Granule-bound Starch Synthase Research Articles

Transcriptomics and starch biosynthesis analysis in leaves and developing seeds of mung bean provide a basis for genetic engineering of starch composition and seed quality.

Mung bean starch is distinguished by its exceptional high amylose content and regulation of starch biosynthesis in leaves and storage tissues, such as seeds, share considerable similarities. Genetic engineering of starch composition and content, requires detailed knowledge of starch biosynthetic gene expression and enzymatic regulation. In this study we applied detailed transcriptomic analyses to unravel the global differential gene expression patterns in mung bean leaves and in seeds during various stages of development. The objective was to identify candidate genes and regulatory mechanisms that may enable generation of desirable seed qualities through the use of genetic engineering. Notable differences in gene expression, in particular low expression of the Protein Targeting to Starch (PTST), starch synthase (SS) 3, and starch branching enzyme1 (SBE1) encoding genes in developing seeds as compared to leaves were evident. These differences were related to starch molecular structures and granule morphologies. Specifically, the starch molecular size distribution at different stages of seed development correlated with the starch biosynthesis gene expression of the SBE1, SS1, granule-bound starch synthases (GBSS) and isoamylase 1 (ISA1) encoding genes. Furthermore, putative hormonal and redox controlled regulation were observed, which may be explained by abscisic acid (ABA) and indole-3-acetic acid (IAA) induced signal transduction, and redox regulation of ferredoxins and thioredoxins, respectively. The morphology of starch granules in leaves and developing seeds were clearly distinguishable and could be correlated to differential expression of SS1. Here, we present a first comprehensive transcriptomic dataset of developing mung bean seeds, and combined these findings may enable generation of genetic engineering strategies of for example starch biosynthetic genes for increasing starch levels in seeds and constitute a valuable toolkit for improving mung bean seed quality.

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.

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.

Grain Chalkiness Is Decreased by Balancing the Synthesis of Protein and Starch in Hybrid Indica Rice Grains under Nitrogen Fertilization.

The important reason for the commercial value of hybrid rice suffering is due to excessive chalkiness, and the biosynthesis of starch and proteins is critical for regulating chalkiness; however, it is currently unclear how the application of N fertilizer affects grains to reduce their chalkiness and improve their quality. The 2019, 2020, and 2021 trials were conducted in a split-plot design, with high and low chalky varieties as the main plot and N fertilizer rate as the split-plot. The effects of fertilization with 75, 150, and 225 kg N ha-1 on the dynamic synthesis of starch, protein, and endogenous hormones and on the amino acid of hybrid indica rice kernels with different degrees of chalkiness were investigated. Grain physiological activity was higher in low-chalky varieties than in high-chalky varieties, and these physiological parameters were strongly associated with chalkiness formation. Higher N fertilization (150 and 225 kg N ha-1) significantly reduced the proportion of chalky grains (8.93-28.02%) and chalkiness (8.61-33.99%) compared with 75 kg N ha-1. Increased N fertilization decreased the activities of granule-bound starch synthase and starch-debranching enzyme, but significantly increased adenosine diphosphate glucose pyrophosphorylase, soluble starch synthase, and starch-branching enzyme activities, synergistically improving glutamate synthetase and glutamine synthetase enzyme activities, which tended to support the synthesis of amylopectin, α-ketoglutarate, and 3-phosphoglyceric acid-derived amino acids in the endosperm cells of the grains; this favored starch and protein accumulation in the grains at 6-30 days after anthesis. Additionally, N application promoted the synthesis of endogenous hormones 1-aminocyclopropane-1-carboxylic acid, gibberellins, and abscisic acid in grains. Hence, N fertilization reduced the rice chalkiness in hybrid indica rice varieties by balancing grain protein and starch composition and enhancing some endogenous hormone synthesis.

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

BackgroundHigher 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.ResultsOur 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.ConclusionsAccording 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.

Effects of elevated atmospheric [CO2] on grain starch characteristics in different specialized wheat.

The increasing atmospheric [CO2] poses great challenges to wheat production. Currently, the response of starch characteristics in different specialized wheat cultivars to elevated [CO2], as well as the underlying physiological and molecular mechanisms remains unclear. Therefore, an experiment was conducted with open-top chambers to study the effects of ambient [CO2] [a(CO2)] and elevated [CO2] [e(CO2)] on photosynthetic performance, yield and starch characteristics of bread wheat (Zhengmai 369, ZM369) and biscuit wheat (Yangmai 15, YM15) from 2020 to 2022. The results demonstrated a significant improvement in photosynthetic performance, yield, amylose and amylopectin content, volume ratio of large granules under e[CO2]. Moreover, e[CO2] upregulated the gene expression and enzyme activities of GBSS (Granule-bound starch synthase) and SSS (Soluble starch synthase), increased starch pasting viscosity, gelatinization enthalpy and crystallinity. Compared to YM15, ZM369 exhibited a higher upregulation of GBSSI, greater increase in amylose content and volume ratio of large granules, as well as higher gelatinization enthalpy and crystallinity. However, ZM369 showed a lower increase in amylopectin content and a lower upregulation of SSSI and SSSII. Correlation analysis revealed amylose and amylopectin content had a positive correlation with GBSS and SSS, respectively, a significant positively correlation among the amylose and amylopectin content, starch granule volume, and pasting properties. In conclusion, these changes may enhance the utilization value of biscuit wheat but exhibit an opposite effect on bread wheat. The results provide a basis for selecting suitable wheat cultivars and ensuring food security under future climate change conditions.

Effect of calcium and magnesium on starch synthesis in maize kernels and its physiological driving mechanism.

The content of kernel starch (STC), which is a fundamental indicator of the nutritional value of maize, is directly correlated with the grain's taste and aroma. Both calcium (Ca) and magnesium (Mg) are critical nutrients that play a significant role in the growth and development of maize, as well as in the synthesis of STC. To determine the physiological driving mechanisms of Ca and Mg effects on the accumulation of STC synthesis in maize kernels and the characteristics of their effects on endogenous hormones and enzymes of STC synthesis in maize leaves, our study applied foliar Ca and Mg fertilizers at various levels to maize prior to pollination. (1) The levels of Ca, Mg, indole-3-acetic acid (IAA), gibberellin (GA), and zeatin riboside (ZR) in maize leaves increased and then decreased after the supplementation of Ca and Mg. They peaked on the 32nd day after pollination. In contrast, the levels of abscisic acid (ABA) initially decreased and then increased. Ca and Mg had a negative correlation with ABA and a positive correlation with IAA, GA, and ZR. (2) As the levels of Ca and Mg increased, correspondingly rose the activities of enzymes responsible for STC synthesis and the content of STC and its components. Principally influencing the synthesis of STC were ABA, IAA, uridine diphosphate-glucose pyrophosphorylase (UDPG), granule-bound starch synthase (GBSS), and soluble starch synthase (SSS). (3) "IAA-UDPG or GBSS-STC" was the predominant physiological regulation pathway of Ca on kernel STC, whereas "IAA-GBSS-STC" was the dominant physiological regulation pathway of Mg on kernel STC. The regulatory impact of STC by UDPG and GBSS was positive, as were the effects of IAA on UDPG and GBSS. In conclusion, the accumulation of kernel starch was significantly enhanced by Ca and Mg supplementation via the modulation of endogenous hormone levels and key enzyme activities. This research identifies a viable approach to improve the nutritional composition of maize.

Physicochemical properties and molecular mechanisms of different resistant starch subtypes in rice.

Resistant starch (RS) can help prevent diabetes and decrease calorie intake and that from plants are the main source of mankind consumption. Rice is many people's staple food and that with higher RS will help health management. A significantly positive correlation exists between apparent amylose content (AAC) of rice and its RS content. In this study, 72 accessions with moderate or high AAC were selected to explore the regulatory mechanisms and physicochemical properties on different proceeding types of rice RS. RS in raw milled rice (RSm), hot cooked rice (RSc), and retrogradation rice (RSr) showed a wide variation and distinct controlling mechanisms. They were co-regulated by Waxy (Wx), soluble starch synthase (SS) IIb and SSI. Besides that, RSm was also regulated by SSIIa and SSIVb, RSc by granule-bound starch synthase (GBSS) II and RSr by GBSSII and Pullulanase (PUL). Moreover, Wx had significant interactions with SSIIa, SSI, SSIIb and SSIVb on RSm, but only the dominant interactions with SSIIb and SSI on RSc and RSr. Wx was the key factor for the formation of RS, especially the RSc and RSr. The genes had the highest expression at 17 days after flowering and were beneficial for RS formation. The longer the chain length of starch, the higher the RS3 content. RSc and RSr were likely to be contained in medium-size starch granules. The findings favor understanding the biosynthesis of different subtypes of RS.

Effects of Post-Anthesis Irrigation on the Activity of Starch Synthesis-Related Enzymes and Wheat Grain Quality under Different Nitrogen Conditions.

To develop optimal management strategies for water and nitrogen fertilizer application in winter wheat cultivation, we conducted a potted experiment to investigate the effects of different irrigation levels and nitrogen fertilizer treatments on the activity of starch synthesis-related enzymes and the grain quality of winter wheat. The potted experiment consisted of three irrigation levels, with the lower limits set at 50-55% (I0), 60-65% (I1), and 70-75% (I2) of the field capacity. In addition, four levels of nitrogen fertilizer were applied, denoted as N0 (0 kg N hm-2), N1 (120 kg N hm-2), N2 (240 kg N hm-2), and N3 (300 kg N hm-2), respectively. The results revealed the significant impacts of irrigation and nitrogen treatments on the activities of key starch-related enzymes, including adenosine diphosphoglucose pyrophosphrylase (ADPG-PPase), soluble starch synthase (SSS), granule-bound starch synthase (GBSS), and starch branching enzymes (SBE) in wheat grains. These treatments also influenced the starch content, amylopectin content, and, ultimately, wheat yield. In summary, our findings suggest that maintaining irrigation at a lower limit of 60% to 65% of the field capacity and applying nitrogen fertilizer at a rate of 240 kg hm-2 is beneficial for achieving both high yield and high quality in winter wheat cultivation.

The Breeding of Waxy Sorghum Using Traditional Three-Line Method and Marker-Assisted Selection

Sorghum (Sorghum bicolor) exhibits drought resistance and environmental adaptability, making it a crucial cereal crop for semi-arid regions. It has a wide range of uses, including as food, feed, brooms, alcohol production, and bioethanol. In particular, Taiwan imports nearly 50,000 tons of sorghum annually, primarily for the production of sorghum liquor. However, the ideal raw material for high-quality sorghum liquor is waxy sorghum, and not all sorghum varieties imported or promoted in Taiwan are of this waxy type. Consequently, there is a shortage of sufficient waxy sorghum raw materials to meet the demands of the Taiwan market. The occurrence of waxy sorghum (wx) is caused by the mutation of granule-bound starch synthase I (GBBS I), and there are currently several known types of mutants, carrying different wxa, wxb, and wxc waxy alleles. Among them, wxc is a novel mutation type, and in native sorghum in Taiwan, individuals with the waxy allele wxc have been found. The three-line method is a commonly used breeding strategy, which simplifies the process of emasculation to obtain hybrid F1 offspring. In this study, imported sorghum variety Liangnuo No.1 (with male sterility), native glutinous sorghum variety SB6 from Taiwan (carrying the wxc waxy allele), and sorghum reference genome variety BTx623 were used as research materials. The goal was to use the three-line method to produce waxy sorghums, including the male sterile line (A-line), male sterile maintenance line (B-line), and fertility-restoring line (R-line). The breeding results showed that by using backcross breeding, molecular-assisted selection, and traditional field selection methods, high-quality three-line materials (A-, B-, R-lines, named CNA1, CNB1 CNR1, respectively) and F1 hybrid (CNH1) with favorable agronomic traits and yield quality were successfully obtained.

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 → 4)-α linear links and (1 → 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.

Effects of 6-Benzyladenine (6-BA) on the Filling Process of Maize Grains Placed at Different Ear Positions under High Planting Density.

Increasing grain weight under dense planting conditions can further improve maize yield. 6-BA is known to be involved in regulating grain development and influencing grain weight. Maize grain development is closely linked to starch accumulation and hormone levels. In this work, the effects of applying 6-BA at the flowering stage under high density on the grain filling characteristics, starch content, starch synthesis critical enzyme activity, and endogenous hormones levels of maize grains (including inferior grains (IGs) and superior grains (SGs)) of two high-yielding summer maize varieties widely cultivated in China were investigated. The findings indicated that applying 6-BA significantly improved maize yield compared to the control, mainly as a result of increased grain weight due to a faster grain filling rate. Additionally, the activities of enzymes associated with starch synthesis, including sucrose synthase (SuSy), ADP-glucose pyrophosphorylase (AGPase), granule-bound starch synthase (GBSS), soluble starch synthase (SSS), and starch branching enzyme (SBE), were all increased following 6-BA application, thus facilitating starch accumulation in the grains. Applying 6-BA also increased the zeatin riboside (ZR), indole-3-acetic acid (IAA), and abscisic acid (ABA) levels, and reduced the gibberellin (GA3) level in the grains, which further improved grain filling. It is worth noting that IG had a poorer filling process than SG, possibly due to the low activities of critical enzymes for starch synthesis and imbalanced endogenous hormones levels. However, IG responded more strongly to exogenous 6-BA than SG. It appears that applying 6-BA is beneficial in improving filling characteristics, promoting starch accumulation by enhancing the activities of critical enzymes for starch synthesis, and altering endogenous hormones levels in the grains, thus improving grain filling and increasing the final grain weight and yield of maize grown under crowded conditions. These results provide theoretical and technical support for the further utilization of exogenous hormones in high-density maize production.

Efficiency of potato genome editing: Targeted mutation on the genes involved in starch biosynthesis using the CRISPR/dMac3-Cas9 system.

Potato (Solanum tuberosum L.) has a tetraploid genome. To make a mutant lacking a specific gene function, it is necessary to introduce mutations into all four gene alleles. To achieve this goal, we developed a powerful genome editing tool, CRISPR/dMac3-Cas9, which installed the translation enhancer dMac3 that greatly increased the translation of the downstream open reading frame. The CRISPR/dMac3-Cas9 system employing three guide RNAs (gRNAs) greatly elevated the frequency of the generation rate of mutation. This system enabled to create the 4-allele mutants of granule-bound starch synthase (GBSS) and starch branching enzyme (SBE). These mutants indicated functionally defective features, suggesting that we succeeded in efficient genome editing of the potato tetraploid genome. Here, we show the effect of the number of gRNAs for efficient mutagenesis of the target gene using the mutants of the GBSS1 gene. CRISPR/dMac3-Cas9 employing three gRNA genes achieved a higher mutation efficiency than the CRISPR/dMac3-Cas9 with two gRNAs, suggesting being influenced by the dose effect of the number of gRNAs at the target region. The alleles of the SBE3 gene contained SNPs that caused sequence differences in the gRNAs but these gRNAs functioned efficiently. However, many rearrangement events and large deletions were induced. These results support the importance of accurate binding of gRNA to the target sequence, which may lead to a hint to avoid the unexpected mutation on the off-target sites.

Spermidine enhances heat tolerance of rice seeds during mid-filling stage and promote subsequent seed germination.

Heat stress is a vital factor which restricts rice seed quality and yield. However, the response mechanism to heat stress in the mid filling stage of rice seed is unclear. In the present study we integrated phenotypic analysis with biochemical, hormone, and gene expression analysis in order to explore technologies for improving rice seeds heat tolerance and subsequent seed germination. Spermidine (Spd) application effectively alleviated the damage of heat stress treatment during mid-filling stage (HTM, 12-20 days after pollination) on seed development, promoted subsequent seed germination and seedlings establishment. Spd significantly increased seed dry weight, starch and amylose contents during seed development under heat stress, and improved seed germinate, seedlings establishment and seedling characteristics during germination time. Biochemical analysis indicated that, HTM significantly decreased the activities of several starch synthase enzymes and led to a decrease in starch content. While Spd treatment significantly enhanced the activities of ADP-glucose pyrophosphorylas and granule-bound starch synthase, as well as the corresponding-genes expressions in HTM rice seeds, resulting in the increases of amylose and total starch contents. In addition, Spd significantly increased the catalase and glutathione reductase activities together with corresponding-genes expressions, and lowered the overaccumulation of H2O2 and malondialdehyde in HTM seeds. In the subsequent seed germination process, HTM+Spd seeds exhibited dramatically up-regulated levels of soluble sugars, glucose, ATP and energy charges. Consistently, HTM+Spd seeds showed significantly increased of α-amylose and α-glucosidase activities as well as corresponding-genes expressions during early germination. Moreover, HTM evidently increased the abscisic acid (ABA) content, decreased the gibberellin (GA) content, and accordingly significantly declined the GA/ABA ratio during early rice seeds germination. However, Spd treatment did not significantly affect the metabolism of GA and ABA in seed germination stage. The present study suggested that Spd treatment could effectively alleviate the negative impact of HTM on seed development and the subsequent seed germination, which might be closely correlated with starch synthesis and antioxidant defense during seed filling period, starch decomposition and energy supply in seed germination period.

Genome-Wide Association Study of Starch Properties in Local Thai Rice.

Rice (Oryza sativa L.) is the main source of energy for humans and a staple food of high cultural significance for much of the world's population. Rice with highly resistant starch (RS) is beneficial for health and can reduce the risk of disease, especially type II diabetes. The identification of loci affecting starch properties will facilitate breeding of high-quality and health-supportive rice. A genome-wide association study (GWAS) of 230 rice cultivars was used to identify candidate loci affecting starch properties. The apparent amylose content (AAC) among rice cultivars ranged from 7.04 to 33.06%, and the AAC was positively correlated with RS (R2 = 0.94) and negatively correlated with rapidly available glucose (RAG) (R2 = -0.73). Three loci responsible for starch properties were detected on chromosomes 1, 6, and 11. On chromosome 6, the most significant SNP corresponded to LOC_Os06g04200 which encodes granule-bound starch synthase I (GBSSI) or starch synthase. Two novel loci associated with starch traits were LOC_Os01g65810 and LOC_Os11g01580, which encode an unknown protein and a sodium/calcium exchanger, respectively. The markers associated with GBSSI and LOC_Os11g01580 were tested in two independent sets of rice populations to confirm their effect on starch properties. The identification of genes associated with starch traits will further the understanding of the molecular mechanisms affecting starch in rice and may be useful in the selection of rice varieties with improved starch.

Cytosine base editors optimized for genome editing in potato protoplasts.

In this study, we generated and compared three cytidine base editors (CBEs) tailor-made for potato (Solanum tuberosum), which conferred up to 43% C-to-T conversion of all alleles in the protoplast pool. Earlier, gene-edited potato plants were successfully generated by polyethylene glycol-mediated CRISPR/Cas9 transformation of protoplasts followed by explant regeneration. In one study, a 3-4-fold increase in editing efficiency was obtained by replacing the standard Arabidopsis thaliana AtU6-1 promotor with endogenous potato StU6 promotors driving the expression of the gRNA. Here, we used this optimized construct (SpCas9/StU6-1::gRNA1, target gRNA sequence GGTC4C5TTGGAGC12AAAAC17TGG) for the generation of CBEs tailor-made for potato and tested for C-to-T base editing in the granule-bound starch synthase 1 gene in the cultivar Desiree. First, the Streptococcus pyogenes Cas9 was converted into a (D10A) nickase (nCas9). Next, one of three cytosine deaminases from human hAPOBEC3A (A3A), rat (evo_rAPOBEC1) (rA1), or sea lamprey (evo_PmCDA1) (CDA1) was C-terminally fused to nCas9 and a uracil-DNA glycosylase inhibitor, with each module interspaced with flexible linkers. The CBEs were overall highly efficient, with A3A having the best overall base editing activity, with an average 34.5%, 34.5%, and 27% C-to-T conversion at C4, C5, and C12, respectively, whereas CDA1 showed an average base editing activity of 34.5%, 34%, and 14.25% C-to-T conversion at C4, C5, and C12, respectively. rA1 exhibited an average base editing activity of 18.75% and 19% at C4 and C5 and was the only base editor to show no C-to-T conversion at C12.

The transcription factors ZmNAC128 and ZmNAC130 coordinate with Opaque2 to promote endosperm filling in maize.

Endosperm filling in maize (Zea mays), which involves nutrient uptake and biosynthesis of storage reserves, largely determines grain yield and quality. However, much remains unclear about the synchronization of these processes. Here, we comprehensively investigated the functions of duplicate NAC-type transcription factors, namely ZmNAC128 and ZmNAC130, in endosperm filling. The gene-edited double mutant zmnac128 zmnac130 exhibits a poorly filled kernel phenotype such that the kernels have an inner cavity. RNA sequencing and protein abundance analysis revealed that the expression of many genes involved in the biosynthesis of zein and starch is reduced in the filling endosperm of zmnac128 zmnac130. Further DNA affinity purification and sequencing combined with chromatin-immunoprecipitation quantitative PCR and promoter transactivation assays demonstrated that ZmNAC128 and ZmNAC130 are direct regulators of three (16-, 27-, and 50-kD) γ-zein genes and six important starch metabolism genes (Brittle2 [Bt2], pullulanase-type starch debranching enzyme [Zpu1], granule-bound starch synthase 1 [GBSS1], starch synthase 1 [SS1], starch synthase IIa [SSIIa], and sucrose synthase 1 [Sus1]). ZmNAC128 and ZmNAC130 recognize an additional cis-element in the Opaque2 (O2) promoter to regulate its expression. The triple mutant zmnac128 zmnac130 o2 exhibits extremely poor endosperm filling, which results in more than 70% of kernel weight loss. ZmNAC128 and ZmNAC130 regulate the expression of the transporter genes sugars will eventually be exported transporter 4c (ZmSWEET4C), sucrose and glucose carrier 1 (ZmSUGCAR1), and yellow stripe-like2 (ZmYSL2) and in turn facilitate nutrient uptake, while O2 plays a supporting role. In conclusion, ZmNAC128 and ZmNAC130 cooperate with O2 to facilitate endosperm filling, which involves nutrient uptake in the basal endosperm transfer layer (BETL) and the synthesis of zeins and starch in starchy endosperm (SE).

Effects of Irrigation and Fertilization Management on Yield and Quality of Rice and the Establishment of a Quality Evaluation System

Yield and rice quality indicators of crops are a direct reflection of the rational irrigation and fertilizer strategy. However, the effects of controlled irrigation (CI) combined with the split application of fertilization managements (straw returning, organic fertilizer, and conventional fertilizer) on rice quality are not clear in southeast China. This study aims at exploring the effects of three fertilization managements applied under CI or flooding irrigation on rice yield, quality, enzyme activity, and soluble sugar content including 43 indicators, to determine the optimal comprehensive evaluation model, management, and representative indexes. The results showed that compared with CF (CI + conventional fertilizer), CS (CI + straw returning) significantly increased yield (27.65%), irrigation water use efficiency (6.20%), chalky grain rate (9.67%), chalkiness (1.83%), protein content (4.29%), and amylose content (0.33%), indicating that CS improved yield and milling quality but decreased cooking and appearance quality. This was mainly because CS promoted the activities of alpha-amylase, ADPG (ADP-glucose pyrophosphorylase), and GBSS (granule-bound starch synthase) and reduced the soluble sugar content in rice. Grey relational degree analysis (GRD), the entropy method (ETM), and TOPSIS (the technique for order preference by similarity to an ideal solution) were used to comprehensively evaluate the rice quality and determined that CS treatments could synergistically improve yield and rice quality. The five indexes (adhesive strength, HPV, ADPG, soluble sugar (leaf), yield) and TOPSIS model can be used as the best indexes and model to evaluate the rice quality. These results could provide scientific management and evaluate practices for high-yield and high-quality rice cultivation, which may be promising for a cleaner production strategy.

Evolutionary and expression analysis of starch synthase genes from Tartary buckwheat revealed the potential function of FtGBSSII‐4 and FtGBSSII‐5 in seed amylose biosynthesis

AbstractThe starch synthase has been identified well as a core enzyme in the starch biosynthesis pathway in monocots. However, only a few starch synthase genes (SSs) are known in dicots. Tartary buckwheat (Fagopyrum tataricum Gaertn.) is one of the dicots‐enriched starch in the seeds, but its starch synthesis pathway remains unclear. Here, 15 SS genes were identified in the Tartary buckwheat genome, including 5 granule‐bound starch synthase (GBSS) and 10 soluble starch synthase (SSS) genes. Evolutionary analysis showed that all FtGBSSs belonged to GBSSII, and its isoforms were more abundant than other dicots due to the segmental duplication events. Expression pattern analysis showed two GBSS genes (FtGBSSII‐4 and FtGBSSII‐5) and three SSS genes (FtSSII‐2, FtSSIII‐1, and FtSSIV‐2) were mainly expressed in seeds, indicating that they may be the dominant isoforms for the starch synthesis in Tartary buckwheat. Besides, 23 transcription factor genes out of 1073 were identified from 17 transcription factor families and highly expressed in seeds. Expression correlation analysis suggested that FtNF‐YB2 may be the promising regulators for FtSSs. These results provide new insights and a theoretical basis for understanding starch synthesis and regulation in Tartary buckwheat and other dicots.