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Article

Overexpression of OsPIN2 Regulates Root Growth and Formation in Response to Phosphate Deficiency in Rice

by
Huwei Sun
1,*,
Xiaoli Guo
1,
Fugui Xu
1,
Daxia Wu
2,3,
Xuhong Zhang
2,3,
Manman Lou
2,3,
Feifei Luo
2,3,
Guohua Xu
2,3 and
Yali Zhang
2,3,*
1
Laboratory of Rice Biology in Henan Province, Collaborative Innovation Center of Henan Grain Crops, College of Agronomy, Henan Agricultural University, Zhengzhou 450002, China
2
State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China
3
Key Laboratory of Plant Nutrition and Fertilization in Low-Middle Reaches of the Yangtze River, Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, China
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2019, 20(20), 5144; https://doi.org/10.3390/ijms20205144
Submission received: 11 September 2019 / Revised: 14 October 2019 / Accepted: 16 October 2019 / Published: 17 October 2019
(This article belongs to the Special Issue Plant Nutrition: Physiological and Metabolic Responses, Molecular Mechanisms and Chromatin Modifications)
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Abstract

:
The response of root architecture to phosphate (P) deficiency is critical in plant growth and development. Auxin is a key regulator of plant root growth in response to P deficiency, but the underlying mechanisms are unclear. In this study, phenotypic and genetic analyses were undertaken to explore the role of OsPIN2, an auxin efflux transporter, in regulating the growth and development of rice roots under normal nutrition condition (control) and low-phosphate condition (LP). Higher expression of OsPIN2 was observed in rice plants under LP compared to the control. Meanwhile, the auxin levels of roots were increased under LP relative to control condition in wild-type (WT) plants. Compared to WT plants, two overexpression (OE) lines had higher auxin levels in the roots under control and LP. LP led to increased seminal roots (SRs) length and the root hairs (RHs) density, but decreased lateral roots (LRs) density in WT plants. However, overexpression of OsPIN2 caused a loss of sensitivity in the root response to P deficiency. The OE lines had a shorter SR length, lower LR density, and greater RH density than WT plants under control. However, the LR and RH densities in the OE lines were similar to those in WT plants under LP. Compared to WT plants, overexpression of OsPIN2 had a shorter root length through decreased root cell elongation under control and LP. Surprisingly, overexpression of OsPIN2 might increase auxin distribution in epidermis of root, resulting in greater RH formation but less LR development in OE plants than in WT plants in the control condition but levels similar of these under LP. These results suggest that higher OsPIN2 expression regulates rice root growth and development maybe by changing auxin distribution in roots under LP condition.

1. Introduction

Phosphorus (P) is essential nutrient for plant growth and development, and its deficiency dramatically affect crop productivity [1,2]. The changes of plant morphology in response to P- deficiency are an important mechanism for crops to optimize growth and productivity [3,4]. Increased root to shoot ratios and root surface areas induced by phosphate (P) deficiency have been reported in several plant species [5]. The changes in root morphology that occur under condition of P deficiency are complex and vary according to the experimental conditions and plant species. For example, a deficiency in P markedly inhibits primary root elongation in Arabidopsis [5,6]. This growth arrest is caused by reduced cell elongation and progressive cessation of cell proliferation in the root meristem that ultimately exhausts the primary root (PR) stem cell niche [7]. In contrast to Arabidopsis, in other plant species such as rice (Oryza sativa L.), elongation of the PRs occurs as a typical response to P deficiency [4,8]. Lateral root (LR) formation is also modulated under P deficiency in Arabidopsis and rice [9,10]. Significant P deficiency induces LR formation in Arabidopsis [7]. Conversely, in rice, the LR density decreases under P deficiency [10]. Meanwhile, the results of several studies have demonstrated root hair (RH) development and elongation in response to P deficiency [3,11,12]. Although the plasticity of root has been well documented in several plant species under P-deficiency. However, the mechanism in the regulatory cascade leading to change in root growth and formation are still not fully understood.
Besides environmental conditions, root growth and formation, are regulated by intrinsic factors such as plant hormones. Auxin is the key phytohormone to be discovered to regulate root growth in plants [4,11,13,14]. Auxin signaling is associated with changes in root morphology caused by P deprivation. As well, P-deprived were shown to undergo primary root growth arrest and show formation of LR with higher auxin levels in plants [3,15]. Most auxin is synthesised in aboveground tissues, such as shoot apices and young leaves, by YUCCA family genes [16,17,18] and then redistributed by auxin-influx carriers, such as AUX1/LAX family proteins, and auxin-efflux carriers, including ABCB/PGP and PIN family proteins [19,20,21].
Several studies have shown that polar auxin transport affects root growth [13,22,23,24,25]. For example, OsPIN1 is involved in auxin-dependent adventitious root emergence in rice [13], and Arabidopsis mutants in AtPIN2 display agravitropic root-growth phenotypes [22,23]. OsPIN2 regulates root elongation and LR formation patterns via the modulation of auxin distribution in rice [25]. In addition, many studies have suggested that polar auxin transport regulates root growth and development in response to P supply [4,11]. In Arabidopsis, OsAUX1 regulates auxin transport from the root apex to the differentiation zone, where this signal promotes root hair elongation when the roots encounter P deficiency [11]. In rice, OsPIN1b is involved in rice SR elongation by regulating root apical meristem activity in response to P deficiency [4]. PIN proteins are the major auxin efflux carriers in plants [19,26]. However, few studies have evaluated the role of PINs in regulating root growth and formation in response to low P condition.
Since auxin are involved in root growth modulated by P deficiency in several plant species. Further research is needed to understand possible links between auxin in the control of root development in response to low P condition. In this study, the role of OsPIN2 in regulating rice root growth in response to P-limiting condition was examined using OsPIN2 OE lines. Rice is an ideal model for auxin transport studies because of its small genome size and the availability of its complete genome sequence [27,28]. Upregulating expression of the OsPIN2 gene in rice under condition of P deficiency revealed that the root growth patterns in these lines were related to elevated root auxin levels. In addition, comparison of the rice WT and OE lines carrying the auxin reporter construct DR5::GUS, following treatment with radiolabelled [3H] IAA and its polar transport, revealed that higher OsPIN2 expression played important roles in regulating root growth and development by modifying auxin transport and distribution in roots under P deficiency.

2. Results

2.1. The Expression of OsPIN2 Was Induced by Low phosphate (LP) Supply

We evaluated the expression pattern of OsPIN2 in rice plants using the GUS reporter gene and qRT-PCR in response to LP condition for 6 h. As shown in Figure 1a, pPIN2::GUS expression was highly expressed in shoot bases, lateral roots and root tips, but weakly in leaves, consistent with the previous results [14,29]. Compared with control treatment, pPIN2::GUS expression level in leaves, shoot bases and roots was increased by LP supply. As well, GUS location of OsPIN2 in root tips were more concentrated in epidermis under LP relative to control condition (Figure 1a). The results of qRT-PCR showed the similar tendency as that of pPIN2::GUS expression (Figure 1b), suggesting the expression of OsPIN2 gene was induced by LP supply.

2.2. Overexpression of OsPIN2 Regulates Auxin Levels in Roots

As previously reported [25], the transcriptional levels of OsPIN2 were significantly increased in OE lines relative to WT plants (Figure S1). In WT plants, the IAA contents in LR regions and root tips (RTs, 0.5 cm) of plants under LP condition were significantly higher than those in plants grown in the control condition, by 65% and 47%, respectively (Figure 2a,b). Expression of the auxin-responsive reporter construct DR5::GUS was increased in the LRs and RTs in the LP condition, a pattern similar to that of IAA levels (Figure 2c). Compared to WT plants, the IAA contents in the LR regions and RTs of the OE lines in the control condition were significantly increased by 1-fold and 85%, respectively, and in the LP condition were significantly increased by 35% and 34%, respectively (Figure 2a,b). The expression results of the auxin-responsive reporter DR5::GUS and GUS enzyme activity were similar to the patterns of the IAA levels (Figure 2c and Figure S2). These results suggested that the root IAA levels were smaller differences between WT and OE lines grown in the LP condition. However, the DR5::GUS levels of root cap were decreased in OE lines and WT under LP relative to WT under control (Figure 2c).

2.3. The Root Architectures of OE Lines Were Less Sensitive Than WT Plants to LP Condition

The experiment of root gravitropic was analyzed in Figure S3. The results indicated that overexpression of OsPIN2 didn’t affect the gravitropic of root, consistent with the result of OsPIN1b overexpression lines [22]. As well, there was little effect of the NPA treatment on the root length of OE lines relative to WT plants (Figure S3). In contrast to WT plants, the root architectures of OE lines were less responsive to LP condition (Figure 3a). SR length of WT plants grown in LP solution increased by 46%, while that of the OE lines increased by approximately 14% (Figure 3b). To verify overexpression of OsPIN2 inhibited root elongation, the length of epidermal cells in the maturity zone were analyzed in OE lines and WT plants (Figure 4a,b). The epidermal cells length in OE lines were shorter than that in WT both under control and LP conditions. The activity of OsCYCB1;1::GUS and the expression of CYCB1;1 gene were stronger under P-deficiency compared to the control (Figure 4c). However, the similar levels of pCYCB1;1::GUS and OsCYCB1;1 gene were observed in OE lines and WT plants under control and LP conditions.
LP corresponded to reductions in LR density of 21%, in the WT (Figure 5a). However, no differences in LR density were recorded in the OE lines between control and LP. The LR density in overexpression of PIN2 was similar to that in WT under LP condition, the differences of roots were consistent with the result of IAA levels in roots. LR formation is dependent on LR primordia. The density of LR primordia significantly increased in WT under control relative to LP supply. As well, the LR primordia were decreased in OE lines relative to WT plants under control, but similar levels were found between WT and OE lines under LP condition (Figure 5b).
The density and length of RH were significantly increased in OsPIN2 overexpression relative to WT plants (Figure 6a–c). Under control treatment, RH density and average length were increased by 3.5- and 2- fold in OE plants relative to WT plants, respectively (Figure 6b,c). However, under LP treatment, in comparison to WT, only RH density were increased by 53% in OE plants. No differences were recorded in RH length between WT and OE lines (Figure 6b,c). These results suggested that overexpression of OsPIN2 increased RH development in rice.
We monitored the expression levels of RH-formative genes in rice plants by qRT-PCR. The expression of RSL4/5//7/9 were increased in OE lines relative to that in WT under control and LP condition (Figure 6d,e). The expression of RSL6 genes was up-regulate in OE lines relative to WT under control but not under LP condition.

2.4. Overexpressing OsPIN2 Regulates Root Development by Modifying Auxin Distribute

The basipetal transport of auxin in WT plants was substantially increased in roots under the LP condition compared to the control condition (Figure 7a), suggesting that the basipetal transport of auxin was promoted by LP stress. In addition, the basipetal transport of auxin was significantly higher in the OE lines than in WT plants under both control and LP conditions. However, the basipetal transport of auxin were smaller differences between WT and OE lines under the LP condition than in the control condition (Figure 7a).
To determine the reasons for the more RHs density in the OE lines, we further analyzed relative expression of auxin-responsive DR5::GUS reporter in transverse roots (1 mm from RT) of WT and OE lines, respectively. Higher DR5::GUS expressed in root epidermis of the OE lines relative to WT plants (Figure 7b,c) regardless of transverse section, suggesting overexpression of OsPIN2 increased auxin distribution in epidermis of root.

3. Discussion

In this study, the level of auxin in WT plants was significantly greater in the LP condition than in the control condition. In addition, greater OsPIN2 expression was involved in the LP-triggered induction of auxin transport changes in rice. The development of LRs and RHs in WT plants in the LP condition appeared similar to that in OsPIN2-overexpressing lines, suggesting that OsPIN2 plays key roles in rice root growth and development in response to LP stress.

3.1. Overexpression of OsPIN2 Affects Root Elongation by Regulating the Elongation of Epidermal Cells that Leave the Root Meristem

The results of several studies have suggested that auxin is involved in root elongation under P deficiency [15]. Likewise, the auxin regulation of rice root elongation maybe related to its levels in root caps. Several studies have shown that both higher and lower auxin levels in root caps inhibit root growth [10,15], but that specific auxin levels in root caps promote root elongation [10,15]. For example, application of IAA was shown to increase auxin levels in root caps but reduce the root length in WT rice plants [15]. Rice plants grown in the presence of 1–1000 nM NAA (an exogenous auxin) was shown to decrease SR length with increasing NAA except at 1 nM, at which concentration a slight increase in SR length was found to occur [10]. Application of 20 µM NPA (a polar auxin transport inhibitor) to the root-shoot junction was shown to increase the root length in WT plants [25], but application of 300 nM NPA to roots reduced the auxin levels in the root caps and inhibited root elongation [4].
Several studies have suggested that OsPIN2 affects root length by regulating auxin transport in the root cap [13,23,25]. In Arabidopsis, the atpin2 mutant displays an agravitropic phenotype with reduced root elongation [23]. However, mutation of OsPIN2 did not affect auxin transport or root length in rice [13]. It is possible that other OsPIN family genes compensate for the function of absent OsPIN2. OsPIN2-overexpressing plants have shown lower auxin accumulation in root caps than WT plants, and their root lengths change less in response to NPA, suggesting that these lines have a strong tolerance for NPA due to increased auxin transport [25]. In this study, in WT plants under the LP condition, the SR length was greater than that under the control condition, and the root cap auxin levels were lower (Figure 2c and Figure 3), consistent with the previous report [4,10]. These results suggest that LP condition cause a decrease in auxin accumulation in the root cap to levels that are ideal for the promotion of SR elongation. However, OsPIN2-overexpressing plants showed shorter SR lengths and lower auxin accumulation in root caps relative to those in WT plants in the LP condition (Figure 2c and Figure 3). These results indicate that overexpression of OsPIN2 inhibit the elongation of SR maybe by reducing auxin accumulation in root caps (Figure 2c).
Root length depends on two basal formation processes: Cell division in the RT meristem, and the final length of cells in the root maturity zone [30]. The activity of meristematic cells in the root meristem affects root elongation [31]. In rice, LP condition increase root meristem activity by regulating the expression of OsCYCB1;1 [8]. In this study, compared to the control condition, the LP condition increased the pCYCB1;1::GUS and OsCYCB1;1 expression levels in the RT and affected the length of mature cells (Figure 3a,b). However, the epidermal cell lengths in the OE lines were shorter than those in WT plants under both control and LP conditions. Similar pCYCB1;1::GUS and OsCYCB1;1 expression levels were observed in the OE lines and WT plants under control and LP conditions (Figure 4c,d). These data show that OsPIN2 overexpression affects root elongation primarily by affecting regulation of the elongation of epidermal cells that leave the root meristem.

3.2. Overexpression of OsPIN2 Regulate RHs and LRs Formation via Change Auxin Distribute in Root Transversal Section

RHs growth are regulated by auxin level and response in plants [11,15,32,33]. Our results showed that, when rice plants facing LP treatment, auxin response reporter DR5::GUS expression was significantly induced (Figure 7). As well, the changing tendency of auxin content under LP treatment paralleled DR5::GUS expression (Figure 7). Correspondingly, induced RH elongation and formation in WT plants under LP treatment were close linked to the auxin content and response.
Increasing evidence found that auixn transport and response genes have been involved in RHs growth. OsARF16, an auxin response factor, promoted RHs elongation in rice [15]. Lower auxin response in roots and shorter RHs were recorded in aux1 mutants under P deficiency [11]. Knockout of AUX3 induced the RHs elongation relative to WT plants [14]. Above results suggested that the mechanisms of auxin influx carrier (AUX) regulated RHs elongation are complex. We found auxin efflux transporter, OsPIN2, participated mainly in RHs formation under control and LP treatments (Figure 6a–c).
Besides auxin, genetic factors play important roles in the development of RHs. RSL Class II family members are expressed in root epidermis and RH cells and as essential regulators of RH development in rice [34]. In this study, compared with WT, the expression of RSL4/5/6/9 were up-regulated in OE lines under control and LP condition (Figure 6). These results suggested that OsRSL family genes play important roles in RHs deveplopment of OE lines. However, the transcriptional level of OsRSL6 was increased in OE lines relative to WT plants under control condition but not under LP. This may be due to the expression of it was induced by LP condition in WT plants (Figure S4). These suggested the five RH-morphogenetic genes may be involved in RHs formation induced by OE plants.
PIN2 plays a pivotal role in transmitting auxin through the peripheral layers of the root meristem and elongation zone [35]. In Arabidopsis and rice, PIN2 localises to the basal side of cortical cells and to the apical sides of epidermal and root cap cells, and mainly regulates root basipetal auxin transport [14,36]. In this study, GUS location of OsPIN2 in root tips were more concentrated in epidermis under LP relative to control condition (Figure 1a). The basipetal transport of auxin was significantly increased in OE lines relative to WT plants in both control and LP conditions (Figure 7a). Due to auxin responsive reporter DR5::GUS closely related to auxin content in plant root, just like discussed above, we investigated DR5::GUS expression level and found that overexpression of OsPIN2 induced higher DR5::GUS expressed in epidermis cell in the OE lines relative to WT plants (Figure 7b). These suggested that overexpression of OsPIN2 increased auxin distribution in epidermis of root.
Auxin distribution in root transversal section affects LRs or RHs development [11,15,32,33,37]. The LR primordia (LRP) are formed from stele sheath cell [38]. However, the RHs are developed from epidermal cell [39]. Compared with WT, overexpression of OsPIN2 increased RHs formation but decreased the LR formation under control treatment (Figure 7b,c). As well, these were smaller differences between WT and OE lines under LP condition relative to control condition (Figure 7b,c). These results suggested that higher expression of OsPIN2 induced RHs development and reduced LRs formation probably by changing auxin distribution between stele and epidermis in rice roots.

4. Materials and Methods

4.1. Plant Materials

We obtained rice (Oryza sativa L.) of the Nipponbare ecotype for this study. Overexpression of the OsPIN2 lines (OE1 and OE2) transgenic lines was performed following the previous methods [25,40].

4.2. Plant Growth

Plants were grown in pots in a greenhouse under natural light at day/night temperatures of 30/18 °C. We transplanted 1-week-old seedlings of uniform size and vigour into holes in a lid placed over the top of each pot. Nutrient solutions varying from one-quarter to full strength were applied for 1 week, followed by full-strength nutrient solution for another week. Pots receiving normal nutrition (control) were filled with and 300 µM P (Control, NaH2PO4), and those receiving P-deficient nutrition were filled with 10 µM P (LP). The full chemical composition of the International Rice Research Institute (IRRI) nutrient solution (pH 5.5) were 1.25 mM NH4NO3, 0.5 mM K2SO4, 1.0 mM CaCl2, 1.0 mM MgSO4·7H2O, 0.5 mM Na2SiO3, 20.0 µM Fe-EDTA, 9.0 µM MnCl2, 0.39 µM (NH4)6Mo7O24, 20.0 µM H3BO3, 0.77 µM ZnSO4, and 0.32 µM CuSO4. The nutrient solution was replaced with fresh solution daily. Each treatment consisted of four replicates arranged in a completely randomised design to avoid edge effects. All experiments included three independent biological replicates.

4.3. Measurement of Root System Architecture

The lengths of seminal roots (SRs) and root hairs (RHs), and the number of lateral roots (LRs), were chosen to study the effects of P on the root system. SR length was measured using a ruler. LR number of SR were calculated with eye. RH measurements were performed using a microscope (MVX10, Olympus) as described previously [41]. The number of RHs were counted on the RH zone of the SR. The LR primordia were observe by GUS (DR5::GUS) dye [37].

4.4. pPIN2::GUS Construct

The pPIN2::GUS fusion construct was transformed into rice plants. Histochemical localisation of pPIN2::GUS activity was performed at 30 min and 37 °C for the root and shoot base, and at 24 h and 37 °C for leaves. Stained tissues were photographed using a stereomicroscope (SZX2-ILLK, Olympus, Tokyo, Japan) and colour charge-coupled device (CCD) camera (Olympus) following the previous method [8,10]. Histochemical localisation was performed for histochemical GUS staining, and the results were photographed using the same microscope/CCD system.

4.5. Determination of IAA Content

Indole-3-acetic acid (IAA) concentrations in the shoot, shoot base, and root were determined as previously described [42]. After measuring fresh weight, each sample was immediately frozen in liquid N2. High-performance liquid chromatography (HPLC) was performed to measure free IAA concentration following the previous method [42]. A standard IAA sample was obtained from Sigma-Aldrich (Sigma, Louis, MO, USA).
The pDR5::GUS fusion construct was transformed into wild-type (WT) plants and overexpressed OsPIN2 transgenic lines (OE1 and OE2) as previously described [25]. Plant issues were subjected to histochemical GUS staining analyses. Histochemical localisation of DR5::GUS activity was performed for the roots at 30 min and 37 °C. Stained tissues were photographed using the SZX2-ILLK microscope and colour CCD system described above.
GUS activity was examined according to previous method [43]. Samples were homogenised in GUS extraction buffer. Fluorometer values were compared with those of a 4-methylumbelliferone dilution series. Protein content was determined with a Bio-Rad protein assay kit using bovine serum albumin as the standard.

4.6. [3H]IAA Transport

Transport away (basipetal) was assayed in root segments 0–3 cm from the root tip. [3H]IAA solution (3 mL) was applied to the root tip placed horizontally on a plastic film. After incubation in a humid, dark environment for 18 h (overnight), root segments were the distal 1 cm from the root tip. [3H]IAA radioactivity was measured in the 2 cm long segments.
[3H]IAA polar transport was assayed as previous described [44]. Five replicate roots were sampled. The [3H]IAA solution contained 0.5 mM [3H]IAA (20 Ci mmol) in 2 % dimethylsulfoxide (DMSO), 25 mM MES (pH 5.2) and 0.25% agar.

4.7. Cortical Cell Length Analysis

The method of cortical cell length analysis as described previously [45]. The cortical cell visualized with a microscope (Olympus SZX16, Olympus) equipped with a colour CCD camera. The average cortical cell length of the maturation zone of seminal roots was obtained using a mixture of cortical cells with eight replicates in the maturation zone (on average per longitudinal section).

4.8. pCYCB1;1::GUS Construct

The pCYCB1;1::GUS fusion construct was transformed into wild-type (WT) plants and overexpressed OsPIN2 transgenic lines (OE1 and OE2). Plants were stained for GUS activity in the root tips for 30 min at 37 °C. The root tips were subjected to histochemical GUS staining and photographed using a stereomicroscope (SZX2-ILLK, Olympus) equipped with a colour CCD camera.

4.9. Quantitative Reverse-Transcription Polymerase Chain Reaction Analyses

Total RNA was isolated from seedling roots, followed by RNA extraction, reverse transcription, and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analyses. Primer sets for the RSLs genes are listed in Table S1.

4.10. Data Analyses

Data were pooled for calculation of means and standard errors (SE) and subjected to one-way analysis of variance (ANOVA), followed by Ryan-Eynot-Gabriel-Welch F test at p < 0.05 to determine the statistical significance of differences between treatments. All statistical evaluations were conducted using SPSS (version 11.0) statistical software (SPSS Inc., Chicago, IL, USA). Different letters indicate significant differences (p < 0.05, ANOVA).

5. Conclusions

This study showed that overexpression of OsPIN2 increases basipetal transport of auxin in roots and changes rice root morphology in response to P supply. And higher OsPIN2 expression playes important roles in regulating root growth and development by modifying auxin transport and distribution in roots under P deficiency in rice.

Supplementary Materials

Supplementary materials can be found at https://www.mdpi.com/1422-0067/20/20/5144/s1.

Author Contributions

H.S. performed experiments; X.G., F.X., D.W., X.Z., M.L., F.L. assisted the experiment, H.S. analyzed data, G.X., Y.Z. designed the experiment and wrote the paper.

Funding

This work was supported by the National Natural Science Foundation of China (No. 31601821 and 31672225). Innovation Scientists and Technicians Troop Construction Projects of Henan Province (94200510003) and Modern Agricultural Industry Technology System Projects of Henan Province (S2012-04-02).

Acknowledgments

The English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, http://www.textcheck.com/certificate/HBdvU9.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

References

  1. Forde, B.; Lorenzo, H. The nutritional control of root development. Plant Soil. 2001, 232, 51–68. [Google Scholar] [CrossRef]
  2. López-Bucio, J.; Cruz-Ramírez, A.; Herrera-Estrella, L. The role of nutrient availability in regulating root architecture. Curr. Opin. Plant Biol. 2003, 6, 280–287. [Google Scholar] [CrossRef]
  3. Jia, H.; Zhang, S.; Wang, L.; Yang, Y.; Zhang, H.; Cui, H.; Shao, H.; Xu, G. OsPht1;8, a phosphate transporter, is involved in auxin and phosphate starvation response in rice. J. Exp. Bot. 2017, 68, 5057–5068. [Google Scholar] [CrossRef] [PubMed]
  4. Sun, H.; Tao, J.; Bi, Y.; Xie, X.; Yoneyama, K.; Zhao, Q.; Xu, G.; Zhang, Y. OsPIN1b is involved in rice seminal root elongation by regulating root apical meristem activity in response to nitrogen- and phosphate-deficiency. Sci. Rep. 2018, 8, 13014. [Google Scholar] [CrossRef] [PubMed]
  5. Gruber, B.D.; Giehl, R.F.H.; Friedel, S.; Wirén, N. Plasticity of the Arabidopsis root system under nutrient deficiencies. Plant Physiol. 2013, 163, 161–179. [Google Scholar] [CrossRef]
  6. Kellermeier, F.; Armengaud, P.; Seditas, T.J.; Danku, J.; Salt, D.E.; Amtmann, A. Analysis of the root system architecture of Arabidopsis provides a quantitative readout of crosstalk between nutritional signals. Plant Cell 2014, 26, 1480–1496. [Google Scholar] [CrossRef]
  7. Péret, B.; Desnos, T.; Jost, R.; Kanno, S.; Berkowitz, O.; Nussaume, L. Root architecture responses: In search of phosphate. Plant Physiol. 2014, 166, 1713–1723. [Google Scholar] [CrossRef]
  8. Sun, H.; Bi, Y.; Tao, J.; Huang, S.; Hou, M.; Xue, R.; Liang, Z.; Xie, X.; Yoneyama, K.; Shen, Q.; et al. Strigolactones are required for nitric oxide to induce root elongation in response to nitrogen and phosphate deficiency in rice. Plant Cell Environ. 2016, 39, 1473–1484. [Google Scholar] [CrossRef]
  9. Ma, W.; Li, J.; Qu, B.; He, X.; Zhao, X.; Li, B.; Fu, X.; Tong, Y. Auxin biosynthetic gene TAR2 is involved in low nitrogen-mediated reprogramming of root architecture in Arabidopsis. Plant J. 2014, 78, 70–79. [Google Scholar] [CrossRef]
  10. Sun, H.; Tao, J.; Liu, S.; Huang, S.; Chen, S.; Xie, X.; Yoneyama, K.; Zhang, Y.; Xu, G. Strigolactones are involved in phosphate and nitrate deficiency-induced root development and auxin transport in rice. J. Exp. Bot. 2014, 65, 6735–6746. [Google Scholar] [CrossRef]
  11. Giri, J.; Bhosale, R.; Huang, G.; Pandey, B.K.; Parker, H.; Zappala, S.; Yang, J.; Dievart, A.; Bureau, C.; Ljung, K.; et al. Rice auxin influx carrier OsAUX1 facilitates root hair elongation in response to low external phosphate. Nat. Commun. 2018, 9, 1408. [Google Scholar] [CrossRef] [PubMed]
  12. Zhang, C.; Simpson, R.; Kim, C.; Warthmann, N.; Delhaize, E.; Dolan, L.; Byrne, M.; Wu, Y.; Ryan, P. Do longer root hairs improve phosphorus uptake? Testing the hypothesis with transgenic Brachypodium distachyon lines overexpressing endogenous RSL genes. New Phytol. 2018, 217, 1654–1666. [Google Scholar] [CrossRef] [PubMed]
  13. Inahashia, H.; Shelleyb, I.; Yamauchia, T.; Nishiuchia, S.; Takahashi-Nosakab, M.; Matsunamie, M.; Ogawa, A.; Noda, Y.; Inukai, Y. OsPIN2, which encodes a member of the auxin efflux carrier proteins, is involved in root elongation growth and lateral root formation patterns via the regulation of auxin distribution in rice. Physiol. Plant. 2018, 164, 216–225. [Google Scholar] [CrossRef] [PubMed]
  14. Wang, L.; Guo, M.; Li, Y.; Ruan, W.; Mo, X.; Wu, Z.; Sturrock, C.; Yu, H.; Lu, C.; Peng, J.; et al. LARGE ROOT ANGLE1, encoding OsPIN2, is involved in root system architecture in rice. J. Exp. Bot. 2018, 69, 385–397. [Google Scholar] [CrossRef] [PubMed]
  15. Shen, C.; Wang, S.; Zhang, S.; Xu, Y.; Qian, Q.; Qi, Y.; Jiang, D. OsARF16, a transcription factor, is required for auxin and phosphate starvation response in rice (Oryza sativa L.). Plant Cell Environ. 2013, 36, 607–620. [Google Scholar] [CrossRef] [PubMed]
  16. Ljung, K.; Bhalerao, R.; Sandberg, G. Sites and homeostatic control of auxin biosynthesis in Arabidopsis during vegetative growth. Plant J. 2001, 28, 465–474. [Google Scholar] [CrossRef] [PubMed]
  17. Stepanova, A.N.; Yun, J.; Robles, L.M.; Novak, O.; He, W.; Guo, H.; Ljung, K.; Alonso, J.M. The Arabidopsis YUCCA1 flavin monooxygenase functions in the indole-3-pyruvic acid branch of auxin biosynthesis. Plant Cell. 2011, 23, 3961–3973. [Google Scholar] [CrossRef]
  18. Zhao, Y. Auxin biosynthesis: A simple two-step pathway converts tryptophan to indole-3-acetic acid in plants. Mol. Plant. 2012, 5, 334–338. [Google Scholar] [CrossRef]
  19. Friml, J. Auxin transport-shaping the plant. Curr. Opin. Plant Biol. 2003, 6, 712. [Google Scholar] [CrossRef]
  20. Blilou, I.; Xu, J.; Wildwater, M.; Willemsen, V.; Paponov, I.; Friml, J.; Heidstra, R.; Aida, M.; Palme, K.; Scheres, B. The PIN auxin efflux facilitator network controls growth and patterning in Arabidopsis roots. Nature 2005, 433, 39–44. [Google Scholar] [CrossRef]
  21. Zazimalova, E.; Murphy, A.; Yang, H.; Hoyerova, K.; Hosek, P. Auxin transporters-why so many? Cold Spring Harb. Perspect. Biol. 2010, 2, a001552. [Google Scholar] [CrossRef] [PubMed]
  22. Xu, M.; Zhu, L.; Shou, H.; Wu, P. A PIN1 family gene, OsPIN1, involved in auxin-dependent adventitious root emergence and tillering in rice. Plant Cell Physiol. 2005, 46, 1674–1681. [Google Scholar] [CrossRef] [PubMed]
  23. Müller, A.; Guan, C.; Gälweiler, L.; Tänzler, P.; Huijser, P.; Marchant, A.; Parry, G.; Bennett, M.; Wisman, E.; Palme, K. AtPIN2 defines a locus of Arabidopsis for root gravitropism control. EMBO J. 1998, 17, 6903–6911. [Google Scholar] [CrossRef] [PubMed]
  24. Shin, H.; Shin, H.S.; Guo, Z.; Blancaflor, E.B.; Masson, P.H.; Chen, R. Complex regulation of Arabidopsis AGR1/PIN2-mediated root gravitropic response and basipetal auxin transport by cantharidin-sensitive protein phosphatases. Plant J. 2005, 42, 188–200. [Google Scholar] [CrossRef] [PubMed]
  25. Chen, Y.; Fan, X.; Song, W.; Zhang, Y.; Xu, G. Over-expression of OsPIN2 leads to increased tiller numbers, angle and shorter plant height through suppression of OsLAZY1. Plant Biotechnol. J. 2012, 10, 139–149. [Google Scholar] [CrossRef] [PubMed]
  26. Wiśniewska, J.; Xu, J.; Seifertová, D.; Brewer, P.; Růžička, K.; Blilou, I.; Rouquie, D.; Benkova, E.; Scheres, B.; Friml, J. Polar PIN localization directs auxin flow in plants. Science 2006, 312, 883. [Google Scholar] [CrossRef]
  27. Feng, Q.; Zhang, Y.; Hao, P.; Wang, S.; Fu, G.; Huang, Y.; Li, Y.; Zhu, J.; Liu, Y.; Hu, X.; et al. Sequence and analysis of rice chromosome 4. Nature 2002, 420, 316–320. [Google Scholar] [CrossRef]
  28. Sasaki, T.; Matsumoto, T.; Yamamoto, K. The genome sequence and structure of rice chromosome 1. Nature 2002, 420, 312–316. [Google Scholar] [CrossRef]
  29. Wang, J.; Hu, H.; Wang, G.; Li, J.; Chen, J.; Wu, P. Expression of PIN genes in rice (Oryza sativa L.): Tissue specificity and regulation by hormones. Mol. Plant. 2009, 2, 823–831. [Google Scholar] [CrossRef]
  30. Scheres, B.; Benfey, P.; Dolan, L. Root devepopment. Arab. Book 2002, 1, e0101. [Google Scholar] [CrossRef]
  31. Blakeslee, J.; Peer, W.; Murphy, A. Auxin transport. Curr. Opin. Plant Biol. 2005, 8, 494–500. [Google Scholar] [CrossRef] [PubMed]
  32. Lee, R.; Cho, H. Auxin, the organizer of the hormonal/environmental signals for root hair growth. Front. Plant Sci. 2013, 4, 448. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  33. Wang, T.; Li, C.; Wu, Z.; Jia, Y.; Wang, H.; Sun, S.; Mao, C.; Wang, X. Abscisic acid regulates auxin homeostasis in rice root tips to promote root hair elongation. Front. Plant Sci. 2017, 8, 1121. [Google Scholar] [CrossRef] [PubMed]
  34. Moon, S.; Cho, L.-H.; Kim, Y.-J.; Gho, Y.-S.; Jeong, H.Y.; Hong, W.-J.; Lee, C.; Park, H.; Jwa, N.-S.; Dangol, S.; et al. RSL class II transcription factors guide the nuclear localization of RHL1 to regulate root hair development. Plant Physiol. 2018, 179, 558–568. [Google Scholar] [CrossRef] [PubMed]
  35. Muday, G.K.; Rahman, A. Auxin transport and the integration of gravitropic growth. Plant Trop. 2008, 3, 47–78. [Google Scholar]
  36. Feraru, E.; Friml, J. PIN polar targeting. Plant Physiol. 2008, 147, 1553–1559. [Google Scholar] [CrossRef] [PubMed]
  37. Sun, H.; Li, J.; Song, W.; Tao, J.; Huang, S.; Chen, S.; Hou, M.; Xu, G.; Zhang, Y. Nitric oxide generated by nitrate reductase increases nitrogen uptake capacity by inducing lateral root formation and inorganic nitrogen uptake under partial nitrate nutrition in rice. J. Exp. Bot. 2015, 66, 2449–2459. [Google Scholar] [CrossRef] [Green Version]
  38. Malamy, J.; Benfey, P. Organization and cell differentiation in lateral root Arabidopsis thaliana. Development 1997, 124, 33–44. [Google Scholar]
  39. Pemberton, L.; Tsai, S.; Lovell, P.; Harris, P. Epidermal patterning in seedling roots of eudicotyledons. Ann. Bot. 2001, 87, 649–654. [Google Scholar] [CrossRef]
  40. Wu, D.; Shen, H.; Yokawa, K.; Baluška, F. Overexpressing OsPIN2 enhances aluminium internalization by elevating vesicular trafficking in rice root apex. J. Exp. Bot. 2015, 66, 6791–6801. [Google Scholar] [CrossRef]
  41. Sun, S.; Gu, M.; Cao, Y.; Huang, X.; Zhang, X.; Ai, P.; Zhao, J.; Fan, X.; Xu, G. A constitutive expressed phosphate transporter, OsPht1;1, modulates phosphate uptake and translocation in phosphate-replete rice. Plant Physiol. 2012, 159, 1571–1581. [Google Scholar] [CrossRef] [PubMed]
  42. Song, W.; Sun, H.; Li, J.; Gong, X.; Huang, S.; Zhu, X.; Zhang, Y.; Xu, G. Auxin distribution is differentially affected by nitrate in roots of two rice cultivars differing in responsiveness to nitrogen nutrients. Ann. Bot. 2013, 112, 1383–1393. [Google Scholar] [CrossRef] [PubMed]
  43. Jia, H.; Ren, H.; Gu, M.; Zhao, J.; Sun, S.; Zhang, X.; Chen, J.; Wu, P.; Xu, G. The phosphate transporter gene OsPht1;8 is involved in phosphate homeostasis in rice. Plant Physiol. 2011, 156, 1164–1175. [Google Scholar] [CrossRef] [PubMed]
  44. Liu, J.; An, X.; Cheng, L.; Chen, F.; Bao, J.; Yuan, L.; Zhang, F.; Mi, G. Auxin transport in maize roots in response to localized nitrate supply. Ann. Bot. 2010, 106, 1019–1026. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  45. Jia, L.; Zhang, B.; Mao, C.; Li, J.; Wu, Y.; Wu, P.; Wu, Z. OsCYTINV1 for alkaline/neutral invertase is involved in root cell development and reproductivity in rice (Oryza sativa L.). Planta 2008, 228, 51–59. [Google Scholar] [CrossRef]
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Figure 1. The expression of OsPIN2 in rice plants. Seedlings were grown in hydroponic media containing normal nutrition (control, 300 μM P) or low P (LP, 10 µM) for 6 h. Bar = 1mm. (a) pPIN2::GUS expression in leaf, shoot base, lateral root and root tip. (b), OsPIN2 expression in roots under control and LP conditions. OsPIN2 expression was normalized to that of OsACT. h = hours. Data are means ± SE of three replicates and bars with different letters indicate significant differences in same organ (p < 0.05, ANOVA). All experiments included three independent biological replicates.
Figure 1. The expression of OsPIN2 in rice plants. Seedlings were grown in hydroponic media containing normal nutrition (control, 300 μM P) or low P (LP, 10 µM) for 6 h. Bar = 1mm. (a) pPIN2::GUS expression in leaf, shoot base, lateral root and root tip. (b), OsPIN2 expression in roots under control and LP conditions. OsPIN2 expression was normalized to that of OsACT. h = hours. Data are means ± SE of three replicates and bars with different letters indicate significant differences in same organ (p < 0.05, ANOVA). All experiments included three independent biological replicates.
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Figure 2. IAA content and histochemical localization of DR5::GUS in the wild type (WT, Nipponbare) and the overexpression of OsPIN2 transgenic plants (OE1/OE2). Seedlings were grown in hydroponic media containing normal nutrition (control, 300 μM P) or low P (LP, 10 µM) for 7 d. (a,b), IAA concentration in lateral root (LR) (a) and root tip (RT) (b). (c), The expression of DR5::GUS. Bar = 1mm. d = days. Data are means ± SE and bars with different letters in the same root zone indicate significant difference at p < 0.05 tested with ANOVA. The red boxes indicate the root cap region. All experiments included three independent biological replicates.
Figure 2. IAA content and histochemical localization of DR5::GUS in the wild type (WT, Nipponbare) and the overexpression of OsPIN2 transgenic plants (OE1/OE2). Seedlings were grown in hydroponic media containing normal nutrition (control, 300 μM P) or low P (LP, 10 µM) for 7 d. (a,b), IAA concentration in lateral root (LR) (a) and root tip (RT) (b). (c), The expression of DR5::GUS. Bar = 1mm. d = days. Data are means ± SE and bars with different letters in the same root zone indicate significant difference at p < 0.05 tested with ANOVA. The red boxes indicate the root cap region. All experiments included three independent biological replicates.
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Figure 3. The morphology of the wild type (WT, Nipponbare) and the overexpression of OsPIN2 transgenic plants (OE1/OE2). Seedlings were grown in hydroponic media containing normal nutrition (control, 300 μM P) or low P (LP, 10 µM). (a) The morphology of rice plants. (b) Seminal root (SR) length. Data are means ± SE and bars with different letters indicate significant differences in the same gene (p < 0.05, ANOVA). All experiments included three independent biological replicates.
Figure 3. The morphology of the wild type (WT, Nipponbare) and the overexpression of OsPIN2 transgenic plants (OE1/OE2). Seedlings were grown in hydroponic media containing normal nutrition (control, 300 μM P) or low P (LP, 10 µM). (a) The morphology of rice plants. (b) Seminal root (SR) length. Data are means ± SE and bars with different letters indicate significant differences in the same gene (p < 0.05, ANOVA). All experiments included three independent biological replicates.
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Figure 4. The epidermal cell lengths in the maturity zone and expression levels of CYCB1;1 in the meristem zone in rice plants. Seedlings were grown in hydroponic medium containing normal nutrition (control, 300 μM P) and low P (LP, 10 µM) for 7 d. (a,b) Epidermal cell length of seminal root, Bar = 500 μm. (c) Cell cycle activity of the root meristem of seminal root, as monitored by the pCYCB1;1::GUS reporter. (d) The expression of OsCYCB1;1 by qRT-PCR. Bar = 1mm. d = days. Data are means ± SE and bars with different letters indicate significant difference at p < 0.05 tested with ANOVA. All experiments included three independent biological replicates.
Figure 4. The epidermal cell lengths in the maturity zone and expression levels of CYCB1;1 in the meristem zone in rice plants. Seedlings were grown in hydroponic medium containing normal nutrition (control, 300 μM P) and low P (LP, 10 µM) for 7 d. (a,b) Epidermal cell length of seminal root, Bar = 500 μm. (c) Cell cycle activity of the root meristem of seminal root, as monitored by the pCYCB1;1::GUS reporter. (d) The expression of OsCYCB1;1 by qRT-PCR. Bar = 1mm. d = days. Data are means ± SE and bars with different letters indicate significant difference at p < 0.05 tested with ANOVA. All experiments included three independent biological replicates.
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Figure 5. Lateral root (LR) density and lateral root (LR) primordia in wild type (WT, Nipponbare) and OsPIN2 overexpression lines (OE1/OE2). Seedlings were grown in hydroponic media containing normal nutrition (control, 300 μM P) or low P (LP, 10 µM). (a) LR density of SR. (b) Lateral root (LR) primordia formation. Bar = 1mm. Data are means ± SE and bars with different letters indicate significant differences in the same gene (p < 0.05, ANOVA). The red arrow indicates the LR primordia. All experiments included three independent biological replicates.
Figure 5. Lateral root (LR) density and lateral root (LR) primordia in wild type (WT, Nipponbare) and OsPIN2 overexpression lines (OE1/OE2). Seedlings were grown in hydroponic media containing normal nutrition (control, 300 μM P) or low P (LP, 10 µM). (a) LR density of SR. (b) Lateral root (LR) primordia formation. Bar = 1mm. Data are means ± SE and bars with different letters indicate significant differences in the same gene (p < 0.05, ANOVA). The red arrow indicates the LR primordia. All experiments included three independent biological replicates.
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Figure 6. Root hairs (RHs) formation and qRT-PCR analysis of genes in wild type (WT, Nipponbare) and OsPIN2 overexpression lines (OE1/OE2). Seedlings were grown in hydroponic media containing normal nutrition (control, 300 μM P) or low P (LP, 10 µM). (a) The morphology of RHs. (b) The density of RHs. (c) Average RHs length. Bar = 0.5mm. (d,e) The gene expression under normal nutrition (d) and low P; (e) for 6 h. Relative mRNA levels were normalised to OsACT. h = hours. Data are means ± SE and bars with different letters indicate significant differences in the same gene (p < 0.05, ANOVA). All experiments included three independent biological replicates.
Figure 6. Root hairs (RHs) formation and qRT-PCR analysis of genes in wild type (WT, Nipponbare) and OsPIN2 overexpression lines (OE1/OE2). Seedlings were grown in hydroponic media containing normal nutrition (control, 300 μM P) or low P (LP, 10 µM). (a) The morphology of RHs. (b) The density of RHs. (c) Average RHs length. Bar = 0.5mm. (d,e) The gene expression under normal nutrition (d) and low P; (e) for 6 h. Relative mRNA levels were normalised to OsACT. h = hours. Data are means ± SE and bars with different letters indicate significant differences in the same gene (p < 0.05, ANOVA). All experiments included three independent biological replicates.
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Figure 7. [3H]IAA transport and histochemical localization of DR5::GUS and root hair (RH) and lateral root primordia (LRP) formation. Seedlings were grown in hydroponic media containing normal nutrition (control; 2.5 mM N, 300 μM P) or low P (LP, 10 µM) for 7 d. (a) IAA transport for acropetal. (b) DR5::GUS distribution in transverse section. (c) RHs and lateral root primordia (LRPs) formation. d = days. Bar = 0.2 mm (b) and Bar = 0.5mm (c). Data are means ± SE and bars with different letters indicate significant differences in the same gene (p < 0.05, ANOVA). All experiments included three independent biological replicates.
Figure 7. [3H]IAA transport and histochemical localization of DR5::GUS and root hair (RH) and lateral root primordia (LRP) formation. Seedlings were grown in hydroponic media containing normal nutrition (control; 2.5 mM N, 300 μM P) or low P (LP, 10 µM) for 7 d. (a) IAA transport for acropetal. (b) DR5::GUS distribution in transverse section. (c) RHs and lateral root primordia (LRPs) formation. d = days. Bar = 0.2 mm (b) and Bar = 0.5mm (c). Data are means ± SE and bars with different letters indicate significant differences in the same gene (p < 0.05, ANOVA). All experiments included three independent biological replicates.
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Sun, H.; Guo, X.; Xu, F.; Wu, D.; Zhang, X.; Lou, M.; Luo, F.; Xu, G.; Zhang, Y. Overexpression of OsPIN2 Regulates Root Growth and Formation in Response to Phosphate Deficiency in Rice. Int. J. Mol. Sci. 2019, 20, 5144. https://doi.org/10.3390/ijms20205144

AMA Style

Sun H, Guo X, Xu F, Wu D, Zhang X, Lou M, Luo F, Xu G, Zhang Y. Overexpression of OsPIN2 Regulates Root Growth and Formation in Response to Phosphate Deficiency in Rice. International Journal of Molecular Sciences. 2019; 20(20):5144. https://doi.org/10.3390/ijms20205144

Chicago/Turabian Style

Sun, Huwei, Xiaoli Guo, Fugui Xu, Daxia Wu, Xuhong Zhang, Manman Lou, Feifei Luo, Guohua Xu, and Yali Zhang. 2019. "Overexpression of OsPIN2 Regulates Root Growth and Formation in Response to Phosphate Deficiency in Rice" International Journal of Molecular Sciences 20, no. 20: 5144. https://doi.org/10.3390/ijms20205144

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