Download Regulation of Phosphate Homeostasis by microRNA in Plants

Regulation of Phosphate Homeostasis by
microRNA in Plants
Tzyy-Jen Chiou1*, Kyaw Aung1,2, Shu-I Lin1,3, Chia-Chune Wu1,
Su-Fen Chiang1, and Chun-Lin Su1
Abstract
Upon phosphate (Pi) starvation, the microRNA miR399 is upregulated and its target gene, a ubiquitin-conjugating E2 enzyme, is downregulated in Arabidopsis.
Accumulation of the E2 transcript is suppressed in transgenic Arabidopsis overexpressing miR399. Transgenic plants accumulated 5 to 6 times the normal Pi level in
shoots and displayed Pi toxicity symptoms associated with increased Pi uptake and
translocation from roots to shoots and retention of Pi in shoots. As well, remobilization
of Pi from old to young leaves was impaired. These results provide the first evidence
that miRNA controls Pi homeostasis by regulating the expression of a component of
the proteolysis machinery in plants.
Phosphorous is one of the mineral nutrients essential for plant growth,
development and reproduction. Not only is it a major component of fundamental macromolecules, such as nucleic acids and phospholipids, but it also
plays an important role in energy transfer and regulation of enzyme reactions and metabolic pathways. Despite the importance of phosphorous in
agricultural production, most of the phosphorous in the soil is unavailable
for plant uptake because of adsorption, precipitation or conversion to organic forms (1). To overcome problems with phosphate (Pi) availability, plants
have evolved a series of adaptive responses to maintain Pi homeostasis. These responses include conservation and remobilization of
internal Pi and enhanced acquisition of external Pi (1, 2) (Figure 1),
which involve rapid and distinct changes in gene expression.
Although many Pi-responsive genes have been reported from largescale expression profiling, the molecular mechanisms involved in
regulating Pi homeostasis remain largely unknown (2). In this
report, we reveal a mechanism by which plants regulate Pi homeFigure 1. Adaptive plant responses under phosphate
deficient environment.
ostasis to adapt to environmental changes in Pi availability. This
mechanism involves the suppression of a ubiquitin-conjugating E2
Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan
Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, Academia
Sinica, Taipei, Taiwan
3
Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan
1
2
75
ACADEMIA SINICA
enzyme by a specific microRNA, miR399.
(7), which supports the cleavage of the E2 transcript
microRNAs (miRNAs) represent a class of
by recognition of miR399. To determine whether
noncoding small RNAs that generally function as
this decrease in E2 mRNA level during Pi starva-
posttranscriptional negative regulators through base
tion was a direct result of negative regulation by
pairing to nearly complementary sequences in the
miR399, our investigation revealed that the expres-
target mRNAs (Figure 2). Recent evidence supports
sion of miR399 in response to Pi starvation was
the notion that miRNAs play a direct role in regulat-
highly induced in Arabidopsis seedlings grown in
ing developmental and physiological processes in
Figure 2. MicroRNA-mediated gene regulation.
plants (3, 4, 5).
miR399 was predicted to target multiple sites
on the 5’ UTR of At2g33770 mRNA encoding a
ubiquitin-conjugating E2 enzyme (6) (Figure 3A).
5’ RACE experiments identified cleaved transcripts
of E2 corresponding to predicted target sequences
Figure 3. Gene Organization and Expression of At2g33770
Encoding a Ubiquitin-conjugating E2 Enzyme. (A) Exons and
5 putative miRNA399 target sites within the second exon are
shown as grey boxes and black bars, respectively. The translation initiation site and the ubiquitin-conjugating conserved
ACADEMIA SINICA
domain (UBC) are indicated. Triangle indicates the T-DNA
Figure 4. Pi Toxicity in miR399-overexpressing Transgenic
insertion site in the SAIL_47_E01 line. (B) and (C), RNA gel
Plants. (A) RNA Gel Blot Analyses of miR399 and E2 in Wild-
blot analyses of miR399 and E2 transcripts in seedlings
type (Wt) and miR399-overexpressing Plants (miR399b,
grown under high Pi (+Pi) or low Pi (-Pi) media. At4, a Pi star-
miR399c and miR399f). (B) Chlorosis and necrosis in the leaf
vation-induced gene was used as a positive control. 5S rRNA
margins of miR399-overexpressing plants. Bar = 1 cm. (C) Pi
and tRNA and 25S and 18S rRNA staining is shown as the
concentration in the roots and shoots of wild-type (Wt) or
loading control.
miR399-overexpressing plants under high Pi.
76
low Pi medium (10 μm KH2PO4) but was undetectable under high Pi conditions (1 mM KH2PO4)
(Figure 3B). The accumulation of miR399 was
accompanied by a decreased level of E2 mRNA in
Pi-starved roots (Figure 3C), which suggests that E2
mRNA was cleaved by the targeting of miR399.
Significantly, this cleavage is directly linked to the
Pi status of the plant. Here, we provide another
example of the participation of miRNA in response
to nutrient deficiency in addition to the induction of
miR395 by sulfate starvation (8).
In order to further understand the function of
miR399, we generated transgenic Arabidopsis
plants overexpressing miR399b, miR399c and
miR399f. In these plants, miR399 was highly
expressed even in the presence of high levels of Pi
under which the expression of miR399 is not normally detectable (Figure 4A). Moreover, the expression of miR399 in transgenic plants was much
stronger than that caused by Pi starvation in wildtype plants. A high level of miR399 accumulation
in transgenic plants indicates the successful expresFigure 5. Increased Pi Uptake in miR399-overexpressing
sion and processing of precursor RNA. In contrast,
Transgenic Plants. (A) Pi uptake activities of wild-type (Wt)
the E2 mRNA level decreased to barely detectable
and miR399-overexpressing plants (miR399b, miR399f).
even in miR399-overexpressing plants grown under
and miR399-overexpressing plants (miR399b, miR399f)
high Pi conditions (Figure 4A). The overproduction
(B) Shoot-to-root ratios of the 33P taken up by wild-type (Wt)
from (A).
of miR399 correlated well with the decreased E2
leaves has been documented in plants with Pi toxic-
mRNA level in transgenic plants, further supporting
ity (9). Taken together, findings suggest that the
the hypothesis that miR399 regulates E2 mRNA
chlorosis phenotype observed in the miR399-over-
abundance.
expressing plants resulted from the overaccumula-
miR399-overexpressing plants grown with a
tion of Pi. Despite the high Pi accumulation in the
high Pi supply showed chlorosis or necrosis on the
leaves of miR399-overexpressing plants, the Pi con-
leaf margins, predominantly in mature leaves
centration in the roots was similar to that in the
(Figure 4B). Interestingly, the Pi concentration in
roots of wild-type plants (Figure 4C).
leaves of these transgenic plants was 5- to 6-fold
We next considered whether overaccumulation
that of wild-type plants (Figure 4C). Phosphorous
of Pi in the leaves resulted from elevated uptake of
usually constitutes approximately 0.2% of plant dry
Pi. Seedlings overexpressing miR399 exhibited
matter and Pi toxicity develops when phosphorus
greater Pi uptake than did wild-type plants (Figure
constitutes more than 1% of plant dry matter (9). In
5A). During Pi uptake, more 33Pi was allocated from
our study, phosphorous levels in the leaves of
roots to shoots in miR399-overexpressing seedlings
miR399-overexpressing plants were 1.8% to 2.0%.
than in wild-type plants, which resulted in a higher
Necrosis or chlorosis on the margins of mature
level of 33Pi in the shoot-to-root ratio (Figure 5B).
77
ACADEMIA SINICA
Figure 6. Distribution of Anthocyanin and Pi within Leaves. (A) 8-day-old seedlings were treated under low Pi medium for 12 days.
The cotyledons of miR399-overexpressing plants (miR399b, miR399c and miR399f) remained green (blue arrowheads) while
those of wild-type plants accumulated a large amount of anthocyanin and underwent senescence (red arrowheads). Bar = 0.5 cm.
(B) After prolonged Pi starvation treatment in a hydroponic system, the young leaves but not the old leaves of miR399-overexpressing plants started to accumulate anthocyanin. Bar = 1 cm. Distribution of Pi in the leaves of wild-type (Wt) and miR399-overexpressing (miR399b, miR399c, miR399f) plants grown under low Pi (C) or high Pi (D).
ACADEMIA SINICA
This observation is consistent with the high Pi accu-
miR399-overexpressing plants remained green
mulation in shoots but normal concentrations in
when young leaves started to show Pi starvation
roots of miR399-overexpressing plants (Figure 4C)
symptoms (accumulation of anthocyanin) (Figure
and indicates that overexpression of miR399 not
6A and 6B). Low levels of anthocyanin accumula-
only affects Pi acquisition by roots but also the allo-
tion were associated with high Pi concentration in
cation of Pi between roots and shoots.
the old leaves of miR399-overexpressing plants
Since Pi is a mobile element, it can remobilize
(Figure 6C). Similar distribution patterns of Pi were
from old to young tissues during senescence or
also observed under high Pi conditions (Figure 6D).
when plants experience Pi deficiency (9). The
Thus, we hypothesized that remobilization of Pi
remobilization of Pi is crucial for supporting the
from old to young leaves could be impaired in
continuous growth and development of young
miR399-overexpressing plants.
leaves. Thus, Pi starvation symptoms typically start
In wild-type plants, the Pi concentration in the
in old rather than young leaves. One of the most
cotyledons and the first 2 leaves decreased, while
recognized symptoms of Pi starvation is old, purple
that in younger leaves increased over the growth
leaves caused by the accumulation of anthocyanin.
period (Figure 7A), which suggests the efficient
Unlike those of wild-type plants, the old leaves of
remobilization of Pi from old to young leaves. In
78
tinually increased during the growth period (Figure
7A). Autoradiograms of leaf image obtained from
pulse-chase labeling experiments also indicated that
strong
33
P signals remained in old leaves of
miR399-overexpressing plants after ten days of
radioactive-free chasing (Figure 7B). High accumulation of 33P radioactivity was associated with
chlorosis or necrosis phenotype on these leaves (red
asterisks in Figure 7B). Quantitative analyses fur-
Figure 7. Remobilization of Pi within Leaves. (A) Changes in Pi
concentration in the leaves of wild-type (dotted lines) or miR399foverexpressing plants (solid lines). (B) Autoradiograms of leaf
image obtained from pulse-chase labeling experiments. The first
2 leaves of wild-type plants are outlined because of faint signals.
Leaves with chlorosis or necrosis phenotypes are marked with
red asterisks. Bar = 1 cm. (C) Change in
33
P distribution in the
leaves of wild-type (Wt) and miR399b-overexpressing plants by
pulse-chase labeling. Also indicated is the amount of 33P accumulated in the apex as a proportion of that in whole shoots.
Figure 8. Phenotypes of At2g33770 (E2) T-DNA Insertion
Line. (A) Chlorosis or necrosis was observed on the leaf
margins of homozygous lines but not in the heterozygous or
azygous lines. (B) RT-PCR analysis confirmed the lack of
expression of E2 in the homozygous T-DNA knockout line.
The leaves of homozygous lines accumulated more Pi than
contrast, the Pi concentrations in cotyledons and the
those of heterozygous or azygous lines (C) and were also
first 2 leaves of miR399-overexpressing plants con-
were all grown under high Pi conditions. Bar = 1 cm.
defective in the remobilization of Pi within leaves (D). Plants
79
ACADEMIA SINICA
Figure 9. Tissue and cellular localization of UBC24 and miR399 by promoter:reporter analyses (A) GUS staining in the vascular
tissues of UBC24 promoter: GUS transgenic plants grown under Pi-sufficient conditions. a: whole seedling; b: cotyledon; c: the
third true leaf; d, e: root; f: flower; g: enlarged receptacle; h: cross section of root. (B) GUS staining (a-m) or GFP florescence (ns) in the miR399 promoter: reporter transgenic plants. All seedlings were grown under Pi-deficient media (-Pi) except those in a,
b, c which were grown under Pi-sufficient media (+Pi). The growth condition and expression driven by different miR399 promoters
was indicated in each panel. a, d, g, j: whole seedling; b, c, e, k: cotyledon; f, l, n: the first true leaf; h, i, o, p, q, r: root; m, s: cross
section of root. Arrow in s indicates the GFP signal in the phloem of root. Red in n is the florescence of chlorophyll. Red florescence of cell walls in q and r results from staining with propidium iodide. Bar = 5 mm in A-a, B-a,-d, -g, -j. Bar = 1 mm in A-b, -c, -f,
B-b, -c, -e, -f, -k, -l, -n. Bar = 100 μm in A-d, -g, B-h, -i, -o, -p. Bar = 50μm in A-e, -h, B-m, -q, -r, -s.
ther revealed little change in the proportion of 33P
obtained
an
E2-null
mutant
distribution in the old leaves of miR399-overex-
(SAIL_47_E01 line) with T-DNA inserted in the
pressing plants during a 6-day chase period (Figure
second exon of the E2 gene (Figure 3A). This
7C). In contrast, the 33P distribution in the old leaves
mutant displayed the phenotypes of Pi toxicity due
(leaf no. 1-4) of wild-type plants decreased 35% to
to overaccumulation of Pi in the shoots (Figure 8A
46% over the same period. Moreover, the propor-
and 8C) and defects in the remobilization of Pi
tion of P distribution in the newly emerging apex
within leaves (Figure 8D), which phenocopy the
of wild-type plants was twice that of miR399-over-
miR399-overexpressing plants. The negative regu-
expressing plants (Figure 7C). Taken together, these
lation of E2 mRNA by miR399s and the identical
data support the view that overexpression of
phenotype of the E2 T-DNA knockout mutant and
miR399 interferes with remobilization of Pi from
the miR399-overexpressing plants establish a direct
old to young leaves.
link between miR399 regulation of E2 and Pi home-
33
ACADEMIA SINICA
We
80
that the expression of miR399 is induced in Pistarved rice and tomato plants, which suggests that
this miRNA-based regulatory system has been conserved across a broad spectrum of angiosperms.
Interestingly, we also revealed that a previously identified Pi overaccumulator, pho2 (10), is
caused by a single nucleotide mutation resulting in
early termination within the E2 gene. Furthermore,
miR399 and UBC24 were co-localized in the vascular cylinder (Figure 9). This observation not only
provides important insights into the interaction
between miR399 and UBC24 mRNA but also supports their systemic function in Pi translocation and
remobilization. A working hypothesis for the regulation of Pi homeostasis via the interaction between
miR399 and E2 is outlined in Figure 10.
In this report, we demonstrate that miR399Figure 10. Working Hypotheses for the Regulation of Pi
Homeostasis by miR399 and E2 in Wild-type (A) and
miR399-overexpressing (B) Plants. The blue solid circles
dependent changes in E2 gene expression affect Pi
acquisition, Pi allocation between roots and shoots
indicate the Pi uptake system in roots. The blue and red
and Pi remobilization within leaves. From these
lines indicate the translocation of Pi from roots to shoots and
results, we conclude that precise control of miR399
remobilization of Pi out of old leaves, respectively. The dotted lines in (B) indicate the impaired Pi remobilization in
expression and its interaction with E2 mRNA is
miR399-overexpressing plants. Greater Pi uptake and trans-
critical to the maintenance of Pi homeostasis within
port activities are presented as large circles and thick lines.
Purple leaves are the initiation sites of Pi starvation symptoms because of accumulated anthocyanin. Pi toxicity is
shown as chlorosis in leaf margins (yellow in B). miR399
and E2 are X and Y, respectively.
ostasis in plants.
Genes encoding miR399 homologues have
been identified in other plant species, and we found
plants. Our results shed light on the molecular
mechanisms that regulate Pi homeostasis. Further
analyses of upstream regulatory pathways of
miR399s and downstream pathways of the E2 protein will provide further insights into the regulatory
network that controls Pi homeostasis in the plant.
The original paper was published in Plant Cell 18 (2006): 412-421.
Plant Physiology 141 (2006): 1000-1011.
References:
1. Raghothama, K. G. (1999) Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 665-693.
2. Poirier, Y. & Bucher, M. (2002) In The Arabidopsis Book, C.R. Somerville and E.M. Meyerowitz, eds (Rockville, MD: The American
Society of Plant Biologists), doi/10.1199/tab.0009, http://www.aspb.org/publications/arabidopsis.
3. Carrington, J. C. & Ambros, V. (2003) Science 301, 336-338.
4. Reinhart, B. J., Weinstein, E. G., Rhoades, M. W., Bartel, B. & Bartel, D. P. (2002) Genes Dev. 16, 1616-1626.
5. Bartel, D. P. (2004) Cell 116, 218-297.
6. Sunkar, R. & Zhu, J.-K. (2004) Plant Cell 16, 2001-2019.
7. Allen, E., Xie, Z., Gustafson, A. M. & Carrington, J. C. (2005) Cell 121, 207-221.
8. Jones-Rhoades, M. W. & Bartel, D. P. (2004) Mol. Cell 14, 787-799.
9. Marschner, H. (1995) Mineral Nutrition of Higher Plants. (Academic Press, London).
10. Delhaize, E. & Randall, P. J. (1995) Plant Physiol. 107, 207-213.
81
ACADEMIA SINICA