MFP1 defines the subchloroplast location of starch granule initiation.

Starch is one of the major carbohydrate storage compounds in plants. The biogenesis of starch granules starts with the formation of initials, which subsequently expand into granules. Several coiled-coil domain-containing proteins have been previously implicated with the initiation process, but the mechanisms by which they act remain largely elusive. Here, we demonstrate that one of these proteins, the thylakoid-associated MAR-BINDING FILAMENT-LIKE PROTEIN 1 (MFP1), specifically determines the subchloroplast location of initial formation. The expression of MFP1 variants "mis"-targeted to specific locations within chloroplasts in Arabidopsis results in distinctive shifts in not only how many but also where starch granules are formed. Importantly, "re" localizing MFP1 to the stromal face of the chloroplast's inner envelope is sufficient to generate starch granules in this aberrant position. These findings provide compelling evidence that a single protein MFP1 possesses the capacity to direct the initiation and biosynthesis machinery of starch granules.

LIKE EARLY STARVATION 1 and EARLY STARVATION 1 promote and stabilize amylopectin phase transition in starch biosynthesis.

Starch, the most abundant carbohydrate reserve in plants, primarily consists of the branched glucan amylopectin, which forms semi-crystalline granules. Phase transition from a soluble to an insoluble form depends on amylopectin architecture, requiring a compatible distribution of glucan chain lengths and a branch-point distribution. Here, we show that two starch-bound proteins, LIKE EARLY STARVATION 1 (LESV) and EARLY STARVATION 1 (ESV1), which have unusual carbohydrate-binding surfaces, promote the phase transition of amylopectin-like glucans, both in a heterologous yeast system expressing the starch biosynthetic machinery and in Arabidopsis plants. We propose a model wherein LESV serves as a nucleating role, with its carbohydrate-binding surfaces helping align glucan double helices to promote their phase transition into semi-crystalline lamellae, which are then stabilized by ESV1. Because both proteins are widely conserved, we suggest that protein-facilitated glucan crystallization may be a general and previously unrecognized feature of starch biosynthesis.

The PRK/Rubisco shunt strongly influences Arabidopsis seed metabolism and oil accumulation, affecting more than carbon recycling.

The carbon efficiency of storage lipid biosynthesis from imported sucrose in green Brassicaceae seeds is proposed to be enhanced by the PRK/Rubisco shunt, in which ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) acts outside the context of the Calvin-Benson-Bassham cycle to recycle CO2 molecules released during fatty acid synthesis. This pathway utilizes metabolites generated by the nonoxidative steps of the pentose phosphate pathway. Photosynthesis provides energy for reactions such as the phosphorylation of ribulose 5-phosphate by phosphoribulokinase (PRK). Here, we show that loss of PRK in Arabidopsis thaliana (Arabidopsis) blocks photoautotrophic growth and is seedling-lethal. However, seeds containing prk embryos develop normally, allowing us to use genetics to assess the importance of the PRK/Rubisco shunt. Compared with nonmutant siblings, prk embryos produce one-third less lipids-a greater reduction than expected from simply blocking the proposed PRK/Rubisco shunt. However, developing prk seeds are also chlorotic and have elevated starch contents compared with their siblings, indicative of secondary effects. Overexpressing PRK did not increase embryo lipid content, but metabolite profiling suggested that Rubisco activity becomes limiting. Overall, our findings show that the PRK/Rubisco shunt is tightly integrated into the carbon metabolism of green Arabidopsis seeds, and that its manipulation affects seed glycolysis, starch metabolism, and photosynthesis.

Determining Protein-Protein Interaction with GFP-Trap Beads.

Protein-protein interactions play a vital role in the cellular physiology of living organisms. Among several available approaches, co-immunoprecipitation (co-IP) has emerged as a reliable method to investigate such interactions. The underlying principle is to retrieve a bait protein from a protein extract using bait-specific antibodies and thereby indirectly capture the interacting partner proteins. However, bait-specific antibodies are not always available, and the genetic fusion of a peptide tag offers an alternative. An extensive range of peptide tags and the tag-specific antibodies are available nowadays. Fluorescent proteins are widely used protein tags for co-IP experiments. In this chapter, we describe a method to co-immunoprecipitate the fluorescently tagged candidate protein with its interacting partners from the crude plant cell extracts using green fluorescent protein (GFP)-trap magnetic beads.

Tuning heterologous glucan biosynthesis in yeast to understand and exploit plant starch diversity.

BACKGROUND: Starch, a vital plant-derived polysaccharide comprised of branched glucans, is essential in nutrition and many industrial applications. Starch is often modified post-extraction to alter its structure and enhance its functionality. Targeted metabolic engineering of crops to produce valuable and versatile starches requires knowledge of the relationships between starch biosynthesis, structure, and properties, but systematic studies to obtain this knowledge are difficult to conduct in plants. Here we used Saccharomyces cerevisiae as a testbed to dissect the functions of plant starch biosynthetic enzymes and create diverse starch-like polymers. RESULTS: We explored yeast promoters and terminators to tune the expression levels of the starch-biosynthesis machinery from Arabidopsis thaliana. We systematically modulated the expression of each starch synthase (SS) together with a branching enzyme (BE) in yeast. Protein quantification by parallel reaction monitoring (targeted proteomics) revealed unexpected effects of glucan biosynthesis on protein abundances but showed that the anticipated broad range of SS/BE enzyme ratios was maintained during the biosynthetic process. The different SS/BE ratios clearly influenced glucan structure and solubility: The higher the SS/BE ratio, the longer the glucan chains and the more glucans were partitioned into the insoluble fraction. This effect was irrespective of the SS isoform, demonstrating that the elongation/branching ratio controls glucan properties separate from enzyme specificity. CONCLUSIONS: Our results provide a quantitative framework for the in silico design of improved starch biosynthetic processes in plants. Our study also exemplifies a workflow for the rational tuning of a complex pathway in yeast, starting from the selection and evaluation of expression modules to multi-gene assembly and targeted protein monitoring during the biosynthetic process.

BETA-AMYLASE9 is a plastidial nonenzymatic regulator of leaf starch degradation.

ß-Amylases (BAMs) are key enzymes of transitory starch degradation in chloroplasts, a process that buffers the availability of photosynthetically fixed carbon over the diel cycle to maintain energy levels and plant growth at night. However, during vascular plant evolution, the BAM gene family diversified, giving rise to isoforms with different compartmentation and biological activities. Here, we characterized BETA-AMYLASE 9 (BAM9) of Arabidopsis (Arabidopsis thaliana). Among the BAMs, BAM9 is most closely related to BAM4 but is more widely conserved in plants. BAM9 and BAM4 share features including their plastidial localization and lack of measurable α-1,4-glucan hydrolyzing capacity. BAM4 is a regulator of starch degradation, and bam4 mutants display a starch-excess phenotype. Although bam9 single mutants resemble the wild-type (WT), genetic experiments reveal that the loss of BAM9 markedly enhances the starch-excess phenotypes of mutants already impaired in starch degradation. Thus, BAM9 also regulates starch breakdown, but in a different way. Interestingly, BAM9 gene expression is responsive to several environmental changes, while that of BAM4 is not. Furthermore, overexpression of BAM9 in the WT reduced leaf starch content, but overexpression in bam4 failed to complement fully that mutant's starch-excess phenotype, suggesting that BAM9 and BAM4 are not redundant. We propose that BAM9 activates starch degradation, helping to manage carbohydrate availability in response to fluctuations in environmental conditions. As such, BAM9 represents an interesting gene target to explore in crop species.

Evolutionary innovations in starch metabolism.

The traditional view of starch metabolism has focused on the multiplicity of enzymes and enzyme isoforms contributing to the production of the constituent polymers, amylopectin and amylose. However, knowledge of these enzymes has not provided a full insight into many aspects of starch biosynthesis. This enzyme-centered view has recently been augmented by the discovery and characterization of novel proteins with proposed regulatory, scaffolding, and interactive roles. This begins to reveal an unprecedented level of complexity beyond mere glucan biosynthesis, enabling us to envisage how starch granules are initiated and grow into specific forms, allowing it to serve biological roles beyond just carbohydrate storage. This review focuses on very recent findings in this vibrant field, highlighting the evolutionary novelty.

STARCH SYNTHASE5, a Noncanonical Starch Synthase-Like Protein, Promotes Starch Granule Initiation in Arabidopsis.

What determines the number of starch granules in plastids is an enigmatic aspect of starch metabolism. Several structurally and functionally diverse proteins have been implicated in the granule initiation process in Arabidopsis (Arabidopsis thaliana), with each protein exerting a varying degree of influence. Here, we show that a conserved starch synthase-like protein, STARCH SYNTHASE5 (SS5), regulates the number of starch granules that form in Arabidopsis chloroplasts. Among the starch synthases, SS5 is most closely related to SS4, a major determinant of granule initiation and morphology. However, unlike SS4 and the other starch synthases, SS5 is a noncanonical isoform that lacks catalytic glycosyltransferase activity. Nevertheless, loss of SS5 reduces starch granule numbers that form per chloroplast in Arabidopsis, and ss5 mutant starch granules are larger than wild-type granules. Like SS4, SS5 has a conserved putative surface binding site for glucans and also interacts with MYOSIN-RESEMBLING CHLOROPLAST PROTEIN, a proposed structural protein influential in starch granule initiation. Phenotypic analysis of a suite of double mutants lacking both SS5 and other proteins implicated in starch granule initiation allows us to propose how SS5 may act in this process.