Mapping the qPC1 locus to a 6.7-kb region

To identify the gene(s) underlying the qPC1 QTL for GPC, we used 190 recombinant inbred lines (RILs) derived from a cross between Zhenshan 97 (ZS97, Oryza sativa L. ssp. indica) and Nanyangzhan (NYZ, O. sativa L. ssp. japonica). qPC1 was mapped to the interval between markers RM472 and RM104 on the long arm of chromosome 1 (Fig. 1a). The allele from ZS97 contributed to increased GPC and total amount of essential amino acids22.

Figure 1: Map-based cloning of the qPC1 QTL and grain protein content of transgenic plants in T 1 . (a) Location of the qPC1 QTL on the genetic linkage map of chromosome 1. (b,c) Fine mapping of the qPC1 region using two mapping populations, with 6,000 and 4,008 plants, respectively. No. of Recs, number of recombinants between qPC1 and indicated molecular marker (b). Numbers in parentheses in c correspond to the numbers of recombinants in the b. (d) Genotypes and phenotypes of the recombinants. The phenotype of each recombinant was determined by progeny testing (Supplementary Table 2). (e) The OsAAP6 coding region of ZS97 was inserted into the vector pCAMBIA1301S under control of the CaMV 35S promoter to prepare the overexpression construct (OX). Arrows represent the direction of PCR primers. (f) The 1.8-kb promoter fragment and 5′-UTR of OsAAP6 from ZS97 with its own coding region was inserted into the vector pCAMBIA1301 to prepare the complementation construct (ZpZc). Arrows represent the direction of PCR primers. (g) The 580 bp cDNA fragment from the fourth exon of OsAAP6 was inserted into the dspCAMBIA1301 vector to generate the RNAi construct. Arrows represent the direction of PCR primers. (h) Grain protein contents of transgenic plants in T 1 . N, number of plants; (+) and (−) indicate transgene-positive and transgene-negative plants, respectively. P-values were produced by two-tailed t-test. Error bars, s.e.m. Full size image

To fine map the qPC1 locus, we backcrossed RIL-105 (one of the RILs containing the chromosome segment RM472–RM104 from NYZ and 78% of the genetic background of ZS97) three times (BC 3 ) to ZS97. BC 3 F 1 heterozygous plants were self-pollinated to develop two BC 3 F 2 populations consisting of 6,000 (population 1) and 4,008 (population 2) individuals, and to establish a pair of near-isogenic lines (NILs), NIL(ZS97) and NIL(NYZ). Analysis of a BC 3 F 2 population of 320 individuals derived by self-pollination of a BC 3 F 1 heterozygote showed that GPC in heterozygous plants was significantly lower than in ZS97 homozygotes but higher than in NYZ homozygotes (Supplementary Table 1), indicating that the ZS97 high-protein content allele of qPC1 was partially dominant.

Fifty-two recombinants between RM472 and RM104 were identified in population 1 and their genotypes at the qPC1 locus were deduced by progeny testing. Using these data, we mapped qPC1 to the interval between PB2 and PB7 (Fig. 1b). Seven recombinants between PB2 and PB7 were detected in population 2 (Fig. 1c). Using markers PB8–PB15, we identified six and two recombinant plants in the intervals PB11–PB12 and PB14–PB15 in the two populations, respectively (Fig. 1c,d). Each recombinant was further phenotyped by progeny testing to deduce the genotype of qPC1 (for example, see Supplementary Table 2). The qPC1 locus co-segregated with marker PB13, located between PB14 and PB15 (Fig. 1c,d).

To convert the genetic map location of qPC1 into a physical map location, we placed the molecular markers onto the physical map of Nipponbare, the reference cultivar for rice genomics. As shown in Supplementary Fig. 1, the PB10–PB15 region was in the same order on the physical map of Nipponbare and on the genetic map derived from the ZS97 × NYZ population (Fig. 1c). In the Nipponbare genome, the distance between PB14 and PB15 is 6.7 kb (Supplementary Fig. 1).

Analysis of the genomic DNA data revealed only one predicted gene (NCBI accession no. Loc_Os01g65670) in the 6.7 kb region and the gene has three predicted full-length complementary DNA (cDNA) sequence (AK121636, AK065286 and AK119529). The sequence of AK121636 includes the full-length cDNA sequence of AK065286 and AK119529, and all of them encode the same amino acid sequence. Thus, this gene (Loc_Os01g65670) was considered the candidate gene for qPC1 (Supplementary Fig. 1). Alignment of the full-length cDNA sequence of AK121636 with the genomic sequence of Nipponbare indicated that qPC1 encodes a putative amino acid transporter belonging to the APC superfamily and has a PF01490 consensus domain ( http://pfam.janelia.org/). Phylogenetic analysis of the qPC1 protein revealed that it is highly homologous to the amino acid permeases (AAPs) family (Supplementary Fig. 2). Interestingly, qPC1 corresponds to OsAAP6 (Loc_Os01g65670), which is highly expressed in seeds and belonging to OsAAT family in rice23.

Confirmation of the qPC1 QTL

Three transformation constructs were prepared to confirm the candidate gene (OsAAP6 (ref. 23)) for the qPC1 QTL. First, a construct for overexpression (OX) contained the coding region of OsAAP6 from ZS97 (high GPC) driven by the CaMV 35S promoter inside the vector pCAMBIA1301 (Fig. 1e); second, a complementation construct (ZpZc) with the promoter and coding regions of OsAAP6 from ZS97 inserted into pCAMBIA1301S (Fig. 1f); and third, an RNAi construct containing a 580-bp PCR fragment from the fourth exon of OsAAP6 inserted into dspCAMBIA1301 in both the sense and antisense orientations (Fig. 1g). Agrobacterium-mediated transformation was used to introduce OX into NYZ (OX(NYZ)) and Zhonghua 11 (ZH11, OX(ZH11)), ZpZc into NYZ (ZpZc(NYZ)), and RNAi into ZS97 (RNAi(ZS97)) and ZH11 (RNAi(ZH11)).

All transgene-positive individuals of OX(NYZ), OX(ZH11) and ZpZc(NYZ) in the T 0 generation had a higher GPC than transgene-negative individuals (Supplementary Fig. 3). Co-segregation analysis between the genotypes and phenotypes in T 1 progenies confirmed these effects: all transgene-positive plants of OX(NYZ), OX(ZH11) and ZpZc(NYZ) had higher GPC than the untransformed controls (Fig. 1h), whereas lower GPC was observed for RNAi(ZS97) and RNAi(ZH11) in both T 0 (Supplementary Fig. 3) and the T 1 (Fig. 1h). Co-segregation analysis of genotypes and expression levels with phenotypes in T 2 progenies confirmed that GPC was significantly (P<0.01) and positively correlated with expression level of OsAAP6 in all three kinds of transformants, OX(NYZ), ZpZc(NYZ) and RNAi(ZS97) (Supplementary Table 3). Transgenic plants showing higher levels of OsAAP6 expression produced larger amounts of grain storage protein (Supplementary Table 3 and Table 1). Thus, our results confirm that the candidate gene (OsAAP6) is the qPC1 QTL.

Table 1 Grain quality traits in NILs and transgenic plants in a 2-year field trial. Full size table

Expression pattern of OsAAP6 and subcellular localization

Sixteen tissues from the NILs were assayed for temporal and spatial expression patterns of OsAAP6 by quantitative reverse transcription–PCR (Fig. 2a). OsAAP6 transcripts were detected in all examined tissues and were most abundant in the endosperms at 5–12 days after flowering (DAF) (Fig. 2a), the same timeframe as reported for transcripts of OsAAP6 (ref. 23), and there were significantly higher transcript levels in the endosperms of NIL(ZS97) than in endosperm of NIL(NYZ) (Fig. 2a).

Figure 2: Expression patterns of OsAAP6 and subcellular localization analysis. (a) The comparative expression pattern of OsAAP6 was determined by quantitative reverse transcription–PCR. RO, ST and FL: root, stem and flag leaf at the heading stage; YL, young leaf; 2YP, 4YP, 6YP, 8YP and 10YP: young panicles 2, 4, 6, 8 and 10 cm in length; H4H and H0H: hulls at 4 and 0 days before heading; H2A, hull at 2 DAF; 5E, 7E, 10E and 12E: endosperms at 5, 7, 10 and 12 DAF. All data are based on three biological replications. Error bars, s.e.m. (b–m) Representative histochemical analysis of tissue expression of GUS transgene under control of the OsAAP6 promoter from ZS97. (b) Endosperm 10 DAF. (c) Seed 10 DAF. (d) Seed 25 DAF. (e) Root. (f) Node. (g) Internode. (h) Hull at 2 days before flowering. (i) Pulvinus. (j,k) Hull cross sections. (l,m) Root cross sections. VT, vascular tissue; OVE, ovular vascular trace ends; OV, ovular vascular region; SV, lateral stylar vascular traces. Scale bars, 1 mm (j), 50 μm (k–m). All transgenic plants were from the T 1 generation, and at least ten independent transformants were subjected to histochemical GUS assays. (n,o) Subcellular localization analysis of OsAAP6. (n) OsAAP6-GFP does not co-localize with SCAMP1-RFP, which is located in the trans-Golgi network and plasma membrane, as a control. (o) Co-localization of OsAAP6-GFP and BiP-RFP, which is located in the ER. Merged images of YFP and bright-field images are also shown. Scale bar, 10 μm. Full size image

We next examined the expression of the β-glucuronidase (GUS) gene under control of the OsAAP6 promoter from ZS97 in transgenic plants. All tissues exhibited GUS activity and the GUS signal was particularly strong in vascular tissues of the hull, root, pulvinus, internode, node, seed and endosperm at 10 and 25 DAF (Fig. 2b–m). GUS activity in the endosperm was more abundant in the ovular vascular trace, ovular vascular trace end and lateral stylar vascular traces than in other positions during grain filling (Fig. 2b). Histochemical GUS studies showed that OsAAP6 was expressed mainly in the root rhizodermis and phloem (Fig. 2l,m). This feature of OsAAP6 closely resembles Arabidopsis AAP family genes and LHT1, which are preferentially expressed in vascular tissues and the root rhizodermiss24,25,26,27.

To investigate the subcellular localization of OsAAP6 protein, we transiently co-expressed OsAAP6 fused to green fluorescent protein (GFP) with various subcellular markers in rice protoplasts28,29,30,31. Co-expression of OsAAP6-GFP and SCAMP1-RFP clearly showed that OsAAP6 was not localized at the plasma membrane or on the trans-Golgi network (Fig. 2n). However, OsAAP6-GFP and the endoplasmic reticulum (ER) marker BiP-RFP overlapped completely when co-expressed in rice protoplasts (Fig. 2o). Therefore, OsAAP6 is localized on the ER, but not on the plasma membrane or in the trans-Golgi network.

Genetic variation in the regulatory region of OsAAP6

To identify the sequence variation between the ZS97 and NYZ alleles of OsAAP6, we compared genomic clones from both parents (Fig. 3a). There were eight nucleotide differences between the two varieties in the open reading frame: seven polymorphisms in the first intron and one synonymous mutation (at 3,813 bp) in the fourth exon (Fig. 3a). In addition, 15 polymorphisms between ZS97 and NYZ were revealed in the 5′-untranslated region (5′-UTR) and promoter region (~1.8 kb). These polymorphisms included a 6-bp indel and 14 upstream single-nucleotide polymorphisms (Fig. 3a). Several types of putative regulatory elements were identified within the polymorphic regions of the OsAAP6 promoter and 5′-UTR, which harbours several cis-regulatory elements involved in transcriptional responses, such as a copper-response element, inr element, ARR1-binding element and other cis-elements (Supplementary Table 4).

Figure 3: Natural variation of OsAAP6 in a 197 accession rice mini-core collection and GUS activities in transgenic plants driven by eight promoter fragments and 5′-UTRs. (a) OsAAP6 gene structure and natural variation between alleles from ZS97 and NYZ. (b) Natural variation of OsAAP6 in 197 rice accessions of a mini-core collection compared with the NILs. (c) Cladogram of eight haplotypes. (d) Protein contents of brown rice in sub-populations A and B; raw data are provided in Supplementary Table 5; n, is the number of accessions. P-values were generated by two-tailed t-tests. Error bars, s.e.m. (e) OsAAP6 transcript levels in the endosperms of Sub1 cultivars with class A and B at 5 DAF; the number of accessions analysed is shown below each bar. The P-value was generated by a two-tailed t-test. Error bars, s.e.m. (f) Diagrams for the four deletions of the OsAAP6 promoter and 5′-UTR fused to the GUS gene. (g) Quantitative analysis of GUS activity in transgenic plants. Y and G indicate young panicles at 2 days before flowering and grains at 5 DAF, respectively. Data were from the transgenic lines planted in a randomized complete block design with three replications. P-values were produced by the Duncan test. Error bars denote s.e.m. Full size image

We sequenced the OsAAP6 5′-UTRs and promoter regions (~1.8 kb), and the coding regions containing the synonymous mutation site, and measured GPC in 197 accessions of the rice mini-core collection originating from a wide geographic range across Asia (Supplementary Table 5). The collection comprised two subpopulations (Sub1 and Sub2) identified as previous population structure analyses32,33. Based on the nucleotide polymorphisms identified between the two parents (Fig. 3a), the sequences of the cultivated varieties were divided into eight haplotypes (Fig. 3b), placed into two groups (A and B) by phylogenetic analysis of the sequences. Five haplotypes were present in Group A (named types 1–5) and three in Group B (types 6–8) (Fig. 3c and Supplementary Table 5). Sub1 with 94 accessions in Group A and 14 accessions in Group B comprised mainly indica cultivars, and Sub2 with 31 accessions in Group A and 58 accessions in Group B were mainly japonica (Fig. 3b,c). Cultivars in Sub1 carrying Group A haplotypes tended to show higher OsAAP6 expression levels in endosperms at 5 DAF and a higher GPC than those having the class B haplotypes (Fig. 3d,e). We also assayed the transcript abundance of OsAAP6 in endosperms at 5 DAF (Fig. 3e) and calculated the correlation between OsAAP6 expression levels and GPC in the indica group. The highly significant correlation (0.65, P<0.01) strongly suggests that expression levels for this gene might be the cause of natural variation in indica GPC. Furthermore, three common nucleotide polymorphisms upstream of the translation start site were identified between Groups A and B (Fig. 3b), and two of the common nucleotide changes (−7 to −12 bp, −32 bp) in the 5′-UTR were in three potential cis-regulatory elements (copper-responsive element, inr element and sulphur-responsive element) (Supplementary Table 4), which are targets for transcriptional activators and a regulator containing an SBP domain, and are involved in a broad range of responses34,35,36,37,38,39,40. Consequently, our results imply that the two common variations in the three potential cis-elements of the OsAAP6 5′-UTR seem to be associated with GPC diversity in the Sub1 population (mainly indica cultivars).

A series of 5′-end deletions of the OsAAP6 promoter from ZS97 and NYZ were fused to the GUS gene (Fig. 3f), and their ability to drive the reporter gene was assessed relative to the full-length ZS97 promoter in transgenic plants. Compared with fragments of −377, −698, −1,226 and −1,814 bp from NYZ, quantitative analysis revealed that the GUS expression levels from ZS97 were significantly increased in young panicles 2 days before flowering and in grains at 5 DAF (Fig. 3g). However, compared with fragments of −698 bp in grains at 5 DAF, the GUS expression levels from the fragments of −377 bp from ZS97 and NYZ were markedly decreased (P<0.01), respectively (Fig. 3g). Thus, the minimal region (−698 to +1) of OsAAP6 may play an important role in regulating the OsAAP6 expression difference between the alleles from ZS97 and NYZ.

Effects of OsAAP6 on grain nutritional quality

To quantify the effects of OsAAP6 on grain quality, we first assayed the quality traits of the NILs and transgenic plants of OX(NYZ), OX(ZH11), ZpZc(NYZ), RNAi(ZS97) and RNAi(ZH11) using data from field trials (Table 1). NIL(ZS97) and transgene-positive plants of OX(NYZ), OX(ZH11) and ZpZc(NYZ) had substantially increased GPC (grain storage proteins, including glutelins, prolamins, globulins and albumins) and amylose contents, coupled with markedly reduced starch content and gel consistency, whereas the reverse was true for RNAi(ZS97) and RNAi(ZH11) (Table 1). There were no significant differences between the NILs and transgenics in other agronomic traits (Supplementary Fig. 4 and Supplementary Table 6).

The total amino acid contents of grains were also determined for the NILs and the transgenic plants. Compared with the corresponding levels in NIL(NYZ) and transgene-negative OX(NYZ) plants, the levels of alanine, leucine, valine, proline, arginine, acidic amino acids and total content of amino acids were significantly increased in NIL(ZS97) and transgene-positive plants, whereas the reverse was true for RNAi (ZH97) (Supplementary Fig. 5). These results strongly suggest that OsAAP6 enhances GPC by increasing grain storage protein (glutelins, prolamins, globulins and albumins) content and the total amount of amino acids, thus improving nutritional quality.

Changes in amino acids of root uptake and distribution

As OsAAP6 was preferentially expressed in the vascular tissues and rhizodermis of roots, we examined the effects of OsAAP6 on root uptake and distribution of various free amino acids. First, roots were submerged in a solution containing a mixture of 20 amino acids, and the depletion of each amino acid in this solution was measured 6 h after incubation. The study was not designed to establish actual rates of amino acid absorption by the plants, but rather to compare the NILs and transgenic plants with respect to this process. NIL(ZS97) and transgene-positive plants of OX(NYZ) had significantly increased threonine, serine, glycine, alanine, proline, acidic amino acids and total amino acids content, coupled with significantly reduced methionine, whereas the opposite was true for RNAi(ZS97) (Fig. 4a–c). The uptake data suggest that OsAAP6 greatly enhanced root absorption of a range of amino acids and displayed substantially higher uptake rates of threonine, serine, glycine, alanine, proline and acidic amino acids.

Figure 4: Amino acid uptake and sap flow assays. (a–c) The effect of OsAAP6 on amino acid uptake by rice roots from NILs (a), RNAi (b) and OX(NYZ) (c). Plants were incubated in solutions containing a mixture of 20 amino acids, each at a concentration of 50 mM; rates of depletion of amino acids from this solution were determined. Root measurements were calculated using fresh weights. (d–f) Amino acid analysis of sap flow of stems from NILs (d), RNAi (e) and OX(NYZ) (f). Aliquots of 20 μl of stem sap were collected and assayed. Insert indicates the total content of amino acids. (+) and (–) indicate transgene-positive and negative T 2 plants, respectively. Significant differences at *P=0.05 and **P=0.01, respectively. All data are based on three biological replications and significant differences are based on two-tailed t-tests. Error bars, s.e.m. Glutamine (Gln) and asparagine (Asn) were hydrolysed to glutamate (Glu) and aspartate (Asp) under acidic conditions; thus, the final content of Glu was exactly the sum of the Gln and Glu contents and the final content of Asp was exactly the sum of the Asn and Asp contents. Full size image

Uptake (or synthesis) of amino acids mainly takes place in mature roots and leaves; they are then exported via the stems to supply the flowers and grains. Sap flow in stems was analysed to determine whether OsAAP6 might play a role in this process. Serine, glycine, alanine, methionine, leucine, glutamate (or glutamine) and total amino acids levels were increased in NIL(ZS97) and transgene-positive plants of OX(NYZ), whereas lower levels were observed for RNAi(ZS97) (Fig. 4d–f).

Free amino acids in flag leaves and hulls were investigated to further determine whether OsAAP6 had any effect on the distribution of free amino acids. NIL(ZS97) and transgene-positive plants of OX(NYZ) showed substantial decreases in serine, alanine, valine, methionine, tyrosine, acidic amino acids and total content of free amino acids in flag leaves (at 10 DAF), whereas the opposite was true for the RNAi(ZS97) (Supplementary Fig. 6a–c). However, in hulls many free amino acids and total content of free amino acids were significantly increased in NIL(ZS97) and transgene-positive plants of OX(NYZ) (at 10 DAF), whereas the reverse was true for the RNAi(ZS97) (Supplementary Fig. 6d–f). Our results therefore suggest that OsAAP6 has effects on the distribution of various amino acids, at least in stems, flag leaves and hulls.

Pleiotropic effects of OsAAP6 on gene expression

To determine whether changes in the accumulation of grain storage proteins and starch were reflected by altered messenger RNA levels, we examined the expression of key genes involved in grain storage materials. The mRNA transcript levels of 41 genes, including 20 involved in grain protein biosynthesis (Fig. 5a, I), 17 related to starch metabolism (12 involved in amylose biosynthesis (Fig. 5a, II) and 5 related to starch degradation (Fig. 5a, III)) in endosperms at 10 DAF were considerably upregulated in OX(NYZ) and NIL(ZS97), in contrast to plants not carrying the transgene and NIL(NYZ). The reverse was true for 37 of the 41 genes in RNAi(ZS97) (Fig. 5a). Furthermore, the transcript levels of three genes involved in amylopectin biosynthesis were markedly downregulated in NIL(ZS97) and OX(NYZ) transgene-positive plants, relative to control plants, and the reverse occurred for these three genes in RNAi(ZS97) (Fig. 5a, IV). These results strongly suggest that OsAAP6 has significant effects on the expression of a large portion of the genes participating in starch and storage protein biosynthesis in developing rice grains.

Figure 5: Expression levels of genes involved in production of storage starch and proteins and transmission electron microscopy. (a) Expression levels of key genes involved on synthesis and storage of endosperm components in transgenic plants (OX(NYZ) and RNAi(ZS97)) in T 2 and NILs. Transgene-positive plants of OsAAP6 and NIL (ZS97) are shown relative to transgene-negative plants and NIL (NYZ), respectively, set as 1. Data are based on three biological replications. All P-values, produced by two-tailed t-tests, were <0.01. Error bar shows s.d. The annotated names of the genes are shown in Supplementary Table 7. (b–m) Ultrastructures of cells in developing endosperm of NILs and transgenic plants at 10 DAF. PBI and PBII, protein bodies I and II; SG, starch granule; ER, endoplasmic reticulum; ECS, extracellular space; CW, cell wall. Scale bars, 2 μm. Full size image

To test whether changes at the transcriptional level affected enzyme behaviour, the activities of five key enzymes involved in starch metabolism were measured. At 10 DAF, NIL(ZS97) and transgene-positive plants of OX(NYZ) had increased activities of ADP-glucose pyrophosphorylase (ADPG-Pase), granule-bound starch synthase (GBSS), soluble starch synthase (SSS) and amylase, coupled with reduced starch branching enzyme (SBE) activity in developing endosperms, whereas the opposite was true for RNAi(ZS97) (Supplementary Fig. 7a–o). These results are consistent with the expression of key genes involved in starch metabolism (Fig. 5a) and the contents of amylose and starch (Table 1).

OsAAP6 enlarges protein bodies

As OsAAP6 greatly enhanced GPC in rice, transmission electron microscopy was employed to determine any effects on protein body (PB) formation. In developing endosperms (at 10 DAF), two types of PBs were readily discernible in ultra-thin sections; prolamin-containing PBI consistently shaped and surrounded by ER, and irregularly shaped glutelin/globulin-containing PBII showing uniform staining (Fig. 5b–m). In the developing endosperms of NIL(ZS97) and OX(NYZ), the mean section areas of PBI and PBII were all expanded, whereas the opposite was true for RNAi(ZS97) (Fig. 5b–g and Supplementary Fig. 8). Interestingly, the ER cisternal space was often dilated in transgene-positive plants of OX(NYZ) and NIL(ZS97), as well as transgene-negative plants of RNAi(ZS97) (Fig. 5h–m, black arrowheads). These results indicate that OsAAP6 has probable effects on the formation of PBs through enlargement.