the amino acid permease 5 (osaap5) regulates tiller ...the amino acid permease 5 (osaap5) regulates...

The Amino Acid Permease 5 (OsAAP5) Regulates TillerNumber and Grain Yield in Rice1

Jie Wang,a Bowen Wu,a Kai Lu,a Qian Wei,a Junjie Qian,a,b Yunping Chen,c and Zhongming Fanga,b,2,3

aCenter of Applied Biotechnology, Wuhan Institute of Bioengineering, Wuhan 430415, ChinabNational Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070,ChinacState Key Laboratory of Hybrid Rice, Engineering Research Center for Plant Biotechnology and GermplasmUtilization of Ministry of Education, College of Life Sciences, Wuhan University, Wuhan 430072, China

ORCID ID: 0000-0002-6921-9874 (Z.F.).

As fundamental nutrients, amino acids are important for rice (Oryza sativa) growth and development. Here, we identified theamino acid permease 5 (OsAAP5), that regulates tiller number and grain yield in rice. The OsAAP5 promoter sequence differedbetween indica and japonica rice varieties. Lower expression of OsAAP5 in the young leaf blade in indica varieties than in japonicavarieties was associated with more tillers in indica than in japonica. Down-regulation of OsAAP5 expression in japonica usingRNA interference (RNAi) and clustered regularly interspaced short palindromic repeats led to increases in tiller number andgrain yield, whereas OsAAP5 overexpression (OE) had the opposite effect. Both a protoplast amino acid uptake assay and HPLCanalysis indicated that more basic (Lys, Arg) and neutral (Val, Ala) amino acids were transported and accumulated in the OElines than in the wild type, but the opposite was observed in the RNAi lines. Furthermore, exogenous application of Lys, Arg,Val, and Ala in the OE lines substantially inhibited tiller bud elongation, but the effect was lost in the RNAi lines. Notably,concentrations of the cytokinins cis-zeatin and dihydrozeatin were much lower in the OE lines than in the wild type, whereasconcentrations in the RNAi lines were higher. Thus, OsAAP5 could regulate tiller bud outgrowth by affecting cytokinin levels,and knockout of OsAAP5 could be valuable for japonica breeding programs seeking high yield and grain quality.

Rice (Oryza sativa) is the staple food for more thanhalf of theworld’s population (Fairhurst andDobermann,2002). According to geographic distribution and geneticvariation, Asian rice is classified into two subspecies,indica and japonica (Liu et al., 2018). Generally, japonicarice has a significantly higher head rice rate, lower de-gree of chalkiness, lower amylose content, and highergel consistency than indica rice; all these characteristicsof japonica contribute to improved grain quality (Fenget al., 2017). In addition, japonica cultivars are more coldtolerant than indica cultivars (Lu et al., 2014; Liu et al.,2018). However, the grain yield of japonica is muchlower than that of indica due to the lower tiller number

of japonica rice (Hu et al., 2015). In recent years, ricevarieties with high tiller numbers have been pursuedby many rice breeders, as these high-tillering varietiestheoretically could producemore effective panicles. Theeffective panicle number per plant, grain number perpanicle, and grain weight per panicle determine thegrain yield of a rice plant (Xing and Zhang, 2010). Thus,increasing japonica rice tiller number is urgent for breed-ing programs to produce japonica cultivars with highyield and good grain quality.Application of nitrogen (N) fertilizer has been repor-

ted as one of the most effective ways to increase tillernumber, because it increases the cytokinin (CK) contentwithin tiller nodes and further enhances tiller bud out-growth (Sakakibara et al., 2006; Liu et al., 2011; Wanget al., 2017). CK content is positively correlated withsoil N content (Takei et al., 2001, 2004), perhaps be-cause the primary product of CK synthesis is the N6-(D2-isopentenyl) adenine nucleotide, which is formedby adenosine phosphate-isopentenyltransferase (IPT).There are seven IPT genes in Arabidopsis (Arabidopsisthaliana), of which IPT3 is upregulated by nitrate(Sakakibara et al., 2006). Another reason may be thatnitric oxide, which is produced as part of N metab-olism, is one of the most extensively used signalingmolecules in living organisms (Schmidt and Walter,1994). It has been reported that nitric oxide coulddirectly interact with transzeatin (tZ) in vivo, creatingnitrated CK species and thereby regulating the CKsignaling pathway (Liu et al., 2013).

1This work was supported by the Ministry of Science andTechnology of the People's Republic of China (Chinese Ministry ofScience and Technology) the National Key Research and 516 Devel-opment Program (2016YFD0100700) and the National NaturalScience Foundation of China (NSFC) (31301250/31701990).

2Author for contact: [email protected] author.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Zhongming Fang ([email protected]).

Z.F. and J.W. designed the experiments, analyzed the data andcompleted the writing; J.W., B.W., and Z.F. performed most of theexperiments; K.L., Q.W., J.Q., and Y.C. provided technical assistanceto J.W.; all authors read and approved the final manuscript.

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Nitrate and ammonium, the two main inorganic Nforms, as well as peptides and amino acids (organicN forms), can be used by plants in soils (Tegeder andRentsch, 2010; Moran-Zuloaga et al., 2015). Plantshave evolved multiple efficient N uptake and trans-port systems to support their growth and develop-ment in environmentswith different forms and amountsof available N. In the rice inorganic N transporter family,the functions of NPF (Nitrate transporter1 [NRT1]/Peptide transporter [PTR] Family) members have beenextensively studied. OsPTR9 (OsNPF8.20; Fang et al.,2013), NRT1.1B (OsNPF6.5; Hu et al., 2015), OsNPF7.2(Wang et al., 2018), and OsNPF7.7 (Huang et al., 2018)could influence rice tiller number by regulating Ncontent. In addition, Gln synthetase1;2, a key enzymeof ammonium assimilation that converts inorganicammonium into Gln in plants, regulates tiller numberin rice by affecting CK level (Funayama et al., 2013;Ohashi et al., 2015, 2017).

In addition to the inorganic N transporters men-tioned above, organic N transporters also play im-portant roles in plant growth and development (Tegederand Rentsch, 2010). Amino acid permeases (AAPs),members of the amino acid transporter family, havebeen extensively functionally studied in plants. InArabidopsis, seven AAP transporters play impor-tant roles in translocating different amino acids fromsource to sink organs (Zhao et al., 2012). AtAAP1functions in root amino acid acquisition and seedyield in Arabidopsis (Lee et al., 2007; Sanders et al.,2009). AtAAP2 is important for amino acid transportfrom the xylem to the phloem (Zhang et al., 2010),whereas AtAAP3 mediates the uptake of neutral andbasic amino acids (Okumoto et al., 2004). AtAAP4has significant involvement in Val transport (Fischeret al., 1995), AtAAP5 transports anionic, neutral,and cationic amino acids (Boorer and Fischer, 1997;Svennerstam et al., 2008), and AtAAP6 affects thecontents of Lys, Phe, Leu, and Asp and regulatesrosette width and seed volume in Arabidopsis (Huntet al., 2010). In addition, AtAAP8 transports acidicamino acids into the endosperm and is importantfor seed yield (Schmidt et al., 2007; Santiago andTegeder, 2016).

Among the 19 AAP transporters (OsAAP1-OsAAP19)in rice, OsAAP3 (Lu et al., 2018) and OsAAP6 (Penget al., 2014) have been reported to influence rice grainyield and quality, respectively, and OsAAP3 mainlytransports Lys and Arg (Taylor et al., 2015). How-ever, whether other OsAAPs are responsible for ricegrowth and development is unclear. In this study,we found that the OsAAP5 promoter sequence wasdivergent between indica and japonica, resulting inhigher expression of OsAAP5 in japonica cultivars,which in turn caused fewer tillers than in indica.Moreover, the down-regulation of OsAAP5 signifi-cantly increased tiller number by decreasing the con-tents of basic amino acids (Lys and Arg) and neutralamino acids (Val and Ala) to maintain higher CK levelsin rice plants.

RESULTS

OsAAP5 Promoter Sequence was Divergent betweenindica and japonica Cultivars

We used Rice Variation Map v2.0, a database forrice genome variation (Chen et al., 2014), to investi-gate the sequences of the OsAAP5 promoter region in306 cultivars from around the world with differenttiller numbers. Among these 306 cultivars, 26 singlenucleotide polymorphisms (SNPs) and 5 insertion/deletions (indels) were detected in 11 variant typesof the OsAAP5 promoter, which were named haplo-types 1 to 11 (Hap1-Hap11; Fig. 1A). Interestingly,Hap1 and Hap2 were mainly found in japonica culti-vars, and Hap3 was mainly found in indica cultivars.A 51-bp insertion was validated in Hap3 by agarosegel electrophoresis (Fig. 1, B and C), which mightbe useful for distinguishing the indica and japonicapromoter types. Additionally, phylogenetic analysisshowed that two clusters existed among the 11 haplo-types, an indica cluster containing Hap3 and a japonicacluster containing Hap1 and Hap2 (Fig. 1D). Theseresults demonstrated divergence in the OsAAP5 pro-moter sequence between indica and japonica cultivars.

The expression pattern ofOsAAP5 in japonica cultivarZH11 (Hap1) showed that OsAAP5was expressed invarious tissues, including the root, tiller basal part,leaf sheath, leaf blade, and young panicle (Fig. 1E).We chose the young leaf blade at the vegetative stageto compare the expression of OsAAP5 in Hap1, Hap2,and Hap3. The results showed that the expression ofOsAAP5 in japonica cultivars (Hap1 and Hap2) wassignificantly higher than that in indica cultivars (Hap3;Fig. 1F). We also detected the expression pattern ofOsAAP5 among Hap1, Hap2, and Hap3 and foundthat japonica haplotypes (Hap1 and Hap2) exhibiteddifferent expression patterns from indica haplotypes(Hap3), especially in the root, tiller basal part, andleaf sheath (Supplemental Fig. S1). Furthermore, wecompared the tiller numbers of indica haplotypes andjaponica haplotypes and found that indica haplotypesproduced more tillers than japonica haplotypes (Fig. 1G).These results demonstrated that japonica accessionswith Hap1 and Hap2 had higher expression ofOsAAP5but fewer tillers than indica accessions with Hap3,indicating a negative association between OsAAP5expression and tiller number.

OsAAP5 Expression Mainly Occurred in VascularAboveground Tissues, and Its Protein Localized to thePlasma Membrane

To further investigate the expression pattern ofOsAAP5, POsAAP5::GUS transgenic plants were pro-duced in the ZH11 background. GUS staining showedthat the GUS signal was particularly strong in the root(Fig. 2A), tiller basal part (Fig. 2B), stem (Fig. 2C), leafsheath (Fig. 2D), and young leaf blade (Fig. 2E) at thevegetative stage and in the young panicle at the

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reproductive stage (Fig. 2F). Additionally, GUS activitywas abundant in all cells in the transverse section of theroot (Fig. 2, G and H) and in the vascular parenchymacells in the leaf sheath (Fig. 2, I and J) and young panicle(Fig. 2K). These results demonstrated that OsAAP5might absorb nutrients through the roots and trans-port nutrients through the vasculature of abovegroundtissues.To investigate the roles of OsAAP5 in response to

various forms of N, ZH11 seedlings were cultured innutrient solution with inorganic N (NO32, NH4+, andNH4NO3) as the sole N source, and the expressionof OsAAP5 was detected in roots and tiller buds. Theresults showed that OsAAP5 expression levels were

up-regulated with increasing N concentrations, exceptunder NH4NO3 treatment (Supplemental Fig. S2, Aand B). These results indicated that the expression ofOsAAP5 could be regulated in response to various Ntreatments, especially in the root.OsAAP5 was predicted to be an amino acid per-

mease with 11 transmembrane domains (SupplementalFig. S3) and possibly localized to the plasma mem-brane. To validate this hypothesis, we fused GFP tothe OsAAP5 C terminus and transiently expressedthis fusion protein in rice protoplasts. The greenfluorescence signal of 35S::GFP was enriched in thecytoplasm and nucleus (Fig. 2L), but coexpressionof OsAAP5-GFP and a plasma membrane mCherry

Figure 1. Sequence divergence in theOsAAP5 promoter regions in 306 rice accessions collected worldwide. A, SNP divergencein the promoters ofOsAAP5. B, Distribution of the 51-bp indel and the average tiller number of some cultivars in riceHap1-Hap3.C, Detection of the 51-bp indel using agarose gel electrophoresis. D, The phylogeny tree ofOsAAP5 promoters was constructedusing the neighbor-joining method with MEGA software (version 5.1). The percentage of replicate trees in which the associatedtaxa clustered together in the bootstrap test (1000 replicates) are shown above the branches. Scale bar = number of base sub-stitutions per site. E, The expression pattern of OsAAP5 in different tissues of ZH11. F, The expression level of OsAAP5 in theyoung leaf blade of rice Hap1-Hap3. G, The average tiller number of all cultivars in rice Hap1-Hap3. Values are means6 SD, andthree replications were performed in each analysis. The letters above the error bars are ranked by Duncan’s multiple range test.Different letters indicate significant differences at P , 0.05.

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OsAAP5 Regulates Rice Tiller Number and Yield

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Figure 2. GUS staining and subcellular localization of OsAAP5. GUS staining of the root tip (A), tiller basal part (B), stem (C), leafsheath (D), young leaf blade (E), and young panicle (F) from the POsAAP5::GUS transgenic plants. The following are also shown:transverse section (G) and its vascular enlargement (H) of a root from the POsAAP5::GUS transgenic plants; transverse section (I)and its vascular enlargement (J) of a leaf sheath from the POsAAP5::GUS transgenic plants; and transverse section of a youngpanicle (K) from the POsAAP5::GUS transgenic plants. VT, vascular tissue; Ep, epidermis; VPC, vascular parenchyma cells. L, FreeGFP expression in rice protoplasts. M, The expression of OsAAP5-GFP, which was coexpressed with plasma membrane proteinOsMCA1 fused with mCherry. Scale bars = 5 mm (A–F), 200 mm (G, I, and K), 50 mm (H and J), and 10 mm (L and M).


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marker showed that OsAAP5 clearly localized to theplasma membrane (Fig. 2M). These results indicatedthat OsAAP5 might play a key role in membranetransport.

OsAAP5 Regulated Rice Tiller Number and Grain Yield byInfluencing Tiller Bud Outgrowth

To uncover the function of OsAAP5 in rice growthand development, we generatedOsAAP5 overexpression(OE) lines and RNA interference (RNAi) lines in thejaponica ZH11 background and validated OsAAP5expression by reverse transcription-quantitative PCR(RT-qPCR) in the young leaf blades of T2 transgeniclines (Fig. 3, A and B). We also detected the expressionof the 18 other OsAAPs in the young leaf blades ofZH11, the OE lines, and the RNAi lines. The resultsshowed that alteration of OsAAP5 expression also af-fected the expression levels ofOsAAP1,OsAAP11, andOsAAP17 (Supplemental Fig. S4), which indicated thatOsAAP5 might coordinate with OsAAP1, OsAAP11,and OsAAP17 to mediate amino acid transport. Inaddition, altered expression of OsAAP5 changed theplant architecture, especially tiller number (Fig. 3A).When compared with that of ZH11, the tiller numberof the RNAi lines increased significantly, but the tillernumber of the OE lines decreased (Fig. 3C). Becauserice grain yield is also determined by two other factors,grain number per panicle and grain weight per panicle(Xing and Zhang, 2010), we investigated additionalyield traits of ZH11, the OE lines, and the RNAi lines.No significant differences in spikelet number per panicle,1000-grain weight, and seed set ratio were observedamong the differential expression lines of OsAAP5(Fig. 3, D–F). When compared with that in ZH11,grain yield per plant in paddy significantly increasedin the RNAi lines but decreased in the OE lines (Fig. 3G).These results indicated that reduced expression ofOsAAP5 could significantly increase tiller number, thusimproving grain yield in ZH11.Rice tiller bud outgrowth is an important factor

determining tiller number (Li et al., 2003). To ex-amine tiller bud development across the differentialexpression lines, seedlings at 7 d after germination(DAG) were cultured with 2.0 mM NH4NO3 underhydroponic conditions for 3 weeks until the tillerbuds began to elongate. At 27 DAG, the seedlingsof the RNAi lines were larger than those of ZH11,and the opposite result was found in the OE lines(Supplemental Fig. S5A). In addition, repressing theexpression of OsAAP5 led to significantly increasedbiomass per plant, including fresh weight and dryweight, compared with that of ZH11 (SupplementalFig. S5C). The tiller buds, especially the second tillerbud, in the RNAi lines were longer than that in ZH11,but no significant difference in bud length was foundbetween the OE lines and ZH11 (Supplemental Fig. S5,B and D). These results demonstrated that OsAAP5affected tiller bud outgrowth and thereby tiller number.

OsAAP5 Might Transport Basic Amino Acids and NeutralAmino Acids in Rice Plants

Because OsAAP5 is an amino acid permease, wedetected the free amino acid concentrations in ZH11,the OE lines, and the RNAi lines to investigate the effectof OsAAP5 expression level on amino acid transport.The analysis of total amino acid concentration indicatedthat OsAAP5 OE significantly increased the total freeamino acid concentration in the tiller basal part, leafsheath, and leaf blade, whereas OsAAP5 repressionexhibited the opposite result (Fig. 4, A–C). We then mea-sured the concentration of each individual amino acid inthe tiller basal part, leaf sheath, and leaf blade of ZH11, theOE lines, and the RNAi lines using HPLC. The concen-trations of basic amino acids (Lys and Arg) and neutralamino acids (Thr, Ala, and Val) in the tiller basal part, leafsheath, and leaf blade of the OE lines were higher thanthose of ZH11, but the opposite result occurred in theRNAi lines (Fig. 4, D–F). In addition, the elevated ex-pression ofOsAAP5 also caused the accumulation of Ser inthe leaf blade but not in the tiller basal part and leaf sheath(Fig. 4, D–F). g-Aminobutyric acid (GABA) functions as asignal that modulates plant growth, development, andstress response (Ramesh et al., 2017); therefore, we alsodetected the content of GABA and found no significantdifference inGABAcontent in lineswithdifferentOsAAP5expression levels (Supplemental Fig. S6). These resultsindicated that OsAAP5 might mediate basic and neutralamino acid transport in rice plants.A protoplast-esculin assay is a method to assay plant

Suc transporters (Rottmann et al., 2018). To further con-firm that OsAAP5mediates basic and neutral amino acidtransport, we performed a protoplast amino acid uptakeassay. Protoplasts were cultured with a fluoresceinisothiocyanate–labeled amino acid (Lys-FITC, Arg-FITC,Val-FITC, Ala-FITC, Ser-FITC, or Pro-FITC) at 1 mM. Toexamine OsAAP5 transport function, we detected thefluorescent signal after 1 h of culture.We detected higherfluorescent cell ratio and higher fluorescence signal in-tensity in the protoplasts of the OE lines cultured withLys-FITC and Arg-FITC than in ZH11 protoplasts, andthe FITC signalwasweaker in theRNAi lines than in ZH11(Fig. 5, A, B, E, and F). The fluorescent signals of proto-plasts culturedwithVal-FITC andAla-FITC for 1 h showedsimilar results to those with the basic amino acids (Fig. 5,C–F). In addition, protoplasts cultured with Ser-FITC(Supplemental Fig. S7, A, C, and D) and Pro-FITC(Supplemental Fig. S7, B, E, and F) showed no significantdifferences in either fluorescent cell ratio or fluorescencesignal intensity among ZH11, the OE lines, and the RNAilines. These results indicated that OsAAP5 might play acrucial role in transporting basic amino acids (Lys andArg)and neutral amino acids (Val and Ala) into rice plant cells.

Exogenous High-Concentration Amino Acids InhibitedTiller Bud Outgrowth

Maintenance of internal amino acid homeostasis inplants is crucial in plant growth and development



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(Lu et al., 2018). Nutrient solutions with added Lys,Arg, Val, and Ala at concentrations of 1.0 mM, 0.2 mM,1.0 mM , and 1.0 mM, respectively, promote tiller budoutgrowth in ZH11, and higher concentrations of theseamino acids than those mentioned above inhibit tillerbud outgrowth (Lu et al., 2018). To further validate theeffects of these amino acids on tiller bud outgrowth

across differentOsAAP5 expression lines, an exogenousamino acid assay was performed. The tiller bud lengthexhibited no significant difference between the OE linesand ZH11. However, the second tiller bud length sig-nificantly increased in the RNAi lines compared withthat in ZH11when the Lys concentration in the nutrientsolution was increased to 1.5 mM (Fig. 6, A and C).

Figure 3. Phenotype analysis ofOsAAP5 transgenic plants grown in paddy field. Whole plant phenotype (A), relative expressionlevels ofOsAAP5 (B), tiller number per plant (n = 30; C), spikelet number per panicle (n = 30; D), 1000-grain weight (n = 30; E),seed setting rate (n = 30; F), and grain yield per plant (n = 30; G) of transgenic plants are shown. OE1-OE3 indicate OsAAP5overexpressing lines, and RNAi1-RNAi3 representOsAAP5-RNAi lines. Scale bar = 10 cm (A). Values are means6 SD, and threereplications were performed in each analysis. Student’s t test (each transgenic line vs. ZH11), *** P , 0.001.


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Figure 4. Effects ofOsAAP5 on amino acid concentration among ZH11, OE, and RNAi lines. Total concentration of amino acidsin tiller basal part (A), leaf sheath (B), and leaf blade (C) of ZH11, OE lines, and RNAi lines. Single concentration of amino acids intiller basal part (D), leaf sheath (E), and leaf blade (F) of ZH11, OE lines, and RNAi lines are also shown. OE-M indicatesOE1-OE3mixed, and RNAi-M indicates RNAi1-RNAi3 mixed. DW, dry weight. Values are means 6 SD, and three replications were per-formed in each analysis. Student’s t test (each transgenic line vs. ZH11), * P , 0.05, ** P , 0.01, *** P , 0.001.



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Figure 5. Protoplast amino acid uptake assay in transgenic plants. Fluorescence was detected after culturing protoplasts withFITC-labeled amino acids for an hour. Green fluorescence images of ZH11, OE, and RNAi lines under Lys-FITC (A), Arg-FITC (B),Ala-FITC (C), and Val-FITC (D) are shown. E, Statistical analysis of the proportion of fluorescence cells. A total of 400 cells werestatistically analyzed. F, Detection of cell fluorescence signal intensity. Fluorescence intensitieswere normalized to the area of therespective cell by ImageJ software, and a total of 100 cells were statistically analyzed. Scale bars = 50mm. Values are means6 SD,


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When the Arg concentration in the nutrient solutionwas elevated to 0.3 mM, a similar result was observed(Fig. 6, B and D). Additionally, when the concentrationsof the neutral amino acids Ala and Val were increasedto 2.0 mM, the tiller bud length, especially that of thesecond tiller bud, significantly increased in the RNAilines compared with ZH11, but no differences in tillerbud length were found between the OE lines and ZH11(Supplemental Fig. S8, A and B). These results dem-onstrated that OsAAP5 might mediate the uptake ofLys, Arg, Val, and Ala and that excessive amino acidaccumulation in plant cells retards rice tiller budelongation.

Altered Expression of OsAAP5 Regulated CK Levelsin Rice

Shoot branching, which is regulated by the planthormones CK, auxin, and strigolactone (SL), is impor-tant for generating diverse plant forms (Gomez-Roldanet al., 2008; Umehara et al., 2008). CK promotes budoutgrowth, whereas auxin and SLs inhibit it (Gomez-Roldan et al., 2008; Domagalska and Leyser, 2011).Recently, both OsGln synthetase1;2 and the nitratetransporter OsNPF7.2 have been reported to affect tillerbud outgrowth through the CK pathway (Ohashi et al.,2017; Wang et al., 2018). This study indicated thatreduced expression of OsAAP5 promoted tiller budoutgrowth. Based on these results, we speculated thatalterations in OsAAP5 expression might affect CKlevels in plant cells. To validate this hypothesis, wemeasured CK-related gene expression in the leafsheath and leaf blade across lines with differentialexpression levels ofOsAAP5. In this study, we detectedtwo genes of the cytokinin oxidase/dehydrogenase(CKX) family, OsCKX2 and OsCKX4, which were repor-ted to regulate tiller number in rice (Gao et al., 2014; Yehet al., 2015). When compared with that in ZH11, leafsheath expression of OsCKX2 significantly increased inthe OE lines, whereas it significantly decreased in theRNAi lines. However, no obvious differences inOsCKX4expression levels occurred among ZH11, the OE lines,and the RNAi lines (Fig. 7C). Additionally, the expres-sion of the type-A response regulator (RR) OsRR1 inresponse to CKs increased in the RNAi lines, whereasit decreased in the OE lines, compared with ZH11(Fig. 7C). These results indicated that the suppressionof OsAAP5 might lead to increased CK content. Sub-sequently, we measured the contents of individualCKs in the leaf sheath and leaf blade in the differentialexpression lines. CK contents, especially tZ, cis-zeatin(cZ), and dihydrozeatin (DZ) contents, were higher inthe leaf sheaths of the RNAi lines than in those ofZH11, but only tZ and DZ contents decreased in the

OE lines compared with ZH11 (Fig. 7A). In the leaf blade,cZ and DZ contents were higher in the RNAi lines andlower in the OE lines than in ZH11 (Fig. 7B). Additionally,the indole-3-acetic acid (IAA) and methyl indole-3-acetate(ME-IAA) levels in the leaf shealths of OE and RNAi lineswere both decreased, but the indole-3-carboxaldehyde(ICA) levels increased, compared with ZH11, respec-tively (Supplemental Fig. S9A). In leaf blade, only ME-IAA content was decreased in RNAi lines, and ICAcontents were both increased in OE and RNAi lines,compared with ZH11, respectively (Supplemental Fig.S9B). These results indicated that the auxin levels in theleaf sheath and leaf blade exhibited no obvious trendswith altered OsAAP5 expression. Moreover, we detec-ted the expression of SL biosynthetic genes (OsD27,OsD17, and OsD10), a perception gene (OsD14), signal-ing genes (OsD3 and OsD53), and another importanttiller-related geneOsFC1 (Ishikawa et al., 2005; Zou et al.,2005; Arite et al., 2007, 2009; Lin et al., 2009; Minakuchiet al., 2010; Yoshida et al., 2012; Jiang et al., 2013; Zhouet al., 2013) in ZH11, the OE lines, and the RNAi lines.However, no significant difference in the expressionlevels of these genes was observed, except that OsD3expression was found to be lower in OE lines andOsD53 expression levels were both higher in the OEand RNAi lines than in ZH11 (Supplemental Fig. S10).These results indicated that OsAAP5 might regulatetiller bud outgrowth through the CK pathway.

Knockout of OsAAP5 in ZH11 Significantly IncreasedTiller Number and Grain Yield

Because OsAAP5 negatively regulated tiller num-ber, we knocked out theOsAAP5 sequence of japonicaZH11 using CRISPR technology to verify the appli-cation value of this gene in rice breeding programs.The target site of OsAAP5 is shown in Fig. 8A. Thesequencing results revealed 2-bp and 1-bp deletionsin the sixth exon of OsAAP5, which caused frameshiftsof OsAAP5 in the OsAAP5-CRISPR lines (OsAAP5-C1and OsAAP5-C2). In the mature stage, OsAAP5-C1and OsAAP5-C2 produced more tillers than ZH11(Fig. 8, B–D). Accordingly, the grain number per plantof OsAAP5-C1 and OsAAP5-C2 were both higher thanthat of ZH11 (Fig. 8, E–G). Statistical analysis revealedthat both tiller number and grain number per plant inOsAAP5-C1 and OsAAP5-C2 significantly increasedcomparedwith those in ZH11 (Fig. 8, H and I), resultingin a significant improvement in rice grain yield perplant in paddy (Fig. 8J). These results demonstratethat knockout of OsAAP5 is valuable for high-yieldrice breeding programs and that the applicationof CRISPR technology is especially important forjaponica improvement.

Figure 5. (Continued.)and three replications were performed in each analysis. Student’s t test (each transgenic line vs. ZH11), * P, 0.05, ** P, 0.01,*** P , 0.001.



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DISCUSSION

OsAAP5 Knockout Is Valuable for japonica Rice Breeding

Indica and japonica, two subspecies of Asian-cultivated rice, are divergent at the physiological, mo-lecular, and biochemical levels (Dan et al., 2014). In theN metabolic pathway, OsNPF6.5 (Hu et al., 2015) andOsAAP3 (Lu et al., 2018) show obvious differentiationbetween indica and japonica rice, and the indica alleles ofboth OsNPF6.5 and OsAAP3 are beneficial to the im-provement of tiller and grain yield in rice. In this study,OsAAP5, a gene found to be highly differentiated be-tween indica and japonica rice, could negatively regulaterice tiller number and grain yield and might, togetherwith OsAAP3 and OsNPF6.5, control plant type differ-ences between indica and japonica rice by affecting Nmetabolism. Because of the high homology between theOsAAP3 andOsAAP5 sequences in rice, we detected theexpression of OsAAP3 in lines with different OsAAP5expression levels and found that changes in the ex-pression level of OsAAP5 do not affect OsAAP3 geneexpression levels. In addition, there was no significantdifference in the expression ofOsAAP5 amongOsAAP3lines with different expression levels (SupplementalFig. S11). These results indicated that the effect ofOsAAP5 on tiller number might be independent ofOsAAP3. Furthermore, knockout of OsAAP5 in theZH11 background using CRISPR technology en-hanced tiller formation and significantly increasedgrain yield (Fig. 8). Based on these findings, we concludethatOsAAP5 plays a role in the difference between indicaand japonica rice plant types. Plant membrane trans-porter genes can be incorporated into plants to enhancecrop yields (Schroeder et al., 2013). Therefore, knockout

of OsAAP5 might be of value in breeding japonica ricewith high yield and grain quality.

OsAAP5 Might Negatively Regulate Rice Tiller BudOutgrowth by Mediating Lys, Arg, Val, and Ala Transport

In this study, the contents of basic amino acids (Lysand Arg) and neutral amino acids (Val and Ala) in theOsAAP5 OE lines significantly increased according toHPLC and protoplast amino acid uptake assays (Figs. 4and 5). To further explore the effects of Lys, Arg, Val,and Ala on rice growth, we performed an exogenousamino acid assaywith ZH11, the OE lines, and the RNAilines. The results revealed that reduced expression ofOsAAP5 significantly promoted second tiller bud out-growth, which might be attributed to the accumulationof Lys, Arg, Val, and Ala within the appropriate con-centration range in the RNAi lines. However, the ex-cessive accumulation of Lys, Arg, Val, and Ala in theOE lines and ZH11 retarded tiller bud outgrowth(Fig. 6; Supplemental Fig. S8).

The accumulation of excessive amino acids in plantcells retards plant growth and development (Lee et al.,2007), and specifically, Lys can inhibit the length of themain root and the tiller bud in Arabidopsis (Yang et al.,2014) and in rice (Lu et al., 2018). Moreover, the rootsystem of the Ataap3 mutant is highly developed andshows a larger number of long main roots and a higherdensity of lateral roots (Marella et al., 2013). Similarly,the Arabidopsis mutant aap2 demonstrates reducedneutral amino acid transport but increased branchnumber and seed yield (Zhang et al., 2010). Our find-ings with respect toOsAAP5were consistent with these

Figure 6. Effects of Lys and Arg on budelongation of seedlings grown in hy-droponic culture. Phenotypes (A) andbud length (C) of ZH11, OE1-OE3,and RNAi1-RNAi3 lines with 1.0 mMNH4NO3 containing 1.5 mM Lys areshown. Phenotypes (B) and bud length(D) of ZH11,OE1-OE3, andRNAi1-RNAi3lines with 1.0 mM NH4NO3 containing0.3 mM Arg are also shown. The tillerbuds were digitally extracted for com-parison. White arrows indicate the firsttiller bud, and red arrows indicate thesecond tiller bud. Scale bars = 3 mm.Values are means 6 SD (n . 15), andthree replications were performed ineach analysis. Student’s t test (eachtransgenic line vs. ZH11), * P , 0.05,** P , 0.01, *** P , 0.001.


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studies. Based on these results, we demonstrated thatOE of OsAAP5 could inhibit bud outgrowth by en-hancing the absorption and accumulation of someamino acids, especially basic (Lys and Arg) and neutralamino acids (Val and Ala).

CK Accelerated Tiller Bud Outgrowth Due to ReducedExpression of OsAAP5

Rice tiller bud outgrowth is an intricate develop-mental process, and it is regulated by phytohormones,such as CK, auxin, and SL (Leyser, 2003; Ferguson andBeveridge, 2009). Elevated CK levels promote tiller budoutgrowth, and CK activity in plants is closely relatedto N availability (Kamada-Nobusada et al., 2013). Thisstudy found that decreased expression of OsAAP5 sig-nificantly increased CK levels in the leaf sheath and leaf

blade, whereas OsAAP5 OE significantly decreasedCK levels (Fig. 7). This study also detected the level ofauxin, which inhibits tiller bud outgrowth; however,the auxin levels in plants with different OsAAP5 ex-pression levels exhibitedno obvious trends (SupplementalFig. S9). These results indicated that OsAAP5 mightregulate rice tiller formation by affecting CK levelsrather than auxin levels in plant cells.SL inhibits tiller bud outgrowth (Domagalska and

Leyser, 2011). This study also detected the expressionlevels of some genes in the SL pathway in ZH11, the OElines, and the RNAi lines to evaluate the effect ofOsAAP5 on SL metabolism. The results demonstratedthat the expression of SL metabolic genes (OsD27,OsD17, OsD10, OsD14, and OsFC1) was not signifi-cantly affected by OsAAP5 expression level. Althoughthe expression of theOsD3was significantly decreasedin OE lines, and OsD53 significantly increased in OE

Figure 7. Effects ofOsAAP5 on CK concentrations. Four CK concentrations were detected in the leaf sheath (A) and leaf blade (B)of ZH11, OE lines, and RNAi lines grown in hydroponic culture. OE-M indicates OE1-OE3mixed, and RNAi-M indicates RNAi1-RNAi3 mixed. IP, isopentenyladenine. The expressions of OsCKX2, OsCKX4, and OsRR1 in tiller basal part (C) of seedlings inhydroponic culture are shown. Values are means 6 SD, and three replications were performed in each analysis. Student’s t test(each transgenic line vs. ZH11), * P , 0.05, ** P , 0.01, *** P , 0.001. DW, dry weight.



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and RNAi lines, no obvious trends were found withaltered OsAAP5 expression (Supplemental Fig. S10).Based on the results above, we conclude that OsAAP5could influence rice tiller bud outgrowth mainly by

regulating CK levels rather than auxin and SL levels,resulting in the observed effects on rice tiller numberand grain yield. However, the mechanism by which theoveraccumulation of amino acids, especially Lys, Arg,

Figure 8. Knockout of OsAAP5 using CRISPR technology significantly improved grain yield in ZH11. A, Sequencing of basedeletion ofOsAAP5-CRISPR lines. Phenotype of ZH11 (B) andOsAAP5-CRISPR lines (C and D) are shown. Grain yield per plantof ZH11 (E) andOsAAP5-CRISPR lines (F and G) are also shown. Tiller number per plant (H), filled grain number per plant (I), andgrain yield per plant (J) were analyzed between ZH11 and OsAAP5-CRISPR lines. Scale bars = 20 cm (B–D) and 5 cm (E–G).Values are means6 SD (n. 20), and three replications were performed in each analysis. Student’s t test (each transgenic line vs.ZH11), ** P , 0.01.


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Val, andAla, in plants decreases the CK level remains tobe studied. Future experiments could explore the rela-tionship between the metabolism of these amino acidsand the CK pathway.

MATERIALS AND METHODS

Sequence Variation in the OsAAP5 Promoter

SNP and indel information on the OsAAP5 promoter of rice (Oryza sativa)cultivars is available on RiceVarMap v2.0 (http://ricevarmap.ncpgr.cn/v2/)according to Zhao et al. (2015). The main haplotypes of the OsAAP5 promoter,Hap1, Hap2, and Hap3, were validated by PCR in corresponding cultivars.Haplotypes with a frequency of 5% or higher (at least fifteen accessions) wereused for an association analysis of tiller number via one-way ANOVA in SPSS.Duncan’s multiple range test was used for multiple comparisons. The primersare listed in Supplemental Table S1.

Plant Materials and Agronomic Trait Analysis

The 1398-bp OsAAP5 (LOC_Os01g65660) complementary DNA (cDNA)was amplified fromZH11 and then inserted downstream of the 35S promoter inthe pCAM1306 vector using Kpn I and Xba I to construct the p35S::OsAAP5plasmid. To construct the OsAAP5-RNAi vector, two 290-bp fragments ofOsAAP5 cDNA were obtained from ZH11 and inserted downstream of theUbi-1 promoter in the vector pTCK303 (Wang et al., 2004) using BamH I/Kpn Iand Spe I/Sac I. All the constructed plasmidswere transformed into japonica ricevariety ZH11 by the Agrobacterium tumefaciens–mediated transformationmethod. Homozygous T2 generation transgenic lines were chosen for furtherstudy. The primers are listed in Supplemental Table S1.

For the basic agronomic trait analysis, rice plants were grown in the paddyfield at the rice experimental station of theWuhan Institute of Bioengineering.Atthe mature stage, 20 plants of each line were randomly chosen for further de-tection. The number of spikelets per paniclewas counted. The 1000-grainweightand grain yield per plant were measured. The seed set ratio was equal to thenumber of grains per plant divided by the number of spikelets per plant.

GUS Activity Analysis and Subcellular Localizationof OsAAP5

For GUS activity analysis, a 2455-bp OsAAP5 promoter fragment was gen-erated from ZH11 and inserted upstream of the GUS coding region using HindIII and BamH I in pCAMBIA1391Z to generate the POsAAP5::GUS plasmid.Then, this plasmid was transformed into japonica rice variety ZH11 to producePOsAAP5::GUS lines. Histochemical GUS assays were conducted as previouslydescribed (Sieburth and Meyerowitz, 1997). First, the samples for GUS stainingwere vacuum infiltrated for 15min and gently fixed in formalin-acetic acid (70%[v/v] ethanol [1:1:18]) at 4°C for 20–30 min. Then, the samples were incubatedin the staining buffer at 37°C overnight. After incubation in a solution of 80%(v/v) ethanol was cleared through, the stained samples were observed using astereomicroscope.

To determine the subcellular localization of OsAAP5, the OsAAP5ORF wasamplified and fused with GFP using Bgl II and Spe I in the pCAM1302 vector togenerate the P35S::OsAAP5-GFP fusion plasmid. Then, the generated plasmidwas transiently expressed in rice protoplasts prepared from etiolated seedlingsof ZH11. The plasma membrane colocalized marker was the rice homologof putative Ca2+-permeable mechanosensitive channels in Arabidopsis(Arabidopsis thaliana), (OsMCA1) fused with mCherry (Kurusu et al., 2012).The fluorescence was observed using a confocal laser scanning microscope.The primers are listed in Supplemental Table S1.

Bud Outgrowth and Amino Acid Analysis

To explore the effects of alteredOsAAP5 expression on seedling growth andbud outgrowth, the seedlings of ZH11 and transgenic lines at 7 DAG werecultured with basic rice nutrient solution (Yoshida et al., 1976). The nutrientsolution was renewed every 3 d. At 30 DAG (the fifth leaf stage), biomass, in-cluding fresh weight and dry weight, and tiller bud length in ZH11, the OElines, and the RNAi lines weremeasured. The leaf sheath and leaf blade of ZH11

and the transgenic lines were prepared for amino acid analysis, phytohormonedetection, and RNA extraction.

For the amino acid analysis, seedlings of ZH11 and the transgenic lines at30DAGwere used. The total free amino acid concentrationwasmeasured by theninhydrin method (Fang et al., 2013). Single amino acid concentrations in theleaf sheath and leaf blade were measured using HPLC (Lu et al., 2018). Ricetissues were extracted with 10 mL 80% (v/v) ethanol at 80°C. A 1-mL aliquot ofeach sample was evaporated to remove the ethanol, redissolved in 1 mL 0.02 MHCl, and subsequently analyzed with HPLC.

The determination of GABA content was performed according to Sansenyaet al. (2017) with appropriate modification. One gram dry powder was takenfrom each sample, dissolved in 5 mL deionized water, and extracted by oscil-lation for 1 h. Then, the supernatant was centrifuged at 12000 rpm for 15 min,and the supernatant was removed and filtered through a 0.45-mm filter mem-brane. Next, 0.5 mL of the filtered sample was taken, and 0.2 mL 0.2 M boratebuffer (pH 9.0), 1 mL 6% (v/v) phenol reagent, and 0.4 mL 9% (w/v) NaClOwere added successively. The mixture was shaken thoroughly, boiled for10 min, and then cooled in the cooling bath until a blue color appeared. Thecontent of GABA was determined by spectrophotometry with a wavelength of645 nm. A standard GABA content curve was prepared according to Sansenyaet al. (2017).

To investigate the effects of single amino acids on tiller bud outgrowth inZH11 and OsAAP5 transgenic lines, seedlings cultured with rice basic nutrientsolution for 14 d were grown in a solution containing 1.5 mM Lys, 0.3 mM Arg,2.0 mM Val, 2.0 mM Ala, and 2.0 mM Thr with the original N decreased to half.The tiller bud length of seedlings was measured from 21 DAG among theOsAAP5 differential expression lines.

Protoplast Amino Acid Uptake Assay

Amino acids with FITC markers (Lys-FITC, Arg-FITC, Val-FITC, Ala-FITC,Pro-FITC, and Ser-FITC) were synthesized by Yuan Peptide BiotechnologyCompany. A protoplast amino acid uptake assay was performed as describedpreviously (Rottmann et al., 2018). Rice protoplasts prepared from etiolatedseedlings of ZH11, and the transgenic lines were kept in 1 mL W5 buffer(154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 5 mM Glc, and 2 mM MES, pH 5.8)supplemented with each FITC-labeled amino acid to a final concentration of1 mM at room temperature in the dark. At 1 h after culture, the protoplasts werecentrifuged at 100g for 5min. After the supernatant was removed carefully witha pipette, the protoplasts were suspended in 1 mLW5 solution and centrifugedagain at 100g for 5min. To fully remove free FITC-marked amino acids from thesolution, the protoplasts were resuspended three times and then observed us-ing a confocal laser scanning microscope.

RNA Extraction and RT-qPCR

Total RNA was isolated from different tissues with TRIzol reagent (Invi-trogen), and cDNA was synthesized using M-MLV reverse transcriptase(Promega). RT-qPCR was performed in the 7500 RT qPCR system (AppliedBiosystems) according to the manufacturer’s instructions. The rice ACTIN gene(LOC_Os03g50885),UBIQUITIN (LOC_Os05g06770), andGAPDH (LOC_Os04g40950)were used as internal references, and the gene expression levels were normal-ized to the geometric average of these internal reference genes. Three biologicalreplicates were performed for each sample. The primers for RT-qPCR are listedin Supplemental Table S1.

Detection of Phytohormones

The leaf sheath and leaf blade of ZH11 and transgenic seedlings at 30 DAGwereobtainedand reduced todrypowder. Then,CK(isopentenyladenine, tZ, cZandDZ) and auxin (IAA,ME-IAAand ICA) contentswere detected byMetWare(http://www.metware.cn/) based on the AB Sciex QTRAP4500 LC-MS/MSplatform. Three replicates of each assay were performed.

Statistical Analysis

A two-tailed t test (each transgenic line vs. ZH11; N treatment N2 or N3 vs.N1; N treatment N5 or N6 vs. N4; N treatment N8 or N9 vs. N7) was performedusing SPSS software (IBM, Inc.) with *, **, and *** indicating significant dif-ferences at P , 0.05, P , 0.01, and P , 0.001, respectively, in Figures 3–8 andSupplemental Figures. S2–S10. For multiple comparisons, one-way ANOVA



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with Duncan’s multiple range test (comparison of all lines) was performedusing SPSS software; different letters indicated significant differences atP , 0.05 in Fig. 1 and Supplemental Fig. S1.

Accession Numbers

Sequence data from this article can be found in the Rice Genome AnnotationProject or GenBank under the following accession numbers: OsAAP5,LOC_Os01g65660; OsACTIN, LOC_Os03g50885; UBIQUITIN, LOC_Os05g06770;and GAPDH, LOC_Os04g40950.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. The expression pattern analysis of OsAAP5 indifferent promoter types.

Supplemental Figure S2. Analysis of OsAAP5 expression under differentnitrogen treatments.

Supplemental Figure S3. Prediction of the transmembrane domain ofOsAAP5.

Supplemental Figure S4. The expression levels of OsAAP5 homologousgenes in ZH11, OE, and RNAi lines.

Supplemental Figure S5. Altered expression of OsAAP5 influenced seed-ling growth and bud outgrowth.

Supplemental Figure S6. Detection of GABA contents in OsAAP5 lineswith different expression levels.

Supplemental Figure S7. Protoplast amino acid uptake assay in transgenicplants.

Supplemental Figure S8. Effects of neutral amino acids on bud elongationof ZH11, OE, and RNAi lines.

Supplemental Figure S9. Effects of OsAAP5 on auxin concentrations.

Supplemental Figure S10. The expression levels of genes related to the SLpathway in ZH11, OE, and RNAi lines.

Supplemental Figure S11. The expression of OsAAP5 in OsAAP3 differ-ential expression lines.

Supplemental Table S1. List of primers used in this study.

Received January 10, 2019; accepted March 5, 2019; published March 19, 2019.

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