Accumulating substantial isomaltulose in transgenic lines
Twenty independent transgenic lines were demonstrated to contain the sucrose isomerase (SI) gene using the polymerase chain reaction (PCR) analysis. A representative chromatogram (Fig. 1a) is shown the detection of sugar profile including glucose, fructose, sucrose, trehalulose, and isomaltulose, by high-performance liquid chromatography (HPLC). Among these transgenic lines, 16 of 20 showed detectable isomaltulose levels. Isomaltulose was accumulated up to 446 mM in stalk juice, which was four-fold higher than the total sugar content of the untransformed Tx430 (118 mM). There were substantial differences in isomaltulose content between transgenic lines (Fig. 1b). As for isomaltulose accumulation, similar patterns were observed in two transgenic populations driven by promoters of A or L (Fig. 1b).
Because of the high specificity of the UQ68J SI for producing isomaltulose [23], trehalulose concentrations in stem juice of most positive samples were below 5.0% of the isomaltulose concentrations in corresponding internodes (Table S1). Most transgenic lines with the vacuole-targeted, silencing-optimized NTPP-68J SI, expression driven by a stem-specific promoter (A or L) [33], were morphologically similar and equivalent in measured growth parameters to the untransformed control Tx430 grown at the vegetative growth stage in the PC2 glasshouse (Fig. S1). Transgenic plants flowered at a similar time as the control Tx430 (Fig. S1).
When the roots and leaves were tested from all the transgenic lines, isomaltulose concentrations were below 5 mM in roots. Isomaltulose content increased with age in leaves to a maximum of 20 mM, which is consistent with the expression patterns for the ‘stem-dominant’ promoters [33, 34]. However, SI enzyme activity could not be detected from cell extracts of transgenic roots or leaves. Despite substantial isomaltulose accumulation in stalks, SI enzyme activity in stalk 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.
Enhancing total sugar content in grain sorghum
The majority of transgenic lines notably increased their total sugar contents compared to the untransformed control, regardless of which promoter was used (Fig. 2). The total sugar content in internode number 4 (from the top) of most lines was in a range of 600 - 1,000 mM, which was equivalent to five to eight folds of the untransformed control. These total sugar contents 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, however, their glucose and fructose contents were similar to the control Tx430 (Fig. 2).
Unexpectedly, some transgenic lines such as L4 and A2 had no detectable isomaltulose but sucrose contents were enhanced five-fold to eightfold when compared to the control Tx430 (Fig. 2).
Accumulating high sugar contents across transgenic stalks
Three transgenic lines with high-sugar contents, A2, A5 driven by A1(A) promoter and L9 driven by LSG2 (L) promoter), were selected for further characterization on sugar profiles of all internodes (Fig. 3) in developmental stages (20 days post anthesis). The control Tx430 accumulated higher sugar content in the first internode (close to panicle) than the rest internodes (Fig. 3a). On the contrary, the transgenic lines accumulated much less sugar in the first internode than the rest internodes (Fig. 3b, 3c, and 3d). Lines A5 and L9 accumulated high levels of isomaltulose in the stalk up to 691 mM in juice from mature internodes (Fig. 3c, 3d). Compared to the control Tx430, the transgenic lines with high yields of isomaltulose did not show commensurable reduction but enhanced levels in stored sucrose concentrations in most internodes (Fig. 3).
Surprisingly, isomaltulose could not be detected in any A2 tissues including all internodes of the stalks, but sucrose content accumulated eightfold higher in A2 than in the control Tx430 (Fig. 3b).
Inheriting high-sugar content in hybrids
The elite sweet sorghum cultivar Rio was selected as a female partner for crossing due to its advantages of large biomass and high-sucrose content in stalks. Crosses were performed with the cytoplasmic male-sterile version of Rio which is used in the sorghum breeding program in Australia. Transgenic line L9 was selected as the male partner based on its isomaltulose accumulation, high total sugar content and normal development in reproductive organs compared to other transgenic lines. Hybrid F1 seeds were harvested from successful crossing.
Thirty seeds of hybrids were sown in pots along with the controls of Rio and Tx430 in the PC2 glasshouse. Another sweet sorghum cultivar R9188, a version of Rio with an extra dwarf gene, hence almost 50 cm shorter, was used as an additional control. Germination and early plant growth were similar to controls, except the progenies of one hybrid seed which did not germinate. Sugar profiles showed that among 29 progenies of the F1 generation, 15 progenies were isomaltulose positive (51.7%) and 14 had no detectable isomaltulose (48.3%), close to the predicted 1:1 ratio (Fig. 4), indicating that hybrid seeds inherited the SI gene sexually from the parent L9.
Within the isomaltulose positive group, three progenies (10.3%), named LR27, LR17, and LR9, converted almost all sucrose into IM (last three bars in Fig. 4); six (20.6%) converted more than 65% of sucrose; two (6.9%) converted about 33% of sucrose; four (13.8%) had less than 1% sucrose converted (Fig. 4). Notably, the enhancement of total sugar content was observed in most isomaltulose positive groups (Fig. 4). The increase of total sugar content in the positive group was from 17% to 57% when compared to the sweet sorghum Rio. The increase ranged from 484% to 932% if compared with the grain sorghum Tx430, which is in agreement with the results of the first transgenic T0 generation (Fig. 2).
Based on isomaltulose production, total sugar content, stalk weight, and seed production, progenies P3 from LR3, P19 from LR19, and P20 from LR20 outperformed the rest and were selected for further characterization. With the parental controls of sweet sorghum Rio, a null segregant progeny P24 from LR24 was also selected as a hybrid control because it was negative of the SI gene and no detectable isomaltulose, with comparative high sugar content. Seeds were produced by self-pollination of the selected progenies and harvested at maturity.
Sugar profiles of the isomaltulose positive plants displayed that they inherited the phenotype of both isomaltulose production and high-sugar accumulation (Fig. 5). In all three SI positive progenies, isomaltulose accumulated at high levels in all internodes along the stalk, plus sucrose stored at comparable levels, resulting in enhancement by up to 69% in total sugar content compared to either the parental or the hybrid control (Fig. 5).
Increasing sugar content and decreasing water content in stalk juice
Carbon partitioning into sugars and fiber was estimated in the selected F2 progenies (P3, P19, and P20) and controls (Rio, and P24). There was more sugar per unit fresh weight (FW) in all internodes of the tested high-sugar progenies along the stalk than the controls (Fig. 6a). In the sweet sorghum Rio and hybrid control progeny P24, the water content was typically constant around 75% along the stalk with a slight increase in the bottom internodes, however, in the stalks of the three high-sugar progenies, water content was significantly lower at around 70% (Fig. 6b). Analysis of variance (ANOVA) with Bonferroni post-tests showed that all three selected SI-positive progenies had significantly increased sugar content and reduced water content (P < 0.001) in all tested internodes, compared with the Rio and hybrid P24 controls. Moreover, there were no significant changes in the fiber content among all samples, which was around 11% in internode tissues (Fig. 6). These results indicated that instead of alteration of fiber and sugar, assimilation was improved and more sugar was stored in the progenies P3, P19, and P20 than the controls. Therefore, the commercially important traits of higher sugar content in juice from the selected progenies are underpinned by increasing the storage of photosynthate as sugars and decreasing water content in the mature stalk.
Increasing photosynthesis in high-sugar hybrid 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 P3, P19, and P20 were higher than the controls Rio, Tx430 and hybrid P24. The increases in electron transport rates measured by chlorophyll fluorescence (reflecting photosynthetic efficiency in photosystem II) and in CO2 assimilation rates were in the range 20% − 35% improved relative to controls at a photosynthetically active radiation (PAR) level. Light response curves from fully expanded leaf 2 are shown as an example (Fig. 7). Analysis of variance (ANOVA) with Bonferroni post-tests showed significant differences (P < 0.001) in both electron transport and CO2 fixation rates in all tested light intensities between any control and P3, P19 or P20. Also, 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 for most of the growth period.
Improving sugar transport in source leaves and sink tissues
Rate of proton gradient-dependent sucrose transport into plasma membrane vesicles (PMV) is an indicator of sucrose uploading in the source leaves [35]. The isolated PMVs from leaf 2 and 3 of the selected high-sugar progenies were 20% − 40% higher than that of controls (null segregant P24, parents Rio and Tx430), indicating the driving power of loading assimilation for transport was improved (Fig. 8a) 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 [36]. Cell wall invertase (CWI) activity is a determinant of the 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 P24, Rio and Tx430 (Fig. 8b), but not in the peripheral vascular-rich zone (Fig. 8c). 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 restricted to the storage parenchyma (Fig. 8d), indicating the abilities on assimilation was increased within the sink tissues of the high-sugar progenies.