Interactions of separately conserved α-(1→6) glucosidases that participate in maize endosperm starch biosynthesis

PLANT PHYSIOLOGY · 2025-09-23 · 2 citations

Chloroplast-containing species possess 2 α-(1→6)-glucosidases that share a common ancestor but were independently acquired by horizontal gene transfer from separate eubacterial donors. The pullulanase-type enzyme (CAZy subfamily GH13_13) and the isoamylase-type enzyme (CAZy subfamily GH13_11) both hydrolyze branch linkages in α-polyglucans. Thus, both enzyme types function as debranching enzymes (DBE) in starch metabolism. As both enzyme types are conserved, distinct selectable functions are expected. This study describes the functional interactions between maize (Zea mays L.) pullulanase1 (ZPU1) and the isoamylase-type enzyme complex comprising the paralogous proteins isoamylase1 (ISA1) and isoamylase2 (ISA2). Mutation of ISA1 or ISA2 caused reduced ZPU1 activity in developing endosperm extracts, and the addition of ISA1 to ZPU1-expressing yeast (Saccharomyces cerevisiae) cells caused increased ZPU1 activity. Specific amino acid substitutions in ISA1 resulted in altered ZPU1 mobility in SDS-PAGE. In vivo protein-protein interaction tests and co-immunoprecipitation revealed that ZPU1 and ISA1 interact in multi-subunit complexes. Maize lines harboring ISA1 mutations, exhibiting a classical low-starch, high-phytoglycogen-accumulation phenotype, were altered by recurrent selection so that kernel appearance reverted to near normal. Extragenic suppression indicated the requirement for ISA1/ISA2 activity had been bypassed. These results are consistent with a functional overlap between the GH13_11 and GH13_13 DBE types and raise the possibility that multiple GH13 proteins, namely ZPU1, ISA1 and ISA2, act together to physically coordinate their hydrolytic activities on precursor α-polyglucans.

Sweet corn phytoglycogen dendrimers as a lyoprotectant for dry‐state protein storage

Journal of Biomedical Materials Research Part A · 2024-06-10 · 3 citations

Abstract Protein biotherapeutics typically require expensive cold‐chain storage to maintain their fold and function. Packaging proteins in the dry state via lyophilization can reduce these cold‐chain requirements. However, formulating proteins for lyophilization often requires extensive optimization of excipients that both maintain the protein folded state during freezing and drying (i.e., “cryoprotection” and “lyoprotection”), and form a cake to carry the dehydrated protein. Here we show that sweet corn phytoglycogens, which are glucose dendrimers, can act as both a protein lyoprotectant and a cake‐forming agent. Phytoglycogen (PG) dendrimers from 16 different maize sources (PG1‐16) were extracted via ethanol precipitation. PG size was generally consistent at ~70–100 nm for all variants, whereas the colloidal stability in water, protein contaminant level, and maximum density of cytocompatibility varied for PG1‐16. 10 mg/mL PG1, 2, 9, 13, 15, and 16 maintained the activity of various proteins, including green fluorescent protein, lysozyme, β‐galactosidase, and horseradish peroxidase, over a broad range of concentrations, through multiple rounds of lyophilization. PG13 was identified as the lead excipient candidate as it demonstrated narrow dispersity, colloidal stability in phosphate‐buffered saline, low protein contaminants, and cytocompatibility up to 10 mg/mL in NIH3T3 cell cultures. All dry protein‐PG13 mixtures had a cake‐like appearance and all frozen protein‐PG13 mixtures had a T g ' of ~ −26°C. The lyoprotection and cake‐forming properties of PG13 were density‐dependent, requiring a minimum density of 5 mg/mL for maximum activity. Collectively these data establish PG dendrimers as a new class of excipient to formulate proteins in the dry state.

Soluble and insoluble α-glucan synthesis in yeast by enzyme suites derived exclusively from maize endosperm

PLANT PHYSIOLOGY · 2023-06-20 · 6 citations

Molecular mechanisms that distinguish the synthesis of semi-crystalline α-glucan polymers found in plant starch granules from the synthesis of water-soluble polymers by nonplant species are not well understood. To address this, starch biosynthetic enzymes from maize (Zea mays L.) endosperm were isolated in a reconstituted environment using yeast (Saccharomyces cerevisiae) as a test bed. Ninety strains were constructed containing unique combinations of 11 synthetic transcription units specifying maize starch synthase (SS), starch phosphorylase (PHO), starch branching enzyme (SBE), or isoamylase-type starch debranching enzyme (ISA). Soluble and insoluble branched α-glucans accumulated in varying proportions depending on the enzyme suite, with ISA function stimulating distribution into the insoluble form. Among the SS isoforms, SSIIa, SSIII, and SSIV individually supported the accumulation of glucan polymer. Neither SSI nor SSV alone produced polymers; however, synergistic effects demonstrated that both isoforms can stimulate α-glucan accumulation. PHO did not support α-glucan production by itself, but it had either positive or negative effects on polymer content depending on which SS or a combination thereof was present. The complete suite of maize enzymes generated insoluble particles resembling native starch granules in size, shape, and crystallinity. Ultrastructural analysis revealed a hierarchical assembly starting with subparticles of approximately 50 nm diameter that coalesce into discrete structures of approximately 200 nm diameter. These are assembled into semi-crystalline α-glucan superstructures up to 4 μm in length filling most of the yeast cytosol. ISA was not essential for the formation of such particles, but their abundance was increased dramatically by ISA presence.

The impact of post-harvest storage on sweet corn aroma

Phytochemistry Letters · 2022 · 21 citations

• Chemistry
• Food science

Genetic Perturbation of the Starch Biosynthesis in Maize Endosperm Reveals Sugar-Responsive Gene Networks

Frontiers in Plant Science · 2022 · 18 citations

• Biology
• Biochemistry
• Genetics

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Characterizing the Physical Properties and Cell Compatibility of Phytoglycogen Extracted from Different Sweet Corn Varieties

Molecules · 2020 · 13 citations

• Chemistry
• Biochemistry

40DAP. Collectively, these data demonstrate that maize phytoglycogen extracts are not uniformly cytocompatible. Rather, maize variant, plant genotype, protein contaminants, and water-binding properties are determinants of phytoglycogen cytotoxicity.

Maize <i>defective kernel5</i> is a bacterial TamB homologue required for chloroplast envelope biogenesis

The Journal of Cell Biology · 2019-06-24 · 37 citations

Chloroplasts are of prokaryotic origin with a double-membrane envelope separating plastid metabolism from the cytosol. Envelope membrane proteins integrate chloroplasts with the cell, but envelope biogenesis mechanisms remain elusive. We show that maize defective kernel5 (dek5) is critical for envelope biogenesis. Amyloplasts and chloroplasts are larger and reduced in number in dek5 with multiple ultrastructural defects. The DEK5 protein is homologous to rice SSG4, Arabidopsis thaliana EMB2410/TIC236, and Escherichia coli tamB. TamB functions in bacterial outer membrane biogenesis. DEK5 is localized to the envelope with a topology analogous to TamB. Increased levels of soluble sugars in dek5 developing endosperm and elevated osmotic pressure in mutant leaf cells suggest defective intracellular solute transport. Proteomics and antibody-based analyses show dek5 reduces levels of Toc75 and chloroplast envelope transporters. Moreover, dek5 chloroplasts reduce inorganic phosphate uptake with at least an 80% reduction relative to normal chloroplasts. These data suggest that DEK5 functions in plastid envelope biogenesis to enable transport of metabolites and proteins.

Effects of long‐term exposure to elevated temperature on <i>Zea mays</i> endosperm development during grain fill

The Plant Journal · 2019-02-12 · 50 citations

Cereal yields decrease when grain fill proceeds under conditions of prolonged, moderately elevated temperatures. Endosperm-endogenous processes alter both rate and duration of dry weight gain, but underlying mechanisms remain unclear. Heat effects could be mediated by either abnormal, premature cessation of storage compound deposition or accelerated implementation of normal development. This study used controlled environments to isolate temperature as the sole environmental variable during Zea mays kernel-fill, from 12 days after pollination to maturity. Plants subjected to elevated day, elevated night temperatures (38°C day, 28°C night (38/28°C])) or elevated day, normal night (38/17°C), were compared with those from controls grown under normal day and night conditions (28/17°C). Progression of change over time in endosperm tissue was followed to dissect contributions at multiple levels, including transcriptome, metabolome, enzyme activities, product accumulation, and tissue ultrastructure. Integrated analyses indicated that the normal developmental program of endosperm is fully executed under prolonged high-temperature conditions, but at a faster rate. Accelerated development was observed when both day and night temperatures were elevated, but not when daytime temperature alone was increased. Although transcripts for most components of glycolysis and respiration were either upregulated or minimally affected, elevated temperatures decreased abundance of mRNAs related to biosynthesis of starch and storage proteins. Further analysis of 20 central-metabolic enzymes revealed six activities that were reduced under high-temperature conditions, indicating candidate roles in the observed reduction of grain dry weight. Nonetheless, a striking overall resilience of grain filling in the face of elevated temperatures can be attributed to acceleration of normal endosperm development.

Maize <i>defective kernel5</i> is a bacterial tamB homolog required for chloroplast envelope biogenesis

bioRxiv (Cold Spring Harbor Laboratory) · 2018-07-18

ABSTRACT Chloroplasts are of prokaryotic origin with a double membrane envelope that separates plastid metabolism from the cytosol. Envelope membrane proteins integrate the chloroplast with the cell, but the biogenesis of the envelope membrane remains elusive. We show that the maize defective kernel5 ( dek5 ) locus is critical for plastid membrane biogenesis. Amyloplasts and chloroplasts are larger and reduced in number in dek5 with multiple ultrastructural defects. We show that dek5 encodes a protein homologous to rice SUBSTANDARD STARCH GRAIN4 ( SSG4 ) and E.coli tamB. TamB functions in bacterial outer membrane biogenesis. The DEK5 protein is localized to the chloroplast envelope with a topology analogous to TamB. Increased levels of soluble sugars in dek5 developing endosperm and elevated osmotic pressure in mutant leaf cells suggest defective intracellular solute transport. Both proteomics and antibody-based analyses show that dek5 chloroplasts have reduced levels of chloroplast envelope transporters. Moreover, dek5 chloroplasts reduce inorganic phosphate uptake with at least an 80% reduction relative to normal chloroplasts. These data suggest that DEK5 functions in plastid envelope biogenesis to enable metabolite transport.

Ovary abortion is prevalent in diverse maize inbred lines and is under genetic control

Scientific Reports · 2018-08-23 · 17 citations

Crop improvement programs focus on characteristics that are important for plant productivity. Typically genes underlying these traits are identified and stacked to create improved cultivars. Hence, identification of valuable traits for plant productivity is critical for plant improvement. Here we describe an important characteristic for maize productivity. Despite the fact mature maize ears are typically covered with kernels, we find that only a fraction of ovaries give rise to mature kernels. Non-developed ovaries degenerate while neighboring fertilized ovaries produce kernels that fill the ear. Abortion occurs throughout the ear, not just at the tip. We show that the fraction of aborted ovaries/kernels is genetically controlled and varies widely among maize lines, and low abortion genotypes are rare. Reducing or eliminating ovary abortion could substantially increase yield, making this characteristic a new target for selection in maize improvement programs.