Isomaltulose was efficiently accumulated in T0 transgenic lines
Twenty independent transgenic lines were demonstrated to contain the sucrose isomerase (SI) gene using the polymerase chain reaction (PCR) analysis. Among these lines, 16 displayed detectable isomaltulose levels by high-performance liquid chromatography (HPLC) in stalks (Fig. 1a). Up to 446 mM isomaltulose was accumulated in stalk juice, which was fourfold higher than the total sugar content of the untransformed T×430. The isomaltulose concentrations were substantially variable among lines (Fig. 1b). Similar patterns were observed in two transgenic populations driven by different promoters of A1 or LSG2 (Fig. 1b).
Because the UQ68J SI gene is highly specific for producing isomaltulose [24], trehalulose concentrations were generally below 5% of the isomaltulose concentrations in the corresponding internodes (Additional file 1: Table S1). The majority of transgenic lines were morphologically similar and equivalent to the untransformed control T×430 in the glasshouse (Additional file 1: Fig. S1). Transgenic plants flowered at a similar time as the control T×430 (Additional file 1: Fig. S1).
The roots and leaves were tested from all the transgenic lines, isomaltulose concentrations were below 5 mM in roots. Isomaltulose concentration increased with age in leaves to a maximum of about 20 mM, which is consistent with the expression patterns for the ‘stem-dominant’ promoters [34, 35]. However, SI enzyme activity could not be detected from cell extracts of transgenic roots or leaves. The negative effect on sorghum growth was not observed due to the small amount of isomaltulose accumulation in roots and leafs (Additional file 1: Fig. S1). Despite substantial isomaltulose accumulation in stalks, SI enzyme activity was below the detection threshold in cell extracts, indicating a short half-life of this protein after delivery into the acidic/proteolytic sucrose storage vacuoles.
Total sugar content was greatly enhanced in T0 transgenic lines
The total sugar content has been notably increased in 20 T0 transgenic lines compared to the wild-type control except two lines (L2, and L24), regardless of which promoter used (A1 or LSG2) (Fig. 2). The total sugar contents in internode number 4 of most lines were in a range of 600 to 1,000 mM, which was equivalent to five to eight folds of the control (Additional file 1: Table S1). These concentrations were comparable or even higher than that of the field-grown sugarcane (normally 600–700 mM). The predominant components of sugar were sucrose and isomaltulose in transgenic lines; meanwhile, their glucose and fructose contents were similar to the parent (Fig. 2). Unexpectedly, some transgenic lines such as L4 and A2 had no detectable isomaltulose but sucrose contents were enhanced fivefold to eightfold when compared to the control T×430 (Fig. 2), regardless of the promoter used.
High sugar contents were accumulated across internodes of elite transgenic stalks
Three transgenic lines, designated A2, A5 (both driven by A1 promoter) and L9 (driven by LSG2 promoter), with high-sugar content were selected for further characterization on sugar profiles in developmental stages. Lines A5 and L9 accumulated high levels of isomaltulose up to 691 mM in juice from mature internodes (Fig. 3c, d). Compared to the control T×430, the transgenic lines with high yields of isomaltulose did not show commensurable reduction but enhanced sucrose content in most internodes (Fig. 2). Surprisingly, isomaltulose could not be detected in any A2 tissues including all internodes of the stalks, but sucrose content accumulated eightfold higher than the level in the control T×430 (Fig. 3b).
Further investigation on T1 progenies of A2, A5, and L9 has been performed and focused on heritability of high sugar content. Up to twelve samples of each progeny have been analyzed. T1 progenies of L9 outperformed the counterparts of A2, and A5 in terms of high heritability. Because no isomaltulose was detected in A2, the phenotype of high sugar content did not transmit to the next generation. T1 progenies of A5 accumulated up to 367 mM isomaltulose and up to 550 mM total sugar content, those sugar contents in T1 progenies were not as high as sugar contents in T0 generation. The results of L9 T1 progeny samples were very promising and displayed high heritability of high sugar content (up to 896 mM in stalk). Positive samples have accumulated much higher sugar content than null-segregants (nil-samples) and the wild-type controls (Additional file 1: Fig. S4).
Real-time PCR was performed on A5, L2, and L9 T1 generation
Quantitative real-time PCR was deployed to determine the SI gene expression in different transgenic lines. The elite transgenic lines, accumulating high isomaltulose, and high total sugar, A5 and L9 were selected. Line L2, with poor isomaltulose accumulation, was chosen for comparison. Non-transgenic T×430 was used as the wild-type control. T1 Positive progenies of A5, L2, and L9 were identified by PCR screening of the SI gene (Additional file 1: Fig. S5). The RT-PCR results revealed that A5 and L9 displayed a relatively high levels of SI gene expression, which are in agreement with their high levels of isomaltulose accumulation. L2 showed comparatively low level of SI gene expression, which aligned with poor isomaltulose accumulation. As expected, no SI gene expression was detected in stalks of the wild-type T×430 (Fig. 4).
High sugar contents were inherited in F1 hybrids
The elite sweet sorghum cultivar R9188, and Rio were selected as female lines for crossing due to their advantages of large biomass and high-sucrose content in stalks. Transgenic lines A5, and L9 were chosen as male lines because of their superior performance on isomaltulose accumulation and high total sugar content. Crosses were performed with the male-sterile lines of R9188, and Rio. Hybrid seeds were harvested from successful crossing.
Thirty seeds of hybrids of Rio X L9 were sown in pots along with the controls of Rio, R9188, and T×430 in the glasshouse. The sweet sorghum cultivar R9188 is another version of Rio with an extra dwarf gene, hence almost 50 cm shorter. Hybrid seed germination and early seedling growth were similar to the controls, except one hybrid seed which did not germinate. Sugar profiles of HPLC showed that among 29 progenies of F1 generation, isomaltulose was detected in 15 progenies (51.7%) and no isomaltulose was detected in the rest of 14 samples (48.3%). The ratio of positive to negative samples was close to the predicted 1:1 ratio (Fig. 5), indicating hybrid seeds inherited the SI gene as a single genetic locus from the parent L9.
Within the 15 isomaltulose positive group, three progenies converted almost all sucrose into isomaltulose; six converted more than 65% of sucrose; two converted about 33% of sucrose; four had less than 1% sucrose converted (Fig. 5). Notably, the improvement of total sugar content was observed in most isomaltulose positive lines (Fig. 5). The increase of total sugar content was on average 37% higher than the sweet sorghum Rio. The increase ranged from 484 to 932% if compared with the grain sorghum T×430, which is in agreement with the results of the T0 generation (Fig. 2).
Another hybrid population of R9188 X L9 were planted as well. It showed similar pattern as the population of Rio X L9. Among 26 F1 population, 12 of them are positive for sucrose isomerase gene (Additional file 1: Table S2). The highest total sugar content at 764 mM was measured in F1 L9R9-20 line and the top isomaltulose content at 565 mM was detected in the F1 L9R9-9 line. By comparison, remarkably higher total sugar contents were monitored in positive SI lines (on average 538 mM) than negative SI lines (on average 342), which means the sugar content has been improved 57.3% thanks to the SI gene. While the average sugar content in the sweet sorghum R9188 and grain T×430 were 261 and 93 mM, respectively. The detail of results was shown in Additional file 1: Table S2.
High sugar contents were inherited in F2 population
Based on isomaltulose concentration, total sugar content, stalk biomass, and seed production, F1 (Rio X L9) progenies LR3, LR19 and LR20 were selected for further characterization. With the parental controls of sweet sorghum Rio, progeny LR24, a null segregant with comparative high sugar content was also selected as a hybrid control. Seeds were produced by self-pollination.
Sugar profiles of the isomaltulose positive plants showed that they inherited the phenotype of high isomaltulose and high sugar content (Fig. 6). In all SI positive progenies, isomaltulose accumulated at high levels in all internodes. In addition, sucrose content were stored at comparable levels (total sugar content up to 812.2 mM), resulting in up to 69% increase of total sugar content compared to the parental (480.6 mM) or the hybrid control (470.9 mM) (Fig. 6).
Sugar content was increased, whereas water content was decreased in F2 stalk juice
Carbon partitioning into sugars and fiber was estimated in the selected F2 progenies and controls. There was more sugar per unit fresh weight (FW) in all internodes of the tested high-sugar progenies than the controls (Fig. 7a). In the sweet sorghum Rio and hybrid null segregant LR24, the water content was typically constant around 75% along the stalk with a slight increase in the bottom internodes; however, in the stalks of three high-sugar progenies, water content was significantly lower around 70% (Fig. 7b). Moreover, there were no significant change in the fiber content among all samples, which was around 11% in internode tissues (Fig. 7). These results indicated that instead of alteration of fiber and sugar, assimilation was improved and more sugar was stored in the progenies LR3, LR19, and LR20 than the controls. Therefore, the commercially important trait of high sugar content in juice from the selected progenies are underpinned by increasing sugar content and decreasing water content in the mature stalk.
Photosynthesis was increased in high sugar F2 lines
Two key physiological characteristics, including photosynthetic electron transport and CO2 assimilation, were examined to understand the mechanisms of enhanced sugar accumulation. Rates of leaf electron transport and CO2 assimilation of the progenies LR3, LR19, and LR20 were higher than the controls Rio, T×430 and hybrid control LR24. The photosynthetic electron transport rate and CO2 assimilation rate were measured by chlorophyll fluorescence (reflecting photosynthetic efficiency in photosystem II). The both rates were improved by 20% to 35% in high sugar content F2 lines compared to the controls at different photosynthetically active radiation (PAR) levels (Fig. 8). Light response curves from the fully expanded leaf 2 are measured (Fig. 8). Moreover, the senescence of the bottom leaves on each stalk of the high sugar progenies was typically delayed by 2–3 weeks, resulting in leaf functional extension in photosynthesis.
Sugar transport was improved in source leaves and sink tissues
Rate of proton gradient-dependent sucrose transport into plasma membrane vesicles (PMV) is an indicator for sucrose uploading in the source leaves [36]. The isolated PMVs from leaf 2 and 3 of the selected high-sugar progenies LR3, LR19, and LR20 were 20% to 40% higher than that of controls (null segregant LR24, parents Rio and T×430), indicating the driving power of loading assimilation for transport was improved (Fig. 9a) in the source leaves of the high-sugar progenies.
Sorghum phloem in a stem vascular bundle is symplasmically isolated from the surrounding parenchyma cells, and the sucrose unloading is apoplasmic [37]. Cell wall invertase (CWI) activity is a determinant of sucrose gradient in the unloading area. In all tested internodes, CWI activities of the central storage parenchyma-rich zone were significantly higher in the high-sugar progenies than in the controls LR24, Rio and T×430 (Fig. 9b), but not in the peripheral vascular-rich zone (Fig. 9c). When the vascular bundles were dissected from the storage parenchyma cells in the central zone of internode 5 and assayed separately, the increased CWI activity in the high-sugar progenies was clearly restricted to the storage parenchyma (Fig. 9d), indicating the abilities on assimilate was increased within the sink tissues of the high-sugar progenies.