Luguang Wu

Characterization of the Highly Efficient Sucrose Isomerase from Pantoea dispersa UQ68J and Cloning of the Sucrose Isomerase Gene

ABSTRACT Sucrose isomerase (SI) genes from Pantoea dispersa UQ68J, Klebsiella planticola UQ14S, and Erwinia rhapontici WAC2928 were cloned and expressed in Escherichia coli. The predicted products of the UQ14S and WAC2928 genes were similar to known SIs. The UQ68J SI differed substantially, and it showed the highest isomaltulose-producing efficiency in E. coli cells. The purified recombinant WAC2928 SI was unstable, whereas purified UQ68J and UQ14S SIs were very stable. UQ68J SI activity was optimal at pH 5 and 30 to 35°C, and it produced a high ratio of isomaltulose to trehalulose (>22:1) across its pH and temperature ranges for activity (pH 4 to 7 and 20 to 50°C). In contrast, UQ14S SI showed optimal activity at pH 6 and 35°C and produced a lower ratio of isomaltulose to trehalulose (<8:1) across its pH and temperature ranges for activity. UQ68J SI had much higher catalytic efficiency; the Km was 39.9 mM, the Vmax was 638 U mg−1, and the Kcat/Km was 1.79 × 104 M−1 s−1, compared to a Km of 76.0 mM, a Vmax of 423 U mg−1, and a Kcat/Km of 0.62 × 104 M−1 s−1 for UQ14S SI. UQ68J SI also showed no apparent reverse reaction producing glucose, fructose, or trehalulose from isomaltulose. These properties of the P. dispersa UQ68J enzyme are exceptional among purified SIs, and they indicate likely differences in the mechanism at the enzyme active site. They may favor the production of isomaltulose as an inhibitor of competing microbes in high-sucrose environments, and they are likely to be highly beneficial for industrial production of isomaltulose.

Characterization of Pantoea dispersa UQ68J: producer of a highly efficient sucrose isomerase for isomaltulose biosynthesis.

Aims:  Isolation, identification and characterization of a highly efficient isomaltulose producer.

Establishment and application of a loop-mediated isothermal amplification (LAMP) system for detection of cry1Ac transgenic sugarcane

To meet the demand for detection of foreign genes in genetically modified (GM) sugarcane necessary for regulation of gene technology, an efficient method with high specificity and rapidity was developed for the cry1Ac gene, based on loop-mediated isothermal amplification (LAMP). A set of four primers was designed using the sequence of cry1Ac along with optimized reaction conditions: 5.25 mM of Mg2+, 4:1 ratio of inner primer to outer primer, 2.0 U of Bst DNA polymerase in a reaction volume of 25.0 μL. Three post-LAMP detection methods (precipitation, calcein (0.60 mM) with Mn2+ (0.05 mM) complex and SYBR Green I visualization), were shown to be effective. The sensitivity of the LAMP method was tenfold higher than that of conventional PCR when using templates of the recombinant cry1Ac plasmid or genomic DNA from cry1Ac transgenic sugarcane plants. More importantly, this system allowed detection of the foreign gene on-site when screening GM sugarcane without complex and expensive instruments, using the naked eye. This method can not only provide technological support for detection of cry1Ac, but can also further facilitate the use of this detection technique for other transgenes in GM sugarcane.

Field performance of transgenic sugarcane expressing isomaltulose synthase

Transgenic sugarcane plants expressing a vacuole-targeted isomaltulose (IM) synthase in seven recipient genotypes (elite cultivars) were evaluated over 3 years at a field site typical of commercial cane growing conditions in the Burdekin district of Australia. IM concentration typically increased with internode maturity and comprised up to 217 mm (33% of total sugars) in whole-cane juice. There was generally a comparable decrease in sucrose concentration, with no overall decrease in total sugars. Sugarcane is vegetatively propagated from stem cuttings known as setts. Culture-derived plants were slower to establish and generally gave shorter and thinner stalks at harvest than those grown from field-sourced setts in the initial field generations. However, after several cycles of field propagation, selections were obtained with cane yields similar to the recipient genotypes. There was no apparent adverse effect of IM accumulation on vigour assessed by stalk height and diameter or other visual indicators including germination of setts and establishment of stools. There was some inconsistency in IM levels in juice, between samplings of the vegetatively propagated transgenic lines. Until the causes are resolved, it is prudent to selectively propagate from stalks with higher IM levels in the initial vegetative field generations. Pol/Brix ratio allowed rapid identification of lines with high IM levels, using common sugar industry instruments. Sucrose isomerase activity was low in these transgenic lines, and the results indicate strong potential to develop sugarcane for commercial-scale production of IM if higher activity can be engineered in appropriate developmental patterns.

Physiological basis for enhanced sucrose accumulation in an engineered sugarcane cell line

Transgenic sugarcane (Saccharum officinarum L. interspecific hybrids) line N3.2 engineered to express a vacuole-targeted sucrose isomerase was found to accumulate sucrose to twice the level of the background genotype Q117 in heterotrophic cell cultures, without adverse effects on cell growth. Isomaltulose levels declined over successive subcultures, but the enhanced sucrose accumulation was stable. Detailed physiological characterisation revealed multiple processes altered in line N3.2 in a direction consistent with enhanced sucrose accumulation. Striking differences from the Q117 control included reduced extracellular invertase activity, slower extracellular sucrose depletion, lower activities of symplastic sucrose-cleavage enzymes (particularly sucrose synthase breakage activity), and enhanced levels of symplastic hexose-6-phosphate and trehalose-6-phosphate (T6P) in advance of enhanced sucrose accumulation. Sucrose biosynthesis by sucrose phosphate synthase (SPS) and sucrose phosphate phosphatase (SPP) was substantially faster in assays conducted to reflect the elevation in key allosteric metabolite glucose-6-phosphate (G6P). Sucrose-non-fermenting-1-related protein kinase 1 (SnRK1, which typically activates sucrose synthase breakage activity while downregulating SPS in plants) was significantly lower in line N3.2 during the period of fastest sucrose accumulation. For the first time, T6P is also shown to be a negative regulator of SnRK1 activity from sugarcane sink cells, hinting at a control circuitry for parallel activation of key enzymes for enhanced sucrose accumulation in sugarcane.

Effects of moist chilling and solid matrix priming on germination of loblolly pine (Pinus taeda L.) seeds

Loblolly pine (Pinus taeda L.) seeds from sources with a mild climate under maritime influence (North Carolina) required shorter moist chilling to achieve maximum germination vigor than seeds from sources with a harsher continental climate (Oklahoma). Solid matrix priming (SMP) for 6 d achieved as much as 60 d of moist chilling to improve rapidity, synchrony and completeness of germination for three of the four families studied. SMP after moist chilling increased the rapidity, synchrony and completeness of germination. The benefit of SMP was greatest for non-stratified seeds and the benefit decreased with length of moist chilling. In general, delaying planting for one week after SMP had minor effects on germination when seeds were kept in the SMP matrix at 4°C. Delayed planting after SMP can increase germination rapidity and synchrony of seeds that have received long moist chilling and reduce the benefit of SMP in non-moist-chilled seeds.

Selection of Suitable Endogenous Reference Genes for Relative Copy Number Detection in Sugarcane

Transgene copy number has a great impact on the expression level and stability of exogenous gene in transgenic plants. Proper selection of endogenous reference genes is necessary for detection of genetic components in genetically modification (GM) crops by quantitative real-time PCR (qPCR) or by qualitative PCR approach, especially in sugarcane with polyploid and aneuploid genomic structure. qPCR technique has been widely accepted as an accurate, time-saving method on determination of copy numbers in transgenic plants and on detection of genetically modified plants to meet the regulatory and legislative requirement. In this study, to find a suitable endogenous reference gene and its real-time PCR assay for sugarcane (Saccharum spp. hybrids) DNA content quantification, we evaluated a set of potential “single copy” genes including P4H, APRT, ENOL, CYC, TST and PRR, through qualitative PCR and absolute quantitative PCR. Based on copy number comparisons among different sugarcane genotypes, including five S. officinarum, one S. spontaneum and two S. spp. hybrids, these endogenous genes fell into three groups: ENOL-3—high copy number group, TST-1 and PRR-1—medium copy number group, P4H-1, APRT-2 and CYC-2—low copy number group. Among these tested genes, P4H, APRT and CYC were the most stable, while ENOL and TST were the least stable across different sugarcane genotypes. Therefore, three primer pairs of P4H-3, APRT-2 and CYC-2 were then selected as the suitable reference gene primer pairs for sugarcane. The test of multi-target reference genes revealed that the APRT gene was a specific amplicon, suggesting this gene is the most suitable to be used as an endogenous reference target for sugarcane DNA content quantification. These results should be helpful for establishing accurate and reliable qualitative and quantitative PCR analysis of GM sugarcane.

Isomaltulose is Actively Metabolized in Plant Cells

Isomaltulose is a structural isomer of sucrose (Suc). It has been widely used as a nonmetabolized sugar in physiological studies aimed at better understanding the regulatory roles and transport of sugars in plants. It is increasingly used as a nutritional human food, with some beneficial properties including low glycemic index and acariogenicity. Cloning of genes for Suc isomerases opened the way for direct commercial production in plants. The understanding that plants lack catabolic capabilities for isomaltulose indicated a possibility of enhanced yields relative to Suc. However, this understanding was based primarily on the treatment of intact cells with exogenous isomaltulose. Here, we show that sugarcane (Saccharum interspecific hybrids), like other tested plants, does not readily import or catabolize extracellular isomaltulose. However, among intracellular enzymes, cytosolic Suc synthase (in the breakage direction) and vacuolar soluble acid invertase (SAI) substantially catabolize isomaltulose. From kinetic studies, the specificity constant of SAI for isomaltulose is about 10% of that for Suc. Activity varied against other Suc isomers, with Vmax for leucrose about 6-fold that for Suc. SAI activities from other plant species varied substantially in substrate specificity against Suc and its isomers. Therefore, in physiological studies, the blanket notion of Suc isomers including isomaltulose as nonmetabolized sugars must be discarded. For example, lysis of a few cells may result in the substantial hydrolysis of exogenous isomaltulose, with profound downstream signal effects. In plant biotechnology, different Vmax and Vmax/Km ratios for Suc isomers may yet be exploited, in combination with appropriate developmental expression and compartmentation, for enhanced sugar yields.

Enhancement of Sugar Yield by Introducing a Metabolic Sink in Sugarcane

Increasing sugar content is a primary objective of sugarcane improvement programs worldwide, since sugar accumulation in sugarcane is well below the theoretical physiological limits. Despite decades of conventional breeding and molecular manipulation, sugar content has remained stagnant in the new varieties for the past few decades. Recent experiments in genetic engineering have shown that sugar content in the transgenic sugarcanes was increased significantly (SugarBooster) by creating a metabolic sink through converting sucrose into slowly digested or inert products in the vacuoles of sugar storage cells. Technically, SugarBooster lines were created by introducing a gene for sucrose isomerase or fructosyl transferase into sugarcane to generate isomaltulose- or fructants-like products. The high-value sugar isomaltulose was accumulated in storage tissues without any decrease in stored sucrose concentration, resulting in up to doubled total sugar concentrations in harvested juice. Plant lines with enhanced sugar accumulation also showed increased photosynthesis, sucrose transport and sink strength. The transgenic sugarcane line engineered to express a vacuole-targeted sucrose isomerase, which shows SugarBooster effects, was also found to accumulate sucrose to twice the concentration of the background genotype in heterotrophic cell cultures, without adverse effects on cell growth. Isomaltulose concentrations declined over successive subcultures, but enhanced sucrose accumulation was stable. Detailed physiological characterization revealed multiple processes altered in the transgenic line in a direction consistent with enhanced sucrose accumulation. Striking differences from the control included reduced extracellular invertase activity, slower extracellular sucrose depletion, lower activities of symplastic sucrose cleavage enzymes (particularly sucrose synthase (SuSy) breakage activity) and enhanced concentrations of symplastic hexose-6-phosphate and trehalose-6-phosphate in advance of enhanced sucrose accumulation. Sucrose biosynthesis by sucrose phosphate synthase (SPS) and sucrose phosphate phosphatase was substantially faster in assays conducted to reflect the elevation in key allosteric metabolite glucose-6-phosphate. Sucrose non-fermenting-1-related protein kinase 1 (SnRK1, which typically activates SuSy breakage activity while down-regulating SPS in plants) was significantly lower in the transgenic line during the period of fastest sucrose accumulation. The SugarBooster effects elucidated from either transgenic plants or suspension-cultured cells provide hints at a control circuitry for parallel activation of key enzymes for enhanced sucrose accumulation in sugarcane. Approaches are discussed for enhancing sugar content by manipulating multiple genes.