Sucrose phosphate synthase (SPS) is a key enzyme in sucrose synthesis, which controls sucrose content in plants. This study was designed to examine the efficacy of the overexpression of SoSPS1 gene on sucrose accumulation and carbon partitioning in transgenic sugarcane. The overexpression of SoSPS1 gene increased SPS activity and sucrose content in transgenic sugarcane leaves. More importantly, the overexpression enhanced soluble acid invertase (SAI) activity concomitant with the increase of glucose and fructose levels in the leaves, whereas sucrose synthase activity exhibited almost no change. In the stalk, a similar correlation was observed, but a higher correlation was noted between SPS activity and sugar content. These results suggest that SPS overexpression has both direct and indirect effects on sugar concentration and SAI activity in sugarcane. In addition, SPS overexpression resulted in a significant increase in plant height and stalk number in some transgenic lines compared to those in non-transgenic control. Taken together, these results strongly suggest that enhancing SPS activity is a useful strategy for improving sugarcane yield.

Sucrose phosphate synthase (SPS) catalyses the transfer of glycosyl group of uridine diphosphate glucose to fructose-6-phosphate to form sucrose-6-phosphate. Plant SPS plays a key role in photosynthetic carbon metabolisms, which activity is modulated by an allosteric activator glucose-6-phosphate (G6P). We produced recombinant sugarcane SPS using Escherichia coli and Sf9 insect cells to investigate its structure-function relationship. When expressed in E. coli, two forms of SPS with different sizes appeared; the larger was comparable in size with the authentic plant enzyme and the shorter was trimmed the N-terminal 20kDa region off. In the insect cells, only enzyme with the authentic size was produced. We purified the trimmed SPS and the full size enzyme from insect cells and found their enzymatic properties differed significantly; the full size enzyme was activated allosterically by G6P, while the trimmed one showed a high activity even without G6P. We further introduced a series of N-terminal truncations up to 171 residue and found G6P-independent activity was enhanced by the truncation. These combined results indicated that the N-terminal region of sugarcane SPS is crucial for the allosteric regulation by G6P and may function like a suppressor domain for the enzyme activity.Keywords: allosteric regulation/carbon metabolism/ recombinant enzyme/sucrose phosphate synthase/ sugarcane.Abbreviations: F6P, fructose-6-phophate; G6P, glucose-6-phosphate SPS, sucrose phosphate synthase; UDP-G, uridine diphosphate glucose.Sucrose is the primary photosynthate in plant leaves and transported to various growing tissues and storage organs for allocation of carbon resources through whole plant body. Sucrose phosphate synthase (SPS) plays a key role in the pathway of sucrose synthesis, which catalyses formation of sucrose-6-phosphate (S6P) by use of fructose-6-phosphate (F6P) and uridine diphosphate-glucose (UDP-G) as substrates (13). The activity of SPS is regulated by diurnal cycles of day/ night (4) and other environmental factors such as osmotic stress (5) and cold temperature (6). Protein phosphorylation of certain serine residues of SPS polypeptide and allosteric control by metabolites, such as glucose-6-phospate (G6P) and inorganic phosphate, are involved in the regulation of SPS activity (7,8). Such molecular characteristics of SPS are believed to be crucial for playing the physiological role of regulating photosynthetic carbon flux into sucrose.Plant SPSs have a molecular mass around 120 kDa and consists of three domains. The central region contains glycosyltransferase domain responsible for the catalytic function of SPS (9). The C-terminal region resembles sucrose phosphate phosphatase and the N-terminal region has no clear similarity with any other proteins. The SPSs from the photosynthetic cyanobacteria (10) and non-photosynthetic bacteria (11) lack the N-terminal domain or both terminal domains of plant SPS, respectively, and unlike plant SPS, their enzyme activity is not allosterically regulated (12,13). This in...

Sugarcane mosaic virus (SCMV) is a plant pathogenic virus of the family Potyviridae that causes chlorosis, stunting and significantly reduced sugar productivity in sugarcane. Pathogen-derived resistance is a method used to develop SCMV-resistant sugarcane by overexpression of viral DNA. In this study, the gene encoding the coat protein (CP) of SCMV was amplified by reverse transcriptase PCR from symptomatic sugarcane leaves and used to generate transgenic sugarcane. Nucleotide sequence analysis of amplified cDNA indicated that the 998-bp-long cDNA, termed ScMVCp cDNA, codes for the CP of SCMV from the PS881 isolate. The ScMVCp cDNA was inserted into the binary vector pRI101-ON with two constructs, a full nucleotide sequence (p927) and a sequence coding for N-terminally truncated protein (p702). The constructs were then introduced into sugarcane using Agrobacterium-mediated transformation. Southern blot analysis showed a single hybridized DNA copy inserted into the genome of transgenic sugarcane lines. The inserted genes were expressed at both the RNA transcript and protein levels in the transgenic sugarcane. The highest expression was found in transgenic lines 10, 11 and 13 from the p927 construct. Artificial infection by the virus showed that p927 generated a higher resistance to virus compared with p702. This resistance was passed on to the second generation of transgenic sugarcane with 100 and 20-40% levels of resistance in the p927 and p702 transgenic lines, respectively. This report shows that the full sequence of the CP gene is required to disrupt viral assembly and packaging, thereby generating resistance to SCMV infection.