Loss of function of SSIIIa and SSIIIb coordinately confers high RS content in cooked rice.

The sedentary lifestyle and refined food consumption significantly lead to obesity, type 2 diabetes, and related complications, which have become one of the major threats to global health. This incidence could be potentially reduced by daily foods rich in resistant starch (RS). However, it remains a challenge to breed high-RS rice varieties. Here, we reported a high-RS mutant rs4 with an RS content of ~10.8% in cooked rice. The genetic study revealed that the loss-of-function SSIIIb and SSIIIa together with a strong Wx allele in the background collaboratively contributed to the high-RS phenotype of the rs4 mutant. The increased RS contents in ssIIIa and ssIIIa ssIIIb mutants were associated with the increased amylose and lipid contents. SSIIIb and SSIIIa proteins were functionally redundant, whereas SSIIIb mainly functioned in leaves and SSIIIa largely in endosperm owing to their divergent tissue-specific expression patterns. Furthermore, we found that SSIII experienced duplication in different cereals, of which one SSIII paralog was mainly expressed in leaves and another in the endosperm. SSII but not SSIV showed a similar evolutionary pattern to SSIII. The copies of endosperm-expressed SSIII and SSII were associated with high total starch contents and low RS levels in the seeds of tested cereals, compared with low starch contents and high RS levels in tested dicots. These results provided critical genetic resources for breeding high-RS rice cultivars, and the evolutionary features of these genes may facilitate to generate high-RS varieties in different cereals.

OsJRL negatively regulates rice cold tolerance via interfering phenylalanine metabolism and flavonoid biosynthesis.

The identification of new genes involved in regulating cold tolerance in rice is urgent because low temperatures repress plant growth and reduce yields. Cold tolerance is controlled by multiple loci and involves a complex regulatory network. Here, we show that rice jacalin-related lectin (OsJRL) modulates cold tolerance in rice. The loss of OsJRL gene functions increased phenylalanine metabolism and flavonoid biosynthesis under cold stress. The OsJRL knock-out (KO) lines had higher phenylalanine ammonia-lyase (PAL) activity and greater flavonoid accumulation than the wild-type rice, Nipponbare (NIP), under cold stress. The leaves had lower levels of reactive oxygen species (ROS) and showed significantly enhanced cold tolerance compared to NIP. In contrast, the OsJRL overexpression (OE) lines had higher levels of ROS accumulation and showed lower cold tolerance than NIP. Additionally, the OsJRL KO lines accumulated more abscisic acid (ABA) and jasmonic acid (JA) under cold stress than NIP. The OsJRL OE lines showed increased sensitivity to ABA compared to NIP. We conclude that OsJRL negatively regulates the cold tolerance of rice via modulation of phenylalanine metabolism and flavonoid biosynthesis.

Recent advances and biotechnological applications of RNA metabolism in plant chloroplasts and mitochondria.

The chloroplast and mitochondrion are semi-autonomous organelles that play essential roles in cell function. These two organelles are embellished with prokaryotic remnants and contain many new features emerging from the co-evolution of organelles and the nucleus. A typical plant chloroplast or mitochondrion genome encodes less than 100 genes, and the regulation of these genes' expression is remarkably complex. The regulation of chloroplast and mitochondrion gene expression can be achieved at multiple levels during development and in response to environmental cues, in which, RNA metabolism, including: RNA transcription, processing, translation, and degradation, plays an important role. RNA metabolism in plant chloroplasts and mitochondria combines bacterial-like traits with novel features evolved in the host cell and is regulated by a large number of nucleus-encoded proteins. Among these, pentatricopeptide repeat (PPR) proteins are deeply involved in multiple aspects of the RNA metabolism of organellar genes. Research over the past decades has revealed new insights into different RNA metabolic events in plant organelles, such as the composition of chloroplast and mitochondrion RNA editosomes. We summarize and discuss the most recent knowledge and biotechnological implications of various RNA metabolism processes in plant chloroplasts and mitochondria, with a focus on the nucleus-encoded factors supporting them, to gain a deeper understanding of the function and evolution of these two organelles in plant cells. Furthermore, a better understanding of the role of nucleus-encoded factors in chloroplast and mitochondrion RNA metabolism will motivate future studies on manipulating the plant gene expression machinery with engineered nucleus-encoded factors.

Higher Food Yields and Lower Greenhouse Gas Emissions from Aquaculture Ponds with High-Stalk Rice Planted.

Aquaculture ponds are an important artificial aquatic system for global food fish production but also are a hot spot of greenhouse gas (GHG) emissions. The GHG mitigation strategy and the underlying mechanism for aquaculture ponds are still poorly understood. In this study, we conducted a 2 year field experiment to determine the effects of planting high-stalk rice (an artificially bred emergent plant for ponds) on GHG emissions from aquaculture ponds. Our results showed that planting high-stalk rice reduced CH4 emission by 64.4% and N2O emission by 76.2% over 2 years. Planting high-stalk rice significantly increased the content of O2 and the abundance of pmoA in the sediment, thus prompting CH4 oxidation in the ponds. The reduction of N2O emission from ponds was attributed to the decreased inorganic nitrogen, amoA-B and nirS in the sediment induced by rice. Furthermore, high-stalk rice culture in the pond increased shrimp yields and gained rice yields, resulting in a significant reduction of yield-scaled global warming potential. Our findings suggest that breeding appropriate emergent aquatic plants is a potential pathway to mitigate GHG emission from aquaculture ponds with more food yields and economic benefits.

Organelle Imaging with Terahertz Scattering-Type Scanning Near-Field Microscope.

Organelles play core roles in living beings, especially in internal cellular actions, but the hidden information inside the cell is difficult to extract in a label-free manner. In recent years, terahertz (THz) imaging has attracted much attention because of its penetration depth in nonpolar and non-metallic materials and label-free, non-invasive and non-ionizing ability to obtain the interior information of bio-samples. However, the low spatial resolution of traditional far-field THz imaging systems and the weak dielectric contrast of biological samples hinder the application of this technology in the biological field. In this paper, we used an advanced THz scattering near-field imaging method for detecting chloroplasts on gold substrate with nano-flatness combined with an image processing method to remove the background noise and successfully obtained the subcellular-grade internal reticular structure from an Arabidopsis chloroplast THz image. In contrast, little inner information could be observed in the tea chloroplast in similar THz images. Further, transmission electron microscopy (TEM) and mass spectroscopy (MS) were also used to detect structural and chemical differences inside the chloroplasts of Arabidopsis and tea plants. The preliminary results suggested that the interspecific different THz information is related to the internal spatial structures of chloroplasts and metabolite differences among species. Therefore, this method could open a new way to study the structure of individual organelles.

Comparative studies on physicochemical properties of three potato varieties different in RS2 and RS3 contents.

BACKGROUND: At present, increasing importance has been attracted to healthy food enriched in resistant starch (RS), which has great benefits in health-promoting. Raw potato has rich RS2, whereas most RS2 may become digestible after gelatinization, resulting in few RS being left in processed potato. Breeding potatoes with high RS2 or RS3 or both can meet the demand for various healthy potato products. RESULTS: There were apparent discrepancies among three potatoes with contrast RS2 and RS3 content in thermal properties, viscosity and digestibility. ZS-5 had the highest RS2 with 50.17% but the lowest RS3 with 3.31%. Meanwhile, ZS-5 had the largest starch granule, the highest proportion of B3, viscosity and hardness, and the highest digestibility. DN303 with the highest content of RS3 (5.08%) had the lowest hardness and fracturability. MG56-42 with both higher RS2 and RS3 content showed the highest resistance to digestion and moderate hardness and fracturability. CONCLUSION: The present study enriches the potential resources and provides a reliable scientific basis for high RS potatoes breeding. The various features of different potatoes make it possible to screen potatoes according to different demands. © 2023 Society of Chemical Industry.

Polymorphisms in cis-elements confer SAUR26 gene expression difference for thermo-response natural variation in Arabidopsis.

The SAUR26 subfamily genes play an important role in conferring variations of thermo-responsiveness of growth architecture among natural accessions of Arabidopsis thaliana. The expression variations are critical for their activity variations, but how expression variations are generated is unknown. We identified genetic loci for gene expression variations through expression genome-wide association study (eGWAS) and investigated their mechanisms through molecular analyses. We found that cis elements are the major determinants for expression variations of SAUR26, SAUR27, and SAUR28. Polymorphisms in the promoter region likely impact PIF4 regulation while those at the 3'UTR affect mRNA stability to generate variations in SAUR26 expression levels. These polymorphisms also differentially affect the mRNA stability of SAUR26 at two temperatures. This study reveals two mechanisms involving cis elements in generating gene expression diversity, which is likely important for local adaptations in Arabidopsis natural accessions.

LAZY2 controls rice tiller angle through regulating starch biosynthesis in gravity-sensing cells.

Rice (Oryza sativa) tiller angle is a key component for achieving ideal plant architecture and higher grain yield. However, the molecular mechanism underlying rice tiller angle remains elusive. We characterized a novel rice tiller angle mutant lazy2 (la2) and isolated the causative gene LA2 through map-based cloning. Biochemical, molecular and genetic studies were conducted to elucidate the LA2-involved tiller angle regulatory mechanism. The la2 mutant shows large tiller angle with impaired shoot gravitropism and defective asymmetric distribution of auxin. We found that starch granules in amyloplasts are completely lost in the gravity-sensing leaf sheath base cells of la2, whereas the seed development is not affected. LA2 encodes a novel chloroplastic protein that can interact with the starch biosynthetic enzyme Oryza sativa plastidic phosphoglucomutase (OspPGM) to regulate starch biosynthesis in rice shoot gravity-sensing cells. Genetic analysis showed that LA2 regulates shoot gravitropism and tiller angle by acting upstream of LA1 to mediate lateral auxin transport. Our studies revealed that LA2 acts as a novel regulator of rice tiller angle by specifically regulating starch biosynthesis in gravity-sensing cells, and established the framework of the starch-statolith-dependent rice tiller angle regulatory pathway, providing new insights into the rice tiller angle regulatory network.

A Trypsin Family Protein Gene Controls Tillering and Leaf Shape in Barley.

Tillering or branching is an important agronomic trait in plants, especially cereal crops. Previously, in barley (Hordeum vulgare) 'Vlamingh', we identified the high number of tillers1 (hnt1) mutant from a γ-ray-treated segregating population. hnt1 exhibited more tillers per plant, narrower leaves, and reduced plant height compared with the wild-type parent. In this study, we show that the hnt1-increased tiller number per plant is caused by accelerated outgrowth of tiller buds and that hnt1 narrower leaves are caused by a reduction in vascular tissue and cell number. Genetic analysis revealed that a 2-bp deletion in the gene HORVU2Hr1G098820 (HvHNT1), encoding a trypsin family protein, was responsible for the hnt1 mutant phenotype. Gene function was further confirmed by transgenic complementation with HvHNT1 and RNA interference experiments. HvHNT1 was expressed in vascular tissue, leaf axils, and adventitious root primordia and shown to negatively regulate tiller development. Mutation of HvHNT1 led to the accumulation of a putative cyclophilin-type peptidyl-prolyl cis/trans-isomerase (HvPPIase), which physically interacts with the HvHNT1 protein in the nucleus of plant cells. Our data suggest that HvHNT1 controls tiller development and leaf width through HvPPIase, thus contributing to understanding of the molecular players that control tillering in barley.

MOS1 Negatively Regulates Sugar Responses and Anthocyanin Biosynthesis in Arabidopsis.

Sugars, which are important signaling molecules, regulate diverse biological processes in plants. However, the convergent regulatory mechanisms governing these physiological activities have not been fully elucidated. MODIFIER OF snc1-1 (MOS1), a modulator of plant immunity, also regulates floral transition, cell cycle control, and other biological processes. However, there was no evidence of whether this protein was involved in sugar responses. In this study, we found that the loss-of-function mutant mos1-6 (mos1) was hypersensitive to sugar and was characterized by defective germination and shortened roots when grown on high-sugar medium. The expression of MOS1 was enhanced by sucrose. Hexokinase 1, an important gene involved in sugar signaling, was upregulated in the mos1 mutant compared to wild-type Col-0 in response to sugar. Furthermore, the mos1 mutant accumulated more anthocyanin than did wild-type Col-0 when grown on high-sugar concentration medium or under high light. MOS1 was found to regulate the expression of flavonoid and anthocyanin biosynthetic genes in response to exogenous sucrose and high-light stress but with different underlying mechanisms, showing multiple functions in addition to immunity regulation in plant development. Our results suggest that the immune regulator MOS1 serves as a coordinator in the regulatory network, governing immunity and other physiological processes.

MOS1 functions closely with TCP transcription factors to modulate immunity and cell cycle in Arabidopsis.

Emerging evidence indicates a close connection between cell-cycle progression and the plant immune responses. In Arabidopsis, MODIFIER OF snc1-1 (MOS1) modulates a number of processes including endoreduplication and plant disease resistance, but the molecular mechanism underlying this modulation was not fully understood. Here, we provide biochemical and genetic evidence that TEOSINTE BRANCHED 1, CYCLOIDEA, PCF1 (TCP) transcription factors TCP15 and its homologues are mediators of MOS1 function in the immune response and are likely to be also involved in cell-cycle control. MOS1 and TCP proteins have a direct physical interaction. They both bind to the promoter of the immune receptor gene SUPRESSOR OF npr1-1, CONSTITUTIVE 1 (SNC1) and modulate its expression and consequently immune responses. MOS1 and TCP15 both affect the expression of cell-cycle genes D-type CYCLIN 3;1 (CYCD3;1), which may mediate the MOS1 function in cell-cycle modulation. In addition, CYCD3;1 overexpression upregulates immune responses, and SNC1 expression. This study investigated and revealed a role for MOS1 in transcriptional regulation through TCP15 and its homologues. This finding suggests the coordination of cell-cycle progression and plant immune responses at multiple levels.

Natural variations of growth thermo-responsiveness determined by SAUR26/27/28 proteins in Arabidopsis thaliana.

How diversity in growth thermo-responsiveness is generated for local adaptation is a long-standing biological question. We investigated molecular genetic basis of natural variations in thermo-responsiveness of plant architecture in Arabidopsis thaliana. We measured the extent of rosette architecture at 22°C and 28°C in a set of 69 natural accessions and determined their thermo-responsiveness of plant architecture. A genome-wide association study was performed to identify major loci for variations in thermo-responsiveness. The SAUR26 subfamily, a new subfamily of SAUR genes, was identified as a major locus for the thermo-responsive architecture variations. The expression of SAUR26/27/28 is modulated by temperature and PIF4. Extensive natural polymorphisms in these genes affect their RNA expression levels and protein activities and influence the thermo-responsiveness of plant architecture. In addition, the SAUR26 subfamily genes exhibit a high variation frequency and their variations are associated with the local temperature climate. This study reveals that the SAUR26 subfamily is a key variation for thermo-responsive architecture and suggests a preference for generating diversity for local adaptation through signaling connectors.

Critical roles of soluble starch synthase SSIIIa and granule-bound starch synthase Waxy in synthesizing resistant starch in rice.

Changes in human lifestyle and food consumption have resulted in a large increase in the incidence of type-2 diabetes, obesity, and colon disease, especially in Asia. These conditions are a growing threat to human health, but consumption of foods high in resistant starch (RS) can potentially reduce their incidence. Strategies to increase RS in rice are limited by a lack of knowledge of its molecular basis. Through map-based cloning of a RS locus in indica rice, we have identified a defective soluble starch synthase gene (SSIIIa) responsible for RS production and further showed that RS production is dependent on the high expression of the Waxya (Wxa ) allele, which is prevalent in indica varieties. The resulting RS has modified granule structure; high amylose, lipid, and amylose-lipid complex; and altered physicochemical properties. This discovery provides an opportunity to increase RS content of cooked rice, especially in the indica varieties, which predominates in southern Asia.

Proteomic insights into floral biology.

The flower is the most important biological structure for ensuring angiosperms reproductive success. Not only does the flower contain critical reproductive organs, but the wide variation in morphology, color, and scent has evolved to entice specialized pollinators, and arguably mankind in many cases, to ensure the successful propagation of its species. Recent proteomic approaches have identified protein candidates related to these flower traits, which has shed light on a number of previously unknown mechanisms underlying these traits. This review article provides a comprehensive overview of the latest advances in proteomic research in floral biology according to the order of flower structure, from corolla to male and female reproductive organs. It summarizes mainstream proteomic methods for plant research and recent improvements on two dimensional gel electrophoresis and gel-free workflows for both peptide level and protein level analysis. The recent advances in sequencing technologies provide a new paradigm for the ever-increasing genome and transcriptome information on many organisms. It is now possible to integrate genomic and transcriptomic data with proteomic results for large-scale protein characterization, so that a global understanding of the complex molecular networks in flower biology can be readily achieved. This article is part of a Special Issue entitled: Plant Proteomics--a bridge between fundamental processes and crop production, edited by Dr. Hans-Peter Mock.

Characterization and comparative profiling of the small RNA transcriptomes in two phases of flowering in Cymbidium ensifolium.

BACKGROUND: Cymbidium ensifolium is one of the most important ornamental flowers in China, with an elegant shape, beautiful appearance, and a fragrant aroma. Its unique flower shape has long attracted scientists. MicroRNAs (miRNAs) are critical regulators in plant development and physiology, including floral development. However, to date, few studies have examined miRNAs in C. ensifolium. RESULTS: In this study, we employed Solexa technology to sequence four small RNA libraries from two flowering phases to identify miRNAs related to floral development. We identified 48 mature conserved miRNA and 71 precursors. These conserved miRNA belonged to 20 families. We also identified 45 novel miRNA which includes 21 putative novel miRNAs*, and 28 hairpin forming precursors. Two trans-acting small interfering RNAs (ta-siRNAs) were identified, one of which was homologous to TAS3a1. TAS3a1 belongs to the TAS3 family, which has been previously reported to target auxin response factors (ARF) and be involved in plant growth and floral development. Moreover, we built a C. ensifolium transctriptome database to identify genes targeted by miRNA, which resulted in 790 transcriptomic target unigenes. The target unigenes were annotated with information from the non-redundant (Nr), gene ontology database (GO), eukaryotic orthologous groups (KOGs) and Kyoto encyclopedia of genes and genomes (KEGG) database. The unigenes included MADS-box transcription factors targeted by miR156, miR172 and miR5179, and various hormone responding factors targeted by miR159. The MADS-box transcription factors are well known to determine the identity of flower organs and hormone responding factors involved in floral development. In expression analysis, three novel and four conserved miRNA were differentially expressed between two phases of flowering. The results were confirmed by RNA-seq and qRT-PCR. The differential expression of two miRNA, miR160 and miR396, targeted ARFs and growth regulating factor (GRF), respectively. However, most of these small RNA were clustered in the uncharacterized group, which suggests there may be many novel small non-coding RNAs yet to be discovered. CONCLUSION: Our study provides a diverse set of miRNAs related to cymbidium floral development and serves as a useful resource for investigating miRNA-mediated regulatory mechanisms of floral development.

Sequence variation and haplotypes of lipoxygenase gene LOX-1 in the Australian barley varieties.

BACKGROUND: Lipoxygenases are a family of enzymes which catalyse the hydroperoxidation of polyunsaturated fatty acids with a cis, cis-1,4-pentadiene to form conjugated hydroperoxydienes. Lipoxygenase-1 (LOX-1) in barley worsens the flavour and foam stability of beer. It has become a major selection criteria for malting quality in the last few years. RESULTS: Lipoxygenase activity was investigated in 41 Australian barley cultivars and advanced breeding lines released since the 1950s; the cultivars differed markedly, ranging from 22.3 to 46.5 U/g. The structural gene and its promoter of lipoxygenase-1 were sequenced from the barley varieties representing different levels of LOX. Based on the analysis of nucleotide and deduced amino acid sequences, two major haplotypes were identified. Barley varieties with lower LOX were classified into three categories based on their pedigrees and sequence variations in the structural gene: (1) barley varieties derived from Canadian varieties with the pre-harvest sprouting susceptible allele, (2) Skiff and Hindmarsh with unique haplotype in the structural gene, and (3) Gairdner and Onslow with an unknown mechanism. CONCLUSION: Lipoxygenase activity has been reduced in the malting barley cultivars in the last 60 years although it is only recognized as a malting quality trait recently. There are clear haplotypes of the lipoxygenase structual gene. The polymorphisms detected in the structural gene can be used to design molecular markers for selection of low LOX haplotype. Other mechanisms also existed for controlling lipoxygenase activity. The results suggest that it is possible to develop barley varieties with lower LOX by combination of low LOX-1 haplotype and other trans-regulation factors.

Disruption of a rice pentatricopeptide repeat protein causes a seedling-specific albino phenotype and its utilization to enhance seed purity in hybrid rice production.

The pentatricopeptide repeat (PPR) gene family represents one of the largest gene families in higher plants. Accumulating data suggest that PPR proteins play a central and broad role in modulating the expression of organellar genes in plants. Here we report a rice (Oryza sativa) mutant named young seedling albino (ysa) derived from the rice thermo/photoperiod-sensitive genic male-sterile line Pei'ai64S, which is a leading male-sterile line for commercial two-line hybrid rice production. The ysa mutant develops albino leaves before the three-leaf stage, but the mutant gradually turns green and recovers to normal green at the six-leaf stage. Further investigation showed that the change in leaf color in ysa mutant is associated with changes in chlorophyll content and chloroplast development. Map-based cloning revealed that YSA encodes a PPR protein with 16 tandem PPR motifs. YSA is highly expressed in young leaves and stems, and its expression level is regulated by light. We showed that the ysa mutation has no apparent negative effects on several important agronomic traits, such as fertility, stigma extrusion rate, selfed seed-setting rate, hybrid seed-setting rate, and yield heterosis under normal growth conditions. We further demonstrated that ysa can be used as an early marker for efficient identification and elimination of false hybrids in commercial hybrid rice production, resulting in yield increases by up to approximately 537 kg ha(-1).

Lipid complexation reduces rice starch digestibility and boosts short-chain fatty acid production via gut microbiota.

In this study, two rice varieties (RS4 and GZ93) with different amylose and lipid contents were studied, and their starch was used to prepare starch-palmitic acid complexes. The RS4 samples showed a significantly higher lipid content in their flour, starch, and complex samples compared to GZ93. The static in vitro digestion highlighted that RS4 samples had significantly lower digestibility than the GZ93 samples. The C∞ of the starch-lipid complex samples was found to be 17.7% and 18.5% lower than that of the starch samples in GZ93 and RS4, respectively. The INFOGEST undigested fractions were subsequently used for in vitro colonic fermentation. Short-chain fatty acids (SCFAs) concentrations, mainly acetate, and propionate were significantly higher in starch-lipid complexes compared to native flour or starch samples. Starch-lipid complexes produced a distinctive microbial composition, which resulted in different gene functions, mainly related to pyruvate, fructose, and mannose metabolism. Using Model-based Integration of Metabolite Observations and Species Abundances 2 (MIMOSA2), SCFA production was predicted and associated with the gut microbiota. These results indicated that incorporating lipids into rice starch promotes SCFA production by modulating the gut microbiota selectively.

Discrepancies in resistant starch and starch physicochemical properties between rice mutants similar in high amylose content.

The content of resistant starch (RS) was considered positively correlated with the apparent amylose content (AAC). Here, we analyzed two Indica rice mutants, RS111 and Zhedagaozhi 1B, similar in high AAC and found that their RS content differed remarkably. RS111 had higher RS3 content but lower RS2 content than Zhedagaozhi 1B; correspondingly, cooked RS111 showed slower digestibility. RS111 had smaller irregular and oval starch granules when compared with Zhedagaozhi 1B and the wild type. Zhedagaozhi 1B showed a B-type starch pattern, different from RS111 and the wild type, which showed A-type starch. Meantime, RS111 had more fa and fb1 but less fb3 than Zhedagaozhi 1B. Both mutants showed decreased viscosity and swelling power when compared with the parents. RS111 had the lowest viscosity, and Zhedagaozhi 1B had the smallest swelling power. The different fine structures of amylopectin between RS111 and Zhedagaozhi 1B led to different starch types, gelatinization properties, paste viscosity, and digestibility. In addition to enhancing amylose content, modifications on amylopectin structure showed great potent in breeding rice with different RS2 and RS3 content, which could meet the increasing needs for various rice germplasms.

Inactivation of SSIIIa enhances the RS content through altering starch structure and accumulating C18:2 in japonica rice.

SSIIIa was the key gene responsible for RS formation in rice endosperm. The higher RS content in ssIIIa mutant has been proposed to be majorly due to the increased amylose-lipid complexes (RS5). However, the formation of RS5 elicited by ssIIIa mutation and the importance of RS5 for total RS content in rice are still unclear. With japonica ssIIIa loss-of-function mutants created by CRISPR/Cas9 gene editing, the effects of SSIIIa mutation on RS5 were furtherly evaluated through investigating the transcriptome and metabolites. Inactivation of SSIIIa caused significant enhancement in amylose and RS content but without depletion in starch reserves. SSIIIa mutation modulated the genes involved in carbohydrate and lipid metabolisms and the redistribution of substances, led to accumulated protein, glucose, fructose, and C18:2. Besides the increased amylose content and altered amylopectin structure, the increased C18:2 contributed greatly to the enhancement in RS content in japonica ssIIIa mutants through complexing with amylose to form RS5, while the existence of lipid counted against the enhancement of RS content in indica rice. RS5 showed discrepant contributions for the total RS in rice with different genetic background. Inactivation of SSIIIa has great potential in improving RS5 content in japonica rice without great yield loss.