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. Among these lines, 16 showed detectable isomaltulose levels by high-performance liquid chromatography (HPLC) in stalk tissues (Fig. 1a). Isomaltulose was accumulated up to 472 mM in stalk juice, which was four-fold higher than the total sugar content of the untransformed Tx430. 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 4% of the isomaltulose concentrations in the corresponding internodes (Table S1). Transgenic lines were morphologically similar and equivalent to the untransformed control Tx430 in the glasshouse (Fig. S1). Transgenic plants flowered at a similar time as the control Tx430 (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 (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.
Enhancing total sugar content in grain sorghum
The total sugar content has been significantly increased in 20 transgenic lines compared to the untransformed control except two lines (L2, and L24), regardless of which promoter used (A1 or LSG2) (Fig. 2). The total sugar content in internode number 4 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 concentrations were comparable or even higher than that of the field-grown sugarcane (normally around 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 parent (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), regardless of the promoter used.
Accumulating high sugar contents across internodes of transgenic stalk
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 down the stalk up to 691 mM in juice from mature internodes (Fig. 3c, d). 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. 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 Tx430 (Fig. 3b).
Further investigation on T1 progenies of A2, A5, and L9 has been performed and focused on heritability of high sugar content. Twelve samples of each progeny have been analysized. T1 progenies of L9 outperformed counterparts of A2, and A5 in terms of high heritability and fertility. Because no isomaltulose was detected in A2, the phenotype of high sugar content did not transmit to the next generation. T1 progenies of A5 did not display full fertility the same as the 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 negative samples (Nil-LG9) and the control (Fig. S4).
Real-time PCR of T1 generation
Quantitative real-time PCR was deployed to determine the SI gene expression in different transgenic lines. The elite transgenic lines, accumulating high isomalutlose, and high total sugar, A5 and L9 were selected. Line L2 , with poor isomaltulose accumulation, was chosen for comparison. Non-transgenic Tx430 was used as the wild-type control. The RT-PCR results revealed that A5 and L9 displayed a relatively high levels of SI gene expression, which is in agreement with their high level of isomaltulose accumulation. L2 showed comparatively low levels of SI gene expression, which aligned with the low level of isomaltulose accumulation. As expected, no SI gene transcript was detected in stalks of the wild-type Tx430 (Fig. 4).
Inheriting high-sugar contents in F1 hybrids
The elite sweet sorghum cultivar R9188, and Rio were selected as female lines for crossing due to its advantages of large biomass and high-sucrose content in stalks. Transgenic lines A5, and L9 were chosen as male line 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. However, transgenic line L9 displayed the normal development in reproductive organs compared to transgenic line A5 which is partially sterile. Rio had stronger and taller stem than R9188 had in the glasshouse. 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 Tx430 in the glasshouse. The sweet sorghum cultivar R9188 is another version of Rio with an extra dwarf gene, hence almost 50 cm shorter. Germination and early plant growth were similar to the 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. 5), indicating hybrid seeds inherited the SI gene sexually from the parent L9 to its progenies.
Within the isomaltulose positive group, three progenies (10.3%) converted almost all sucrose into IM; 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. 5). Notably, the enhancement of total sugar content was observed in most isomaltulose positive groups (Fig. 5). The increase of total sugar content in the positive group was on average of 37% 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 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 isomerse gene gene (Fig. S5). The highest total sugar content at 764 mM was measured in F1 LR920 line and the best isomaltulose content at 565 mM was detected in the F1 LR99 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% because of the SI gene. While the average sugar content in the sweet sorghum R9188 and grain Tx430 were 261 and 93 mM respectively. The detail of results was shown in (Table S2).
Inheriting high-sugar contents in F2 populations
Based on isomaltulose production, total sugar content, stalk biomass, and seed production, F1 (Rio X L9) progenies LR3, 19 and 20 were selected for further characterization. With the parental controls of sweet sorghum Rio, progeny 24, a null segregant with comparative high sugar content was also selected as a hybrid control. Seeds were produced by self-pollination of the selected progenies.
Sugar profiles of the isomaltulose positive plants showed that they inherited the phenotype of both isomaltulose production and high-sugar accumulation (Fig. 6). In all three SI positive progenies, isomaltulose accumulated at high levels in all internodes along the stalk, plus sucrose stored at comparable levels (total sugar content up to 812.2 mM), resulting in enhancement by up to 69% in total sugar content compared to either the parental (480.6 mM) or the hybrid control (470.9 mM) (Fig. 6).
Increasing sugar content and water content in 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 along the stalk than the controls (Fig. 7a). In the sweet sorghum Rio and hybrid control 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. 7b). Moreover, there were no significant changes 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 P3, P19, and P20 than the controls. Therefore, the commercially important traits of higher sugar concentration 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. 8). 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 for sucrose uploading in the source leaves [36]. 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. 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 P24, Rio and Tx430 (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.