Waxy Gene Research Articles

Adjusting the amylose content of semi-glutinous japonica rice by genome editing of uORF6 in the Wx gene

Amylose content, the key determinant of rice eating and cooking quality, is regulated primarily by the Waxy (Wx) gene. We adjusted the amylose content and transparency of semi-glutinous japonica rice carrying the Wxmp allele by genome editing of upstream open reading frame 6 (uORF6) of Wx.

In-silico characterization and expression analysis of the Waxy gene in rice (Oryza sativa L.).

In-silico characterization and expression analysis of the Waxy gene in rice (Oryza sativa L.).

Deciphering the Role of Waxy Gene Mutations in Enhancing Rice Grain Quality.

Amylose content (AC) stands as a pivotal determinant of rice grain quality, primarily governed by the Waxy gene (Wx). The allelic variation within this gene, particularly the presence of the Wxmp allele derived from the ancestral Wxmq allele, significantly influences AC and is prevalent among soft japonica rice varieties in southern China. Although both alleles are associated with lower AC, there remains a paucity of detailed understanding regarding the interplay between specific functional single nucleotide polymorphisms (SNPs) within these alleles and the overarching rice grain quality. To investigate this, we engineered three distinct transgenic rice lines, each harboring the Wxmp, Wxmq, or Wxb-5c alleles in the background of the glutinous rice cultivar Nip(wx). This suite of transgenic rice lines showcased varying degrees of grain transparency inversely correlated to AC, which in turn influenced other physicochemical properties of the rice grains, such as taste value of cooked rice, gel consistency, and starch pasting properties. Additionally, analyses of gene expression and enzyme activity revealed that the functional SNPs, Ex4-53G to A and Ex5-53T to C, lead to a decline in the activity of granule-bound starch synthase I (GBSSI) without altering expression levels.

Creating a zero amylose barley with high soluble sugar content by genome editing.

Amylose biosynthesis is strictly associated with granule-bound starch synthase I (GBSSI) encoded by the Waxy gene. Mutagenesis of single bases in the Waxy gene, which induced by CRISPR/Cas9 genome editing, caused absence of intact GBSSI protein in grain of the edited line. The amylose and amylopectin contents of waxy mutants were zero and 31.73%, while those in the wild type were 33.50% and 39.00%, respectively. The absence of GBSSI protein led to increase in soluble sugar content to 37.30% compared with only 10.0% in the wild type. Sucrose and β-glucan, were 39.16% and 35.40% higher in waxy mutants than in the wild type, respectively. Transcriptome analysis identified differences between the wild type and waxy mutants that could partly explain the reduction in amylose and amylopectin contents and the increase in soluble sugar, sucrose and β-glucan contents. This waxy flour, which showed lower final viscosity and setback, and higher breakdown, could provide more option for food processing.

Population Structure and Genetic Diversity of Shanlan Landrace Rice for GWAS of Cooking and Eating Quality Traits.

The Shanlan landrace rice in Hainan Province, China, is a unique upland rice germplasm that holds significant value as a genetic resource for rice breeding. However, its genetic diversity and its usefulness in rice breeding have not been fully explored. In this study, a total of eighty-four Shanlan rice, three typical japonica rice cultivars, and three typical indica rice cultivars were subjected to resequencing of their genomes. As a result, 11.2 million high-quality single nucleotide polymorphisms (SNPs) and 1.6 million insertion/deletions (InDels) were detected. Population structure analysis showed all the rice accessions could be divided into three main groups, i.e., Geng/japonica 1 (GJ1), GJ2, and Xian/indica (XI). However, the GJ1 group only had seven accessions including three typical japonica cultivars, indicating that most Shanlan landrace rice are different from the modern japonica rice. Principal component analysis (PCA) showed that the first three principal components explained 60.7% of the genetic variation. Wide genetic diversity in starch physicochemical parameters, such as apparent amylose content (AAC), pasting viscosity, texture properties, thermal properties, and retrogradation representing the cooking and eating quality was also revealed among all accessions. The genome-wide association study (GWAS) for these traits was conducted and identified 32 marker trait associations in the entire population. Notably, the well-known gene Waxy (Wx) was identified for AAC, breakdown viscosity, and gumminess of the gel texture, and SSIIa was identified for percentage of retrogradation and peak gelatinization temperature. Upon further analysis of nucleotide diversity in Wx, six different alleles, wx, Wxa, Wxb, Wxin, Wxla/mw, and Wxlv in Shanlan landrace rice were identified, indicating rich gene resources in Shanlan rice for quality rice breeding. These findings are expected to contribute to the development of new rice with premium quality.

Crop Evolution of Foxtail Millet.

Studies on the domestication, genetic differentiation, and crop evolution of foxtail millet are reviewed in this paper. Several genetic studies were carried out to elucidate the genetic relationships among foxtail millet accessions originating mainly from Eurasia based on intraspecific hybrid pollen semi-sterility, isozymes, DNA markers, and single-nucleotide polymorphisms. Most studies suggest that China is the center of diversity of foxtail millet, and landraces were categorized into geographical groups. These results indicate that this millet was domesticated in China and spread over Eurasia, but independent origin in other regions cannot be ruled out. Furthermore, the evolution of genes was reviewed (i.e., the Waxy gene conferring amylose content in the endosperm, the Si7PPO gene controlling polyphenol oxidase, the HD1 and SiPRR37 genes controlling heading time, the Sh1 and SvLes1 genes involved in grain shattering, and the C gene controlling leaf sheath pigmentation), and the variation and distribution of these genes suggested complex patterns of evolution under human and/or natural selection.

Mapping of the Waxy Gene in Brassica napus L. via Bulked Segregant Analysis (BSA) and Whole-Genome Resequencing

Plant cuticular wax is the covering of the outer layer of the plant. It forms a protective barrier on the epidermis of plants and plays a vital role like a safeguard from abiotic and biotic stresses. In the present study, Brassica napus L. materials with and without wax powder were observed. Genetic analysis showed that the separation ratio of waxy plants to waxless plants was 15:1 in the F2 population, which indicated that the wax powder formation was controlled by two pairs of genes. In order to identify the candidate genes associated with the wax powder trait of B. napus L., bulked segregant analysis (BSA) was performed. The homozygous waxy plants, the homozygous waxless plants, and plants from three parents were selected for establishing five DNA pools for genome-wide resequencing. The results of the resequencing showed that the site associated with wax powder trait was located in the region of 590,663–1,657,546 bp on chromosome A08. And 48 single nucleotide polymorphisms (SNPs) were found between the DNA sequences of waxy plants and waxless plants in this region. These SNPs were distributed across 16 gene loci. qRT-PCR analysis was conducted for the 16 candidate genes and three genes (BnaA08g01070D, BnaA08g02130D, and BnaA08g00890D) showed significantly differential expression between waxy and waxless parents. BnaA08g01070D and BnaA08g02130D were significantly down-regulated in the waxless parent, while BnaA08g00890D was significantly up-regulated in the waxless parent. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed that the BnaA08g02130D gene was enriched in lipid biosynthetic or metabolic processes. All the results in our study would provide valuable clues for exploring the genes involved in wax powder development.

Segregation of molecular markers associated with Bph3 gene in BC5F2 population derived from Ciherang rice variety as the recipient parent

Brown planthopper (BPH), an important insect pest of rice, can cause severe damage and significant yield loss. An efficient and environmentally friendly method to control this pest is by planting resistant varieties. The brown planthopper resistance gene Bph3 from Rathu Heenati variety is closely linked to the Waxy gene that regulates amylose synthesis, the primary determinant of rice eating and cooking quality. The purposes of this study were to analyze the segregation ratio of SSR markers associated with Bph3 gene and to identify individual plants carrying homozygous allele for Bph3 gene in a BC5F2 population derived from a backcross that used Ciherang as the recurrent recipient parent. Analyses using RM589, RM586, and RM588 markers linked to Bph3 gene and RM190 marker linked to the Waxy gene indicated that the ratio of genotype segregation for each marker related to Bph3 gene did not deviate from the expected ratio with the χ 2 value was 0.956, 0.587, and 1.467 for RM589, RM586, and RM588, respectively (χ2 table = 5.991, α=0,05). All individual plants had a homozygous allele from Ciherang for Waxy gene based on the RM190 marker. As many as 25 individual plants which have homozygous Bph3 allele from Rathu Heenati and homozygous Waxy allele from Ciherang can be further developed as promising BPH-resistant rice varieties with eating and cooking quality similar to Ciherang.

Structure characteristics of mutation sites in two waxy alleles from Yunnan waxy maize (Zea mays L. var. certaina Kulesh) landraces.

A large number of waxy maize landraces are distributed in Yunnan and surrounding areas, and abundant waxy alleles of different types are distributed in these landraces. The identification of waxy alleles is helpful to the protection and utilization of these waxy landraces. This study introduced structure characteristics of waxy genes from two specific landraces of Yunnan, Zinuoyumi and Myanmar Four-Row Wax. Zinuoyumi has two waxy alleles wx-Cin4 and wx-Cin4-2; Myanmar Four-Row Wax has three waxy alleles wx-D10, wx-Reina and wx-D11. The wx-Cin4-2 and wx-D11 are two types of waxy alleles first reported in this study. The wx-Cin4-2 has two mutation sites, deletion of 30 bp in exon 10, insertion of a 1,267 bp non-long terminal repeat (non-LTR) retrotransposon Cin4 in intron 10, and 13 bp extra sequence were found at 5' end of the Cin4; the mutation site of wx-D11 is a 1,082 bp deletion from exons 11 to 14 of the waxy gene and is replaced with a 72 bp filler sequence. This study enriched the type of waxy allele from Yunnan waxy maize landraces and further discussed the molecular basis for the formation of mutation sites of wx-Cin4-2 and wx-D11.

Relationships between starch molecular components and eating and cooking qualities of rice using single-segment substitution lines with different Wx loci

Waxy (Wx) gene in rice endosperm synthesizes amylose and amylopectin extra-long chain (ELC). Five rice lines with different Wx locus single-segment substitutions under the same genetic background were investigated for relationships of their starch molecular components and eating and cooking qualities (ECQs) including pasting, thermal, textural and sensory properties. Rice lines with different Wx loci exhibited different ECQs, and their starches had different apparent amylose contents (AACs), true amylose contents (TACs), ELC contents (ELCCs), and amylopectin chain length distributions. The AAC was correlated positively to hardness and cohesiveness of cooked rice, and negatively to adhesiveness and palatability of cooked rice and breakdown viscosity of rice flour. The TAC was correlated positively to gelatinization temperature range and negatively to gelatinization temperature and enthalpy. ELCC showed a positive relationship with pasting peak time of rice flour and chewiness of cooked rice, and could potentially be used as an important reference for rice ECQ evaluation. This study could help the applications of Wx loci in rice quality breeding.

A glossy mutant in onion (Allium cepa L.) shows decreased expression of wax biosynthesis genes.

Cuticular wax is a characteristic feature of land plants that provides protection against both biotic and abiotic stresses. In this study, a glossy mutant lacking an epicuticular wax layer was identified in the γ-irradiated M2 mutant population of the onion cultivar Bhima Super. The inheritance of the mutant's glossy phenotype was determined to be recessive and single locus. Scanning electron microscopy analysis showed poor accumulation of wax crystals in the glossy mutant, concentrated near the stomata. The plant height, number of leaves per plant, and stomatal parameters of the mutant were similar to the wild-type. RNA-seq was used to comprehend the expression variations of waxy cuticle-related genes in the glossy mutant and its wild-type waxy cultivars. Differential gene expression analysis of the RNA-seq data revealed that the genes involved in wax biosynthesis, such as AcCER1, AcCER26, AcMAH1, and AcWSD1, were downregulated by 2.72, 1.74, 2.59 and 2.12-fold, respectively, in the glossy mutant respectively. The expression patterns of these four unigenes were validated using semi-quantitative RT-PCR. The glossy mutant displayed a substantial 3.5-fold reduction in cuticular wax load compared to the wild-type due to the significant downregulation of these wax biosynthesis genes. These findings represent early advancements in understanding the molecular mechanisms of wax biosynthesis in onions. Furthermore, they provide a foundation for utilizing the glossy mutant trait in breeding programmes to enhance stress and pest resilience.

Molecular insights on the origin and development of waxy genotypes in major crop plants.

Starch is a significant ingredient of the seed endosperm with commercial importance in food and industry. Crop varieties with glutinous (waxy) grain characteristics, i.e. starch with high amylopectin and low amylose, hold longstanding cultural importance in some world regions and unique properties for industrial manufacture. The waxy character in many crop species is regulated by a single gene known as GBSSI (or waxy), which encodes the enzyme Granule Bound Starch Synthase1 with null or reduced activity. Several allelic variants of the waxy gene that contribute to varying levels of amylose content have been reported in different crop plants. Phylogenetic analysis of protein sequences and the genomic DNA encoding GBSSI of major cereals and recently sequenced millets and pseudo-cereals have shown that GBSSI orthologs form distinct clusters, each representing a separate crop lineage. With the rapidly increasing demand for waxy starch in food and non-food applications, conventional crop breeding techniques and modern crop improvement technologies such as gene silencing and genome editing have been deployed to develop new waxy crop cultivars. The advances in research on waxy alleles across different crops have unveiled new possibilities for modifying the synthesis of amylose and amylopectin starch, leading to the potential creation of customized crops in the future. This article presents molecular lines of evidence on the emergence of waxy genes in various crops, including their genesis and evolution, molecular structure, comparative analysis and breeding innovations.

The Arabidopsis thaliana integrin-like gene AT14A improves drought tolerance in Solanum lycopersicum.

Using effective genes to improve crop stress tolerance through genetic engineering is an important way to stabilize crop yield and quality across complex climatic environments. Integrin-like AT14A, as a continuum of the cell wall-plasma membrane-cytoskeleton, functions in the regulation of cell wall synthesis, signal transduction, and the response to stress. In this study, AT14A was overexpressed in Solanum lycopersicum L. In transgenic plants, both chlorophyll content and net photosynthetic rate increased. Physiological experiments suggested that the proline content and antioxidant enzyme (superoxide dismutase, catalase, peroxidase) activities of the transgenic line were significantly higher than those of wild-type plants under stress, which contributed to the enhanced water retention capacity and free radical scavenging ability of the transgenic line. Transcriptome analysis revealed that AT14A enhanced drought tolerance by regulating waxy cuticle synthesis genes, such as 3-ketoacyl-CoA synthase 20 (KCS20), non-specific lipid-transfer protein 2 (LTP2), antioxidant enzyme system genes peroxidase 42-like (PER42), and dehydroascorbate reductase (DHAR2). AT14A regulates expression of Protein phosphatase 2C 51 (PP2C 51) and ABSCISIC ACID-INSENSITIVE 5 (ABI5) to participate in ABA pathways to enhance drought tolerance. In conclusion, AT14A effectively improved photosynthesis and enhanced drought tolerance in S. lycopersicum.

The kinase OsSK41/OsGSK5 negatively regulates amylose content in rice endosperm by affecting the interaction between OsEBP89 and OsBP5.

Amylose content (AC) is the main factor determining the palatability, viscosity, transparency, and digestibility of rice (Oryza sativa) grains. AC in rice grains is mainly controlled by different alleles of the Waxy (Wx) gene. The AP2/EREBP transcription factor OsEBP89 interacts with the MYC-like protein OsBP5 to synergistically regulate the expression of Wx. Here, we determined that the GLYCOGEN SYNTHASE KINASE 5 (OsGSK5, also named SHAGGY-like kinase 41 [OsSK41]) inhibits the transcriptional activation activity of OsEBP89 in rice grains during amylose biosynthesis. The loss of OsSK41 function enhanced Wx expression and increased AC in rice grains. By contrast, the loss of function of OsEBP89 reduced Wx expression and decreased AC in rice grains. OsSK41 interacts with OsEBP89 and phosphorylates four of its sites (Thr-28, Thr-30, Ser-238, and Thr-257), which makes OsEBP89 unstable and attenuates its interaction with OsBP5. Wx promoter activity was relatively weak when regulated by the phosphomimic variant OsEBP89E -OsBP5 but relatively strong when regulated by the nonphosphorylatable variant OsEBP89A -OsBP5. Therefore, OsSK41-mediated phosphorylation of OsEBP89 represents an additional layer of complexity in the regulation of amylose biosynthesis during rice grain development. In addition, our findings provide four possible sites for regulating rice grain AC via precise gene editing.

Evolutionary insight into the invasive allopolyploidy Spartina anglica inferred from multiple chloroplast DNA and nuclear Waxy gene

Polyploidy has played an important role in the Spartina system. However, the genomic changes following polyploidy evolution are complex and poorly understood, especially in the recent allopolyploid species Spartina anglica. In the present study, we used the nuclear Waxy gene in conjunction with multiple chloroplast DNA genes (trnK intron + matK, rps16-trnK, rps16 intron, trnV-ndhC, trnT-trnL, ndhF, ccsA, and ycf3) to investigate the evolution of Waxy homoeologs and chloroplast genes following allopolyploid formation in S. anglica. Our results revealed that most of the chloroplast regions analyzed were conserved and undistinguishable from the maternal parent (Spartina alterniflora); however, some regions (trnK intron + matK and rps16-trnK) have already undergone some changes and are slightly differentiated from the maternal parent. In the nuclear Waxy gene, at least four copies were expected in S. anglica; however, only two homoeologous copies of maternal origin were recovered, which are evolving under selective constraints. Our results indicate that, despite the presumed genetic uniformity of S. anglica, some structural genome changes have occurred in Korean individuals, and thus, the S. anglica genome may be more dynamic than previously thought. This provides more insights into the evolutionary history of Spartina species and may have implications for the success and diversification of S. anglica.

CRISPR/Cas9-mediated editing of Wx and BADH2 genes created glutinous and aromatic two-line hybrid rice.

Amylose content (AC) is one of the physicochemical indexes of rice quality, which is largely determined by the Waxy (Wx) gene. Fragrance in rice is favored because it adds good flavor and a faint scent. Loss of function of the BADH2 (FGR) gene promotes the biosynthesis of 2-acetyl-1-pyrroline (2AP), which is the main compound responsible for aroma in rice. Here, we used a CRISPR/Cas9 system to simultaneously knock out Wx and FGR genes in 1892S and M858, which are the parents of an indica two-line hybrid rice, Huiliangyou 858 (HLY858). Four T-DNA-free homozygous mutants (1892Swxfgr-1, 1892Swxfgr-2, M858wxfgr-1, and M858wxfgr-2) were obtained. The 1892Swxfgr and M858wxfgr were crossed to generate double mutant hybrid lines HLY858wxfgr-1 and HLY858wxfgr-2. Size-exclusion chromatography (SEC) data indicated that true AC of the wx mutant starches ranged from 0.22 to 1.63%, much lower than those of the wild types (12.93 to 13.76%). However, the gelatinization temperature (GT) of the wx mutants in backgrounds of 1892S, M858, and HLY858 were still high, and showed no significant differences with the wild type controls. The aroma compounds 2AP content in grains of HLY858wxfgr-1 and HLY858wxfgr-2 were 153.0 μg/kg and 151.0 μg/kg, respectively. In contrast, 2AP was not detected in grains of HLY858. There were no significant differences in major agronomic traits between the mutants and HLY858. This study provides guidelines for cultivation of ideal glutinous and aromatic hybrid rice by gene editing.

Three Starch Synthase IIa (SSIIa) Alleles Reveal the Effect of SSIIa on the Thermal and Rheological Properties, Viscoelasticity, and Eating Quality of Glutinous Rice

Glutinous rice accumulates amylose-free starch and is utilized for rice cakes and crackers, owing to the loss of the Waxy gene which encodes granule-bound starch synthase I (GBSSI). Starch synthase IIa (SSIIa) elongates amylopectin chains with a degree of polymerization (DP) of 6-12 to 13-24 and greatly influences starch properties. To elucidate the relationship between the branch length of amylopectin and the thermal and rheological properties, viscoelasticity, and eating quality of glutinous rice, three allelic near isogenic lines with high, low, or no SSIIa activity were generated (designated as SS2a wx, ss2aL wx, and ss2a wx, respectively). Chain length distribution analyses revealed that ss2a wx exhibited the highest short chain (DP < 12) number and lowest gelatinization temperature, whereas SS2a wx showed the opposite results. Gel filtration chromatography showed that the three lines contained essentially no amylose. Viscoelasticity analyses of rice cakes stored at low temperature for different durations revealed that ss2a wx maintained softness and elasticity for up to 6 days, while SS2a wx hardened within 6 h. Sensory evaluation was consistent with mechanical evaluation. The relationship of amylopectin structure with the thermal and rheological properties, viscoelasticity, and eating quality of glutinous rice is discussed.

Generating waxy rice starch with target type of amylopectin fine structure and gelatinization temperature by waxy gene editing

Waxy rice, which lacks amylose, is an important variant in rice, and its starches have been widely used. New waxy rice varieties generated via the CRISPR/Cas9 gene-editing system is described. Herein, four waxy rice starches with different physicochemical properties were successfully obtained by the CRISPR/Cas9 editing Waxy (Wx) gene. The results showed that the amylose content (AC) of wx mutant starches ranged from 0.26 to 1.78 %, and CZBwx1 starches had the best gel consistency and highest water solubility among all wx mutants. Mutations of Wxb allele produced more short-chains (degree of chain polymerization (DP) 6–11), and less medium- and long-chains (DP12–70) than that of Wxa, while the AC of Wxa allele mutants was higher than the mutations of Wxb allele. The gelatinization temperature (GT) of wxa mutant starches was higher than that of wxb mutant starches. Moreover, we found that the GT and amylopectin fine structure type of waxy rice starch did not change after Wx gene editing. Based on these findings, it is possible to produce waxy rice starch with different physicochemical properties, containing target GT and chain length distributions of amylopectin.

Molecular characterization and phylogenetic analysis of novel Wx genes from Psathyrostachys juncea and Eremopyrum bonaepartis

The ratio of amylose/amylopectin can greatly influence the end-use quality of wheat products, and amylose synthesis only needs the granule-bound starch synthase I that is encoded by the Waxy (Wx) gene. Hence, the Wx allele variability is a vital determinant of amylose synthesis. In this study, four novel Wx genes were isolated from two diploid Triticeae species. Their sequence variations were analyzed, which showed that the polymorphism among the Wx-F1 alleles was low; only seven single nucleotide polymorphisms (SNPs) and one amino acid (AA) residue change (Arg541 ? Cys) were detected in the exon regions, and this change in Wx-F1b was predicted to have deleterious effects on protein function. The two Wx-Ns1 alleles in this study were different from two published alleles obtained using three overlapping primer sets. The polymorphism among the four Wx-Ns1 alleles was high, and 37 SNPs and ten AA residue changes were found, while all of those substitutions were neutral. However, one substitution (Leu237 ? Pro) between Wx-H1 and Wx-Ns1 was predicted that would probably affect the protein function. Divergence time analysis showed that Wx-Ns1a diverged from Wx-Ns1b about 1.06 MYAs. Phylogenetic analysis indicated that Wx-F1 were more closely related to Wx-O1 from Henrardia persica, Wx-R1 from Secale cereale ssp. cereale, and Wx-Xe1 from Eremopyrum triticeum, while Wx-Ns1 were more closely related to Wx-H1 from Hordeum vulgare ssp. vulgare and Wx-Hch1 from Hordeum chilense. These genes may be important candidates for wheat quality improvement.

Fine-tuning grain amylose contents by genome editing of Waxy cis-regulatory region in rice.

Rice grain amylose contents (ACs) is a key quantitative trait influencing eating and cooking quality. Regulating the expression level of Waxy, a key gene controlling ACs, and in turn fine-tuning the grain ACs, is an ideal approach to improve grain quality of rice varieties. Based on CRISPR/Cas9 genome editing technology, we designed eight targets in the cis-regulatory region of Wxa background, screened phenotypic changes of the transgenic lines and generated eight novel Waxy alleles with altered grain ACs. Among the eight alleles, we found that a 407-bp non-homologous substitution (NHS) in the 5'UTR-intron caused by genome editing regulated Waxy expression and decreased grain ACs by 2.9%. Moreover, embedding the 407-bp NHS into the cis-regulatory region of Wxb allele can also affect gene activity. Our work suggested the effect of 5'UTR-intron on Waxy gene expression regulation, and provided a potentially useful allele in breeding that can finely adjust rice grain ACs.