Determination of Glucan Chain Length Distribution of Glycogen Using the Fluorophore-Assisted Carbohydrate Electrophoresis (FACE) Method.

Glycogen particles are branched polysaccharides composed of linear chains of glucosyl units linked by α-1,4 glucoside bonds. The latter are attached to each other by α-1,6 glucoside linkages, referred to as branch points. Among the different forms of carbon storage (i.e., starch, ß-glucan), glycogen is probably one of the oldest and most successful storage polysaccharides found across the living world. Glucan chains are organized so that a large amount of glucose can quickly be stored or fueled in a cell when needed. Numerous complementary techniques have been developed over the last decades to solve the fine structure of glycogen particles. This article describes Fluorophore-Assisted Carbohydrate Electrophoresis (FACE). This method quantifies the population of glucan chains that compose a glycogen particle. Also known as chain length distribution (CLD), this parameter mirrors the particle size and the percentage of branching. It is also an essential requirement for the mathematical modeling of glycogen biosynthesis.

Facilitating gene editing in potato: a Single-Nucleotide Polymorphism (SNP) map of the Solanum tuberosum L. cv. Desiree genome.

Genome editing is a powerful tool for plant functional genomics allowing for multiallelic targeted mutagenesis. The recent development of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated protein 9 (Cas9) systems for gene editing in plants allows for simple, cost-effective introduction of site-specific double-stranded DNA breaks. The nuclear genomes of a homozygous doubled-monoploid potato clone (DM) and a heterozygous diploid clone (RH) have been sequenced in 2011. However, common potato cultivars display a highly heterozygous autotetraploid genome thus complicating target design for tetra-allelic gene editing. Here, we report on the SNP physical map of the widely used Solanum tuberosum L. cv. Desiree and on the position of the diverse indels providing an essential tool for target design in genome editing approaches. We used this tool for designing a specific gRNA and successfully knocking-out a newly discovered starch synthase gene (SS6) in potato. Resequencing data are publicly available at the Sequence Read Archive of the NCBI (accession number: PRJNA507597) and will represent a valuable resource for functional genomic studies of various metabolic pathways, cell and plant physiology as well as high-throughput reverse genetics in potato.

Intra-Sample Heterogeneity of Potato Starch Reveals Fluctuation of Starch-Binding Proteins According to Granule Morphology.

Starch granule morphology is highly variable depending on the botanical origin. Moreover, all investigated plant species display intra-tissular variability of granule size. In potato tubers, the size distribution of starch granules follows a unimodal pattern with diameters ranging from 5 to 100 µm. Several evidences indicate that granule morphology in plants is related to the complex starch metabolic pathway. However, the intra-sample variability of starch-binding metabolic proteins remains unknown. Here, we report on the molecular characterization of size-fractionated potato starch granules with average diameters of 14.2 ± 3.7 µm, 24.5 ± 6.5 µm, 47.7 ± 12.8 µm, and 61.8 ± 17.4 µm. In addition to changes in the phosphate contents as well as small differences in the amylopectin structure, we found that the starch-binding protein stoichiometry varies significantly according to granule size. Label-free quantitative proteomics of each granule fraction revealed that individual proteins can be grouped according to four distinct abundance patterns. This study corroborates that the starch proteome may influence starch granule growth and architecture and opens up new perspectives in understanding the dynamics of starch biosynthesis.

The Solanum tuberosum GBSSI gene: a target for assessing gene and base editing in tetraploid potato.

KEY MESSAGE: The StGBSSI gene was successfully and precisely edited in the tetraploid potato using gene and base-editing strategies, leading to plants with impaired amylose biosynthesis. Genome editing has recently become a method of choice for basic research and functional genomics, and holds great potential for molecular plant-breeding applications. The powerful CRISPR-Cas9 system that typically produces double-strand DNA breaks is mainly used to generate knockout mutants. Recently, the development of base editors has broadened the scope of genome editing, allowing precise and efficient nucleotide substitutions. In this study, we produced mutants in two cultivated elite cultivars of the tetraploid potato (Solanum tuberosum) using stable or transient expression of the CRISPR-Cas9 components to knock out the amylose-producing StGBSSI gene. We set up a rapid, highly sensitive and cost-effective screening strategy based on high-resolution melting analysis followed by direct Sanger sequencing and trace chromatogram analysis. Most mutations consisted of small indels, but unwanted insertions of plasmid DNA were also observed. We successfully created tetra-allelic mutants with impaired amylose biosynthesis, confirming the loss of function of the StGBSSI protein. The second main objective of this work was to demonstrate the proof of concept of CRISPR-Cas9 base editing in the tetraploid potato by targeting two loci encoding catalytic motifs of the StGBSSI enzyme. Using a cytidine base editor (CBE), we efficiently and precisely induced DNA substitutions in the KTGGL-encoding locus, leading to discrete variation in the amino acid sequence and generating a loss-of-function allele. The successful application of base editing in the tetraploid potato opens up new avenues for genome engineering in this species.

Proteome Analysis of Potato Starch Reveals the Presence of New Starch Metabolic Proteins as Well as Multiple Protease Inhibitors.

Starch bound proteins mainly include enzymes from the starch biosynthesis pathway. Recently, new functions in starch molecular assembly or active protein targeting were also proposed for starch associated proteins. The potato genome sequence reveals 77 loci encoding starch metabolizing enzymes with the identification of previously unknown putative isoforms. Here we show by bottom-up proteomics that most of the starch biosynthetic enzymes in potato remain associated with starch even after washing with SDS or protease treatment of the granule surface. Moreover, our study confirmed the presence of PTST1 (Protein Targeting to Starch), ESV1 (Early StarVation1) and LESV (Like ESV), that have recently been identified in Arabidopsis. In addition, we report on the presence of a new isoform of starch synthase, SS6, containing both K-X-G-G-L catalytic motifs. Furthermore, multiple protease inhibitors were also identified that are cleared away from starch by SDS and thermolysin treatments. Our results indicate that SS6 may play a yet uncharacterized function in starch biosynthesis and open new perspectives both in understanding storage starch metabolism as well as breeding improved potato lines.

The Chlamydomonas mex1 mutant shows impaired starch mobilization without maltose accumulation.

The MEX1 locus of Chlamydomonas reinhardtii was identified in a genetic screen as a factor that affects starch metabolism. Mutation of MEX1 causes a slow-down in the mobilization of storage polysaccharide. Cosegregation and functional complementation analyses were used to assess the involvement of the Mex1 protein in starch degradation. Heterologous expression experiments performed in Escherichia coli and Arabidopsis thaliana allowed us to test the capacity of the algal protein in maltose export. In contrast to the A. thaliana mex1 mutant, the mutation in C. reinhardtii does not lead to maltose accumulation and growth impairment. Although localized in the plastid envelope, the algal protein does not transport maltose efficiently across the envelope, but partly complements the higher plant mutant. Both Mex orthologs restore the growth of the E. coli ptsG mutant strain on glucose-containing medium, revealing the capacity of these proteins to transport this hexose. These findings suggest that Mex1 is essential for starch mobilization in both Chlamydomonas and Arabidopsis, and that this protein family may support several functions and not only be restricted to maltose export across the plastidial envelope.

Oil Accumulation in Transgenic Potato Tubers Alters Starch Quality and Nutritional Profile.

Plant storage compounds such as starch and lipids are important for human and animal nutrition as well as industry. There is interest in diverting some of the carbon stored in starch-rich organs (leaves, tubers, and cereal grains) into lipids in order to improve the energy density or nutritional properties of crops as well as providing new sources of feedstocks for food and manufacturing. Previously, we generated transgenic potato plants that accumulate up to 3.3% triacylglycerol (TAG) by dry weight in the tubers, which also led to changes in starch content, starch granule morphology and soluble sugar content. The aim of this study was to investigate how TAG accumulation affects the nutritional and processing properties of high oil potatoes with a particular focus on starch structure, physical and chemical properties. Overall, TAG accumulation was correlated with increased energy density, total nitrogen, amino acids, organic acids and inorganic phosphate, which could be of potential nutritional benefit. However, TAG accumulation had negative effects on starch quality as well as quantity. Starch from high oil potatoes had lower amylose and phosphate content, reduced peak viscosity and higher gelatinization temperature. Interestingly, starch pasting properties were disproportionately affected in lines accumulating the highest levels of TAG (>2.5%) compared to those accumulating only moderate levels (0.2-1.6%). These results indicate that optimized engineering of specialized crops for food, feed, fuel and chemical industries requires careful selection of traits, and an appropriate level of transgene expression, as well as a better understanding of starch structure and carbon partitioning in plant storage organs.

Rapid and sensitive quantification of C3- and C6-phosphoesters in starch by fluorescence-assisted capillary electrophoresis.

Phosphate groups are naturally present in starch at C3- or C6-position of the glucose residues and impact the structure of starch granules. Their precise quantification is necessary for understanding starch physicochemical properties and metabolism. Nevertheless, reliable quantification of Glc-3-P remains laborious and time consuming. Here we describe a capillary electrophoresis method for simultaneous measurement of both Glc-6-P and Glc-3-P after acid hydrolysis of starch. The sensitivity threshold was estimated at the fg scale, which is compatible with the analysis of less than a µg of sample. The method was validated by analyzing antisense potato lines deficient in SBEs, GWD or GBSS. We show that Glc-3-P content is altered in the latter and that these variations do not correlate with modifications in Glc-6-P content. We anticipate the method reported here to be an efficient tool for high throughput study of starch phosphorylation at both C3- and C6-position.

Long-Distance Transport of Thiamine (Vitamin B1) Is Concomitant with That of Polyamines.

Thiamine (vitamin B1) is ubiquitous and essential for cell energy supply in all organisms as a vital metabolic cofactor, known for over a century. In plants, it is established that biosynthesis de novo is taking place predominantly in green tissues and is furthermore limited to plastids. Therefore, transport mechanisms are required to mediate the movement of this polar metabolite from source to sink tissue to activate key enzymes in cellular energy generating pathways but are currently unknown. Similar to thiamine, polyamines are an essential set of charged molecules required for diverse aspects of growth and development, the homeostasis of which necessitates long-distance transport processes that have remained elusive. Here, a yeast-based screen allowed us to identify Arabidopsis (Arabidopsis thaliana) PUT3 as a thiamine transporter. A combination of biochemical, physiological, and genetic approaches permitted us to show that PUT3 mediates phloem transport of both thiamine and polyamines. Loss of function of PUT3 demonstrated that the tissue distribution of these metabolites is altered with growth and developmental consequences. The pivotal role of PUT3 mediated thiamine and polyamine homeostasis in plants, and its importance for plant fitness is revealed through these findings.

Branching patterns in leaf starches from Arabidopsis mutants deficient in diverse starch synthases.

This is the first report on the cluster structure of transitory starch from Arabidopsis leaves. In addition to wild type, the molecular structures of leaf starch from mutants deficient in starch synthases (SS) including single enzyme mutants ss1-, ss2-, or ss3-, and also double mutants ss1-ss2- and ss1-ss3- were characterized. The mutations resulted in increased amylose content. Clusters from whole starch were isolated by partial hydrolysis using α-amylase of Bacillus amyloliquefaciens. The clusters were then further hydrolyzed with concentrated α-amylase of B. amyloliquefaciens to produce building blocks (α-limit dextrins). Structures of the clusters and their building blocks were characterized by chromatography of samples before and after debranching treatment. While the mutations increased the size of clusters, the reasons were different as reflected by the composition of their unit chains and building blocks. In general, all mutants contained more of a-chains that preferentially increased the number of small building blocks with only two chains. The clusters of the double mutant ss1-ss3- were very large and possessed also more of large building blocks with four or more chains. The results from transitory starch are compared with those from agriculturally important crops in the context that to what extent the Arabidopsis can be a true biotechnological reflection for starch modifications through genetic means.

Strategies for vitamin B6 biofortification of plants: a dual role as a micronutrient and a stress protectant.

Vitamin B6 has an essential role in cells as a cofactor for several metabolic enzymes. It has also been shown to function as a potent antioxidant molecule. The recent elucidation of the vitamin B6 biosynthesis pathways in plants provides opportunities for characterizing their importance during developmental processes and exposure to stress. Humans and animals must acquire vitamin B6 with their diet, with plants being a major source, because they cannot biosynthesize it de novo. However, the abundance of the vitamin in the edible portions of the most commonly consumed plants is not sufficient to meet daily requirements. Genetic engineering has proven successful in increasing the vitamin B6 content in the model plant Arabidopsis. The added benefits associated with the enhanced vitamin B6 content, such as higher biomass and resistance to abiotic stress, suggest that increasing this essential micronutrient could be a valuable option to improve the nutritional quality and stress tolerance of crop plants. This review summarizes current achievements in vitamin B6 biofortification and considers strategies for increasing vitamin B6 levels in crop plants for human health and nutrition.

Recycling of pyridoxine (vitamin B6) by PUP1 in Arabidopsis.

Vitamin B6 is a cofactor for more than 140 essential enzymatic reactions and was recently proposed as a potent antioxidant, playing a role in the photoprotection of plants. De novo biosynthesis of the vitamin has been described relatively recently and is derived from simple sugar precursors as well as glutamine. In addition, the vitamin can be taken up from exogenous sources in a broad range of organisms, including plants. However, specific transporters have been identified only in yeast. Here we assess the ability of the family of Arabidopsis purine permeases (PUPs) to transport vitamin B6. Several members of the family complement the growth phenotype of a Saccharomyces cerevisiae mutant strain impaired in both de novo biosynthesis of vitamin B6 as well as its uptake. The strongest activity was observed with PUP1 and was confirmed by direct measurement of uptake in yeast as well as in planta, defining PUP1 as a high affinity transporter for pyridoxine. At the tissue level the protein is localised to hydathodes and here we use confocal microscopy to illustrate that at the cellular level it is targeted to the plasma membrane. Interestingly, we observe alterations in pyridoxine recycling from the guttation sap upon overexpression of PUP1 and in a pup1 mutant, consistent with the role of the protein in retrieval of pyridoxine. Furthermore, combining the pup1 mutant with a vitamin B6 de novo biosynthesis mutant (pdx1.3) corroborates that PUP1 is involved in the uptake of the vitamin.

Integrated functions among multiple starch synthases determine both amylopectin chain length and branch linkage location in Arabidopsis leaf starch.

This study assessed the impact on starch metabolism in Arabidopsis leaves of simultaneously eliminating multiple soluble starch synthases (SS) from among SS1, SS2, and SS3. Double mutant ss1- ss2- or ss1- ss3- lines were generated using confirmed null mutations. These were compared to the wild type, each single mutant, and ss1- ss2- ss3- triple mutant lines grown in standardized environments. Double mutant plants developed similarly to the wild type, although they accumulated less leaf starch in both short-day and long-day diurnal cycles. Despite the reduced levels in the double mutants, lines containing only SS2 and SS4, or SS3 and SS4, are able to produce substantial amounts of starch granules. In both double mutants the residual starch was structurally modified including higher ratios of amylose:amylopectin, altered glucan chain length distribution within amylopectin, abnormal granule morphology, and altered placement of α(1→6) branch linkages relative to the reducing end of each linear chain. The data demonstrate that SS activity affects not only chain elongation but also the net result of branch placement accomplished by the balanced activities of starch branching enzymes and starch debranching enzymes. SS3 was shown partially to overlap in function with SS1 for the generation of short glucan chains within amylopectin. Compensatory functions that, in some instances, allow continued residual starch production in the absence of specific SS classes were identified, probaby accomplished by the granule bound starch synthase GBSS1.

Starch granule initiation in Arabidopsis requires the presence of either class IV or class III starch synthases.

The mechanisms underlying starch granule initiation remain unknown. We have recently reported that mutation of soluble starch synthase IV (SSIV) in Arabidopsis thaliana results in restriction of the number of starch granules to a single, large, particle per plastid, thereby defining an important component of the starch priming machinery. In this work, we provide further evidence for the function of SSIV in the priming process of starch granule formation and show that SSIV is necessary and sufficient to establish the correct number of starch granules observed in wild-type chloroplasts. The role of SSIV in granule seeding can be replaced, in part, by the phylogenetically related SSIII. Indeed, the simultaneous elimination of both proteins prevents Arabidopsis from synthesizing starch, thus demonstrating that other starch synthases cannot support starch synthesis despite remaining enzymatically active. Herein, we describe the substrate specificity and kinetic properties of SSIV and its subchloroplastic localization in specific regions associated with the edges of starch granules. The data presented in this work point to a complex mechanism for starch granule formation and to the different abilities of SSIV and SSIII to support this process in Arabidopsis leaves.

Overlapping functions of the starch synthases SSII and SSIII in amylopectin biosynthesis in Arabidopsis.

BACKGROUND: The biochemical mechanisms that determine the molecular architecture of amylopectin are central in plant biology because they allow long-term storage of reduced carbon. Amylopectin structure imparts the ability to form semi-crystalline starch granules, which in turn provides its glucose storage function. The enzymatic steps of amylopectin biosynthesis resemble those of the soluble polymer glycogen, however, the reasons for amylopectin's architectural distinctions are not clearly understood. The multiplicity of starch biosynthetic enzymes conserved in plants likely is involved. For example, amylopectin chain elongation in plants involves five conserved classes of starch synthase (SS), whereas glycogen biosynthesis typically requires only one class of glycogen synthase. RESULTS: Null mutations were characterized in AtSS2, which codes for SSII, and mutant lines were compared to lines lacking SSIII and to an Atss2, Atss3 double mutant. Loss of SSII did not affect growth rate or starch quantity, but caused increased amylose/amylopectin ratio, increased total amylose, and deficiency in amylopectin chains with degree of polymerization (DP) 12 to DP28. In contrast, loss of both SSII and SSIII caused slower plant growth and dramatically reduced starch content. Extreme deficiency in DP12 to DP28 chains occurred in the double mutant, far more severe than the summed changes in SSII- or SSIII-deficient plants lacking only one of the two enzymes. CONCLUSION: SSII and SSIII have partially redundant functions in determination of amylopectin structure, and these roles cannot be substituted by any other conserved SS, specifically SSI, GBSSI, or SSIV. Even though SSIII is not required for the normal abundance of glucan chains of DP12 to DP18, the enzyme clearly is capable of functioning in production such chains. The role of SSIII in producing these chains cannot be detected simply by analysis of an individual mutation. Competition between different SSs for binding to substrate could in part explain the specific distribution of glucan chains within amylopectin.

Further evidence for the mandatory nature of polysaccharide debranching for the aggregation of semicrystalline starch and for overlapping functions of debranching enzymes in Arabidopsis leaves.

Four isoforms of debranching enzymes are found in the genome of Arabidopsis (Arabidopsis thaliana): three isoamylases (ISA1, ISA2, and ISA3) and a pullulanase (PU1). Each isoform has a specific function in the starch pathway: synthesis and/or degradation. In this work we have determined the levels of functional redundancy existing between these isoforms by producing and analyzing different combinations of mutations: isa3-1 pu1-1, isa1-1 isa3-1, and isa1-1 isa3-1 pu1-1. While the starch content strongly increased in the isa3-1 pu1-1 double mutant, the latter decreased by over 98% in the isa1-1 isa3-1 genotype and almost vanished in triple mutant combination. In addition, whereas the isa3-1 pu1-1 double mutant synthesizes starch very similar to that of the wild type, the structure of the residual starch present either in isa1-1 isa3-1 or in isa1-1 isa3-1 pu1-1 combination is deeply affected. In the same way, water-soluble polysaccharides that accumulate in the isa1-1 isa3-1 and isa1-1 isa3-1 pu1-1 genotypes display strongly modified structure compared to those found in isa1-1. Taken together, these results show that in addition to its established function in polysaccharide degradation, the activity of ISA3 is partially redundant to that of ISA1 for starch synthesis. Our results also reveal the dual function of pullulanase since it is partially redundant to ISA3 for degradation and to ISA1 for synthesis. Finally, x-ray diffraction analyses suggest that the crystallinity and the presence of the 9- to 10-nm repetition pattern in starch precisely depend on the level of debranching enzyme activity.