Demonstration of improved plant growth and biomass production by At1g73160 promoter-controlled root-preferential expression of the AtGA20ox gene

Background: Overexpression of the GA20 oxidase gene has been a recent trend for improving plant growth and biomass. Constitutive expression of GA20ox has successfully improved plant growth and biomass in several plant species. However, the constitutive expression of this gene causes side-effects, such as reduced leaf size and stem diameters. To avoid these effects, different tissue-specic promoters were identied and employed for GA20ox overexpression. In this study, we demonstrated the potential of At1g, a root-preferential promoter, for GA20ox expression to enhance plant biomass in tobacco and Melia azedarach. Results: We examined the utility of the At1g promoter to drive the expression of a GUS (β-1,4-glucuronidase) reporter and the GA20ox gene in tobacco and Melia azedarach. Histochemical GUS assays in tobacco showed that At1g was a root-preferential promoter whose expression was particularly strong in root tips. The ectopic expression of the AtGA20ox gene under the control of the At1g promoter showed improved plant growth and biomass of both tobacco and M. azedarach transgenic plants compared to wild-type (WT) control plants. Stem length as well as stem and root fresh weights increased by up to 1.5-3 fold in transgenic tobacco and 2-fold in transgenic M. azedarach. Both tobacco and M. azedarach transgenic plants showed increases in the root xylem width and xylem over phloem ratio by 50%–100% compared to WT plants. Importantly, no signicant differences in the leaf shape or size were observed between the At1g::AtGA20ox transgenic and WT plants. Moreover, transgenic M. azedarach showed a 135% increase in stem diameter although no change was found in transgenic tobacco. Conclusions: These results demonstrate the great utility of the At1g promoter for driving the AtGA20ox gene to induce growth and biomass improvements in woody plants and potentially some other plant species. of At1g::AtGA20ox.

low numbers of adventitious roots, a reduced stem diameter, a small leaf area and delayed owering due to the hyper-accumulation of bioactive gibberellins [5,9,12,20,22,23]. These undesirable phenotypes were eliminated when the 35S promoter was replaced by a non-constitutive promoter such as CAD4 or DX15 [3,20]. Thus, the recent trend has been identifying and employing suitable promoters to drive GA20ox expression in order to achieve desirable phenotypes.
The At1g promoter, a 614 bp upstream sequence of the At1g73160 gene, was previously isolated from chromosome 1 of Arabidopsis thaliana [24]. This Arabidopsis genome locus encodes the UDPglycosyltransferase superfamily protein. The report showed that the At1g73160 promoter was predicted to drive root-speci c expression based on sequence information and GUS assays. This promoter contains several different root motifs such as ASF1 MOTIF CAMV, ARFAT, OSE1, OSE2, RAV1AAT, SURE core and TAPOX1, which are involved in root tissue expression. As the At1g promoter was used to drive the GUS gene, GUS staining was observed only in Arabidopsis roots, with the primary root displaying stronger GUS expression than the secondary roots. However, the expression patterns of this promoter have not been fully characterized at the molecular level for the whole Arabidopsis plant. Therefore, additional research is needed to validate the function of the At1g promoter and its utility in biotechnological applications and basic research.
Melia azedarach Linn is a native forestry plant in Asia, including Vietnam. This plant grows fast and, adapts well to a variety of soil and climatic conditions. M. azedarach is grown in all ecological regions in Asia as a multifunctional timber tree. Due to its termite resistance and durability, M. azedarach wood could be used in construction and furniture as well as input material for energy production. In addition, M. azedarach leaves can be used as an insecticide [25], and some of their limonoid compounds show potential for human cancer treatment [26,27]. Therefore, M. azedarach is considered an important plant species for forestry development strategies, not only in Vietnam but also in many other countries. Our previous works succeeded in growth and biomass production improvements of this plant. However, the transgenic plants showed weak stems and narrow leaves as the 35S promoter was used to guide GA20ox gene [3]. These undesirable phenotypes of transgenic M. azedarach plants were minimized and eliminated as the 35S promoter was replaced by a xylem speci c promoter CAD4 [28]. In the current study, we demonstrated the great utility of the root-preferential At1g promoter to control AtGA20ox gene expression for plant growth and biomass improvement. We rst analysed and ascertained the rootpreferential expression of the At1g promoter in transgenic tobacco using a GUS (β-1,4-glucuronidase) reporter gene as a proof of concept for non-host plant. Then, we used this promoter to guide the ectopic AtGA20ox expression and found desirable plant morphological alterations, including accelerated plant growth and increased biomass production in both transgenic tobacco and M. azedarach. Importantly, these desirable phenotypes were consistently observed in all At1g::AtGA20ox transgenic plants compared to non-transgenic plants.

Results
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. Speci c primers for AtGA20ox were used to con rm 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 signi cant 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 1.25fold 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 3 rd tobacco internode with toluidine blue showed no signi cant 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 cm 2 in the 35S::AtGA20ox transgenic plants compared to 285 cm 2 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. 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).

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 modi cations (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 con rm 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.

Discussions
To overcome the various undesirable limitations of using a constitutive promoter, the employment of a tissue-speci c promoter instead has become the approach of choice and has achieved more desirable results. Jeon and colleagues used the DX15 promoter (a xylem tissue-speci c promoter) to drive PdGA20ox1 gene expression in transgenic poplar plants, which enhanced plant growth with the partial elimination of the unwanted phenotype [20]. However, the leaf area and root mass of these transgenic plants were still lower than those of the WT plants. In addition, other studies using root-speci c promoters for AtGA20ox overexpression demonstrated its superiority over the constitutive promoters [30][31][32]. In agreement with previous reports, the At1g::AtGA20ox transgenic plants showed enhanced plant growth and root development without comprising leaf area and owering time compared to the WT. Therefore, our results here illustrate the superiority of using tissue-preferential promoters instead of constitutive promoters in overexpressing AtGA20ox for plant biomass improvement.
Prior to our study, Vijaybhaskar and colleagues indicated that the At1g promoter was a root-speci c promoter based on GUS assays in Arabidopsis [24]. However, no transgene expression at the transcription levels under the control of the At1g promoter had been characterized before. In our research, we visually observed GUS activity only in the root tips of At1g::AtGA20ox transgenic tobacco instead of the entire root, different from that reported by Vijaybhaskar et al. [24]. This difference in GUS expression may be due to the differences in promoter activities from plant to plant. For example, the rolD promoter was described as a root-speci c promoter in tobacco [33]. Nevertheless, this promoter showed a strong expression in both the roots and shoots of pea and Gladiolus plants [34,35]. In the report by Vaughan et al., the FaRB7 promoter showed root-speci c expression in strawberry but constitutive expression in tobacco plants [36]. In our work, the qRT-PCR results exhibited expression of AtGA20ox throughout the entire plant under the control of the At1g promoter (Fig. 6c). In these transgenic plants, the transcription levels were in the descending order of root tips, root elongation zones, stems and leaves. This result agrees with that of Chen et al., who used a "root-speci c" promoter GmPrP2 for soybean transgenic plants [30]. Their qRT-PCR results showed that the transgene was expressed strongly not only in roots but also in stems, leaves, owers, seeds and hypocotyls, suggesting a root-preferential rather than a rootspeci c expression. A more recent study reported that At1g was a root-speci c promoter in Arabidopsis, mainly based on GUS assays [24]. However, transcription analysis, which is more sensitive than reporter gene analysis, was not conducted in their study. Here, we have demonstrated that At1g is a rootpreferential promoter with its expression level varying in different plant tissues. Thus, our study has provided more insightful information about the At1g promoter function, employing not only reporter genes but also the gene of interest.
Previously, different root-speci c promoters had been isolated and analysed for spatiotemporal expression using reporter genes [24,30,33,35,37]. However, very few researchers later observed the effect of root-speci c expression of their target genes on the phenotype, growth and development of transgenic lines as Jeong et al. reported [38]. Most publications on root-speci c promoters have been con ned to the analysis of promoter expression through the GUS gene [24,30,35,37,39]. In our study, we evaluated AtGA20ox expression and function under the control of the At1g promoter. The gene expression pro le was highly correlated with plant morphology and biomass production, which included enhanced stem and root growth and improved biomass production. The desired phenotype could be explained by a moderate level of GA20ox expression in the transgenic lines since a root speci c promoter was utilized [3,12,22]. Moderate bioactive gibberellin levels promote plant cells to enlarge and divide, enhancing plant growth. This is in comparison to the observed plant morphology when the constitutive promoter 35S was used to drive AtGA20ox gene. The activity of bioactive gibberellins may affect plant tissues differently, causing changes in certain tissues such as the xylem zone [40]. Furthermore, the increased plant growth and biomass production of At1g::AtGA20ox overexpression transgenic lines may be due to the speci city of the At1g promoter, which enhances root system development, resulting in better nutrient absorption e ciencies [38,41]. The desirable phenotypes observed in both tobacco and M. azedarach demonstrated the great potential of this promoter for gene-preferential expression in root tissue for plant biomass improvement. Additional research is necessary to evaluate the effects of At1g::AtGA20ox root-preferential expression on the drought tolerance of transgenic plants.

Conclusions
Our results showed that the At1g promoter drove strong expression of the AtGA20ox target gene in roots.
The root system of the transgenic lines was better developed than in the control lines, in terms of both volume and weight. The stems and leaves also expressed the AtGA20ox gene, but to a much lesser degree. This minimized the undesirable phenotypes caused by the strong expression of target genes in all parts of transgenic lines with the 35S promoter. The At1g promoter has great utility in genetic engineering to improve growth and biomass or to enhance stress tolerance of woody plants and potentially some other plant species.

Materials And Methods
Plant materials and growth conditions

Vector construction
The speci c forward and reverse primers (At1gF/At1gR) were designed based on the sequence of the At1g73160 Arabidopsis promoter [24] (Table S1) and used to amplify this promoter from Arabidopsis genomic DNA. The AtGA20ox gene was ampli ed from Arabidopsis thaliana genomic DNA using primer pairs speci c to this gene sequence [3] (Table S1). The ampli ed AtGA20ox gene and the At1g promoter were con rmed by Sanger sequencing and inserted into the binary vector pBI121. To do so, the 35S promoter region of pBI121 was replaced with the At1g promoter as a HindIII/SmaI fragment while the GUS gene was replaced by the AtGA20ox gene, resulting in the transformation construct pBI121/At1g::AtGA20ox vector (Fig. S1, S2).

Tobacco and M. azedarach transformation
Leaves of tobacco in vitro seedlings were used as explants for Agrobacterium-mediated transformation following the protocol of Topping (1998) with modi cations [42]. Brie y, a single A. tumefaciens C58 colony carrying the transformation constructs was cultured on LB medium [43] supplemented with appropriate antibiotics and kept at 28°C, 250 rpm until the bacterial density reached an OD 650 of 0.6. The bacterial cells were harvested by centrifugation at 5,000 rpm at room temperature for 10 min and then suspended in ½ MS liquid medium containing 200 µM acetosyringone (Sigma-Aldrich, Inc.) (inoculation medium). The in vitro tobacco leaf disks (1×1 cm 2 ) were prepared and soaked in the bacterial suspension with gentle agitation for 30 min. The infected leaf disks were placed on the co-cultivation media (CCM-K) at 25±2 °C in the dark for 2 days. After co-cultivation, the leaf disks were washed with sterile water, blotted by lter papers and cultured on selection medium (SM-K) containing 150 mg/L kanamycin. Healthy shoots that formed from the leaf disks were transferred to root induction medium (RM-K). Rooted plants were moved to mixed soil under greenhouse conditions.
For M. azedarach transformation, sterilized seeds were germinated on MS medium [44] containing 3% (w/v) sucrose and 7.5 g/L agar in 250 ml glass asks. The cultures were kept in the dark at 24 ± 1°C for 2 days and then transferred to a 14 h photoperiod. After 2 weeks on seed germination medium, the hypocotyl was cut into 1 cm fragments and used for Agrobacterium-mediated transformation following the procedure described by Dong et al. [28].

Con rmation of transgene integration and expression
Genomic DNA was isolated from young leaves following the CTAB method [45] and was used for PCRs using speci c forward and reverse primers GA20ox-F/R (Table S1) to con rm the integrated transgenes. The PCRs were performed as follows: Initial denaturation at 94ºC for 5 min, 35 cycles of denaturation at 94ºC for 30 s, annealing at 55ºC for 30 s, and extension at 72ºC for 50 s, followed by a nal extension at 72ºC for 7 min. Then, the PCR products were analysed by electrophoresis using 0.8% agarose gels.
To con rm transgene expression, total RNA was extracted from the root tips, roots (non-tip roots), stems and leaves of 45-day-old tobacco and 90-day-old M. azedarach plants using TRIzol™ reagent following the manufacturer's protocol (Thermo Fisher Scienti c-Promega, Madison, WI, USA). The RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scienti c) was used to synthesize the cDNA from the total isolated RNA. Semi-quantitative RT-PCR was performed with 30 cycles to analyse the expression of the AtGA20ox gene using the Nicotiana tabacum Actin or M. azedarach GADPH gene as an internal control. qRT-PCR was carried out utilizing the Advanced TM Universal SYBR® Green Supermix. The primers for RT-PCR and qRT-PCR are given in Table S1. Gene expression levels were normalized and analysed using three clonal plants of each event as biological replicates.

Plant growth measurements
These transgenic lines were in vitro propagated to generate multiple plants for phenotyping. At1g::AtGA20ox transgenic and wild-type (WT) plants were transferred to pots of mixed soil under greenhouse conditions, and their stem length was measured every 5 days for tobacco and every 15 days for M. azedarach plants. Stem length was measured from the stem above ground to the top of the shoot without removing any leaves. Other morphological characteristics such as stem and root fresh weights, stem diameters, and leaf areas were also measured on the tobacco 45 days after being transferred to the greenhouse. Stem fresh weight only measures the weight of the stem, excluding leaf weight. The tobacco leaf area is measured by the average area of three tested leaves (the 3 rd , 4 th and 5 th leaves from the top) of each plant. The stem diameter is measured at a position 10 cm above the ground. For M. azedarach, the measurements were taken 90 days after being transferred to the greenhouse. The data were collected from at least 5 propagated plants from each transgenic line.

Histological analysis
Stem and root cross-sections of tobacco and M. azedarach plants were collected and histologically analysed. The cross-sections were stained with 0.05% toluidine blue for 1 min. Images were observed and captured by a Meiji Techno MA151/MT05 C-mount 0.5× Microscope (at 4×, 10× and 40×). Xylem and phloem width were identi ed from the images using the method described by Jeon et al. [20].

Data analysis
A complete random design was applied to the experimental units under tissue culture and greenhouse conditions. All treatments were conducted in at least three replicates. Data were collected and analysed by SPSS software Version 20 (Chicago, IL) and Microsoft Excel.

Declarations
This research was supported by the Vietnamese national grants to NP to Develop and evaluate fastgrowing transgenic Melia azedarach Linn for forestry plantation.

Availability of data and materials
All of the data used in this study are mentioned within the article and its additional les.
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Competing interests
The authors declare no competing nancial interest.

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