Starch-branching enzyme IIa is required for proper diurnal cycling of starch in leaves of maize

Starch-branching enzyme (SBE), a glucosyl transferase, is required for the highly regular pattern of α-1,6 bonds in the amylopectin component of starch. In the absence of SBEIIa, as shown previously in the sbe2a mutant of maize (Zea mays), leaf starch has drastically reduced branching and the leaves exhibit a severe senescence-like phenotype. Detailed characterization of the maize sbe2a mutant revealed that SBEIIa is the primary active branching enzyme in the leaf and that in its absence plant growth is affected. Both seedling and mature sbe2a mutant leaves do not properly degrade starch during the night, resulting in hyperaccumulation. In mature sbe2a leaves, starch hyperaccumulation is greatest in visibly senescing regions but also observed in green tissue and is correlated to a drastic reduction in photosynthesis within the leaf. Starch granules from sbe2a leaves observed via scanning electron microscopy and transmission electron microscopy analyses are larger, irregular, and amorphous as compared with the highly regular, discoid starch granules observed in wild-type leaves. This appears to trigger premature senescence, as shown by an increased expression of genes encoding proteins known to be involved in senescence and programmed cell death processes. Together, these results indicate that SBEIIa is required for the proper diurnal cycling of transitory starch within the leaf and suggest that SBEIIa is necessary in producing an amylopectin structure amenable to degradation by starch metabolism enzymes.     

Maize starch-branching enzyme isoforms and amylopectin structure. In the absence of starch-branching enzyme IIb, the further absence of starch-branching enzyme Ia leads to increased branching

Previous studies indicated that the deficiency of starch-branching enzyme (SBE) Ia in the single mutant sbe1a::Mu (sbe1a) has no impact on endosperm starch structure, whereas the deficiency of SBEIIb in the ae mutant is well known to reduce the branching of starch. We hypothesized that in maize (Zea mays) endosperm, the function of SBEIIb is predominant to that of SBEIa, and SBEIa would have an observable effect only on amylopectin structure in the absence of SBEIIb. To test this hypothesis, the mutant sbe1a was introgressed into lines containing either wx (lacking the granule-bound starch synthase GBSSI) or ae wx (lacking both SBEIIb and GBSSI) in the W64A background. Both western blotting and zymogram analysis confirmed the SBEIa deficiency in sbe1a wx and sbe1a ae wx, and the SBEIIb deficiency in ae wx and sbe1a ae wx. Using zymogram analysis, no pleiotropic effects of sbe1a genes on SBEIIa, starch synthase, or starch-debranching enzyme isoforms were observed. High-performance size exclusion chromatography analysis shows that the chain-length profiles of amylopectin as well as beta-limit dextrin were indistinguishable between wx and sbe1a wx, whereas significant differences for both were observed between ae wx and sbe1a ae wx, suggesting an effect of SBEIa on amylopectin biosynthesis that is observable only in the absence of SBEIIb. The amylopectin branch density and the average number of branches per cluster were both higher in endosperm starch from sbe1a ae wx than from ae wx. These results indicate possible functional interactions between SBE isoforms that may involve enzymatic inhibition. Both the cluster repeat distance and the distance between branch points on the short intracluster chains were similar for all genotypes however, suggesting a similar pattern of individual SBE isoforms in cluster initiation and the determination of branch point location.     

Mutation of the maize sbe1a and ae genes alters morphology and physical behavior of wx-type endosperm starch granules

In maize, three isoforms of starch-branching enzyme, SBEI, SBEIIa, and SBEIIb, are encoded by the Sbe1a, Sbe2a, and Amylose extender (Ae) genes, respectively. The objective of this research was to explore the effects of null mutations in the Sbe1a and Ae genes alone and in combination in wx background on kernel characteristics and on the morphology and physical behavior of endosperm starch granules. Differences in kernel morphology and weight, starch accumulation, starch granule size and size distribution, starch microstructure, and thermal properties were observed between the ae wx and sbe1a ae wx plants but not between the sbe1a wx mutants when compared to wx. Starch from sbe1a ae wx plants exhibited a larger granule size with a wider gelatinization temperature range and a lower endotherm enthalpy than ae wx. Microscopy shows weaker iodine staining in sbe1a ae wx starch granules. X-ray diffraction revealed A-type crystallinity in wx and sbe1a wx starches and B-type in sbe1a ae wx and ae wx. This study suggests that, while the SBEIIb isoform plays a dominant role in maize endosperm starch synthesis, SBEI also plays a role, which is only observable in the presence of the ae mutation.     

Sbe2p and sbe22p, two homologous Golgi proteins involved in yeast cell wall formation

The cell wall of fungal cells is important for cell integrity and cell morphogenesis and protects against harmful environmental conditions. The yeast cell wall is a complex structure consisting mainly of mannoproteins, glucan, and chitin. The molecular mechanisms by which the cell wall components are synthesized and transported to the cell surface are poorly understood. We have identified and characterized two homologous yeast proteins, Sbe2p and Sbe22p, through their suppression of a chs5 spa2 mutant strain defective in chitin synthesis and cell morphogenesis. Although sbe2 and sbe22 null mutants are viable, sbe2 sbe22 cells display several phenotypes indicative of defects in cell integrity and cell wall structure. First, sbe2 sbe22 cells display a sorbitol-remediable lysis defect at 37 degrees C and are hypersensitive to SDS and calcofluor. Second, electron microscopic analysis reveals that sbe2 sbe22 cells have an aberrant cell wall structure with a reduced mannoprotein layer. Finally, immunofluorescence experiments reveal that in small-budded cells, sbe2 sbe22 mutants mislocalize Chs3p, a protein involved in chitin synthesis. In addition, sbe2 sbe22 diploids have a bud-site selection defect, displaying a random budding pattern. A Sbe2p-GFP fusion protein localizes to cytoplasmic patches, and Sbe2p cofractionates with Golgi proteins. Deletion of CHS5, which encodes a Golgi protein involved in the transport of Chs3p to the cell periphery, is lethal in combination with disruption of SBE2 and SBE22. Thus, we suggest a model in which Sbe2p and Sbe22p are involved in the transport of cell wall components from the Golgi apparatus to the cell surface periphery in a pathway independent of Chs5p.     

The Characterization of Modified Starch Branching Enzymes: Toward the Control of Starch Chain-Length Distributions

Starch is a complex branched glucose polymer whose branch molecular weight distribution (the chain-length distribution, CLD) influences nutritionally important properties such as digestion rate. Chain-stopping in starch biosynthesis is by starch branching enzyme (SBE). Site-directed mutagenesis was used to modify SBEIIa from Zea mays (mSBEIIa) to produce mutants, each differing in a single conserved amino-acid residue. Products at different times from in vitro branching were debranched and the time evolution of the CLD measured by size-exclusion chromatography. The results confirm that Tyr352, Glu513, and Ser349 are important for mSBEIIa activity while Arg456 is important for determining the position at which the linear glucan is cut. The mutant mSBEIIa enzymes have different activities and suggest the length of the transferred chain can be varied by mutation. The work shows analysis of the molecular weight distribution can yield information regarding the enzyme branching sites useful for development of plants yielding starch with improved functionality.     

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 SBEIIb activity. The predicted amino acid sequence of Sbe2a cDNA matches the N-terminal sequence of SBEIIa 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.     

Starch branching enzyme IIb in wheat is expressed at low levels in the endosperm compared to other cereals and encoded at a non-syntenic locus

Studies of maize starch branching enzyme mutants suggest that the amylose extender high amylose starch phenotype is a consequence of the lack of expression of the predominant starch branching enzyme II isoform expressed in the endosperm, SBEIIb. However, in wheat, the ratio of SBEIIb and SBEIIa expression are inversely related to the expression levels observed in maize and rice. Analysis of RNA at 15 days post anthesis suggests that there are about 4-fold more RNA for SBE IIa than for SBE IIb. The genes for SBE IIa and SBE IIb from wheat are distinguished in the size of the first three exons, allowing isoform-specific antibodies to be produced. These antibodies were used to demonstrate that in the soluble fraction, the amount of SBE IIa protein is two to three fold higher than SBIIb, whereas in the starch granule, there is two to three fold more SBE IIb protein amount than SBE IIa. In a further difference to maize and rice, the genes for SBE IIa and SBE IIb are both located on the long arm of chromosome 2 in wheat, in a position not expected from rice–maize–wheat synteny.     

A Putative Gene sbe3-rs for Resistant Starch Mutated from SBE3 for Starch Branching Enzyme in Rice (Oryza sativa L.)

Foods high in resistant starch (RS) are beneficial to prevent various diseases including diabetes, colon cancers, diarrhea and chronic renal or hepatic diseases. Elevated RS in rice is important for public health since rice is a staple food for half of the world population. A japonica mutant ‘Jiangtangdao 1’ (RS = 11.67%) was crossed with an indica cultivar ‘Miyang 23’ (RS = 0.41%). The mutant sbe3-rs that explained 60.4% of RS variation was mapped between RM6611 and RM13366 on chromosome 2 (LOD = 36) using 178 F₂ plants genotyped with 106 genome-wide polymorphic SSR markers. Using 656 plants from four F_3∶4 families, sbe3-rs was fine mapped to a 573.3 Kb region between InDel 2 and InDel 6 using one STS, five SSRs and seven InDel markers. SBE3 which codes for starch branching enzyme was identified as a candidate gene within the putative region. Nine pairs of primers covering 22 exons were designed to sequence genomic DNA of the wild type for SBE3 and the mutant for sbe3-rs comparatively. Sequence analysis identified a missense mutation site where Leu-599 of the wild was changed to Pro-599 of the mutant in the SBE3 coding region. Because the point mutation resulted in the loss of a restriction enzyme site, sbe3-rs was not digested by a CAPS marker for SpeI site while SBE3 was. Co-segregation of the digestion pattern with RS content among 178 F₂ plants further supported sbe3-rs responsible for RS in rice. As a result, the CAPS marker could be used in marker-assisted breeding to develop rice cultivars with elevated RS which is otherwise difficult to accurately assess in crops. Transgenic technology should be employed for a definitive conclusion of the sbe3-rs.     

Functional interactions between heterologously expressed starch-branching enzymes of maize and the glycogen synthases of Brewer's yeast

Starch-branching enzymes (SBEs) catalyze the formation of alpha(1-->6) glycoside bonds in glucan polymers, thus, affecting the structure of amylopectin and starch granules. Two distinct classes of SBE are generally conserved in higher plants, although the specific role(s) of each isoform in determination of starch structure is not clearly understood. This study used a heterologous in vivo system to isolate the function of each of the three known SBE isoforms of maize (Zea mays) away from the other plant enzymes involved in starch biosynthesis. The ascomycete Brewer's yeast (Saccharomyces cerevisiae) was employed as the host species. All possible combinations of maize SBEs were expressed in the absence of the endogenous glucan-branching enzyme. Each maize SBE was functional in yeast cells, although SBEI had a significant effect only if SBEIIa and SBEIIb also were present. SBEI by itself did not support glucan accumulation, whereas SBEIIa and SBEIIb both functioned along with the native glycogen synthases (GSs) to produce significant quantities of alpha-glucan polymers. SBEIIa was phenotypically dominant to SBEIIb in terms of glucan structure. The specific branching enzyme present had a significant effect on the molecular weight of the product. From these data we suggest that SBEs and GSs work in a cyclically interdependent fashion, such that SBE action is needed for optimal GS activity; and GS, in turn, influences the further effects of SBE. Also, SBEIIa and SBEIIb appear to act before SBEI during polymer assembly in this heterologous system.     

控制水稻胚乳淀粉合成代谢若干关键酶基因对花后高温的响应表达

以稻米品质温度敏感型的早籼稻品种嘉早935为材料,利用人工气候箱控温试验和实时荧光定量PCR技术,探讨了不同灌浆温度(日均温分别为22和32 ℃)处理下胚乳淀粉分支酶(SBE)、淀粉去分支酶(DBE)和淀粉合酶(SS)的10个同工型基因(sbe1、sbe3、sbe4、pul、isa1、isa2、isa3、Wx、sss1和sss2a)的相对表达量差异及动态变化特征.结果表明: 淀粉合成相关功能基因对水稻灌浆期高温胁迫的响应表达方式存在明显差异,而且因同工型的类型而不同.在高温处理下,sbe1和sbe3的相对表达量显著下降,二者属于SBE类基因中对高温胁迫较敏感的主要同工型;DBE基因中,pul属于高表达的同工型,而且其对高温胁迫响应比isa1、isa2和isa3敏感;在Wx、sss1和sss2a中,sss2a的相对表达量显著低于sss1和Wx, 但sss2a和sss1对高温胁迫响应比Wx敏感,因此二者可能也是高温胁迫对胚乳淀粉结构进行调控的重要位点,尤其在水稻灌浆的中后期发挥重要作用.     

High-throughput linkage analysis of Mutator insertion sites in maize

Insertional mutagenesis is a cornerstone of functional genomics. High-copy transposable element systems such as Mutator ( Mu ) in maize ( Zea mays ) afford the advantage of high forward mutation rates but pose a challenge for identifying the particular element responsible for a given mutation. Several large mutant collections have been generated in Mu -active genetic stocks, but current methods limit the ability to rapidly identify the causal Mu insertions. Here we present a method to rapidly assay Mu insertions that are genetically linked to a mutation of interest. The method combines elements of MuTAIL (thermal asymmetrically interlaced) and amplification of insertion mutagenized sites (AIMS) protocols and is applicable to the analysis of single mutants or to high-throughput analyses of mutant collections. Briefly, genomic DNA is digested with a restriction enzyme and adapters are ligated. Polymerase chain reaction is performed with TAIL cycling parameters, using a fluorescently labeled Mu primer, which results in the preferential amplification and labeling of Mu -containing genomic fragments. Products from a segregating line are analyzed on a capillary sequencer. To recover a fragment of interest, PCR products are cloned and sequenced. Sequences with lengths matching the size of a band that co-segregates with the mutant phenotype represent candidate linked insertion sites, which are then confirmed by PCR. We demonstrate the utility of the method by identifying Mu insertion sites linked to seed-lethal mutations with a preliminary success rate of nearly 50%.     

Creation of a potato mutant lacking the starch branching enzyme gene StSBE3 that was generated by genome editing using the CRISPR/dMac3-Cas9 system

The potato tuber starch trait is changed depending on the composition of amylose and amylopectin. The amount of amylopectin is determined by the activity of the starch branching enzymes SBE1, SBE2, and SBE3 in potato. SBE3, a homolog of rice BEI, is a major gene that is abundant in tubers. In this study, we created mutants of the potato SBE3 gene using CRISPR/Cas9 attached to the translation enhancer dMac3. Potato has a tetraploid genome, and a four-allele mutant of the SBE3 gene is desired. Mutations in the SBE3 gene were found in 89 of 126 transformants of potato plants. Among these mutants, 10 lines contained four mutant SBE3 genes, indicating that 8% efficiency of target mutagenesis was achieved. These mutants grew normally, similar to the wild-type plant, and yielded sufficient amounts of tubers. The potato starch in these tubers was similar to that of the rice BEI mutant. Western blot analysis revealed the defective production of SBE3 in the mutant tubers, suggesting that these transformants were loss-of-function mutants of SBE3.     

Protein phosphorylation in amyloplasts regulates starch branching enzyme activity and protein-protein interactions

Protein phosphorylation in amyloplasts and chloroplasts of Triticum aestivum (wheat) was investigated after the incubation of intact plastids with gamma-(32)P-ATP. Among the soluble phosphoproteins detected in plastids, three forms of starch branching enzyme (SBE) were phosphorylated in amyloplasts (SBEI, SBEIIa, and SBEIIb), and both forms of SBE in chloroplasts (SBEI and SBEIIa) were shown to be phosphorylated after sequencing of the immunoprecipitated (32)P-labeled phosphoproteins using quadrupole-orthogonal acceleration time of flight mass spectrometry. Phosphoamino acid analysis of the phosphorylated SBE forms indicated that the proteins are all phosphorylated on Ser residues. Analysis of starch granule-associated phosphoproteins after incubation of intact amyloplasts with gamma-(32)P-ATP indicated that the granule-associated forms of SBEII and two granule-associated forms of starch synthase (SS) are phosphorylated, including SSIIa. Measurement of SBE activity in amyloplasts and chloroplasts showed that phosphorylation activated SBEIIa (and SBEIIb in amyloplasts), whereas dephosphorylation using alkaline phosphatase reduced the catalytic activity of both enzymes. Phosphorylation and dephosphorylation had no effect on the measurable activity of SBEI in amyloplasts and chloroplasts, and the activities of both granule-bound forms of SBEII in amyloplasts were unaffected by dephosphorylation. Immunoprecipitation experiments using peptide-specific anti-SBE antibodies showed that SBEIIb and starch phosphorylase each coimmunoprecipitated with SBEI in a phosphorylation-dependent manner, suggesting that these enzymes may form protein complexes within the amyloplast in vivo. Conversely, dephosphorylation of immunoprecipitated protein complex led to its disassembly. This article reports direct evidence that enzymes of starch metabolism (amylopectin synthesis) are regulated by protein phosphorylation and indicate a wider role for protein phosphorylation and protein-protein interactions in the control of starch anabolism and catabolism.     

Cas9‐mediated mutagenesis of potato starch‐branching enzymes generates a range of tuber starch phenotypes

We investigated whether Cas9‐mediated mutagenesis of starch‐branching enzymes (SBEs) in tetraploid potatoes could generate tuber starches with a range of distinct properties. Constructs containing the Cas9 gene and sgRNAs targeting SBE1, SBE2 or both genes were introduced by Agrobacterium‐mediated transformation or by PEG‐mediated delivery into protoplasts. Outcomes included lines with mutations in all or only some of the homoeoalleles of SBE genes and lines in which homoeoalleles carried several different mutations. DNA delivery into protoplasts resulted in mutants with no detectable Cas9 gene, suggesting the absence of foreign DNA. Selected mutants with starch granule abnormalities had reductions in tuber SBE1 and/or SBE2 protein that were broadly in line with expectations from genotype analysis. Strong reduction in both SBE isoforms created an extreme starch phenotype, as reported previously for low‐SBE potato tubers. HPLC‐SEC and ¹H NMR revealed a decrease in short amylopectin chains, an increase in long chains and a large reduction in branching frequency relative to wild‐type starch. Mutants with strong reductions in SBE2 protein alone had near‐normal amylopectin chain‐length distributions and only small reductions in branching frequency. However, starch granule initiation was enormously increased: cells contained many granules of <4 μm and granules with multiple hila. Thus, large reductions in both SBEs reduce amylopectin branching during granule growth, whereas reduction in SBE2 alone primarily affects numbers of starch granule initiations. Our results demonstrate that Cas9‐mediated mutagenesis of SBE genes has the potential to generate new, potentially valuable starch properties without integration of foreign DNA into the genome.