The Waxy Gene Has Pleiotropic Effects on Hot Water-Soluble and -Insoluble Amylose Contents in Rice (Oryza sativa) Grains.

Rice (Oryza sativa) is a cereal crop with a starchy endosperm. Starch is composed of amylose and amylopectin. Amylose content (AC) is the principal determinant of rice quality, but varieties with similar ACs can still vary substantially in their quality. In this study, we analyzed the total AC (TAC) and its constituent fractions, the hot water-soluble amylose content (SAC) and hot water-insoluble amylose content (IAC), in two sets of related chromosome segment substitution lines of rice with a common genetic background grown in two years. We searched for quantitative trait loci (QTLs) associated with SAC, IAC, and TAC and identified one common QTL (qSAC-6, qIAC-6, and qTAC-6) on chromosome 6. Map-based cloning revealed that the gene underlying the trait associated with this common QTL is Waxy (Wx). An analysis of the colors of soluble and insoluble starch-iodine complexes and their λmax values (wavelengths at the positions of their peak absorbance values) as well as gel permeation chromatography revealed that Wx is responsible for the biosynthesis of amylose, comprising a large proportion of the soluble fractions of the SAC. Wx is also involved in the biosynthesis of long chains of amylopectin, comprising the hot water-insoluble fractions of the IAC. These findings highlight the pleiotropic effects of Wx on the SAC and IAC. This pleiotropy indicates that these traits have a positive genetic correlation. Therefore, further studies of rice quality should use rice varieties with the same Wx genotype to eliminate the pleiotropic effects of this gene, allowing the independent relationship between the SAC or IAC and rice quality to be elucidated through a multiple correlation analysis. These findings are applicable to other valuable cereal crops as well.

Insights into wheat-starch biosynthesis from two-dimensional macromolecular structure.

Starch structure is often characterized by the chain-length distribution (CLD) of the linear molecules formed by breaking each branch-point. More information can be obtained by expanding into a second dimension: in the present case, the total undebranched-molecule size. This enables answers to questions unobtainable by considering only one variable. The questions considered here are: (i) are the events independent which control total size and CLD, and (ii) do ultra-long amylopectin (AP) chains exist (these chains cannot be distinguished from amylose chains using simple size separation). This was applied here to characterize the structures of one normal (RS01) wheat and two high-amylose (AM) mutant wheats (an SBEIIa knockout and an SBEIIa and SBEIIb knockout). Absolute ethanol was used to precipitate collected fractions, then size-exclusion chromatography for total molecular size and for the size of branches. The SBEIIa and SBEIIb mutations significantly increased AM and IC contents and chain length. The 2D plots indicated the presence of small but significant amounts of long-chain amylopectin, and the asymmetry of these plots shows that the corresponding mechanisms share some causal effects. These results could be used to develop plants producing improved starches, because different ranges of the chain-length distribution contribute independently to functional properties.

Functional Characterization of the MeSSIII-1 Gene and Its Promoter from Cassava.

Soluble starch synthases (SSs) play important roles in the synthesis of cassava starch. However, the expression characteristics of the cassava SSs genes have not been elucidated. In this study, the MeSSIII-1 gene and its promoter, from SC8 cassava cultivars, were respectively isolated by PCR amplification. MeSSIII-1 protein was localized to the chloroplasts. qRT-PCR analysis revealed that the MeSSIII-1 gene was expressed in almost all tissues tested, and the expression in mature leaves was 18.9 times more than that in tuber roots. MeSSIII-1 expression was induced by methyljasmonate (MeJA), abscisic acid (ABA), and ethylene (ET) hormones in cassava. MeSSIII-1 expression patterns were further confirmed in proMeSSIII-1 transgenic cassava. The promoter deletion analysis showed that the -264 bp to -1 bp MeSSIII-1 promoter has basal activity. The range from -1228 bp to -987 bp and -488 bp to -264 bp significantly enhance promoter activity. The regions from -987 bp to -747 bp and -747 bp to -488 bp have repressive activity. These findings will provide an important reference for research on the potential function and transcriptional regulation mechanisms of the MeSSIII-1 gene and for further in-depth exploration of the regulatory network of its internal functional elements.

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.

OsLESV and OsESV1 promote transitory and storage starch biosynthesis to determine rice grain quality and yield.

Transitory starch is an important carbon source in leaves, and its biosynthesis and metabolism are closely related to grain quality and yield. The molecular mechanisms controlling leaf transitory starch biosynthesis and degradation and their effects on rice (Oryza sativa) quality and yield remain unclear. Here, we show that OsLESV and OsESV1, the rice orthologs of AtLESV and AtESV1, are associated with transitory starch biosynthesis in rice. The total starch and amylose contents in leaves and endosperms are significantly reduced, and the final grain quality and yield are compromised in oslesv and osesv1 single and oslesv esv1 double mutants. Furthermore, we found that OsLESV and OsESV1 bind to starch, and this binding depends on a highly conserved C-terminal tryptophan-rich region that acts as a starch-binding domain. Importantly, OsLESV and OsESV1 also interact with the key enzymes of starch biosynthesis, granule-bound starch synthase I (GBSSI), GBSSII, and pyruvate orthophosphote dikiase (PPDKB), to maintain their protein stability and activity. OsLESV and OsESV1 also facilitate the targeting of GBSSI and GBSSII from plastid stroma to starch granules. Overexpression of GBSSI, GBSSII, and PPDKB can partly rescue the phenotypic defects of the oslesv and osesv1 mutants. Thus, we demonstrate that OsLESV and OsESV1 play a key role in regulating the biosynthesis of both leaf transitory starch and endosperm storage starch in rice. These findings deepen our understanding of the molecular mechanisms underlying transitory starch biosynthesis in rice leaves and reveal how the transitory starch metabolism affects rice grain quality and yield, providing useful information for the genetic improvement of rice grain quality and yield.

Genome-Wide Identification and Expression Analysis of the Starch Synthase Gene Family in Sweet Potato and Two of Its Closely Related Species.

The starch synthase (SS) plays important roles in regulating plant growth and development and responding to adversity stresses. Although the SS family has been studied in many crops, it has not been fully identified in sweet potato and its two related species. In the present study, eight SSs were identified from Ipomoea batatas (I. batata), Ipomoea trifida (I. trifida), and Ipomoea trlioba (I. trlioba), respectively. According to the phylogenetic relationships, they were divided into five subgroups. The protein properties, chromosomal location, phylogenetic relationships, gene structure, cis-elements in the promoter, and interaction network of these proteins were also analyzed; stress expression patterns were systematically analyzed; and real-time polymerase chain reaction (qRT-PCR) analysis was performed. Ipomoea batatas starch synthase (IbSSs) were highly expressed in tuber roots, especially Ipomoea batatas starch synthase 1 (IbSS1) and Ipomoea batatas starch synthase 6 (IbSS6), which may play an important role in root development and starch biosynthesis. At the same time, the SS genes respond to potassium deficiency, hormones, cold, heat, salt, and drought stress. This study offers fresh perspectives for enhancing knowledge about the roles of SSs and potential genes to enhance productivity, starch levels, and resistance to environmental stresses in sweet potatoes.

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 ß-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 ß-glucan contents. This waxy flour, which showed lower final viscosity and setback, and higher breakdown, could provide more option for food processing.

GBSS mutations in an SBE mutated background restore the potato starch granule morphology and produce ordered granules despite differences to native molecular structure.

Potato starch with mutations in starch branching enzyme genes (SBEI, SBEII) and granule-bound starch synthase gene (GBSS) was characterized for molecular and thermal properties. Mutations in GBSS were here stacked to a previously developed SBEI and SBEII mutation line. Additionally, mutations in the GBSS gene alone were induced in the wild-type variety for comparison. The parental line with mutations in the SBE genes showed a âˆ¼ 40 % increase in amylose content compared with the wild-type. Mutations in GBSS-SBEI-SBEII produced non-waxy, low-amylose lines compared with the wild-type. An exception was a line with one remaining GBSS wild-type allele, which displayed ∼80 % higher amylose content than wild-type. Stacked mutations in GBSS in the SBEI-SBEII parental line caused alterations in amylopectin chain length distribution and building block size categories of whole starch. Correlations between size categories of building blocks and unit chains of amylopectin were observed. Starch in GBSS-SBEI-SBEII mutational lines had elevated peak temperature of gelatinization, which was positively correlated with large building blocks.

Exogenous abscisic acid (ABA) improves the filling process of maize grains at different ear positions by promoting starch accumulation and regulating hormone levels under high planting density.

BACKGROUND: Higher planting densities typically cause a decline in grain weight, limiting the potential for high maize yield. Additionally, variations in grain filling occur at different positions within the maize ear. Abscisic acid (ABA) is important for grain filling and regulates grain weight. However, the effects of exogenous ABA on the filling process of maize grains at different ear positions under high planting density are poorly understood. In this study, two summer maize hybrids (DengHai605 (DH605) and ZhengDan958 (ZD958)) commonly grown in China were used to examine the effects of ABA application during the flowering stage on grain filling properties, starch accumulation, starch biosynthesis associated enzyme activities, and hormone levels of maize grain (including inferior grain (IG) and superior grain (SG)) under high planting density. RESULTS: Our results showed that exogenous ABA significantly increased maize yield, primarily owing to a higher grain weight resulting from an accelerated grain filling rate relative to the control. There was no significant difference in yield between DH605 and ZD958 in the control and ABA treatments. Moreover, applying ABA promoted starch accumulation by raising the activities of sucrose synthase, ADP-glucose pyrophosphorylase, granule-bound starch synthases, soluble starch synthase, and starch branching enzyme in grains. It also increased the levels of zeatin riboside, indole-3-acetic acid, and ABA and decreased the level of gibberellin in grains, resulting in more efficient grain filling. Notably, IG exhibited a less efficient filling process compared to SG, probably due to lower starch biosynthesis associated enzyme activities and an imbalance in hormone contents. Nevertheless, IG displayed greater sensitivity to exogenous ABA than SG, suggesting that appropriate cultural measures to improve IG filling may be a viable strategy to further increase maize yield. CONCLUSIONS: According to our results, spraying exogenous ABA could effectively improve grain filling properties, accelerate starch accumulation by increasing relevant enzyme activities, and regulate hormone levels in grains, resulting in higher grain weight and yield of maize under high planting density. Our findings offer more evidence for using exogenous hormones to improve maize yield under high planting density.

The elite eating quality alleles Wxb and ALKb are regulated by OsDOF18 and coordinately improve head rice yield.

Head rice yield (HRY) measures rice milling quality and determines final grain yield and commercial value. Here, we report that two major quantitative trait loci for milling quality in rice, qMq-1 and qMq-2, represent allelic variants of Waxylv/Waxyb (hereafter Wx) encoding Granule-Bound Starch Synthase I (GBSSI) and Alkali Spreading Value ALKc/ALKb encoding Soluble Starch Synthase IIa (SSIIa), respectively. Complementation and overexpression transgenic lines in indica and japonica backgrounds confirmed that Wx and ALK coordinately regulate HRY by affecting amylose content, the number of amylopectin branches, amyloplast size, and thus grain filling and hardness. The transcription factor OsDOF18 acts upstream of Wx and ALK by activating their transcription. Furthermore, rice accessions with Wxb and ALKb alleles showed improved HRY over those with Wxlv and ALKc. Our study not only reveals the novel molecular mechanism underlying the formation of HRY but also provides a strategy for breeding rice cultivars with improved HRY.

White and Gray Matter Abnormality in Burning Mouth Syndrome Evaluated with Diffusion Tensor Imaging and Neurite Orientation Dispersion and Density Imaging.

PURPOSE: Burning mouth syndrome (BMS) is defined by a burning sensation or pain in the tongue or other oral sites despite the presence of normal mucosa on inspection. Both psychiatric and neuroimaging investigations have examined BMS; however, there have been no analyses using the neurite orientation dispersion and density imaging (NODDI) model, which provides detailed information of intra- and extracellular microstructures. Therefore, we performed voxel-wise analyses using both NODDI and diffusion tensor imaging (DTI) models and compared the results to better comprehend the pathology of BMS. METHODS: Fourteen patients with BMS and 11 age- and sex-matched healthy control subjects were prospectively scanned using a 3T-MRI machine using 2-shell diffusion imaging. Diffusion tensor metrics (fractional anisotropy [FA], mean diffusivity [MD], axial diffusivity [AD], and radial diffusivity [RD]) and neurite orientation and dispersion index metrics (intracellular volume fraction [ICVF], isotropic volume fraction [ISO], and orientation dispersion index [ODI]) were retrieved from diffusion MRI data. These data were analyzed using tract-based spatial statistics (TBSS) and gray matter-based spatial statistics (GBSS). RESULTS: TBSS analysis showed that patients with BMS had significantly higher FA and ICVF and lower MD and RD than the healthy control subjects (family-wise error [FWE] corrected P < 0.05). Changes in ICVF, MD, and RD were observed in widespread white matter areas. Fairly small areas with different FA were included. GBSS analysis showed that patients with BMS had significantly higher ISO and lower MD and RD than the healthy control subjects (FWE-corrected P < 0.05), mainly limited to the amygdala. CONCLUSION: The increased ICVF in the BMS group may represent myelination and/or astrocytic hypertrophy, and microstructural changes in the amygdala in GBSS analysis indicate the emotional-affective profile of BMS.

Development of a model for granule-bound starch synthase I activity using free-energy calculations.

Starch is a branched polymer of glucose with two components, both of which have (1 â†’ 4)-α linear links and (1 â†’ 6)-α branch points: amylopectin, of high molecular weight with many short branches, and amylose, of lower molecular weight and only a few long-chain branches. Granule-bound starch synthase I (GBSSI) is one of the main enzymes controlling amylose synthesis and chain-length distribution. As production of different GBSSI mutants is time-consuming and laborious, molecular dynamics (MD) simulations are used here to predict the binding of different GBSSI mutants to a representative amylose fragment. The simulations were atomistic, with explicit solvent and docking, a method successfully used to understand the binding of wild-type GBSSI to amylose fragments. The binding of GBSSI to G5 (a pentasaccharide amylose fragment) is combined with free-energy calculations employing a thermodynamic integration method to predict the effects of mutations on enzyme activity. Ten GBSSI mutants with different enzyme activities were analyzed to find the structural and energy changes among different single amino-acid mutants and their possible relationship to starch characteristics. Comparing the structural changes and the relative binding free energy of G5 to the wild type GBSSI and GBSSI mutants, it was found that mutants with negative binding energy (lower than -2.0 kcal/mol) are more likely to have higher enzyme activity and amylose content compared to the wild type. This theoretical paper used simulations and robust free energy calculations to interpret in planta data with potential predictions as to what mutants might be generated to give desired properties. This study can be used to help develop grains with improved functional properties.

Impact of dormancy periods on some physiological and biochemical indices of potato tubers.

Background: Storage of potato tubers is an essential stage of the supply chain, from farm to consumer, to efficiently match supply and demand. However, the quality and yield of potatoes are influenced by physiological changes during storage. Methods: This study tested the physiological and biochemical indices in three potato varieties (YunSu 108, YunSu 304 and YunSu 306) during their dormancy periods. Results: Three potato varieties with different dormancy periods were used to follow changes in starch, protein and several enzymes during storage. The starch and sugar content of the long-dormant variety (YunSu 108, LDV) were stable, whereas those of the short-dormant variety (YunSu 306, SDV) were variable. Starch synthase activity in the three varieties was initially high, then decreased; the starch content of LDV was relatively stable, that of the medium-dormant variety (YunSu 304, MDV) increased with storage time and peaked at sprouting, and that of SDV was low but variable. The sucrose synthase activity of LDV was significantly higher (p < 0.05) than MDV and SDV in the middle storage period. Two spikes were observed in the invertase activity of SDV, whereas those of MDV and LDV were stable. The reducing sugar content of LDV increased significantly before sprouting, that of MDV slowly decreased and that of SDV dropped sharply. During the whole storage period, pectinase activity in LDV did not change significantly, whereas pectinase in MDV and SDV decreased. The cellulase and protein contents initially increased and then decreased in LDV, and steadily decreased in MDV and SDV. Conclusion: The metabolic indices related to starch and sugar in the LDV were relatively stable during storage, whereas those of the SDV varied greatly. SDV showed increased sucrose, reducing sugars and cellulose; LDV PCA plots clustered in the positive quadrant of PC1 and the negative quadrant of PC2, with increased protein, sucrose synthase and starch; MDV had increased soluble starch synthase.

Granule-bound starch synthase in plants: Towards an understanding of their evolution, regulatory mechanisms, applications, and perspectives.

Amylose content (AC) is a significant quality trait in starchy crops, affecting their processing and application by the food and non-food industries. Therefore, fine-tuning AC in these crops has become a focus for breeders. Granule-bound starch synthase (GBSS) is the core enzyme that directly determines the AC levels. Several excellent reviews have summarized key progress in various aspects of GBSS research in recent years, but they mostly focus on cereals. Herein, we provide an in-depth review of GBSS research in monocots and dicots, focusing on the molecular characteristics, evolutionary relationships, expression patterns, molecular regulation mechanisms, and applications. We also discuss future challenges and directions for controlling AC in starchy crops, and found simultaneously increasing both the PTST and GBSS gene expression levels may be an effective strategy to increase amylose content.

Soluble and insoluble α-glucan synthesis in yeast by enzyme suites derived exclusively from maize endosperm.

Molecular mechanisms that distinguish the synthesis of semi-crystalline α-glucan polymers found in plant starch granules from the synthesis of water-soluble polymers by nonplant species are not well understood. To address this, starch biosynthetic enzymes from maize (Zea mays L.) endosperm were isolated in a reconstituted environment using yeast (Saccharomyces cerevisiae) as a test bed. Ninety strains were constructed containing unique combinations of 11 synthetic transcription units specifying maize starch synthase (SS), starch phosphorylase (PHO), starch branching enzyme (SBE), or isoamylase-type starch debranching enzyme (ISA). Soluble and insoluble branched α-glucans accumulated in varying proportions depending on the enzyme suite, with ISA function stimulating distribution into the insoluble form. Among the SS isoforms, SSIIa, SSIII, and SSIV individually supported the accumulation of glucan polymer. Neither SSI nor SSV alone produced polymers; however, synergistic effects demonstrated that both isoforms can stimulate α-glucan accumulation. PHO did not support α-glucan production by itself, but it had either positive or negative effects on polymer content depending on which SS or a combination thereof was present. The complete suite of maize enzymes generated insoluble particles resembling native starch granules in size, shape, and crystallinity. Ultrastructural analysis revealed a hierarchical assembly starting with subparticles of approximately 50 nm diameter that coalesce into discrete structures of approximately 200 nm diameter. These are assembled into semi-crystalline α-glucan superstructures up to 4 µm in length filling most of the yeast cytosol. ISA was not essential for the formation of such particles, but their abundance was increased dramatically by ISA presence.

CRISPR/Cas9-mediated multiple guide RNA-targeted mutagenesis in the potato.

CRISPR/Cas9 technology has become the most efficient method for genome editing in many plant species, including important industrial crops such as potatoes. This study used three target regions (T1, T2, and T3) in gbss exon I, whose sequences were first inserted into the BbsI sites in the appropriate guide RNA (gRNA) vector (pEn-Chimera, pMR203, pMR204, and pMR205), and then localized between the AtU6 promoter and the gRNA scaffold sequence. Expression vectors were constructed by introducing gRNA genes into the pMR287 (pYUCas9Plus) plasmids using the MultiSite Gateway system by attR and attL sites. The three target regions of mutant potato lines were analyzed. The use of CRISPR/Cas9-mediated multiple guide RNA-targeted mutagenesis allowed tri- or tetra-allelic mutant potato lines to be generated. Multiple nucleotide substitutions and indels within and around the three target sites caused a frameshift mutation that led to a premature stop codon, resulting in the production of gbss-knockout plants. Mutation frequencies and analysis of mutation patterns suggested that the stably transformed Cas9/multiple guide RNA expression constructs used in this study can induce targeted mutations efficiently in the potato genome. Full knockout of the gbss gene was analyzed by CAPS, Sanger sequencing and iodine staining. The present study demonstrated successful CRISPR/Cas9-mediated multiple guide RNA-targeted mutagenesis in the potato gbss gene by Agrobacterium-mediated transformation, resulting in an amylose-free phenotype.

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.

Further insight into the involvement of PII1 in starch granule initiation in Arabidopsis leaf chloroplasts.

The control of starch granule initiation in plant leaves is a complex process that requires active enzymes like Starch Synthase 4 and 3 (SS4 or SS3) and several noncatalytic proteins such as Protein Involved in starch Initiation 1 (PII1). In Arabidopsis leaves, SS4 is the main enzyme that control starch granule initiation, but in its absence, SS3 partly fulfills this function. How these proteins collectively act to control the initiation of starch granules remains elusive. PII1 and SS4 physically interact, and PII1 is required for SS4 to be fully active. However, Arabidopsis mutants lacking SS4 or PII1 still accumulate starch granules. Combining pii1 KO mutation with either ss3 or ss4 KO mutations provide new insights of how the remaining starch granules are synthesized. The ss3 pii1 line still accumulates starch, while the phenotype of ss4 pii1 is stronger than that of ss4. Our results indicate first that SS4 initiates starch granule synthesis in the absence of PII1 albeit being limited to one large lenticular granule per plastid. Second, that if in the absence of SS4, SS3 is able to initiate starch granules with low efficiency, this ability is further reduced with the additional absence of PII1.

Efficient Accumulation of Amylopectin and Its Molecular Mechanism in the Submerged Duckweed Mutant.

Large-scale use of fossil fuels has brought about increasingly serious problems of environmental pollution, development and utilization of renewable energy is one of the effective solutions. Duckweed has the advantages of fast growth, high starch content and no occupation of arable land, so it is a promising starchy energy plant. A new submerged duckweed mutant (sub-1) with abundant starch accumulation was obtained, whose content of amylopectin accounts for 84.04% of the starch granules. Compared with the wild type (Lemna aequinoctialis), the branching degree of starch in sub-1 mutant was significantly increased by 19.6%. Chain length DP 6-12, DP 25-36 and DP > 36 of amylopectin significantly decreased, while chain length DP 13-24 significantly increased. Average chain length of wild-type and sub-1 mutant starches were greater than DP 22. Moreover, the crystal structure and physical properties of starch have changed markedly in sub-1 mutant. For example, the starch crystallinity of sub-1 mutant was only 8.94%, while that of wild-type was 22.3%. Compared with wild type, water solubility of starch was significantly reduced by 29.42%, whereas swelling power significantly increased by 97.07% in sub-1 mutant. In order to further analyze the molecular mechanism of efficient accumulation of amylopectin in sub-1 mutant, metabolome and transcriptome were performed. The results showed that glucose accumulated in sub-1 mutant, then degradation of starch to glucose mainly depends on α-amylase. At night, the down-regulated ß-amylase gene resulted in the inhibition of starch degradation. The starch and sucrose metabolism pathways were significantly enriched. Up-regulated expression of SUS, AGPase2, AGPase3, PYG, GPI and GYS provide sufficient substrate for starch synthesis in sub-1 mutant. From the 0H to 16H light treatment, granule-bound starch synthase (GBSS1) gene was inhibited, on the contrary, the starch branching enzyme (SBE) gene was induced. Differential expression of GBSS1 and SBE may be an important reason for the decrease ratio of amylose/amylopectin in sub-1 mutant. Taken together, our results indicated that the sub-1 mutant can accumulate the amylopectin efficiently, potentially through altering the differential expression of AGPase, GBSS1, SBE, and BAM. This study also provides theoretical guidance for creating crop germplasm with high amylopectin by means of synthetic biology in the future.