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.

Pleiotropic Effects of miR5504 Underlying Plant Height, Grain Yield and Quality in Rice.

MicroRNAs (miRNAs) are known to play critical roles in regulating rice agronomic traits through mRNA cleavage or translational repression. Our previous study indicated that miR5504 regulates plant height by affecting cell proliferation and expansion. Here, the two independent homozygous mir5504 mutants (CR1 and CR2) and overexpression lines (OE1 and OE2) were further used to investigate the functions of miR5504. The panicle length, 1000-grain weight and grain yield per plant of miR5504-OE lines were identical to those of Nipponbare (NIP), but the 1000-grain weight of mir5504 mutants was reduced by about 10% and 9%, respectively. Meanwhile, the grain width and thickness of mir5504 mutants decreased significantly by approximately 10% and 11%, respectively. Moreover, the cytological results revealed a significant decrease in cell number along grain width direction and cell width in spikelet in mir5504, compared with those in NIP. In addition, several major storage substances of the rice seeds were measured. Compared to NIP, the amylose content of the mir5504 seeds was noticeably decreased, leading to an increase of nearly 10 mm in gel consistency (GC) in mir5504 lines. Further investigation confirmed that LOC_Os08g16914 was the genuine target of miR5504: LOC_Os08g16914 over-expression plants phenocopied the mir5504 mutants. This study provides insights into the role of miR5504 in rice seed development.

Genome-wide association analysis and transgenic characterization for amylose content regulating gene in tuber of Dioscorea zingiberensis.

BACKGROUND: Amylose, a prebiotic found in yams is known to be beneficial for the gut microflora and is particularly advantageous for diabetic patients' diet. However, the genetic machinery underlying amylose production remains elusive. A comprehensive characterization of the genetic basis of amylose content in yam tubers is a prerequisite for accelerating the genetic engineering of yams with respect to amylose content variation. RESULTS: To uncover the genetic variants underlying variation in amylose content, we evaluated amylose content in freshly harvested tubers from 150 accessions of Dioscorea zingibensis. With 30,000 high-quality single nucleotide polymorphisms (SNP), we performed a genome-wide association analysis (GWAS). The population structure analysis classified the D. zingiberensis accessions into three groups. A total of 115 significant loci were detected on four chromosomes. Of these, 112 significant SNPs (log10(p) = 5, q-value < 0.004) were clustered in a narrow window on the chromosome 6 (chr6). The peak SNP at the position 75,609,202 on chr6 could explain 63.15% of amylose variation in the population and fell into the first exon of the ADP-glucose pyrophosphorylase (AGPase) small subunit gene, causing a non-synonymous modification of the resulting protein sequence. Allele segregation analysis showed that accessions with the rare G allele had a higher amylose content than those harboring the common A allele. However, AGPase, a key enzyme precursor of amylose biosynthesis, was not expressed differentially between accessions with A and G alleles. Overexpression of the two variants of AGPase in Arabidopsis thaliana resulted in a significantly higher amylose content in lines transformed with the AGPase-G allele. CONCLUSIONS: Overall, this study showed that a major genetic variant in AGPase probably enhances the enzyme activity leading to high amylose content in D. zingiberensis tuber. The results provide valuable insights for the development of amylose-enriched genotypes.

Combining genome-wide association study and linkage mapping in the genetic dissection of amylose content in maize (Zea mays L.).

KEY MESSAGE: Integrated linkage and association analysis revealed genetic basis across multiple environments. The genes Zm00001d003102 and Zm00001d015905 were further verified to influence amylose content using gene-based association study. Maize kernel amylose is an important source of human food and industrial raw material. However, the genetic basis underlying maize amylose content is still obscure. Herein, we used an intermated B73 × Mo17 (IBM) Syn10 doubled haploid population composed of 222 lines and a germplasm set including 305 inbred lines to uncover the genetic control for amylose content under four environments. Linkage mapping detected 16 unique QTL, among which four were individually repeatedly identified across multiple environments. Genome-wide association study revealed 17 significant (P = 2.24E-06) single-nucleotide polymorphisms, of which two (SYN19568 and PZE-105090500) were located in the intervals of the mapped QTL (qAC2 and qAC5-3), respectively. According to the two population co-localized loci, 20 genes were confirmed as the candidate genes for amylose content. Gene-based association analysis indicated that the variants in Zm00001d003102 (Beta-16-galactosyltransferase GALT29A) and Zm00001d015905 (Sugar transporter 4a) affected amylose content across multi-environment. Tissue expression analysis showed that the two genes were specifically highly expressed in the ear and stem, respectively, suggesting that they might participate in sugar transport from source to sink organs. Our study provides valuable genetic information for breeding maize varieties with high amylose.

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.

Silicon nutrition improves the quality and yield of rice under dry cultivation.

BACKGROUND: Dry cultivation of rice is a water-saving, emission reduction and labor-saving rice farming method. However, the development of rice under dry cultivation is hampered by the limitations of dry cultivation on rice yield and rice quality. We hypothesized that additional silicon (Si) would be a measure to address these limitations or challenges. RESULTS: In the present study, we set up field trials with three treatments: flooded cultivation (W), dry cultivation (D) and dry cultivation plus Si. Yield and quality were reduced under D treatment compared to W treatment. The addition of Si promoted root development, increased plant height and leaf area, increased photosynthetic enzyme activity, net photosynthetic rate and SPAD values, and increased biomass under dry crop conditions. Under the drought conditions, silica up-regulated the expression of AGPSI, SBEI, SBEIIb, SSI and SSII-1 genes and the activities of ADP-glucose pyrophosphorylase (AGPase), soluble starch synthetase (SSS) and starch branching enzyme (SBE) enzymes, which reduced protein, amylose, chalkiness percentage and chalkiness degree, increased brown rice rate, milled rice rate and head milled rice rate, and also improved rice quality. In addition, the increase of AGPase, SSS and SBE enzyme activities promoted the filling rate and the number of spikes was guaranteed, whereas the yield was improved by promoting the seed setting rate and 1000-grain weight. CONCLUSION: The results of the present study indicate that adding appropriate amounts of Si fertilizer can improve the yield and quality of rice under dry cultivation by regulating source supply capacity and grain starch synthesis. © 2023 Society of Chemical Industry.

Sticking to starch.

Not all starches in the human diet are created equal: "resistant starches" are consolidated aggregates of the α-glucan polysaccharides amylose and amylopectin, which escape digestion by salivary and pancreatic amylases. Upon reaching the large intestine, resistant starches become fodder for members of the human gut microbiota, impacting the metabolism of both the symbionts and the host. In a recent study, Koropatkin et al. provided new molecular insight into how a keystone bacterium in the human gut microbiota adheres to resistant starches as a prelude to their breakdown and fermentation.

Sas20 is a highly flexible starch-binding protein in the Ruminococcus bromii cell-surface amylosome.

Ruminococcus bromii is a keystone species in the human gut that has the rare ability to degrade dietary resistant starch (RS). This bacterium secretes a suite of starch-active proteins that work together within larger complexes called amylosomes that allow R. bromii to bind and degrade RS. Starch adherence system protein 20 (Sas20) is one of the more abundant proteins assembled within amylosomes, but little could be predicted about its molecular features based on amino acid sequence. Here, we performed a structure-function analysis of Sas20 and determined that it features two discrete starch-binding domains separated by a flexible linker. We show that Sas20 domain 1 contains an N-terminal ß-sandwich followed by a cluster of α-helices, and the nonreducing end of maltooligosaccharides can be captured between these structural features. Furthermore, the crystal structure of a close homolog of Sas20 domain 2 revealed a unique bilobed starch-binding groove that targets the helical α1,4-linked glycan chains found in amorphous regions of amylopectin and crystalline regions of amylose. Affinity PAGE and isothermal titration calorimetry demonstrated that both domains bind maltoheptaose and soluble starch with relatively high affinity (Kd ≤ 20 µM) but exhibit limited or no binding to cyclodextrins. Finally, small-angle X-ray scattering analysis of the individual and combined domains support that these structures are highly flexible, which may allow the protein to adopt conformations that enhance its starch-targeting efficiency. Taken together, we conclude that Sas20 binds distinct features within the starch granule, facilitating the ability of R. bromii to hydrolyze dietary RS.

Alternative splicing of OsGS1;1 affects nitrogen-use efficiency, grain development, and amylose content in rice.

Excessive nitrogen fertilizer application is harmful to the environment and reduces the quality of cereal crops. Maintaining crop yields under low nitrogen (LN) conditions and improving quality are important goals for cereal crop breeding. Although the effects of nitrogen assimilation on crop nitrogen-use efficiency (NUE) have been intensively studied, natural variations of the key assimilation genes underlying grain development and quality are largely unclear. Here, we identified an NUE-associated gene, OsGS1;1, encoding glutamine synthase, through genome-wide association analysis, followed by validation experiments and functional analysis. Fifteen single-nucleotide polymorphisms in the OsGS1;1 region led to alternative splicing that generated two functional transcripts: OsGS1;1a and OsGS1;1b. The elite haplotype of OsGS1;1 showed high OsGS1;1b activity, which improved NUE, affected grain development, and reduced amylose content. The results show that OsGS1;1, which is induced under LN conditions, affects grain formation by regulating sugar metabolism and may provide a new avenue for the breeding of high-yield and high-quality rice (Oryza sativa).

Utilization of natural alleles for heat adaptability QTLs at the flowering stage in rice.

BACKGROUND: Heat stress threatens rice yield and quality at flowering stage. In this study, average relative seed setting rate under heat stress (RHSR) and genotypes of 284 varieties were used for a genome-wide association study. RESULTS: We identified eight and six QTLs distributed on chromosomes 1, 3, 4, 5, 7 and 12 in the full population and indica, respectively. qHTT4.2 was detected in both the full population and indica as an overlapping QTL. RHSR was positively correlated with the accumulation of heat-tolerant superior alleles (SA), and indica accession contained at least two heat-tolerant SA with average RHSR greater than 43%, meeting the needs of stable production and heat-tolerant QTLs were offer yield basic for chalkiness degree, amylose content, gel consistency and gelatinization temperature. Chalkiness degree, amylose content, and gelatinization temperature under heat stress increased with accumulation of heat-tolerant SA. Gel consistency under heat stress decreased with polymerization of heat-tolerant SA. The study revealed qHTT4.2 as a stable heat-tolerant QTL that can be used for breeding that was detected in the full population and indica. And the grain quality of qHTT4.2-haplotype1 (Hap1) with chalk5, wx, and alk was better than that of qHTT4.2-Hap1 with CHALK5, WX, and ALK. Twelve putative candidate genes were identified for qHTT4.2 that enhance RHSR based on gene expression data and these genes were validated in two groups. Candidate genes LOC_Os04g52830 and LOC_Os04g52870 were induced by high temperature. CONCLUSIONS: Our findings identify strong heat-tolerant cultivars and heat-tolerant QTLs with great potential value to improve rice tolerance to heat stress, and suggest a strategy for the breeding of yield-balance-quality heat-tolerant crop varieties.

Evaluation of altered starch mutants and identification of candidate genes responsible for starch variation in wheat.

BACKGROUND: Induction of mutation through chemical mutagenesis is a novel approach for preparing diverse germplasm. Introduction of functional alleles in the starch biosynthetic genes help in the improvement of the quality and yield of cereals. RESULTS: In the present study, a set of 350 stable mutant lines were used to evaluate dynamic variation of the total starch contents. A megazyme kits were used for measuring the total starch content, resistant starch, amylose, and amylopectin content. Analysis of variance showed significant variation (p < 0.05) in starch content within the population. Furthermore, two high starch mutants (JE0173 and JE0218) and two low starch mutants (JE0089 and JE0418) were selected for studying different traits. A multiple comparison test showed that significant variation in all physiological and morphological traits, with respect to the parent variety (J411) in 2019-2020 and 2020-2021. The quantitative expression of starch metabolic genes revealed that eleven genes of JE0173 and twelve genes of JE0218 had consistent expression in high starch mutant lines. Similarly, in low starch mutant lines, eleven genes of JE0089 and thirteen genes of JE0418 had consistent expression in all stages of seed development. An additional two candidate genes showed over-expression (PHO1, PUL) in the high starch mutant lines, indicating that other starch metabolic genes may also contribute to the starch biosynthesis. The overexpression of SSII, SSIII and SBEI in JE0173 may be due to presence of missense mutations in these genes and SSI also showed overexpression which may be due to 3-primer_UTR variant. These mutations can affect the other starch related genes and help to increase the starch content in this mutant line (JE0173). CONCLUSIONS: This study screened a large scale of mutant population and identified mutants, could provide useful genetic resources for the study of starch biosynthesis and genetic improvement of wheat in the future. Further study will help to understand new genes which are responsible for the fluctuation of total starch.

Overexpression of the ZmSUS1 gene alters the content and composition of endosperm starch in maize (Zea mays L.).

MAIN CONCLUSION: ZmSUS1 increases the amylose content of maize by regulating the expression of Shrunken2 (Sh2) and Brittle2 (Bt2) which encode the size subunits of endosperm ADP-glucose pyrophosphorylase, and Granule bound starchsynthase1 (GBSS1) and Starch synthase1 (SS1). Cereal crops accumulate starch in seeds as an energy reserve. Sucrose Synthase (SuSy) plays an important role in grain starch synthesis. In this study, ZmSUS1 was transformed into maize inbred line KN5585, and transgenic plants were obtained. Compared with the non-transgenic negative control, the content and activity of SuSy were significantly increased, the amylose content in mature seeds of transgenic maize increased by 41.1-69.2%, the total starch content increased by 5.0-13.5%, the 100-grain weight increased by 19.0-26.2% and the average diameter of starch granules increased by 10.8-17.2%. These results indicated that overexpression of ZmSUS1 can significantly improve the traits of maize seeds and obtain new lines with high amylose content. It was also found that the overexpression of ZmSUS1 may increase the amylose content by altering the expression of endosperm ADP-glucose pyrophosphorylase (AGPase) subunits Shrunken2 (Sh2) and Brittle2 (Bt2). Moreover, the ectopic expression of ZmSUS1 also affected the expression of Granule bound starch synthase1 (GBSS1) and Starch synthase1 (SS1) which encode starch synthase. This study proved the important role of ZmSUS1 in maize starch synthesis and provided a new technology strategy for improving maize starch content and yield.

Improving rice eating and cooking quality by enhancing endogenous expression of a nitrogen-dependent floral regulator.

Improving rice eating and cooking quality (ECQ) is one of the primary tasks in rice production to meet the rising demands of consumers. However, improving grain ECQ without compromising yield faces a great challenge under varied nitrogen (N) supplies. Here, we report the approach to upgrade rice ECQ by native promoter-controlled high expression of a key N-dependent floral and circadian clock regulator Nhd1. The amplification of endogenous Nhd1 abundance alters rice heading date but does not affect the entire length of growth duration, N use efficiency and grain yield under both low and sufficient N conditions. Enhanced expression of Nhd1 reduces amylose content, pasting temperature and protein content while increasing gel consistence in grains. Metabolome and transcriptome analyses revealed that increased expression of Nhd1 mainly regulates the metabolism of carbohydrates and amino acids in the grain filling stage. Moreover, expression level of Nhd1 shows a positive relationship with grain ECQ in some local main cultivars. Thus, intensifying endogenous abundance of Nhd1 is a promising strategy to upgrade grain ECQ in rice production.

The OsNAC24-OsNAP protein complex activates OsGBSSI and OsSBEI expression to fine-tune starch biosynthesis in rice endosperm.

Starch accounts for up to 90% of the dry weight of rice endosperm and is a key determinant of grain quality. Although starch biosynthesis enzymes have been comprehensively studied, transcriptional regulation of starch-synthesis enzyme-coding genes (SECGs) is largely unknown. In this study, we explored the role of a NAC transcription factor, OsNAC24, in regulating starch biosynthesis in rice. OsNAC24 is highly expressed in developing endosperm. The endosperm of osnac24 mutants is normal in appearance as is starch granule morphology, while total starch content, amylose content, chain length distribution of amylopectin and the physicochemical properties of the starch are changed. In addition, the expression of several SECGs was altered in osnac24 mutant plants. OsNAC24 is a transcriptional activator that targets the promoters of six SECGs; OsGBSSI, OsSBEI, OsAGPS2, OsSSI, OsSSIIIa and OsSSIVb. Since both the mRNA and protein abundances of OsGBSSI and OsSBEI were decreased in the mutants, OsNAC24 functions to regulate starch synthesis mainly through OsGBSSI and OsSBEI. Furthermore, OsNAC24 binds to the newly identified motifs TTGACAA, AGAAGA and ACAAGA as well as the core NAC-binding motif CACG. Another NAC family member, OsNAP, interacts with OsNAC24 and coactivates target gene expression. Loss-of-function of OsNAP led to altered expression in all tested SECGs and reduced the starch content. These results demonstrate that the OsNAC24-OsNAP complex plays key roles in fine-tuning starch synthesis in rice endosperm and further suggest that manipulating the OsNAC24-OsNAP complex regulatory network could be a potential strategy for breeding rice cultivars with improved cooking and eating quality.

Genetic Engineering of Starch Biosynthesis in Maize Seeds for Efficient Enzymatic Digestion of Starch during Bioethanol Production.

Maize accumulates large amounts of starch in seeds which have been used as food for human and animals. Maize starch is an importantly industrial raw material for bioethanol production. One critical step in bioethanol production is degrading starch to oligosaccharides and glucose by α-amylase and glucoamylase. This step usually requires high temperature and additional equipment, leading to an increased production cost. Currently, there remains a lack of specially designed maize cultivars with optimized starch (amylose and amylopectin) compositions for bioethanol production. We discussed the features of starch granules suitable for efficient enzymatic digestion. Thus far, great advances have been made in molecular characterization of the key proteins involved in starch metabolism in maize seeds. The review explores how these proteins affect starch metabolism pathway, especially in controlling the composition, size and features of starch. We highlight the roles of key enzymes in controlling amylose/amylopectin ratio and granules architecture. Based on current technological process of bioethanol production using maize starch, we propose that several key enzymes can be modified in abundance or activities via genetic engineering to synthesize easily degraded starch granules in maize seeds. The review provides a clue for developing special maize cultivars as raw material in the bioethanol industry.

Targeted Delivery of Butyrate Improves Glucose Homeostasis, Reduces Hepatic Lipid Accumulation and Inflammation in db/db Mice.

Butyrate produced by the gut microbiota has beneficial effects on metabolism and inflammation. Butyrate-producing bacteria are supported by diets with a high fiber content, such as high-amylose maize starch (HAMS). We investigated the effects of HAMS- and butyrylated HAMS (HAMSB)-supplemented diets on glucose metabolism and inflammation in diabetic db/db mice. Mice fed HAMSB had 8-fold higher fecal butyrate concentration compared to control diet-fed mice. Weekly analysis of fasting blood glucose showed a significant reduction in HAMSB-fed mice when the area under the curve for all five weeks was analyzed. Following treatment, fasting glucose and insulin analysis showed increased homeostatic model assessment (HOMA) insulin sensitivity in the HAMSB-fed mice. Glucose-stimulated insulin release from isolated islets did not differ between the groups, while insulin content was increased by 36% in islets of the HAMSB-fed mice. Expression of insulin 2 was also significantly increased in islets of the HAMSB-fed mice, while no difference in expression of insulin 1, pancreatic and duodenal homeobox 1, MAF bZIP transcription factor A and urocortin 3 between the groups was observed. Hepatic triglycerides in the livers of the HAMSB-fed mice were significantly reduced. Finally, mRNA markers of inflammation in liver and adipose tissue were reduced in mice fed HAMSB. These findings suggest that HAMSB-supplemented diet improves glucose metabolism in the db/db mice, and reduces inflammation in insulin-sensitive tissues.

Novel quality features to expand durum wheat applications.

Durum wheat represents a staple food in the human diet owing to its nutritional and technological features. In comparison to common wheat, durum wheat has higher tolerance to biotic and abiotic stresses. However, its production and culinary uses are limited compared to common wheat. Therefore, significant attention was attributed to upgrading the key quality of durum wheat (i.e., hardness, protein, starch and color). This review intends to put the spotlight on the modification of these properties to create new functionalities suiting a wider range of food applications based on critical compilation of scientific publications. Targeting specific genes has been shown to be a valuable strategy to design novel wheat varieties with higher nutritional value (e.g., high amylose), improved technological properties (e.g., higher glutenin content), attractive appearance (e.g., colored wheat) and new uses (e.g., soft durum wheat for breadmaking). Further efforts are still needed to find efficient ways to stabilize and maintain these properties. © 2022 Society of Chemical Industry.

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

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

Structural elements determining the transglycosylating activity of glycoside hydrolase family 57 glycogen branching enzymes.

Glycoside hydrolase family 57 glycogen branching enzymes (GH57GBE) catalyze the formation of an α-1,6 glycosidic bond between α-1,4 linked glucooliogosaccharides. As an atypical family, a limited number of GH57GBEs have been biochemically characterized so far. This study aimed at acquiring a better understanding of the GH57GBE family by a systematic sequence-based bioinformatics analysis of almost 2500 gene sequences and determining the branching activity of several native and mutant GH57GBEs. A correlation was found in a very low or even no branching activity with the absence of a flexible loop, a tyrosine at the loop tip, and two ß-strands.

Protein targeting to starch 1, a functional protein of starch biosynthesis in wheat (Triticum aestivum L.).

KEY MESSAGE: TaPTST1, a wheat homolog of AtPTST1 containing CBM can interact with GBSSI and regulate starch metabolism in wheat endosperm. In cereal endosperm, native starch comprising amylose and amylopectin is synthesized by the coordinated activities of several pathway enzymes. Amylose in starch influences its physio-chemical properties resulting in several human health benefits. The Granule-Bound Starch Synthase I (GBSSI) is the most abundant starch-associated protein. GBSSI lacks dedicated Carbohydrate-binding module (CBM). Previously, Protein Targeting To Starch 1 (PTST1) was identified as a crucial protein for the localization of GBSSI to the starch granules in Arabidopsis. The function of its homologous protein in the wheat endosperm is not known. In this study, TaPTST1, an AtPTST1 homolog, containing a CBM and a coiled-coil domain was identified in wheat. Protein-coding nucleotide sequence of TaPTST1 from Indian wheat variety 'C 306' was cloned and characterized. Homology modelling and molecular docking suggested the potential interaction of TaPTST1 with glucans and GBSSI. The TaPTST1 expression was higher in wheat grain than the other tissues, suggesting a grain-specific function. In vitro binding assays demonstrated different binding affinities of TaPTST1 for native starch, amylose, and amylopectin. Furthermore, the immunoaffinity pull-down assay revealed that TaPTST1 directly interacts with GBSSI, and the interaction is mediated by a coiled-coil domain. The direct protein-protein interaction was further confirmed by bimolecular fluorescence complementation assay (BiFC) in planta. Based on our findings we postulate a functional role for TaPTST1 in starch metabolism by targeting GBSSI to starch granules in wheat endosperm.