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Key message Suppression of starch branching enzymes 1 and 2 in cassava leads to increased resistant starch content through the production of high-amylose and modification of the amylopectin structure. Cassava ( Manihot esculenta Crantz) is a starchy root crop used for human consumption as a staple food and industrial applications. Starch is synthesized by various isoforms of several enzymes. However, the function of starch branching enzymes (SBEs) in starch biosynthesis and mechanisms of starch regulation in cassava have not been understood well. In this study, we aimed to suppress the expression of SBEs in cassava to generate starches with a range of distinct properties, in addition to verifying the functional characteristics of the SBEs. One SBE1 , two SBE2 , and one SBE3 genes were classified by phylogenetic analysis and amino acid alignment. Quantitative real-time RT-PCR revealed tissue-specific expression of SBE genes in the tuberous roots and leaves of cassava. We introduced RNAi constructs containing fragments of SBE1 , SBE2 , or both genes into cassava by Agrobacterium -mediated transformation, and assessed enzymatic activity of SBE using tuberous roots and leaves from these transgenic plants. Simultaneous suppression of SBE1 and SBE2 rendered an extreme starch phenotype compared to suppression of SBE2 alone. Degree of polymerization of 6–13 chains in amylopectin was markedly reduced by suppression of both SBE1 and SBE2 in comparison to the SBE2 suppression; however, no change in chain-length profiles was observed in the SBE1 suppression alone. The role of SBE1 and SBE2 may have functional overlap in the storage tissue of cassava. Simultaneous suppression of SBE1 and SBE2 resulted in highly resistant starch with increased apparent amylose content compared to suppression of SBE2 alone. This study provides valuable information for understanding starch biosynthesis and suggests targets for altering starch quality.

Key message High levels of two major starch synthases, SSIIa and GBSSI, in ss3a ss4b double mutant rice alter the starch structure but fail to recover the polygonal starch granule morphology. The endosperm starch granule is polygonal in wild-type rice but spherical in double mutant japonica rice lacking genes encoding two of the five major Starch synthase (SS) isozymes expressed in endosperm, SSIIIa and SSIVb. Japonica rice naturally has low levels of SSIIa and Granule-bound SSI (GBSSI). Therefore, introduction of active SSIIa allele and/or high-expressing GBSSI allele from indica rice into the japonica rice mutant lacking SS isozymes can help elucidate the compensatory roles of SS isozymes in starch biosynthesis. In this study, we crossed the ss3a ss4a double mutant japonica rice with the indica rice to generate three new rice lines with high and/or low SSIIa and GBSSI levels, and examined their starch structure, physicochemical properties, and levels of other starch biosynthetic enzymes. Lines with high SSIIa levels showed more SSI and SSIIa bound to starch granule, reduced levels of short amylopectin chains (7 ≤ DP ≤ 12), increased levels of amylopectin chains with DP > 13, and consequently higher gelatinization temperature. Lines with high GBSSI levels showed an increase in amylose content. The ADP-glucose content of the crude extract was high in lines with low or high SSIIa and low GBSSI levels, but was low in lines with high GBSSI. Addition of high SSIIa and GBSSI altered the starch structure and physicochemical properties but did not affect the starch granule morphology, confirming that SSIIIa and SSIVb are key enzymes affecting starch granule morphology in rice. The relationship among SS isozymes and its effect on the amount of substrate (ADP-glucose) is discussed.

Rice grain yield and quality are predicted to be highly vulnerable to global warming. Five genotypes including heat-tolerant and susceptible checks, a heat-tolerant near-isogenic line and two hybrids were exposed to control (31 °C/23 °C, day/night), high night-time temperature (HNT; 31 °C/30 °C), high day-time temperature (HDT; 38 °C/23 °C) and high day- and night-time temperature (HNDT; 38 °C/30 °C) treatments for 20 consecutive days during the grain-filling stage. Grain-filling dynamics, starch metabolism enzymes, temporal starch accumulation patterns and the process of chalk formation were quantified. Compensation between the rate and duration of grain filling minimized the impact of HNT, but irreversible impacts on seed-set, grain filling and ultimately grain weight were recorded with HDT and HNDT. Scanning electron microscopy demonstrated irregular and smaller starch granule formation affecting amyloplast build-up with HDT and HNDT, while a quicker but normal amylopast build-up was recorded with HNT. Our findings revealed temporal variation in the starch metabolism enzymes in all three stress treatments. Changes in the enzymatic activity did not derail starch accumulation under HNT when assimilates were sufficiently available, while both sucrose supply and the conversion of sucrose into starch were affected by HDT and HNDT. The findings indicate differential mechanisms leading to high day and high night temperature stress-induced loss in yield and quality. Additional genetic improvement is needed to sustain rice productivity and quality under future climates.

During starch metabolism, the phosphorylation of glucosyl residues of starch, to be more precise of amylopectin, is a repeatedly observed process. This phosphorylation is mediated by dikinases, the glucan, water dikinase (GWD) and the phosphoglucan, water dikinase (PWD). The starch-related dikinases utilize ATP as dual phosphate donor transferring the terminal γ-phosphate group to water and the β-phosphate group selectively to either C6 position or C3 position of a glucosyl residue within amylopectin. By the collaborative action of both enzymes, the initiation of a transition of α-glucans from highly ordered, water-insoluble state to a less order state is realized and thus the initial process of starch degradation. Consequently, mutants lacking either GWD or PWD reveal a starch excess phenotype as well as growth retardation. In this review, we focus on the increased knowledge collected over the last years related to enzymatic properties, the precise definition of the substrates, the physiological implications, and discuss ongoing questions.

Starch is the principle constituent of potato tubers and is of considerable importance for food and non-food applications. Its metabolism has been subject of extensive research over the past decades. Despite its importance, a description of the complete inventory of genes involved in starch metabolism and their genome organization in potato plants is still missing. Moreover, mechanisms regulating the expression of starch genes in leaves and tubers remain elusive with regard to differences between transitory and storage starch metabolism, respectively. This study aimed at identifying and mapping the complete set of potato starch genes, and to study their expression pattern in leaves and tubers using different sets of transcriptome data. Moreover, we wanted to uncover transcription factors co-regulated with starch accumulation in tubers in order to get insight into the regulation of starch metabolism. We identified 77 genomic loci encoding enzymes involved in starch metabolism. Novel isoforms of many enzymes were found. Their analysis will help to elucidate mechanisms of starch biosynthesis and degradation. Expression analysis of starch genes led to the identification of tissue-specific isoenzymes suggesting differences in the transcriptional regulation of starch metabolism between potato leaf and tuber tissues. Selection of genes predominantly expressed in developing potato tubers and exhibiting an expression pattern indicative for a role in starch biosynthesis enabled the identification of possible transcriptional regulators of tuber starch biosynthesis by co-expression analysis. This study provides the annotation of the complete set of starch metabolic genes in potato plants and their genomic localizations. Novel, so far undescribed, enzyme isoforms were revealed. Comparative transcriptome analysis enabled the identification of tuber- and leaf-specific isoforms of starch genes. This finding suggests distinct regulatory mechanisms in transitory and storage starch metabolism. Putative regulatory proteins of starch biosynthesis in potato tubers have been identified by co-expression and their expression was verified by quantitative RT-PCR.

Ostreococcus tauri is a picoalga that contains a small and compact genome, which resembles that of higher plants in the multiplicity of enzymes involved in starch synthesis (ADP-glucose pyrophosphorylase, ADPGlc PPase; granule bound starch synthase, GBSS; starch synthases, SSI, SSII, SSIII; and starch branching enzyme, SBE, between others), except starch synthase IV (SSIV). Although its genome is fully sequenced, there are still many genes and proteins to which no function was assigned. Here, we identify the OT_ostta06g01880 gene that encodes CBM20CP, a plastidial protein which contains a central carbohydrate binding domain of the CBM20 family, and a coiled coil domain at the C-terminus that lacks catalytic activity. We demonstrate that CBM20CP has the ability to bind starch, amylose and amylopectin with different affinities. Furthermore, this protein interacts with OsttaSSIII-B, increasing its binding to starch granules, its catalytic efficiency and promoting granule growth. The results allow us to postulate a functional role for CBM20CP in starch metabolism in green algae. Key message CBM20CP, a plastidial protein that has a modular structure but lacks catalytic activity, regulates the synthesis of starch in Ostreococcus tauri .

Since the role of intestinal microbiota in metabolism was understood, the importance of dietary components such as fibres and prebiotics, which affect the modulation of microbiota, has been increasing day by day. While all prebiotic components are considered dietary fibre, not every dietary fibre is considered a prebiotic. While fructooligosaccharides, galactooligosaccharides, inulin, and galactans are considered prebiotics, other fermentable carbohydrates are considered candidate prebiotic components based on in vitro and preclinical studies. Resistant starch, one of such carbohydrates, is considered a potential prebiotic component when it is made resistant to digestion naturally or chemically. In this review, both in vitro and in vivo studies in which the prebiotic capacity of type II, type III, and type IV resistant starch isolated from food and produced commercially was assessed were analyzed. According to the results of current studies, certain types of resistant starch are thought to have a high prebiotic capacity, and they may be candidate prebiotic components although positive results have not been achieved in all studies. Key points • Resistant starch is undigested in the small intestine and is fermented in the large intestine. • Resistant starch fermentation positively affects the growth of Bifidobacterium and Lactobacillus. • Resistant starch can be considered a prebiotic ingredient.

sugary enhancer1 (se1) is a naturally occurring mutant allele involved in starch metabolism in maize endosperm. It is a recessive modifier of sugary1 (su1) and commercially important in modern sweet corn breeding, but its molecular identity and mode of action remain unknown. Here, we developed a pair of near-isogenic lines, W822Gse (su1-ref/su1-ref se1/se1) and W822GSe (su1-ref/su1-ref Se1/Se1), that Mendelize the se1 phenotype in an su1-ref background. W822Gse kernels have lower starch and higher water soluble polysaccharide and sugars than W822GSe kernels. Using high-resolution genetic mapping, we found that wild-type Se1 is a gene Zm00001d007657 on chromosome 2 and a deletion of this gene causes the se1 phenotype. Comparative metabolic profiling of seed tissue between these 2 isolines revealed the remarkable difference in carbohydrate metabolism, with sucrose and maltose highly accumulated in the mutant. Se1 is predominantly expressed in the endosperm, with low expression in leaf and root tissues. Differential expression analysis identified genes enriched in both starch biosynthesis and degradation processes, indicating a pleiotropic regulatory effect of se1. Repressed expression of Se1 and Su1 in RNA interference-mediated transgenic maize validates that deletion of the gene identified as Se1 is a true causal gene responsible for the se1 phenotype. The findings contribute to our understanding of starch metabolism in cereal crops.

plays a vital role in regulating floret development and grain shape, but whether it regulates rice grain quality still remains largely unknown. Therefore, we used comprehensive molecular genetics, plant biotechnology, and functional omics approaches, including phenotyping, mapping-by-sequencing, target gene seed-specific RNAi, transgenic experiments, and transcriptomic profiling to answer this biological and molecular question. Here, we report the characterization of the 'Oat-like rice' mutant, with poor grain quality, including chalky endosperms, abnormal morphology and loose arrangement of starch granules, and lower starch content but higher protein content in grains. The poor grain quality of Oat-like rice was found to be caused by the mutated allele through mapping-by-sequencing analysis and transgenic experiments. OsMADS1 protein is highly expressed in florets and developing seeds. Both OsMADS1-eGFP and OsMADS1 -eGFP fusion proteins are localized in the nucleus. Moreover, seed-specific RNAi of also caused decreased grain quality in transgenic lines, such as the Oat-like rice. Further transcriptomic profiling between Oat-like rice and Nipponbare grains revealed that regulates gene expressions and regulatory networks of starch and storage protein metabolisms in rice grains, hereafter regulating rice quality. In conclusion, our results not only reveal the crucial role and preliminary mechanism of in regulating rice grain quality but also highlight the application potentials of and the target gene seed-specific RNAi system in improving rice grain quality by molecular breeding.

The molecular mechanism that initiates the synthesis of starch granules is poorly understood. Here, we discovered two plastidial proteins involved in granule initiation in Arabidopsis thaliana leaves. Both contain coiled coils and a family-48 carbohydrate binding module (CBM48) and are homologs of the PROTEIN TARGETING TO STARCH (PTST) protein; thus, we named them PTST2 and PTST3. Chloroplasts in mesophyll cells typically contain five to seven granules, but remarkably, most chloroplasts in ptst2 mutants contained zero or one large granule. Chloroplasts in ptst3 had a slight reduction in granule number compared with the wild type, while those of the ptst2 ptst3 double mutant contained even fewer granules than ptst2. The ptst2 granules were larger but similar in morphology to wild-type granules, but those of the double mutant had an aberrant morphology. Immunoprecipitation showed that PTST2 interacts with STARCH SYNTHASE4 (SS4), which influences granule initiation and morphology. Overexpression of PTST2 resulted in chloroplasts containing many small granules, an effect that was dependent on the presence of SS4. Furthermore, isothermal titration calorimetry revealed that the CBM48 domain of PTST2, which is essential for its function, interacts with long maltooligosaccharides. We propose that PTST2 and PTST3 are critical during granule initiation, as they bind and deliver suitable maltooligosaccharide primers to SS4.