Jack C. Shannon

Genetics and physiology of starch development

Publisher Summary This chapter reviews nonmutant starch granule composition and development, and focuses on genetic mutants and how they have been useful in understanding the complexity of polysaccharide biosynthesis and development. Plant species, which are important sources of commercial starch production are focused on, especially maize, because of the many known endosperm mutants of maize, which affect polysaccharide biosynthesis. The information gained applies to other species, and these effects are illustrated in the chapter. General trends in the genetics and physiology of starch development have been illustrated with examples. Starch is a common constituent of higher plants and a source of carbohydrates. Starch in chloroplasts is transitory and accumulates during the light period and is utilized during the dark. Storage starch accumulates in reserve organs during one phase of the plants lifecycle and is utilized at another time. Starches from reserve organs of many plants are important in commerce. Although considerable effort has been directed at characterizing the enzymes involved in starch synthesis, the role of these enzymes and other factors in determining subtle variations in starch granule structure and starch fine structure remain largely unknown. Variations in granule structure can be associated with plant species, cultivars of a species, the environment in which a cultivar is grown, and genetic mutations.

Independent Genetic Control of Maize Starch-Branching Enzymes IIa and IIb (Isolation and Characterization of a Sbe2a cDNA)

In maize (Zea mays L.) three isoforms of starch-branching enzyme (SBEI, SBEIIa, and SBEIIb) are involved in the synthesis of amylopectin, the branched component of starch. To isolate a cDNA encoding SBEIIa, degenerate oligonucleotides based on domains highly conserved in Sbe2 family members were used to amplify Sbe2-family cDNA from tissues lacking SBIIb activity. The predicted amino acid sequence of a Sbe2a cDNA matches the N-terminal sequence of SBIIa protein purified from maize endosperm. The size of the mature protein deduced from the cDNA also matches that of SBEIIa Features of the predicted protein are most similar to members of the SBEII family; however, it differs from maize SBEIIb in having a 49-amino acid N-terminal extension and a region of substantial sequence divergence. Sbe2a mRNA levels are 10-fold higher in embryonic than in endosperm tissue, and are much lower than Sbe2b in both tissues. Unlike Sbe2b, Sbe2a-hybridizing mRNA accumulates in leaf and other vegetative tissues, consistent with the known distribution of SBEIIa and SBEIIb activities.

EVOLUTIONARY CONSERVATION AND EXPRESSION PATTERNS OF MAIZE STARCH BRANCHING ENZYME I AND IIB GENES SUGGESTS ISOFORM SPECIALIZATION

Expression of the maize (Zea mays L.) starch branching enzyme (SBE) genes Sbe1 and Sbe2 were characterized during kernel development and in vegetative tissues. The onset of Sbe1 and Sbe2 expression during endosperm development was similar to that of other genes involved in starch biosynthesis (Wx, Sh2 and Bt2). However, the expression of Sbe2 peaked earlier than that of Sbe1 in developing endosperm and embryos resulting in a shift in the ratio of Sbe1 to Sbe2 relative message levels during kernel and embryo development. Transcripts hybridizing to the Sbe2 probe were not detectable in leaves or roots which nonetheless have SBEII enzymatic activity, suggesting that there may be another divergent SBEII-like gene(s) in maize. A similar expression pattern is shared between the maize genes and related genes in pea, which together with their evolutionary conservation, suggests that the SBE isoforms may play unique roles in starch biosynthesis during plant development.

Nucleotides and Nucleotide Sugars in Developing Maize Endosperms (Synthesis of ADP-Glucose in brittle-1)

As part of an in vivo study of carbohydrate metabolism during development of Zea mays L. kernels, quantities of nucleotides and nucleotide sugars were measured in endosperm extracts from normal, the single-mutant genotypes shrunken-1 (sh1), shrunken-2 (sh2), and brittle-1 (btl}, and the multiple-mutant genotypes sh1bt1, sh2bt1, and sh1sh2bt1. Results showed that bt1 kernels accumulated more than 13 times as much adenosine 5[prime] diphospho-glucose (ADP-Glc) as normal kernels. Activity of starch synthase in bt1 endosperm was equal to that in endosperm extracts from normal kernels. Thus the ADP-Glc accumulation in bt1 endosperm cells was not due to a deficiency in starch synthase. ADP-Glc content in extracts of sh1bt1 endosperms was similar to that in bt1, but in extracts of the sh2bt1 mutant kernels ADP-Glc content was much reduced compared to bt1 (about 3 times higher than that in normal). Endosperm extracts from sh1sh2bt1, kernels that are deficient in both ADP-Glc pyrophosphorylase (AGPase) and sucrose synthase, had quantities of ADP-Glc much lower than in normal kernels. These results clearly indicate that AGPase is the predominant enzyme responsible for the in vivo synthesis of ADP-Glc in bt1 mutant kernels, but Suc synthase may also contribute to the synthesis of ADP-Glc in kernels deficient in AGPase.

Chapter 3 – Genetics and Physiology of Starch Development

Publisher Summary This chapter reviews nonmutant starch granule composition and development, and focuses on genetic mutants and how they have been useful in understanding the complexity of polysaccharide biosynthesis and development. Plant species, which are important sources of commercial starch production are focused on, especially maize, because of the many known endosperm mutants of maize, which affect polysaccharide biosynthesis. The information gained applies to other species, and these effects are illustrated in the chapter. General trends in the genetics and physiology of starch development have been illustrated with examples. Starch is a common constituent of higher plants and a source of carbohydrates. Starch in chloroplasts is transitory and accumulates during the light period and is utilized during the dark. Storage starch accumulates in reserve organs during one phase of the plants lifecycle and is utilized at another time. Starches from reserve organs of many plants are important in commerce. Although considerable effort has been directed at characterizing the enzymes involved in starch synthesis, the role of these enzymes and other factors in determining subtle variations in starch granule structure and starch fine structure remain largely unknown. Variations in granule structure can be associated with plant species, cultivars of a species, the environment in which a cultivar is grown, and genetic mutations.

Single Kernel Sampling Method for Maize Starch Analysis While Maintaining Kernel Vitality

ABSTRACT A nondestructive protocol for maize kernel starch sampling was developed, enabling starch preparation from a single kernel for analysis of starch structure while also maintaining the vitality of the seed. To develop the single kernel sampling (SKS) method, maize genotypes varying in starch structure including ae, wx, su2, du and normal in the W64A inbred line were used. Crude endosperm material was removed from the kernel crown, soaked, ground, washed, and dissolved in 90% DMSO. The sample represented ≈10% of the total kernel. Endosperm starch was also isolated from the same genotypes by a standard multikernel isolation (MKI) method. Starches isolated by the two methods were debranched and analyzed by high-performance size-exclusion chromatography (HPSEC) and fluorophore-assisted carbohydrate electrophoresis (FACE). HPSEC and FACE showed similar results for the two sampling methods for degree of polymerization (DP) ≤ 50. We concluded that the material obtained by SKS could be used for identifying...

[22] Isolation of amyloplasts from developing endosperm of maize (Zea mays L.)

Publisher Summary This chapter describes a method for the isolation of amyloplasts from developing endosperm of maize. Probably the most critical stage in the isolation of intact amyloplasts from maize endosperm is the release of the amyloplasts from the cells. Amyloplasts containing a single large starch granule, such as potato and maize, is very prone to rupture during even low-gravity centrifugation. Although castor bean endosperm protoplasts were ruptured with little damage to plastids by passage through nylon mesh, the presence of large starch granules in the maize amyloplasts creates a special problem. For example, during maize endosperm protoplast rupture the starch granules quickly settle and cake on the surface of the nylon mesh. The trypsin treatment is shown to increase the rate of maize amyloplast membrane rupture and thus resulted in a low estimate of percentage intactness. Dihydroxyacetone phosphate (DHAP) readily crosses the maize amyloplast membrane and is converted to starch.

The Relationship between Pyrophosphatase and Branching Enzyme Activity with Amyloplast Size in Maize Endosperm

Summary This study aimed to clarify the relationship between amyloplast size (age) and activities of alkaline pyrophosphatase (PPase) and branching enzyme (BE) within developing Zea mays L. endosperm cells. PPase and BE activities per starch granule were increased when the organelle grew in size, although decreased when calculated per granule surface area. The amyloplast specific PPase and the stromal marker enzyme BE had identical distribution (percent of total activity) in the stroma. The amount of free Pi increased in the stroma with the increased granule size up to 8 μ in diameter (about one third of the final size) and then remained steady. The maximum Pi content at this granule size was 5.2 mmol per 10 6 amyloplasts (about 5.5 fmol per granule) and PPase activity released about 1 nmol min -1 per 10 6 amyloplasts. Attempts to detect endogenous PPi in amyloplasts were unsuccessful.