Development of a visible marker trait based on leaf sheath-specific anthocyanin pigmentation applicable to various genotypes in rice.

To overcome a limitation to the breeding of autogamous crops, recurrent selection using transgenic male sterility (RSUTMS) has been proposed. In this system, negatively or positively selectable marker traits are required along with dominant transgenic male sterility. Anthocyanin pigmentation is an excellent marker trait. Two regulatory genes for MYB and bHLH and a structural gene for DFR are required for anthocyanin pigmentation in rice. Therefore, to apply anthocyanin pigmentation as a marker trait in various rice genotypes, coordinated expression of the three genes is required. In this study, we developed a leaf sheath-specific promoter and introduced three genes-DFR and C1/Myb, driven by the 35S promoter, and OsB2/bHLH, driven by the leaf sheath-specific promoter-into the rice genome. Leaf sheath-specific pigmentation was confirmed in all seven genotypes tested, which included japonica and indica cultivars. Analysis of genome sequence data from 25 cultivars showed that the strategy of conferring leaf sheath-specific anthocyanin pigmentation by introduction of these three genes would be effective for a wide range of genotypes and will be applicable to RSUTMS.

Introduction

Genetically fixed cultivars of autogamous crops are generally bred through two-way crosses and genetic fixation by selfing, followed by selection. However, the applicability of this method is critically limited by the number of available parental materials, the frequency of genetic recombination, and continuity of breeding effect (Fujimaki 1980). To overcome these limitations, recurrent selection using transgenic male sterility (RSUTMS) was proposed (Tanaka 2010). RSUTMS is a novel breeding system based on outcrossing in autogamous crop species that effectively makes use of genomic information, such as the genomic selection. To establish this breeding system, the development of dominant transgenic male sterility (TMS) is required to eliminate self-pollination. Application of dominant TMS has the advantage that efficient and continuous outcrossing can be realized due to the high frequency (theoretically 50%) of male sterile individuals. A gene cassette carrying a negatively or positively selectable marker with dominant TMS is also required to distinguish the male sterile individuals from fertile ones at the juvenile stage. Recently, anther-specific gene promoters, which can generate male sterility in rice (Oryza sativa), have been developed (Abe , Akasaka ). Gene cassettes for TMS and the selectable marker need to be functional as dominant traits in plants. Because RSUTMS requires the handling of many individuals, a visible marker is desirable that can be easily recognized without special equipment or processing at the juvenile stage.

Anthocyanin pigmentation has been used as a visible marker in the maize haploid inducer system to effectively select haploid seeds and seedlings through the elimination of colored diploids (Nanda and Chase 1965, Rotarenco ). Because the mechanism of anthocyanin pigmentation is well understood (see below), pigmentation patterns could be controlled in an organ- and/or growth stage–specific manner. Anthocyanins are predominantly accumulated in epidermal cells of purple-leaf rice, and they absorb visible radiation between 400 and 700 nm, with a maximum of about 530 nm (Peng ). In maize, Pietrini and Massacci (1998) estimated that 43% of light energy was absorbed by the anthocyanin pigments in leaves with 35 times higher amount of anthocyanins than control leaves. Thus, anthocyanin pigmentation has a negative effect on photosynthesis and, therefore, plant growth in the field. Therefore, to use anthocyanin pigmentation as an easily recognized marker trait in rice, it is necessary to accumulate the pigments in organs other than the leaf blade, which is the main photosynthetic organ.

The anthocyanin pigmentation trait in maize has been well investigated over the last century (Emerson 1921). It is controlled by two regulatory gene families: C1/Purple plant (Pl), encoding R2R3 MYB-related transcription factors (TFs), and R/B, encoding basic helix-loop-helix (bHLH) MYC-type TFs. A combination of the MYB and bHLH proteins coordinately activates transcription of a set of genes for anthocyanin biosynthesis. Moreover, the expression patterns of both Myb and bHLH genes are tissue and developmental stage specific (Dooner , Grotewold , Petroni and Tonelli 2011, Schwinn ). Although typical field-grown rice cultivars have green leaves, several accumulate anthocyanins and/or proanthocyanidins in various tissues, including leaf blades, sheaths, pericarps, and apiculi. Studies of the genetics of anthocyanin biosynthesis and accumulation in rice also have a long history (Morinaga 1938, Takezaki 1921); they have revealed that the pigmentation of various rice tissues is controlled mainly by three types of dominant genes: C ( Chromogen), A (Activator), and their tissue-specific regulators including P (Purple), Pl (Purple leaf), etc. (Nagao and Takahashi 1963, Takahashi 1957). Further genetic analyses have uncovered additional loci that determine tissue-specific distribution and accumulation of anthocyanins and proanthocyanidins (Reddy 1996, Sakamoto , Takahashi 1982). In the C-A-P/Pl system of cultivated rice, the C gene corresponds to OsC1, a homolog of maize C1 (Reddy , Saitoh ). Two independent genes from the Pl alleles have been identified; they encode R/B (bHLH) homologs (Hu , Sakamoto ). Furukawa demonstrated that the A gene encodes dihydroflavonol 4-reductase (DFR) in the anthocyanin biosynthesis pathway. Many modern Japanese rice cultivars, including ‘Nipponbare’, have a loss-of-function allele of the DFR gene (Furukawa , Nakai , Sun ). Various mutations in the OsC1 gene have also been found in rice (Saitoh , Sun ). Zhu demonstrated the accumulation of anthocyanins in the endosperm of rice highly expressing six structural genes from coleus and the above two TF genes (C1 and R/B) from maize. In light of these facts, the accumulation of anthocyanins in the leaf blades and sheaths of rice is likely to require the introduction of at least three genes encoding DFR, MYB, and bHLH, preferably under the control of a tissue-specific promoter(s).

In this study, we first focused on the leaf sheath, as it is an organ in which it is easy to detect pigmentation and which can accumulate anthocyanins with little negative influence on photosynthesis. Next, we developed a leaf sheath–specific promoter by using an expression profile database of rice (Akasaka ). Then, by simultaneously introducing three genes: the DFR and maize C1/Myb genes driven by the CaMV 35S promoter and the OsB2/bHLH gene driven by a leaf sheath–specific promoter into the rice genome, we conferred anthocyanin pigmentation of the target organ (leaf sheath) in various japonica and indica rice genotypes. Finally, we discuss the potential applications of this ‘three genes introduction strategy’.

Materials and Methods

Plant materials

Seven rice cultivars (japonica: ‘Koshihikari’, ‘Kusahonami’, ‘Momiroman’, ‘Nipponbare’, and ‘Tachiaoba’; indica: ‘Kasalath’ and ‘Tachisugata’) were used in this study. All plants were grown in commercial soil (Bonsol No. 1, Sumitomo Chemical, Tokyo, Japan) under simplified Biotron Breeding System conditions (Tanaka ) or in a closed greenhouse, at 28°C in the daytime and 22°C at night.

Screening for leaf sheath–specific promoters

The leaf sheath–specific promoters were searched for using the method described in Akasaka . Sixteen candidate genes specifically expressed in the leaf sheath were selected in the rice expression profile database RiceXPro (Sato , 2013; http://ricexpro.dna.affrc.go.jp). From these candidates, three representative genes with no or extremely low expression levels in other tissues were selected. Approximately 2-kb sequences upstream of these three genes were cloned and the fragments were used as leaf sheath–specific promoters (LSSP) 01, 02, and 03 (Table 1).

Table 1

Leaf sheath–specific promoters developed in this study

Promoter	RAP locus ID	Accession no. of cDNA	Putative gene product	Level of gene expression (RiceXPro)
LSSP01	Os08g0460000	AK065284	Similar to Germin-like protein 1 precursor	200,920
LSSP02	Os02g0308400	AK104655	Beta-Ig-H3/fasciclin domain containing protein	62,520
LSSP03	Os06g0639800	AK106068	Cytochrome P450 family protein	35,004

Polymerase chain reaction

Genomic DNA was isolated from leaves according to Edwards . LSSP01, LSSP02, and LSSP03 sequences with restriction sites (AscI or HindIII and XbaI) were amplified from genomic DNA of ‘Nipponbare’ using the corresponding sets of specific primers (Supplemental Table 1). Point mutations were introduced in LSSP01 and LSSP02 PCR to eliminate restriction sites (Supplemental Table 1). PCR amplification was performed using PrimeSTAR GXL DNA Polymerase (Takara Bio, Shiga, Japan) in a MyCycler thermal cycler (Bio-Rad, Hercules, CA, USA) under the following conditions: initial denaturation at 94°C for 5 min; 30 cycles of 98°C for 15 s, 55°C for 30 s, and 68°C for 2 min; and final extension at 68°C for 5 min.

To detect the inserted gene in transformants, PCR was performed using the primers HPT-F01 and HPT-R01 (Supplemental Table 1) under the following conditions: initial denaturation at 94°C for 5 min; 30 cycles of 98°C for 15 s, 55°C for 30 s, and 68°C for 50 s, and final extension at 68°C for 5 min.

Vector construction and rice transformation

ZmC1 (Acc. No. AF320613), a C1 family gene (Dooner ), OsB2 (Acc. No. AB021080), an R family gene (Sakamoto ) and OsDFR (Acc. No. AB003496), an anthocyanin synthesis gene (Furukawa , Nakai ) were used in this study.

The LSSP01, LSSP02, and LSSP03 promoters were individually fused to OsB2. These fragments and the double terminator DT (35S terminator + nos terminator; Luo and Chen 2007) were introduced into the pUC198AM sub-plasmid (Kuroda ). The CaMV 35S promoter (35SP) and DT were fused with each of OsB2, ZmC1, and OsDFR, and each of the resulting fragments was also introduced into pUC198AM. These gene cassettes were removed from the sub-plasmid using AscI and MluI restriction enzymes and introduced into the pZH2B binary vector (Kuroda ) in various combinations (Fig. 1). Vector p35SP::B2/C1 carried two gene cassettes for anthocyanin pigmentation: 35SP::OsB2::DT and 35SP::ZmC1::DT. Vector p35SP::DFR/B2/C1 carried three cassettes: 35SP::OsB2::DT, 35SP::ZmC1::DT, and 35SP::ZmDFR::DT. The pOsLSSP01, pOsLSSP02, and pOsLSSP03 constructs contained cassettes carrying 35SP::OsDFR::DT, 35SP::ZmC1::DT, and LSSP (01, 02, or 03, respectively)::OsB2::DT (Fig. 1). To generate pLSSP03::GUS, we fused the GUS gene from pIG121-Hm (Acc. No. AB489142; Ohta ) with the LSSP03 promoter, and introduced this fragment and DT into pZH2B (Fig. 1).

Fig. 1

Structure of the T-DNA region in the binary vectors p35SP::B2/C1, p35SP::DFR/B2/C1, pOsLSSP01, pOsLSSP02, pOsLSSP03, and pLSSP03::GUS. 35SP: promoter from the CaMV 35S RNA gene; mHPT: modified hygromycin phosphotransferase gene with deleted restriction enzyme sites; Tnos: terminator from the nopaline synthase gene; LSSP: leaf sheath–specific promoter; OsB2: B2 gene from O. sativa; DT: double terminator from the CaMV 35S RNA and nopaline synthase genes; ZmC1: C1 gene from Z. mays; OsDFR: dihydroflavonol 4-reductase gene from O. sativa; GUS: β-glucuronidase gene; LB and RB: left and right borders, respectively, of the T-DNA; XbaI: XbaI restriction enzyme sites used in Southern blot analysis. Arrows indicate HPT-specific primers (HPT-F01 and HPT-R01).

All of the plasmids were introduced into Agrobacterium tumefaciens strain EHA105 (Hood ), and rice was transformed as described in Ozawa (2009). The T1 seedlings were grown on MS medium (Murashige and Skoog 1962) under continuous white fluorescent light at 27°C; 3 weeks after germination, seedlings were transferred to a closed greenhouse.

GUS assay

GUS assay was performed as described in Konagaya with minor modifications; plant tissues were incubated for 24 h at 37°C in GUS assay solution and bleached with 70% ethanol.

Southern blot analysis

Southern blot assay was performed as in Abe with minor modifications. Genomic DNA was isolated from leaves of ‘Nipponbare’ and T0 plants carrying pOsLSSP03 using the CTAB method (Murray and Thompson 1980). Genomic DNA (10 μg) was digested with XbaI. A 529-bp fragment of the HPT gene was amplified with HPT-F01 and HPT-R01 primers (primer positions are indicated in Fig. 1) and used as a probe.

Genome sequence comparison of structural genes for anthocyanin biosynthesis

The presumed anthocyanin synthesis pathway is shown in Fig. 2. The Rice Annotation Project Database (RAP-DB, Sakai ; http://rapdb.dna.affrc.go.jp/) was used for searching the genes, and the open reading frame (ORF) sequences of retrieved genes were compared in TASUKE (Kumagai ; http://ricegenomes.dna.affrc.go.jp/RiceGenomes/) with the next generation sequence (NGS) data (Table 2). The NGS data from five genotypes—‘Kasalath’, ‘Koshihikari’, ‘Nipponbare’, ‘Tachiaoba’, and ‘Tachisugata’—are available and were used in this analysis. In addition, the NGS data from ‘Taichung 65’ (‘T65’), which has anthocyanin pigmentation owing to the introgression of DFR, OsC1, and OsB2 (Hirose ), and Oryza rufipogon ‘W0120’, which has anthocyanin pigmentation in the anthers (Kurata and Yamazaki 2006; http://www.shigen.nig.ac.jp/rice/oryzabase/top/top.jsp), were used. The NGS data from 16 rice genotypes (11 temperate japonica, 2 tropical japonica, 3 indica; selected on the basis of NGS data quality (average sequencing depth > 25)) were used to check the natural variation of the ORFs of the structural genes related to anthocyanin biosynthesis (Table 2). In addition, NGS data from ‘IR 8’ and ‘IR 64’ were used despite their lower read depth because these cultivars are historically important.

Fig. 2

Anthocyanin synthesis pathway in ‘Nipponbare’ assumed from NGS data. PAL: phenylalanine ammonia-lyase; C4H: cinnamate 4-hydroxylase; 4CL: 4-coumarate-CoA ligase; CHS: chalcone synthase; CHS2: chalcone synthase 2; CHI: chalcone isomerase; F3H: flavanone 3-hydroxylase; F3′H: flavanone 3′-hydroxylase; DFR: dihydroflavonol reductase; ANS: anthocyanidin synthase. *At least one gene encodes a functional enzyme in ‘Nipponbare’ and some other cultivars (see Table 2 for details).

Table 2

Estimation of the functions of the enzyme genes related to anthocyanin biosynthesis on the basis of NGS data

Cultivar/strain	Variety	PAL	C4H	4CL	CHS	CHS	CHS	CHS2	CHI	CHI	CHI	F3H	F3H	F3H	F3′H	DFR	ANS	Accession number
		Os02g0626100	Os05g0320700	Os04g0683700	Os11g0530600	Os07g0501100	Os04g0103900	Os07g0214900	Os12g0115700	Os11g0116300	Os03g0819600	Os04g0667200	Os10g0536400	Os04g0662600	Os10g0320100	Os01g0633500	Os01g0372500
Nipponbarea	TEJ	Functional	Functional	Functional	Functionald	Functional	Functionald	Functionald	Functional	L.O.F. (t)	Functional	DRA004358
Koshihikaria	TEJ	Functional	Functional	Functional	Functionald	Functional	Functionald	Functionald	Functional	L.O.F. (k)	Functional	DRA004358
Tachiaobaa	TEJ	Functional	Functional	Functional	Functionald	Functional	Functionald	Functionald	Functional	L.O.F. (t)	Functional	DRP002297
Taichung 65	TEJ	Functional	Functional	Functional	Functionald	Functional	Functionald	Functionald	Functional	L.O.F. (t)	Functional	SRX982231
(T65)b
Kasalatha	IND	Functional	Functional	Functional	Functionald	Functional	Functionald	Functionald	Functional	Est. F	Functional	DRX093434
Tachisugataa	IND	Functional	Functional	Functional	Functionald	Functional	Functionald	Functionald	Functional	Est. F	Functional	DRP002297
W0120c	O. rufipogon	Functional	Functional	Functional	Functionald	Functional	Functionald	Functionald	Functional	Functional	Functional	DRR001186
Hinohikari	TEJ	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	L.O.F. (k)	Est. F	DRX093430
Hitomebore	TEJ	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	L.O.F. (k)	Est. F	DRX093427
Hoshinoyume	TEJ	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	L.O.F. (t)	Est. F	DRX093426
Fanny	TEJ	Est. F	Est. F	Est. F	Est. F	A.A.S.	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	SRP023273
Gohyakumangoku	TEJ	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	L.O.F. (t)	Est. F	DRA000897
Kameji	TEJ	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	L.O.F. (t)(k)	Est. F	DRA000897
Norin 8	TEJ	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	L.O.F. (t)	Est. F	DRA000897
Omachi	TEJ	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	L.O.F. (t)	Est. F	DRA000307
Tenkomori	TEJ	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	L.O.F. (k)	Est. F	DRX093429
Tentakaku	TEJ	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	L.O.F. (k)	Est. F	DRX093428
Yamadanishiki	TEJ	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	L.O.F. (t)	Est. F	DRA000897
Moroberekan	TRJ	A.A.S.	Est. F	Est. F	Est. F	A.A.S.	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	DRA000897
Parao	TRJ	Est. F	Est. F	Est. F	Est. F	A.A.S.	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	SRP023273
Suweon 258	IND	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	DRP002297
Hokuriku 193	IND	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	A.A.S.	Est. F	Est. F	Est. F	Est. F	Est. F	DRP002297
Takanari	IND	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	A.A.S.	Est. F	Est. F	Est. F	Est. F	Est. F	DRP002297
IR8	IND	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	A.A.S.	Est. F	Est. F	Est. F	Est. F	Est. F	SRP023273
IR64	IND	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	Est. F	A.A.S.	Est. F	Est. F	Est. F	Est. F	Est. F	SRP023273

Cultivars in which anthocyanin accumulation was recovered by the introduction of three genes, DFR, maize C1, and OsB2, in this study.

Hirose achieved anthocyanin accumulation by introducing OsB2 into an isogenic line in the T65 genetic background that contained the DFR and OsC1 loci from ‘Murasakiine’ (a purple-leaf rice cultivar).

Anthocyanin pigmentation was observed in the stigma of this accession (https://shigen.nig.ac.jp/rice/oryzabase/imageGallery/list?fqTags=tagForFacet%3AFLOWER).

Anthocyanin pigmentation in these strains, which have three isoforms, indicates that at least one of the genes is functional.

TEJ, temperate japonica; IND, indica; TRJ. tropical japonica.

Est. F, allele estimated to be functional; these genes have no mutations affecting amino acid sequences.

A.A.S., allele with amino acid substitution(s) in comparison with the presumed functional allele.

L.O.F., loss-of-function allele.

(t) and (k) indicate the alleles carrying ‘Toride 1’-type and ‘Koshihikari’-type loss-of-function mutations, respectively, in the DFR gene (Nakai ).

Results

Anthocyanin pigmentation in calluses with introduced OsDFR, ZmC1, and OsB2 genes driven by the CaMV 35S promoter

First, we introduced each construct (OsDFR, ZmC1, and OsB2) under the control of the CaMV 35S promoter (35SP) into ‘Nipponbare’ separately. However, no anthocyanin pigmentation was observed in any calluses or regenerated plants. We also introduced the tandem cassette vector p35SP::B2/C1 (Fig. 1), in which ZmC1 and OsB2 are driven by 35SP, but no anthocyanin pigmentation was observed (Fig. 3). However, when we introduced the p35SP::DFR/B2/C1 vector, which contains the OsDFR, ZmC1, and OsB2 cassettes in tandem, all controlled by 35SP, calluses with and without anthocyanin appeared 2 weeks after Agrobacterium infection. However, anthocyanin-containing calluses transferred onto regeneration medium rapidly turned brown, stopped cell division, and did not re-differentiate.

Fig. 3

Anthocyanin accumulation in transformed calluses of seven rice cultivars carrying p35SP::B2/C1 or p35SP::DFR/B2/C1. Calluses were transformed with Agrobacterium harboring the indicated binary vector and cultured under hygromycin selection for 4 weeks. In the case of the p35SP::B2/C1 vector, anthocyanin pigmentation was observed only in indica cultivars (‘Kasalath’ and ‘Tachisugata’) Red arrowheads indicate calluses with anthocyanin pigmentation.

The p35SP::B2/C1 vector was also introduced into six other cultivars (‘Kasalath’, ‘Koshihikari’, ‘Kusahonami’, ‘Momiroman’, ‘Tachiaoba’, and ‘Tachisugata’). Only the indica cultivars (‘Kasalath’ and ‘Tachisugata’) accumulated anthocyanin in calluses (Fig. 3). On the other hand, when the p35SP::DFR/B2/C1 vector was introduced into these six cultivars, the calluses of all of them accumulated anthocyanin (Fig. 3). However, similar to ‘Nipponbare’, no regenerated plants could be obtained from anthocyanin-containing calluses.

Features of T0 transgenic calluses and regenerated plants

To avoid anthocyanin pigmentation in calluses and to prevent the negative effect of accumulated anthocyanin on photosynthesis, we explored novel leaf sheath–specific promoters, which we named LSSP01, 02 and 03 (Table 1), and used them to construct the binary vectors pOsLSSP01, pOsLSSP02, and pOsLSSP03, respectively (Fig. 1). These vectors had three gene cassettes: (1) LSSP01, 02 or 03 and OsB2; (2) 35SP and ZmC1; and (3) 35SP and OsDFR. These vectors were introduced into ‘Nipponbare’.

Calluses and regenerated plants carrying these vectors showed different phenotypes. Most calluses carrying pOsLSSP01 had anthocyanin pigmentation; the regeneration rate was clearly inferior to that of ‘Nipponbare’. All plants regenerated from these calluses showed anthocyanin pigmentation in the whole body and inferior growth phenotype (Fig. 4A, 4B). Calluses and regenerated plants carrying pOsLSSP02 showed normal phenotype and no accumulation of anthocyanin (Fig. 4C, 4D). Among the calluses carrying pOsLSSP03, a few accumulated anthocyanin (Fig. 4E); in regenerated plants, anthocyanin accumulation was observed only in the leaf sheath (Fig. 4F, 4G). The pOsLSSP03 vector was also introduced into six other cultivars. The resultant calluses of all cultivars showed anthocyanin accumulation. The regenerated plants accumulated anthocyanin in the leaf sheath (Supplemental Fig. 1); in comparison with ‘Nipponbare’, anthocyanin pigmentation was stronger in ‘Momiroman’ and ‘Kasalath’ and similar or weaker in the other cultivars. Anthocyanin pigmentation was very clear at the juvenile phase but weakened later (Fig. 4F, 4G, Supplemental Fig. 1). In some cultivars, anthocyanin pigmentation was also observed in the ligules, auricles, apiculi, and stigmata (Supplemental Fig. 1).

Fig. 4

Transgenic calluses after 4 weeks of hygromycin selection and regenerated T0 plants carrying pOsLSSP01, pOsLSSP02, or pOsLSSP03. (A) Most calluses carrying pOsLSSP01 were purple. (B) Regenerated pOsLSSP01 plants had anthocyanin pigmentation in various organs and a severe growth defect. (C, D) All pOsLSSP02 calluses and regenerated plants accumulated no anthocyanin and grew normally. (E, F, G) Some of the pOsLSSP03 calluses accumulated low amounts of anthocyanin, and the regenerated plants showed anthocyanin pigmentation of the leaf sheaths.

Analysis of the tissue specificity of the LSSP03 promoter in rice revealed GUS staining in a few calluses and only in the leaf sheath of regenerated plants (Fig. 5); this pattern coincided with anthocyanin pigmentation in calluses and regenerated plants carrying pOsLSSP03.

Fig. 5

GUS expression in (A) T0 calluses and (B) a seedling transformed with pLSSP03::GUS.

Genetic segregation and phenotypes of T1 pOsLSSP03 transgenic plants

Southern blot analysis was carried out to select regenerated plants carrying pOsLSSP03 in one locus. A single fragment was detected in the T0 plant pOsLSSP03#16 and multiple fragments were detected in the T0 plants pOsLSSP03#20 and #34 (Supplemental Fig. 2). In T1 seedlings derived from pOsLSSP03#16, segregation of leaf sheath–specific anthocyanin pigmentation was observed (Fig. 6A, 6B), and anthocyanin pigmentation coincided with the presence of the transgene detected by PCR (Fig. 6C).

Fig. 6

Transgene identification by leaf sheath–specific anthocyanin pigmentation in pOsLSSP03 plants. (A, B) pOsLSSP03#16 T1 seedlings (A) with or (B) without anthocyanins in their leaf sheaths. (C) Genomic PCR analysis of T1 plants transformed with pOsLSSP03. M: 1-kbp DNA Ladder One marker (Nacalai Tesque, Kyoto, Japan); Purple LS: plants with purple leaf sheath; Green LS: plants with green leaf sheath; Vector: pOsLSSP03 vector as a positive control; Nipponbare: a negative control. The location of mHPT-specific primers is indicated in Fig. 1.

Some of the T1 plants with the transgene showed poorer growth than the wild type, but others showed similar growth (Fig. 7). In T2 populations derived from T1 plants with poorer growth, all plants showed anthocyanin pigmentation. In T2 populations derived from T1 plants with wild-type growth, genetic segregation of anthocyanin pigmentation was observed (Supplemental Table 2).

Fig. 7

Growth of a pOsLSSP03 transgenic plant (T1). Left: ‘Nipponbare’; right: pOsLSSP03#16 plant (T1). Scale bar, 10 cm.

Natural variations in the ORFs of enzyme genes related to anthocyanin biosynthesis

A RAP-DB search revealed 16 loci involved in anthocyanin biosynthesis, including 4 CHS loci (including CHS2), 3 CHI loci, and an F3H locus (Fig. 2, Table 2).

In all cultivars tested, anthocyanin pigmentation was induced by DFR and the two TFs; this means that the cultivars have active alleles of all genes for enzymes of the anthocyanin biosynthesis pathway except DFR. We used the NGS data from five cultivars (‘Kasalath’, ‘Koshihikari’, ‘Nipponbare’, ‘Tachiaoba’, and ‘Tachisugata’), ‘T65’, and the O. rufipogon accession ‘W0120’, which we defined as the “seven primary genotypes” having the active anthocyanin biosynthesis pathway (other than DFR). We compared the other 18 genotypes with the “seven primary genotypes” in TASUKE and detected many natural variations in the ORFs of 15 loci except DFR, but these variations (SNPs and indels) were included in the “seven primary genotypes”. No mutations that could be responsible for the loss of enzymatic activity, such as insertions, deletions, or frame shifts, were detected (Table 2).

Discussion

The cause of inferior growth and regeneration in highly pigmented rice calluses

Transgenic rice calluses constitutively overexpressing OsB2, ZmC1, and OsDFR genes accumulated high amounts of anthocyanins (Fig. 3), but they grew more slowly than wild-type calluses and hardly regenerated. Flavonoids, including anthocyanins, are synthesized via the phenylpropanoid pathway and accumulated in vacuoles. Under stress or senescence, these phenolic compounds can be oxidized into corresponding quinones by polyphenol oxidases (PPO; generally localized in plastids) or type III peroxidases (POD; localized in the vacuoles and apoplasts) (Pourcel , Takahama 2004). Such quinones are highly reactive with phenols, amino acids, or proteins, and generate a complex mixture of brown products (Pourcel ). Oxidative browning in cultured cells often results in growth retardation and inhibition of plant regeneration (Dong , Jones and Saxena, 2013, Laukkanen ).

All regenerated plants carrying pOsLSSP01 accumulated anthocyanin in the whole body and grew poorly (Fig. 4B). Anthocyanin biosynthesis and accumulation are increased by various environmental factors, including UV, high light intensity, low temperature, and depletion of sucrose and other nutrients (Petroni and Tonelli 2011, Pourcel ). Anthocyanin biosynthesis is also induced by jasmonate (JA) (De Geyter , Qi , Schweizer ), and activation of the anthocyanin biosynthesis pathway could upregulate genes for JA biosynthesis and signaling (Pourcel ). Activation of JA biosynthesis and signaling inhibits plant growth by inhibiting cell cycle progression (Noir , Pauwels , Świa̧tek , Zhang and Turner 2008) and photosynthesis (Attaran ). Therefore, when anthocyanin pigmentation is used as a marker trait, accumulation of the pigments needs to be limited to tissue(s) where it is easily detectable other than leaf blades, which are the primary sites of photosynthesis.

Phenotypic variation among the transformants

The degree of anthocyanin pigmentation in the leaf sheath depended on the genotype. Pigmentation was stronger in ‘Momiroman’ and ‘Kasalath’ than in ‘Nipponbare’. Depending on the cultivar, pigmentation was also observed in ligules, auricles, apiculi, and stigmata (Supplemental Fig. 1).

In rice, pigmentation of various tissues is controlled mainly by the C, A, and P/Pl genes (Nagao and Takahashi 1963, Takahashi 1957). Several genetic variants of the TF genes C and Pl have been found. The OsC1 (C) gene has 13 haplotypes, of which 9 are non-functional (Sun ). Allelic variations were also found in Pl (OsB1/B2) genes (Sakamoto , Shih ). Tissue and temporal specificity and the levels of expression of the genes for MYB and bHLH play important roles in anthocyanin pigmentation and its distribution (Consonni , Ludwig , Sakamoto , Schwinn ). This complexity confers extensive natural variation in the pigmentation profiles to a wide range of genotypes. In maize, the regulation of the anthocyanin pathway involves a direct interaction of MYB and bHLH (Goff ). In rice, Sun showed that the S1/bHLH protein, which is also called OsB2 or Kala4 (Oikawa , Sakamoto ), interacts with OsC1/MYB, and that the two factors coordinately activate the expression of the DFR/A gene through direct binding to its promoter.

Because ‘Kasalath’ has lost OsC1 (C) function (Sun ), we assumed that its strong anthocyanin pigmentation might be caused by the interaction between its own P/Pl gene or the other endogenous genes of TFs in its genome and the introduced ZmC1 gene. Similarly, ‘Nipponbare’ has no functional OsC1 and DFR genes (Furukawa , Nakai , Sun ), the intermediate pigmentation might be caused by the interactions between the introduced genes and endogenous TFs. Especially, there are many genes for the bHLH family in the rice genome (RAP-DB, Sakai ; http://rapdb.dna.affrc.go.jp/) and some of them regulate the tissue specificity of anthocyanin biosynthesis in the presence of the active DFR and MYB TF(s). The differences in the degree and site of anthocyanin pigmentation among cultivars are likely due to the interaction between the introduced and these endogenous TFs.

Anthocyanin pigmentation was clearly observed at the juvenile stage, and became faint at later stages. Because the interaction between bHLH and MYB is required for anthocyanin pigmentation, this decrease might be due to the differences in expression in terms of micro-tissue specificity and timing between OsB2 driven by LSSP03 and ZmC1 and OsDFR driven by the 35SP.

Inferior growth of plants homozygous for pOsLSSP03

No segregation of anthocyanin pigmentation was observed in the T2 populations derived from T1 plants that showed inferior growth (Supplemental Table 2), likely because the T1 plants were homozygous for the transgenes. The segregation in the T2 populations derived from T1 plants with normal growth likely resulted from the heterozygosity of the T1 plants. Homozygosity of the transgene that inhibited growth in the T2 population was likely caused by the negative effect of the 35SP–driven Myb gene and its dose effect. Generally, overexpression of TF genes inhibits cell and plant growth, as it disturbs the coordinated expression of many genes. Overexpression of the Myb gene in Arabidopsis thaliana and Brassica napus inhibits growth (Cominelli , Ren ). Anthocyanin pigmentation may also inhibit photosynthesis and therefore plant growth by hindering absorption of light (Pietrini and Massacci 1998).

To develop useful rice lines for practical RSUTMS, it will be necessary to consider alternative promoters to replace 35SP. RSUTMS would allow allogamous-based breeding even in autogamous crops such as rice. In this system, dominant male sterility and genes for the marker trait are maintained as heterozygotes in the progeny of crosses with other materials (Tanaka 2010). Therefore, in RSUTMS, the inferior growth observed in homozygotes for the transgenes for anthocyanin pigmentation will not be a significant issue.

Toward the application of a dominant visible marker trait in RSUTMS

A marker trait for RSUTMS needs to be applicable to many genetically diverse genotypes. In this study, application of the three genes introduction strategy conferred leaf sheath pigmentation in all seven genotypes tested (Supplemental Fig. 1). Since anthocyanin pigmentation using LSSP03 and the three genes introduction strategy can be applied for efficiently discriminating the male sterile individuals from fertile ones at the juvenile stage before transplanting to the field. However, anthocyanin pigmentation in the leaf sheath of some indica cultivars (Reddy ) would interfere with accurate detection. For such materials, it is necessary to reconsider the tissue specificity of the pigmentation.

By comparing the NGS data of 18 cultivars, we found only a few differences between 16 of them and the above 7 genotypes, with no mutations that could result in a loss of function. Although this result does not guarantee the function of all anthocyanin biosynthesis pathway–related genes, the upstream part of the pathway is also involved in the biosynthesis of important secondary metabolites, including lignin, catechins, and flavonoids, and a loss of function of these genes would cause serious growth defects (Huang ) or a clear phenotype such as the golden hull trait in rice (Hong ). Therefore, we believe that the three genes introduction strategy will be effective for widely diverse genotypes. Scrutinizing the sequences of the genes of founder materials by Sanger sequencing or NGS before starting the crossings for RSUTMS should provide practical reassurance.

The three gene introduction strategy described here can be further improved, for example by exchange improved promoter driven Myb from 35SP. Male sterility gene cassettes have been developed (Abe , Akasaka ), and the development of a positive or negative selectable marker trait is the last important step toward the effective application of RSUTMS. The three genes introduction strategy will be ideal for the introduction of a dominant visible marker trait that can be used as a positive or negative selectable marker. If efficient outcrossing could be realized with easy identification of the male-sterile individuals, even autogamous crops would be able to perform outcrossing-based breeding using genomic information, as in maize, and the application of this strategy to RSUTMS will help to overcome the limitations in rice breeding.