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. 2019 Mar 29;180(2):952–965. doi: 10.1104/pp.18.01385

OsPINOID Regulates Stigma and Ovule Initiation through Maintenance of the Floral Meristem by Auxin Signaling1

Meng Xu a,2, Ding Tang b,2, Xinjie Cheng a, Jianxiang Zhang a, Yujie Tang a, Quandan Tao a, Wenqing Shi b, Aiqing You c, Minghong Gu a, Zhukuan Cheng b,3, Hengxiu Yu a,3,4
PMCID: PMC6548252  PMID: 30926655

The OsPID-mediated auxin signaling pathway maintains the floral meristem and thereby regulates stigma and ovule initiation in rice.

Abstract

Stigma and ovule initiation is essential for sexual reproduction in flowering plants. However, the mechanism underlying the initiation of stigma and ovule primordia remains elusive. We identified a stigma-less mutant of rice (Oryza sativa) and revealed that it was caused by the mutation in the PINOID (OsPID) gene. Unlike the pid mutant that shows typical pin-like inflorescences in maize (Zea mays) and Arabidopsis (Arabidopsis thaliana), the ospid mutant does not display any defects in inflorescence development and flower initiation, and fails to develop normal ovules in most spikelets. The auxin activity in the young pistil of ospid was lower than that in the wild-type pistil. Furthermore, the expression of most auxin response factor genes was down-regulated, and OsETTIN1, OsETTIN2, and OsMONOPTEROS lost their rearrangements of expression patterns during pistil and stamen primordia development in ospid. Moreover, the transcription of the floral meristem marker gene, OSH1, was down-regulated and FLORAL ORGAN NUMBER4, the putative ortholog of Arabidopsis CLAVATA3, was up-regulated in the pistil primordium of ospid. These results suggested that the meristem proliferation in the pistil primordium might be arrested prematurely in ospid. Based on these results, we propose that the OsPID-mediated auxin signaling pathway plays a crucial role in the regulation of rice stigma and ovule initiation by maintaining the floral meristem.

In angiosperms, flower organs are essential for the diploid-to-haploid state transition and syngamy that reconstitutes a diploid genome through the processes of meiosis and fertilization, respectively (Walbot and Evans, 2003; Coelho et al., 2007). Flowers consist of sepals, petals, stamens, and carpels. The four distinct types of organs show orderly development in four distinct whorls (Coen and Meyerowitz, 1991). Previous studies in two eudicots, Arabidopsis (Arabidopsis thaliana) and snapdragon (Antirrhinum majus), established a proposed genetic ABCDE model to explain how the flower organs are specified (Coen and Meyerowitz, 1991; Theissen and Saedler, 2001; Robles and Pelaz, 2005; Causier et al., 2010; Rijpkema et al., 2010). Although the specification of most flower organs is well explained by this model, the specification of the stigma and ovule remains elusive.

The shoot meristem is an indeterminate proliferative organ that generates new organ primordia by cell division and differentiation (Reinhardt et al., 2000). Floral meristems (FM) are derived from shoot meristems and give rise to flowers and the floral organs (Smyth et al., 1990). The FM is similar to the shoot meristem but has a different fate. The FM terminates its stem cell fate appropriately after all flower organs have been specified (Clark et al., 1997). The auxin maximum in the meristem results in polar auxin transport driven by the polar subcellular localization of the major auxin efflux carriers, the PIN-FORMED (PIN) family of proteins (Benková et al., 2003). In the meristem, auxin maxima form not only at the peripheral zone to promote new organ initiation and differentiation but also at the center zone to maintain stem cell identity in the meristem (Vernoux et al., 2011; Luo et al., 2018).

The AUXIN RESPONSE FACTORs (ARFs) play pivotal roles in auxin signaling transduction by regulating the expression of auxin response genes (Salehin et al., 2015). ARFs bind to specific auxin response elements in the promoters of their target genes (Korasick et al., 2014; Chandler, 2016). Studies in Arabidopsis have shown that ARF3/ETTIN (ETT) and ARF5/MONOPTEROS (MP) have complicated functions in regulating stem cell identity in the FM (Zhao et al., 2010; Liu et al., 2014; Zhang et al., 2018). Indeed, ETT participates in FM maintenance and determinacy by indirectly regulating cytokinin biosynthesis, which plays a pivotal role in the activation of the stem cell maintenance gene WUSCHEL (WUS; Sessions et al., 1997; Han et al., 2014; Schaller et al., 2014; Zhang et al., 2018). Moreover, ETT is also involved in the establishment of abaxial-adaxial polarity in organ development. Mutations in ETT cause abaxial identity defects and apical-basal patterning defects in the gynoecium and an enlarged stigma in Arabidopsis (Sessions and Zambryski, 1995; Sessions et al., 1997; Pekker et al., 2005). In rice (Oryza sativa), OsETT1/OsARF15 and OsETT3/OsARF3 are involved in the adaxial-abaxial polarity in anther patterning (Toriba et al., 2010). The MP protein is not only located in differentiated cells at the peripheral zone but also in undifferentiated cells in the central zone (Zhao et al., 2010). MP can respond to auxin maxima at the central zone and indirectly promote WUS expression to stabilize stem cell homeostasis (Luo et al., 2018). In addition, MP also has a crucial role in promoting flower primordia initiation (Yamaguchi et al., 2013). Mutations in MP cause defects in lateral floral primordia initiation, resulting in the naked inflorescence phenotype in Arabidopsis, similar to pin1 mutants (Przemeck et al., 1996). In rice, the function of OsMP/OsARF11 has yet to be reported.

Polar localization of the PIN protein depends on its phosphorylation state that is mediated by a Ser/Thr protein kinase, PINOID (PID), which, together with the PP2A phosphatase, acts antagonistically on PIN apical-basal targeting (Friml et al., 2004; Kaplinsky and Barton, 2004; Michniewicz et al., 2007). Moreover, PID can affect PIN activity by phosphorylating PIN at specific phosphosites, and the activation of PIN is crucial for auxin efflux (Zourelidou et al., 2014). In Arabidopsis, pin1 mutant plants show a strong defect in lateral primordia initiation, and they produce pin-like inflorescences (Okada et al., 1991). Mutations in PID also cause a pin-like inflorescence phenotype (Bennett et al., 1995; Christensen et al., 2000; Haga et al., 2014). In maize (Zea mays), BARREN INFLORESCENCE2 (BIF2) encodes the ortholog of PID, and the bif2 mutant displays a pin-like inflorescence phenotype (McSteen et al., 2007; Pressoir et al., 2009). Constitutive overexpression of OsPID, the PID ortholog in rice, caused abnormal stamens and pistils in transgenic plants (Morita and Kyozuka, 2007). These results suggest that auxin signaling is crucial for organ morphogenesis, although the molecular mechanism remains elusive.

The genes that specifically control stigma and simultaneously ovule initiation have not been reported in plants. In this study, we found that OsPID is crucial for stigma and ovule initiation and the establishment of anther adaxial-abaxial polarity in rice. OsPID has an important role in auxin efflux in an activating OsPIN1 manner, which acts to form an optimal local auxin concentration in the pistil primordium. This auxin signaling is required to maintain the stem cell identity of FM. Moreover, OsETT1, OsETT2, and OsMP may play important roles in the regulation of stigma and ovule initiation via the auxin signaling response in the pistil primordium. These results shed light on the mechanism of stigma and ovule initiation in rice.

RESULTS

Characterization of a Stigma-Less Rice Mutant

A stigma-less mutant was identified from the progeny of a japonica rice variety, Wuxiangjing 9, treated with 60Co γ-rays. The phenotype of this mutant closely resembles that of the wild type during the vegetative stage (Fig. 1A). However, a truncated pistil without stigmas and six inward-curving anthers were observed in mature spikelets of the mutant plants (Fig. 1E). The mature pollen grains of the mutant can be stained with iodine-potassium iodide solution (Fig. 1F). When pistils of the wild-type plants were pollinated with pollen from the mutant, they set grains normally, indicating that the microspores are viable in this mutant. In the segregating population, plants with a normal phenotype and stigma-less mutants were present in a 3:1 ratio, suggesting that this mutant harbors a recessive mutation in a gene involved in stigma development (308:92, χ2 = 0.75, P > 0.05).

Figure 1.

Figure 1.

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Identification of the ospid mutant. A, Morphological comparison of wild type (left) and ospid mutant (right) plants. B, Schematic representation of the OsPID gene and the mutations in the ospid-1, ospid-2, and ospid-3 alleles. Coding regions are shown as black boxes, and untranslated regions are shown as gray boxes. C, The ospid mutant shows no defects in the inflorescence. Left, Wild type; right, ospid. D, The ospid mutant shows no defects in the spikelet. Left, Wild type; right, ospid. Bars = 2 mm. E, Comparison of floral organs between the wild type (top) and ospid (bottom). Left, Wild-type and ospid flowers shown with the palea and lemma removed. Bars = 1,000 μm. Middle, The ospid mutant has smaller, inward-curving anthers compared with the wild type. Bars = 500 μm. Right, The ospid mutant has a truncated pistil with no stigma. Bars = 200 μm. F, Pollen grains from wild-type (top) and ospid (bottom) plants were stained with 1% (w/v) iodine-potassium iodide solution. Bars = 100 μm. G, Anthers from wild-type (top) and ospid (bottom) plants stained red with Alexander’s stain (Alexander, 1969). Bars = 500 μm.

Isolation of PID in Rice

To characterize the molecular defect in the ospid-1 mutant, we performed map-based cloning to isolate the gene. A mapping population was constructed by crossing a mutant plant with an indica variety, 9311. A total of 1,023 F2 and F3 plants with mutant phenotypes were used for gene mapping. Linkage analysis mapped the target gene to the long arm of chromosome 12. The target gene was further delimited to a 90-kb chromosomal region between 26.02 and 26.11 Mb (Supplemental Fig. S1). According to the Rice Genome Annotation Project database (http://rice.plantbiology.msu.edu/cgi-bin/gbrowse/rice), there are 11 putative genes in this region. Comparative sequence analysis revealed a 34-bp deletion in the coding region of LOC_Os12g42020. The deletion was predicted to cause mistranslation of LOC_Os12g42020 at the C terminus (Supplemental Fig. S2B).

The target gene, LOC_Os12g42020, is predicted to encode a Ser/Thr protein kinase and has no introns (Fig. 1B). Sequence analysis showed that LOC_Os12g42020 shares a high degree of similarity with PID from Arabidopsis (Christensen et al., 2000; Supplemental Figs. S3 and S4). Indeed, Morita and Kyozuka (2007) had previously characterized LOC_Os12g42020 as OsPID, based on the results of a phylogenetic analysis. We therefore named the mutant ospid-1.

Additionally, we identified another two mutant alleles of ospid-1 using a map-based cloning approach (Fig. 1B). Both the additional allelic mutants have identical plant phenotypes to ospid-1, particularly with respect to the truncated pistils and the absence of stigmas (Supplemental Fig. S2A). In the ospid-2 mutant, we found that the T at nucleotide position 631 is deleted, which leads to premature termination of translation. In the ospid-3 mutant, we found an A insertion between nucleotides 1,265 and 1,266, which causes a predicted reading frame shift and mistranslation of the mRNA (Fig. 1B; Supplemental Fig. S2B). We crossed ospid-1 heterozygous plants with ospid-2 heterozygotes and found that the F1 hybrids carrying the two different OsPID mutations had abnormal pistils without stigmas. These results further confirm that OsPID is the target gene and that mutation results in the observed ospid-1 phenotype.

Mutations in OsPID Lead to Defects in Stigma Primordia Initiation and Disturbances in Anther Patterning

To further explore the reasons why the ospid mutant fails to form normal stigmas, we performed scanning electron microscopy analysis to observe the developmental progression of the pistil in mutant and wild-type flowers. A previous study divided the developmental sequence of the spikelet into several different stages (Ikeda et al., 2004). In this study, we focused on stages Sp6 to Sp8, and we subdivided stage Sp8 into Sp8a (early) and Sp8b (late) to facilitate the analysis. In the wild type, six rod-like stamen primordia are formed in the whorl, and a pistil primordium is formed in the center of the spikelet at stage Sp6 (Fig. 2A). Subsequently, the carpel primordium differentiates from the lemma side of the FM at stage Sp7, and the FM remains on the palea side. At the same time, the stamen differentiates into filaments and anthers. At this point, it is apparent that four protuberant early anther locules are formed at the four corners of the anther and appear butterfly shaped (Fig. 2A). At stage Sp8a, the ovule primordium is differentiated from the FM and is enclosed by the carpel. Accompanied by ovule primordium initiation, two stigma primordia are generated from the palea side of the carpel, and the establishment of anther patterning is accompanied by the formation of a bilaterally symmetrical structure with four lobes (Fig. 2A). The anther includes two thecae that are linked by connective tissue symmetrically. Each theca contains two locules: the one on the abaxial side is longer at the base and the other on the adaxial side is shorter (Fig. 2, A and B). Stage Sp8a is also recognized as the stage when the FM loses indeterminacy (Ikeda et al., 2004). At stage Sp8b, the two stigmas protrude, and papillae are formed on the epidermis of the stigmas (Fig. 2A).

Figure 2.

Figure 2.

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Stigma primordia initiation, ovule formation, and anther patterning are severely affected in the ospid mutant. A, Scanning electron micrographs of young spikelets from wild-type (top) and ospid (bottom) plants at several critical stages of development (Sp6, Sp7, Sp8a, and Sp8b). Bars = 50 μm. B, Transverse sections of young pistils and anthers from wild-type (top) and ospid (bottom) plants were stained with 0.25% (w/v) Toluidine Blue O. Red arrowheads indicate the vascular bundles leading to the stigma. Bars = 20 μm. C, Observation of the mature embryo sac in ovaries from the wild type (left) and the ospid mutant (center and right) by confocal microscopy. The ospid mutant produces two types of ovary: normal (center) and ovule-less (right). Bars = 50 μm. D, Histogram showing the number of normal and ovule-free ovaries in wild-type and ospid plants. AN, Antipodal cell nuclei; Ca, carpel; Cal, carpel-like tissue; EN, egg nucleus; Le, lemma; Pa, palea; PN, polar nuclei; St, stamen; Sti, stigma.

In the ospid mutant, the morphological features of the pistil primordium are normal at stages Sp6 and Sp7, but stamen primordia appear irregular in shape rather than rod like at stage Sp6 and appear butterfly shaped at stage Sp7, compared with the wild type. At stage Sp8a, when the carpel encloses its internal tissues, no papillary stigma primordium is generated from the palea side of the carpel in ospid. Mutant stamens form two inward-curving anther locules on the abaxial side and two severely defective anther locules on the adaxial side (Fig. 2A). Finally, the ospid mutant forms six defective anthers and a truncated pistil without stigmas (Figs. 1E and 2A).

The ospid Mutant Developed Vasculature-Free Carpels and Abaxial-Adaxial Extremely Asymmetrically Developed Anthers

Analysis of transverse sections indicated that each theca of the mutant anthers formed only one normal locule on the abaxial side, and the one on the adaxial side either was smaller than the wild type with fewer and developmentally delayed microspores or failed to form the characteristic four-layered anther walls and microspores (Fig. 2B). The cytological observations also found that the pistil of ospid failed to form the vasculatures leading to the stigmas. In the wild type, when the early carpel differentiated from FM at stage Sp7, no vasculature was formed. Subsequently, accompanied by two stigma primordia initiating from carpel, the cells under stigma primordia began to differentiate and form early immature vasculatures at stage Sp8a (Fig. 2B). At stage Sp8b, two vasculatures leading to the stigmas were obviously observed in carpel. No obvious difference was detected in FM and carpel at stages Sp6 and Sp7 between ospid and the wild type. Nevertheless, vasculature failed to differentiate from the carpel of ospid at stage Sp8a, and no vasculature was observed at stage Sp8b. Accordingly, the obvious difference between the wild type and ospid appeared from the Sp8a stage. The vasculatures leading to stigmas have never been formed in the ospid mutant, suggesting that the stigma primordia failed to initiate rather than developmental defect or arrest (Fig. 2B).

The Pistil of the ospid Mutant Sometimes Failed to Form a Normal Embryo Sac

To further determine whether the ospid mutant can form normal ovules, we observed the mature embryo sac via confocal microscopy of the ovaries. In the wild type, the mature pistil forms an eight-nucleate embryo sac that contains two synergid nuclei, one egg nucleus, two polar nuclei, and three antipodal cell nuclei (Fig. 2C). Interestingly, 21.6% (19/88) of the pistils formed eight-nucleate embryo sacs in ospid, but 78.4% (69/88) of the pistils failed to form an embryo sac. Instead, it was occupied by carpel-like tissue (Fig. 2, C and D).

OsPID Gene Expression Pattern

To determine the expression pattern of OsPID, we performed reverse transcription quantitative PCR (RT-qPCR) analysis, GUS staining, and RNA in situ hybridization. RT-qPCR analysis showed that OsPID is strongly expressed in the young inflorescence and that expression declines in the older inflorescence (Fig. 3, A and B). GUS staining results were consistent with the results of RT-qPCR analysis (Supplemental Fig. S5). The GUS signals reached a peak in young inflorescences and young spikelets, and the signal decreased as inflorescence and spikelet development progressed. Indeed, stigma primordium initiation and the establishment of anther patterning overlapped with the OsPID expression peak.

Figure 3.

Figure 3.

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Expression pattern of OsPID and subcellular localization of the OsPID protein. A, Spatial and temporal expression analyses of OsPID in wild-type tissues by RT-qPCR. B, RT-qPCR analysis of OsPID expression was performed in wild-type inflorescences at different developmental stages. Data represent means ± sd. All experiments were repeated three times in terms of one sample used and three experiments performed. C, In situ analysis of OsPID expression in wild-type spikelets at different developmental stages. Bars = 200 μm. D, Subcellular localization of OsPID in rice protoplasts. GFP was fused in frame to either the C-terminal or N-terminal end of OsPID. The nuclear protein marker OsMADS3 was fused to red fluorescence protein (mCherry). Bars = 10 μm. Ca, Carpel; DIC, differential interference contrast; P0.5, 0.5-cm-long panicle; P1, 1-cm-long panicle; P2, 2-cm-long panicle; P3, 3-cm-long panicle; P4, 4-cm-long panicle. P5, 5-cm-long panicle; P10, mixed panicles greater than 10 cm in length; Pa, palea; Le, lemma; St, stamen.

RNA in situ hybridization was performed using a digoxigenin-labeled antisense RNA probe from the 3′ end of the OsPID gene. The results showed that OsPID is transiently expressed in young spikelets. OsPID is expressed strongly in the stamen primordia and the pistil primordium at stage Sp6. Subsequently, the OsPID expression level decreased. OsPID expression was detected in the peripheral zone of the stamen primordium tip and at both sides of the pistil primordium during its development. At stage Sp7, OsPID expression was further reduced, and only a weak signal was detected at the stamen tip and at the palea side of the carpel. After stage Sp8, the OsPID signal was beneath the detection threshold in the spikelet (Fig. 3C). These results suggest that OsPID plays a pivotal role in promoting pistil and stamen development.

OsPID Localizes to the Nucleus and Cytoplasm

To determine the subcellular localization of OsPID, we made a DNA construct in which the GFP was fused in frame to OsPID under the control of the Cauliflower mosaic virus (CaMV) 35S promoter, and this was used in a transient expression assay in rice protoplasts. The OsMADS3-mCherry fusion protein was used as a nuclear localization marker in the assay (Gao et al., 2013). We found that OsPID localized to the nucleus and cytoplasm regardless of whether the GFP was fused to the N terminus or the C terminus of the OsPID protein (Fig. 3D).

The ospid Mutation Reduces Auxin Activity in the Young Pistil

In Arabidopsis, AtPIN1, a PIN class of auxin efflux carrier proteins, mediates polar auxin transport to control organ initiation. The polarity of the subcellular distribution of AtPIN1 is mediated by PID. To further investigate whether OsPIN1 polarity is affected in the ospid mutant, we performed an immunocytochemical assay to analyze the immunofluorescence signals of OsPIN1 in the pistil primordium at developmental stage Sp6. However, there was no obvious difference in OsPIN1 polar subcellular distribution in the pistil primordium between ospid and the wild type (Fig. 4A). Nevertheless, PID can affect PIN activity, and the activation of PIN is crucial for auxin efflux (Zourelidou et al., 2014). Local auxin distribution in the young pistil of ospid would be different from the wild type if the activation of OsPIN1 was affected in ospid. To test this hypothesis, the widely used DR5 reporter system, which consists of a synthetic auxin-responsive promoter driving the GUS gene, was used to visualize auxin activity gradients (Ulmasov et al., 1997; Benková et al., 2003; Friml et al., 2003; Michniewicz et al., 2007). Compared with the wild type, auxin activity in the young pistil was dramatically decreased to the point that it was below the level of detection in ospid (Fig. 4B; Supplemental Fig. S6). Thus, we can conclude that ospid may perturb OsPIN1 activity and affect polar auxin transport in the pistil primordia. These results suggest that auxin activity plays an important role in the development of pistil primordia.

Figure 4.

Figure 4.

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Auxin activity is reduced in the pistil primordium of ospid. A, Immunolocalization of OsPIN1 using the α-OsPIN1 antibody in pistil primordia from the wild type (left) and the ospid mutant (right). Bars = 100 μm. B, Auxin activity in spikelet primordia and young pistils from the wild type (left) and ospid (right) is shown by GUS staining. Auxin activity in the ospid young pistil is less than in the wild type. GUS gene expression was driven by the DR5 promoter. Bars = 200 μm. C, RT-qPCR analysis shows that expression of the auxin response factor genes OsEET1, OsETT2, OsETT3, and OsMP is down-regulated significantly in the ospid young inflorescence. Data represent means ± sd. All experiments were repeated three times in terms of one sample used and three experiments performed. Student’s t test: **, P < 0.01 and *, P < 0.05. Ca, Carpel; Ov, ovule; Sp, spikelet primordium; St, stamen; Sti, stigma.

Spatial Expression Patterns of OsETT1, OsETT2, and OsMP Are Perturbed in Stamen and Pistil Primordia of ospid

Auxin activity is reduced in ospid. To further explore the role of auxin signaling in stamen patterning and stigma initiation, we examined the expression patterns of OsETT1, OsETT2, OsETT3, and OsMP in stamen and pistil primordia. Only the expression pattern of OsETT3 showed no obvious difference between the wild type and ospid (Supplemental Fig. S7). The expression patterns of OsETT1, OsETT2, and OsMP were altered in ospid. The spatial expression patterns of OsETT1, OsETT2, and OsMP showed marked rearrangements during stamen and pistil development in the wild type, but this rearrangement did not occur in ospid (Figs. 5–7).

Figure 5.

Figure 5.

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The expression pattern of OsETT1 is perturbed in ospid. A, In situ expression analysis of OsETT1 in spikelets from wild-type (left) and ospid (right) plants at different developmental stages. The arrowheads indicate three OsETT1 expression domains in the wild-type carpel and one in the ospid carpel. Ca, Carpel; Le, lemma; Pa, palea; St, stamen. Bars = 100 μm. B and C, Micrographs and diagrams showing the rearrangement of the OsETT1 expression patterns between wild-type (left) and ospid (right) stamens (B) and carpels (C) at different developmental stages. Dashed regions in the micrographs indicate stamens (B) and carpels (C). The arrowheads indicate three OsETT1 expression domains in the wild-type carpel and one in the ospid carpel. Blue areas represent the OsETT1 transcription signal. Bars = 100 μm.

Figure 7.

Figure 7.

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The expression pattern of OsMP during the early stages of spikelet development, and kinetic analysis of OsETT1, OsETT2, and OsMP. A, In situ expression analysis of OsMP in wild-type (left) and ospid mutant (right) spikelets at different developmental stages. Top row, spikelets; center row, higher magnification of the carpel from the spikelet in the top row; bottom row, higher magnification of the pistil from the spikelet in the top row. Dashed regions in micrographs indicate carpels (center row) and stamens (bottom row). The arrowheads show the three OsMP expression domains in the wild-type carpel and the single domain in the ospid mutant carpel. Ca, Carpel; Le, lemma; Pa, palea; St, stamen. Bars = 100 μm. B, Gene expression kinetic analysis of OsETT1, OsETT2, and OsMP in response to IAA. Data represent means ± sd. All experiments were repeated three times in terms of one sample used and three experiments performed. Student’s t test: **, P < 0.01 and *, P < 0.05.

In the wild type, expression of OsETT1 is restricted to the abaxial regions of stamen primordia and the palea side of pistil primordium at stage Sp6. OsETT1-specific transcripts were detected on both the adaxial and abaxial sides of the initial axis in stamens at stage Sp7. The OsETT1 expression domain in pistil primordium was expanded. After the initiation of the ovule primordium at stage Sp8a, the OsETT1 expression level declined markedly and formed three expression domains at the palea side of pistil primordium: one behind the ovule primordium and two that correspond to regions of stigma primordia. At this stage, OsETT1 transcripts are restricted to the marginal regions between two thecae (Fig. 5). In ospid, we found that OsETT1 transcription was down-regulated markedly compared with the wild type. We failed to detect the expression domains in the stamen primordia and pistil primordium at stages Sp6 and Sp7. Interestingly, at stage Sp8a, the one OsETT1 expression domain behind the ovule primordium was detectable only in a small number of ospid mutant spikelets (Fig. 5), but we were unable to detect the other two domains at the carpel margins in all spikelets of ospid (Supplemental Fig. S8A). OsETT1 seems to be expressed relatively strongly at the adaxial side of ospid stamen primordia.

In the wild type, OsETT2 showed diffuse expression in the stamen and pistil primordia at stage Sp6. The expression level increased and was restricted to the palea side of the pistil primordium at stage Sp7. Subsequently, OsETT2 expression resolved into two domains, which corresponded to the regions of stigma primordia at stage Sp8a. OsETT2 transcription in the stamen was detected at the back of each anther locule and surrounded the initial axis at stage Sp8a (Fig. 6). In ospid, OsETT2 expression was down-regulated compared with the wild type. In particular, we were unable to detect any obvious OsETT2 signal in the pistil primordium of ospid. We also found that the OsETT2 expression patterns were perturbed during stamen development (Fig. 6).

Figure 6.

Figure 6.

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The expression pattern of OsETT2 is perturbed in ospid. A, In situ expression analysis of OsETT2 in wild-type (left) and ospid mutant (right) spikelets at different developmental stages. The arrowheads indicate two OsETT2 expression domains in the wild-type carpel. Ca, Carpel; Le, lemma; Pa, palea; St, stamen. Bars = 100 μm. B and C, Micrographs and diagrams showing the rearrangement of the OsETT2 expression pattern in stamens (B) and carpels (C) from the wild type (left) and the ospid mutant (right) at different developmental stages. Dashed regions in micrographs indicate stamens (B) and carpels (C). The arrowheads indicate three OsETT2 expression domains in the wild-type carpel and one in the ospid carpel. Blue areas represent the OsETT2 transcription signal. Bars = 100 μm.

In the wild type, OsMP expression domains were found to correspond to vascular bundles in stamens. OsMP transcription was diffuse throughout the entire pistil primordium at stage Sp7. Subsequently, the signal for OsMP in pistil primordium gradually condensed, forming three expression domains similar to what was observed for OsETT1: one domain was behind the ovule primordium and two corresponded to the stigma primordia (Fig. 7A). In ospid, OsMP signals were much weaker than in the wild type. This was especially true in the pistil primordium at stage Sp8a, where the one expression domain behind the ovule primordium was detectable only in a small number of ospid spikelets (Fig. 7A) but the other two domains at the carpel margins were not detected in all pistil primordia of ospid (Supplemental Fig. S8B). These results indicate that OsETT1, OsETT2, and OsMP may play important roles in rice flower development.

OsETT1, OsETT2, and OsMP Are Auxin Regulated

To test whether OsETT1, OsETT2, and OsMP are auxin regulated, analysis of the gene expression kinetic responding to auxin signal was performed. Ten-day-old seedlings of the wild type were treated with the exogenous auxin indole-3-acetic acid (IAA), and untreated seedlings were used as a control. The treated and untreated seedlings were harvested within a certain interval of time. The kinetic analysis showed that expression levels of OsETT1, OsETT2, and OsMP were dramatically up-regulated within 1 h. The expression of these genes increased significantly and was maintained for at least 4 h (Fig. 7B). These results indicate that OsETT1, OsETT2, and OsMP can respond to auxin signal rapidly.

Most ARFs Are Down-Regulated in the ospid Mutant

There are 25 ARF genes in the rice genome (Wang et al., 2007). We examined the expression of these ARF genes in early inflorescence in both the wild type and ospid using an RT-qPCR assay. Although we were unable to detect expression of OsARF13 and OsARF20, the other 23 genes were found to be expressed in early inflorescence at various levels. Our results showed that only OsARF1 and OsARF7 were expressed at comparable levels in the wild type and ospid and that other ARF expression levels of ospid were lower than in the wild type. Moreover, 15 ARFs were significantly down-regulated in the early inflorescence of ospid (Supplemental Fig. S9); these genes included OsETT1, OsETT2, and OsMP (Fig. 4C). These results are consistent with the low auxin activity in the early pistil of ospid. Nevertheless, the functions of these ARF genes that show different expression patterns between the wild type and the ospid mutant in flower development will require further study.

The FM Arrests Prematurely in the ospid Pistil Primordium

Previous studies have shown that ETTIN and MP have pivotal roles in FM maintenance (Liu et al., 2014; Luo et al., 2018; Zhang et al., 2018). The expression patterns of OsETT1, OsETT2, and OsMP were perturbed in the pistil primordium of ospid. The ovule initiates directly from the FM in rice (Colombo et al., 2008), but the ovule failed to form in most ospid mutant pistils. Based on this evidence, we speculate that the FM may have lost its homeostasis in the pistil primordium of ospid. To test this hypothesis, we analyzed the spatial expression pattern of OSH1, which is a molecular marker of meristematic indeterminate cells in rice (Sato et al., 1996; Kurakawa et al., 2007). In the wild type, expression of OSH1 is restricted to the palea side of pistil primordium at stage Sp6 (Fig. 8A). After the carpel is formed at stage Sp7, OSH1 transcription is reduced dramatically because the FM is exhausted by initiating the ovule primordium (Yamaki et al., 2011). Consistent with our hypothesis, the OSH1 transcription signal in ospid was weaker than in the wild type at stage Sp6 (Fig. 8A). In Arabidopsis, the stem cell population is strictly maintained by WUS and CLAVATA3 (CLV3; Mayer et al., 1998; Schoof et al., 2000). We also analyzed the expression patterns of the FLORAL ORGAN NUMBER4 (FON4) gene, the putative ortholog of Arabidopsis CLV3, and MOC3, the putative ortholog of Arabidopsis WUS (Chu et al., 2006; Suzaki et al., 2006; Lu et al., 2015; Tanaka et al., 2015). The FON4 transcription signal was detectable in the ospid mutant carpel at stage Sp7, while the transcript of FON4 could not be detected in the wild type during the same stage Sp7 (Fig. 8B). We were unable to detect the transcription signal of MOC3 in either the wild type or the ospid mutant. It may be possible that some other unknown gene exerts the function of WUS to maintain stem cell identity in rice FM rather than MOC3. RT-qPCR analysis also showed that OSH1 expression was down-regulated significantly (Fig. 8C) and FON4 expression was up-regulated significantly (Fig. 8D) in the early inflorescence of ospid. The defect of stigma primordium and ovule in ospid may result from premature arrest of FM. Although ovules sometimes form normally, suggesting that the machinery for ovule formation is intact in ospid, ovules may fail to form if the meristem arrests too soon. These results suggest that the meristem proliferation arrests prematurely during pistil primordium development in ospid.

Figure 8.

Figure 8.

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The expression patterns of OSH1 and FON4 during the early stages of spikelet development. A, In situ expression analysis of OSH1 in wild-type (left) and ospid (right) spikelets at stages Sp6 and Sp7. Ca, Carpel; Le, lemma; Pa, palea; St, stamen. Bars = 100 μm. B, In situ expression analysis of FON4 in wild-type (left) and ospid (right) spikelets at stage Sp7. Bars = 20 μm. C, RT-qPCR analysis showing that OSH1 expression is down-regulated significantly in the ospid young inflorescence. D, RT-qPCR analysis showing that FON4 expression is up-regulated significantly in the ospid young inflorescence. Data represent means ± sd. All experiments were repeated three times in terms of one sample used and three experiments performed. Student’s t test: **, P < 0.01.

The Mechanism Controlling Stigma Development May Differ between Rice and Arabidopsis

In Arabidopsis, four genes, HECATE1 (HEC1), HEC2, HEC3, and SPATULA (SPT), have been identified as being important for stigma development. The gynoecium completely lacks stigmatic tissue in the hec1 hec2 hec3 triple mutant (Gremski et al., 2007; Schuster et al., 2015). The spt mutant affects apical carpel fusion and has a reduced amount of stigmatic tissue (Heisler et al., 2001; Alvarez and Smyth, 2002). To investigate whether rice shares a similar regulatory mechanism, we analyzed the expression patterns of OsHEC1, OsHEC2, OsHEC3, and OsSPT genes, which are the rice putative orthologs of Arabidopsis HEC1, HEC2, HEC3, and SPT, respectively. In the wild type, we could not detect any expression of OsHEC1, OsHEC2, and OsSPT in the pistil primordium by RNA in situ hybridization analysis. Only OsHEC3 could be detected in the pistil primordium, but there was no obvious difference between ospid and the wild type (Fig. 9). The mechanism(s) controlling stigma development in rice may differ from that in Arabidopsis.

Figure 9.

Figure 9.

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In situ expression analysis of OsHEC1, OsHEC2, OsHEC3, and OsSPT in spikelets at stage Sp8a. Expression signals of OsHEC1 (A), OsHEC2 (B), and OsSPT (D) could not be detected in young spikelets from both the wild type (left) and the ospid mutant (right). OsHEC3 (C) was expressed in young spikelets but showed no obvious difference between the wild type (left) and ospid (right). Ca, Carpel; Le, lemma; Pa, palea; St, stamen. Bars = 100 μm.

DISCUSSION

The Function of OsPID in Rice

Previous studies have indicated that PID controls polar auxin transport by phosphorylating PIN proteins (Friml et al., 2004; Kaplinsky and Barton, 2004; Michniewicz et al., 2007). Morita and Kyozuka (2007) showed that OsPID may be involved in the control of polar auxin transport. We have not found the alteration of OsPIN1 polarity in the pistil or stamen primordia of ospid. However, the auxin concentration is reduced in the young pistil of ospid. PID can also affect PIN activity by phosphorylating PIN at specific phosphosites, and the activation of PIN is crucial for auxin efflux (Zourelidou et al., 2014). Taken together, OsPID may exert its function by affecting the OsPIN1 activity. Precise local auxin concentration formation in FM is driven by OsPIN1, and it is crucial for stigma and ovule initiation.

Mutations in PID produce pin-like inflorescences in both Arabidopsis and maize (Bennett et al., 1995; McSteen et al., 2007). Moreover, mutations in ZmPID can suppress the increased vegetative branch (tiller) phenotype of the tb1 mutant, indicating that ZmPID has a role in vegetative axillary meristem development (McSteen et al., 2007). Nevertheless, defects in the production of vegetative branches in pid mutants have not been reported in Arabidopsis. Previous studies of gene expression patterns showed that OsPID is also expressed in axillary meristems during vegetative development in rice, implying that OsPID may have a role in tiller development (McSteen et al., 2007; Morita and Kyozuka, 2007). However, distinct to Arabidopsis and maize, tiller numbers and inflorescences of ospid show no significant differences compared with the wild type. The palea and lemma of ospid also show no significant differences compared with the wild type. The ospid mutant shows severe defects in stigma and ovule initiation and anther patterning. The function of PID is conserved, suggesting that the stigma and ovule may be lateral organs in rice. The distinct effects of mutations in PID in rice, maize, and Arabidopsis reveal that the locations and developmental stages in which PID functions differ among these species. It is possible that an additional homolog of OsPID exerts PID function in inflorescence development or that the roles of OsPID in inflorescence development are masked by redundant partners.

OsPID Regulates Stigma and Ovule Initiation by Maintaining Stem Cell Identity through Auxin Signaling

In most angiosperms, the FM is completely exhausted after producing a pair of carpels. In fact, the FM is no longer in existence after the carpels are generated. Stigmas and ovules differentiate from the carpels. In this case, the initiation of the stigma and ovule are regulated by the carpel, and this process is independent of the FM (Colombo et al., 2008). In petunia (Petunia hybrida), the FM is exhausted following production of the placenta. Thus, initiation of the stigma and ovule are also independent of the FM in petunia (Angenent and Colombo, 1996; Colombo et al., 2008). Stigmas and ovules can be formed on ectopic carpelloid structures in Arabidopsis floral development mutants (Bowman et al., 1989; Drews et al., 1991; Jack et al., 1992; Mizukami and Ma, 1992). This phenomenon can also be observed in petunia (Angenent et al., 1993, 1995). Therefore, in Arabidopsis and petunia, the stigma and ovule are considered to be part of the carpel (Yamaki et al., 2011). However, in rice, the FM can be maintained in the carpel until ovule initiation. The ovule is differentiated directly from the FM as a lateral organ in rice (Yamaki et al., 2011). Ovules cannot form on ectopic carpels in rice mutants in which the stamens are homeotically converted to carpels, such as in superwoman1 and osmads13 mutant plants (Nagasawa et al., 2003; Dreni et al., 2007). Here, we found that ospid failed to form stigmas always and ovules sometimes. FM lost its homeostasis in the pistil primordium of ospid. Accordingly, we propose that stigma, as is the case of ovule, is regulated by FM in rice.

The FM may be arrested prematurely in the ospid pistil primordium. Indeed, auxin plays a pivotal role in the maintenance of the FM and primordium initiation by integrating auxin response factors into the WUS-CLV feedback pathway (Vernoux et al., 2010; Yamaguchi et al., 2013; Luo et al., 2018). Previous studies have shown that ETT and MP are involved in FM maintenance in Arabidopsis (Zhao et al., 2010; Liu et al., 2014; Luo et al., 2018; Zhang et al., 2018). In this study, we showed that the expression patterns of OsETT1, OsETT2, and OsMP are perturbed in the pistil and stamen primordia of ospid and that expression of these genes was down-regulated significantly. Moreover, the stages from Sp6 to Sp8a are critical for the FM fate transition. During this developmental period, the FM begins to lose its indeterminate fate as it differentiates into the ovule, and FM is eventually exhausted at stage Sp8 (Yamaki et al., 2011). Therefore, we speculate that the changes in the expression patterns of OsETT1, OsETT2, and OsMP in the ospid mutant may cause premature arrest of FM and lead to failure of the stigma and ovule to initiate from the abnormal FM.

In addition, OsETT1 is a marker gene for adaxial-abaxial polarity in stamen development (Toriba and Hirano, 2014). The spatial expression patterns of OsETT1 and OsETT2 were altered during stamen development in ospid, suggesting that OsETT1 and OsETT2 may play pivotal roles in establishing the adaxial-abaxial polarity of the stamen.

It is also possible that the changes in expression pattern of OsETT1, OsETT2, and OsMP may arise secondarily from altered patterning or from alteration of the FM. Low auxin activity may be the prime cause to the alteration of patterning and the FM. Similar alteration of anther patterning was also observed in the osett1 mutant (Toriba et al., 2010). The changes in expression pattern of OsETT1, OsETT2, and OsMP more likely resulted from the altered auxin signaling.

Based on the results from both previous studies and our findings, we propose a framework for the translation of a local auxin signal into stigma and ovule initiation (Fig. 10). OsPIN1 may be activated by OsPID, which acts to form a local auxin maximum in the pistil primordium. The auxin maximum signaling is required for maintenance of stem cell identity in the FM. The ARF genes OsETT1, OsETT2, and OsMP may be involved in maintenance of the FM through the response to the auxin maximum signal. Thus, auxin signaling is essential for initiation of the stigma and ovule primordia from the FM. Additionally, OsETT1 and OsETT2 are also involved in the regulation of anther adaxial-abaxial polarity by responding to the auxin signal in the stamen primordium.

Figure 10.

Figure 10.

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Proposed model for the role of OsPID in stigma and ovule initiation and the establishment of adaxial-abaxial polarity in the anther. OsPID regulates the activity of OsPIN1 in the pistil and stamen primordia. OsPIN1 promotes auxin transport, leading to a high-concentration auxin pool in the pistil and stamen primordia. The auxin signaling is essential for stigma and ovule initiation and anther adaxial-abaxial polarity. In pistil primordium, the ARF genes OsMP, OsETT1, and OsETT2 may have roles in maintaining stem cell identity by responding to auxin signaling. The stigma and ovule are differentiated from FM. In stamen primordium, OsETT1 and OsETT2 respond to auxin signaling to regulate the establishment of adaxial-abaxial polarity in anther. The blue ovals represent areas of high auxin concentration. Positive interactions are denoted by arrows.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

The ospid-1 mutant was isolated from the offspring of a japonica rice (Oryza sativa) variety, Wuxiangjing 9, that had been treated with 60Co γ-rays. The ospid-1 mutant was crossed with an indica rice variety, 9311, to construct a genetic mapping population. The ospid-2 and ospid-3 mutants were isolated from the offspring of a japonica rice variety, Yandao 8, which also had been irradiated with 60Co γ-rays. The ospid-2 and ospid-3 mutants were also crossed with 9311 to produce the mapping populations for map-based gene cloning. All plant materials were grown in paddy fields under normal rice cultivation conditions.

Map-Based Cloning of OsPID

To map OsPID, sequence-tagged site markers were developed based on sequence differences between 9311 and Nipponbare from the data published at https://www.ncbi.nlm.nih.gov/. DNA sequencing was used to identify the target gene after fine-mapping of ospid-1. Primer pairs P1, P2, and P3 were used to detect the genomic switch in ospid-1, ospid-2, and ospid-3, respectively. The primers used in this study are listed in Supplemental Table S1.

Morphological and Cellular Analyses

Fresh young panicles from both the wild type and the ospid mutant were fixed in Carnoy’s solution (ethanol:glacial acetic acid, 3:1). For histological analysis of the anther and pistil, the samples were dehydrated in a graded ethanol series, embedded in Technovit 7100 resin (Hereaus Kulzer), and polymerized at 37°C. The samples were transversely sectioned into 4-μm slices with a Leica microtome and stained with 0.25% (w/v) Toluidine Blue O (Chroma Gesellschaft Schmidt). The slides were observed with an Olympus BX51 microscope and photographed with a digital camera.

Scanning Electron Microscopy

Young panicles were fixed in FAA solution (glacial acetic acid:formalin:50% [v/v] ethanol, 1:1:18) at 4°C overnight, dehydrated in a graded ethanol series, and substituted with 100% ethanol. A critical-point drier (Hitachi HCP-2) was used to dry the samples. The samples were dissected using a microscope (S8APO; Leica Microsystems), sputter coated with platinum, and observed with a scanning electron microscope (S-3000N; Hitachi High-Technologies).

Embryo Sac Confocal Laser Scanning Microscopic Observations

Mature ovaries were collected from flowering spikelets and fixed in FAA solution for at least 24 h. The ovaries were hydrated sequentially in a graded ethanol series and substituted with distilled water. The ovaries were then stained with 10 mg L−1 eosin B (C20H6N2O9Br2Na2, formula weight = 624.1) solution (dissolved in 4% [w/v] Suc) for 10 to 12 h at room temperature. The samples were posttreated in 2% (w/v) aluminum potassium sulfate for 20 min. The samples were then rinsed with distilled water and dehydrated in a graded ethanol series into 100% ethanol. Subsequently, the dehydrated samples were transferred into a mixture of absolute ethanol and methyl salicylate (1:1) for 1 h and then cleared in pure methyl salicylate solution for 1 h. The samples were observed with a Zeiss confocal laser scanning microscope (Carl Zeiss Meta 510).

Phylogenetic Tree Construction and Multiple Sequence Alignments

The amino acid sequence of OsPID was used as the query to perform PSI-BLAST searches (https://www.ebi.ac.uk/Tools/sss/psiblast/). Target sequences were downloaded and used to construct neighbor-joining trees with MEGA5 software (Tamura et al., 2011). Multiple sequence alignments were performed using the online toolkit MAFFT (https://toolkit.tuebingen.mpg.de/mafft), and the results were visualized using ESPRIPT3 (http://espript.ibcp.fr/ESPript/ESPript/).

Subcellular Protein Localization

The coding sequence of OsPID was cloned into the pJIT163-GFP vector (GFP:GFP) to generate OsPID-GFP and GFP-OsPID protein fusion constructs for transfection into rice protoplasts. The coding sequence of OsMADS3 was fused in frame to mCherry to generate an OsMADS3-mCherry fusion construct. The OsPID-GFP and OsMADS3-mCherry plasmids were cotransfected into rice protoplasts. After incubation in the dark at 28°C for 20 h, fluorescence signals were observed with a confocal laser scanning microscope (Carl Zeiss Meta 510).

Gene Expression Kinetic Analysis

The seeds of Yandao 8 were grown in a plastic tunnel seedbed on one-half-strength Murashige and Skoog (1/2 MS) liquid medium. After 10 d of growth, the seedlings with similar height were transplanted to 1/2 MS liquid medium supplemented with IAA (final concentration of 100 μm). The seedlings with similar height on 1/2 MS liquid medium without IAA were used as controls. The seedlings were harvested at the following time points after IAA treatment: 0, 1, 2, 4, 6, 8, 10, and 12 h. The samples were used for gene expression kinetic analysis.

RT-qPCR Analysis

Total RNA was isolated from roots, stems, leaf blades, seedlings, and young panicles of rice plants using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. A 4-μg sample of RNA was reverse transcribed into first-strand cDNA using an oligo(dT)18 primer and moloney murine leukemia virus (M-MLV) reverse transcriptase (Invitrogen; Thermo Fisher Scientific catalog no. 28025013). The primers used for RT-qPCR analysis are given in Supplemental Table S1. The rice Ubiquitin gene was used as the internal control for normalizing gene expression. RT-qPCR assays were performed on a Bio-Rad CFX96 using Fast Evagreen qPCR Master Mix (Biotium) with the following thermal cycler profile: 94°C for 3 min; followed by 25 to 30 cycles of 94°C for 30 s, 57°C for 30 s, and 72°C for 30 s; with a final extension at 72°C for 10 min. All experiments were repeated three times in terms of one sample used and three experiments performed. Student’s t test was used for statistical analysis.

Immunofluorescence

For protein immunolocalization, fresh young panicles were fixed in 100% methanol at −20°C overnight and embedded in Steedman’s wax, which is composed of polyethylene glycol 400 distearate and 1-hexadecanol (Sigma-Aldrich). After rehydration, 6-mm sections were pretreated for 0.5 h with 2% (w/v) BSA in phosphate-buffered saline (PBS) for 1 h and incubated overnight with the anti-OsPIN1 antiserum diluted 1:500 in PBS containing 0.1% (w/v) BSA. After three washes in PBS containing 0.1% (v/v) Tween 20, the panicle sections were incubated for 1 h with the secondary goat anti-rat antibody diluted 1:1,000 in PBS supplemented with 0.1% (w/v) BSA. After additional rinses in PBS plus 0.1% (v/v) Tween 20, sections were imaged with an A2 fluorescence microscope with a micro-CCD camera (Zeiss). Anti-OsPIN1 antibodies were raised against the peptide KPKYPLPASNAAPM.

In Situ Hybridization and GUS Staining

Fresh panicles, which were still embedded in the flag leaves, were dissected from plants and fixed in FAA solution. To make probes for the rice OsPID, OsETT1, OsETT2, OsETT3, OsMP, OSH1, OsHEC1, OsHEC2, OsHEC3, and OsSPT genes, cDNA-specific fragments were amplified with the primers listed in Supplemental Table S1 and cloned into the pEASY-Blunt simple vector (Transgen Biotechnology). Nonradioactive in situ hybridization was performed using a previously described method (Ito et al., 2004). Details of the methods used for fixation of plant tissues, embedding in paraffin, and in situ hybridization can be found at http://www.its.caltech.edu/∼plantlab/protocols/insitu.html. The promoter region (5,159 bp) of OsPID was amplified and inserted into pMDC163-GUS in front of the GUS reporter gene. The OsPID promoter-GUS construct was used for Agrobacterium tumefaciens-mediated transformation of the japonica rice variety Yandao 8. Dr5::GUS plasmid was donated by Jiayang Li’s laboratory at the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under the following accession numbers: OsPID, XM_015763606; BIF2, NP_001106051; PID, NP_181012; OsPIN1, XP_015627099; OSH1, XM_015773906; OsMP, XM_026025267; OsETT1, XM_026025637; OsETT2, XM_015791948; OsETT3, XM_026021030; FON4, XM_015761256; MOC3, XM_015779885; OsHEC1, XM_026019952; OsHEC2, XM_015755510; OsHEC3, XM_015794125; and OsSPT, XM_026026213.

Supplemental Data

The following supplemental materials are available.

Footnotes

1

This work was supported by the Ministry of Science and Technology of the People's Republic of China (2016YFD0102001), the National Natural Science Foundation of China (31872859 and 31670313), a key project of the Jiangsu Education Department of China (15KJA180010), the Yangzhou Science and Technology Bureau of China (YZ2017059), and the Jiangsu Higher Education Institutions of China (PAPD).

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