Transcriptional response of rice flag leaves to restricted external phosphorus supply during grain filling in rice cv. IR64
Transcriptional response of rice flag leaves to restricted external phosphorus supply during grain filling in rice cv. IR64
Kwanho Jeong 0 1 2
Omar Pantoja 1 2
Abdul Baten 0 1 2
Daniel Waters 1 2
Tobias Kretzschmar 0 1 2
Matthias Wissuwa 1 2
Cecile C. Julia 0 1 2
Sigrid Heuer 1 2
Terry J. Rose 0 1 2
0 Southern Cross Plant Science, Southern Cross University , Australia, 2 Southern Cross GeoScience , Southern Cross University, Australia, 3 Instituto de BiotecnologA?o?a, Universidad Nacional AutoA? noma de M eA?xico , Cuernavaca, Morelos, Mexico, 4 ARC ITTC for Functional Grains , Charles Sturt University , Wagga Wagga NSW , Australia , 5 Genotyping Services Laboratory, International Rice Research Institute (IRRI) , Metro Manila, Philippines, 6 Crop , Livestock and Environment Division, Japan International Research Center for Agricultural Sciences , Ohwashi, Tsukuba, Ibaraki , Japan , 7 Department of Plant Biology and Crop Sciences, Rothamsted Research , West Common, Harpenden, Herts , United Kingdom
1 Editor: Prasanta K. Subudhi, Louisiana State University College of Agriculture , UNITED STATES
2 Current address: AgResearch, Grasslands Research Centre , Palmerston North , New Zealand
Plant phosphorus (P) remobilisation during leaf senescence has fundamental implications for global P cycle fluxes. Hypothesising that genes involved in remobilisation of P from leaves during grain filling would show altered expression in response to P deprivation, we investigated gene expression in rice flag leaves at 8 days after anthesis (DAA) and 16 DAA in plants that received a continuous supply of P in the nutrient solution vs plants where P was omitted from the nutrient solution for 8 consecutive days prior to measurement. The transcriptional response to growth in the absence of P differed between the early stage (8 DAA) and the later stage (16 DAA) of grain filling. At 8 DAA, rice plants maintained production of energy substrates through upregulation of genes involved in photosynthesis. In contrast, at 16 DAA carbon substrates were produced by degradation of structural polysaccharides and over 50% of highly upregulated genes in P-deprived plants were associated with protein degradation and nitrogen/amino acid transport, suggesting withdrawal of P from the nutrient solution led to accelerated senescence. Genes involved in liberating inorganic P from the organic P compounds and vacuolar P transporters displayed differential expression depending on the stage of grain filling stage and timing of P withdrawal.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: This research work was supported by
funding from the Global Rice Science Partnership
(GRiSP) New Frontiers Research Project. O.P. was
supported by a Sabbatical Fellowship by
DGAPAUNAM. The funders had no role in study design,
data collection and analysis, decision to publish, or
preparation of the manuscript.
An estimated 5.7 billion ha of global crop-lands are deficient in bioavailable phosphorus (P)
]. To obtain high yield and maintain soil fertility, regular inputs of P fertiliser, and other
nutrients, are required. The efficiency with which applied P fertiliser is utilised by crops is,
however, generally low. Much of the applied P fertiliser becomes `fixed' with iron (Fe) and
aluminium (Al) oxides and hydroxides in acid soils, and as calcium (Ca) complexes in alkaline
Competing interests: The authors have declared
that no competing interests exist.
], which is problematic given that rock phosphate utilised by most P-fertilisers is a finite
natural resource [
A further factor that contributes to unsustainable P use in agriculture is the removal of an
estimated 12 million tonnes of P in harvested agricultural produce each year [
], of which
little is recycled back to fields . Most of the P removed in harvested produce is in the grains
of cereal crops [
], mostly in the form of the anti-nutrient phytate which cannot be digested by
humans and other monogastric animals [
]. Reducing P levels in the grains of cereals would
minimise the amount of P removed from fields at harvest and, if crop stubble is retained in the
field, the P fertiliser requirements of subsequent crops would be reduced in the long term [
Recent studies have identified critical periods of P remobilisation from leaves and loading
into developing grains of rice (Oryza sativa L.) [
], however, our understanding of the
molecular regulation of this process is limited [10?13]. Using rice as a model, we recently investigated
whether a subset of genes involved in the well described molecular response of plants to P
starvation [14?16] were involved in the remobilisation of P from senescing flag leaves to
developing rice grains [
]. Three purple acid phosphatases (OsPAP3, OsPAP9b and OsPAP10a) that
have been implicated in the P starvation response were significantly upregulated during the
phase of rapid P remobilisation from flag leaves (15 days after anthesis; DAA) compared to 6
DAA when flag leaf P levels were relatively stable [
]. Additionally, three genes not previously
associated with the P starvation response, OsPAP26, SPX-MFS1 and SPX-MFS2, showed
expression profiles consistent with an involvement in P remobilisation from senescing flag
]. Consistent with these findings, the Arabidopsis homologue of OsPAP26, AtPAP26,
was shown to be involved in P remobilisation from senescing Arabidopsis leaves [
addition, data from recent studies suggest that SPX genes may encode vacuolar P transporters
] and two VPTs were recently identified in rice as influx (OsSPX-MFS1) and efflux
(OsSPX-MFS3) transporters [19?21].
While the expression profiles of the OsPAPs and OsSPX-family genes were consistent with
a role in remobilisation of P from senescing rice flag leaves [
], the absence of any specific P
treatment prevented us from establishing a definitive link with P remobilisation. In a
subsequent study, we found that P from specific leaf P pools was remobilised prematurely following
the permanent withdrawal of P from the nutrient solution at anthesis or 8 DAA [
]. The data
suggested that the demand for P in developing grains led to premature remobilisation of P
from flag leaves in the absence of an external (nutrient solution) P supply.
The aim of the present study was to investigate whether the premature remobilisation of P
in flag leaves when external P supply was withdrawn during grain filling corresponded to
changes in expression of:
1. previously identified genes putatively related to P remobilisation during senescence
including OsPAPs and OsSPX genes from the study of Jeong et al.[
2. novel genes that have not previously been associated with P remobilisation from senescing
This was tested using an RNA-seq approach to compare gene expression in flag leaves of
rice at specific time points during grain filling from plants that had a continuous P supply
until maturity and from plants that had P permanently withdrawn (for 8 d) from the
hydroponic solution at anthesis and at 8 DAA, respectively. We used the rice mega-variety IR64 that
has been well-characterised genetically and is cultivated on more than 10 million ha across the
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Materials and methods
Experimental design and overview
Rice plants (cv. IR64) were grown to maturity in hydropic culture solution under adequate P
supply (0.75 mg P day-1), supplied until maturity, or P permanently withdrawn from the
nutrient solution at anthesis or 8 DAA. The amount of P provided in the nutrient solution for
`adequate' P supply was determined in a preliminary experiment, and was defined as the
minimum supply of P required for obtaining maximum grain yields. Data from the
preliminary experiment, along with the flag leaf photosynthesis and P mobilisation data, are presented
in Jeong et al. [
], and key data pertinent to this paper are presented in S1 Table.
Plant growth and experimental design
Evenly sized rice seeds (cv. IR64) were sterilised with HClO3 for 2 min and germinated in
Petri dishes in the dark at 30?C for 2 d. The germinated rice seeds were transferred to a mesh
floating on a hydroponic nutrient solution containing 1 mM calcium (CaCl2) and 36 ?M iron
(Fe EDTA). After 10 days, the solution was changed to half strength Yoshida solution [
without P, in which the plants were grown for a further 2 weeks. Nutrient solutions were
replaced after 1 week. After 2 weeks growing on the floating mesh, two evenly sized seedlings
were transplanted into 5 L pots wrapped with aluminium foil containing full strength Yoshida
solution without P. Yoshida nutrient solution was changed and the pH adjusted to 5.2?5.5
each week. Rice plants were grown under temperature-controlled conditions in a glasshouse at
Southern Cross University (Lismore, NSW, Australia) with a mean day/night air temperature
of 29?C/21?C and relative humidity (RH) of 75%.
Plants received 0.75 mg of P per day per pot by application of 17.5 ml of P stock solution
(150 mg P L-1) every 3.5 days to the nutrient solution. Phosphorus withdrawal treatments were
applied during the reproductive growth phase as shown in S1 Fig. Each treatment was
replicated three times. Anthesis was defined as the state when 50% of panicles had at least 50% of
florets with visible anthers. A set of six panicles that reached the booting stage simultaneously
were tagged and their flag leaves were harvested for RNA extraction and RNA-seq analysis.
Total RNA extraction, library construction and sequencing
Based on flag leaf photosynthesis and P mobilisation data (S1 Table) [
], two sets of samples
that had received `adequate' P supply were selected for RNA-seq analysis. The first set
consisted of flag leaf samples harvested at 8 DAA from plants where P supply had been withdrawn
at anthesis (T8) and of the corresponding control plants (P supplied continuously; C8). The
second set consisted of leaf samples from plants harvested at 16 DAA where P supply had been
withdrawn at 8 DAA (T16) and plants of the corresponding control treatment (P supplied
continuously; C16) (S1 Fig). These two time-points were chosen because in response to P
deprivation, photosynthesis was unaffected in T8 while it was significantly impaired in T16 (S1 Table)
Total RNA was extracted using the RNeasy Mini kit (Qiagen, Victoria, Australia) according
to the manufacturer's instructions. After extraction, total RNA was quantified with a
Nanodrop (ND1000, Labtech, Paris, France) and the quality of RNA was examined on a 2100
Bioanalyzer (Agilent Technologies, California, US). Based on the quantity and quality control of
RNA samples, high quality RNA samples were selected for library construction. Samples that
did not meet the criteria (i.e. 2.0 260 / 280 ratio, 7.0 RIN number) were re-extracted. Library
construction using Truseq RNA V2 kit (Illumina, California, US) and RNA sequencing
(RNAseq) using Hi-seq 2500 system (Illumina) were carried out by Macrogen (Seoul, Korea).
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Analysis of RNA-seq data
RNA-seq data analysis was performed as described in Jeong et al. [
]. Fastq files were filtered
for adapter sequence, poly-N stretches and low-quality reads using FASTQC [
] and the
BBDuk module of the BBMap software package (https://sourceforge.net/projects/bbmap/,
version 35.51). Bowtie version 2.2.4 [
] was used to index the rice genome (IRGSP 1.0). Clean
high quality reads were mapped using the splice aware RNASeq aligner program TopHat
version 2.1.0 [
]. The Ensembl Plants (http://plants.ensembl.org/Oryza_sativa/Info/Annotation)
O. sativa cv. Nipponbare (ssp. japonica) reference genome annotation was utilised. TopHat
identified the exon?exon junctions and produced the read vs genome alignment in BAM
(Binary Alignment Map) format. Cufflinks [
] then used the TopHat-generated alignment to
assemble a set of reference-based transcripts. Finally, the CuffDiff module of Cufflinks was
used to identify differentially expressed genes between samples. Raw sequencing data have
been uploaded to SRA (Sequence Read Archive) database (accession number: SRP134062).
A total of 304 million 101 bp paired end reads generated more than 96% of high quality
reads across all libraries and about 87.6% of those high quality reads were mapped to the
reference rice genome (S2 Table).
Gene expression levels were normalised using the FPKM (Fragments Per Kilobase of
transcript per Million mapped reads) method. Differentially expressed genes (DEGs) were
calculated by comparing the FPKM values between T8 vs C8 and T16 vs C16 using three biological
replicates for each treatment. Significant DEGs were defined as those genes that were up or
downregulated with log2 fold change > 1 or < -1, respectively, while significance threshold
was set at a false discovery rate (FDR) corrected p value of < 0.05.
Gene identification, annotation and classification
A total of 400 DEGs, the 100 most differentially up- and down-regulated genes from both T8
and T16, were categorised based on their descriptions, annotated functions and data from the
literature (S3 and S4 Tables). After sorting based on log2 fold change the annotations were
retrieved from the rice annotation project database (RAP-DB; http://rapdb.dna.affrc.go.jp/
index.html), rice locus identifier search (http://rice.plantbiology.msu.edu/analyses_search_
locus.shtml) and RiceXpro global gene expression profile (http://ricexpro.dna.affrc.go.jp/
category-select.php). The functional annotation of DEG products was cross referenced with
the functional protein association network String (version 10.0; http://string-db.org/). To
annotate and classify transcription factors (TFs), plant transcription factor database version
3.0 (PlantTFDB; http://planttfdb.cbi.pku.edu.cn/) was used.
Examination of key genes in the P starvation response
We assumed that the withdrawal of P from the nutrient solution during grain filling would not
simply elicit the well-known P starvation response observed when young plants are deprived of
P. To confirm this, we specifically examined the expression of 11 key genes (OsPHO1, OsPHO2,
OsPHR1, OsPHR2, OsSPX2, OsSPX3, OsSPX5, OsPT5, OsPAP23, OsIPS1 and OsACP1) that have
been widely reported to respond to P starvation in young rice plants (Table 1) [16, 29?32].
Expression of specific genes of interest from earlier studies
We previously observed that 32 genes, including P starvation response genes (PSRs), OsPAPs,
rice P transporters (OsPTs), and OsSPX and OsSPX-MFS family genes, were upregulated in
rice flag leaves at 15 DAA when leaves were acting as a P source [
]. We further investigated
the expression of these 32 genes (hereafter referred to as the `32 P-remobilisation related genes
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(PRGs)'; see list in S5 Table) in the present study at 8 DAA and 16 DAA in both treatment (P
withdrawn) and control samples. Further, given the critical role of Pi transporter family 1
(PHT1) genes in Pi uptake from roots and remobilisation in vegetative tissues [
expression of PHT1 genes (OsPT1 to OsPT13) was analysed at 8 DAA and 16 DAA.
Global gene expression data and differential gene expression
Three biological replicates for each library (T8, C8, T16 and C16) were analysed using
RNAseq. The average number of high quality reads of three replicates from each library, T8, C8,
T16 and C16, were 24 ? 0.35, 25 ? 4.84, 26 ? 1.37 and 23 ? 0.97 million, respectively. An
average of 87.6 ? 0.69% of high quality reads from each replicate were mapped to the rice genome
(S2 Table) and distinct DEGs retrieved based on log2 fold change (log2 > 1 or < -1). At 8
DAA, 1,515 and 2,044 genes were up and downregulated, respectively, while 715 and 1,157
genes were up and downregulated, respectively, at 16 DAA (Fig 1). Subsequently, the DEGs
common to both treatments were identified, with 102 DEGs upregulated at both 8 and 16
DAA, while 306 DEGs were downregulated at both 8 and 16 DAA (Fig 1). The number of
DEGs that were upregulated at 8 DAA but downregulated at 16 DAA was 142, and 103 DEGs
were upregulated at 16 DAA but downregulated at 8 DAA (Fig 1).
Expression of key P starvation induced genes
We assumed that P withdrawal would not simply elicit the up-regulated expression of PSR genes.
In order to verify this, the expression of 11 highly and consistently expressed PSR genes was
investigated. Seven of the 11 key PSR genes examined, including the transcription factors OsPHR1 and
OsPHR2, were either not differentially expressed or were downregulated in T8 and T16 (Table 1).
Only OsPHO2 was upregulated in both T8 and T16, with log2 fold changes of 0.52 and 0.57,
respectively, while OsIPS1, OsPAP23 and OsSPX5 were upregulated in T8 (Table 1).
Expression of 32 PRGs putatively involved in P remobilisation during grain filling
In an earlier study [
], we found that the expression profiles of 32 P-related genes (PRGs)
were consistent with a possible role in remobilisation of P from senescing rice flag leaves,
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Fig 1. Venn diagram of total DEGs and overlapping DEGs (log2 fold change > +1 or < -1, P value < 0.05).
however, the absence of any specific P treatment precluded any definitive conclusions. Three
of the 32 PRGs (OsPAP15, OsPAP10a and OsPAP1d) were significantly upregulated in T8
while five genes (OsSPX-MFS1, OsSPX-MFS3, OsPHO1;1, OsPAP9b and OsCAX1a) were
downregulated (Table 2). In T16, only one gene (OsSPX-MFS3) was upregulated while three
genes (OsPT20, OsPT19 and LOC_Os03g15530) were downregulated (Table 2). Most of the 32
? Gene expression in 20 genes out of 32 PRGs did not change upon P withdrawal
Positive and negative values indicate upregulation and downregulation, respectively.
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Fig 2. The 100 most highly upregulated (A) and downregulated (B) genes in T8. The number in the chart indicates the number of genes that are assigned in the
PRGs were not differentially expressed; only OsSPX-MFS1 was highly downregulated in T8
with a log2 fold change of -3.13 (Table 2). OsSPX-MFS2 and OsSPX-MFS4 were not
differentially expressed in flag leaves during grain filling under P withdrawal (S2 Fig).
Analysis of the 100 most highly up-regulated and down-regulated DEGs in T8 and T16
In order to obtain information on the metabolic pathways or processes that were responding
to P-withdrawal, we decided to analyse the 100 most highly up/downregulated genes in T8 and
T16 on flag leaves under P withdrawn between different time-points during rice grain filling.
Highly upregulated genes at 8 DAA in response to P-withdrawal. More than 50% of the
upregulated genes in T8 were classified within the nucleic acid metabolism, photosynthesis,
detoxification and protein translation groups (Fig 2A). Twenty-five of the genes upregulated
in T8 were assigned to nucleic acid metabolism (Fig 2A). Most of these were broadly associated
with DNA replication and RNA splicing in the chloroplast, for example, ribonucleotide
reductase (RNRL1; LOC_Os06g07210) and chloroplastic group IIA intron splicing facilitator CRS1
(LOC_Os05g47850). Additionally, six genes encoding for proteins containing
pentaricopeptide repeat (PPR) domains, four encoding for proteins with a glycine rich (GRP) domain
involved in RNA recognition and RNA polymerases, that together are involved in RNA
transcription and editing in chloroplast, were also upregulated in T8 (S3 Table). Nine DEGs were
assigned to photosynthesis (Fig 2A). Among these were photosystem II (PSII) oxygen evolving
complex protein PsbQ family protein (LOC_Os02g36850, with a log2 fold change of 2.6). The
light-harvesting chlorophyll a/b-binding proteins from photosystem I (PSI) precursor (Lhca6,
LOC_Os09g26810), ferredoxin-type domain containing protein (LOC_Os07g30670) and
aldose 1-epimerase family protein (AEP; LOC_Os03g53710), with log2 fold changes of 3.0, 4.0
and 3.2, respectively (S3 Table).
Genes involved in detoxification processes were also upregulated in T8 (Fig 2A), including
thioredoxin and oxidoreductase proteins that are involved in detoxification of reactive oxygen
species (ROS) in chloroplasts (S3 Table). In addition, four genes associated with nitrogen (N)
remobilisation were upregulated, including the urea transporter, OsTIP4;1 (LOC_Os05g14240)
and aspartate carbamoyltransferase (LOC_Os08g15030), that catalyses the first committed step
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in pyrimidine biosynthesis, the condensation of l-aspartate and carbamoyl phosphate to form
N-carbamyl-L-aspartate and inorganic phosphate (S3 Table).
Five ribosomal and translational proteins were upregulated and were assigned to protein
translation and several other upregulated DGEs were assigned to protein related processes
such as protein synthesis, phosphorylation and ubiquitination (Fig 2A). Descriptions of other
genes that were upregulated in T8 are given in S3 Table.
Highly down regulated genes at 8 DAA in response to P-withdrawal. Twenty-seven of
the genes that were downregulated in T8 were TFs, five of which participate in protein
synthesis regulation. For example, OsbHLH148 (LOC_Os03g53020) acts on an initial response of
jasmonate-regulated gene expression toward drought tolerance; OsDof2 (LOC_Os01g15900) is
associated with plant growth, leaf expansion, grain size and flowering time in rice. The heat
stress transcription factor, OsHsfC2a (LOC_Os02g13800) was downregulated in T8. The
WRKY transcription factors OsWRKY21 (LOC_Os01g60640) that putatively regulates a
transcriptional repressor of the gibberellin signalling pathways, and OsWRKY71, associated with
the defensive reaction to rice blast disease were also downregulated in T8 (Fig 2B and Table 3).
Numerous phytohormone-related TFs were also downregulated in T8, including ethylene
response genes (eight AP2/ERF domain TFs), jasmonate signalling related TFs (four Tify
domain TFs and 1 helix-loop-helix DNA binding domain TF) and a further seven DEGs
associated with auxin, gibberellin and other phytochemicals (terpenoids and alkaloids) (Fig 2B and
S3 Table). A further eight DEGs associated with abiotic/biotic stress responses were
downregulated in T8 (Fig 2B and S3 Table). Genes associated to carbon metabolism were found to be
downregulated, like OsINV3 (LOC_Os02g01590), OsUGlcAE3 (LOC_Os02g54890),
sugar/inositol transporter (LOC_Os12g32760) and OsSWEET2b (LOC_Os01g50460). Those coding for
a glycosyl hydrolase (OsXTH1; LOC_Os04g51460), transferase (LOC_Os03g47530) and
OsPCS13 (LOC_Os03g18910) were assigned to cell wall degradation and were downregulated
in T8 (Fig 2B).
Highly upregulated genes at 16 DAA in response to P-withdrawal. More than 50% of
upregulated genes in T16 were grouped into protein ubiquitination, N remobilisation, TFs,
detoxification, stress response and cellular hydrolysing processes (Fig 3A). The upregulated
genes in T16 were quite distinct from those upregulated in T8, despite both group of plants
having been P deprived for the same length of time (i.e. 8 d), which indicates that the responses
are influenced by plant development stage. A total of 10 DEGs were annotated to protein
ubiquitination, and they were mainly zinc finger domain containing proteins (Fig 3A and S4
Table). Several peptide transporters and an aquaporin protein (OsTIP4;1), which are involved
in urea transport, were upregulated in addition to four upregulated DEGs associated with
releasing ammonium or with the degradation of glutamine, that together were assigned to N
remobilisation (Fig 3A).
Nine TFs were upregulated in T16, mostly from the MYB family (Fig 3A and Table 4),
which are associated with a range of processes including protein synthesis, protein
ubiquitination and plant growth and stress response (Table 4). Eight upregulated DEGs were associated
with abiotic/biotic stress response, including universal stress protein (LOC_Os05g28740) and
hypothetical protein (Os05g0355450 ?no MSU ID matched) that showed 42% identity to the
universal stress protein in Zea mays. These two genes were highly expressed in T16, with
FPKM values of 1,252 and 1,056, respectively (S4 Table). Eight genes coding for proteins
involved in detoxification of ROS were upregulated, and these included thioredoxin (TRX),
glutaredoxin (OsGRX23), peroxidase and FAD dependent oxidoreductase (Fig 3A and S4
Interestingly, two enzymes assigned to carbohydrate metabolism, beta-amylase
(LOC_Os10g41550) and beta-fructofuranosidase (invertase: LOC_Os09g08120), were
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Plant growth and stress response
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upregulated in T16 (Fig 3A and S4 Table). Similarly, four DEGs assigned to cell wall
degradation, including two glycosyl hydrolases (OsXTH15 [LOC_Os06g22919 and
LOC_Os05g31140]) and glycosyltransferase (LOC_Os08g38710), were upregulated in T16,
which indicates that xyloglycan was catalysed, probably corresponding to cell wall degradation
(Fig 3A and S4 Table). In contrast to the nine photosynthesis related genes upregulated in T8,
Fig 3. The 100 most highly upregulated (a) downregulated (b) in T16. The number in the chart indicates the number of genes that are assigned in the
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only one photosystem II 10 kDa polypeptide (LOC_Os07g05365) was upregulated in T16, and
one phosphatase involved in phosphoryl transfer (LOC_Os03g49440) was upregulated (Fig
Highly downregulated genes at 16 DAA in response to P-withdrawal. Eleven of the
downregulated DGEs in T16 were assigned to protein synthesis and nine out of eleven were
specifically related to heat shock protein (Hsp) (S4 Table). In addition, three TFs in the HSF
(heat shock transcription factor) family were also downregulated (Fig 3B and Table 5). As
occurred in T8, several ERF family TFs that are involved in ethylene response and a bHLH
family TF that is involved in jasmonate signalling were also downregulated in T16 (Table 5).
Ten DEGs assigned to abiotic/biotic stress were also downregulated in T16 (Fig 3B and S4
Four DEGs involved in photosynthesis were downregulated, three of which were early
light-induced proteins (LOC_Os01g14410, LOC_Os07g08150, LOC_Os07g08160; Fig 3B and
Notably, six genes associated with seed storage proteins, including glutelin and cupin
(LOC_Os03g31360, LOC_Os01g55690, LOC_Os02g25640, LOC_Os06g09820, LOC_Os07g38630,
LOC_Os06g03390), were downregulated in T16. OsGRX2, a glutaredoxin, was downregulated, in
addition to a cytosolic ascorbate peroxidase (OsAPx2), an oxidoreductase (OsAKR2), a thioredoxin
protein (LOC_Os02g56900) and heavy metal-associated domain (LOC_Os10g38870) that are
involved in detoxification processes (Fig 3B). The downregulation of the glucose-6-phosphate/
phosphate translocator, OsGPT2-3 (LOC_Os07g33910) and the sucrose transporter OsSWEET13
(LOC_Os12g29220) indicates the downregulation of carbon metabolism in T16 (Fig 3B).
Expression of Pi transporters in flag leaves during grain filling
It has been well documented that Pi transporter family 1 (Pht 1; OsPT 1?13) genes are involved
in Pi uptake and distribution [
], so the expression of these genes was examined in flag
leaves during grain filling. Only four OsPT genes (OsPT1, OsPT4, OsPT5 and OsPT8) were
expressed at detectable levels (i.e. FPKM > 1). The expression of OsPT1 was the highest in
both treatments at both time points (FPKM > 40), followed by OsPT8 (FPKM > 10), while
OsPT4 and OsPT5 had lower expression levels (FPKM < 10) (Fig 4). Notably, OsPT1, OsPT4,
OsPT5 and OsPT8 were downregulated in T8, whereas they were not differentially expressed at
16 DAA (Fig 4).
Zinc finger/CCCH transcription factor, putative, expressed
CCT/B-box zinc finger protein, putative, expressed
MYB family transcription factor, putative, expressed
myb-like DNA-binding domain containing protein, putative, expressed
MYB family transcription factor, putative, expressed
MYB family transcription factor, putative, expressed
MYB family transcription factor, putative, expressed
MYB family transcription factor, putative, expressed
B-box zinc finger family protein, putative, expressed
AP2 domain containing protein, expressed
AP2 domain containing protein, expressed
Dehydration-responsive element-binding protein, putative, expressed
AP2 domain containing protein, expressed
Basic helix-loop-helix family protein, putative, expressed
C2H2 zinc finger protein, expressed
HSF-type DNA-binding domain containing protein, expressed
HSF-type DNA-binding domain containing protein, expressed
Heat stress transcription factor B-1, putative, expressed
No apical meristem protein, putative, expressed
While the response of young plants to P deprivation is well documented at the physiological
and molecular levels, little is known about the physiological and molecular regulation of P
mobilisation from senescing leaves during grain filling. We previously identified several genes
that showed expression profiles consistent with a role in P remobilisation from senescing rice
flag leaves, but in the absence of any specific P treatment, it was not possible to definitively
conclude their involvement in the liberation and transport of P from leaves during grain filling
Fig 4. The expression of the PHT 1 family of rice P transporters in rice flag leaves during grain filling. P values
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]. While the treatments imposed in the current study involved deprivation of P for 8 d
(either from anthesis to 8 DAA or from 8 DAA to 16 DAA), the absence of any consistent
upregulation of the 11 key PSR genes in T8 or T16 clearly demonstrates that the response
observed was distinct from the typical P deprivation response observed in younger plants
under P starvation (Table 1) and by the same token, emphasising the different metabolic states
present in young and mature plants.
Different response to P withdrawal depending on grain filling stage
Not only did the withdrawal of P from the nutrient solution during grain filling elicit a
different transcriptional response to that from young plants, but a distinct difference between the
response to 8 d of P deprivation during the early stage of grain filling (8 DAA) and the
response to 8 d of P deprivation at later stage (16 DAA) was observed. At 8 DAA, a total of 34
upregulated DEGs assigned to nucleic acid metabolism and photosynthesis were upregulated,
while at 16 DAA, only two nucleic acid metabolism genes were upregulated, and four
photosynthesis related genes were actually downregulated in T16 (Figs 2 and 3). Downregulation of
photosynthesis related genes is in accordance with the photosynthetic rate data from these
plants?reported in Jeong et al. [
]?where the withdrawal of P supply from anthesis to 8
DAA did not significantly reduce photosynthesis activity compared to the control plants
(14.5 ?mol CO2 m-2 s-1), while withdrawal of P supply at 8 DAA significantly reduced this
activity to 7.5 ?mol CO2 m-2 s-1 at 16 DAA compared to the control plants (14.8 ?mol CO2 m-2
s-1) (S1 Table). However, it is possible that the response observed is specific to the cultivar
studied (cv. IR64) and further research is needed to determine whether this response is
observed in a wider range of rice cultivars.
Response to P withdrawal at early stage of grain filling (8DAA)
At 8DAA, PPR proteins and RNA polymerase (OsRpoTp) were highly upregulated, indicating
activation of RNA metabolic processes in the chloroplast [35?38], that together wth the
upregulation of the whirly TF (LOC_Os06g05350) (S3 Table), indicate the correct functioning of
chloroplasts in T8 [
]. We also observed significant upregulation of photosystem II (PSII)
oxygen evolving complex protein PsbQ family protein which stabilises PsbP binding, thereby
contributing to the maintenance of the catalytic manganese (Mn) cluster of the water
oxidation machinery [
]. In addition, the significant upregulation of an aldose 1-epimerase family
protein (AEP; LOC_Os03g53710), indicated the activating of energy metabolism, since AEP
participates in the conversion of D-galactose to D-glucose 6-phosphate by either the Leloir
pathway or the pyrophosphorylase pathway [
]. This pathway is required since galactose itself
cannot be used for glycolysis directly. Taking these observations together, it appears that the
deprivation of P from anthesis to 8 DAA in plants that received adequate P supply during
vegetative growth induced a plant transcriptional response aimed at sustaining photosynthesis by
upregulation of genes directly involved in chloroplast and thylakoid functioning. This would
enable continued production of carbon substrates to be used in glycolysis and the TCA cycle
for sustained growth and to meet the carbon demands of the developing grains (Fig 5). The
most notable changes in gene expression among the 32 PRGs (those identified in the study of
Jeong et al. [
]) were the upregulation of three OsPAP genes (OsPAP15, OsPAP10a and
OsPAP1d) and downregulation of OsSPX-MFS1 and OsSPX-MFS3 (Table 2). Given that
OsPAP genes are known to play a role in liberating P from organic P sources [
17, 30, 42, 43
and OsSPX-MFS1 and OsSPX-MFS3 function as phosphate influx and efflux transporters in
rice, respectively [
], it is possible that P liberated from organic P sources was sufficient to
meet the competing P demands of developing grains and concurrent leaf photosynthetic
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Fig 5. Hypothesised response to P withdrawal at the cellular level during grain filling (A) at 8 DAA and (B) 16 DAA based on differentially expressed genes.
processes without requiring vacuolar P. Notably, the P demand of developing grains is
relatively low during this period; most P is loaded into grains from around 6 DAA to 15 DAA in a
sigmodal accumulation pattern [
], and hence, only a small proportion is accumulated in
the grains by 8 DAA. The downregulation of both OsSPX-MFS1 and OsSPX-MFS3 supports
the notion that plants had sufficient cytoplasmic P without the need to remove/use any P
stored in the vacuole (Table 2). Regardless, the data clearly support results from our earlier
] that implicated OsPAP15, OsPAP10a and OsPAP1d in the mobilisation of P from
Response to P withdrawal from 8 to 16 days after anthesis
In contrast to the response from plants to P deprivation between anthesis and 8 DAA (T8), the
overall response of plants to P deprivation between 8 DAA and 16 DAA (T16) appeared to be
accelerated leaf senescence. This involved downregulation of photosynthesis-related genes
including early light induced proteins (LOC_Os07g08150, LOC_Os07g08160 and
LOC_Os01g14410) and OsPsbS2 which is a light harvesting complex gene within PSII [
Parallel to these changes, the upregulation of polysaccharide degradation associated genes
such as beta-amylase (OsBAM5), beta-fructofuranosidase (OsCIN8) for degrading of storage
polysaccharides, and two glycosyl hydrolases (defensin OsDEFL9) and a glycosyltransferase
(LOC_Os08g38710) for degrading of structural polysaccharide (cell wall) were also observed
(Fig 5 and S4 Table). The large number of upregulated DEGs assigned to protein
ubiquitination and N remobilisation that indicated an increase in protein degradation [
], was also
consistent with accelerated leaf senescence in T16. Interestingly, the three OsPAP genes that were
upregulated in T8 also showed high expression levels in the control plants at 16 DAA (Fig 6),
suggesting that some degree of leaf senescence had already been initiated in these plants.
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Fig 6. The expression of three OsPAPs in rice flag leaves during grain filling. P values
OsSPX-MFS3, which plays a role in the efflux of P from the vacuole to maintain a constant
cytoplasmic P level  (Fig 5), was upregulated in T16 (Table 2). This may suggest that plants
were using vacuolar P stores to meet a critical P deficit in cells to maintain their function, or to
meet the high P demands of developing grains, however, leaf P fraction data from these plants
indicated no difference in flag leaf inorganic P (Pi) content between T16 plants and control
]. This anomaly may be due to a lack of resolution in the leaf P fraction data due to
inherent variability among biological replicates or in the methods used for fractionation. The
expression pattern of OsSPX-MFS3, however, is indicative of a role in P mobilisation from
senescing leaves, and further study of the function of this gene during grain filling is
Phosphorus is remobilised from old tissues to young tissues via the phloem [
predominantly as Pi [
]. Transport of Pi into the phloem is thought to occur mostly through the
action of members of the PHT1 family of transporters. In rice, six PHT1 family transporters
(OsPT1, OsPT2, OsPT6, OsPT8, OsPT9 and OsPT10) have been shown to play a role in P
remobilisation in the shoots of young plants [48?51], but the function of these transporters
during leaf senescence has not been fully resolved [
]. The absence of any upregulation of
PHT family transporters in T8 or T16 plants may suggest that their induction is not necessary
and that the basal activity present in control plants is enough to satisfy Pi transport along the
A recent study also revealed that a node specific transporter, sulfate transporter-like
phosphorus distribution transporter (SPDT) plays an important role in P remobilisation from old
tissues to young tissues through nodes in rice, with a 20% decrease in grain P content observed
in grains of an spdt mutant [
]. No differential expression of SPDT was found in our data
since SPDT is a gene specifically expressed in nodes (S3 Fig). Similar reductions in grain P
concentration were observed in rice mutants where the sulfate transporter OsSULTR3;3 was
knocked out [
]. While SPDT and OsSULTR3;3 provide potential targets for manipulation of
grain P content, resolving the role of three OsPAPs (OsPAP1d, OsPAP10a and OsPAP15) and
OsSPX-MFS3 in flag leaves during grain filling may lead to additional targets for manipulation
of genes to reduce P concentrations in the grains of rice.
15 / 19
S1 Fig. Schematic diagram of experimental design and timing of permanent P withdrawal from the nutrient solution.
S2 Fig. The expression of four OsSPX-MFSs in rice flag leaves during grain filling. Statisti
cal analysis was calculated individually by the sample T8 vs C8, T16 vs C16. P values <
0.001, no marks = non-significant.
S3 Fig. The expression of SPDT in rice flag leaves during grain filling. Statistical analysis
was calculated individually by the sample T8 vs C8, T16 vs C16. P values < 0.001, no
marks = non-significant.
S1 Table. Concentration of total phosphorus and phosphorus fractions in flag leaves, and photosynthetic rate in sampled flag leaves.
S2 Table. Sequencing reads and mapping statistics.
S3 Table. The list of 100 of most each up and downregulated differential expressed genes in T8.
S4 Table. The list of 100 of most each up and downregulated differential expressed genes in T16.
S5 Table. 32 PRGs analysed in the present study.
We would like to thank Dr. Hedia Tnani for help with the additional bioinformatical analysis.
O.P. was supported by a Sabbatical Fellowship by DGAPA-UNAM.
Conceptualization: Kwanho Jeong, Daniel Waters, Sigrid Heuer, Terry J. Rose.
Data curation: Kwanho Jeong, Omar Pantoja, Abdul Baten, Cecile C. Julia.
Formal analysis: Kwanho Jeong.
Funding acquisition: Matthias Wissuwa, Terry J. Rose.
Investigation: Kwanho Jeong, Cecile C. Julia.
Methodology: Kwanho Jeong, Omar Pantoja, Daniel Waters, Tobias Kretzschmar, Terry J.
Project administration: Terry J. Rose.
Software: Kwanho Jeong, Abdul Baten.
Supervision: Daniel Waters, Matthias Wissuwa, Sigrid Heuer, Terry J. Rose.
16 / 19
Validation: Daniel Waters, Terry J. Rose.
Writing ? original draft: Kwanho Jeong, Daniel Waters, Terry J. Rose.
Writing ? review & editing: Kwanho Jeong, Omar Pantoja, Abdul Baten, Daniel Waters,
Tobias Kretzschmar, Matthias Wissuwa, Cecile C. Julia, Sigrid Heuer, Terry J. Rose.
17 / 19
18 / 19
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