(Received for publication, July 26, 1995; and in revised form, October 23, 1995)
From the
A cDNA corresponding to an mRNA which accumulates in germinating
rice seeds in response to the phytohormone abscisic acid was isolated
by differential hybridization. Northern blotting indicated that the
mRNA also accumulates in vegetative tissues in response to treatment
with abscisic acid and to osmotic stress. Sequencing identified a major
open reading frame encoding a novel protein of 27.4 kDa. The identity
of the open reading frame was confirmed by comparing the translation
products of cellular, hybrid-selected, and in vitro transcribed RNAs and by immunoprecipitation. Western blotting of
cellular extracts indicated that the protein is associated with
microsomal or membrane fractions. Data base searches indicated that it
contains a conserved Ca-binding, EF-hand motif and
that related proteins are similarly expressed in Arabidopsis
thaliana. A fusion protein purified from Escherichia coli containing the putative EF-hand region was shown to bind
Ca
in blot binding assays. These data identify a
novel gene family encoding proteins involved in the response of plants
to abscisic acid and osmotic stress.
Plants growing in many environments must respond and adapt to
osmotic stress caused by drought, salinity, and temperature extremes.
Stress tolerance involves changes in gene expression and solute
metabolism(1, 2, 3, 4) . The
complexity of these responses indicates that they may be mediated by
multiple signaling pathways, one of which is modulated by the
phytohormone abscisic acid (ABA)()(5, 6) .
Although varied, plant responses to ABA indicate that the hormone acts
as an osmoregulatory signal which inhibits normal growth, and
potentiates stress tolerance or adaptation (reviewed in (7) and (8) ). For example, ABA levels rise
developmentally during late embryogenesis and enhance dormancy and
desiccation tolerance(9, 10) . Similarly, osmotic
stress in vegetative tissues leads to increased ABA levels which
regulate stomatal closure, thereby reducing transpiration, and which
promote the expression of genes involved in stress
tolerance(11, 12) .
Molecular studies of ABA action
focus on the mechanism(s) by which the hormone regulates specific gene
expression and on the functions of these ABA-responsive genes. Two
specific components in the ABA signaling pathway(s) are known. One is a
transactivator which regulates ABA-responsive genes in seed
tissues(13, 14) , perhaps in concert with basic
leucine zipper proteins binding to a G-box
element(15, 16) . The other encodes a
Ca-modulated, protein phosphatase 2C which may
integrate ABA and Ca
signals with
phosphorylation-dependent responses in seed and vegetative
tissues(17, 18) . The involvement of this phosphatase
supports evidence indicating that Ca
acts as a
messenger in ABA mediated processes(19, 20) .
The
contribution of downstream, ABA-responsive genes to an integrated set
of physiological responses to ABA or osmotic stress remains obscure, in
part because many of these genes encode proteins of unknown function.
Some of them may function as molecular chaperones during
dehydration(21) , in osmolyte metabolism(22) , or in
regulatory processes such as nuclear protein trafficking(23) .
We describe here another family of ABA-responsive genes encoding novel
proteins with an EF-hand, Ca-binding
domain(24) . The properties and patterns of regulation of these
proteins appear to be similar among distantly related species,
indicating that they are involved in the response of plants to ABA and
osmotic stress.
The EF-hand region was
synthesized by PCR using Vent polymerase with SalI
linker-primers: (5`-TATTATGTCGACGTGTACCATCCTGAAGGAACC, nucleotides
234-257) (5`-TATTATGTCGACTTACGAAACAATCACGTTGAAGCCCAA, nucleotides
369-392). The resulting fragment, encoding residues
Ser-Leu
was cloned in frame at the C
terminus of the E. coli maltose-binding protein (MBP) (36) in the SalI site of the pMal-c2 expression vector
(New England Biolabs), and the orientation was confirmed by sequencing.
Transformants obtained in strain TB1 were grown in LB medium containing
0.2% glucose and 100 µg/ml ampicillin at 37 °C to an A
of 0.5. The culture was induced with 0.3
mM IPTG and incubated for 2.5 h. The fusion protein was
purified by amylose-affinity chromatography according to the
manufacturer's instructions (New England Biolabs).
pMal-2c-encoded MBP and a fusion between MBP and the hippocalcin
EF-hand 1 (37) were similarly produced as negative and positive
Ca
-binding controls, respectively.
Ca
-binding blots were performed after
standard protocols (38) with
Ca
from DuPont NEN.
The open reading frame of RAB16 (25) was synthesized by PCR and cloned into the expression
vector pET-11a (Novagen). The recombinant RAB16 protein was expressed
in E. coli, purified by ion exchange chromatography, ()and used as a control protein on Western blots. Protein
concentrations were determined from A
values and
from values obtained in bicinchoninic acid assays (Pierce).
Microsomal preparations from control and ABA-treated
seedling leaves were obtained by modification of the method of Halkier
and Møller (39) . The hormone was applied by spray
wetting four times with 100 µM ABA over a 48-h period.
Freshly harvested leaves were homogenized in 5 (w/v) microsome
extract buffer (250 mM sucrose, 100 mM Tricine (pH
7.9), 50 mM NaCl, 2 mM EDTA, 2 mM dithiothreitol, 5 mg/ml polyvinylpolypyrrolidone, 5 µg/ml
antipain and leupeptin) in a kitchen blender. The homogenate was
filtered through 600- and 100-µm nylon screens and centrifuged 20
min at 10,000
g. The supernatant was collected and
centrifuged for 60 min at 264,000
g. The pellet was
resuspended in 0.5 ml of microsome buffer, and aliquots were subjected
to SDS-PAGE. Separated proteins were transferred to polyvinylidene
difluoride membranes and blocked with 1% (w/v) nonfat dry milk.
Anti-IgG horseradish peroxidase conjugate was used as the secondary
antibody. Protein concentrations were determined as described above.
When necessary, the protein samples were precipitated with 80% acetone
and resuspended to remove impurities before determination of protein
concentrations.
Figure 1: Sequence of the cDNA and encoded protein. The start (nucleotide 84) and stop (nucleotide 816) codons and a putative polyadenylation signal(1017) are underlined.
The longest ORF encodes a protein with a
molecular mass of 27.4 kDa. We call this protein EFA27 (EF-hand,
abscisic acid responsive, see below). Two lines of evidence indicate
that it is not secreted. First, the N-terminal sequence does not
possess the characteristics of a secretory peptide(40) .
Second, translation of in vitro transcribed EFA27 RNA with
reticulocyte lysate and dog pancreatic membranes did not produce
detectable product cleavage, although E. coli -lactamase
used as a positive control was completely processed (data not shown).
Figure 2: Northern blot analysis of EFA27 mRNA levels in rice seed and vegetative tissues. 20 µg of total RNA/lane. A, steady-state mRNA levels in developing embryo and endosperm quarter-seeds harvested 10 days after flowering (DAF) (lanes 1 and 4), 20 DAF (lanes 2 and 5), 30 DAF (lanes 3 and 6). mRNA levels in shoots of hydroponically grown plants (lane 7) transferred to media containing 200 mM NaCl for 6 h (lane 8) or 24 h (lane 9) or desiccated at room temperature for 6 h (lane 10) or 24 h (lane 11). B, mRNA levels in shoots of hydroponic plants before (lane 1) or 6 h (lane 2), 24 h (lane 3), and 48 h (lane 4) after spray application of 50 µM ABA. mRNA levels in roots of hydroponic plants (lane 5) or 6 h (lane 6), 24 h (lane 7), and 48 h (lane 8) after the addition of 50 µM ABA to the medium.
Two lines of evidence indicate that EFA27 mRNA also accumulated in response to the phytohormone ABA in embryo and/or seedling tissues. First, the cDNA was isolated by differential screening for ABA-induced mRNAs from a library constructed with poly(A) RNA from ABA-treated, germinating embryos(25) . Second, in vitro translation experiments indicated that EFA27 mRNA accumulated in germinating embryos following ABA treatment (Fig. 3, see below). To determine whether the expression of the mRNA could also be induced by ABA in vegetative tissues, the hormone was added to the hydroponic medium at a final concentration of 50 µM. Fig. 2B (lanes 1-8) indicates that the mRNA accumulated some 20-30-fold in both roots and shoots after 6 h of treatment. EFA27 mRNA accumulated in shoots for up to 48 h, although its accumulation in roots was transient and was not detectable after 24 h (Fig. 2B, lanes 4-7).
Figure 3: SDS-PAGE of translation products of germinating seed poly(A) RNA and of RNA transcribed in vitro from the cDNA. Products of poly(A) RNA from embryo quarter-seeds incubated for 20 h in the absence (CON, lane 1) or presence (ABA, lane 2) of 25 µM ABA. Translation products of poly(A) RNA selected by hybridization to the cDNA (HS, lane 3). Translation products of RNA in vitro transcribed from the full-length cDNA (FL, lane 4). The arrow in lane 3 indicates the polypeptide with the same electrophoretic mobility as the major product in lane 4.
In conclusion, the Northern blot data indicated that EFA27 accumulates in tissues in response to osmotic stress and ABA. Its pattern of expression is therefore similar to that of RAB16, a member of another family of ABA-responsive genes(25, 41) .
Figure 4: SDS-PAGE of translation products of poly(A) RNA from germinating seeds immunoprecipitated with antisera against EFA27 and RAB16 fusion proteins. Products of poly(A) RNA from embryo quarter-seeds incubated for 20 h in the absence (CON, lane 1) or presence (ABA, lane 2) of 25 µM ABA. The same products immunoprecipitated with antibody against EFA27 (CON, lane 3; ABA, lane 4), or antibodies against RAB16 used as a positive control (CON, lane 5; ABA, lane 6). Molecular mass markers are indicated at left in kilodaltons.
The BLAST algorithm (27) identified several partial cDNAs from Arabidopsis
thaliana encoding proteins with similarity to EFA27. While we have
yet to confirm these cDNA sequences, it is unlikely that the correction
of potential inaccuracies in them would significantly decrease
similarities between the proteins they encode and EFA27. An alignment
between them and EFA27 is shown in Fig. 5A. The
N-terminal region of EFA27 is most similar to Z27053, while its
C-terminal region is most similar to Z29900. Z27053 and Z29900 may
therefore represent 5` and 3` regions of a single mRNA. The comparison
also shows that the signature sequence of the tyrosine phosphorylation
site is conserved in Z17677. Of the three signatures for casein kinase
2 phosphorylation on serine, one (Ser) is conserved in
Z29900 while the two threonine sites (Thr
and
Thr
) are not overlapped by the Arabidopsis cDNAs.
Figure 5: Sequence alignment of EFA27 and open reading frames translated from data base sequences. A, the amino acid sequence of EFA27 compared with those encoded by the longest open reading frames of four Arabidopsis partial cDNAs in the EMBL data base of expressed sequence tags: EMBL account no. Z27053 (ATTS1925), Z17677 (ATTS0251), Z47401 (ATTS4423), and Z29900 (ATTS2344). Residues identical or conserved between EFA27 and one other sequence are capitalized, and the consensus is given for residues showing identity with EFA27. EFA27 and Z29900 stop codons are noted with an asterisk. B) comparison of the EF-hand motif in EFA27 with related proteins. EMBL Z27503 and Z17677 are as noted in A; PIR S22503 is Arabidopsis calmodulin 3 (EF-hand 1); EF-hand is the conserved EF-hand motif (24) where n is hydrophobic, * is any residue, and x, y, and z contain oxygen in their side chains.
The data bases were then searched for sequences similar
to regions of EFA27 with the greatest similarity to these Arabidopsis cDNAs. The region
Ser-Tyr
was thereby found to contain
the EF-hand consensus sequence, a conserved
Ca
-binding motif(24, 28) . Fig. 5B shows an alignment of EFA27 with the Arabidopsis sequences, EF-hand 1 of Arabidopsis calmodulin 3(43) , and the EF-hand consensus(24) .
A secondary structure algorithm (29) predicted the conserved
EF-hand, helix-turn/loop-helix structure for all of these sequences.
The ability of the EFA27 EF-hand domain to bind Ca
was tested in a protein blot assay.
To this end, EFA27 residues 51-103 were fused in frame with the E. coli MBP, and the ability of the fusion protein to bind
Ca
was examined following affinity
purification from E. coli extracts, SDS-PAGE, and protein
blotting(38) . Fig. 6A shows the Coomassie-stained
pattern following SDS-PAGE of 0.5 and 5 µg of MBP (lanes 1 and 2, negative control), MBP/EFA27 EF-hand (lanes 3 and 4), and of MBP/hippocalcin EF-hand 1 (lanes 5 and 6, positive control)(37) . Fig. 6B shows that, as expected,
Ca
was not
bound by MBP, but was bound by both of the EF-hand fusion proteins.
This suggests that the EF-hand sequence of EFA27 and presumably those
encoded by the Arabidopsis cDNAs bind Ca
under physiological conditions.
Figure 6:
SDS-PAGE of maltose-binding fusion
proteins and demonstration of Ca
-binding
by the EFA27 EF-hand region. A, Coomassie Blue staining of E. coli-produced maltose-binding protein (0.5 µg, lane
1; 5 µg, lane 2), of a fusion between MBP and the
EFA27 EF-hand region, MBP/EFA27 (0.5 µg, lane 3;
5 µg lane 4), and of a fusion between MBP and EF-hand of
hippocalcin, MBP/HIP-EF1, used as a positive control (0.5
µg, lane 5; 5 µg lane 6; 37). Molecular mass
markers are indicated at the left in kilodaltons. B,
Ca
-binding blot assay of the same
proteins.
The apparent insolubility of
the heterologous GST/EFA27 fusion protein in E. coli could be
due to the accumulation of improperly folded or aggregated recombinant
protein. Alternatively, hydrophobic regions of EFA27 could make the
fusion protein insoluble. The sequence of EFA27 was therefore analyzed
with algorithms which predict hydropathy and secondary structure. Two
secondary structure algorithms predicted that residues
Leu-Tyr
may form an
-helical,
trans-membrane domain(29, 30) . To examine the
possibility that EFA27 is associated with membranes, antibodies against
GST/EFA27 were used to probe cell extracts and fractions prepared from
ABA-treated quarter-seeds and seedling leaves (Fig. 7A). These experiments detected EFA27 among
total, SDS-solubilized proteins from ABA-treated quarter-seeds (Fig. 7A, lane 2). It was not detected in a
supernatant protein fraction solubilized with an aqueous buffer, but
was detected among the insoluble proteins pelleted by centrifugation (Fig. 7A, lanes 3 and 4). In extracts
of ABA-treated seedling leaves, EFA27 was detected in the homogenate (lane 5) and in a fraction derived from it containing
microsomal membranes (lane 7), but not in the soluble fraction (lane 6). As a control, the same fractions were probed with an
antibody against RAB16, a soluble, ABA-responsive protein(25) .
As expected, RAB16 was detected in total and soluble fractions of
quarter-seeds and leaves (Fig. 7B, lane 3 and 6), but not in insoluble or microsomal membrane fractions (Fig. 7B, lanes 4 and 7). These
experiments suggest that EFA27 is not a soluble, cytosolic protein and
that it may associate in vivo with membranes or with
membrane-associated proteins.
Figure 7: Western blot of ABA-treated quarter-seeds and seedlings. A, blot incubated with anti-EFA27 antibody. Control, polyacrylamide powder (200 µg) containing the GST/EFA27 fusion protein from E. coli (C, lane 1), total protein (70 µg) from quarter-seeds (T, lane 2), soluble and insoluble, pelleted proteins (70 µg) from quarter-seeds (S, lane 3; P, lane 4), total microsome homogenate (70 µg) from seedlings (T, lane 5), soluble proteins and microsomal proteins from seedlings (70 µg) (S, lane 6; M lane 7). Molecular mass markers are indicated at the left in kilodaltons. B, blot incubated with anti-RAB16 antibody(25) . Positive control, recombinant RAB16 (0.5 µg) (C, lane 1), the same fractions as in A. (lanes 2-7).
We are studying the molecular mechanism(s) of action of the plant hormone ABA. Part of this work entails the characterization of ABA-responsive genes with the aim of understanding the functions of the proteins they encode. Here we present the characterization of a rice cDNA isolated by differential screening of a library derived from ABA-treated, germinating embryo quarter-seeds. Northern blotting indicated that it corresponds to an mRNA which accumulates during late embryogenesis, and which accumulates in germinating seed and vegetative tissues in response to ABA treatment. The mRNA also accumulates in vegetative tissues in response to salt stress or desiccation. This pattern of expression is similar to that of other previously characterized ABA-responsive genes(25) .
The cDNA encodes a
novel protein of 27 kDa which does not exhibit homology to previously
described ABA or osmotic stress-responsive plant proteins. However, its
sequence is similar to several polypeptides encoded by A.
thaliana-expressed sequence tags (partial-length cDNAs) isolated
from a library derived from dry seed mRNAs. These similarities include
a region containing an EF-hand, a Ca-binding motif.
We have therefore initially called the protein EFA27 (EF-hand,
ABA-responsive, 27 kDa). To our knowledge, it is the first example of
an EF-hand protein whose expression is responsive to ABA and osmotic
stress. Hybridization selection and in vitro translation
experiments suggested that several other proteins related to EFA27 show
similar patterns of expression. These findings indicate that EFA27 is a
member of a small gene family whose members are expressed in response
to ABA and osmotic stress in phylogenetically distant species.
The
EFA27 EF-hand region was shown to bind Ca
using binding blots. Like other EF-hands, the EFA27 motif is
predicted to form a helix-loop-helix structure, and the most highly
conserved residues of the EF-hand motif are also present in the EFA27
EF-hand. Invariant Asp and Glu residues forming part of a
Ca
coordination site are present in positions 10 and
21. In addition, oxygen containing side chains in positions 12, 14, and
18 are also often involved in Ca
coordination either
directly or via a water molecule. Other invariant residues are also
conserved in the EFA27 motif including Gly at position 15, which
permits a sharp bend in the Ca
-binding loop and a
hydrophobic residue (Val) in position 17, which attaches the
Ca
-binding loop to the hydrophobic core of the
structure. Most EF-hands also have a conserved pattern of hydrophobic
residues in the two helices at positions 2, 5, 6, 9, 22, 25, 26, and
29. These are involved in structural stabilization and contribute to
packing EF-hands against each other in proteins in which they are
repeated. The EFA27 EF-hand has such hydrophobic residues in some, but
not all, of these positions. This incomplete pattern may be allowed
because EFA27 contains only a single EF-hand. The pattern of
hydrophobicity is also less conserved in the EF-hand of another
protein, G14 from Arabidopsis. This protein also has a single
EF-1 hand and has been shown to bind
Ca
in vitro(44) .
EF-hand proteins have been
characterized from many organisms and function in regulating a variety
of cellular activities. Ca may function as a
cytosolic second messenger by modulating the activity of EF-hand
proteins in the cytosol or in membranes facing the cytosol. In plant
cells, cytosolic Ca
levels change in response to ABA
treatment (45) , and an EF-hand protein phosphatase was
recently shown to be part of an ABA-signaling
pathway(17, 18) . These findings clearly implicate
Ca
as a second messenger in ABA-dependent signaling.
The presence of an EF-hand in EFA27 suggests that its activity or
function may be modulated by changes in Ca
levels
effected by upstream signaling events. EFA27 activity or function may
also be modulated by phosphorylation at tyrosine kinase and/or casein
kinase 2 sites in the C-terminal region. For example, phosphorylation
has been shown to affect the Ca
-binding properties of
proteins, including lipocortins (46) and possibly the Arabidopsis 14-3-3 protein homologue GF14(44) .
Furthermore, phosphorylation at casein kinase 2 signature sites has
been shown to affect an activity of another ABA responsive protein,
RAB16(23) . This suggests that phosphorylation by this class of
kinase may be involved in the post-translational modification of
ABA-responsive proteins.
EFA27 was detected by Western blotting in
SDS-soluble and in membrane-enriched cellular fractions. The EFA27
sequence was examined for the presence of membrane spanning regions.
Two algorithms(20, 29) predicted that residues
Leu-96-Tyr-116 may form a hydrophobic, -helical membrane
spanning region. This region contains no charged side chains and has a
high content of Leu, Phe, and Val, residues which make up about 50% of
the residues of transmembrane protein regions (47) . Membrane
spanning regions were also predicted by the algorithm in the
corresponding regions of the Arabidopsis homologues.
Application of the positive inside rule (48) indicates that the
regions of EFA27 and one of the Arabidopsis polypeptides
(Z17677) C-terminal to these putative membrane anchors would be
cytosolic. This suggests that the EFA27 N-terminal and EF-hand are
extracellular or intraorganellar while all of the polypeptides
potential phosphorylation sites are cytosolic. While speculative, such
a structure would be consistent with a role for EFA27 in some form of
Ca
modulated membrane or transmembrane signaling. We
are conducting further immunological and biochemical studies to
determine the cell type and subcellular localization of EFA27 in
plants.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X89891[GenBank].