Abstract
Phosphate is one of the major nutrients of growth, development, and reproduction of crop plants and functions in energy metabolism, signal transduction cascades, and regulates enzymatic activities. To understand uptake and usage of this nutrient in Oryza sativa (rice), a model crop plant, global studies on this family is more effective. Here, we conducted phylogenomic analyses of 26 rice and 19 Arabidopsis phosphate transporters (PHT) reported from previous studies, by integrating various meta-expression data to the phylogenic tree context. Subsequently, of four subfamilies, the PHT1 subfamily was a high affinity phosphate transporter, which functioned under low concentrations of phosphorous in soil, while the others (i.e., PHT2, PHT3, and PHT4) were low-affinity phosphate transporter subfamilies. Most members of the PHT1 in rice and Arabidopsis, in contrast to the other transporter subfamilies, showed significant induction under phosphate starvation, and the responses were more obvious in the roots. These results indicated that the functions of PHT1 phosphate transporters in rice and Arabidopsis were well conserved in response to phosphate starvation. We confirmed significant upregulation of seven PHT1 subfamily genes in rice under phosphate starvation, by RT-PCR, indicating that the high affinity phosphate transporters played important roles in the uptake of phosphate under phosphate deficiency. The regulatory network of OsPT4 belonged to the PHT1 subfamily based on RiceNet analysis, suggesting clues for further analyses. Our study showed the significance of at least seven PHT1 subfamily members, which could improve the efficiency of phosphate use in rice, as a model crop plant.
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Key words: Rice phosphate transporter, Phosphate deficiency, Phylogenetic analysis, Meta-expression analysis
INTRODUCTION
Phosphorus (P) is a basic component of cellular structures. It functions in energy metabolism and signal transduction cascades and regulates enzymatic activities (
Schachtman et al. 1998;
Lambers et al. 2006). As an essential macronutrient, supplementation with P is required for normal plant growth, development (especially root development), and reproduction. Phosphorus deficiency is widespread in all major rice ecosystems. The phosphate content of fertilizer is limited and is an un-renewable resource. Cultivated crop plants use only approximately 20%–30% of the applied phosphate as fertilizers, with the remaining phosphate retained in the soil, causing pollution of freshwater reserves (
López- Arredondo et al. 2014). The phosphate transporter (PT) is the first gate to uptake phosphate (Pi) from the soil. Genome sequencing projects in a model plant and in major crop plants revealed that 19 PT genes exist in Arabidopsis and 26 in rice (
Raghothama 1999;
Liu et al. 2011). Phylogenetic analyses of rice and Arabidopsis PHTs suggests four subgroups of PHTs (
Rausch and Bucher 2002).
The PHT1 subfamily includes high affinity Pi transporters, which are included in a major facilitator super family (
Raghothama 2000;
Koyama et al. 2005). The PHT2 subfamily is structurally similar to members of the PHT1 family, but most of them are preferentially expressed in the shoot (
Daram et al. 1999;
Versaw and Harrison 2002). The PHT3 subfamily is highly conserved within the mitochondrial transporter family, and the PHT4 subfamily genes are expressed in both roots and leaves. All PHT4 proteins mediate Pi transport in yeast, with high affinity (
Guo et al. 2008;
Cubero et al. 2009). Of these subfamilies, PHT1 transporters are high affinity Pi transporters, and the other transporters are low-affinity Pi transporters (
Muchhal et al. 1996;
Smith et al. 1997). Regarding the PHT1 members, the functions of nine Arabidopsis (
AtPHT1;1-9) and 13 rice (
OsPT1-13) genes have been elucidated. Regarding PHT2, one Arabidopsis (
AtPHT2;1) and one rice PHT2 (
OsPT14) gene has been elucidated. Regarding PHT3, three Arabidopsis (
AtPHT3;1-3) and six rice PHT3 (
OsPT15-20) genes have been elucidated. Regarding PHT4, six Arabidopsis (
AtPHT4;1-6) and six rice PHT4 (
OsPT21-26) genes have been elucidated. The PHT1 family has the largest number of members, and all members exhibit high sequence similarities with each other (
Chiou et al. 2001). Most of the functionally characterized PHTs belong to PHT1, indicating its major contribution to uptake of phosphate from soil for plant growth. Although several PHTs have been studied, remaining PHTs needs further functional studies in rice, which is a model crop plant. Global analysis for this family in rice will be useful in providing clues for functional studies.
Here, we carried out phylogenomic analysis of the rice PHT family by integrating transcriptome data to the phylogenomic context. This analysis provides the functional information of individual members based on integrated transcriptome data. In addition, comparative phylogenomic analyses of rice and Arabidopsis PHT families suggested the functional conservancy of PHT subfamilies between two species and featured elements for further studies. Detailed data analysis and discussion will be presented.
MATERIALS AND METHODS
Plant growth and phosphate starvation treatment
The studies were conducted using
Oryza sativa (rice) Japonica cultivar ‘Dongjin’ variety seedlings grown for 7 days, and 21 days on Pi-sufficient (0.320 mM Pi) or -deficient Yoshida solution (
Yoshida et al. 1976) after germination and growth on Yoshida media for 2 weeks. The pH of the culture solution was adjusted to 5.5 using 10 M NaOH. The Yoshida solution was replaced with new solution every 3 days. In all the hydroponic culture experiments, seedlings were directly grown in two kinds of Yoshida solution (8 L) with an 8-hour light (30°C)/16-hour dark (22°C) photo-period in the growth chambers (Younghwa Science, Daegu, Republic of Korea).
To perform phylogenomic analysis of PTs between rice and
Arabidopsis thaliana, we collected 26 rice family members from the Rice Genome Annotation Project (RGAP,
http://rice.plantbiology.msu.edu/) and 19 Arabidopsis family members from the TAIR (
Lamesch et al. 2012). In cases of gene loci having multiple gene models (transcripts), we selected a representative transcript encoding the PT proteins for each locus as suggested in RGAP and TAIR. Multiple amino acid sequences of 45 PT proteins were aligned using the ClustalX program, version 2.0.11 (
Thompson et al. 2002). A joint unrooted tree was generated using MEGA7 with the neighbor-joining method. Bootstrap values tested by 1,000 replicates were indicated in each branch (
Kumar et al. 2016). We developed a phylogenic tree with rice and Arabidopsis PTs, and rice and Arabidopsis PT proteins were divided into four clusters as previously classified (Supplementary Fig. S1).
To conduct the comparative transcriptome analyses, we used RNA-seq data produced under phosphate starvation for rice (
Secco et al. 2013) and microarray data for Arabidopsis (
Woo et al. 2012). The former had the root and shoot samples under phosphate starvation for 1 hour, 6 hours, 24 hours, 1 day, 3 days, 7 days, or 21 days, and the latter were under phosphate starvation for 10 days. In addition, when samples were under phosphate starvation for 21 days (rice) and 10 days (Arabidopsis), 0.320 mM phosphate for rice samples was resupplied for 1 hour, 6 hours and 24 hours, and full-strength MGRL for Arabidopsis samples was resupplied for 3 days. Log
2-fold changes of phosphate starvation (-Pi) over MOCK (control), and phosphate resupply (Pi re) over phosphate starvation (-Pi) were calculated and used for generating green to red heat maps. Log
2 intensity values in each sample were used to generate blue to yellow heat maps.
Roots and shoots of rice seedlings were frozen in liquid nitrogen and ground with a Tissue Lyser II (Qiagen, Hilden, Germany). RNAs were extracted with the RNA iso-Plus kit (Takara Bio, Kyoto, Japan) according to the manufacturer’s protocol. First-strand cDNAs were synthesized from total RNA using reverse transcriptase (Takara Bio). To confirm phosphate starvation inducible expression patterns by RT-PCR, the PCR products were loaded on 1% agarose gels and the images were captured by a camera. The gene expression levels were compared by the band density after normalization of initial variation in sample concentration by
OsUbi5, and the quality of samples under phosphate starvation was evaluated by using primer sets for sulfoquinovosyldiacylglycerol 2 (OsSQD2, LOC_Os01g04920) and phosphate transporter 6 (OsPT6, LOC_Os08g45000), which are phosphate starvation marker genes (
Gho et al. 2018). The PCR cycle conditions used were 95°C for 30 seconds, 57°C for 30 seconds, and 72°C for 1 minute 30 seconds for 24–33 cycles. Quantitative real-time PCR was performed using a SYBR PremixExTaq kit (TaKaRa Biomedicals,
http://www.takara-bio.com/) on a LightCycler480 machine (Roche Diagnostics,
http://www.roche.com), according to the manufacturer’s instructions. Semi-quantitative RT-PCR and quantitative real-time PCR was performed using the gene specific primers listed in Supplementary Table S1.
To estimate the regulatory model of high affinity PT1s in rice, we predicted the protein-protein interaction network using the PRIN tool (
http://bis.zju.edu.cn/prin/). We also found 68 interacting proteins with
OsPT4 out of queried OsPHT1 subfamily members and integrated the MapMan terms to this network using Cytoscape, version 3.0 (
Demchak et al. 2014).
RESULTS
Morphological appearance for the duration of phosphate treatment in rice
To determine the morphological appearance of rice seedlings, we grew
O.
sativa Japonica cultivar ‘Dongjin’ seedlings for 7 days (
Fig. 1A) or 21 days (
Fig. 1B) on Pi-sufficient or Pi-deficient media after growing the germinated seedlings for 2 weeks using Yoshida solution. Our data showed that Pi was particularly important during the early root development of rice (
Fig. 1A). The numbers of leaves and tillers were reduced. The lengths of shoots were also shorter. The young leaves appeared to be healthy, but the older leaves turned brown (
Fig. 1A). To test the phosphate response of those samples, we checked the expression patterns of phosphate marker genes,
OsPT6 and
OsSQD2 (
Gho et al. 2018) (
Fig. 1C). We found that two marker genes were significantly upregulated under phosphate starvation for 7 days and 21 days after normal growth for 2 weeks, qualifying the samples as being under phosphate starvation for 7 days and 21 days.
Liu et al. (2011) recently performed a phylogenetic analysis of the PT family members of rice (26 genes) and Arabidopsis (19 genes). However, the biological roles of PT genes have not been well defined. In this study, we tried to provide biological functions for rice and Arabidopsis PT genes and functional conservancy between rice and Arabidopsis PT genes. To accomplish this goal, we first developed a phylogenic tree of 26 rice and 19 Arabidopsis PTs, and found that there were four subgroups in rice and Arabidopsis PTs (Supplementary Fig. S1). The PT1 consisted of 13 proteins in rice and nine proteins in Arabi dopsis; PT2, one in rice and one in Arabidopsis; PT3, six in rice and three in Arabidopsis; and PT4, six in rice and six in Arabidopsis. PT1 was a high affinity Pi transporter, and the others were low-affinity Pi transporters (Supplementary Fig. S1). In addition, most PT1 members were expected to be localized in the plasma membrane. These high affinity PTs might be major mediators of Pi uptake at the rice root/soil interface during phosphate starvation.
Transcriptome analysis of the phosphate transporter family under phosphate starvation
To determine the functional implications of rice and Arabidopsis PTs, we incorporated global gene expression patterns into the phylogenic context of each species. We used RNA-seq data analysis for
O. sativa Japonica cultivar ‘Dongjin’ shoot and root samples, and Affymetrix data analysis for Arabidopsis shoot and root samples under phosphate starvation, showing that the high affinity transporter (PHT1) in both species showed very obvious induction under phosphate starvation with root preferential expression patterns, while the low-affinity transporter did not show these preferential expression patterns. PHT2 and PHT4 genes in rice and Arabidopsis showed shoot preferred expression, while PHT3 showed ubiquitous expression patterns (
Fig. 2). Together, these data indicated that the functions of all PHTs in rice and Arabidopsis were conserved in response to phosphate starvation.
Our primary interest involved enhancing phosphate use associated with PT in rice. We tried to validate meta-expression patterns under phosphate starvation using RT-PCR analysis. To accomplish this goal, we used samples tested in
Fig. 1. In total, expression patterns of 13 PHT1 family genes were tested after evaluating the samples with phosphate response marker genes and internal controls in rice. We confirmed the inducible expression patterns under phosphate starvation for seven PHT1 family genes in rice (
Fig. 3). These PHT1 genes were therefore primary targets for further application.
To estimate the molecular function of
OsPT4, we first analyzed the predicted interacting proteins with OsPT4 using the PRIN database (
http://bis.zju.edu.cn/prin/). We found that OsPT4 interacted with 68 proteins (Supplementary Tables S2 and S3). The interactors were further classified into diverse functional groups using the MapMan tool. Of these interactors, three were related to hormone response such as abscisic acid (ABA) and brassinosteroid (BR), seven to lipid metabolism, eight to transporters, seven to DNA synthesis, four to RNA processing, two to signaling, 14 to protein modification, three to secondary metabolism, and 14 to other processes (
Fig. 4). Among these interactors, we had more interest in the signaling, transcriptional regulation, and protein modification processes. Our analysis was very simple, and was effective in quickly identifying regulatory genes associated with OsPT4. A similar strategy can be used for other PT genes, and further analyses will clarify the functions based on these possibilities.
DISCUSSION
P is a basic component of cellular structures, functions in energy metabolism and signal transduction cascades, and regulates enzymatic activities, especially in a vital role in plant reproduction, of which grain production is an important result. But P deficiency is widespread in all major rice ecosystems and is the major growth-limiting factor. Importantly, when phosphate is applied as a fertilizer in soil, it is rapidly immobilized owing to fixation and microbial activity. Cultivated plants therefore use only approximately 20%–30% of the applied phosphate, and the rest is lost, eventually causing water eutrophication (
López- Arredondo et al. 2014). Research to increase phosphorus uptake will not only help to reduce fertilizer usage, but will also help to improve crop productivity. PHTs are the primary targets in better understanding the uptake and usage of this nutrient in plants (
Gho et al. 2018).
To better characterize the PHT family, we conducted phylogenomic analyses of 26
O. sativa and 19 Arabidopsis PHTs reported from previous studies, by integrating various meta-expression data to a phylogenic tree context. Phylogenomic analyses integrating large amounts of transcription data have been used to estimate functional redundancy among the same family members, as well as to predict the biological function of individual family members. In this study, the functions of PHT family members were also effectively suggested by using this integrating analysis tool on the backbone of the phylogenetic tree. The phylogenetic tree was generated using MEGA7 by the neighbor-joining method for relationships between PT family genes in rice and Arabidopsis. The identified Pi transporters were classified into four families: PHT1, PHT2, PHT3, and PHT4 (
Rausch and Bucher 2002). The four families in rice were in accordance with the four PHT families in Arabidopsis. PHT1 consisted of 13 proteins in rice; the PHT2 consisted of one protein; PHT3 consisted of six proteins; and PHT4 consisted of six proteins. PHT1 was a high affinity Pi transporter and the others were low-affinity Pi transporters. RNA-seq data analysis for rice cultivar ‘Dongjin’ shoots and roots and Affymetrix data analysis for Arabidopsis shoots and roots under phosphate starvation revealed that the high affinity transporter showed an obvious induction under phosphate starvation with root preferential expression patterns, but the low affinity did not (Supplementary Fig. S1).
Most members of PHT1 are expected to be localized in the plasma membrane. High affinity transporters might be major mediators of Pi uptake at the rice root/soil interface during phosphate starvation. The PHT1 transporters were high affinity Pi transporters and were included in the major facilitator superfamily. The majority of plant PTs belong to the PHT1 family, and all members of the PHT1 family exhibit high sequence similarities with each other. Most of the PHT1 genes are strongly expressed in roots, but PT1, PT4, and PT8 were also expressed in the shoots. PHT2 is structurally similar to members of the PHT1 family, and was preferentially expressed in the shoot, especially in the rosette leaves. In Arabidopsis, the low-affinity transporter PHT2;1 was located in chloroplasts, and a
pht2;1 mutation reduced Pi transport into the chloroplast and decreased Pi allocation throughout the whole plant (
Versaw and Harrison 2002). The PHT3 family was highly conserved within the mitochondrial transporter family. The phosphate transported into the matrix by the mitochondrial carrier family (PHT3) is either used for ATP synthesis or exchanges back to the cytosol using the dicarboxylate transporter (
Stappen and Krämer 1994;
Wohlrab and Briggs 1994). PHT4 genes are expressed in both roots and leaves. Other groups suggest roles for PHT4 proteins in the transport of Pi between the cytosol and chloroplasts, heterotrophic plastids, and the Golgi apparatus (
Cubero et al. 2009). PHT2 and PHT4 of rice and Arabidopsis showed preferred expression in shoots and leaves and PHT3 showed ubiquitous expression patterns. Overall, our data indicated that the functions of PTs in rice and Arabidopsis were well conserved in response to phosphate starvation.
ACKNOWLEDGEMENTS
This work was supported by grants from the Next-Generation BioGreen 21 Program (PJ01366401 and PJ01369001 to KHJ), and the Rural Development Administration, Republic of Korea.
ACKNOWLEDGEMENTS
This work was supported by grants from the Next-Generation BioGreen 21 Program (PJ01366401 and PJ01369001 to KHJ), and the Rural Development Administration, Republic of Korea.
Fig. 1Morphological appearance under phosphate starvation and a schematic diagram of samples used in transcriptome analysis.
Fig. 2Comparative expression analysis of rice and Arabidopsis phosphate transporter families under phosphate starvation.
Fig. 3Verification of expression in high affinity phosphate transporter families under phosphate starvation using reverse transcriptase-polymerase chain reaction.
Fig. 4Regulatory model of OSPT4 using the predicted protein-protein interaction network and MapMan terms.
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