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First published online August 25, 2006
doi: 10.1242/10.1242/dev.02535
1 Division of Bioscience, Graduate School of Natural Science and Technology,
Okayama University, Okayama 700-8530, Japan.
2 Division of Biological Sciences, Graduate School of Science, Hokkaido
University, N10, W8, Sapporo 060-0810, Japan.
Author for correspondence (e-mail:
perfect{at}cc.okayama-u.ac.jp)
Accepted 17 July 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Arabidopsis thaliana, Polyamine, Spermine, Stem elongation, Upstream ORF
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Heby and Persson, 1990). These
low-molecular-weight compounds are positively charged at physiological pH and
bind to negatively charged molecules, e.g. nucleic acids, acidic phospholipids
and various types of proteins (Cohen,
1998). The biosynthesis of putrescine occurs from the processing
of ornithine and/or arginine, and is regulated by the enzymes ornithine
decarboxylase (ODC) and arginine decarboxylase (ADC), respectively. Subsequent
reactions convert putrescine into spermidine and spermine, and these are
catalyzed by spermidine and spermine synthases, which add propylamino groups
from decarboxylated S-adenosylmethionine, catalyzed by
S-adenosylmethionine decarboxylase (SAMDC)
(Pegg, 1988).
Studies of higher plants have shown that polyamines play important roles in
a wide range of developmental processes, such as embryogenesis, floral
development, fruit ripening, senescence, and response to environmental
stresses (Evans and Malmberg,
1989; Galston and
Kaur-Sawhney, 1990; Bouchereau
et al., 1999). The acaulis 5 (acl5) mutant of
Arabidopsis thaliana is defective in a spermine synthase and exhibits
a severe dwarf phenotype, suggesting that spermine is a novel regulator of
stem elongation (Hanzawa et al.,
2000). Spermine has also been identified as a signal mediator of
the defense responses in tobacco
(Takahashi et al., 2003;
Takahashi et al., 2004).
Moreover, transgenic potatoes that express the antisense SAMDC gene
under the control of the CaMV 35S promoter with a duplicated enhancer region
exhibit stunted growth with highly branched stems and short internodes
(Kumar et al., 1996).
Overexpression of the oat ADC gene in transgenic tobacco plants
results in short internodes, thin stems and leaves, leaf necrosis and short
roots (Masgrau et al., 1997).
However, the molecular mechanisms by which polyamines control plant growth
remain unknown.
The Arabidopsis genome has two genes encoding spermidine synthase,
SPDS1 and SPDS2, and two genes encoding spermine synthase,
ACL5 and SPMS (Hanzawa
et al., 2002; Panicot et al.,
2002) (see Fig. S1 in the supplementary material). The spds1
spds2 double loss-of-function mutant shows an embryo lethal phenotype
(Imai et al., 2004b). The
spms mutant shows no aberrant phenotype, whereas acl5 spms
double mutants contain no endogenous spermine but display a dwarf phenotype
that is identical to the acl5 single mutant
(Imai et al., 2004a). The
ACL5 gene is upregulated by auxin, whereas the SPMS gene is
responsive to abscisic acid (Hanzawa et
al., 2002). Moreover, SPMS interacts with SPDS1 and SPDS2 to form
`metabolon' complexes, whereas ACL5 does not interact with either of these
proteins (Panicot et al.,
2002). Measurement of the polyamine levels in acl5 and
spms mutants has also revealed that SPMS is a principal
contributor to spermine biosynthesis in vivo
(Imai et al., 2004a). These
findings thus suggest that ACL5 is specifically required for stem
elongation.
To further elucidate the role of ACL5 during stem elongation, we identified extragenic suppressors of the acl5 mutant and designated these as suppressor of acaulis (sac) mutants. We show that the sac51-d mutation disrupts a short upstream open reading frame (uORF) of the SAC51 gene, which encodes a basic helix-loop-helix (bHLH) transcription factor. Our results suggest that ACL5 is involved in the translational control of the SAC51 gene.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), dim (Takahashi
et al., 1995), spms-1
(Imai et al., 2004a) and
transgenic lines from the Columbia (Col-0) ecotype. The axr2-1 and
gai-1 (Koornneef et al.,
1985) mutants were obtained from the Arabidopsis Biological
Resource Center. acl5-1 of the Col-0 ecotype was generated by more
than seven crosses of the original acl5-1 strain of the Ler
ecotype (Hanzawa et al., 1997)
to wild-type Col-0.
Plants were grown under continuous fluorescent light at 22°C on
rock-wool bricks supplemented with vermiculite, or on 0.8% (w/v) agar plates
containing MS salts (pH 5.8) and 3% sucrose, after surface sterilization of
seeds. Heat-shock treatments of plants were performed as described previously
(Matsuhara et al., 2000).
Mutagenesis and screens for sac mutants
Mutagenesis of the acl5-1 seeds with ethylmethane sulfonate (EMS)
was performed as described previously
(Takahashi et al., 1992).
Briefly, approximately 20,000 acl5-1 seeds were surface-sterilized,
hydrated, treated with 0.2% EMS (Sigma, St Louis, MO, USA) for 14 hours and
washed extensively. The M1 plants were then divided into 10 pools and
self-pollinated. Approximately 2000 M2 seeds from each pool were grown for
suppressor screening. Putative suppressors showing recovery from the
acl5-1 dwarf phenotype were used for further analysis after being
backcrossed three times to acl5-1 of either the Ler or Col-0
ecotypes.
Mapping and cloning
Each of the sac acl5 mutants of the Ler ecotype was
crossed to acl5-1 of the Col-0 ecotype. Genomic DNA was extracted
from F2 plants showing the acl5 phenotype and analyzed for
co-segregation with respect to cleaved amplified polymorphic sequence (CAPS),
simple sequence length polymorphism (SSLP) and single nucleotide polymorphism
(SNP) markers (Konieczny and Ausubel,
1993; Bell and Ecker,
1994). These markers were derived from the Arabidopsis Information
Resource (TAIR;
http://www.arabidopsis.org).
The CAPS markers MDC12Dra, MHJ24Dde, MSJ1Eco and MSJ1Hha were developed for
mapping the SAC51 locus by using the primer sequences shown in
Table 1. DNA sequences were
determined from PCR products using a 377 DNA sequencer (Applied Biosystems,
Foster City, CA).
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) of the
ACL5 locus. The dCAPS primers used for genotyping the ACL5
locus were ACLdF and ACLdR, which produce a single PCR fragment from
acl5-1 and two cleaved PCR fragments from ACL5, after
XhoI digestion and resolution on an 8% acrylamide gel. All of the
selected F2 plants (dim/dim acl5-1/+) were then subjected to growth
assays and were further crossed to acl5-1 to determine the
SAC genotype. F2 plants that segregated with no progeny of the
acl5 phenotype in the F3 generation of this testcross were
consequently identified as homozygous sac mutants and their growth
data were calculated. The genotypes of the SPMS locus in the sac
acl5 spms mutants were confirmed by PCR with SPMS-specific
primers (Imai et al., 2004a).
For primer sequences, see Table
2.
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-TIP, ACL, and
SPMS, were performed as described previously
(Hanzawa et al., 1997;
Hanzawa et al., 2002). A
gene-specific probe for SAC51 was prepared by PCR from the wild-type
genomic DNA using 51F1 and 51R1 primers.
Reverse transcription reactions were done with 1 µg of DNase-treated
total RNA and 2.5 pmol oligo(dT) primer in 20-µl reactions using an RNA LA
PCR Kit (Takara, Kyoto, Japan), according to the manufacturer's protocol.
Quantitative PCR was performed in a DNA Engine Opticon2 System (Bio-Rad,
Hercules, CA) using gene-specific primers. Triplicate PCR reactions were
averaged. The primers 51F2 and GUSR were used for detecting expression of
SAC51-GUS and sac51-d-GUS chimeric transcripts. Relative
transcript levels in all samples were normalized using ACTIN8
(An et al., 1996). For primer
sequences, see Table 2.
Plasmid construction and plant transformation
For recapitulation of the sac51-d phenotype, a 4.1-kb genomic
fragment encompassing from 990 bp upstream of the SAC51 transcription
start site to 723 bp downstream of the SAC51 stop codon was amplified
from the sac51-d allele with 51F3 and 51R2 primers, digested with
BglII, and cloned into the BamHI site of pBI101 (Clontech,
Palo Alto, CA), resulting in pSAC51R. For generating the SAC51
5'-leader deletion construct, the 990-bp SAC51 promoter
fragment was amplified with primers 51F4 and 51R3, digested with ClaI
and XbaI, and inserted into the ClaI/XbaI-digested
pBI101, resulting in pSAC51pro. The SAC51 coding sequence was
amplified from genomic DNA using 51F5 and 51R1 primers, subcloned into pGEM-T
Easy (Promega, Madison, WI), and further transferred as a
SpeI-digested fragment to the XbaI-digested pSAC51pro,
resulting in pSAC51
5'. For heat shock-inducible SAC51
expression, the same SpeI-digested SAC51 fragment was
transferred to the heat-shock cassette Ti-vector pTT101
(Matsuhara et al., 2000),
resulting in pHS-SAC515'. For heat shock-inducible SPMS
expression, the SPMS cDNA was amplified with SPMSF and SPMSR primers,
subcloned into pGEM-T Easy, and further transferred as a SacI
fragment to pTT101, resulting in pHS-SPMS. These Ti constructs were used to
transform acl5-1 in the Col-0 background. The acl5-1 mutant
carrying the HS-ACL5 construct was previously described
(Hanzawa et al., 2000).
For GUS expression analysis, the 990-bp SAC51 promoter fragment and the 5'-leader region of either the wild-type SAC51 or sac51-d transcripts were amplified by PCR with 51F3 and 51R4 primers, digested with BglII, and cloned into the BamHI site of pBI101. The construct that contains a point mutation in the SAC51 uORF (sac51-C549A-GUS) was generated by a two-step mutagenesis protocol. PCR amplification was first performed using 51F3/mut-C549AR and mut-C549AF/51R4 primer pairs. The PCR products were subjected to a second round of amplification with 51F3 and 51R4, and cloned into pGEM-T Easy. After checking the sequence, the BglII-digested fragment was cloned into pBI101 as described above.
Transformation of Arabidopsis was carried out using the floral dip
method (Bechtold and Pelletier,
1998) with the Agrobacterium strain C58C1. Transformants
were selected in MS agar plates containing 50 µg ml-1 kanamycin.
Independent transgenic lines that segregated 3:1 for the kanamycin-resistance
marker in the T2 generation were further selected to isolate progeny that were
homozygous for the transgene.
Microscopy
Inflorescence stems of 6-week-old plants were fixed overnight in 50%
ethanol, 5% formaldehyde and 5% acetic acid. The samples were then dehydrated
through an ethanol series and embedded in Technovit 7100 resin (Heraeus
Kulzer, Wehrheim, Germany). Sections (8 µm) were stained with 0.1%
Toluidine blue for 15 seconds.
GUS assays
Histochemical and fluorometric GUS assays were performed as described
previously (Jefferson et al.,
1987). For histochemical analysis, samples were prefixed in 90%
acetone at room temperature for 20 minutes. Protein content was determined
using the Bradford assay (Bio-Rad) to compare activity to protein units, and
each experiment was repeated three times.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). We
found that the restoration of these organ sizes is paralleled by that of the
plant height in sac51-d acl5-1 and sac52-d acl5-1 double
mutants (Fig. 2A,
Table 3). The sac53-d
allele is more effective in restoring the stem length (
61% recovery),
than in restoring the stem diameter (31% recovery), rendering sac53-d
acl5-1 plants more slender than wild-type plants. Interestingly, in
sac54-d acl5-1 plants, the pedicel length is fully restored but the
stem length is only partially restored (39% recovery). Microscopic
observations of stem longitudinal sections revealed that the recovery of plant
height is attributable to cell lengths in all four classes of sac
acl5 mutants (Fig. 2B,
Table 3). Our genetic
segregation data revealed that all sac single mutants show no obvious
phenotype in the presence of the wild-type ACL5 gene (data not
shown).
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). This phenotype is suppressed in sac acl5 mutants,
in accordance with the recovery of the plant height
(Fig. 2C). The abnormalities in
xylem development and lignin accumulation in acl5-1 are reminiscent
of those observed in transgenic Arabidopsis plants that
constitutively express ATHB8, a gene encoding a class III
homeodomain-leucine zipper (HD-Zip III) transcription factor
(Baima et al., 2001). The
HD-Zip III genes have been implicated in the regulation of vascular
differentiation (Baima et al.,
1995; Baima et al.,
2001; Zhong et al.,
1999; Ohashi-Ito and Fukuda,
2003), leaf polarity
(McConnell et al., 2001) and
meristem initiation (Otsuga et al.,
2001; Green et al.,
2005). We prepared RNA samples from 12-day-old seedlings of wild
type, acl5, and four classes of sac acl5 plants, and
examined the transcript levels of all five HD-Zip III genes
(Prigge et al., 2005) by
quantitative real-time RT-PCR. Our results revealed that the expression of
each of these genes is upregulated in acl5-1 but is restored to
normal levels in sac acl5 mutants, in parallel with the degree of
recovery of plant height during the adult stage of growth
(Fig. 2D).
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-TIP, which encodes a vacuolar aquaporin,
tonoplast intrinsic protein (Hanzawa et
al., 1997). To examine the effect of each sac allele on
the expression of these genes, northern blot analysis was performed with total
RNA isolated from flower buds with apical inflorescence meristems of
6-week-old plants. The levels of both the EXGT-A1 and
-TIP transcripts were restored in sac acl5 mutants
(Fig. 3). We also examined
acl5-1 transcript levels in each sac acl5 mutant. In
contrast to EXGT-A1 and -TIP, the acl5-1
transcript level is upregulated in acl5-1, probably as a result of a
negative-feedback control of ACL5 expression
(Hanzawa et al., 2000). The
acl5-1 transcript levels were restored in accordance with the degree
of recovery of the plant height in sac acl5 plants
(Fig. 3). The recovery in
acl5-1 transcript levels was also detected at the seedling stage,
prior to the manifestation of morphological phenotypes (data not shown).
SPMS transcript levels were not affected in any of the sac
acl5 mutants.
sac mutants do not suppress hormone-related dwarf phenotypes
To determine whether sac mutations are general suppressors of
hormone-related dwarf phenotypes or not, the sac mutants were crossed
to an auxin-resistant mutant, axr2, a GA-insensitive mutant,
gai, and a BR-requiring mutant, dim. Because axr2-1
and gai-1 alleles are dominant, we crossed their respective mutants
with sac acl5 and measured the plant height in the F1 generation.
sac dim double mutants were identified as homozygotes for both
alleles. Our results revealed that none of the sac alleles suppresses
the dwarf phenotypes of axr2, gai and dim mutants
(Fig. 4A).
acl5-1 spms-1 plants have no detectable levels of endogenous
spermine but are morphologically indistinguishable from acl5-1
(Imai et al., 2004a). To
examine the effect of sac alleles on stem elongation in a background
of complete spermine depletion, we generated sac acl5 spms triple
mutants. All four of the sac mutants suppressed the acl5
phenotype to a similar degree in either the presence or absence of
SPMS (Fig. 4B).
SAC51 encodes a bHLH protein
We chose the sac51-d allele for further analysis. Fine mapping
experiments placed the SAC51 locus within a 60-kb region of the P1
clone MSJ1 (Fig. 5A). All of
the genes in this region were amplified by PCR from homozygous
sac51-d plants and their sequences were compared with those of the
wild-type Ler. We detected a single C- to-T mutation in the
At5g64340 gene (Fig.
5B). A genomic fragment encompassing from 990 bp upstream of the
transcription start site to 723 bp downstream of the stop codon of
At5g64340 was cloned from sac51-d plants and introduced into
acl5-1 in the Col-0 background by Agrobacterium-mediated
transformation. The resulting transgenic plants displayed the wild-type
phenotype in four independent lines (see Fig. S2 in the supplementary
material), confirming that At5g64340 is indeed the SAC51
gene.
The full-length SAC51 cDNA in the GenBank database is 2,472 bp in
length and is separated by three introns. The longest ORF encodes a protein of
348-amino acids with an estimated molecular mass of 37.8 kDa. The SAC51
protein contains a basic helix-loop-helix (bHLH) domain in its C-terminal half
(Fig. 5C) and has been
designated as AtbHLH142 in the compilation of Toledo-Ortiz et al.
(Toledo-Ortiz et al., 2003).
SAC51 shows a high sequence similarity over the entire length of the protein
(57.3% identity) to AtbHLH143 (At5g09460). The bHLH sequence homologies
between SAC51, At5g09460 and three known bHLH proteins in Arabidopsis
are shown in Fig. 5D. Most
known plant bHLH proteins also exhibit homology outside of the bHLH domain,
such as in the MYB-interacting domain of the R proteins
(Goff et al., 1992;
Abe et al., 1997) or the PAS
domain of PIF3 (Kay, 1997).
However, such domains are not conserved in SAC51.
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Northern analysis revealed that SAC51 shows high expression in stems, roots and flowers, but little or no expression in siliques (Fig. 6A). The sac51-d transcript level was markedly higher in sac51-d acl5-1 and sac51-d seedlings than was the SAC51 transcript level in either wild-type or acl5-1 seedlings (Fig. 6B; data not shown). Other sac mutations did not affect the SAC51 transcript level (Fig. 6B).
The SAC51 5'-leader deletion restores the acl5 phenotype
The mutation found in the SAC51 uORF suggested that its effects
were manifested through the altered translation of the main ORF encoding the
bHLH protein. To examine whether deletion of the SAC51
5'-leader can also serve as a gain-of-function allele and restore the
acl5 phenotype, we made the SAC515'
construct, which contains the SAC51 coding sequence fused with its
own promoter, but lacks the 5'-leader, and introduced it into
acl5-1. We confirmed in all six independent transgenic lines that,
although not completely, the construct restored the acl5 dwarf
phenotype. The SAC515' construct had no effect
on the growth of wild-type plants. Moreover, overexpression of SAC51
under the control of a heat-shock gene promoter in acl5-1 also
partially restored the phenotype in response to heat-shock treatments of the
plants (see Fig. S2 in the supplementary material).
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To examine whether disrupting the peptide sequence of the SAC51 uORF is crucial for suppression of the acl5 phenotype, we generated another GUS construct carrying a C-to-A substitution at the site of the sac51-d mutation (sac51-C549A-GUS). This causes a Gln-to-Lys substitution of the fourth amino acid of the fourth uORF (Fig. 7H). This construct was introduced into wild-type plants and was found to have no obvious effect on transcription and translation of the GUS reporter gene, when compared with the wild-type SAC51 construct (Fig. 7G). The results were reproduced in three independent transformants for each construct.
ACL5 may be involved in the translational activation of SAC51
To address possible regulatory interactions between ACL5 and
SAC51, we introduced both SAC51-GUS and sac51-d-GUS
constructs into acl5-1 and sac51-d acl5-1 mutants by
crossing experiments, and examined the GUS expression in these
mutants. The GUS activity driven by the SAC51-GUS construct in
acl5-1 and sac51-d acl5-1 seedlings was about 40% of the
levels in the wild-type background, whereas the steady-state levels of the
GUS transcript were unaffected in these mutant seedlings. Thus, the
GUS translational efficiency in acl5-1 and sac51-d acl5-1
was estimated to be 38.8% and 48.2%, respectively, of the levels in the
wild-type background (Fig. 7G).
The steady-state levels of the GUS transcript from the
sac51-d-GUS construct were markedly increased in acl5-1 and
sac51-d acl5-1, as in the wild-type background. The GUS activities in
these mutants were consequently increased but reached to about 60% of the
activities in the wild-type background. These results suggest that
ACL5 is required for full activation of SAC51
translation.
The fact that ACL5 encodes spermine synthase suggests a role of
spermine in the translational activation of SAC51. A previous study
reported that the acl5 phenotype is restored by heat-shock treatments
of acl5-1 plants carrying the HS-ACL5 construct
(Hanzawa et al., 2000). Thus,
we finally tested whether or not overexpression of another spermine synthase
gene, SPMS, can rescue the acl5 phenotype with the heat
shock-inducible HS-SPMS construct. However, the results showed no
effect of heat shocks on the growth of acl5-1 plants carrying the
HS-SPMS construct, suggesting that ACL5 and SPMS
take on different roles in the same cell (see Fig. S2B in the supplementary
material).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), the HS-SPMS construct had no effect on the
growth of acl5 plants carrying the construct (see Fig. S2B in the
supplementary material). Determination of the subcellular localization of ACL5
and SPMS, and/or the identification of an ACL5-interacting protein, will be
needed for further understanding of the functional difference between these
two spermine synthases.
We observed that the transcript levels of the HD-Zip III class homeobox
genes were increased in acl5-1, even at vegetative stages, prior to
the visible manifestation of the acl5 phenotype
(Fig. 2D). These transcript
levels were restored in each sac acl5 mutant, and restoration
correlated with the degree of recovery in plant height, suggesting a close
association between HD-Zip III gene expression and reduced stem elongation.
The Arabidopsis HD-Zip III class genes are mainly expressed in the
procambium and developing vascular tissues
(Baima et al., 1995;
Zhong and Ye, 1999;
Ohashi-Ito and Fukuda, 2003;
Prigge et al., 2005).
Overexpression of a member of this class, ATHB8, promotes xylem
formation and results in a strong reduction in plant height
(Baima et al., 2001). This may
be a consequence of the accelerated differentiation of the primary
vasculature, which in turn provokes the anticipated transition to secondary
growth. Because acl5-1 stem internodes also have abnormal vascular
structures following the overproduction of xylem tissues, the defect in stem
elongation in acl5-1 might be attributable to the increased
transcript levels of the HD-Zip III class genes, resulting in abnormal
vascular differentiation.
A recent study of the thickvein (tkv) mutant, a new
allele of ACL5, suggested that a defect in polar auxin transport is
responsible for the tkv phenotype that is characterized by the
abnormal vasculature in the leaves and stem internodes, and that
ACL5/TKV functions in a mechanism that defines the boundaries between
veins and the non-vein regions through correct polar auxin transport in
provascular cells (Clay and Nelson,
2005). ACL5 expression is upregulated by auxin and by the
acl5-1 mutation itself (Hanzawa
et al., 2000). Moreover, the increased acl5-1 transcript
levels are restored in each of the sac mutants, in parallel with the
suppression of the dwarf phenotype (Fig.
3). Taken together, these findings suggest that the defect in the
polar auxin transport system in the acl5-1 provascular cells causes a
local increase in the auxin levels, which in turn results in the
overproliferation of provascular cells and in the increased expression of the
acl5-1 and HD-Zip III genes. If this is indeed the case, the
sac alleles identified would restore the polar auxin transport system
in accordance with the recovery of vascular differentiation.
Posttranscriptional control of SAC51 by its uORF
The SAC51 transcript has a long 5'-leader containing five
short ORFs upstream of its bHLH protein coding sequence
(Fig. 5B). The sac51-d
dominant allele was found to have a point mutation in one of these uORFs,
which introduces a premature stop codon. Although uORFs occur relatively
infrequently in eukaryotic mRNAs, their occurrence is more frequent in
growth-related genes such as oncogenes
(Kozak, 1987;
Kozak, 1991). uORFs are known
to impede the translational initiation of their downstream coding ORFs, and
this translational repression is released under appropriate conditions
(Morris and Geballe, 2000).
For example, the yeast GCN4 gene contains four uORFs. These allow the
ribosome to reach the start codon of the main ORF only when cells are under
amino acid starvation and need to express the GCN4 protein
(Hinnebusch, 1990). Such
uORF-mediated translational regulation has been investigated in several plant
genes including Opaque 2 (Lohmer
et al., 1993) and Lc
(Wang and Wessler, 1998) in
maize, and SAMDC (Hanfrey et al.,
2002) and ATB2 (Wiese
et al., 2004) in Arabidopsis. Our experiments with
transgenic plants carrying GUS reporter fusion constructs revealed
that the sac51-d sequence increases both the transcript levels and
the efficiency of translation of the GUS reporter gene
(Fig. 7G). We also confirmed
that the stem growth of acl5-1 was partially restored by
transformation with the SAC51 5'-leader deletion construct
(SAC5') and the heat shock-inducible SAC51 construct
(HS-SAC515') (see Fig. S2 in the supplementary material). Thus,
we conclude that the sac51-d allele deregulates a posttranscriptional
control of the SAC51 gene and that the resulting overproduction of
the SAC51 bHLH protein is responsible for the suppression of the acl5
phenotype in sac51-d acl5-1. This is consistent with the dominant
characteristics of sac51-d. A mutation similar to sac51-d
has been also reported for a uORF of the Arabidopsis ATR1 gene, which
encodes a MYB transcription factor (Bender
and Fink, 1998). The dominant atr1D allele was identified
as an altered tryptophan regulation mutant, with increased expression of a
target of ATR1, ASA1, which encodes the anthranilate synthase 
subunit.
Why are the steady-state levels of both sac51-d and
sac51-d-GUS transcripts much higher than those of SAC51 and
SAC51-GUS transcripts in respective plant lines
(Fig. 6A,
Fig. 7G)? The most likely
possibility is that the sac51-d and sac51-d-GUS transcripts
are stabilized by increased translation of their main ORF. Some studies with
other plant systems have shown that the increased translation has the
potential to protect a transcript from degradation
(Sullivan and Green, 1993;
Abler and Green, 1996). It is
less likely that the sac51-d transcript is stabilized by the
alteration its own secondary structure with the C-to-T substitution, because
the C-to-A substitution at the site of the sac51-d mutation had no
effect on the GUS expression.
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).
The presence of such a conserved uORF suggests that all of these bHLH proteins
are under a common posttranscriptional control mechanism. However, we have so
far identified no peptide sequences homologous to those of the SAC51
uORFs in other plant species in the database. In Neurospora crassa,
the arg-2 gene encoding an arginine biosynthetic enzyme contains an
evolutionarily conserved uORF. This uORF encodes the 24-amino-acid arginine
attenuator peptide (AAP) that confers negative translational regulation in
response to increased arginine levels (Luo
and Sachs, 1996). At high arginine concentrations, AAP causes
ribosomes to stall at the uORF termination codon and create a blockade of
additional ribosome scanning. The peptide sequences encoded by uORFs are
crucial regulators of some genes, including the cytomegalovirus gpUL4
(Alderete et al., 1999) and the
mammalian SAMDC (Raney et al.,
2000), whereas those of the above mentioned GCN4 gene are
relatively unimportant for its function
(Hinnebusch, 1996;
Hinnebusch, 2000). A more
detailed mutagenic analysis of the SAC51 5'-leader sequence
will be needed to determine whether the function of the SAC51 uORF is
sequence-dependent or not.
A model for ACL5-dependent translational regulation of SAC51
One important question is whether ACL5 acts in the control of
SAC51 expression. A key finding of our experiments is that the
translational efficiency of the SAC51-GUS construct was decreased in
acl5-1 and sac51-d acl5-1
(Fig. 7G). This implies that
the translational efficiency of the SAC51 main ORF depends at least
in part on the ACL5 function. As polyamines are known to promote
various stages of protein biosynthesis
(Tabor and Tabor, 1984), it is
possible that spermine is directly involved in the translational control of
SAC51. In bacteria, spermidine facilitates the formation of the
translation initiation complex (Yoshida et
al., 1999). By contrast, in the mammalian SAMDC gene,
polyamines may directly participate in an interaction between its uORF-encoded
peptide and a constitutive component of the translation machinery, which leads
to the inhibition of ribosome activity
(Mize and Morris, 2001). We
found that the GUS translational efficiency of the sac51-d-GUS gene
in the wild-type background was decreased to about 60% in acl5-1 and
sac51-d acl5-1 (Fig.
7G), indicating that ACL5 enhances SAC51
translation independently of the regulation by its uORFs. Taken together, we
propose a model whereby ACL5 acts, either directly or indirectly, as
a translational activator of SAC51, and probably of its homologs
(Fig. 8). In this model, we
also hypothesize that the premature termination of a 53-amino-acid polypeptide
encoded by the fourth uORF of SAC51 facilitates the release of the
ribosome from the uORF and the reinitiation of translation at the main ORF in
sac51-d. The restoration of the acl5-1 transcript level in
sac51-d acl5-1 plants, suggests that the negative-feedback control of
ACL5 expression does not require spermine itself. This feedback
control might involve auxin homeostasis, as discussed above, or downstream
components of either SAC51 or its homologs. This model is supported
by the histochemical localization of SAC51-GUS expression in
transgenic plants (Fig. 7B-F),
as SAC51 shows high expression in the shoot apex and vascular tissues
of young leaves and roots, a pattern similar to the expression domain of
ACL5 (Clay and Nelson,
2005).
|
), SAC51 might be continuously accumulated and degraded below
detectable levels. In conclusion, our findings shed light on uORF-mediated posttranscriptional control in plant development for the first time. The identification of other SAC genes, and the downstream targets of SAC51, will further elucidate the exact roles of ACL5 and SAC51 during stem internode elongation.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/18/3575/DC1
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Present address: Department of Biological Sciences, Graduate School of
Science, The University of Tokyo, Tokyo 113-0033, Japan
|
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