Download Phosphate Utilization Efficiency Correlates with Expression of Low

Phosphate Utilization Efficiency Correlates with
Expression of Low-Affinity Phosphate Transporters and
Noncoding RNA, IPS1, in Barley1[W]
Chun Y. Huang*, Neil Shirley, Yusuf Genc, Bujun Shi, and Peter Langridge
Australian Centre for Plant Functional Genomics, University of Adelaide, Glen Osmond, South Australia
5064, Australia (C.Y.H., N.S., B.S., P.L.); and School of Agriculture, Wine, and Food, University of Adelaide,
Adelaide, South Australia 5005, Australia (Y.G.)
Genetic variation in phosphorus (P) efficiency exists among wheat (Triticum aestivum) and barley (Hordeum vulgare) genotypes,
but the underlying mechanisms for the variation remain elusive. High- and low-affinity phosphate (Pi) PHT1 transporters play
an indispensable role in P acquisition and remobilization. However, little is known about genetic variation in PHT1 gene
expression and association with P acquisition efficiency (PAE) and P utilization efficiency (PUE). Here, we present quantitative
analyses of transcript levels of high- and low-affinity PHT1 Pi transporters in four barley genotypes differing in PAE. The
results showed that there was no clear pattern in the expression of four paralogs of the high-affinity Pi transporter HvPHT1;1
among the four barley genotypes, but the expression of a low-affinity Pi transporter, HvPHT1;6, and its close homolog
HvHPT1;3 was correlated with the genotypes differing in PUE. Interestingly, the expression of HvPHT1;6 and HvPHT1;3 was
correlated with the expression of HvIPS1 (for P starvation inducible; noncoding RNA) but not with HvIPS2, suggesting that
HvIPS1 plays a distinct role in the regulation of the low-affinity Pi transporters. In addition, high PUE was found to be
associated with high root-shoot ratios in low-P conditions, indicating that high carbohydrate partitioning into roots occurs
simultaneously with high PUE. However, high PUE accompanying high carbon partitioning into roots could result in low PAE.
Therefore, the optimization of PUE through the modification of low-affinity Pi transporter expression may assist further
improvement of PAE for low-input agriculture systems.
Phosphorus (P) is an essential macronutrient for
plant growth and development. The availability of P is
often low in soil, and a large amount of inorganic
phosphate (Pi) fertilizers is applied to achieve high
crop yields. Wheat (Triticum aestivum) and barley
(Hordeum vulgare) production uses approximately 46%
of the P fertilizers applied to cereals (FAO Fertilizer
and Plant Nutrition Bulletin 17; http://www.fao.org).
Extensive fertilization with P leads to rapid depletion
of nonrenewable P resources and contributes to environmental pollution (Vance et al., 2003). P deficiency in
cereal crops is widespread and causes significant yield
reductions (Elliott et al., 1997; Gahoonia and Nielsen,
2004). The improvement of P efficiency in winter cereals
through breeding is crucial for sustainable agriculture
1
This work was supported by the Grains Research and Development Corporation (to C.Y.H., N.S., B.S., P.L.), the Australian
Research Council (to C.Y.H., N.S., B.S., P.L.), the South Australian
Government (to C.Y.H., N.S., B.S., P.L.), and the University of Adelaide
(to C.Y.H., N.S., Y.G., B.S., P.L.).
* Corresponding author; e-mail [email protected].
au.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Chun Y. Huang ([email protected]).
[W]
The online version of this article contains Web-only data.
www.plantphysiol.org/cgi/doi/10.1104/pp.111.178459
and world food security (Godfray et al., 2010; Parry and
Hawkesford, 2010).
There are many definitions of P efficiency in the
literature (Gourley et al., 1993; Hammond et al., 2009;
Wang et al., 2010), but it can generally be divided into
P acquisition efficiency (PAE) and P utilization efficiency (PUE). PAE refers to the ability of the plant to
mobilize P from poorly soluble sources and/or to take
up the soluble Pi available in the soil solution (Narang
et al., 2000), whereas PUE is the amount of biomass
produced per unit of acquired P (Ozturk et al., 2005;
Wang et al., 2010). PAE is considered to be a major
component of overall P efficiency (Ozturk et al., 2005;
Ismail et al., 2007; Ramaekers et al., 2010), but PUE can
influence PAE. P supply to plants is often fluctuating;
therefore, P remobilization within the plant is critical
for plant survival (Drew and Saker, 1984; Marschner
and Cakmak, 1986; Jeschke et al., 1997; Vance et al.,
2003; Huang et al., 2008). Remobilization and translocation of P from shoots to roots can lead to a larger root
system, and hence greater exploitation of limited soil
P resources for more P. However, the effect of PUE
and simultaneous carbohydrate partitioning on PAE
(Hermans et al., 2006; Liao et al., 2008) is not clear
(Manske et al., 2001; Wang et al., 2010).
PHT1 Pi transporters play a critical role in Pi acquisition from soil solution and Pi remobilization within
the plant. Nine members of the PHT1 gene family have
been found in the Arabidopsis (Arabidopsis thaliana)
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Huang et al.
genome (Mudge et al., 2002) and 13 in rice (Oryza
sativa; Paszkowski et al., 2002). Functional characterization shows that some of the PHT1 members, such as
AtPHT1;1, AtPHT1;4 (Shin et al., 2004), and OsPHT1;6
(Ai et al., 2009), are high-affinity transporters while
others are low-affinity transporters, such as OsPHT1;2
(Ai et al., 2009). Eight PHT1 genes have been described
in barley (Smith et al., 1999). The promoter::reporter
fusions of HvPHT1;1 and HvPHT1;2 show that reporters are expressed in root hairs, epidermal and
cortex cells, and vascular tissues of roots (Schünmann
et al., 2004; Glassop et al., 2005). Recently, it was
confirmed that HvPHT1;1 is a high-affinity Pi transporter with a Km of 1.9 mM when expressed in Xenopus
laevis oocytes (Preuss et al., 2011), suggesting that
HvPHT1;1 is involved in Pi acquisition. HvPHT1;6 is
expressed in both shoots and roots (Huang et al., 2008)
and localized in the leaf phloem tissue (Rae et al.,
2003). It displays unsaturated uptake in a millimolar
range of Pi concentrations when expressed in oocytes
(Preuss et al., 2010), suggesting that HvPHT1;6 is
involved in Pi retranslocation. HvPHT1;7 is specifically expressed in green anthers (Druka et al., 2006),
and HvPHT1;8 is a mycorrhiza-specific gene (Glassop
et al., 2005). HvPHT1;3 and HvPHT1;4 are almost
identical in nucleotide sequence. HvPHT1;3 is specifically expressed in roots (Smith et al., 1999). However,
the functions of HvPHT1;3 and HvPHT1;4 in Pi homeostasis are not clear. Only one wheat PHT1 gene,
TaPHT1;2D1, has been characterized so far (Miao et al.,
2009), although several TaPHT1 genes are reported
(Davies et al., 2002; Miao et al., 2009). Whole genomic
sequences of barley and wheat are not yet available,
which has hindered progress toward the functional
characterization of PHT1 genes and their regulation in
barley and wheat.
Pi acquisition and translocation are highly regulated
processes. Transcriptional activation of Pi transporter
genes is believed to be the major control point in
Pi transporter activity (Raghothama, 1999; FrancoZorrilla et al., 2004). IPS (for induced by phosphate
starvation) genes have been shown to be involved in P
homeostasis (Shin et al., 2006; Franco-Zorrilla et al.,
2007; Doerner, 2008; Hammond and White, 2008; Liu
et al., 2010). They are a class of non-protein-coding
RNAs. Overall sequence identities among IPS genes
are relatively low, but 22- to 24-nucleotide sequences
are highly conserved among plant species (Hou et al.,
2005; Shin et al., 2006). IPS genes are expressed primarily in vascular tissues of roots and shoots (Hou
et al., 2005; Shin et al., 2006). Five IPS genes are found
in the Arabidopsis genome (Franco-Zorrilla et al.,
2007), and two IPS genes are present in monocotyledonous species such as rice, maize (Zea mays), and
barley (Hou et al., 2005). The Arabidopsis loss-offunction mutant at4 shows high P accumulation in
shoots (Shin et al., 2006). Inversely, overexpression of
AtIPS1 and At4 decreases P accumulation in shoots
(Franco-Zorrilla et al., 2007), indicating that IPS genes
are involved in Pi remobilization and translocation.
MicroRNAs represent a class of noncoding small
RNAs that generally function as posttranscriptional
negative regulators through base pairing to nearly
complementary sequences in target mRNAs (JonesRhoades et al., 2006). miR399 is the first microRNA
found to be involved in Pi homeostasis (Sunkar and
Zhu, 2004; Fujii et al., 2005; Bari et al., 2006; Chiou
et al., 2006). miR399-guided degradation of mRNA of
the target gene PHO2, encoding for a ubiquitin E2
conjugase, regulates Pi homeostasis in the plant (Aung
et al., 2006; Bari et al., 2006; Mallory and Bouché, 2008).
IPS genes are also involved in the regulation of the
miR399-PHO2 pathway (Doerner, 2008). Incomplete sequence complementarity between AtIPS1 and AtmiR399
forms an RNA duplex with a mismatch loop, which
inhibits the degradation of AtPHO2 mRNA (FrancoZorrilla et al., 2007). AtPHO2 regulates the expression
of AtPHT1;8 and AtPHT1;9, and controls the remobilization and translocation of Pi via an unknown process
(Aung et al., 2006; Bari et al., 2006; Chiou et al., 2006;
Doerner, 2008).
Considerable genetic variation in PAE and PUE has
been reported in a wide range of barley and wheat
genotypes (Gahoonia and Nielsen, 1996; Manske et al.,
2001; Ozturk et al., 2005; Liao et al., 2008; George et al.,
2011), but there have been few studies on the genetic
variation in expression levels of Pi transporters and
the relationships between the expression of Pi transporters and PAE or PUE (Ramaekers et al., 2010). To
address this issue, we used barley, a diploid relative of
hexaploid wheat, as a model system for winter cereals
to study the functions of PHT1 genes involved in the
acquisition and remobilization of Pi (Preuss et al.,
2010, 2011). In this report, we show that differences in
expression levels of four paralogs of the high-affinity
Pi transporter gene HvPHT1;1, are not apparent
among four barley genotypes differing in PAE, but
there is a large difference in the expression of the lowaffinity Pi transporter HvPHT1;6 and a close homolog,
HvPHT1;3. In addition, the expression of HvPHT1;6
and HvPHT1;3 correlates with the expression of
HvIPS1 and PUE. The implications of these findings
in the improvement of PAE for barley and wheat are
discussed.
RESULTS
Plant Growth, P Concentration, PAE, and PUE among
Four Distinct Barley Genotypes
Growing plants in a soil is essential for the evaluation of PAE because it allows the efficient mechanisms operating at the root-soil interface to be
functional. These mechanisms include greater exploitation of soil volume by roots, including root
hairs, root exudates, and microbial activities. These
morphological and biochemical changes can increase
P availability by mobilizing sparingly soluble mineral
P and organic P sources (Gahoonia and Nielsen, 1997;
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Genetic Variation in PHT1 and IPS Expression
Bates and Lynch, 2001; Lambers et al., 2006). We grew
barley plants in fine sand mixed with calcium carbonate to simulate calcareous sandy soil. Four distinct
barley genotypes (Clipper, Sahara, Pallas, and a hairless mutant, brb, derived from Pallas) were selected.
These four barley genotypes were grown in the simulated calcareous sandy soil with three rates of P. At
high Pi supply (75 mg P kg21; referred to as P75), shoot
dry weights of Pallas, brb, and Sahara were significantly higher than for Clipper, but there were no
differences between Pallas, brb, and Sahara (Fig. 1A).
At moderately low P supply (22.5 mg P kg21; referred
to as P22.5), shoot dry weights of all four genotypes
were significantly reduced compared with those at P75
(Fig. 1A). Shoot dry weight of Pallas was significantly
higher than that of brb, Clipper, or Sahara. When P
supply was further reduced (7.5 mg P kg21; referred to
as P7.5), a further reduction in shoot dry weight was
observed for all four genotypes, but the shoot dry
weight of Pallas was significantly higher than that of
brb and Sahara and the shoot dry weight of Clipper
was significantly higher than that of Sahara (Fig. 1A).
Root growth on a fresh weight basis was reduced by
the low rates of P supply, but the reduction was much
less than for shoot growth (Fig. 1, A and B). There was
no significant interaction between P rates and genotypes. The root fresh weight of Pallas on average was
significantly higher than that of the three other
genotypes, but no differences were found among
brb, Clipper, and Sahara. These results indicate that the
low rates of P supply have a smaller impact on root
growth than shoot growth. A genotypic difference in
root growth was found between Pallas and brb but not
between Clipper and Sahara.
An increase in root-shoot ratio is often observed in
plants grown at low P supply and is attributed to
increased carbohydrate partitioning to the root (Zhu
et al., 2002; Hermans et al., 2006; Liao et al., 2008).
Root-shoot ratios on a fresh weight basis increased as P
supply decreased in all genotypes (Fig. 1C). At low P
supply (P7.5), root-shoot ratios of brb and Sahara were
significantly higher than those of Pallas and Clipper,
while the difference between Pallas and Clipper was
not significant. At moderately low P supply (P22.5), a
similar trend to that at low P supply (P7.5) was observed (Fig. 1C). At high P supply (P75), root-shoot
ratios in brb and Clipper were significantly higher than
that in Sahara and the root-shoot ratio of Pallas was
between these two groups (Fig. 1C).
P concentrations of shoots in all four genotypes at
P75 were above the critical level of 4,000 mg P g21 dry
weight (Fig. 2A), which is required for normal plant
growth (Reuter and Robison, 1997; George et al., 2011).
Genotypic differences in P concentrations of shoots
were observed at P75. Clipper had a significantly
higher P concentration than the other genotypes, and
the P concentration in Pallas was higher than that in
brb or Sahara. When P supply was reduced to P22.5, P
concentrations of shoots were reduced to a level below
the critical level (Fig. 2A). The shoot P concentration of
Figure 1. Shoot dry weight and root fresh weight of four barley
genotypes. A, Shoot dry weight. B, Root fresh weight. C, Root-shoot
ratio. Barley seedlings were grown in a calcareous sandy soil with three
rates of P (7.5, 22.5, and 75 mg P kg21 sand, referred to as P7.5, P22.5,
and P75, respectively). Plants were harvested at day 16 after seed imbibition. Means and SE values of four replicates are presented. There are
significant differences in shoot dry weight and root-shoot ratio for
interactions of P rate 3 genotype (P , 0.001). Error bars indicate LSD
(LSD0.05) for shoot dry weight and root-shoot ratio. There are no
significant differences in root fresh weight for interactions of P rate 3
genotype (P = 0.25).
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Huang et al.
action between P rates and genotypes. There was a
significant difference among the four genotypes. The
shoot P content was significantly higher in Pallas and
Clipper than in brb or Sahara, but no difference
between Pallas and Clipper was seen. These results
indicate that Pallas and Clipper are efficient in P
acquisition and that brb and Sahara are inefficient in P
acquisition.
When biomass production per unit of P in shoots of
four genotypes was calculated for PUE, there were no
significant differences at the high P supply (P75)
among the four genotypes (Fig. 2C). The low rates of
P supply at P22.5 and P7.5 increased PUE in all four
genotypes, but differences in PUE were found among
the four genotypes. The genotypes low in PAE (brb and
Sahara) had significantly higher PUE than those with
high PAE (Pallas and Clipper) at both P22.5 and P7.5
(Fig. 2C). These results show that all genotypes increase PUE when P supply is limited for optimal
growth, and the genotypes with greater PUE at low
P conditions have a higher root-shoot ratio (Fig. 1C).
This suggests that high remobilization of P occurs
simultaneously with enhanced carbohydrate partitioning into roots.
Identification of Additional HvPHT1;1 Paralogs in the
Barley Genome
Figure 2. P concentration, P content, and PUE of shoots in four barley
genotypes. A, P concentration of shoots. B, P content of shoots. C, PUE
of shoots. Barley seedlings were grown in a calcareous sandy soil with
three rates of P (7.5, 22.5, and 75 mg P kg21 sand, referred to as P7.5,
P22.5, and P75, respectively). Plants were harvested at day 16 after
seed imbibition. Means and SE values of four replicates are presented.
There are significant differences in P concentrations and PUE for
interactions of P rate 3 genotype (P , 0.009). Error bars indicate LSD
(LSD0.05) for P concentrations and PUE. There are no significant
differences in shoot P content for interactions of P rate 3 genotype (P =
0.39).
Clipper remained higher than in the other genotypes,
and Pallas had a significantly higher P concentration
than either brb or Sahara. When P supply further
decreased to P7.5, P concentrations of shoots were
further reduced in all four genotypes (Fig. 2A). The
shoot P concentration of Pallas remained higher than
that of brb or Sahara. The shoot P concentration of
Clipper was also higher than that of Sahara but similar
to that of Pallas.
Shoot P content, a measure of PAE (Ozturk et al.,
2005), was significantly reduced at the two low rates of
P supply (Fig. 2B), but there was no significant inter-
Two paralogs of HvPHT1;1 genes have been described by Smith et al. (1999). We isolated two additional HvPHT1;1 paralogs from a Haruna Nijo bacterial
artificial chromosome (BAC) library (Saisho et al., 2007)
using a 388-bp probe derived from the coding sequence
of HvPHT1;1. The first gene encoding a protein was
identical to HvPHT1;1 (Fig. 3A) but differed in the 3#
untranslated region (UTR). We designated this new
HvPHT1 gene as HvPHT1;9 (GenBank accession no.
AM904733). The transcript of HvPHT1;9 had a 56nucleotide deletion in the 3# UTR compared with that
of HvPHT1;1 (Fig. 3B). The second new HvPHT1;1
paralog is similar to HvPHT1;2 and is designated as
HvPHT1;10 (GenBank accession no. FN392213). The
protein sequence of HvPHT1;10 differs only by four
amino acid residues from HvPHT1;2 (Fig. 3A).
Database searches identified an ortholog of HvPHT1;9
in wheat (GenBank accession no. BJ277773), which also
has a 30-nucleotide deletion in the 3# UTR compared
with that of TaPHT1;1 (GenBank accession no.
CD871730). No Brachypodium sequences similar to either HvPHT1;1 or HvPHT1;9 were identified (Supplemental Fig. S1).
Database searches using the coding sequences of
HvPHT1;2 and HvPHT1;10 identified four orthologs
in wheat and one ortholog in Brachypodium. All five
orthologs of HvPHT1;2/HvPHT1;10 are 525 amino acid
residues in length. The four wheat orthologs differ from
each other only by two amino acid residues (data not
shown). One of the four wheat orthologs (GenBank
accession no. AJ344240) was used to represent
TaPHT1;2 in the phylogenetic analysis of PHT1 proteins
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Genetic Variation in PHT1 and IPS Expression
Figure 3. Alignment of four HvPHT1 proteins and
partial nucleotide sequences of HvPHT1;1 and
HvPHT1;9. A, Alignment of four HvPHT1 proteins. Amino acid residues that differ among four
HvPHT1 proteins are shown, and the residue
numbers are indicated above. B, Alignment of
nucleotide sequences of HvPHT1;1 and HvPHT1;9
in the 3# UTR. The nucleotide deletion in
HvPHT1;9 is shown as X, and specific primers for
quantitative RT-PCR of HvPHT1;1 and HvPHT1;9
are underlined. Accession numbers are as follows:
AY188394 (HvPHT1;1), AY187020 (HvPHT1;2),
AM904733 (HvPHT1;9), and FN392213
(HvPHT1;10).
(Supplemental Fig. S1). The phylogenetic tree showed
that protein sequences of HvPHT1;1/HvPHT1;9,
HvPHT1;2, HvPHT1;10, TaPHT1;1/TaPHT1;9, TaPHT1;2,
and BdPHT1;2 form a closely related clade (Supplemental Fig. S1), suggesting that HvPHT1;1/HvPHT1;9
and TaPHT1;1/TaPHT1;9 originated from HvPHT1;10/
HvPHT1;2 and TaPHT1;2.
Expression of Two HvIPS Genes among Four
Barley Genotypes
IPS genes are highly responsive to P deficiency, and
their transcript levels are a good biomarker for P
deficiency responses (Liu et al., 1997; Martı́n et al., 2000;
Wasaki et al., 2003). IPS transcripts also interact with
miR399s, regulating P translocation (Franco-Zorrilla
et al., 2007). Therefore, the transcript levels of two
barley IPS genes (HvIPS1 and HvIPS2) in roots were
determined for four barley genotypes. At the sufficient
P supply (P75), a moderate level of HvIPS2 transcripts
(4 to 7 3 106 normalized copies mg21 RNA) was
detected in each of the four genotypes (Fig. 4). Lower
P supply enhanced transcript levels of HvIPS2 in all
four genotypes to 64 to 74 3 106 normalized copies mg21
RNA at P22.5 and 85 to 109 3 106 normalized copies
mg21 RNA at P7.5 (Fig. 4). There was no significant
interaction in the transcript levels of HvIPS2 between
P rates and genotypes, but there was a significant difference at all P rates among the genotypes. The transcript level of HvIPS2 in brb was significantly higher
at all P rates than that in Pallas, and the transcript level
of HvIPS2 in Sahara was marginally higher at all P rates
than that in Clipper.
In contrast, the transcript levels of HvIPS1 at the high
P supply (P75) were hardly detectable in any barley
genotype (Fig. 4). When P supply was reduced to the
moderately low level (P22.5), the transcript levels of
HvIPS1 in brb and Sahara were sharply increased to
approximately 30 3 106 normalized copies mg21 RNA
and 80 3 106 normalized copies mg21 RNA, respectively. However, the transcript levels of HvIPS1 in Pallas
and Clipper at P22.5 increased only to less than 3 3 106
normalized copies mg21 RNA (Fig. 4). A further reduction in P supply to P7.5 increased the transcript level of
HvIPS1 only slightly in brb, Pallas, and Clipper, but the
transcript level of HvIPS1 in Sahara fell sharply relative
to that at P22.5 (Fig. 4). These results show that HvIPS1
and HvIPS2 in all four genotypes are highly inducible
under the low rates of P supply, indicating that the
plants grown at P22.5 are moderately P deficient and
those at P7.5 are severely P deficient. There is a basal
level of HvIPS2 expression in the roots of the P-adequate
plants but hardly any HvIPS1 transcripts in the roots
of the P-adequate plants (Fig. 4). A large variation in
the expression of HvIPS1 is found between the
P-acquisition-efficient genotypes, Pallas and Clipper,
and the P-acquisition-inefficient genotypes, brb and
Sahara, especially at the moderately low P supply
(P22.5), but this is not seen for HvIPS2 expression.
Expression of Four HvPHT1;1 Paralogs in Four
Barley Genotypes
At P75, a moderate level of HvPHT1;1 transcripts (3
to 12 3 106 normalized copies mg21 RNA) was detected
in the roots of all four genotypes (Fig. 5). The transcript
level of HvPHT1;1 in brb was the highest among the
four genotypes, and the transcript level of HvPHT1;1
in Sahara was higher than that in Clipper or Pallas
(Fig. 5). The decrease in P supply to P22.5 increased
HvPHT1;1 transcripts in all four genotypes, especially
for Sahara (Fig. 5). Sahara had a much higher level of
HvPHT1;1 transcripts than the three other genotypes.
The transcript level of HvPHT1;1 in brb was significantly higher than that in Pallas but was similar to that
in Clipper. When P supply decreased to P7.5, the
transcript levels of HvPHT1;1 further increased in
all four genotypes (Fig. 5A). The transcript level of
HvPHT1;1 in Sahara remained higher than in the three
other genotypes, but there was no significant difference among Pallas, brb, and Clipper (Fig. 5).
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Huang et al.
similar size was present in Sahara. Instead, a larger PCR
product was observed in Sahara (Supplemental Fig. S2).
The sequence analysis of this larger PCR product of
HvPHT1;9 from Sahara revealed that it was identical to
that of HvPHT1;1. Therefore, the higher transcript levels
of HvPHT1;1 detected in Sahara relative to the other
genotypes (Fig. 5) could result from combined gene
products of both HvPHT1;1 and HvPHT1;9.
The transcript levels of HvPHT1;2 (2 to 9 3 106
normalized copies mg21 RNA) at P75 were detected in
the roots of all four genotypes (Fig. 5). The moderately
low P supply (P22.5) increased the transcript levels of
HvPHT1;2 in all four genotypes to more than 4 times
those at P75. The low P supply (P7.5) further increased
the transcript levels of HvPHT1;2 in Pallas, brb, and
Clipper, but the transcript levels of HvPHT1;2 were
slightly reduced in Sahara (Fig. 5). There was a significant difference in the transcript level of HvPHT1;2
among the four genotypes. brb and Clipper had much
higher transcript levels of HvPHT1;2 than Pallas and
Sahara across all three rates of P supply (Fig. 5).
The expression of HvPHT1;10 was similar to that
of HvPHT1;2 in Pallas and brb but was different in
Clipper and Sahara (Fig. 5). The transcript levels of
Figure 4. Transcript levels of two HvIPS genes in roots of four barley
genotypes. Barley seedlings were grown in a calcareous sandy soil with
three rates of P (7.5, 22.5, and 75 mg P kg21 sand, referred to as P7.5,
P22.5, and P75, respectively). Plants were harvested at day 16 after
seed imbibition. Quantitative real-time RT-PCR was used to determine
transcript levels in roots. The means (n = 4) of normalized copies mg21
RNA and SE are presented. Log-transformed data were used in the
statistical analysis for comparisons of means. There is a significant
difference in the expression of HvIPS1 for interactions of P rate 3
genotype (P , 0.001) but not in the expression of HvIPS2 (P = 0.16).
Much lower levels of HvPHT1;9 transcripts (0.4 to
2.3 3 106 normalized copies mg21 RNA) were detected
in the roots of Pallas, brb, and Clipper at the sufficient P
supply (P75) but not in the roots of Sahara at any P rate
(Fig. 5). The low rates of P supply at P22.5 and P7.5
increased the transcript levels of HvPHT1;9 in Pallas, brb,
and Clipper, but the transcript levels were approximately 3 times lower than those of HvPHT1;1 (Fig. 5). A
genotypic difference in the transcript level of HvPHT1;9
was found at P75 but not at the two low P rates. brb had
a significantly higher level of HvPHT1;9 than Pallas or
Clipper at P75. PCR amplification of genomic DNA
using HvPHT1;9-specific primer pairs generated PCR
products of HvPHT1;9 from Pallas, Clipper, and Haruna
Nijo (note that the Haruna Nijo BAC library was used to
obtain HvPHT1;9 sequence), but no PCR product of
Figure 5. Transcript levels of four paralogs of HvPHT1;1 in roots of four
barley genotypes. Barley seedlings were grown in a calcareous sandy
soil with three rates of P (7.5, 22.5, and 75 mg P kg21 sand, referred
to as P7.5, P22.5, and P75, respectively). Plants were harvested at day
16 after seed imbibition. Quantitative real-time RT-PCR was used to
determine the levels of transcripts in roots. The means (n = 4) of normalized
copies mg21 RNA and SE are presented. Log-transformed data were used in
the statistical analysis for comparisons of means. There are significant
differences in the expression of HvPHT1;1, HvPHT1;9, and HvPHT1;2
for interactions of P rate 3 genotype (P , 0.001 for all three genes) but
not for HvPHT1;10 (P = 0.49).
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Genetic Variation in PHT1 and IPS Expression
HvPHT1;10 in Clipper and Sahara were at least twice
as much as those of HvPHT1;2 at all three rates of
P. There was no difference in the transcript level of
HvPHT1;10 between Clipper and Sahara across three
rates of P supply (Fig. 5).
The transcript levels of the four HvPHT1;1 paralogs
reveal that all four genes are highly responsive to P
deficiency. Although there were genotypic differences
in the expression of the four HvPHT1;1 paralogs, the
expression pattern was not apparent between two
P-acquisition-inefficient genotypes and two P-acquisitionefficient genotypes.
Expression of HvPHT1;6 and HvPHT1;3 in Four
Barley Genotypes
At sufficient P supply (P75), the transcript levels of
HvPHT1;6 in Pallas, brb, and Clipper were less than
0.5 3 106 normalized copies mg21 RNA, but a higher
transcript level (9.3 3 106 normalized copies mg21
RNA) was detected in Sahara (Fig. 6). When moderately low P (P22.5) was supplied, the transcript levels
of HvPHT1;6 increased to 9 3 106 normalized copies
mg21 RNA in brb and 20 3 106 normalized copies mg21
RNA in Sahara (Fig. 6). However, the transcript levels
of HvPHT1;6 increased only to 0.7 to 1.3 3 106 normalized copies mg21 RNA in Pallas and Clipper (Fig.
6). With low P supply (P7.5), the transcript level of
HvPHT1;6 was further increased in brb to approximately 20 3 106 normalized copies mg21 RNA but was
greatly reduced in Sahara relative to that at P22.5 (Fig.
6). The transcript levels of HvPHT1;6 in Pallas and
Clipper were also increased at P7.5 but remained
lower than 3.0 3 106 normalized copies mg21 RNA.
HvPHT1;3 protein has the same number of amino acid
residues as HvPHT1;6. HvPHT1;3 protein is more similar to HvPHT1;6 (77%) than the four HvPHT1;1 paralogs (70% identity). The transcript levels of HvPHT1;3
almost mimicked those of HvPHT1;6 (Fig. 6). A large
difference in the expression of HvPHT1;6 and HvPHT1;3
was evident between the P-acquisition-efficient and
P-acquisition-inefficient genotypes (Fig. 6). Interestingly,
the difference in the expression of HvPHT1;6 and
HvPHT1;3 is significantly correlated with the expression
of HvIPS1 under the low P rates (P7.5 and P22.5) across
the four genotypes (r = 0.91 and r = 0.95 at degrees of
freedom = 30, respectively; P , 0.001) but not with that
of HvIPS2. The P-acquisition-inefficient genotypes,
brb and Sahara, had much higher expression levels of
HvPHT1;3, HvPHT1;6, and HvIPS1 in roots under the
low-P conditions than the P-acquisition-efficient genotypes, Pallas and Clipper (Figs. 4 and 6).
Stability of RNA Duplexes Formed between Conserved
24-Nucleotide Motifs of HvIPS1 or HvIPS2
and HvmiR399s
The conserved 24-nucleotide motif of HvIPS1 differs
from that of HvIPS2 by six nucleotides in the 11th to
16th nucleotide positions, which correspond to the
Figure 6. Transcript levels of HvPHT1;6 and HvPHT1;3 in roots of four
barley genotypes. Barley seedlings were grown in a calcareous sandy
soil with three rates of P (7.5, 22.5, and 75 mg P kg21 sand, referred to
as P7.5, P22.5, and P75, respectively). Plants were harvested at day 16
after seed imbibition. Quantitative real-time RT-PCR was used to
determine the levels of transcripts in roots. The means (n = 4) of normalized copies mg21 RNA and SE are presented. Log-transformed data
were used in the statistical analysis for comparisons of means. There are
significant differences in the expression of HvPHT1;6 and HvPHT1;3
for interactions of P rate 3 genotype (P , 0.001).
10th to 14th nucleotide positions of HvmiR399s from
the 5# end (Fig. 7, A and B). These HvmiR399s were
recently identified by deep sequencing of short reads
of P-deficient barley plants of Pallas (Schreiber et al.,
2011). By using RNAhybrid software (Rehmsmeier
et al., 2004), minimum free energy (MFE) was calculated for the incomplete double-stranded RNA molecules formed between the conserved 24-nucleotide
motif of HvIPS1 or HvIPS2 and HvmiR399s (Fig. 7, A
and B). The HvIPS1 motif with HvmiR399s could form
an RNA duplex with a mismatch loop of two nucleotides only at the 11th to 12th nucleotide positions of
HvmiR399s, and the RNA duplexes possess MFE more
negative than 230.9 kcal mol 21 (Fig. 7, A and C). In
contrast, the 24-nucleotide motif of HvIPS2 with
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Huang et al.
between HvIPS1 and HvmiR399s are more stable than
those between HvIPS2 and HvmiR399s.
We also determined the transcript levels of a barley
PHO2 ortholog, HvPHO2. Two primer pairs were used
for quantitative reverse transcription (RT)-PCR amplification of both 3# and 5# ends of the HvPHO2 mRNA
sequence, similar to the measurement of AtPHO2
described by Bari et al. (2006), given that HvPHO2
has a long mRNA (approximately 4,000 nucleotides)
and multiple miR399 target sites in the 5# UTR (data not
shown). When a pair of 5# end primers was used
(HvPHO2-5), the transcript levels of HvPHO2 in roots
were low in all four genotypes at the high rate of P
supply (P75), but the transcript level of HvPHO2 in
Clipper was significantly higher than in the three other
genotypes (Fig. 8). When P supply was reduced to
P22.5, the transcript levels of HvPHO2 were significantly increased in brb and Sahara but no change was
found in Pallas and Clipper (Fig. 8). The further decrease in P supply to P7.5 enhanced the transcript levels
of HvPHO2 in brb, Clipper, and Sahara to a similar level
without much change in Pallas. In comparison, the
transcript levels of HvPHO2 measured using a pair of
primers at the 3# end (HvPHO2-3) were similar to those
of HvPHO2-5 in response to P rates and genotypic
differences, except for a higher transcript level (Fig. 8).
These results indicate that the transcript levels of
HvPHO2 in brb, Clipper, and Sahara are increased in
the low P supply, and Pallas appears to be less responsive to P supply.
DISCUSSION
Genetic Variation in P-Acquisition Efficiency in Four
Distinct Barley Genotypes
Figure 7. Nucleotide sequence alignment of conserved motifs of
HvIPS1 and HvIPS2 with known HvmiR399 members and MFE of
RNA duplexes. A, Alignment of the conserved motif of HvIPS1 with
known HvmiR399 members. B, Alignment of the conserved motif of
HvIPS2 with known HvmiR399s. C, An example of the structure of the
RNA duplex formed between HvIPS1 (in red) and HvmiR399b (in
green). D, An example of the structure of the RNA duplex formed
between HvIPS2 (in red) and HvmiR399b (in green). MFE and the
structures of RNA duplexes were obtained with RNAhybrid software
(see “Materials and Methods”).
HvmiR399s could form an RNA duplex with a mismatch loop of five nucleotides at the 10th to 11th
nucleotide positions of HvmiR399s and a second loop
at the 12th to 15th nucleotide positions. The RNA
duplexes possess MFE less negative than 227.1 kcal
mol21 except for HvmiR399d (Fig. 7, B and D). A higher
MFE value was predicted in the RNA duplex formed
between HvIPS1 or HvIPS2 and HvmiR399d (Fig. 7, A
and B), suggesting that HvIPS1 and HvIPS2 are more
effective in sequestering HvmiR399d than the other
HvmiR399s. Nevertheless, the RNA duplexes formed
Three of the four genotypes used in this study have
a diverse genetic background. Clipper is an Australian
barley cultivar, Sahara is a landrace originating from
Algeria (Karakousis et al., 2003), Pallas is a northern
European barley cultivar (Gahoonia et al., 2001), and
the fourth genotype, brb, is a root-hairless mutant
derived from Pallas (Gahoonia et al., 2001). Shoot P
content can be used as an indication of P acquisition
efficiency (Manske et al., 2001; Zhu et al., 2002; Ozturk
et al., 2005). The higher P content of shoots in Pallas
and Clipper than in brb and Sahara indicates that Pallas
and Clipper are P-acquisition-efficient genotypes
whereas brb and Sahara are P-acquisition-inefficient
genotypes. Our results are consistent with a previous
study of Clipper and Sahara where they were grown in
a calcareous soil (Zhu et al., 2002). A similar result was
also observed for soil-grown Pallas and brb (Gahoonia
and Nielsen, 2003). Reduced root surface area in brb
is likely to be responsible for its low PAE (Gahoonia
and Nielsen, 2003). However, it is not known what is
responsible for the low PAE in Sahara. It is noteworthy
that PUE in the P-acquisition-inefficient Sahara and
brb is greater than that in the P-acquisition-efficient
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Genetic Variation in PHT1 and IPS Expression
Figure 8. Transcript levels of HvPHO2 in roots of four barley genotypes.
Barley seedlings were grown in a calcareous sandy soil with three rates of
P (7.5, 22.5, and 75 mg P kg21 sand, referred to as P7.5, P22.5, and P75,
respectively). Plants were harvested at day 16 after seed imbibition.
Quantitative real-time RT-PCR was used to determine the levels of
transcripts in roots. The means (n = 4) of normalized copies per mg21
RNA and SE are presented. There are significant differences in the
expression of HvPHO2-5 and HvPHO2-3 for interactions of P rate 3
genotype (P = 0.006).
Clipper and Pallas under low-P conditions (Fig. 2, B and
C). This shows that greater PUE could have an adverse
effect on PAE due to a concomitant increase in carbohydrate partitioning into roots, indicated by higher rootshoot ratios (Fig. 1C).
Expression Levels of the Genes Related to High-Affinity
Pi Transporters in P-Acquisition-Inefficient Genotypes
May Not Be the Limiting Factor
All four paralogs of the high-affinity Pi transporter,
HvPHT1;1, were expressed in Pallas, brb, and Clipper,
whereas expression of only three of the four HvPHT1;1
paralogs could be detected in Sahara (Fig. 5) due to the
identical sequences in the 3# UTR between HvPHT1;9
and HvPHT1;1 (Figs. 3B and 5; Supplemental Fig. S2).
Our recent work revealed that HvPHT1;1 is a high-
affinity Pi transporter with a Km value of 1.9 mM when
expressed in Xenopus oocytes (Preuss et al., 2011).
Higher transcript abundance of HvPHT1;1 is found in
the root hair zone than in the root tip (Preuss et al.,
2011). The expression pattern of HvPHT1;1 in root
tissues overlaps with HvPHT1;2 (Schünmann et al.,
2004; Glassop et al., 2005), suggesting that both
HvPHT1;1 and HvPHT1;2 are involved in P acquisition. The two newly identified paralogs of HvPHT1;1
(HvPHT1;9 and HvPHT1;10) are either identical or
very similar in protein sequences to HvPHT1;1 and
HvPHT1;2 (Fig. 3A) and highly responsive to P deficiency (Fig. 5), suggesting that they have a role in P
acquisition similar to HvPHT1;1 and HvPHT1;2. Orthologs (BdPHT1;2, TaPHT1;2, TaPHT1;1, and TaPHT1;9)
group with the four barley HvPHT1;1 paralogs in the
phylogenetic tree (Supplemental Fig. S1), suggesting
that the PHT1 genes involved in P acquisition are
conserved in Brachypodium and the Triticeae lineage.
There is no apparent pattern in the expression levels
of the four HvPHT1;1 paralogs between two P-acquisition-inefficient genotypes (Sahara and brb) and the
two P-acquisition-efficient genotypes (Clipper and
Pallas) under low-P conditions (Fig. 5). A similar result
from two contrasting rice genotypes differing in PAE is
also reported using a microarray analysis of seven
OsPHT1 genes (Ismail et al., 2007). These data suggest
that transcript levels of the high-affinity Pi transporters
in P-acquisition-inefficient genotypes may not be a
limiting factor if it is assumed that the high-affinity
PHT1 is regulated predominantly at the transcript level.
Interestingly, the absence of root hairs did not reduce the expression levels of any of the four HvPHT1;1
paralogs. Increased expression levels of HvPHT1;2 and
HvPHT1;10 in brb relative to Pallas under the low P
supply (Fig. 5) could be due to enhanced systemic Pi
signaling initiated by plant low-P status (Figs. 2A and
4). However, the increased expression levels of the
four HvPHT1;1 paralogs were also observed in brb
relative to Pallas under the high P supply (Fig. 5),
when plants have adequate P (Fig. 2A), and there was
no increased expression of HvIPS1 (Fig. 4). This suggests that the interception of external Pi signal (Yang
and Finnegan, 2010) may be impaired due to the
absence of root hairs in brb.
Expression Levels of the Genes Related to Low-Affinity
Pi Transporters Are Correlated with PUE
HvPHT1;6 is a low-affinity Pi transporter (Preuss
et al., 2010). It is expressed in both shoots and roots
(Huang et al., 2008) and especially in phloem tissues of
the leaf (Rae et al., 2003), suggesting that HvPHT1;6
plays a role in Pi remobilization in the whole plant.
The protein sequence of HvPHT1;3 is more similar to
HvPHT1;6 than to the HvPHT1;1 paralogs. HvPHT1;3
is grouped to the clade containing OsPHT1;8, BdPHT1;3,
and TaPHT1;3 with a high bootstrap value (Supplemental Fig. S1). OsPHT1;8 and the low-affinity Pi
transporter OsPHT1;2 have been shown to be involved
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Huang et al.
in Pi retranslocation and regulated by the miR399PHO2 signaling pathway (Wang et al., 2009; Liu et al.,
2010). In addition, the expression of HvPHT1;3 in
roots mimics that of HvPHT1;6 (Fig. 6). These data
suggests that HvPHT1;3 may play a role in Pi remobilization in roots similar to HvPHT1;6. Higher expression of HvPHT1;6 and HvPHT1;3 is associated with
higher PUE (Figs. 2C and 6). PUE has been proposed
to be a bottleneck for further improvement of P efficiency in modern crop cultivars (Wang et al., 2010).
Interestingly, the P-acquisition-inefficient genotype
Sahara is a landrace, whereas P-acquisition-efficient
Clipper is a modern cultivar. Higher remobilization of
internal P to metabolically active tissues such as
growing root tips and shoot meristems under low-P
conditions could promote root growth for access to
unexplored P resources in soil while maintaining
shoot growth. This would lead to higher PAE. However, higher remobilization of internal P could be
associated with higher carbohydrate partitioning into
roots, as indicated by higher root-shoot ratios in the
P-inefficient genotypes (Fig. 1C). A higher root-shoot
ratio is also found in P-inefficient wheat genotypes
(Liao et al., 2008). Reduced shoot growth would decrease the production of carbohydrates and consequently have an adverse effect on PAE. Therefore,
optimization of Pi remobilization may be necessary for
high PAE and high biomass production.
Closely related orthologs (BdPHT1;3/BdPHT1;6
and TaPHT1;3/TaPHT1;6) of HvPHT1;3 and HvPHT1;6
were present in both Brachypodium and wheat (Supplemental Fig. S1), suggesting that the low-affinity Pi
transporters are conserved within Brachypodium and
the Triticeae lineage. Understanding the function and
regulation of HvPHT1;3 and HvPHT1;6 could contribute to further improvement of PAE in both barley and
other winter cereals.
Correlation in the Expression of HvPHT1;3 and HvPHT1;6
with HvIPS1
The 24-nucleotide motifs are conserved in HvIPS1
and HvIPS2 (Fig. 7, A and B), and both genes were
highly responsive to P deficiency (Fig. 4). However,
genetic variation in gene expression was observed only
for HvIPS1. The expression of HvIPS1 is highly correlated
with the expression of HvPHT1;3 and HvPHT1;6 in the
four barley genotypes tested under low-P conditions
(Figs. 4 and 6), suggesting that HvIPS1 plays a unique
role in Pi remobilization.
The conserved motifs are more variable in two barley
IPS genes than those of Arabidopsis (Franco-Zorrilla
et al., 2007). The calculated MFE values of RNA duplexes formed between HvIPS1 or HvIPS2 and known
HvmiR399s (Fig. 7, A and B) suggest that HvIPS1 is more
effective in sequestering HvmiR399s than HvIPS2. Consequently, HvIPS1 could be more effective in the protection of HvPHO2 against HvmiR399-guided cleavage.
In contrast to Arabidopsis, MFE values predicted for
RNA duplexes formed between 24-nucleotide motifs
of AtIPS genes and AtmiR399s were similar among five
AtIPS genes (data not shown). This suggests that mRNA
of all five AtIPS genes may have comparable effectiveness in sequestering AtmiR399s as that shown for AtIPS1
(Franco-Zorrilla et al., 2007). The transcript levels of
the miR399 target gene HvPHO2 were generally higher
in the low-P conditions (Fig. 8) and were parallel to
the transcript levels of HvIPS1 to some extent (Fig. 4),
suggesting that the miR399-PHO2 signaling pathway
is conserved in barley. AtPHO2 has been shown to be
involved in the regulation of PHT1 genes such as
AtPHT1;8 and AtPHT1;9 in Arabidopsis (Aung et al.,
2006; Bari et al., 2006) and OsPHT1;2 and OsPHT1;8
in rice (Wang et al., 2009). Therefore, it is likely that
HvPHT1;6 and HvPHT1;3 are also subject to the regulation of the miR399-PHO2 signaling pathway.
Notably, there are some differences in the transcript
levels of PHO2 among Arabidopsis, rice, and barley.
The transcript levels of HvPHO2 in roots are generally
higher when the transcript levels of HvIPS1 reach the
highest values in severely P-deficient plants except for
Sahara (Fig. 8), whereas the expression of AtPHO2 in
roots is decreased with an increase of AtIPS genes
(Aung et al., 2006; Bari et al., 2006; Franco-Zorrilla et al.,
2007). There is no change in the transcript levels of
OsPHO2 in low-P conditions relative to high-P conditions when OsIPS1 and OsIPS2 are increased (Zhou
et al., 2008). miR399-guided cleavage can decrease
transcript levels of PHO2, while IPS transcripts can
protect HvPHO2 mRNA against miR399-guided degradation (Franco-Zorrilla et al., 2007). A number of
factors could have effects on the transcript levels of
PHO2, including (1) PHO2 transcription rates under
different P conditions, (2) PHO2 mRNA turnover rate,
(3) the abundance of different members of the miR399
family (Lin et al., 2008), (4) the abundance of transcripts
from different IPS genes, which vary in the conserved
24-nucleotide motif (Hou et al., 2005; Franco-Zorrilla
et al., 2007) and the formation of incomplete doublestranded RNA complexes with different stability (Fig. 7,
A and B), and (5) four to five complementarity sites for
miR399 present in the 5# UTR of PHO mRNA (FrancoZorrilla et al., 2007), since different members of the
miR399 family may have preference for these sites
(Allen et al., 2005). The observed differences in the
transcript levels of PHO2 between the three species
could be due to any combination of these factors or
other factors. Further studies are obviously required to
elucidate the difference. Despite the differences in the
transcript levels of PHO2 among these three species, the
correlation in the expression levels between IPS and
PHT1 genes related to Pi retranslocation is consistent
across the three species. In addition, although the expression levels of HvPHT1;3 and HvPHT1;6 are highly
correlated with those of HvIPS1 (Figs. 4 and 6), the
correlation in the expression between HvPHT1;3/
HvPHT1;6 and HvPHO2 is not as tight as that with
HvIPS1, especially in Sahara at P7.5 (Figs. 6 and 8), suggesting that additional mechanisms may be involved
in the regulation of PHO2 and downstream PHT1 genes
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Genetic Variation in PHT1 and IPS Expression
in Pi remobilization. There are other regulators involved
in P signaling (Lin et al., 2009, 2010). It has been shown
that OsSPX1 and OsPHR2 are involved in the regulation
of low-affinity Pi transporters such as OsPHT1;2 (Wang
et al., 2009; Liu et al., 2010). Sugars as a systemic signal
can also regulate the expression of Pi transporters
(Hammond and White, 2008). Therefore, the transcript
levels of HvPHT1;3 and HvPHT1;6 could be modulated by additional regulators. A further analysis of
barley orthologs of OsSPX1 and OsPHR2 could shed
light on the regulation of HvPHT1;3 and HvPHT1;6 via
the miR399-PHO2 pathway.
In summary, Pi retranslocation within the plant is
an important mechanism in plant adaptation to variable P supply. Low-affinity Pi transporters are responsible for Pi retranslocation within the plant. The higher
expression of the low-affinity Pi transporter genes,
HvPHT1;6/HvPHT1;3, could lead to greater PUE, but
the concomitant increase in carbohydrate partitioning
into roots could have an adverse effect on carbohydrate production and PAE. Therefore, optimization of
PUE in plants may be required for high PAE and high
yield in low-input agriculture systems.
MATERIALS AND METHODS
Plant Growth and P Analysis
Four barley (Hordeum vulgare) genotypes, Pallas, brb (a mutant of Pallas),
Clipper, and Sahara, previously shown to differ in P efficiency (Zhu et al.,
2002; Gahoonia and Nielsen, 2003) were used in our experiments. All seeds
were multiplied in a potting mix for similar nutrient contents. The weight of
seeds used in the experiment was similar, in the range of 52 to 54 mg seed21 for
three genotypes (Pallas, brb, and Clipper), but smaller for Sahara (45 mg
seed21). The P content of Pallas, brb, Clipper, and Sahara was 259, 212, 285, and
193 mg P seed21, respectively.
Siliceous sand was used in the experiments. Basal nutrients and plant
growth conditions were as described by Genc et al. (2007). Calcium carbonate
powder (0.5%, w/w) was added in the sand to simulate calcareous sandy soil
and to reduce P availability (Ryan et al., 1985; Westermann, 1992). Three
P rates (7.5, 22.5, and 75 mg P kg21 sand as KH2PO4) were used in the
experiments for severely P-deficient, moderately P-deficient, and P-adequate
plants, respectively.
Plants were harvested at 16 d after seed imbibition. At harvest, shoots of
two plants in each pot were cut above the soil surface, and the fresh weight of
each plant was recorded. The shoots of one plant were oven dried for shoot
dry weight and nutrient analysis, and the shoots of the other plant were frozen
in liquid nitrogen for transcript analysis. Shoot P was determined with
inductively coupled plasma emission spectrometry (Zarcinas et al., 1987). The
roots of two plants in each pot were washed free of sand particles and
separated. The excess water in roots was removed with tissue paper, and the
fresh weight of roots was recorded. The roots of one plant were frozen
immediately in liquid nitrogen for transcript analysis, and the roots of the
other plant were used for measurements of root morphology.
roots was prepared using Trizol reagent according to the manufacturer’s
instructions (Invitrogen) and treated with DNase I (Ambion). Then, RNA
integrity was checked on an agarose gel. Two micrograms of total RNA from
roots was used to synthesize cDNA with SuperScript III reverse transcriptase
(Invitrogen). The transcript levels of four control genes (barley a-tubulin, heat
shock protein 70, glyceraldehyde-3-phosphate dehydrogenase, and cyclophilin) were determined for all cDNA samples, and the most similar three of
these four genes were used as normalization controls. Normalization was
carried out as described by Vandesompele et al. (2002) and Burton et al. (2008)
for the transcript levels of all root cDNA samples. Four biological replicates
were used for transcript analysis. Three technical replicates were conducted
for each cDNA sample. The normalized copies mg21 RNA were used to
represent transcript levels. The primer sequences for all genes determined are
listed in Supplemental Table S1.
MFE of RNA Duplexes
The RNAhybrid software (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/
submission.html) was used with default settings to calculate MFE of RNA
duplexes formed between HvIPS genes and HvmiR399s (Rehmsmeier
et al., 2004).
Statistical Analysis
The experimental setup was a completely randomized block design with
four replications. Data of plant growth, P nutrition, and transcripts were
analyzed using the Genstat Statistical Program (version 11.1; VSN International). The log-transformed data were used in analysis when treatment
effects were larger than 10 times. The LSD at P = 0.05 was used for comparisons
of means.
Sequence data from this article can be found in the GenBank/EMBL data
libraries under accession numbers AM904733 for HvPHT1;9 and FN392213
for HvPHT1;10.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Phylogenetic tree of PHT1 Pi transporters in
Arabidopsis, rice, Brachypodium, barley, and wheat.
Supplemental Figure S2. PCR amplification of genomic DNA of four
barley genotypes using HvPHT1;9-specific primers.
Supplemental Table S1. Primer sequences used for quantitative, real-time
RT-PCR analysis of transcript levels.
ACKNOWLEDGMENTS
We are grateful to Dr. Julie Hayes for valuable comments on the manuscript, Dr. Iver Jakobsen for providing the seeds of Pallas and brb, and John
Toubia, Margaret Pallotta, and Hui Zhou for technical assistance.
Received April 18, 2011; accepted May 20, 2011; published May 23, 2011.
Identification of Two New Paralogs of HvPHT1;1
LITERATURE CITED
A 388-bp probe derived from the coding sequence of HvPHT1;1 (GenBank
accession no. AY188394; 945–1,332 bp) was used for screening a Haruna Nijo
BAC library (Saisho et al., 2007). Two unique BAC clones were isolated, one
containing HvPHT1;10 and the other containing HvPHT1;1, HvPHT1;9, and
HvPHT1;2. The two BAC clones were sequenced, and contigs were assembled
for HvPHT1;9 and HvPHT1;10.
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phasing during trans-acting siRNA biogenesis in plants. Cell 121:
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Bari R, Datt Pant B, Stitt M, Scheible WR (2006) PHO2, microRNA399, and
Real-Time Quantitative RT-PCR
RNA isolation and real-time quantitative RT-PCR analysis of transcripts
were conducted as described by Preuss et al. (2011). Briefly, total RNA from
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