NaCl Facilitates Cell Wall Phosphorus Reutilization in Abscisic Acid Dependent Manner in Phosphorus Deficient Rice Root

YANG Xiaozheng ,LIU Yusong ,HUANG Jing ,TAO Ye,WANG Yifeng,SHEN Renfang,ZHU Xiaofang

(1State Key Laboratory of Soil and Sustainable Agriculture,Institute of Soil Science,Chinese Academy of Science,Nanjing 210008,China;2University of Chinese Academy of Sciences,Beijing 100049,China;3College of Land Resources and Environment,Jiangxi Agricultural University,Nanchang 330045,China;4State Key Laboratory of Rice Biology,China National Rice Research Institute,Hangzhou 311400,China)

Abstract: Phosphorus (P) starvation in rice facilitates the reutilization of root cell wall P by enhancing the pectin content.NaCl modulates pectin content,however,it is still unknown whether NaCl is also involved in the process of pectin regulated cell wall P remobilization in rice under P starved conditions. In this study,we found that 10 mmol/L NaCl increased the shoot and root biomasses under P deficiency to a remarkable extent,in company with the elevated shoot and root soluble P contents in rice.Further analysis indicated that exogenous NaCl enhanced the root cell wall P mobilization by increasing the pectin methylesterase activity and uronic acid content in pectin suggesting the involvement of NaCl in the process of cell wall P reutilization in P starved rice roots.Additionally,exogenous NaCl up-regulated the expression of P transporter OsPT6, which was induced by P deficiency,suggesting that NaCl also facilitated the P translocation prominently from root to shoot in P starved rice.Moreover,exogenous abscisic acid (ABA) can reverse the NaCl-mediated mitigation under P deficiency,indicating the involvement of ABA in the NaCl regulated root cell wall P reutilization.Taken together,our results demonstrated that NaCl can activate the reutilization of root cell wall P in P starved rice,which is dependent on the ABA accumulation pathway.

Key words: abscisic acid;cell wall;NaCl;phosphorus transporter;phosphorus deficiency;remobilization

Phosphorus (P) is an important macroelement that is critical for the components of cell structure,including phospholipids,membranes,nucleic acids and adenosine triphosphates.In addition,P is also required for plant growth and development (Shukla et al,2017).Although large amount of P exists in the soil,the availability of inorganic P is limited due to their conversion to organic matters by microorganisms or their binding to cations (Wu et al,2013;Ali et al,2019),which results in 30% of the world’s arable soils suffering from P deficiency (Carstensen et al,2018).To enhance the crop yields in P-deficient soils,mineral P fertilizers are used.However,as a major pool of phosphate fertilizer,phosphate rock is an irreplaceable resource(Beardsley,2011),and thus,it is necessary to understand how plants survive in the absence of P,and also to breed new crop varieties in adaptation to low P environments.

To survive under P-deficient conditions,a set of strategies have been employed by plants.The typical strategies include inhibiting primary root elongation,increasing lateral roots,growing more root hairs(Desnos,2008;Wu et al,2013;Zribi et al,2021),forming ‘root clusters’ (Lynch and Brown,2008) or symbiotic relation with rhizosphere microorganisms(Zhang et al,2014).Among them,the most typical strategy is to reconstruct the root morphology and to facilitate the liberate of the soil insoluble P through producing rhizosphere exudates including phenolics(Sarker and Karmoker,2011),organic acids (Hernández and Munne-Bosch,2015;Wu et al,2018),and phosphatases (Abram et al,2009).In addition,another strategy is to reutilize the internal stored P.Vacuole is the major intracellular reservoir for excess P.When rice responds to P deficiency,P is released from the vacuole to the cytoplasm through the action of a pair of vacuolar P efflux transporters OsVPE1 (vacuolar processing enzyme) and OsVPE2 (Xu et al,2019).Besides vacuole,cell wall is also recognized as the repository of P in P starved rice (Zhu et al,2016).Recently,it has been found that nitric oxide (NO)-ethylene,jasmonic acid (JA)-NO and abscisic acid(ABA) regulated the P deficiency pathway in rice dependent on the regulation of pectin content,as pectin plays a key role in the release of insoluble P from rice cell walls (Tao et al,2022).

Sodium (Na) is a constraint on crop productivity,and a large portion of the world’s land remains in a state of high salinity (Rengasamy,2006;Munns and Tester,2008).Salinity (NaCl) inhibits the growth of plants through affecting ion balance,generation of reactive oxygen species (ROS),and etc (Zhu,2001;Trifil et al,2013).However,as early as 1950s,Na was also regarded as nutrition for tomato,and the application of 1 mmol/L Na improves the dry weight of tomato (Woolley,1957).Then,this beneficial effect of Na was further demonstrated inAtriplex,PanicumandKochiagenera (Brownell and Crossland,1972),and wheat (Box and Schachtman,2000).Furthermore,inTheobroma cacao,Na+can partially substitute for K+by increasing the photosynthesis,mineral nutrition and water use efficiency (Gattward et al,2012),and inEucalyptus grandistrees and cotton,Na has the ability to replace K (Ali et al,2006;Battie-Laclau et al,2013).NaCl treatedArabidopsis thalianacells decrease hemicellulose content (Zhu et al,2017a),while NaCl treated maize,tomato and tobacco cells increase pectin content (Shedletzky et al,1992;Zhong and L?uchli,1993;McCann et al,1994;Schmohl and Horst,2000).Moreover,NaCl can alleviate the P deficient phenotype of barley by promoting photosynthetic activity and regulating the selective uptake of nutrient ions (Zribi et al,2012).However,the underlying mechanism of beneficial effect of Na is still elusive.

As pectin level has a good correlation with the growth performance in P starved rice (Zhu et al,2015,2016),and NaCl participates in regulating the pectin content (Schmohl and Horst,2000),an assumption was proposed that whether there is a crosslink between P deficiency and exogenous NaCl.Here,using a rice cultivar Nipponbare (Nip),we demonstrated that exogenous NaCl accelerated root cell wall P reutilization and promoted the root to shoot P translocation through enhancing the transcription level ofOsPT6in P starved rice,a process that works dependent on the accumulation of ABA.

RESULTS

NaCl (10 mmol/L) significantly mitigates P deficiency in rice

To investigate the role of exogenous NaCl in P deficient rice,the growth performances of Nip seedlings that grown under different NaCl concentrations were conducted.As shown in Fig.1,the root and shoot biomasses were reduced by P deficiency.It was interesting that the growth inhibition induced by P deficiency was significantly alleviated after adding of exogenous NaCl.With the increased treated time,the alleviating effect of NaCl was gradually obvious,indicating the significant role of NaCl under P deficiency.As rice plants exposed to 10 mmol/L NaCl showed best growth among the P deficient treated rice(Fig.1-B and -C),a concentration of 10 mmol/L NaCl was selected for further investigation.

NaCl increases root cell wall P reutilization in P starved rice

To explore the underlying mechanism of how NaCl alleviates P deficiency in rice,we first measured the soluble P contents in shoot and root of 7-day-old Nip seedlings treated with or without 10 mmol/L NaCl in P-sufficient (+P) or P-deficient (-P) solution for 7 d.As shown in Fig.2,more shoot and root soluble P contents were found in the -P+NaCl treated rice than those in the -P treated rice alone,while there was no significant difference between the +P+NaCl and +P treatments,and this increased effect was the most remarkable when treated for 7 d compared with those for 1 and 4 d,indicating that exogenous NaCl significantly elevated the soluble P contents in the P starved rice shoots and roots.In addition,the increased degrees of the transcription levels ofOsSPX1,OsSPX3,OsSPX5andOsSPX6by P deficiency were all reduced with the application of NaCl,further suggested the mitigated role of NaCl in P starved rice(Fig.3).

Fig.1.Effects of different concentrations of NaCl on growth performance of rice seedlings under P-sufficient (+P) and P-deficient (-P) conditions.

Fig.2.Effects of 10 mmol/L NaCl on soluble P contents in root (A) and shoot (B) of rice under P-sufficient (+P) and P-deficient (-P)conditions for 1,4 and 7 d.

Fig.3.Effects of 10 mmol/L NaCl on transcription levels of OsSPX1 (A),OsSPX3 (B),OsSPX5 (C) and OsSPX6 (D) in rice roots under P-sufficient (+P) and P-deficient (-P) conditions for 7 d.

Fig.4.Effects of 10 mmol/L NaCl on P contents in cell wall (A and F) and pectin (B and G),uronic acid contents in pectin (C and H),pectin methylesterase (PME) activities (D and I) as well as pectin methyl esterification degrees (E and J) of rice roots and shoots under P-sufficient (+P) and P-deficient (-P) conditions for 1,4 and 7 d.

Since NaCl can regulate pectin content (Schmohl and Horst,2000),and pectin content contributes significantly to the cell wall alleviated P deficiency in rice (Tao et al,2022),we extracted cell wall materials for further analysis.As shown in Fig.4-A and -B,less P was found in the -P+NaCl treated rice root cell walls than in the -P treatment rice,especially for the pectin fraction,and the differences were more significant with the increased time of treatment,although no remarkable difference was observed in the shoot cell walls at all the treated time (Fig.4-F and-G),which suggested that NaCl can accelerate the releasing of the cell wall stored P in the P starved rice.Furthermore,when compared with the -P treatment alone,the -P+NaCl treatment caused significant reduction in the degree of pectin methyl esterification,a symptom that was obviously observed after 4 and 7 d treatments in the rice roots.In fact,the degree of pectin methyl esterification was negatively correlated with the enhancement of uronic acid content in the pectin and the pectin methylesterase (PME) activity in the rice roots (Fig.4-C to -E),suggesting both of them contributed significantly to the reutilization of the cell wall P and made the soluble P more available.

NaCl facilitates root to shoot P translocation

Since shoot soluble P content was also enhanced with the addition of exogenous NaCl in the P starved rice(Fig.2-B),and both PME activity and pectin content in the shoot cell walls were also reported to contribute to the increased shoot soluble P concentration significantly(Zhu et al,2019),we extracted the shoot cell walls for further analysis.It was interesting that no remarkable difference was observed for P content in the shoot cell walls (Fig.4-F),especially for the P content in the pectin (Fig.4-G),uronic acid content (Fig.4-H),PME activity (Fig.4-I),and pectin methyl esterification degree (Fig.4-J),regardless of the -P and -P+NaCl treatments at all the treated time,implying the enhanced shoot soluble P by exogenous NaCl might attribute to the increased P translocation from root to shoot,rather than the shoot cell wall P reutilization itself (Figs.2 and 4).To further verify this hypothesis,xylem P content was measured.As expected,in the P starved rice,NaCl enhanced the xylem P content (Fig.5),indicating the increased shoot soluble P content was relied on P transportation from root to shoot.

Then,to detect how NaCl affects the translocation of P,the transcription levels ofOsPT2,OsPT6andOsPT8that are required for root to shoot P transportation were detected (Ai et al,2009;Wang et al,2014).As expected,the transcription levels ofOsPT2,OsPT6andOsPT8were all induced by P deficiency,but only the transcription level ofOsPT6was further significantly up-regulated in the -P+NaCl treated rice roots (Fig.6),indicating thatOsPT6contributed greatly to theNaCl prompted P transportation from root to shoot.

Fig.5.Effects of 10 mmol/L NaCl on rice xylem P content in P-sufficient (+P) and P-deficient (-P) solution.

ABA participates in NaCl accelerated cell wall P reutilization process in P starved rice

Next,which signal participated in the NaCl accelerated the reusing of the cell wall stored P? As NaCl can respond to abiotic stress inArabidopsis thalianaby acting upstream of ABA (Zhu et al,2017a),ABA might be involved in this NaCl mediated cell wall P reutilization.Therefore,we firstly detected the ABA accumulation in rice roots.It was interesting that the-P treatment markedly decreased the root ABA level,and this reduction was further aggravated with exogenous NaCl supply (Fig.7-A),revealing that ABA might take part in the NaCl mediated cell wall P reutilization.Then,to further elucidate the relationships between NaCl and ABA,ABA was also applied exogenously.It was interesting that NaCl alleviated P deficiency can be reversed through adding of ABA(Fig.7-B and -C),strongly indicating that ABA was involved in the process that NaCl mediated cell wall P utilization,as illustrated in Fig.8.

DISCUSSION

NaCl alleviates P deficiency inhibited growth in rice plants

Fig.6.Effects of 10 mmol/L NaCl on transcripts of OsPT2 (A),OsPT6 (B) and OsPT8 (C) in P-sufficient (+P) and P-deficient (-P) solution in rice roots for 7 d.

Fig.7.Effects of NaCl on root abscisic acid (ABA) content (A),as well as ABA on root (B) and shoot (C) soluble P concentrations in P-sufficient (+P) and P-deficient (-P) solution with or without 10 mmol/L NaCl in rice.

Fig.8.Proposed diagram of action pattern of NaCl in P starved rice.

In plants,more and more evidences have demonstrated the role of NaCl in response to abiotic stress,such as regulating the uptake of plant micronutrient including zinc (Zn),copper (Cu) and manganese (Mn) (Neves-Piestun and Bernstein,2005),increasing root cell wall iron (Fe) inA.thaliana(Zhu et al,2017a),stimulating P uptake (Roberts et al,1984),alleviating the adverse effects of P deficiency on barley (Zribi et al,2012) andAeluropus littoralis(Zribi et al,2021).In this study,we elucidated the effects of NaCl on the improvement of P homeostasis in P starved rice,a process that is mainly dependent on two strategies.One is to facilitate the root cell wall P remobilization by elevating the PME activity and uronic acid content in pectin (Fig.4),the other is to up-regulate the expression ofOsPT6and increase the root to shoot P transportation (Fig.6).As a result,the growth inhibitions of root and shoot induced by P deficiency in rice were alleviated (Figs.1 and 2).

NaCl promotes root cell wall P reutilization in P deficient rice

Since root cell wall fixed P accounts for about half of total root P in P deficient rice (Zhu et al,2018),any strategy that accelerates the reusing of the cell wall P is important to improve the P nutrition in P starved rice.For example,Arabidopsismutantamos1(loss function ofAMMONIUM-OVERLY-SENSITIVE1) releases more root cell wall P than its wild type under P deficient conditions (Yu et al,2016).In this study,less P was fixed in the -P+NaCl treated root cell wall and pectin than in the -P treated rice (Fig.4-A and -B),although there was almost no difference in shoot cell wall between the -P+NaCl and -P treatments (Fig.4-F),indicating that the reutilization of cell wall P accelerated by NaCl only occurred in roots.

How dose NaCl accelerate this reutilization? As cell wall is the first barrier to counteract with abiotic stress such as toxicity element aluminium (Al)/cadmium (Cd)stress (Zhu et al,2012a,b),or beneficial element Fe/P deficiency (Zhu et al,2012a,2016),it mainly consists of cellulose,hemicellulose and pectin.Different from cellulose that has no ability to bind metal cations,both pectin and hemicellulose are able to bind cations.For example,both pectin and hemicellulose are involved in binding of cations including Al (Yang et al,2011;Sun et al,2016),and hemicellulose is associated with the ABA facilitated root cell wall Fe reutilization whenArabidopsisis coped with Fe deficiency (Lei et al,2014).However,under P deficient conditions,only pectin plays a role in the cell wall P reutilization process.For instance,Ae and Shen (2002) demonstrated that groundnut owns a strong ability to absorption P from P deficient soil as groundnut root cell wall possesses the ‘contact reaction’ to pectin.Polygalacturonic acid,a component of pectin,can be in conjunction with Al3+/Fe3+and follow by releasing of P from clay minerals (Nagarajah et al,1970).Zhu et al (2015)further demonstrated that pectin has the ability to reuse the cell wall P,as its negative charges (-COO--)can directly bind to cations including Fe3+,and PO43-is further caught in a linkage of -COO-Fe-PO4.Increased pectin content can bind to Fe more tightly as the increased -COO-facilitates the release of pectintrapped PO43-,hence,more soluble P is available and any approach that regulates the pectin content will increase the reutilization process of cell wall P.In fact,pectin content can be regulated by a series of hormones or signal molecules,such as NO,ethylene,ABA and JA (Zhu et al,2016,2017b,2018;Tao et al,2022).Under the P deficient conditions,more pectin content was found after the addition of NaCl (Fig.4-C),indicating the involvement of the pectin in this NaCl accelerated cell wall P reutilization.Furthermore,pectin is constituted of rhamnogalacturonans I,rhamnogalacturonans II,galactose,homogalacturonan(HGA),arabinose,and etc (Cosgrove,2005).Among them,HGA is synthesized in the Golgi body as the form of methyl esterification and then secretes into the cell wall.However,with the catalysis action of PME,the pectin undergoes a dimethyl esterification process that the methyl group is removed from the carboxyl group (Ostatek-Boczynski et al,1995),thus,the number of negatively charged groups is increased,and also the ability of the pectin to bind cations is enhanced (Willats et al,2006).The higher the activity of PME,the more the negative charge pectin owns,and the more the P released from the cell wall.Here,NaCl application can significantly enhance the activity of PME in the P deficient rice root cell wall (Fig.4-D),further confirming the action of NaCl in reutilizing the rice root cell wall P through facilitating pectin synthesis and enhancing PME activity.

Additionally,besides reutilization of cell wall P,the increased expression levels ofOsPT2,OsPT6andOsPT8are also important in P starved rice (Zhu et al,2017a,2018).OsPT6andOsPT8,widely expressed in various organs,have high affinities for P and are essential for maintaining the P homeostasis in plants(Jia et al,2011).OsPT2,highly expressed in vascular bundle cells of primary roots,has low affinity for P,and is required for P transportation (Ai et al,2009).Accordingly,NaCl can markedly up-regulate the transcription level ofOsPT6under P limited conditions(Fig.6),in accordance with the increased P content in xylem sap (Fig.5),indicating that NaCl indeed promoted the root to shoot P transportation in P starved rice.Moreover,SPX proteins are reported to contain SPX domains,and six homologous genes(namedSPX1-6) are identified in rice.Among them,SPX1plays a pivotal role in P sensing process (Wang et al,2014).SPX3andSPX5function as the repressors of PHR2 (phosphate starvation response regulator 2)to maintain P homeostasis and signaling (Shi et al,2014).Zhong et al (2018) demonstrated that SPX6,served as a negative regulated factor in inhibiting the binding of PHR2 to phosphate starvation-induced (PSI)genes,can also hinder the transport of PHR2 to the nucleus in P starved rice.In this study,the expression levels ofOsSPX1,OsSPX3,OsSPX5andOsSPX6were all induced by P deficiency,and the magnitude of this increment was reduced after the application of NaCl (Fig.3),further suggesting the mitigated role of NaCl in the P starved rice.

ABA might be involved in NaCl induced cell wall P reutilization process in P deficient rice

As NaCl can regulate ABA accumulation (Hong et al,2007;Zhu et al,2017a),and ABA affects the cell wall P reusing process (Zhu et al,2018),a question then raised whether ABA is participated in the process of NaCl accelerated cell wall P remobilization.It was exciting that exogenous NaCl further aggravated the reduction of ABA accumulation induced by P deficiency(Fig.7-A),which was further verified with the fact that the mitigated role of NaCl in the P starved rice disappeared after the application of exogenous ABA(Fig.7-B and -C).

In conclusions,we proposed a working mode in Fig.8.When rice is subjected to P deficiency,exogenously applied NaCl significantly decreases the accumulation of endogenous ABA,which in turn decreases the pectin methyl esterification degree and increases the PME activity and the pectin content to reuse the cell wall fixed P,then up-regulates the expression ofOsPT6to promote the root to shoot P transportation.Finally,the growth of rice that cultivated under the P limited conditions is improved.

METHODS

Rice material and growth condition

Oryza sativasubsp.japonicacv.Nipponbare was used.After being sterilized with 1% NaClO,rice seeds were washed three times with deionized water and immersed in pure water for 1 d,and then germinated on a float in 0.5 mmol/L CaCl2solution with pH 5.5.Germinated seeds with uniform buds were cultivated in Kimura B solution with pH 5.5 for 7 d (Tao et al,2022).Finally,similar seedlings were transferred to +P solution(Kimura B) and -P solution (withdrawing NaH2PO4·2H2O from the Kimura B solution) in the presence or absence of NaCl with concentrations of 0,2,10 and 50 mmol/L for 7 d.The nutrient solution was updated every 2 d.Light intensity and relative humidity for rice growth were set at 400 μmol/(m2·s) and 60%,respectively (Zhu et al,2018).

To detect the crosslink among NaCl,ABA and the reutilization process of the cell wall stored P,7-day-old rice seedlings with uniform growth performance were transplanted to 0.5 μmol/L ABA treatment with or without 10 mmol/L NaCl(Zhu et al,2018).All the above experiments were conducted under +P and -P solution.

Determination of soluble P content

Rice shoots and roots were rinsed with ultrapure water,weighed,ground into powder in liquid nitrogen,and incubated with 4 mL of 5 mmol/L H2SO4.At 24 h later,centrifuged the mixtures at 12 000 ×gfor 8 min,and mixed the supernatant(400 μL) with ammonium molybdate (200 μL) that was composed of 15% ascorbic acid (pH 5.0) for 30 min.At last,the absorbance of the mixtures at 650 nm was measured according to Zheng et al (2009).

Measurement of xylem sap P concentration

For collection of the xylem sap,rice shoots were cut.After removed the first dropped sap,the remaining sap was gathered for 2 h.The collected xylem sap was recorded and diluted with deionized water to make up the total volume to 2 mL.Inductively coupled plasma atomic emission spectroscopy (ICP-AES;FisonsARLAccuris,Ecublens,Switzerland) was used to detect the P concentration (Che et al,2016).

Isolation of cell wall and pectin

First,rice roots were harvested and ground into powder.Second,added 75% ethanol to the powder,and 20 min later,the mixtures were centrifugated at 13 000 ×gfor 8 min.The pellets were further extracted by acetone,methanol:chloroformmethyl(1:1) and methanol,respectively.Finally,the surplus material after centrifugation was regarded as cell wall and stored in a 4 °C freezer for the following experiments.Then,weighed around 3 mg cell wall and mixed with 1 mL boiling ultrapure water.At 60 min later,centrifuged the mixtures at 13 000 ×gfor 8 min,and collected the supernatants.After that,added 1 mL boiling ultrapure water to the sediment and repeated the above steps 3 times.The combined supernatant was referred to as pectin.

Determination of P content in pectin

Absorbed pectin extract 100 μL and added 100 μL ammonium molybdate that was composed of 15% ascorbic acid (pH 5.0)and 800 μL ddH2O.Then,mixed them sufficiently to render for 30 min in the dark.Finally,measured the absorbance of the mixtures at 650 nm.

Measurement of uronic acid content in pectin

In general,uronic acid content is regarded as pectin content(Blumenkrantz and Asboe-Hansen,1973).To measure uronic acid content,pectin solution (200 μL) was mixed with 1 mL of 98% H2SO4that consisted of 1 250 μmol/L Na2B4O7·10H2O in boiling water for 5 min.After cooling down,the mixtures were mixed with 20 μL of 0.15%m-hydroxydiphenyl.Finally,the absorbance at 520 nm was measured using the galacturonic acid as the equivalent (Zhu et al,2018).

Measurement of pectin methyl esterification degree

To measure pectin methyl esterification degree,25 μL of 4 mol/L NaOH was added to 125 μL pectin extract,and shaked it sufficiently.After saponification at 37 °C for 30 min,50 μL of 2 mol/L HCl was added to neutralize the mixture and next added 400 μL of 200 mmol/L phosphoric acid buffer (pH 7.5)and 10 μL alcohol oxidase.After incubation at 30 °C for 10 min,800 μL of 5 g/L purpald (Aladdin,Shanghai,China,dissolved in 0.5 mol/L NaOH) was added.The absorbance value at 550 nm was measured after further incubation at 30 °C for 30 min.Degree of pectin methyl esterification (%)=Pectin methyl group content/ (Pectin methyl group content+Pectin uronic acid content) × 100 (Klavons and Bennett,1986).

Measurement of PME activity

First,around 3 mg cell wall was weighed,and then extracted by 1 mL extraction buffer (1 mol/L NaCl in 10 mmol/L Tris buffer with pH 6.0) at 4 °C.Second,after shaking the mixtures for 60 min and centrifuged at 12 000 ×gfor 10 min,50 μL collected supernatant was transferred to microplate together with 10 μL alcohol oxidase and 0.1 mL reaction solution (640 μg/mL pectin in 200 mmol/L PBS solution with pH 7.5) at 37 °C.Finally,at 10 min later,0.2 mL of 500 mmol/L NaOH that composed of purpald with a concentration of 5 mg/mL was applied to measure the absorbance at 550 nm using methanol as the equivalent (Yang et al,2008).

Determination of cell wall P content

Around 5 mg cell wall was extracted by 2 mL of 6.25% HCl in oscillators for 1 d.Next,the samples were centrifuged at 13 000 ×gfor 15 min,and ICP-AES was used to detect the P content in the supernatant (Tao et al,2022).

Detection of ABA accumulation in roots

Enzyme Linked Immunosorbent Assay (ELISA) kit (Phytohormones Research Institute of China Agricultural University,Beijing,China) was used to measure ABA content.First,rice roots were washed with PBS buffer,weighed,and ground into powder.After adding 1 mL PBS buffer,samples were centrifugated at 12 000 ×g.Then,50 μL collected supernatant or standards were added to 96 well plate to mix with 50 μL detection A working solution at 37 °C for 1 h.After washed for 3 times,the samples were added with 100 μL working solution of reagent B at 37 °C for another 45 min.The plate was washed,followed by adding 90 μL solution containing substrate at 37 °C.Finally,at 15 min later,50 μL stop solution was added and the absorbance was measured at 450 nm.

qRT-PCR analysis

First,the reagent Iso plus (TaKaRa,Japan) was utilized to isolate RNA,and the RNA was then reverse transcribed into cDNA by the PrimeScript RT kit (TOYOBO,Japan).qRT-PCR was performed according to Tao et al (2022).The 2-ΔΔCTmethod was utilized to detect the transcription levels of the tested genes(Livak and Schmittgen,2001).The experiments were performed in three replicates with gene-specific primers: 5＇-TCCGGTGG ATCTTCATGCTTACCT-3＇ and 5＇-ATGGACCATTGCGAC GAGTCTTCT-3＇ forOsActin;5＇-GAAGTTTGGGAAGAGC CTGAGT-3＇ and 5＇-TGTAGTTGAGGGCGCTGTAGTT-3＇ forOsSPX1;5＇-TGCAGTCCATCCGATCCG-3＇ and 5＇-ATGTGT ATGTATGTTCTCTACCACG-3＇ forOsSPX3;5＇-CGACGAG CTGCAACATT-3＇ and 5＇-CAAGAACCATTGGTATTGATC-3＇forOsSPX5;5＇-TCTGCGCTGCGAAATCTG-3＇ and 5＇-TTGA AAGCCAAAACACGTATG-3＇ forOsSPX6;5＇-GACGAGAC CGCCCAAGAAG-3＇ and 5＇-TTTTCAGTCACTCACGTCGA GAC-3＇ forOsPT2;5＇-TATAACTGATCGATCGAGACCAG AG-3＇ and 5＇-TGGATAGCCAGGCCAGTTATATATC-3＇ forOsPT6;5＇-AGAAGGCAAAAGAAATGTGTGTTAAAT-3＇ and 5＇-AAAATGTATTCGTGCCAAATTGCT-3＇ forOsPT8.

Statistical analysis

Each group of experiments was repeated for three times.Oneway analysis of variance was utilized to test and compare the data with the Duncan’s multiple range test.Statistical differences in mean values atP＜ 0.05 were indicated by different lowercase letters on the histogram.

ACKNOWLEDGEMENTS

This study was supported by the Foundation for Distinguished Young Scholars of Jiangsu Province,China (Grant No.BK20190050),Zhejiang Provincial Natural Science Foundation of China (Grant No.LY22C130004),and the Youth Innovation Promotion Association of Chinese Academy of Sciences (Grant No.2015250).