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Plant Physiol. (1999) 120: 827-832
Expansins Are Conserved in Conifers and Expressed in Hypocotyls
in Response to Exogenous Auxin1
Keith W. Hutchison*,
Patricia B. Singer,
Stephanie McInnis,
Carmen Diaz-Sala2, and
Michael S. Greenwood
Department of Biochemistry, Microbiology, and Molecular Biology
(K.W.H., P.B.S., S.M., C.D.-S.) and Department of Forest Ecosystem
Science (M.S.G.), The University of Maine, Orono, Maine 04469
 |
ABSTRACT |
Differential
display reverse transcription-polymerase chain reaction was used to
detect the induction of gene expression during adventitious root
formation in loblolly pine (Pinus taeda) after treatment
with the exogenous auxin indole-3-butyric acid. A BLAST search of the
GenBank database using one of the clones obtained revealed very strong
similarity to the  -expansin gene family in angiosperms. A
near-full-length loblolly pine -expansin sequence was obtained using
5 - and 3-rapid amplification of cDNA end cloning, and the deduced
amino acid sequence was highly conserved relative to those of
angiosperm expansins. Northern analysis indicates that -expansin
mRNA expression increases 50- to 100-fold in the base of hypocotyl stem
cuttings from loblolly pine seedlings in response to indole-3-butyric
acid, with peak expression occurring 24 to 48 h after induction.
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INTRODUCTION |
Conifers show a decline in the ability to form adventitious roots
that is associated with maturation, an age-related developmental process that also affects reproductive competence, morphology, and
growth rate (Poethig, 1990 ; Greenwood and Hutchison, 1993). The rate
and the extent of the loss of rooting ability are species dependent.
For example, in eastern larch we showed that the frequency of cuttings
that root declines from 100% to 50% during the course of 20 years
(Greenwood et al., 1989). In contrast, loss of rooting ability occurs
abruptly and early in loblolly pine (Pinus taeda). Greenwood
and Weir (1995) showed that 20-d-old hypocotyl cuttings from loblolly
pine root readily within 3 weeks in the presence of exogenous auxin.
However, woody cuttings from 1- to 2-year-old plants root poorly even
after 2 to 3 months. In the latter case, exogenous auxin has little
effect. We have extended the observations of Greenwood and Weir (1995)
and shown that, whereas hypocotyl cuttings made from 20- or 50-d-old
seedlings rapidly form adventitious roots, epicotyl cuttings from
50-d-old seedlings root poorly, if at all, and only after 2 to 3 months (Diaz-Sala et al., 1996). This rapid decline in rooting
ability in loblolly pine provides us the opportunity to develop a woody
plant model system for the study of adventitious rooting, specifically,
and maturation, generally.
Rooting of loblolly pine hypocotyl cuttings is dependent on the
addition of exogenous auxin and is inhibited by the auxin polar
transport inhibitor NPA. However, NPA must be applied during the first
2 d of root induction. By d 3 the cuttings are fully committed to
root formation and are insensitive to NPA inhibition. Despite the clear
role for auxin in adventitious rooting, auxin uptake and metabolism do
not account for the differences in the ability of hypocotyls and
epicotyls to form adventitious roots. Uptake and polar transport
of exogenous auxin was comparable in rooting-competent hypocotyls and
rooting-incompetent epicotyls. Furthermore, there was no difference in
auxin tissue distribution and metabolism (Diaz-Sala et al.,
1996).
During the first 2 d of the rooting process the cambium layer of
the hypocotyl dedifferentiates into parenchyma cells in both hypocotyls
and epicotyls. Although some mitotic figures can be detected within 2 to 4 d, localized rapid cell division is not seen until d 6 and
then only in hypocotyl cuttings. Cells competent to form roots are
confined to the vascular parenchyma region of the hypocotyl,
immediately centrifugal to the resin canals. These observations suggest
that the decline in rooting ability is a result of a loss of cells
capable of fully responding to the induction of adventitious root
formation by auxin. It is not known whether this is due to a loss of a
specific cell type, the inability of individual cells to perceive auxin
signals that are specific for root meristem organization, or the
suppression of gene expression needed for cells to enter the
root-formation pathway.
To extend our knowledge of the induction of adventitious roots in
loblolly pine, we sought genes whose expression in hypocotyl cuttings
responded in the first 24 to 48 h to the application of exogenous
auxin. Using differential display RT-PCR (Liang and Pardee, 1992) we
have identified a gene family whose mRNA levels increase in hypocotyl
cuttings in response to the application of the auxin IBA. This gene
family is the loblolly pine homolog of the -expansins, which have
previously been found in monocots and dicots (McQueen-Mason et al.,
1992; Shcherban et al., 1995). Recently, expansins were thoroughly
reviewed (Cosgrove, 1996, 1997, 1998). They are thought to be
responsible for acid-induced loosening of cellulose-hemicellulose
networks (McQueen-Mason et al., 1992; Rayle and Cleland, 1992;
Cosgrove and Li, 1993; McQueen-Mason, 1995). Their expression in
angiosperms has been reported in rapidly growing areas of the plant
(McQueen-Mason et al., 1992; Cosgrove and Li, 1993; Cho and Kende,
1997) and in ripening fruit (Rose et al., 1997). We report here that in
loblolly pine the -expansin-related sequences are induced in
nongrowing regions of the stem prior to the resumption of cell division
leading to adventitious roots.
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MATERIALS AND METHODS |
Plant Material
Growth of seedlings and induction of rooting in hypocotyl or
epicotyl stem segments were previously described (Diaz-Sala et al.,
1996). Hypocotyl cuttings were made from 20-d-old seedlings by severing
the hypocotyl above its base, 3.5 cm from the cotyledons. Epicotyl
cuttings were made from 50-d-old seedlings by severing the seedling
above the cotyledons, 3.5 cm from the apex. All of the needles except
for the apical tuft were removed. Cuttings were place in distilled
water either with or without 10 µM IBA. IBA- plus
NPA-treated samples were placed in distilled water with IBA (10 µM) and NPA (10 µM).
RNA Extraction
The bottom 0.5 cm was removed from the cuttings and quick
frozen in liquid N2. The stem segments from
cuttings from 30 seedlings were pooled for each time point and
treatment. Total RNA was extracted as previously described (Hutchison
et al., 1990).
Differential Display RT-PCR
Differential display RT-PCR was performed using a modification of
the method of Liang and Pardee (1992), as described by Rhodes and Van
Beneden (1996). All primers were obtained from Operon Technologies
(Alameda, CA). Total RNA extracted from the stem segments was
pretreated with DNase I (10 units/25 µg RNA) in 100 mM
Tris-HCl (pH 8.3), 500 mM KCl, and 15 mM
MgCl2 for 30 min at 37°C. After the segments
were treated with DNase I the RNA was extracted once with DEPC-treated
water-saturated phenol:chloroform (3:1) and once with chloroform. The
RNA was then precipitated by adding 0.1 volume of 3 M
sodium acetate (pH 5.2) and 3 volumes of 100% ethanol. Samples were
centrifuged in a microfuge (Brinkmann) for 45 min at 4°C. After
washing with 80% ethanol the pellet was redissolved in DEPC-treated
water.
Total RNA (0.2 µg) plus the T11GC primer (1.5 µM) was heated to 80°C for 5 min in DEPC-treated water
and cooled to 37°C for 5 min. The heated and reannealed RNA-primer
mixtures were reverse transcribed in a 20-µL reaction containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 1 mM DTT, 500 µM each dNTP, 5 units of RNasin (Promega), and 300 units
of Superscript reverse transcriptase (GIBCO-BRL) for 60 min at 37°C.
Reactions were stopped by heating at 80°C for 10 min.
The cDNA was amplified in a 20-µL PCR containing 1× PCR buffer
(Perkin-Elmer), 1.5 mM MgCl2, 5 µM each dNTP, 80 µM
-35S-dATP (5 µCi, Amersham), 0.5 µM each of primers OPD9 (5-CTCTGGAGAC-3) and OPD10
(5-GGTCTACACC-3), and 0.3 unit of AmpliTaq DNA polymerase (Perkin-Elmer). Cycling conditions were: initial denaturation at 94°C
for 5 min, then 94°C for 90 s; 38°C for 40 s; and 72°C for 60 s for 40 cycles. After a final extension at 72°C for 5 min, the reactions were cooled to 4°C.
The PCR reactions were electrophoresed through a 6% denaturing
acrylamide gel, and the bands were detected by autoradiography. PCR
fragments were extracted from the gel by soaking the gel slice in
sterile distilled water at 4°C overnight and by boiling for 15 min.
The DNA was ethanol precipitated, redissolved in sterile distilled
water, and reamplified using the above conditions. The reamplified PCR
fragments were cloned into a TA-cloning vector prepared from
SmaI digest pUC19 DNA, as described by Marchuk et al.
(1991).
Full-Length cDNA Clones
A near-full-length cDNA clone was constructed in a two-step
procedure. First, 5-RACE clones were made essentially as described by
Harvey and Darlison (1991). First-strand cDNA was reverse transcribed from total RNA using the anchor primer T11GC. A poly(A) tail was then
added to the 3 end of the first cDNA using terminal transferase (New
England Biolabs). The DNA was amplified with the Promega primer adapter
(5-GTCGACTCTAGATTTTTTTTTTTTTTT-3; Xba-PA) and primer based on the
DD21.4-1 sequence (5-GCATTTCATAGCAGG-3, Exp3) and cloned as described
above. 3-RACE clones were made essentially as described by Schaefer
(1995). First-strand cDNA was reverse transcribed from total RNA as
described above, except the Xba-PA primer was used. PCR amplification
was carried out with Xba-PA for the 3 primer and a primer designed
from the 5-RACE clone sequence (5-CCGGATCCTTCAGATCTCCCTGATTCTC-3;
Ptexp75RI) for the 5 primer and Xba-PA for the 3 primer. The
amplified fragment was ligated into the EcoRI and
XbaI sites of pBluescript (Stratagene).
DNA Sequencing and Analysis
DNA was sequenced by the University of Maine DNA Sequencing
Facility (Orono) using the ABI-Prism Dye Terminator Cycle Sequencing Ready Reaction kit with Amplitaq DNA Polymerase, FS (Perkin-Elmer). The
reaction products were resolved and the sequence was determined using an ABI 373A DNA Stretch Sequencer. DNA sequences were
analyzed using the Wisconsin Package software (version 8, Genetics
Computer Group, Madison, WI). PCR primers were designed using Primer3
(Rozen and Skaletsky, 1997). The location of the signal peptide
cleavage site was determined with SignalP (Nielsen et al., 1997).
Northern Blots
Northern blots were performed as previously described (Hutchison
et al., 1990). Expansin mRNA was detected using the insert from clone
DD21.4-1 as a probe. The actin probe was from clone pAc20.20, a
loblolly pine partial cDNA clone obtained by 3-RACE (accession no.
AF085331; C. Diaz-Sala and K.W. Hutchison, unpublished data). 18S rRNA
levels were detected using a larch 18S rRNA probe, as previously
described (Hutchison et al., 1990). Radioactive DNA probes were made
from PCR-amplified inserts of the respective clones according to the
method of Feinberg and Vogelstein (1983). Autoradiograms were
exposed for 5 to 9 d using a DuPont Lightning Plus intensifying
screen. RNA levels were quantitated by scanning the autoradiograms with
a densitometer (LKB, Bromma, Sweden) or by using ImagePC software
(Scion, Frederick, MD).
| |
RESULTS AND DISCUSSION |
Expansin-Related Sequences Are Detected in Loblolly Pine Seedlings
undergoing Adventitious Root Formation
To search for genes involved in adventitious rooting we used
differential display RT-PCR to detect transcripts expressed after 24 h in auxin-treated hypocotyl stem segments but not found in untreated hypocotyl segments. Two clones (pDD21.3.3 and pDD21.4.1) were
obtained from a gel-purified differential display-RT-PCR band that
conformed to these criteria. These clones were sequenced and were 231 bp, exclusive of the RAPD primer-binding site. The sequence for clone
pDD21.4.1 was deposited in the GenBank database (accession no. U64889).
A BLAST search (Altschul et al., 1990) of the database showed a high
level of similarity to the -expansin gene family of monocots and
dicots (Shcherban et al., 1995; and below).
To obtain a full-length cDNA sequence a 5-RACE was first constructed.
The sequence of this clone can be found in the GenBank/EMBL/DDBJ databases (accession no. U64895). A unique primer was designed that
annealed to the 5 end of the loblolly pine expansin cDNA and used in a
3-RACE reaction. This resulted in a PCR fragment of approximately 1 kb, which was subsequently cloned and sequenced. The complete sequence
and translation of clone pCPEx7-8 is given in Figure
1 and has been deposited in
the GenBank database (accession no. AF085330). The putative protein is
translated from the 5-most ATG. The total length of the sequence minus
the poly(A) tail is 999 nucleotides.
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| Figure 1.
The DNA and deduced amino acid sequence for a
loblolly pine expansin. The expansin cDNA sequence was submitted to the
GenBank/EMBL DNA databases (accession no. AF085330). The putative
translation initiation codon is underlined. The translation termination
codon is indicated by an asterisk. Nucleotide positions are given by
numbers to the left of the DNA sequence. Amino acid positions are
indicated by numbers to the right of the amino acid sequence. The
sequence includes the 5 primer used for 3-RACE cloning but does not
include additional 5 residues found in a 5-RACE clone (accession no.
U64895) from which the 5 PCR primer was derived.
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Based on a phylogenetic analysis of angiosperm expansin sequences,
Shcherban et al. (1995) proposed that the expansins represented an
ancient gene lineage and would be found in other land plants. Our
results confirm this hypothesis. The predicted amino acid sequence of
the loblolly pine -expansin protein is highly conserved relative to
those from the angiosperms Arabidopsis, cucumber, and rice (Fig.
2). There is little similarity in the
first 27 residues of the peptide sequence. This segment constitutes
the signal peptide identified using SignalP (Nielsen et al.,
1997), and the predicted N terminus of the mature peptide at Y28 is one amino acid downstream of that found for a mature -expansin protein (CuExS1) from cucumber (Shcherban et al., 1995). The mature peptide shows an average sequence identity of 80% to the rice, cucumber, and
Arabidopsis -expansins. The evolutionary relationship of the
loblolly pine expansin to that of other plants was shown by Cho and
Kende (1997), who used a partial cDNA sequence that we previously
submitted to the GenBank database (accession no. U64892). Although we
have not yet purified the protein, the strong sequence conservation
with known -expansins suggests that the loblolly pine expansins will
be functionally equivalent. Preliminary genetic data suggest that there
may be three family members that are expressed in hypocotyls in
response to auxin treatment (S. McInnis, Z. Wang, and K.W.
Hutchison, unpublished data).
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| Figure 2.
Alignment of loblolly pine amino acid sequence for
expansin with angiosperm expansin sequences. Deduced amino acid
sequences for expansins from loblolly pine (accession no. AF085330),
Arabidopsis (accession no. U30481), cucumber (accession no. U30382),
and rice (accession no. U85246) were aligned using the Pileup
program from the Wisconsin Package DNA sequence. Output was
produced using the program Boxshade
(www.isrec.isb-sib.ch/software/BOX_form.html). Amino acids printed
in inverse represent positions where at least 50% of the residues are
identical. Amino acids that are similar to the consensus are shaded.
The location of the putative N terminus of the mature peptide from
loblolly pine as predicted by SignalP (Nielsen et al., 1997) is
indicated by an arrow ( ).
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Expression of Expansin Sequences during Adventitious Rooting
Expansin mRNA levels increased during the early stages of
induction of adventitious root formation in response to the application of exogenous auxin (Fig. 3A). RNA was
extracted at different times from hypocotyl and epicotyl stem segments
in the presence and absence of IBA. The stem segments were fully
elongated before they were exposed to IBA. Northern blots were probed
using clone pDD21.4.1. Peak expression was achieved in 24 h, after
which there was a slow decline in expansin mRNA levels. Nevertheless,
after 5 d there was still a significant level of expansin mRNA in
hypocotyl segments undergoing rooting (data not shown). In a second
experiment, RNA was extracted from samples at 0 and 24 h in the
presence and absence of IBA. In this experiment, with more RNA loaded
onto the gel, a low level of expansin expression could be detected at 0 and 24 h in the absence of auxin (Fig. 3B). However, densitometric measurement of the autoradiogram indicated that exogenous auxin increases expansin mRNA levels by 50- to 100-fold. The low level of
expansin expression may represent constitutive expression of the
sequences or induction by the endogenous pool of auxin, which accumulates at the base of the cutting.
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| Figure 3.
Northern blot of hypocotyl RNA from loblolly pine
seedling cuttings. A, RNA was extracted from the base of hypocotyl
cuttings and placed in either distilled water ( ) or distilled water
plus 10 mM IBA (+) for the times indicated. One microgram
of total RNA was electrophoresed in a 1% agarose gel, northern
blotted, and hybridized with the clone pDD21.4.1. After exposure to
x-ray film, the filter was erased and rehybridized with an 18S rRNA
probe from eastern larch and with a loblolly pine partial cDNA clone
for actin (accession no. AF085331), as described by Hutchison et al.
(1990). B, RNA extracted from hypocotyl cuttings at the times indicated
in an independent rooting experiment. Four micrograms of RNA was used
per sample, and the filter was hybridized with the larch 18S rRNA probe
and the expansin probe.
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The large increase in expansin mRNA levels in response to exogenous
auxin is not a result of a generalized increase in transcription. Probing the same blot with a loblolly pine actin probe showed an
overall increase in actin mRNA levels during the course of the
experiment and a slight increase in response to auxin addition (Fig.
3A). We also showed that expression of neither Phe ammonia lyase nor a
cyclin-dependent kinase is auxin induced in the bases of hypocotyl and
epicotyl cuttings and that actin expression may be inhibited by auxin
in epicotyls (Greenwood et al., 1997). The importance of the small
increase in actin mRNA levels in response to auxin is therefore
difficult to assess at this time.
Regulation of expansin expression appears to be complex, although some
of the complexity may be due to different regulatory schemes for
different members of the expansin gene family. Rose et al. (1987)
reported the up-regulation of one tomato expansin by ethylene during
fruit ripening. In rice expansin expression in the internode region is
increased by GA (Cho and Kende, 1997). In this case, three different
family members showed a response to GA addition, but the kinetics of
the response was unique for each sequence. Downes and Crowell (1998)
reported that a  -expansin from cotton responds to the addition of
cytokinins. Our observation that in pine expansins increase in response
to exogenous auxin during adventitious root formation adds yet another
hormone and developmental circumstance to the list of controls of the
expansin gene family. An increase of expansin expression in response to auxin in cucumber hypocotyls has also been reported (Shieh et al.,
1997).
Fleming et al. (1997) showed that topical application of expansin to
tomato apical meristems could induce localized tissue expansion and
morphogenesis of leaf primordia. These data suggest a role for expansin
in organogenesis. It is possible that expansin is playing a similar
role in adventitious root formation. This is most likely to occur
during the earliest events in adventitious rooting in the
rooting-competent cells within the stem. Expansin expression increases
during and is maximal within the first 2 d of rooting induction
and coincides with the observed dedifferentiation of cambial cells. An
additional correlation is that the auxin-dependent 100-fold increase in
expansin levels occurs during the first 2 d of the rooting
process, a time when the rooting process is also sensitive to NPA
inhibition (Diaz-Sala et al., 1996).
Cosgrove (1996) proposed that there are a variety of cell- and
organ-specific expansins that can respond to a variety of unique environmental and hormonal signals. We do not know whether the patterns
of expansin expression that we observe are the result of a coordinated
expression of all members of a gene family or only some members.
Experiments are in progress to test these alternatives. Work is also in
progress to look at the cellular distribution of expansin expression in
the cuttings and at the level of expansin protein. These data
demonstrate that expansin is found in gymnosperms, as well as the
angiosperms, is highly conserved, and mRNA levels respond to auxin.
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FOOTNOTES |
1
This work was supported by a grant from the
Maine Agriculture and Forestry Experiment Station (K.W.H.) and the
North Carolina State Loblolly and Slash Pine Rooted Cutting Project
(M.S.G.). This is Maine Agriculture and Forestry Experiment Station
publication no. 2321.
2
Present address: Department of Biologica
Vegetal, Universidad Alcala de Henares, 28871 Alcala de Henares,
Madrid, Spain.
*
Corresponding author; e-mail keithh{at}maine.edu; fax
1-207-581-2801.
Received January 13, 1999;
accepted March 30, 1999.
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ABBREVIATIONS |
Abbreviations:
DEPC, diethyl pyrocarbonate.
IBA, indole-3-butyric acid.
NPA, N-(1-naphthyl)phthalamic
acid.
RACE, rapid amplification of cDNA ends.
RT-PCR, reverse
transcription-PCR.
| |
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Top
Abstract
Introduction