Root-preferential expression of At1g promoter
Overexpression of AtGA20ox gene in tobacco under the controls of different promoters
The GUS gene under the control of CaMV35S or At1g promoter was cloned into the binary vector pBI121 which was used to transform tobacco cultivar K326 using Agrobacterium mediated method (Fig. S3). Transgenic tobacco plants of 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 leaves, stems and roots of 35S::GUS transgenic lines (Fig. 1). In contrast, GUS expression was only found in roots, particularly in root tips, of At1g::GUS transgenic tobacco plants. In addition, expression of GUS gene was stronger in root tips of At1g::GUS than 35S::GUS transgenic tobacco. These results demonstrated the root-preferential expression of At1g promoter in tobacco, especially in root tips.
We further characterized At1g::AtGA20ox and 35S::AtGA20ox transgenic tobacco plants. Specific primers for AtGA20ox were used to confirm the presence of this transgene in transgenic lines. All tested plants showed clear PCR bands (1.2 kb) in the gel running results (Fig. 2a) indicating the transgene integration in tobacco genome. We used RT-PCR to determine transcript levels of AtGA20ox in different tissue including roots, stems and leaves of transgenic lines. A higher transcript level of AtGA20ox was found in 35S::AtGA20ox than that in At1g::AtGA20ox transgenic lines in all tested tissues. We also compared AtGA20ox expression levels in different tissues of transgenic tobacco. The 35S::AtGA20ox transgenic lines showed no significant difference in AtGA20ox expression among roots, leaves and stems. However, AtGA20ox expressions varied in root, stem and leaf tissue (in this descending order) of At1g::AtGA20ox transgenic plants with roots especially root tips showing the highest expression (Fig. 2c). These results indicated the At1g promoter controlled root-preferential expression of At1g::AtGA20ox.
Growth and developmental characterizations of AtGA20ox tobacco plants
Previous studies showed that an 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 35S promoter exhibited longer stems than WT tobacco (p = 0.015-0.037). In particularly, either At1g-7 and At1g-9 or 35S-2 and 35S-5 showed 1.5-fold increase in the stem length compared to WT plants except that line At1g-5 had about 1.25-fold increase in stem length (Fig. 3b). Both 35S and At1g transgenic tobacco were taller than WT plants. However, stem diameter of 35S transgenic tobacco, particularly 35S-5, was smaller than WT plants (p = 0.031). On the other hand, there was no observable difference in stem diameter between At1g::AtGA20ox and WT plants (Fig. 3c). As a result, stem fresh weights of At1g::AtGA20ox lines were greater than 35S::AtGA20ox transgenic lines (Fig. 3d). Of these At1g::AtGA20ox lines, At1g-9 showed the highest increase in stem diameter (~110%) and stem fresh weight (~200%) compared to 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 35S::AtGA20ox transgenic tobacco and 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 much more stem fresh weight than 35S::AtGA20ox transgenic and WT plants (p = 0.013-0.043) (Fig. 3d). In addition, the 35S::AtGA20ox lines had smaller and curling leaves than WT plants (Fig. 4a). The leaf area of these lines were around 210 cm2 in 35S::AtGA20ox transgenic plants as compared to 285 cm2 in WT plants (p = 0.012, 0.017) (Fig. 4b). Despite this there was no observable difference in leaf area between At1g::AtGA20ox and WT plants. Finally, under greenhouse conditions, all 35S::AtGA20ox transgenic tobacco lines showed a delayed flowering as compared to WT plants (Fig. S4). Their flower buds failed to emerge within 100 days (from 104 days to 107 days) after planting as 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 At1g::AtGA20ox transgenic tobacco and WT plants (Fig. S4). 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 WT plants (Fig. 5). Of these transgenic plants, At1g::AtGA20ox transgenic lines showed a large increase in the root diameter by up to 2.2-fold (At1g-9) as compared to WT plants (p = 0.011). In contrast the root diameter of 35S::AtGA20ox lines was only 1.25-fold increase compared to WT (Fig. 5c). Analysis of root cross section displayed a faster root development in AtGA20ox transgenic plants with bigger xylem zones and higher xylem/phloem ratios than WT (Fig. 5d, e, f). The xylem width of At1g::AtGA20ox roots was from 2- to 2.5-fold increase whereas that of 35S::AtGA20ox roots was 1.2- to 1.5-fold, compared to 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 35S::AtGA20ox transgenic lines, while that of WT was only 1.45 (Fig. 5f). Consequently, the root fresh weight of At1g::AtGA20ox transgenic tobacco (from 11.4 g to 16.8 g in three tested lines) increased from 2 to nearly 4 times compared to 35S::AtGA20ox (7 g and 7.7 g) and WT plants (4.7 g), respectively (Fig. 5b).
Root-preferential expression of At1g::AtGA20ox in woody plant (M. azedarach)
Expression of AtGA20ox in transgenic M. azedarach
Transgenic M. azedarach of At1g::AtGA20ox were generated using Agrobacterium-mediated method (Dong et al. 2011) with modifications (Fig. S5). About 250 hypocotyl fragments were used for inoculation and more than 30 transgenic lines were produced on selection medium. We randomly selected 13 lines to confirm the presence of AtGA20ox gene. Of these, 12 plants showed the expected PCR bands in gel run indicating the integration of AtGA20ox gene in M. azedarach genome (Fig. 6a). Semi-quantitative RT-PCR and gel-run were performed using GADPH gene as an internal control to evaluate AtGA20ox transcript level in transgenic M. azedarach (Fig. 6b). All tested plants showed transcript levels of AtGA20ox gene compared to WT plants. In addition, the expression levels of AtGA20ox gene were higher in root than in the leaf and stem of all M. azedarach transgenic lines. Different tissues of transgenic line At1g-1 including leaves, stems, roots and root tips were also analyzed by quantitative real-time-PCR to examine the expression patterns of AtGA20ox gene which were highly consistent with those in At1g::AtGA20ox transgenic tobacco. The higher expression level of AtGA20ox was in root tissue, especially root tips (Fig. 6c), which was as high as 1.4- and 2-fold compared to that in root elongation zone and young stem. The AtGA20ox expression in leaves was much lower than other tissues. Together, these results again indicated the preferential-root expression of At1g promoter.
Increased stem and root growth
At three months under greenhouse conditions, all tested transgenic plants exhibited a faster growth than WT plants (Fig. 7). The stem length increased two-fold in transgenic plants compared to 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 more xylem cell number in transgenic M. azedarach than in WT plants (Fig. 7f, g). As a result, transgenic lines At1g-1, At1g-5 and At1g-14 displayed around two-fold increase in the stem fresh weight as compared to WT plants (Fig. 7e). This result was highly consistent with that of transgenic AtGA20ox tobacco.
Similar to the observations on 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 around 1249 µm (se = 69.36). The xylem cell number increased from 60 in WT plants to up to 93 in At1g::AtGA20ox plants (p = 0.013) (Fig. 8e, f). Furthermore, the xylem/phloem ratio was much higher in transgenic lines than WT plants (Fig. 8d, g). Importantly, transgenic M. azedarach plants showed over two-fold increase in root fresh weight compared to 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 a great utility of At1g::AtGA20ox root-preferential expression for root growth and root biomass production of M. azedarach and potentially other woody plants.