, including apple
(Md, P[N] = 1.8e259; Watillon et al., 1993),
Arabidopsis small peptide (At, P[N] = 5.8e247; Orozco
et al., 1993), and tobacco small peptide (Nt, P[N] = 4.8e240; accession no. U35111).
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The relative numbers of Dgrca clones in both libraries
indicated that the corresponding mRNA was not a rare-class message in
D. glomerata nodules. Initially, a 400-bp partial cDNA clone of Dgrca was isolated from approximately 400 clones picked
at random. A screen of approximately 1.2 × 105 recombinant phage from a second library
yielded over 20 separate clones that hybridized to a probe consisting
of the partial Dgrca cDNA insert. The cDNAs ranged in size
from 0.7 to 1.8 kb. The 3
-untranslated region varied in length; among
five of the longest clones examined, two were found to contain 41 additional nucleotides immediately upstream of the
poly(A+) tail, suggesting that multiple
polyadenylation signals in Dgrca are utilized.
Two Size Classes of Dgrca RNA Are Present in Nodules
Dgrca mRNA was expressed in nodules and in
photosynthetic organs of D. glomerata, including leaves,
flowers, and immature fruits (Fig. 2A).
Dgrca mRNA was not detected in the root samples. In the
photosynthetic organs Dgrca mRNA was estimated to be 1700 nucleotides long. This corresponded to the size of the full-length cDNA
clone (1634 bp), which was similar to Rubisco activase mRNAs from other
plants (1650-1900 nucleotides). The data indicate that the
1700-nucleotide mRNA band represented the mature, fully spliced message.
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| Figure 2.
Expression of Dgrca mRNA in various
organs of D. glomerata (A), in nodules with or without
light treatment (B, lanes Ne, Nw, and
Nw) and in oligo(dT)-cellulose fractions of nodule
RNA (B, lanes A and A+). Sizes in nucleotide of rRNA species are
shown on the right of each panel. A, Total RNA from leaves (lane L),
flowers (lane Fl), immature fruits (lane Fr), roots (lane R), and
nodules harvested 4 to 11 weeks after Frankia
inoculation (N4-N11) was hybridized to
radiolabeled Dgrca cDNA insert. B, Total RNA from
nodules harvested from excised roots (lane Ne) or from
roots of whole plants (lanes Nw) after treatment with white
light (Lt) or darkness (Dk) were hybridized to the full-length
Dgrca cDNA probe. Total N11 RNA (lane
N11), root RNA (lane R), and leaf RNA (lane L) are included
for a comparison. The unbound fraction of total N11 RNA
(lane A) and the bound N11 RNA fraction (lane A+) after
oligo(dT)-cellulose treatment were also hybridized to the full-length
Dgrca cDNA probe. C, Total RNA from roots (lane R),
N4 nodules, or N7 nodules was hybridized to a
radiolabeled intron-A probe. The asterisk indicates the predicted
position of the 1700-nucleotide mRNA species. For A and C and the first
seven lanes of B, approximately 5 µg of total RNA was loaded into
each lane; for lane A+ in B, 2.5 µg of poly(A+) RNA was
used. Autoradiography was carried out with preflashed Kodak XAR 5 film
at 80°C for 16.5 h (A), 6 d (B), and 5 d (C).
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The two distinct size classes of Dgrca transcripts, 3000 and
1700 nucleotides, were observed in nodules harvested 4 to 11 weeks
after Frankia inoculation (Fig. 2A). Only the
1700-nucleotide mRNA was detected in the photosynthetic organs, e.g.
the flower, immature fruit, and leaves. Roots showed no detectable
levels of either species. The 3000-nucleotide species was 2- to 5-fold more abundant than the 1700-nucleotide mRNA in total RNA from nodules
harvested 11 weeks after inoculation (data not shown). The relative
abundance of the 3000-nucleotide mRNA was reduced in the older
(17-week) nodule samples, but was not altered significantly by exposure
to white light (Fig. 2B, lanes 1-3). However, this light treatment was
sufficient to cause faint greening in the mature nodules closest to the
light source (data not shown).
To determine whether both size classes of mRNA were polyadenylated,
poly(A+) and poly(A)
fractions were hybridized to the Dgrca cDNA insert in gel
blots (Fig. 2B, lanes 7 and 9). Whereas no hybridization was detected in the poly(A) fraction, both the 3000- and
1700-nucleotide species were observed in the
poly(A+) fraction. The abundance ratio,
quantified by phosphor imaging (data not shown), was nearly 1:1 in the
nodule poly(A+) RNA fraction, as compared with
5:1 in total RNA. Attempts to obtain a 3-kb cDNA clone from several
different poly(A+) RNA preparations, using random
amplification of cDNA 3 ends with two different primer pairs were
unsuccessful.
Dgrca Is a Single-Copy Gene in D. glomerata
To determine the size of the rca gene family in
D. glomerata, we hybridized total DNA to the 400-bp partial
cDNA insert. A single 8.2-kb EcoRI fragment and a single
4.7-kb HindIII fragment (Fig.
3A) were radiolabeled; there was no
evidence of two polymorphic Dgrca genes. We therefore
conclude that Rubisco activase is encoded by a single gene or by a
small, conserved gene family in D. glomerata, as in other
plant species (Wernecke and Ogren, 1989; Wernecke et al., 1989; Rundle
and Zielinski, 1991; Qian and Rodermel, 1993).
Occurrence of Intronic Sequences in the Dgrca Gene and
3000-Nucleotide mRNA
We were interested in whether the Dgrca gene was
interrupted by introns that could account for the additional mass of
the 3000-nucleotide mRNA. We examined Dgrca gene sequences
from Arabidopsis (Orozco et al., 1993), spinach (Wernecke et al.,
1989), and barley (Rundle and Zielinski, 1991). All three genes have
four to six introns at conserved positions within the coding regions. A
1.2-kb fragment was amplified from the D. glomerata genome
using PCR primers specific to the Dgrca coding region (Table
I). This Dgrca gene segment contained three stretches of
nucleotides, 96, 201, and 128 bp in length, that were not present in
the Dgrca cDNA (Fig. 4A). The
positions of these AT-rich stretches with respect to the coding region
were identical to those of the introns in the rca genes from
the other plants (Fig. 4B). Therefore, we refer to these genomic
sequences as introns A, B, and C.
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| Figure 4.
A, The coding sequence (uppercase letters) of the
Dgrca genomic DNA segment is interrupted by at least
three intronic sequences (lowercase letters), designated Intron A,
Intron B, and Intron C. B, The positions of these introns were highly
conserved with respect to the Arabidopsis, spinach, and barley genes.
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To generate hybridization probes, genomic DNA template was amplified
with PCR primers annealing to coding sequences that immediately flanked
intron A and intron C (Table I). In both RNA and DNA blots, the intron
C PCR product hybridized to a wide number of species, possibly because
it carried a repetitive sequence (data not shown). However, intron A
hybridized to a single 8.2-kb EcoRI DNA fragment (Fig. 3B)
and hybridized weakly to the 3000-nucleotide mRNA in blots of total
nodule RNA (Fig. 2C).
Dgrca and DgrbcL mRNA Localization Patterns
The mature Frankia-infected zone of the D. glomerata nodule consisted of a compact region of expanded host
cells filled with Frankia vesicles arrayed peripherally
around a central vacuole (Fig. 5, A and
E). The mature tissue was distinguished by expression of
nifH, seen as silver grains localized over the
Frankia vesicles in the infected cells (Fig. 5, B and F).
The youngest of these cells relative to the developmental gradient of
the infection zone are indicated by white arrows in Figure 5, E through
H. Beginning in the cells of the early infection zone, the host
cytoplasm contained multiple nuclei (Fig. 5, A-C).
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| Figure 5.
In situ localization of Dgrca,
DgrcbL, and nifH mRNA in
D. glomerata nodule sections. A, Bright-field micrograph
of the interface between Frankia-infected cells and
mature, uninfected cells. Bar = 15 µm (for A-C). B,
Bright-field micrograph of nifH expression in
Frankia-infected cells. Silver grains indicating
hybridization to antisense probes appear as black specks, particularly
dense over the Frankia vesicles in the infected cells
(white asterisk). C, Bright-field micrograph showing dense
accumulations of silver grains indicating hybridization of the
Dgrca antisense probe to nuclei of infected cells
(arrowheads). Some Dgrca transcripts are detected in
adjacent younger cells of the early infection stage. Mature, uninfected
cells show scattered silver grains, with no significant accumulation of
Dgrca mRNA. D, Bright-field micrograph of a longitudinal
nodule section hybridized to the Dgrca antisense probe.
The relatively large nuclei in the meristem are darkly stained with
toluidine blue, but do not have silver grains. Infected cells
accumulated Dgrca transcripts, whereas uninfected cells
showed much less hybridization. Bar = 30 µm. E, Bright-field
micrograph of a longitudinal section. The Frankia
vesicles stained red are arrayed around central vacuoles in the mature,
infected cells. Bar = 500 µm (for E-H). F, Dark-field
micrograph showing nifH expression.
Hybridization of transcripts to the nifH
antisense probe, apparent as white specks, delineates the onset of
N2 fixation in mature, infected cells (white arrows in
E-H) within the developmental gradient of the infection zone.  , A
zone of senescence where nifH expression is
reduced; the corresponding area in E has a vesiculated appearance
because of degradation of the endosymbiont. G, Dark-field micrograph of
DgrbcL expression in the zone before N2
fixation and in mature, infected cells. DgrbcL mRNA was
less abundant in the zone of senescence. H, Dark-field micrograph of
Dgrca expression in the infected cells and in
surrounding uninfected cortical cells. nu, Nuclei; UN, uninfected
cells; F, Frankia-infected cells; am, amyloplasts in
uninfected cells; p, periderm; m, meristem; INF, infected cells; nr,
base of a nodule root.
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In contrast, the in situ expression pattern of Dgrca mRNA
was complex. High levels of expression of Dgrca were
observed in Frankia-infected cells, especially at early
stages of vesicle differentiation before the onset of
N2 fixation (Fig. 5, C, D, and G). The punctate
appearance of the Dgrca signal (Fig. 5G) was attributed to
the concentration of message in the nuclei of infected cells (Fig. 5C).
Dgrca message was detected at lower levels in adjacent
(younger) cells in the early stages of Frankia infection.
Some Dgrca RNA was also detected in the nuclei of mature, infected cells expressing nifH and in the cortical cells
immediately adjacent to infected cells (Fig. 5H). No Dgrca
expression was observed in the nodule-lobe meristem (Fig. 5D) or in the
nodule-root meristem (data not shown). The nuclei in the meristem
and in the cells underlying the periderm in Figure 5D stained brightly
with toluidine blue, but silver grains did not accumulate.
Hybridization with the Dgrca sense probe was negative (data
not shown).
DgrbcL mRNA was most abundant in the infected cells of the
zone preceding N2 fixation (Fig. 5G). It was also
detected in mature, infected cells and the uninfected cortical cells
lying between the infected cells and nodule periderm. In both infected
and uninfected cells, silver grains indicating DgrbcL
expression were not densely clustered over a particular region of the
cell, but appeared to be distributed throughout the cytoplasm. In
carpels the DgrbcL antisense probe hybridized to
photosynthetic parenchyma cells as expected, but not to immature,
nonphotosynthetic anthers (data not shown). Hybridization of
Dgrca, DgrbcL, and nifH was somewhat reduced in a region where Frankia vesicles were undergoing
senescence (Fig. 5, E-H; see legend). Neither Dgrca nor
DgrbcL antisense probes hybridized to periderm or vascular
tissue.
Rubisco Activase and Other Proteins Associated with the
Photosynthetic Apparatus Do Not Accumulate in D. glomerata Nodules
We performed western analyses to determine whether proteins of
Rubisco activase, Rubisco large subunit, and the OEC33 were present in
nodule extracts. All three proteins were detected in leaf extracts, but
not in nodule or root extracts (Fig. 6).
With polyclonal antibodies made to soybean Rubisco large subunit, a strong protein band was seen in leaf extracts at 50 to 60 kD (Fig. 6,
left panel), representing the native form of the protein. We detected a
protein of 40 kD in D. glomerata leaf extracts with antibodies made to tobacco Rubisco activase (Fig. 6, middle panel). This band was similar in size to Rubisco activase small polypeptides from other plant species (41-44 kD). Antibodies made to OEC33 from pea
detected a 33-kD protein in the leaves of D. glomerata (Fig.
6, right panel).
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| Figure 6.
Western analyses for the Rubisco large
subunit (left), Rubisco activase (middle), and OEC33 (right). Ten
microliters of extracts of D. glomerata nodule, root,
and leaf protein was partitioned on SDS-PAGE before reaction with
antibodies (diluted as indicated). For Rubisco, 17, 2.0, and 59 µg of
protein from nodule, root, and leaf were used, respectively; for
Rubisco activase and OEC33, 32, 33, and 91 µg of protein from nodule,
root, and leaf were used, respectively. Protein standards (in
kilodaltons) are shown at the right.
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DISCUSSION |
We identified an mRNA encoding Rubisco activase in symbiotic root
nodules of D. glomerata. Similarities between
Dgrca and Rubisco activases from other organisms were
evident in comparisons of their nucleotide and amino acid sequences,
mRNA expression patterns in various organs, sizes of the gene families,
and positions and sizes of three introns. Rubisco activase is conserved
in photosynthetic organisms across a wide range of genera, including
lower eukaryotes (Roesler and Ogren, 1990; Li et al., 1993), and has no
reported alternative functions. A Rubisco activase-like GA-binding
protein has been identified (Komatsu et al., 1996), but the deduced
amino acid sequence of this gene shares limited identity with that of Dgrca, as well as with Rubisco activases from other species.
Rubisco activase functions as a heterodimer in most higher plants, with
the exception of a monomeric form in maize (Salvucci et al., 1987).
Large and small isoforms of Rubisco activase reported in Arabidopsis,
spinach (Wernecke et al., 1989), and barley (Rundle and Zielinski,
1991) arise from alternative splicing of a single mRNA. In other
species such as tobacco, the polypeptides are encoded by separate genes
(Qian and Rodermel, 1993). In our RNA blots we detected two
Dgrca transcripts expressed in D. glomerata
nodules, a full-length, mature Dgrca mRNA of 1700 nucleotides, and an unusually large (3000 nucleotides), abundant
transcript. The 1300-nucleotide size differential between these mRNAs
was much greater than the difference of approximately 300 nucleotides
that results from alternative splicing in other species. In addition,
five separate Dgrca cDNA clones that were examined had
identical 3 sequences. Therefore, we found no evidence from either
northern blots or from limited nucleotide sequence data to indicate
that the alternative splicing of Rubisco activase mRNA observed in
other species is occurring for Dgrca in D. glomerata nodules. Because Dgrca appears to be a
single-copy gene, the 3000-nucleotide species is not likely to arise
from transcription of a second gene.
Hybridization of the large transcript to intronic DNA suggested either
that it was a form of heteronuclear RNA or that it represented an
anomalous, incompletely spliced message. This possibility was supported
by in situ localization of the Dgrca RNA primarily to nuclei
of infected cells. Because the D. glomerata nodule is indeterminate, a developmental gradient from meristem to
N2-fixing infected cells is present in the mature
nodule, at least until 6 weeks after inoculation. Both mRNA species
were observed in nodules up to 11 weeks postinoculation. The relatively
low level of hybridization to the intron-A probe in RNA blots and the
absence of intron-A hybridization in in situ experiments was attributed to its relatively short length (96 bp of intron A-specific sequence) and its AT-rich nature. Nucleotide sequence data of a 3-kb cDNA clone
would reveal the additional sequences accounting for the larger
transcript, but our attempts to obtain such a clone using random
amplification of cDNA 3 ends were unsuccessful. We do not know whether
the 3000-nucleotide Dgrca transcript represents a novel form
of alternatively spliced mRNA or whether it is translated. If it is
translated, it must give rise to a protein that is not detected by the
Rubisco activase antiserum used in our experiments.
The 3000-nucleotide RNA species was recovered from an
oligo(dT)-cellulose column, indicating that at least a portion of this size population was polyadenylated. The ratio of the 3000-nucleotide species to the 1700-nucleotide species decreased from 5:1 in total nodule RNA preparations to about 1:1 in the nodule
poly(A+) fraction, suggesting either that a
significant portion of the 3000-nucleotide species did not have a
poly(A+) tail and was labile, or that the
3000-nucleotide poly(A+) RNA was unstable during
fractionation. Accumulation of unspliced or incompletely spliced
heteronuclear RNA has been postulated to result from changes in the
organization of the nucleus or from changes in splicing efficiency
associated with promoter structure (Cramer et al., 1997), with
heteronuclear RNA structure (for review, see Simpson and Filipowicz,
1996), or with the availability or action of components of the
spliceosome complex (e.g. the SR proteins; Cáceres et al.,
1997). Because of the complexity of the splicing apparatus and
the splicing process, we have not pursued splicing as the biological
basis for the presence of the 3000-nucleotide RNA.
Retention of transcripts within the nucleus has been reported for
specific genes in a variety of organisms. Early expression of a male
germ line-specific gene, Mst40, in spermatocytes of third
larval instars of Drosophila melanogaster is associated with
the nucleus; Mst40 mRNA was detected later in the cytoplasm (Russell and Kaiser, 1994). The D. melanogaster Hsr-omega
gene, located in a heat-shock puff, undergoes alternative
transcriptional termination in response to heat shock and other
stimuli, generating a longer, nuclear-localized form of the transcript,
which is polyadenylated and in which the introns are retained (Hogan et
al., 1994). The authors suggest that the nuclear transcript may
interact with proteins or have a regulatory role; for Dgrca,
however, this remains to be determined. The O2
(Opaque-2)
transcript, encoding a bZIP transcriptional regulator of zein storage
protein synthesis in maize endosperm, is normally found in both the
nucleus and cytoplasm (Dolfini et al., 1992). In one mutant an
intron-containing O2 transcript accumulates in the nucleus.
The efficiency of splicing of the waxy transcript in rice
endosperm appears to vary naturally among cultivars, some of which
accumulate a large pre-mRNA containing an intron (Wang et al., 1995).
As expected, the titers of granule-bound starch synthase (Waxy protein)
correlate directly with the level of mature waxy mRNA and
inversely with that of the pre-mRNA. Genetic data indicate that
splicing of the waxy intron is governed by cis-acting elements. It is curious that all of the
intron-containing transcripts described above occur in organs or cells
that are undergoing endoreduplication and, hence, share a common
feature with the mature infected cells of D. glomerata
nodules. Our findings suggest that the D. glomerata
symbiotic nodule provides the signals or trans-acting
factors necessary for transcription of Dgrca. However, other
molecular components that are needed for efficient processing of the
Dgrca transcript or for its transport from the nucleus do
not appear to be functional or present in appropriate amounts.
The mature, 1700-nucleotide Dgrca mRNA is expected to be
translatable. Our western-blot data indicate that Rubisco activase protein is present in leaves of D. glomerata but not in
nodules or roots. The absence of detectable protein in nodules could
result from a block in the export of mRNA to the cytoplasm, from a
block in translation of the mRNA, and/or from rapid protein turnover. Low amounts of protein in tissues showing high levels of the
corresponding transcript have been reported for a number of genes,
including fbp1, a MADS-box transcription factor in stamens
of wild-type petunia (Angenent et al., 1992); Sh1, which
encodes an isoform of Suc synthase in maize embryos (Chourey and
Taliercio, 1994); and an mRNA for S-adenosyl Met synthetase
in poplar (Mijnsbrugge et al., 1996). In the case of an mRNA encoding
the small subunit of ADP-Glc pyrophosphorylase, relatively low levels
of the small subunit of ADP-Glc pyrophosphorylase in potato leaves are
attributed to its instability in the absence of the large subunit
(Nakata and Okita, 1996). Arrest of translation can also occur in
response to environmental conditions, as observed for a mitochondrial
mRNA-encoding adenine nucleotide translocator in young maize roots
after O2 deprivation (Fennoy and
Bailey-Serres, 1995).
In some species rbcL mRNA accumulates in etioplasts before
greening (Tobin and Silvethorne, 1985; Berry et al., 1990); thus its
presence in plastids of nonphotosynthetic organs is not without precedent. The localization of Dgrca transcripts coincided
with that of the rbcL mRNA, but the former were most
abundant in the nuclei of cells of the early infection zone, whereas
the latter was abundant in the cytoplasm of mature, vesicle-containing
cells, typically those expressing nitrogenase, leghemoglobin, Gln
synthetase, and other proteins involved in N2 and
C assimilation and O2 partitioning. Dgrca expression was also localized at low levels to the
cytoplasm of uninfected cortical cells adjacent to the periderm and to
cells surrounding the vascular cylinder. Differential expression
patterns for Dgrca and DgrbcL can be expected,
because these genes are transcribed in different subcellular
compartments and are independently regulated in other developmental
systems (Reski, 1994). The observed expression patterns may therefore
reflect differences in the induction of nuclear- and plastid-coded
genes or in plastid number and distribution within the nodule.
Nodule expression of Dgrca deviated from the organ-specific,
light-regulated expression for rca genes observed in leaves
of spinach (Orozco and Ogren, 1993) and in Arabidopsis (Liu et al., 1996). Dgrca nodule expression also deviated from the
coordinate expression of other photosynthetic genes during
photomorphogenesis and diurnal fluxes in photosynthetic
capacity. White light treatment had no significant effect on the
overall abundance of Dgrca mRNA or on the relative abundance
of the two size classes of Dgrca transcripts in symbiotic
root nodules. Circadian regulation of rca genes such as that
observed in tomato (Martino-Catt and Ort, 1992) and Arabidopsis (Liu et
al., 1996) has not yet been examined in Dgrca nodules.
Because neither Rubisco activase nor Rubisco proteins accumulated to
detectable levels in D. glomerata nodules, a role for these
proteins in photosynthetic C reduction, oxygenase activity, or
N2 accumulation in the nodule is unlikely. In
stem nodules of Sesbania rostrata, photosynthetically
competent chloroplasts were observed in cortical cells adjacent to
N2-fixing infected cells, suggesting that they
may have a role in C assimilation (James et al., 1996). In D. glomerata nodules, however, the absence of OEC33, a protein
associated with the O2-evolving complex of the
photosynthetic apparatus, suggests that Dgrca expression is not accompanied by the assembly of a functional photosynthetic apparatus or by additional chloroplast development.
The nodule greening and autofluorescence attributed to chlorophyll that
we have observed in some mature D. glomerata nodules (P.A.
Okubara and A.M. Berry, unpublished data) occurs much later after
Frankia inoculation than does Dgrca mRNA
expression. Although the relative abundance of 3000-nucleotide mRNA was
not altered significantly by white light treatment, this treatment was
sufficient to cause faint greening in the mature nodules closest to the
light source (data not shown). We therefore postulate that the greening represents a distinct phenomenon not necessarily related to the induction of Dgrca transcription. The greening of organs
derived from roots seems anomalous, yet development of functional
chloroplasts in the pith cells of young poplar twigs (van Cleve et al.,
1993) has been reported. Such cases of chloroplast differentiation may reflect the recruitment of stem-like traits during the development or
evolution of certain organs, including the D. glomerata
nodule. Greening has also been observed in some multinucleated giant
cells derived from tomato roots after they have been infected with root knot nematodes from the genus Meloidogyne (V. Williamson,
personal communication). The differentiation of certain root knots
appears to be accompanied by chlorophyll accumulation, presumably
through the ectopic expression of photosynthesis-associated
mRNAs. The molecular or cellular basis for the unusual accumulation of
Dgrca transcripts in nodules may well be revealed as
progress is made in understanding the extensive cellular activity,
strong induction of N2 metabolic pathways, and
other phenomena that characterize nodule development.
| |
FOOTNOTES |
1
This work was supported by the California
Agricultural Experiment Station (project no. 6258 to A.M.B.) and by a
Katherine Esau Fellowship from the University of California, Davis, to
K.P.
2
Present address: U.S. Department of Agriculture,
Agricultural Research Station, Western Regional Research Center, 800 Buchanan Street, Albany, CA 94710.
3
Present address: Albrecht-von-Haller-Institut
für Pflanzenwissenschaften, Abteilung Biochemie der Pflanze,
Untere Karlspüle 2, 37073 Göttingen, Germany.
*
Corresponding author; e-mail amberry{at}ucdavis.edu; fax
1-530-752-1819.
Received November 3, 1998;
accepted March 19, 1999.
| |
ABBREVIATIONS |
Abbreviation:
OEC33, oxygen-evolving complex 33-kD subunit.
| |
ACKNOWLEDGMENTS |
The authors thank David Neale (U.S. Department of Agriculture
Forest Service, Pacific Southwest, PWS, Davis, CA) for advice and use
of facilities in construction of the first D. glomerata cDNA
library; David Gilchrist, Chris Mau, and other members of the Center
for Engineering Plants for Resistance Against Pathogens, Davis, CA, for
use of the facilities for construction of the second cDNA library, for
autoradiography, and for sequence alignments; Rich Jorgensen
(University of California, Davis) for the use of his PCR
facilities; Dean Lavelle (University of California, Davis) for
diligent and expert sequencing of the Dgrca full-length cDNA and the Dgrca gene segment; Tony van Kampen (Wageningen
Agricultural University) for sequencing of Dgrca clones;
Michael E. Salvucci for the gift of Rubisco activase antibodies and for
insightful discussions; Steve Theg for the gift of OEC33 antibodies;
and Susan Swensen and Beth Mullin (University of Tennessee, Knoxville) for pDgRcbL.
| |
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Top
Abstract
Introduction