NARROW LEAF 1 Research Articles

The catalytic triad of rice NARROW LEAF1 involves H234.

NARROW LEAF1 (NAL1) exerts a multifaceted influence on leaf morphology and crop yield. Recent crystal study proposed that histidine 233 (H233) is part of the catalytic triad. Here we report that unlike suggested previously, H234 instead of H233 is a component of the catalytic triad alongside residues D291 and S385 in NAL1. Remarkably, residue 233 unexpectedly plays a pivotal role in regulating NAL1's proteolytic activity. These findings establish a strong foundation for utilizing NAL1 in breeding programs aimed at improving crop yield.

A novel Effective Panicle Number per Plant 4 haplotype enhances grain yield by coordinating panicle number and grain number in rice

Increasing effective panicle number per plant (EPN) is one approach to increase yield potential in rice. However, molecular mechanisms underlying EPN remain unclear. In this study, we integrated map-based cloning and genome-wide association analysis to identify the EPN4 gene, which is allelic to NARROW LEAF1 (NAL1). Overexpression lines containing the Teqing allele (TQ) of EPN4 had significantly increased EPN. NIL-EPN4TQ in japonica (geng) cultivar Lemont (LT) exhibited significantly improved EPN but decreased grain number and flag leaf size relative to LT. Haplotype analysis indicated that accessions with EPN4-1 had medium EPN, medium grain number, and medium grain weight, but had the highest grain yield among seven haplotypes, indicating that EPN4-1 is an elite haplotype of EPN4 for positive coordination of the three components of grain yield. Furthermore, accessions carrying the combination of EPN4-1 and haplotype GNP1-6 of GNP1 for grain number per panicle showed higher grain yield than those with other allele combinations. Therefore, pyramiding of EPN4-1 and GNP1-6 could be a preferred approach to obtain high yield potential in breeding.

PhNAL1 Is Involved in Regulating Branch Development of Petunia

The branch number of plants is an important agronomic trait that directly influences the ornamental characters and production costs of ornamental plants. Shoot branching has always been a hot topic for Petunia hybrida. During our research, we isolated the homologous gene of narrow-leaf 1 (NAL1), denoted as PhNAL1. The expression level of PhNAL1 was higher in leaves and axils than in roots, stems, and flowers. Pertinent to shoot apex removal and 6-benzyladenine treatments, both interventions demonstrated a suppressive effect on the expression of PhNAL1. Through subcellular localization analysis, we found that PhNAL1 predominantly localized in the nucleus. By using RNA interference targeting PhNAL1, we induced a noticeable increase in branch number while concurrently reducing plant height of petunia. These findings demonstrate that PhNAL1 is involved in regulating branch development within petunia. This study provides genetic resources for the subsequent cultivation of new cultivars of petunia endowed with distinct branching characteristics.

The gain-of-function mutation blf13 in the barley orthologue of the rice growth regulator NARROW LEAF1 is associated with increased leaf width.

Canopy architecture in cereals plays an important role in determining yield. Leaf width represents one key aspect of this canopy architecture. However, our understanding of leaf width control in cereals remains incomplete. Classical mutagenesis studies in barely identified multiple morphological mutants, including those with differing leaf widths. Of these, we characterized the broad leaf13 (blf13) mutant in detail. Mutant plants form wider leaves due to increased post-initiation growth and cell proliferation. The mutant phenotype perfectly co-segregated with a missense mutation in the HvHNT1 gene which affected a highly conserved region of the encoded protein, orthologous to the rice NARROW LEAF1 (NAL1) protein. Causality of this mutation for the blf13 phenotype is further supported by correlative transcriptomic analyses and protein-protein interaction studies showing that the mutant HvNHT1 protein interacts more strongly with a known interactor than wild-type HvHNT1. The mutant HvHNT1 protein also showed stronger homodimerization compared with wild-type HvHNT1, and homology modelling suggested an additional interaction site between HvHNT1 monomers due to the blf13 mutation. Thus, the blf13 mutation parallels known gain-of-function NAL1 alleles in rice that increase leaf width and grain yield, suggesting that the blf13 mutation may have a similar agronomic potential in barley.

Serine protease NAL1 exerts pleiotropic functions through degradation of TOPLESS-related corepressor in rice.

NARROW LEAF 1 (NAL1) is a breeding-valuable pleiotropic gene that affects multiple agronomic traits in rice, although the molecular mechanism is largely unclear. Here, we report that NAL1 is a serine protease and displays a novel hexameric structure consisting of two ATP-mediated doughnut-shaped trimeric complexes. Moreover, we identified TOPLESS-related corepressor OsTPR2 involved in multiple growth and development processes as the substrate of NAL1. We found that NAL1 degraded OsTPR2, thus modulating the expression of downstream genes related to hormone signalling pathways, eventually achieving its pleiotropic physiological function. An elite allele, NAL1A, which may have originated from wild rice, could increase grain yield. Furthermore, the NAL1 homologues in different crops have a similar pleiotropic function to NAL1. Our study uncovers a NAL1-OsTPR2 regulatory module and provides gene resources for the design of high-yield crops.

Dissecting the genetic basis of heterosis in elite super-hybrid rice.

Y900 is one of the top hybrid rice (Oryza sativa) varieties, with its yield exceeding 15 t·hm-2. To dissect the mechanism of heterosis, we sequenced the male parent line R900 and female parent line Y58S using long-read and Hi-C technology. High-quality reference genomes of 396.41 Mb and 398.24 Mb were obtained for R900 and Y58S, respectively. Genome-wide variations between the parents were systematically identified, including 1,367,758 single-nucleotide polymorphisms, 299,149 insertions/deletions, and 4,757 structural variations. The level of variation between Y58S and R900 was the lowest among the comparisons of Y58S with other rice genomes. More than 75% of genes exhibited variation between the two parents. Compared with other two-line hybrids sharing the same female parent, the portion of Geng/japonica (GJ)-type genetic components from different male parents increased with yield increasing in their corresponding hybrids. Transcriptome analysis revealed that the partial dominance effect was the main genetic effect that constituted the heterosis of Y900. In the hybrid, both alleles from the two parents were expressed, and their expression patterns were dynamically regulated in different tissues. The cis-regulation was dominant for young panicle tissues, while trans-regulation was more common in leaf tissues. Overdominance was surprisingly prevalent in stems and more likely regulated by the trans-regulation mechanism. Additionally, R900 contained many excellent GJ haplotypes, such as NARROW LEAF1, Oryza sativa SQUAMOSA PROMOTER BINDING PROTEIN-LIKE13, and Grain number, plant height, and heading date8, making it a good complement to Y58S. The fine-tuned mechanism of heterosis involves genome-wide variation, GJ introgression, key functional genes, and dynamic gene/allele expression and regulation pattern changes in different tissues and growth stages.

Large Vascular Bundle Phloem Area 4 enhances grain yield and quality in rice via source-sink-flow.

In rice (Oryza sativa L.), vascular bundle phloem tissue in the panicle neck is vital for the transport of photosynthetic products from leaf to panicle and is positively associated with grain yield. However, genetic regulation of the single large vascular bundle phloem area (LVPA) in rice panicle neck tissue remains poorly understood. In this study, we carried out genome-wide association analysis of LVPA in the panicle neck using 386 rice accessions and isolated and characterized the gene LVPA4, which is allelic to NARROW LEAF1 (NAL1). Phenotypic analyses were carried out on the near-isogenic line (NIL) NIL-LVPA4LT in the high-yielding indica (xian) cultivar Teqing and on overexpression lines transformed with a vector carrying the Lemont alleles of LVPA4. Both NIL-LVPA4LT and LVPA4 overexpression lines exhibited significantly increased LVPA, enlarged flag leaf size, and improved panicle type. NIL-LVPA4LT had a 7.6%-9.6% yield increase, mainly due to the significantly higher filled grain number per panicle, larger vascular system for transporting photoassimilates to spikelets, and more sufficient source supply that could service the increased sink capacity. Moreover, NIL-LVPA4LT had improved grain quality compared with Teqing, which was mainly attributed to substantial improvement in grain filling, especially for inferior spikelets in NIL-LVPA4LT. The single-nucleotide variation in the third exon of LVPA4 was associated with LVPA, spikelet number, and leaf size throughout sequencing analysis in 386 panels. The results demonstrate that LVPA4 has synergistic effects on source capacity, sink size, and flow transport and plays crucial roles in rice productivity and grain quality, thus revealing the value of LVPA4 in rice breeding programs for improved varieties.

The APC/CTAD1-WIDE LEAF 1-NARROW LEAF 1 pathway controls leaf width in rice.

Leaf morphology is one of the most important features of the ideal plant architecture. However, the genetic and molecular mechanisms controlling this feature in crops remain largely unknown. Here, we characterized the rice (Oryza sativa) wide leaf 1 (wl1) mutant, which has wider leaves than the wild-type due to more vascular bundles and greater distance between small vascular bundles. WL1 encodes a Cys-2/His-2-type zinc finger protein that interacts with Tillering and Dwarf 1 (TAD1), a co-activator of the anaphase-promoting complex/cyclosome (APC/C) (a multi-subunit E3 ligase). The APC/CTAD1 complex degrades WL1 via the ubiquitin-26S proteasome degradation pathway. Loss-of-function of TAD1 resulted in plants with narrow leaves due to reduced vascular bundle numbers and distance between the small vascular bundles. Interestingly, we found that WL1 negatively regulated the expression of a narrow leaf gene, NARROW LEAF 1 (NAL1), by recruiting the co-repressor TOPLESS-RELATED PROTEIN and directly binding to the NAL1 regulatory region to inhibit its expression by reducing the chromatin histone acetylation. Furthermore, biochemical and genetic analyses revealed that TAD1, WL1, and NAL1 operated in a common pathway to control the leaf width. Our study establishes an important framework for understanding the APC/CTAD1-WL1-NAL1 pathway-mediated control of leaf width in rice, and provides insights for improving crop plant architecture.

Partially functional NARROW LEAF1 balances leaf photosynthesis and plant architecture for greater rice yield.

NARROW LEAF1 (NAL1) is an elite gene in rice (Oryza sativa), given its close connection to leaf photosynthesis, hybrid vigor, and yield-related agronomic traits; however, the underlying mechanism by which this gene affects these traits remains elusive. In this study, we systematically measured leaf photosynthetic parameters, leaf anatomical parameters, architectural parameters, and agronomic traits in indica cultivar 9311, in 9311 with the native NAL1 replaced by the Nipponbare NAL1 (9311-NIL), and in 9311 with the NAL1 fully mutated (9311-nal1). Leaf length, width, and spikelet number gradually increased from lowest to highest in 9311-nal1, 9311, and 9311-NIL. In contrast, the leaf photosynthetic rate on a leaf area basis, leaf thickness, and panicle number gradually decreased from highest to lowest in 9311-nal1, 9311, and 9311-NIL. RNA-seq analysis showed that NAL1 negatively regulates the expression of photosynthesis-related genes; NAL1 also influenced expression of many genes related to phytohormone signaling, as also shown by different leaf contents of 3-Indoleacetic acid, jasmonic acid, Gibberellin A3, and isopentenyladenine among these genotypes. Furthermore, field experiments with different planting densities showed that 9311 had a larger biomass and yield advantage under low planting density compared to either 9311-NIL or 9311-nall. This study shows both direct and indirect effects of NAL1 on leaf photosynthesis; furthermore, we show that a partially functional NAL1 allele helps maintain a balanced leaf photosynthesis and plant architecture for increased biomass and grain yield in the field.

Genetic basis of vascular bundle variations in rice revealed by genome-wide association study

The vascular bundles play important roles in transportation of photoassimilate, and the number, size, and capacity of vascular bundles influence the transportation efficiency. Dissecting the genetic basis may help to make better use of naturally occurring vascular bundle variations. Here, we conducted a genome-wide association study (GWAS) of the vascular bundle variations in a worldwide collection of 529 Oryza sativa accessions. A total of 42 and 93 significant association loci were identified in the neck panicle and flag leaf, respectively. The introgression lines showing extreme values of the target traits harbored at least one GWAS signal, indicating the reliability of the GWAS loci. Based on the data of near-isogenic lines and transgenic plants, Grain number, plant height, and heading date7 (Ghd7) was identified as a major locus for the natural variation of vascular bundles in the neck panicle at the heading stage. In addition, Narrow leaf1 (NAL1) was found to influence the vascular bundles in both the neck panicle and flag leaf, and the effects of the major haplotypes of NAL1 were characterized. The loci or candidate genes identified would help to improve vascular bundle system in rice breeding.

Construction of a High-Density Genetic Map Based on SLAF Markers and QTL Analysis of Leaf Size in Rice.

Leaf shape is an important agronomic trait for constructing an ideal plant type in rice, and high-density genetic map is facilitative in improving accuracy and efficiency for quantitative trait loci (QTL) analysis of leaf trait. In this study, a high-density genetic map contained 10,760 specific length amplified fragment sequencing (SLAF) markers was established based on 149 recombinant inbred lines (RILs) derived from the cross between Rekuangeng (RKG) and Taizhong1 (TN1), which exhibited 1,613.59 cM map distance with an average interval of 0.17 cM. A total of 24 QTLs were detected and explained the phenotypic variance ranged from 9% to 33.8% related to the leaf morphology across two areas. Among them, one uncloned major QTL qTLLW1 (qTLL1 and qTLLW1) involved in regulating leaf length and leaf width with max 33.8% and 22.5% phenotypic variance respectively was located on chromosome 1, and another major locus qTLW4 affecting leaf width accounted for max 25.3% phenotypic variance was mapped on chromosome 4. Fine mapping and qRT-PCR expression analysis indicated that qTLW4 may be allelic to NAL1 (Narrow leaf 1) gene.

Narrow leaf 1 (NAL1) regulates leaf shape by affecting cell expansion in rice (Oryza sativa L.)

The narrow leaf1 (nal1) mutant of rice (Oryza sativa L.) exhibits a narrow leaf phenotype. Previous studies have shown that NAL1 modulates leaf size by affecting vein patterning and cell division; however, the underlying mechanism remains unclear. Here, we report that the nal1 mutant shows reduced size of the leaf abaxial epidermal cells and culm parenchyma cells compared with the wild type (WT), indicating that NAL1 also regulates cell expansion. To understand the molecular mechanism of the reduced cell size phenotype, leaves of 40-day-old nal1 mutant and WT seedlings were subjected to RNA-Seq analysis, which has identified 4277 differentially expressed genes (DEGs) between WT and the nal1 mutant. Gene ontology (GO) enrichment analysis revealed a large number of genes down-regulated in the nal1 mutant were involved in cell wall formation. Furthermore, the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that NAL1-regulated DEGs, such as ARFs and SAURs, were mapped in auxin signal transduction and auxin-regulated cell expansion pathways. A combination of RNA-Seq analysis and gene expression validation using RT-qPCR suggested that NAL1 is involved in the regulation of auxin-mediated acid growth in rice. These results indicate that, in addition to controlling cell division, NAL1 controls leaf width, at least partially, through its effect on cell expansion, probably via the acid growth mechanism.

Cas9 protein variant VQR recognizes NGAC protospacer adjacent motif in rice

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system is the third-generation genome editing tools that was developed and widely used in recent years. However, Streptococcus pyogenes Cas9 (SpCas9) in this system could only recognize NGG PAM (protospacer adjacent motif), which largely restricts the range of genome editing. The VQR (D1135V/R1335Q/T1337R) variant of SpCas9 could recognize NGAA, NGAG and NGAT PAMs in rice. However, whether VQR variant could recognize NGAC PAM remains unclear. In this study, three low editing efficiency sites of the VQR variant, NAL1-Q1, NAL1-Q2 and LPA1-Q, were selected for genome editing using the improved CRISPR/VQR system. The improved CRISPR/VQR system effectively edited these target sites, and the gene editing efficiency was 9.75%, 43.90% and 29.26% respectively. To ensure the recognition of NGAC PAM by the improved CRISPR/VQR system, two NGAC PAM containing sites (NAL-C and GL1-C) in the NARROW LEAF 1 (NAL1) for leaf length and GLOSSY1 (GL1) genes for wax biosynthesis were selected for genome editing in rice in this study, and 57 transgenic plants were obtained. The PCR amplification and sequencing results showed that 27 plants (47.36%) had mutation in the NAL1-C site, 44 plants (77.19%) had mutation in the GL1 gene, and 26 plants (45.61%) had mutation in the NAL-C and GL1-C sites. Further analysis revealed that there were four types of mutations caused by the CRISPR/VQR system, respectively for the hybrid mutation, biallelic mutation, chimeric mutation and homozygous mutations. Among them, heterozygous mutation and biallelic mutation were dominant changes. These results indicated that the improved CRISPR/VQR system could efficiently edit the NGAC PAM sites of the rice and produce abundant mutant types. This study provides a theoretical basis for NGAC PAM editing in rice and other related plants.

Short and narrow flag leaf1, a GATA zinc finger domain-containing protein, regulates flag leaf size in rice (Oryza sativa)

BackgroundThe flag leaf of rice (Oryza sativa L.) is an important determinant of plant type characteristics and grain yield. Identification of flag leaf mutants of rice is crucial to elucidate the molecular mechanism of flag-leaf development, and for exploitation of rice germplasm resources.ResultsIn this study, we describe a mutant designated short and narrow flag leaf 1 (snfl1). Histological analysis showed that the length of epidermal cells and number of longitudinal veins were decreased in the flag leaf of the snfl1 mutant. Map-based cloning indicated that a member of the GATA family of transcription factors is a candidate gene for SNFL1. A single-nucleotide transition at the last base in the single intron of snfl1 led to variation in alternative splicing and early termination of translation. Complemented transgenic plants harbouring the candidate SNFL1 gene rescued the snfl1 mutant. Analysis of RT-PCR and the SNFL1 promoter by means of a GUS fusion expression assay showed that abundance of SNFL1 transcripts was higher in the culm, leaf sheath, and root. Expression of the SNFL1-GFP fusion protein in rice protoplasts showed that SNFL1 was localized in nucleus.ConclusionsWe conclude that SNFL1 is an important regulator of leaf development, the identification of which might have important implications for future research on GATA transcription factors.

Variation in the regulatory region of FZP causes increases in secondary inflorescence branching and grain yield in rice domestication.

Inflorescence branching is a key agronomic trait determining rice yield. The primary branch of the ancestral wild rice (Oryza rufipogon Griff.) bears few grains, due to minimal secondary branching. By contrast, Oryza sativa cultivars have been selected to produce large panicles with more secondary branches. Here we showed that the CONTROL OF SECONDARY BRANCH 1 (COS1) gene, which is identical to FRIZZY PANICLE (FZP), plays an important role in the key transition from few secondary branches in wild rice to more secondary branches in domesticated rice cultivars. A 4-bp tandem repeat deletion approximately 2.7 kb upstream of FZP may affect the binding activities of auxin response factors to the FZP promoter, decrease the expression level of FZP and significantly enhance the number of secondary branches and grain yield in cultivated rice. Functional analyses showed that NARROW LEAF 1 (NAL1), a trypsin-like serine and cysteine protease, interacted with FZP and promoted its degradation. Consistently, downregulating FZP expression or upregulating NAL1 expression in the commercial cultivar Zhonghua 17 increased the number of secondary branches per panicle, grain number per panicle and grain yield per plant. Our findings not only provide insights into the molecular mechanism of increasing grain number and yield during rice domestication, but also offer favorable genes for improving the grain yield of rice.

Partial loss-of-function of NAL1 alters canopy photosynthesis by changing the contribution of upper and lower canopy leaves in rice

Little is known about the genetic basis of leaf and canopy photosynthesis. Here we aimed to detect novel quantitative trait loci (QTL) controlling photosynthesis by increasing leaf nitrogen content (LNC) per leaf area and analysed its effect on leaf and canopy photosynthesis. To identify QTL that increase photosynthetic rate in leaves, we screened chromosome segment substitution lines (CSSLs) of Oryza sativa ssp. japonica cultivar Koshihikari and O. sativa ssp. indica cultivar Nona Bokra using LNC per leaf area as the phenotype indicator. Locus leaf nitrogen content on chromosome four (qLNC4) is associated with increased LNC and photosynthetic rate per leaf area. Moreover, a non-synonymous amino acid substitution was identified in the NARROW LEAF 1 (NAL1) gene located in the qLNC4 region. This NAL1 allele increases LNC and photosynthetic rate per leaf area in flag leaves but does not increase whole-leaf photosynthesis. This NAL1 allele also increases light capture and whole-leaf nitrogen content of the lower leaves and is associated with slower senescence in flag leaves. These results suggest that this NAL1 allele does not increase whole-leaf photosynthesis but plays a role in regulating spatial and temporal trade-offs among traits at the whole-plant level.

Genetic analysis of rice mutants responsible for narrow leaf phenotype and reduced vein number.

Leaves are a major site for photosynthesis and a key determinant of plant architecture. Rice produces thin and slender leaves, which consist of the leaf blade and leaf sheath separated by the lamina joint. Two types of vasculature, the large and small vascular bundles, run in parallel, together with a strong structure, the midrib. In this paper, we examined the function of four genes that regulate the width of the leaf blade and the vein number: NARROW LEAF1 (NAL1), NAL2, NAL3 and NAL7. We backcrossed original mutants of these genes with the standard wild-type rice, Taichung 65. We then compared the effect of each mutation on similar genetic backgrounds and examined genetic interactions of these genes. The nal1 single mutation and the nal2 nal3 double mutation showed a severe effect on leaf width, resulting in very narrow leaves. Although vein number was also reduced in the nal1 and nal2 nal3 mutants, the small vein number was more strongly reduced than the large vein number. In contrast, the nal7 mutation showed a milder effect on leaf width and vein number, and both the large and small veins were similarly affected. Thus, the genes responsible for narrow leaf phenotype seem to play distinct roles. The nal7 mutation showed additive effects on both leaf width and vein number, when combined with the nal1 single or the nal2 nal3 double mutation. In addition, observations of inner tissues revealed that cell differentiation was partially compromised in the nal2 nal3 nal7 mutant, consistent with the severe reduction in leaf width in this triple mutant.

Natural Variation in the Flag Leaf Morphology of Rice Due to a Mutation of the NARROW LEAF 1 Gene in Oryza sativa L.

We investigated the natural variations in the flag leaf morphology of rice. We conducted a principal component analysis based on nine flag leaf morphology traits using 103 accessions from the National Institute of Agrobiological Sciences Core Collection. The first component explained 39% of total variance, and the variable with highest loading was the width of the flag leaf (WFL). A genome-wide association analysis of 102 diverse Japanese accessions revealed that marker RM6992 on chromosome 4 was highly associated with WFL. In analyses of progenies derived from a cross between Takanari and Akenohoshi, the most significant quantitative trait locus (QTL) for WFL was in a 10.3-kb region containing the NARROW LEAF 1 (NAL1) gene, located 0.4 Mb downstream of RM6992. Analyses of chromosomal segment substitution lines indicated that a mutation (G1509A single-nucleotide mutation, causing an R233H amino acid substitution in NAL1) was present at the QTL. This explained 13 and 20% of total variability in WFL and the distance between small vascular bundles, respectively. The mutation apparently occurred during rice domestication and spread into japonica, tropical japonica, and indica subgroups. Notably, one accession, Phulba, had a NAL1 allele encoding only the N-terminal, or one-fourth, of the wild-type peptide. Given that the Phulba allele and the histidine-type allele showed essentially the same phenotype, the histidine-type allele was regarded as malfunctional. The phenotypes of transgenic plants varied depending on the ratio of histidine-type alleles to arginine-type alleles, raising the possibility that H(233)-type products function differently from and compete with R(233)-type products.

Genetic factors determining varietal differences in characters affecting yield between two rice (Oryza sativa L.) varieties, Koshihikari and IR64

The yield of Koshihikari, a Japanese premium rice variety, is relatively lower than that of modern high yielding varieties. IR64 carries several well-known genes such as GS3, an important gene for grain size, sd-1, a semi-dwarf gene, and NARROW LEAF1 (NAL1), a gene for small, narrow flag leaves. In this study, we used two sets of chromosome segment substitution lines (CSSLs), from Koshihikari and IR64, and attempted to evaluate the genetic factors that cause differences between parents by analyzing the function of chromosome regions affecting a trait (CRATs). For 28 traits, we identified 312 CRATs in the Koshihikari background and 275 in the IR64 background. In these, donor alleles had positive effects in 84 and 103 CRATs, respectively. Among these, the CRAT related to GS3 and those for grain number expanded the potential sink size in Koshihikari, although this did not affect final yield. The combination of CRATs that enhances source ability may increase grain yield. Although the sd-1 gene might improve resistance to lodging, the yield of CSSLs with sd-1 decreased by 28.7 %. These results suggest that the smaller biomass conferred by sd-1 might reduce canopy photosynthesis. In the Koshihikari background, the CRAT related to NAL1 and those located on chr. 6 increased SPAD value but had the opposite effect on leaf size. Two CRATs that were detected on chr. 6 and 7 increased leaf area without any effect on the SPAD value. The combination of these CRATs for area and SPAD value might improve source ability.

Genetic Architecture of Natural Variation in Rice Chlorophyll Content Revealed by a Genome-Wide Association Study

Chlorophyll content is one of the most important physiological traits as it is closely related to leaf photosynthesis and crop yield potential. So far, few genes have been reported to be involved in natural variation of chlorophyll content in rice (Oryza sativa) and the extent of variations explored is very limited. We conducted a genome-wide association study (GWAS) using a diverse worldwide collection of 529 O. sativa accessions. A total of 46 significant association loci were identified. Three F2 mapping populations with parents selected from the association panel were tested for validation of GWAS signals. We clearly demonstrated that Grain number, plant height, and heading date7 (Ghd7) was a major locus for natural variation of chlorophyll content at the heading stage by combining evidence from near-isogenic lines and transgenic plants. The enhanced expression of Ghd7 decreased the chlorophyll content, mainly through down-regulating the expression of genes involved in the biosynthesis of chlorophyll and chloroplast. In addition, Narrow leaf1 (NAL1) corresponded to one significant association region repeatedly detected over two years. We revealed a high degree of polymorphism in the 5′ UTR and four non-synonymous SNPs in the coding region of NAL1, and observed diverse effects of the major haplotypes. The loci or candidate genes identified would help to fine-tune and optimize the antenna size of canopies in rice breeding.