Root-preferential expression of the At1g promoter
Overexpression of the AtGA20ox gene in tobacco under the control of different promoters
The GUS gene under the control of the CaMV35S or At1g promoter was cloned into the binary vector pBI121, which was used to transform the tobacco cultivar K326 using the Agrobacterium mediated method (Fig. S3a). Transgenic tobacco plants of the 35S::GUS and At1g::GUS constructs were randomly selected for GUS assays. The GUS gene expression indicated by histochemical blue staining was observed in all tested tissues, including the leaves, stems and roots of the 35S::GUS transgenic lines (Fig. 1, Fig. S4). In contrast, GUS expression was only found in the roots, particularly in the root tips, of the At1g::GUS transgenic tobacco plants. In addition, expression of the GUS gene was stronger in the root tips of At1g::GUS than the 35S::GUS transgenic tobacco. These results demonstrated the root-preferential expression of the At1g promoter in tobacco, especially in the root tips.
We further characterized the At1g::AtGA20ox and 35S::AtGA20ox transgenic tobacco plants. Specific primers for AtGA20ox were used to confirm the presence of this transgene in the transgenic lines. All tested plants showed clear PCR bands (1176 bp) in the agarose gels (Fig. S3b), indicating the transgene integration into the tobacco genome. We used RT-PCR to determine the transcript levels of AtGA20ox in different tissues, including the root tips, roots (non-tip roots), stems and leaves of the two representative transgenic lines from At1g::AtGA20ox and 35S::AtGA20ox constructs (Fig. 2). The 35S::AtGA20ox transgenic lines showed no significant differences in AtGA20ox expression among the roots, leaves and stems. However, the AtGA20ox expression varied in the root, stem and leaf tissue (in this descending order) of the At1g::AtGA20ox transgenic plants with the roots, especially the root tips, showing the highest expression. These results indicate the At1g promoter controls root-preferential expression of At1g::AtGA20ox.
Growth and developmental characterizations of AtGA20ox tobacco plants
Previous studies showed that overexpression of AtGA20ox increased cell division and elongation and resulted in other morphological alterations in different plant species [12, 17, 29]. In this present study, all AtGA20ox transgenic lines carrying either At1g or the 35S promoter exhibited longer stems than the WT tobacco (p = 0.015-0.037). In particular, either At1g-7 and At1g-9 or 35S-2 and 35S-5 showed 1.5-fold increases in the stem length compared to WT plants except for the line At1g-5, which had about a 1.25-fold increase in stem length (Fig. 3b). Both the 35S and At1g transgenic tobacco were taller than the WT plants.
However, the stem diameter of the 35S transgenic tobacco, particularly 35S-5, was smaller than the WT plants (p = 0.031). On the other hand, there was no observable difference in stem diameter between the At1g::AtGA20ox and WT plants (Fig. 3c). As a result, the stem fresh weights of the At1g::AtGA20ox lines were greater than the 35S::AtGA20ox transgenic lines (Fig. 3d). Of these At1g::AtGA20ox lines, At1g-9 showed the greatest increase in stem diameter (~110%) and stem fresh weight (~200%) compared to the WT plants (Fig. 3c, d).
Histological analysis of stem cross-sections of the 3rd tobacco internode with toluidine blue showed no significant difference in xylem width and cell number between the 35S::AtGA20ox transgenic tobacco and the WT plants. However, there were increases in both xylem width and cell number in certain At1g::AtGA20ox lines (Fig. 3e, f, g). As a result, these At1g::AtGA20ox transgenic tobacco lines had had a higher stem fresh weight than the 35S::AtGA20ox transgenic and WT plants (p = 0.013–0.043) (Fig. 3d).
In addition, the 35S::AtGA20ox lines had smaller, curling leaves relative to the WT plants (Fig. 4a). The leaf area of these lines was approximately 210 cm2 in the 35S::AtGA20ox transgenic plants compared to 285 cm2 in the WT plants (p = 0.012, 0.017) (Fig. 4b). Despite this, there was no observable difference in leaf area between the At1g::AtGA20ox and WT plants.
Finally, under greenhouse conditions, all 35S::AtGA20ox transgenic tobacco lines showed a delayed flowering compared to the WT plants (Fig. S5). Their flower buds failed to emerge within 100 days (from 104 days to 107 days) after planting compared to the flower bud emergence of WT plants approximately 90 days after planting (from 89 days to 92 days). Interestingly, there was no significant difference in flowering time between the At1g::AtGA20ox transgenic tobacco and WT plants (Fig. S5). Together, these results demonstrated the desirable phenotypes of the At1g::AtGA20ox transgenic plants.
Root growth and morphology of transgenic tobacco
All tested transgenic tobacco plants showed a faster root growth than the WT plants (Fig. 5). Of these transgenic plants, the At1g::AtGA20ox transgenic lines showed a large increase in the root diameter by up to 2.2-fold (At1g-9) compared to the WT plants (p = 0.011). In contrast, the root diameter of the 35S::AtGA20ox lines was only 1.25-fold increased compared to WT (Fig. 5c). Analysis of the root cross-section displayed a faster root development in the AtGA20ox transgenic plants with larger xylem zones and higher xylem/phloem ratios than the WT (Fig. 5d, e, f). The xylem width of the At1g::AtGA20ox roots was 2- to 2.5-fold increased whereas that of the 35S::AtGA20ox roots was increased 1.2- to 1.5-fold, compared to the WT roots (Fig. 5e). The xylem/phloem ratio was from 1.9 to 2.2 in At1g::AtGA20ox and from 1.5 to 1.75 in the 35S::AtGA20ox transgenic lines, while that of the WT was only 1.45 (Fig. 5f). Consequently, the root fresh weight of the At1g::AtGA20ox transgenic tobacco (from 11.4 g to 16.8 g in the three tested lines) increased from 2 to nearly 4 times compared to the 35S::AtGA20ox (7 g and 7.7 g) and WT plants (4.7 g), respectively (Fig. 5b).
Root-preferential expression of At1g::AtGA20ox in a woody plant (M. azedarach)
Expression of AtGA20ox in transgenic M. azedarach
Transgenic M. azedarach of At1g::AtGA20ox were generated using the Agrobacterium-mediated method (Dong et al. 2011) with modifications (Fig. S6a). Approximately 250 hypocotyl fragments were used for inoculation, and more than 30 transgenic lines were produced on the selection medium. We randomly selected 13 lines to confirm the presence of the AtGA20ox gene. Of these, 12 plants showed the expected PCR bands on agarose gels, indicating the integration of the AtGA20ox gene in the M. azedarach genome (Fig. S6b). Semi-quantitative RT-PCR and agarose gels were performed using the GADPH gene as an internal control to evaluate the AtGA20ox transcript level in transgenic M. azedarach (Fig. 6a). All tested plants showed increased transcript levels of the AtGA20ox gene compared to the WT plants. In addition, the expression levels of the AtGA20ox gene were higher in the root than in the leaf and stem of all M. azedarach transgenic lines. Different tissues of the transgenic line At1g-1, including leaves, stems, roots and root tips, were also analysed by quantitative real-time-PCR to examine the expression patterns of the AtGA20ox gene, which were highly consistent with those in At1g::AtGA20ox transgenic tobacco. The highest expression level of AtGA20ox was in the root tissue, especially the root tips (Fig. 6b), which was elevated 1.4- and 2-fold compared to that in the root elongation zone and young stem. The AtGA20ox expression in the leaves was much lower than in the other tissues. Together, these results again indicated the preferential root expression of the At1g promoter.
Increased stem and root growth
At three months under greenhouse conditions, all tested transgenic plants exhibited a faster growth rate than the WT plants (Fig. 7). The stem length increased two-fold in the transgenic plants compared to the WT plants (Fig. 7c). In addition, the stem diameter was greater in all tested At1g::AtGA20ox transgenic M. azedarach (p = 0.036-0.04) (Fig. 7d). Stem cross-sectional analysis showed a large xylem zone and greater xylem cell numbers in the transgenic M. azedarach than in the WT plants (Fig. 7f, g). As a result, the transgenic lines At1g-1, At1g-5 and At1g-14 displayed around a two-fold increase in the stem fresh weight compared to the WT plants (Fig. 7e). This result is highly consistent with that of the transgenic AtGA20ox tobacco.
Similar to the observations of At1g::AtGA20ox tobacco, all tested transgenic M. azedarach exhibited a faster root growth and bigger root system than WT plants (Fig. 8). The bigger xylem zone and more numbers of xylem cells were observed in all transgenic plants. The xylem zone of transgenic plants varied from 1622 µm (At1g-14, p = 0.03) to 1847 µm (At1g-5, p = 0.016), while that for WT plants was approximately 1249 µm (se = 69.36). The xylem cell number increased from 60 in the WT plants to up to 93 in the At1g::AtGA20ox plants (p = 0.013) (Fig. 8e, f). Furthermore, the xylem/phloem ratio was much higher in the transgenic lines than the WT plants (Fig. 8d, g). Importantly, the transgenic M. azedarach plants showed an over two-fold increase in root fresh weight compared to the WT (Fig. 8b). The highest root fresh weight was obtained in transgenic line At1g-5 (p = 0.044), which had the largest xylem zone and the highest xylem cell number. Consequently, all transgenic plants had a much higher dry root weight (Fig. 8c). These results demonstrated the great utility of At1g::AtGA20ox root-preferential expression for root growth and root biomass production of M. azedarach and potentially other woody plants.