research paper pectin enhances rice (oryza sativa) root...

Journal of Experimental Botany, Vol. 66, No. 3 pp. 1017–1024, 2015doi:10.1093/jxb/eru461 Advance Access publication 20 December, 2014

ReseaRch PaPeR

Pectin enhances rice (Oryza sativa) root phosphorus remobilization

Xiao Fang Zhu1, Zhi Wei Wang1, Jiang Xue Wan1, Ying Sun1, Yun Rong Wu1, Gui Xin Li2, Ren Fang Shen3 and Shao Jian Zheng1,*1 State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences; Zhejiang University, Hangzhou 310058, China2 College of Agronomy and Biotechnology, Zhejiang University, Hangzhou 310058, China3 State Key Laboratory of Soil and Sustainable Agriculture, China Institute of Soil Science, Chinese Academy of Science, Nanjing 210008, China

* To whom correspondence should be addressed. E-mail: [email protected]

Received 12 June 2014; Revised 15 October 2014; Accepted 21 October 2014

Abstract

Plants growing in phosphorus (P)-deficient conditions can either increase their exploration of the environment (hence increasing P uptake) or can solubilize and reutilize P from established tissue sources. However, it is currently unclear if P stored in root cell wall can be reutilized. The present study shows that culture of the rice cultivars ‘Nipponbare’ (Nip) and ‘Kasalath’ (Kas) in P-deficient conditions results in progressive reductions in root soluble inorganic phos-phate (Pi). However, Nip consistently maintains a higher level of soluble Pi and lower relative cell wall P content than does Kas, indicating that more cell wall P is released in Nip than in Kas. P-deficient Nip has a greater pectin and hemicellulose 1 (HC1) content than does P-deficient Kas, consistent with the significant positive relationship between pectin and root-soluble Pi levels amongst multiple rice cultivars. These observations suggest that increased soluble Pi might result from increased pectin content during P starvation. In vitro experiments showed that pectin releases Pi from insoluble FePO4. Furthermore, an Arabidopsis thaliana mutant with reduced pectin levels (qua1-2), has less root soluble Pi and is more sensitive to P deficiency than the wild type (WT) Col-0, whereas NaCl-treated WT plants exhibit both an increased root pectin content and an elevated soluble Pi content during P-starvation. These observa-tions indicate that pectin can facilitate the remobilization of P deposited in the cell wall. This is a previously unknown mechanism for the reutilization of P in P-starved plants.

Key words: Phosphorus deficiency, cell wall, pectin, hemicellulose, rice, Arabidopsis.

Introduction

As an essential macronutrient, phosphorus (P) plays impor-tant roles in plant growth and development. P is a constituent of many cellular molecules (e.g. nucleic acids, phospholipids, ATP, etc) and is also a pivotal regulator of many biological processes (e.g. energy transfer, protein activation, carbon and amino acid metabolism; Marschner, 1995). Approximately 67% of the world’s potential arable land is estimated to be P-deficient soil, and this deficiency places limitations on crop growth and productivity (Gong et al., 2011). Furthermore, although P is relatively abundant in many soils, the majority

of it is not available to most plants. In general, plants have two strategies to maximize P uptake and tolerate P deficiency. First, plants enhance P uptake by adopting morphological changes (e.g. the production of more lateral roots and root hairs or an increase in root biomass; Watanabe et al., 2006). These changes enable the plant to explore increased soil vol-umes and thus access limited bioavailable P sources. In addi-tion, plant roots secrete organic acids (Ae et al., 1990; Otani et al., 1996; Zhu et al., 2012b), phosphatases (Hall, 1969; Pammenter and Woolhouse, 1975), and other substances

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(Otani et al., 1996) that release P from insoluble P sources in soil. Second, plants can improve the efficiency with which they use previously assimilated P. For example, phosphohy-drolases may function as a P-recycling mechanism in sec-ondary metabolism (Gong et al., 2011). In addition, acid phosphatase (APase) and ribonuclease (RNase) can mobi-lize organic P in established tissues, and this mobilized P can then be translocated to younger, growing tissues (Yun and Kaeppler, 2001). Nevertheless, cellular mechanisms for reu-tilization of the insoluble P deposited in the plant cell wall remain relatively unexplored.

The plant cell wall is composed mainly of cellulose and of matrix polysaccharides including pectin and hemicelluloses (Cosgrove, 2005). Ae et al. (1996) previously reported that, following growth in conditions of low P availability, the cell wall polysaccharides of groundnut roots exhibit P-solubilizing activity. Subsequently, Gessa et al. (1997) found that the three-dimensional arrangement of the carboxyl groups in the d-galacturonic acid component of the pectic network of plant cell walls has a high affinity for Fe3+, and that this net-work may possess the ability to solubilize Fe-P and Al-P. This finding is supported by a previous report from Nagarajah et al. (1970), who found that polygalacturonic acid can form a complex with Fe3+ or Al3+ through phosphate ligand exchange, resulting in the desorption of P from clay minerals. These previous studies therefore suggested that cell wall poly-saccharides might have some involvement in the reutilization of P sequestered within the plant cell wall. Related to this suggestion, we recently found that P deficiency decreases the pectin and hemicellulose 1 content of A. thaliana cell walls (Zhu et al., 2012b). We therefore decided to determine if the modification of cell wall components consequent upon P deficiency could affect P remobilization within the plant.

Rice (Oryza sativa) is not only a worldwide staple crop, but also an important monocot plant model. Using two typi-cal rice cultivars, ‘Nipponbare’ (Nip) and ‘Kasalath’ (Kas), which were shown to have different sensitivity to P defi-ciency (Gamuyao et al, 2012), this study showed a correla-tion between cell wall pectin and soluble inorganic phosphate (Pi) contents. This relationship was then further verified in other rice cultivars and by studying a pectin-deficient mutant of A. thaliana. Our study proposes a novel mechanism for the remobilization of P from the cell wall under P-deficient conditions.

Materials and methods

Plant material and growth conditionsRice (Oryza sativa) sp. japonica ‘Nipponbare’ (Nip) and indica ‘Kasalath’ (Kas) cultivars were initially used in this study, with subsequent study of thirteen additional rice cultivars: Azucena, Zhezhen, Taizao, Huanghuazhan, Erjiufen, Zhongsierhao, Huhan 1B, Wuyunjing, Jiahua No.1, MH63, Widetype, 9311, and xiushui110. Seeds were transferred to an incubator at 25 ºC for ger-mination. Germinated seeds were then transferred to the following treatments: +P (complete nutrient solution), –P (complete nutri-ent solution lacking P). The complete nutrient solution contained 114.25 mg l–1 NH4NO3, 50.375 mg l

–1 NaH2PO4·2H2O, 89.25 mg l–1

K2SO4, 110.75 mg l–1 CaCl2, 405 mg l

–1 MgSO4·7H2O, 1.875 mg l–1

MnCl2·4H2O, 0.0925 mg l–1 (NH4)6Mo7O24, 1.1675 mg l

–1 H3BO3, 0.04375 mg l–1 ZnSO4·7H2O, 0.03875 mg l

–1 CuSO4, 34.75 mg l–1

FeSO4·7H2O, 46.53 mg l–1 EDTA–Na2, solutions were renewed every

3 d. All experiments were conducted in a growth chamber with a 14-h/26 ºC day and a 10-h/23 ºC night regime, a light intensity of 400 µmol m–2 s–1 and a relative humidity of 60%. All experiments in this study were conducted independently at least twice.

Arabidopsis thaliana (Columbia ecotype, Col-0, WT) and qua1-2 were also used. For growth on plates, following surface steriliza-tion, seeds were germinated in Petri dishes containing agar-solid-ified nutrient medium. The nutrient medium consisted of the following macronutrients: KNO3, 606.6 mg l

–1; Ca(NO3)2, 944.6 mg l–1; MgSO4, 246 mg l

–1; NH4H2PO4, 11.513 mg l–1, and the follow-

ing micronutrients: Fe(Ⅲ)–EDTA, 21.06 mg l–1; KCl, 3.7275 mg l–1; H3BO3, 0.7729 mg l

–1; MnSO4, 0.16902 mg l–1; CuSO4, 0.12484 mg

l–1; ZnSO4, 0.28756 mg l–1; H2MoO4, 0.017997 mg l

–1; NiSO4 0.026285 mg l–1, according to Murashige-Skoog salts (Murashige and Skoog, 1962). The Petri dishes were placed at 4 ºC for 2 d for vernalization, and then transferred to an environmentally controlled growth chamber, positioned vertically as in Yang et al. (2011), and maintained at a temperature of 24 ºC, a light intensity of 140 µmol m–2 S–1 and a 16/8 h day/night rhythm. Growth con-ditions were the same for all experiments except where otherwise specified.

For hydroponic culture, A. thaliana seedlings were first asepti-cally germinated on the above solid Murashige and Skoog medium. Two weeks later, young plantlets were placed on vermiculite for an additional 3 weeks in an environmentally controlled growth cham-ber. Seedlings of similar rosette diameters were then transferred to nutrient solution containing the above mentioned Murashige-Skoog salts for another week. The plants were subsequently subjected to the following treatments: +P (complete nutrient solution), –P (com-plete nutrient solution lacking P), NaCl (complete nutrient solution with additional 5850 mg l–1 NaCl), and –P+NaCl (complete nutrient solution lacking P with additional 5850 mg l–1 NaCl). In –P nutri-ent solution, the lack of P also resulted in the lack of NH4

+ as P was applied as NH4H2PO4, thus NH4

+ was compensated by the same concentration of NH4NO3.

Effect of P deficiency on Arabidopsis growthSeedlings (WT Col-0 and qua1-2) with a root length about 1 cm long were transferred to Petri dishes containing agar-solidified nutrient medium (with or without P) for 10 d, following which the photo-graphs were taken.

Determination of soluble inorganic phosphate (Pi) concentrationsRice and A. thaliana roots and leaves were weighed and homoge-nized with a mortar and pestle in liquid nitrogen. Pi was extracted with 4 ml of 5 % (v/v) sulphuric acid (5 M) solution. Following cen-trifugation at 12 000 g, 400 µl of the supernatant was transferred and mixed with a 200 µl aliquot of 15 % (w/v) fresh ascorbic acid (pH 5.0) dissolved in ammonium molybdate. The mixture was then incu-bated at 37 ºC for 30 min and absorbance at 650 nm was recorded. Pi concentration was calculated using fresh weight normalization (Zheng et al., 2009).

Cell wall extraction and fractionationExtraction of crude cell wall materials and subsequent fractionation of cell wall components were performed according to Zhong and Läuchli (1993) with minor modifications according to Yang et al. (2011). Roots were ground up with mortar and pestle in liquid nitro-gen, homogenized with 75% ethanol for 20 min in ice-cold water, centrifuged at 8000 rpm for 10 min, following which the superna-tant was removed. The pellets were then homogenized and washed sequentially with acetone, with methanol:chloroform at a ratio of

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1:1, and with methanol, each wash for 20 min each, all supernatants being removed following centrifugation. The remaining pellets (i.e. the cell wall materials) were dried and stored at 4 ºC for further use.

Pectin was extracted from the prepared cell wall materials (about 2 mg) by extraction three times with 1-ml hot water at 100 ºC for 1 h each. The supernatants were combined in a 5-ml tube follow-ing centrifugation at 12 000 rpm for 10 min. The residue was subse-quently extracted twice with 1-ml 4% (w/v) KOH and 0.02% (w/v) KBH4 at room temperature for 12 h. The two supernatants were then combined in a 2-ml tube, and centrifuged at 12 000 rpm for 10 min to obtain hemicellulose fraction 1 (HC1). Finally, the residue was further extracted twice with 1-ml 24% (w/v) KOH and 0.02% (w/v) KBH4 at room temperature for 12 h. The two supernatants were combined in a 2-ml tube, and then centrifuged at 12 000 rpm for 10 min to obtain the hemicellulose fraction 2 (HC2).

Uronic acid and total polysaccharide measurementPectin uronic acid content was assayed according to Blumenkrantz and Asboe-Hansen (1973), using galacturonic acid (Sigma) as standard. Briefly, 200 µl pectin extract was incubated with 1 ml 98% H2SO4 (containing 0.0125M Na2B4O7·10H2O) at 100 ºC for 5 min. Following chilling, 20 µl M-hydro-dipheny (0.15%) was applied to the solution, which was then left standing for 20 min at room tem-perature before the absorbance was measured spectrophotometri-cally at 520 nm.

Total polysaccharide contents of the hemicellulose fractions were determined by the phenol sulfuric acid method (Dubois et al., 1956), and expressed as glucose equivalents. Briefly, 200 µl hemicellulose 1 (HC1) extracts were incubated with 1 ml 98% H2SO4 and 10 µl 80% phenol at room temperature for 15 min before incubation at 100 ºC for 15 min. Following chilling, absorbance was measured spectro-photometrically at 490 nm.

P retention in cell wall materialsAs plant P status changes with respect to growth phases, and to reflect relative differences between the +P and –P treatments, we calculated the percentage of P contained in the cell wall in the –P treatment to that in the +P treatment. To determine the P retention in cell walls, 5 mg cell wall materials were placed in a 2-ml Eppendorf tube filled with 1 ml 2 N HCl, following which the solution was shaken on a rotary shaker for 3 d. The samples were then centrifuged at 23 000g and the supernatant collected for P concentration determination by inductively coupled plasma atomic emission spectroscopy (ICP-AES; Fisons ARL Accuris, Ecublens, Switzerland).

Root exudate collection and pH measurementFor organic acid and pH analysis, Nip and Kas were first grown in complete nutrient solution for 4 weeks, following which seedlings were transferred to +P or –P nutrient solution for a further week. The solutions were renewed every 3 d, whilst the pH was measured daily with a pH electrode (Sartorius, Gottingen, Germany). Following 6 d of culture, roots were exposed to 55.5 mg l–1 CaCl2 solution (pH 5.6) for 24 h to collect root exudates. Roots were harvested, then washed with deionized water, and their fresh weights were recorded.

Measurement of organic acid effluxRoot exudates (as above) were first passed through a cation exchange column (16 mm×14 cm) filled with 5 g Amerlite IR-120B resin (H+ form, Muromachi chemical, Tokyo, Japan), and then through an anion exchange column filled with 1.5 g Dowex 1 × 8 resin (100–200 mesh, formate form). Organic acid anions retained in the anion exchange resin were eluted with 15 ml 1 M HCl, and the eluent was concentrated to dryness using a rotary evaporator at 40 ºC. The residue was re-dissolved in 1 ml 1200.3 mg l–1 NaOH and filtered (0.2 µm) before analysis. Organic acid anions were detected by ion chromatography (ICS 3000; Dionex) equipped with an IonPac AS11 anion-exchange analytical column (4 × 250 mm) and a guard column (4 × 50 mm). The mobile phase was 1200 mg l–1 NaOH at a flow rate of 0.6 ml min–1.

Statistical analysisEach treatment had at least three replicates, each experiment was repeated at least twice, and one set of representative data are shown in the results. Data were analysed by one-way ANOVA and the means were compared by Student’s t test. Different letters on the histograms indicate that the means were statistically different at P

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significant in the shoot except at day 7 (Fig. 1B). Therefore, in the following experiments, we mainly focus on the change of root P.

Root cell walls were extracted and the P retained in the cell walls was measured. Nearly 50% of total P was present in the cell walls of both rice cultivars. As P content is changeable in different stage of plant growth, we calculated the percentage of P contained in the cell wall in the –P treatment to that in the +P treatment at the same treatment time to make it compara-ble between different cultivars. Nip always exhibited a lower –P/+P percentage than did Kas as indicated by the dynamics of change in cell wall P content over time of exposure to P deficiency, especially within the 3 weeks of P-deficient treat-ment (Fig. 2). As on the start day of the treatment the per-centage is 100 for both cultivars, it means that much more P was released from the cell wall of Nip on the first 3 weeks than that from Kas. As the largest difference between Kas and Nip was observed on the 7th day of treatment, all subse-quent experiments were performed at this time point.

P deficiency was associated with a decreased pectin con-tent in Kas, whereas Nip pectin content was unaffected by

P starvation (Fig. 3A; uronic acid levels provide a measure-ment of pectin content). Thus, the root cell walls of P-starved Nip plants have a greater pectin content than the roots of P-starved Kas plants (Fig. 3A). In contrast, whereas the hemicellulose 1 (HC1) and hemicellulose 2 (HC2) uronic acid contents of both Nip and Kas were relatively unaffected by P starvation (Fig. 3B, C), the HC1 content indicated by total sugar content decreased during P starvation in Kas but increased in Nip, whereas the HC2 content was relatively unaffected (Fig. 4).

The above described changes in soluble Pi level and cell wall polysaccharide content upon P starvation are suggestive of a general relationship between the two. To test this possi-bility, an array of 15 different rice cultivars were used (Fig. 5), and a general positive correlation between root-soluble Pi and pectin contents was found (this correlation was significant if the cultivar Huanghuazhan was excluded from the regression analysis; Fig. 5A). In contrast, no relationship was observed with HC1 content (Fig. 5B), suggesting that root-soluble Pi level is related to cell wall pectin content.

It has previously been reported that the secretion of organic acids may affect the capacity for cell wall binding of cations

Fig. 2. Retention of P in the cell walls of roots of P-starved Kas and Nip plants. Seedlings were exposed to P-sufficient or P-deficient nutrient solutions for 5 weeks. P retained in the cell wall was determined from the percentage of the cell wall P content of plants grown in P-starvation conditions (–P) versus the cell wall P content of plants grown in P sufficient conditions (+P). Data are means±SD (n=4). Asterisks indicate a significant difference at P


such as Al (Li et al., 2009). However, there was no significant differences in either pH value (dependent on proton extru-sion) or organic acid (malate, citrate, and oxalate) secretion between Nip and Kas (irrespective of P status; Fig. 6), sug-gesting that organic acid efflux and acidification are unlikely to promote root P mobilization during P starvation.

We conducted in vitro experiments to test whether pectin can release Pi from an insoluble P source and found that pectin can release Pi from FePO4 in vitro (Fig. 7). To verify the relationship between pectin and soluble Pi levels, and to determine if this effect is more generally characteristic of plant roots (i.e. rather than being a specific characteristic of rice roots), we next tested the relationship in the dicots model plant Arabidopsis thaliana. The qua1-2 mutant has a reduced cell wall pectin content (Bouton et al., 2002), and it has altered responses to the P status of the growth medium. In P-deficient conditions, the roots of qua1-2 mutants were shorter than those of WT (Col-0) controls, whereas in con-trol P conditions qua1-2 mutant roots were longer than those of WT controls (Fig. 8A). Consistent with these findings, in P-deficient conditions, the soluble Pi content of qua1-2 mutant roots was less than that of WT although the soluble

Pi content in the qua1-2 mutant was much higher than that of WT in the +P conditions, which might be due to the lower fixation of Pi in the cell wall in the mutant under P-sufficient condition (Fig. 8B). Furthermore, as it was reported that NaCl treatment can increase pectin content in the cell wall (Schmohl and Horst, 2000), we treated the Arabidopsis plants with 100 mM 5850 mg l–1 NaCl too, and found that the NaCl-treated Col-0 can elevate the soluble Pi content concomitant with the increment of the pectin content under P-deficient conditions (Fig. 9), further demonstrating the role of pectin in the remobilization of P in cell wall.

Discussion

It has been known for some time that plants remobilize previ-ously assimilated P as a strategy for coping with P starvation (Raghothama and Karthikeyan, 2005). For example, when plants are suffering from P deficiency, organic and inorganic P in existing cells, tissues, and organs is decomposed and sol-ubilized and thus released for reutilization in newly growing tissues of the plant. Accordingly, older leaves tend to senesce more rapidly during P starvation, because their reutilized P

Fig. 5. Identification of a correlation between root soluble inorganic phosphate (Pi) and cell wall pectin contents. (A) Root soluble Pi content correlates with uronic acid determined pectin content. (B) Root soluble Pi content does not correlate with HC1 content. Fifteen different rice cultivars were analysed (HuangHuaZhan was treated as an outlier in A). Data are means±SD (n=4).

Fig. 6. Proton extrusion (A) and organic acid secretion (B) are shown for four-week-old seedlings grown in the presence or absence of P. Data are means±SD (n=4).

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is translocated to the newly growing tissues. In contrast, root integrity needs to be maintained to allow systematic uptake and transport of water and nutrients to the shoots. It is there-fore potentially important for plants to maximize the utili-zation of P stored in the non-metabolically active parts of the roots (such as the cell wall) in conditions where soil P availability cannot meet the requirements for root growth. Here, we have determined that root cell wall P is a source of reutilized P during P starvation, and that pectin plays a role in releasing P from that source. As expected, about half of the P was present in the matrix space in the cell walls of rice root (Oryza sativa), thus indicating that cell wall P is indeed a potential source of P remobilization during P starvation. All

these results demonstrated for the first time that P deposited in the cell wall can be reutilized and that cell wall pectin con-tributes to P remobilization. The conclusions are based on the following observations.

First, root soluble Pi content correlates positively with root pectin content in Nip, Kas, and a further group of 15 assayed rice cultivars. Before the P reutilization, stored P needs to be resolubilized, and this is why we initially measured changes in root soluble Pi during the course of a 5-week period of P starvation in Nip and Kas, showing that whilst the soluble Pi level in the roots of both varieties decreased with time, the soluble Pi content of Nip was always higher than that of Kas (Fig. 1A), whereas the relative amount of P retained in the cell wall of –P roots (versus +P roots) was always higher in Kas (Fig. 2). These initial observations suggested that the P deposited in the cell wall of Nip is more easily solubilized and hence contributes to the higher soluble Pi of Nip. As plant cell wall is composed of a matrix of polysaccharides, with cellulose, hemicellulose, and pectin being major compo-nents, the levels of pectin and hemicelluloses were examined. Although there was no difference between Nip and Kas at the start of the treatment, Nip had significantly higher pectin and HC1 contents after one week of –P treatment (Figs 3, 4). These observations suggested that the higher pectin and HC1 contents of Nip might be related to the higher soluble Pi content of Nip. However, although HC1 content correlates well with Al accumulation in A. thaliana, rice, and triticale (Liu et al., 2008; Yang et al, 2008; Zhu et al., 2012a), and Cd retention in rice and A. thaliana (Xiong et al, 2009; Zhu et al; 2012b), there was no significant correlation between HC1 and root soluble Pi contents among the 15 assayed rice cultivars (Fig. 5). However, a strong positive correlation was

Fig. 8. The phenotype of WT (Col-0) and the qua1-2 mutant. A. thaliana seedlings grown in P-deficient (–P) or P-sufficient (+P) conditions (A), soluble inorganic phosphate (Pi) content of roots (B), and shoots (C). Seedlings about 1 cm in length were grown on P-deficient or P-sufficient media for a further 10 d. Data are means±SD (n=4). Columns with different letters are significantly different at P


observed between the pectin and soluble Pi contents of these rice cultivars (Fig. 5), thus further confirming the relationship between pectin and soluble Pi content in roots of P-starved plants. However, Zhu et al. (2012b) previously reported a sig-nificant decrease of pectin content in P-starved A. thaliana, which is consistent with Kas, and this may be due to different effects of P deficiency on pectin content in different plant spe-cies or cultivars within the same species.

Second, pectin can release Pi from insoluble P sources. Pectin has previously been shown to be a cation adsorber. The negatively charged carboxylic groups of pectin have a particularly high affinity for Al3+, Fe3+, and Cd2+ (Blamey et al., 1990; Chang et al., 1999; Zhu et al., 2012b), an affin-ity which even exceeds the capacity to bind Ca2+ (for review, see Rengel and Zhang, 2003). PO4

3– also has high affinity for Fe3+, Al3+, and Ca2+, resulting in the formation of insolu-ble FePO4, AlPO4, and Ca3(PO4)2. For example, AlPO4 is thought to be deposited in the apoplast of buckwheat when there is Al3+ in the growth medium (Zheng et al., 2005). Moreover, the carboxylate group (–COO–) in pectin can che-late Fe3+, and PO4

3– can be further trapped with a linkage of –COO–Fe–PO4. Increasing –COO

– will bind Fe more tightly to the pectin and facilitate the release of pectin-trapped PO4

3–. We tested this hypothesis by incubating insoluble FePO4 with pectin, and found that a significant amount of Pi was released owing to the chelation of pectin –COO– with Fe3+ (Fig. 7). It is therefore reasonable to propose that the higher the pectin content of the cell wall of a rice cultivar, the greater the ability of that cultivar to solubilize insoluble P from the cell wall will be; in other words, the change of the pectin content in cell wall seems to be an adaptive response to P deficiency. This is further supported by our observed correlation between pectin and soluble Pi contents amongst different rice cultivars (Fig 5).

Third, pectin content is positively related to soluble Pi con-tent and P sensitivity in A. thaliana. Zhu et al (2012b) previ-ously showed that P deficiency decreases the pectin content of A. thaliana, Here, the pectin-reduced mutant qua1-2 dis-played both increased sensitivity to P deficiency and a sig-nificantly reduced root soluble Pi content under P-deficient conditions (Fig. 8). In addition, as reported by Schmohl and

Horst (2000), treatment of maize suspension cells with NaCl causes an increase in cell wall pectin content, and consequent increase in Al bound to the cell wall, and in cell sensitivity to aluminium stress. Therefore, after A. thaliana was treated with NaCl, both pectin and soluble Pi contents were signifi-cantly increased (Fig. 9), further confirming the positive rela-tionship between pectin content and soluble Pi in the roots.

Finally, secretion of neither protons nor organic acids from rice roots was detected under –P conditions. It has pre-viously been reported that P deficiency can induce the extru-sion of protons and the secretion of organic acids (Johnson et al., 1996; Neumann et al., 1999; Dinkelaker et al., 1989; Zhu et al., 2012b). Organic acids such as citric and piscidic acids can form complexes with Fe3+, resulting in the release of Pi from minerals (Gardner, 1983; Ae et al., 1990 1993). The present study rules out the involvement of proton extru-sion or organic acid secretion in the cell wall P remobilization mechanism.

Intriguingly, Kas has actually been reported to be more (not less) tolerant of P deficiency than Nip, because it con-tains a Kas-specific gene, PHOSPHORUS STARVATION TOLERANCE 1 (PSTOL1) (Gamuyao et al., 2012). OsPSTOL1 is expressed in root primordia and encodes a Ser/Thr protein kinase that acts as an enhancer of root growth. Overexpression of PSTOL1 can enhance root growth and thus increases rice grain yield in P-deficient soils. Kas prob-ably has a more vigorous root system when soil P availabil-ity is limited, and this increased root system vigour probably increases P uptake because it increases the volume of soil explored. However, when a rice plant enters the reproduc-tion stage, it will transport most of the photosynthates to the grains, and much less is available for root growth to explore more P sources in soils. This present study has defined an additional mechanism for tolerance of P deficiency, wherein pectin enhances the mobilization of P deposited in the root cell wall, a mechanism which is more powerful in Nip than in Kas. We propose that these two mechanisms are comple-mentary routes to the achievement of the same final goal, the exploitation of both external and internal resources to meet the plant demand for P under conditions of P-supply limitation.

Fig. 9. Root soluble inorganic phosphate (Pi) content (A), shoot soluble Pi content (B), and pectin uronic acid content (C) of WT (Col-0). A. thaliana seedlings exposed to P-deficient nutrient solution for 1 week in the presence and absence of 5850 mg l–1 NaCl. Data are means±SD (n=4). Columns with different letters are significantly different at P

1024 | Zhu et al.

In conclusion, the present study for the first time demon-strates that cell wall pectin enhances P remobilization in rice grown in conditions where P availability is limited. In those varieties where root cell wall pectin content is relative higher under low available P, the higher pectin content helps to sol-ubilize P fixed in the cell wall, thus making it available for reutilization.

AcknowledgementsThis research was supported by the “973” programme (2014CB441002), Natural Science Foundation of China (31372127), Program for Innovative Research Team in Universities (IRT1185), and the Fundamental Research Funds for the Central Universities. Thanks are given to Nicholas P. Harberd from Oxford University for his critical reading and editing of the text and the three anonymous reviewers for their valuable comments to improve the quality of our work.

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