Inducing Expression of the BAS1 Gene via the SAG12 Promoter to Delay Flower and Plant Senescence in Transgenic Petunia hybrida

Background: Brassinosteroids (BRs) are essential hormones that play crucial roles in plant growth, reproduction and response to abiotic and biotic stress. Results: In transgenic Petunia hybrida , resulting in short stature, dark green leaves, and slowed aging.We demonstrate that the exogenous expression of the SAG12-BAS1 gene results in delayed senescence of flowers. SAG12-BAS1 transgenic lines, grown in the vegetative state, exhibited a range of phenotypic changes, including dark green leaves, short stature, delayed senescence, increased flower bud counts, branching, reduced internode lengths, and delayed flowering. SAG12-BAS1 transgenic expression increased the activity of protective enzymes, reduced malondialdehyde content, and increased chlorophyll content and soluble sugar accumulation in plants. Expression of senescence genes was increased in the transgenic Petunia hybrida compared to wild-type plants. Conclusions: Our finding suggests that BAS1 could be used as a potential candidate gene regulate plant flower senescence and prolong flower longevity. of histochemical of of the gene Transgenic until the Transgenic from of Gene expression analysis showed that expression of the PhARR2 gene that regulates cytokinin and the average expression of Petunia hybrida hybrida cytokinin receptor histidine protein kinase (PhHK) were significantly increased by 2.35 times and 2.41 times, respectively, in transgenic plants compared to WT plants. Expression of Petunia hybrida PhGA2ox1 and PhGA2ox3 were significantly higher 11 times and 3 times, respectively) in transgenic compared to WT plants (P 0.01). . The response factor (PHARF4) gene transgenic WT Our experimental results show that the BAS1 gene of Petunia hybrida can affect senescence signals and activity of the protective enzymes SOD, POD, and CAT in leaves. SOD, POD, and CAT activities in transgenic Petunia hybrida leaves increased by 82.74 %, 131.80 %, and 135 %, respectively, compared to WT Petunia hybrida . During plant senescence, the expression ofthe SAG12-BAS1 gene improved the activity of protective enzymes and enhanced oxygen scavenging activity in the plant. In contrast, MDA content was 46 % lower in transgenic Petunia hybrida compared to WT, which suggests that the cell membranes of transgenic Petunia hybrida were not damaged. In addition, the chlorophyll and soluble sugar content in transgenic Petunia hybrida leaves were higher than in WT plants.


Background
The BAS1 gene encodes a cytochrome P450 monooxygenase with hydroxylase activity, which catalyzes the hydroxyl hydroxylation of brassinosteroid (BR) C-26, leading to the reduction or loss of BR physiological activity [1,2] . As adult plants, BR mutants exhibit essentially the opposite phenotype of mutants lacking the red light receptor, phyB. The BR mutants are dark green, slow-growing, and have blade shrinkage and short stems. In addition, BAS1 mutants exhibit delayed senescence [3] . Overexpression of the transgenic BAS1 gene can significantly reduce the BR content in plants, resulting in shrinking leaves, slowed leaf aging, and short stems [4] . The leaf senescence induced by BAS1 overexpression is very similar to the phenotype resulting from overexpression of cytokinin in tobacco plants. To observe cytokinin-induced phenotypic changes, scientists used heat shock [5,6,7] , hypothermia induction, copper induction, wounding [8] , and light induction [9] to drive cytokinin expression.
Isopentenyltransferase (ipt) catalyzes the rate-limiting step for cytokinin synthesis. Gan and Amasino [10] demonstrated that a specific developmental response could be elicited through precise control of ipt expression. In the Gan study, the SAG12 promoter activated ipt expression only at the onset of senescence. This activation of ipt expression resulted in inhibition of the senescence process. Inhibition of leaf senescence by ipt expression led to attenuation of the senescence-specific promoter, thus preventing cytokinin overproduction that would interfere with other aspects of development. In SAG12-ipt tobacco, leaf senescence was effectively controlled without other developmental abnormalities [10] . Subsequently, this strategy was successfully used in rice [11] , cauliflower [12] , Petunia hybrida [ 13] , lettuce [14] , and Brassica chinensis [15] . For example, leaf senescence was retarded in mature 60 day old lettuce plants that exhibited normal morphology with no significant differences in head diameter or the fresh weight of leaves and roots.
Using the senescence-specific SAG12 promoter to drive BAS1 expression in tobacco, Yao [16] found that SAG12-BAS1 transgenic and wild-type plant growth were similar. However, transgenic tobacco leaves exhibited delayed aging, manifested mainly as dark green plant leaves, increased chlorophyll content, increased protective enzymatic activity, and increased cytokinin content. The SAG12-BAS1 gene experiments laid the foundation for the study of flower senescence.
Flower senescence represents the last stage of floral development and results in wilting or abscission of whole flowers or flower parts [17] . The length of the flowering period is related to many factors, including temperature, nutrition, and other external environmental factors. However, these strategies are external to the plants and are timeconsuming and laborious. Genetic engineering can rapidly incorporate stable new genetic material resulting in prolonged flowering. Leaves from cytokinin overproducing BAS1 transgenic plants display prolonged chlorophyll retention and delayed senescence [16]. In the present study, a plasmid, containing the SAG12-BAS1 gene was inserted into Agrobacterium strains, and, subsequently, was used to transform Petunia hybrida leaf discs, resulting in delayed senescence. These transgenic plants were used to develop senescent Petunia hybrida for horticultural purposes.

Plant materials
All Petunia hybrida plants (Shanghai 'lvyu' Gardening Co., Ltd.)were grown in a greenhouse at the Institute of Agro-Bioengineering, Guizhou University, Guiyang (China), The PCR primers were synthesized by Shanghai Yingjun Biotechnology Co., Ltd. Other chemical reagents used are domestic or imported analytically pure.

Plasmids constructions
All DNA manipulations were performed essentially as described by Green and Sambrook [18] . Binary plasmid pSH737 contained a replication origin of wide-range host, one kanamycin resistance gene, NPTII (neomycin phosphotransferase I, from Tn903) for bacterial selection, and one chimeric gene bearing GUS reporter gene (from Escherichia coli) with an neomycin phosphotransferase II (NPTII, from Tn5) selectable gene for plant expression and selection respectively (Supplementary Figure 1). The chimeric gene GUS was driven by the cauliflower mosaic virus 35S promoter (CaMV 35S). Plasmid pSH-737 was provided by the Institute of Agro-Bioengineering (Guizhou University). Plasmid pSH737-SAG12-BAS1 was transferred into Agrobacterium tumefaciens strain LBA4404 [19] .
Colonies resistant to kanamycin were selected to ensure the presence of all plasmids by enzyme digestion and PCR amplification.

Plants transformation and transgenic plant detection
Petunia hybrida leaves were prepared for transformation using a leaf disk transformation procedure [20] . For co-cultivation, Agrobacterium containing pSH737-SAG12-BAS1 was suspended in MS liquid medium, and the OD 600 was adjusted to 0.6, the Petunia hybrida leaves were dipped into the Agrobacterium solution for 8 min and dried on sterilized filter paper before they were placed on the co-cultivation medium (MS+1.0 mg L − 1 ZT) for 2 d at (25±2)°C in dark. Then, the leaves were transferred onto the selective regeneration medium (MS + 2.0 mg L -1 ZT+100 mg L -1 Timentin +100 mg L -1 Kanamycin). Regenerated shoots were transferred to fresh medium biweekly. When the shoots were 3-5 cm, they were separated from the calli and transferred onto rooting medium (1/2 MS +100 mg L -1 Timentin +100 mg L -1 Kanamycin), rooted shoots were transplanted into pots. according to previously described methods [21] Extraction of Petunia hybrida DNA and GUS histochemical staining of Petunia hybrida plants was conducted, and the presence of the Plant growth conditions and morphological analysis of the transgenic phenotype Petunia hybridas were used for this experiment because this is an ornamental plant with distinct vegetative and generative growth stages that are controlled by the photoperiod.
Transgenic BAS1 Petunia hybrida lines and WT cultivars were transplanted to 10 cm pots and maintained in a growth chamber at 25 °C during the day (16 h) and 20 °C at night.
Plants were allowed to acclimate to the growth chamber conditions for two weeks and then exposed to either vegetative growth conditions or generative growth conditions. Ten plants from each WT and transgenic line were allowed to progress to flower senescence to determine flower longevity. Plant development and morphological changes were observed and recorded.

Flower Senescence Evaluations and Senescence of excised leaves and flowers
Flower senescence was visually rated during natural senescence and following pollination [22] . One day before anthesis (flower opening), flower corollas were slit with a sharp razor blade, and anthers were removed to prevent self-pollination. On the day of Maleic Dialdehyde (MDA) content was determined using the thiobarbituric acid (TBA)based colorimetric method, as described byHeath and Packer [23] . Chlorophyll concentration was assayed prior to the start of dark conditions and after a significant loss of chlorophyll was detected in the WT tissue. Each transgenic and WT line was tested in triplicate and the experiment was repeated three times. Specific chlorophyll concentration was determined using WT and transgenic leaves obtained from each treatment plate of the previously described chlorophyll concentration study. Leaves were blotted dry and 100 mg of tissue from each sample was placed in a 1.5 ml microcentrifuge tube. The samples were re-suspended in 80 % acetone, ground with a disposable pestle, and incubated in darkness for 30 min. Total chlorophyll (g L -1 ) was determined using absorbance at 645 and 663 nm according to the equation: 20.2 A645 + 8.02 A663 [24] .
Determination of soluble sugar content was conducted as described [25] . Fresh plant leaves (0.5 g) were placed in 80 % alcohol (4 ml) and carefully ground into a homogenate.
The homogenate was transferred into a centrifuge tube and incubated at 80 °C. The homogenate was stirred for 30 min and then centrifuged for 10 min (6000 g). The supernatant was transferred to a 10 ml graduated test tube with 2 ml of 80 % alcohol.
Activated carbon (0.5 g) was added to the supernatant and decolorized in a water bath at 80 °C for 30 min. The volume was adjusted to 10 ml and the sample was filtered (diluted 10-fold or 20-fold). The sugar extract (1 ml) was transferred to a clean tube, 5 ml of anthraquinone reagent was added, and the mixture was boiled for 10 min. After cooling, absorbance was measured at a wavelength of 625 nm. The sugar percentage in each sample was calculated based on a standard curve. The standard curve was generated as follows. A standard glucose solution was serially diluted to final concentrations of 0, 5, 10, 20, 40, 60, and 80(g /L -3 ). Absorbance was measured as described above, and then linear regression was used to calculate the sugar content in each sample. The sugar percentage was calculated using the following equation:  Table S2).

Statistical analysis
The study was performed independently three times and each result shown in the figures was expressed as the mean ± standard deviation (SD). Excel 2017 software (Microsoft, Redmond, USA) and SPSS Statistics 22.0 software (SPSS Inc., Chicago, IL, USA) were used for statistical analyses. Samples were compared using one-way ANOVA followed by Duncan's multiple range posthoc test. Samples were considered significantly different at P <0.05.

Results
Agrobacterium-mediated transformation of transgenic Petunia hybrida GUS histochemical staining of wild-type and transgenic Petunia hybrida plant leaves at the 3-5 leaf stage showed that GUS activity was not detected in wild-type Petunia hybrida plants, whereas GUS activity was detected in Kan resistant Petunia hybrida plants (Fig.   1a). Target bands were not amplified in wild-type Petunia hybrida plants, but the expected 419 nucleotide bands were observed in transgenic plants (Fig. 1b). This demonstrated that the exogenous BAS1 gene had been successfully integrated into the genomes of Kan resistant Petunia hybrida plants, and 29 strains of SAG12-BAS1 gene Petunia hybrida were obtained.

Morphological analysis of transgenic Petunia hybrida plants expressing PSAG12-BAS1
Wild-type and transgenic Petunia hybrida were grown under the same conditions, and three phenotypes of transgenic Petunia hybrida were observed (Fig.2a). The transgenic plant, Phenotype 1, and the wild-type phenotypes were similar(Experiment requires phenotype 1 follow-up experiment, Such as TP12,TP28,TP38, etc.), and the expression of the senescence promoter was normal. The SAG12-BAS1 transgenic plants (Phenotype 2and Phenotype 3) exhibited shorter stature, slower growth, darker green leaves, and delayed aging compared to wild-type (WT) plants. Phenotype 3exhibited the darkest green leaves.
The promoters in Phenotype 2and Phenotype 3 may not have been strictly controlled, similar to ipt overexpression phenotypes in transgenic plants (Fig.2a) [4] . Analysis of the expression of BAS1 gene in three transgenic plants is shown in Figure 2b. Expression of the BAS1 gene was similar in the Phenotype 1 and Phenotype 2 plants, while BAS1 gene expression was higher in Phenotype 3 (Fig.2b).

Discussion
Our results show transgenic and WT Petunia hybrida plant morphology, physiology, biochemical metabolism, and the regulation of genes involved in senescence. BAS1, part of C-26-hydroxylase, is an important brassinosteroid inactivating gene [26][27][28][29] , which affects the content of active brassinosteroids in plants. The overexpression of plant brassinosteroids affects plant phenotype, but the mechanism of BAS1 action on plant senescence is not completely understood.
Our experimental results show that the BAS1 gene of Petunia hybrida can affect senescence signals and activity of the protective enzymes SOD, POD, and CAT in leaves. Overexpression of Type A members of the Arabidopsis response regulator (ARR) gene family, including ARR4, ARR5, ARR6, ARR7, can inhibit ARR6 transcription, and stable overexpression of ARR8 in transgenic plants inhibits the cytokinin response, suggesting that type A ARR can negatively regulate the cytokinin pathway [30] . Additional research indicates that multiple mutants of A-ARR are suppressed in response to exogenous cytokinins [31] . ARR15 is highly cytotoxic in response to cytokinin because His-Asp phosphorylation inhibits negative feedback regulation [32] . In the present study, expression of PHARR4 in transgenic plants was increased to 2.35 times that of WT plants, indicating that ARR4 appears to positively regulate cytokinin levels.
The cytokinin receptor histidine kinases, AHK2, AHK3, and CREI/AHK4/WOODEN LEG (WOL), bind to cytokinins and autophosphorylate [33] . These receptor histidine kinases then transfer the phosphate group from a histidine residue, which is conserved in the kinase domain, to an aspartate residue, which is conserved in the signal receiving region.
Phosphoric acid groups are transferred to cytoplasmic Arabidopsis histidinephosphotransfer proteins (AHPs) and these AHPs subsequently enter the nucleus and transfer the phosphate groups to a series of ARR. This regulates the downstream cytokinin response and results in a series of biochemical effects that regulate plant growth and development [34] . The experimental results show that the average expression of PHAHK is Some studies have shown that ERF1 can regulate the expression of ACS3, ACO, and ACO2 in plants, and thereby enhance the biosynthesis of ethylene in transgenic plants [36][37][38][39] .
This suggests that transcription termination factors may play an important role in regulating plant ethylene biosynthesis [40,41] . The homeodomain-leucine Zipper (HD-Zip) is a type of transcription factor that is unique to higher plants and belongs to a class of homeodomain (HD) transcription factors [42] . For conserved HD, the HD carboxyl terminal is tightly linked to the leucine zipper domain (LZ). Research suggests that these transcription factors mainly regulate the development of plants, including the development of vascular tissues and trichome [43] . These factors are also involved in the    Total chlorophyll content (a) and soluble sugar content (b) measured in leaves from WT and 4 different transgenic plants after 90 days of plant growth. Data are presented as means ± SD (n=3). Asterisks indicate significant differences between wild-type (WT) and transgenic lines (, TP12, TP28, TP38) (*P < 0.05; **P < 0.01).

Supplementary Files
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