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Article

ZmGRAS46 Negatively Regulates Flowering Time in Arabidopsis thaliana

by
Honglin Zhang
1,
Zhenzhong Jiang
2,
Peng Jiao
1,
Yang Zhao
1,
Bai Gao
1,
Siyan Liu
1,
Shuyan Guan
1,3,* and
Yiyong Ma
1,3
1
College of Agronomy, Jilin Agricultural University, Changchun 130118, China
2
College of Life Sciences, Jilin Agricultural University, Changchun 130118, China
3
Joint International Research Laboratory of Modern Agricultural Technology, Ministry of Education, Jilin Agricultural University, Changchun 130118, China
*
Author to whom correspondence should be addressed.
Agronomy 2024, 14(1), 155; https://doi.org/10.3390/agronomy14010155
Submission received: 15 December 2023 / Revised: 6 January 2024 / Accepted: 8 January 2024 / Published: 10 January 2024
(This article belongs to the Section Plant-Crop Biology and Biochemistry)
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Versions Notes

Abstract

:
Flowering is an essential process in plant development, and there are six major flowering pathways: the photoperiodic pathway, gibberellin pathway, vernalization pathway, age pathway, autonomous pathway, and temperature pathway. In this study, we screened the transcriptome sequencing of early flowering mutants from the laboratory for the significantly differentially expressed ZmGRAS46, which belongs to the DELLA subfamily of the GRAS family. DELLA is involved in the gibberellin pathway to regulate plant flowering. However, it is not clear whether ZmGRAS46 is involved in the gibberellin pathway which regulates plant flowering; therefore, in this experiment, we investigated the regulatory role of this gene in Arabidopsis flowering by overexpressing ZmGRAS46. It was found that overexpression of ZmGRAS46 in Arabidopsis promotes the formation of rosette leaves and flower buds and delays flowering time in Arabidopsis, and experiments have shown that ZmGRAS46 represses the expression of FLOWERING LOCUS T (FT), SUPPRESSOR OF CONSTANS1 (SOC1), CONSTANS (CO), and LEAFY (LFY). Our results indicated the possibility that ZmGRAS46 represses flowering through the CO-FT-SOC1-mediated photoperiodic flowering pathway. The delayed flowering phenotype of overexpressing ZmGRAS46 Arabidopsis could be rescued by applying GA3. The experimental results indicate that ZmGRAS46 depends on the GA3 pathway to regulate flowering in Arabidopsis.

1. Introduction

Flowering occurs in the apical meristematic tissue of the stem of plants. With the occurrence of a long evolution, plants have a mechanism for sensing and responding to changes in the external environment. Among the many mechanisms, the regulatory mechanism of flowering has a critical position [1]. Arabidopsis thaliana (Arabidopsis thaliana (L.) Heynh.), one of the model plants in plant molecular, cellular, and evolutionary biology, is favorable for cultivation, has a self-fertilizing reproductive system, has a relatively short generation time, and has easy access to germplasm material. Many recent research advances have been made to regulate flowering by mediating different signaling pathways. The autonomous flowering pathway enables Arabidopsis to promote flowering independently of day length by inhibiting the central flowering deterrent FLOWERING LOCUS C (FLC) [2]. Diurnal temperature changes regulate photoperiodic flowering by altering the pattern of FT accumulation [3]. ARGONAUTE5 physically and functionally interacts with miR156 to regulate flowering in Arabidopsis by mediating the aging pathway [4]. BraVRG is a new cabbage protein promoting Arabidopsis flowering through vernalization induction [5]. Gibberellin regulates flowering in Arabidopsis by mediating DELLA-BRAHMA-NF-YC [6]. Among them, the DELLA protein plays a significant role as a central regulator in gibberellin signaling [7].
DELLA proteins belong to a conserved subfamily of GRAS proteins, and the GRAS family of transcription factors has essential roles in plant growth and development, signal transduction, axillary bud and root development, mycorrhiza and rhizoma formation, and stress response. An analysis of the evolutionary tree of GRAS protein family members in Arabidopsis thaliana and the conserved structural domains of the proteins showed that there are 12 conserved sequences at the C-terminal end of GRAS proteins and several conserved structural domains at the N-terminal end, including the conserved DELLA structural domain [8,9]. DELLA proteins can regulate flowering by interacting with several transcription factors in the leaf and stem tip to regulate their activities [10]. Gomez shows that DELLA proteins are new players in the control of seed size and suggests that the modulation of DELLA-dependent pathways can improve crop yield [11]. Different expression patterns of the oilseed rape BnaDELLA gene were detected under biotic and abiotic stresses, suggesting that regulating BnaDELLA expression in oilseed rape is a promising approach to improving oilseed rape stress tolerance and harvest indexes [12]. RGL2 is one of the DELLA proteins, and studies have shown that RGL2 has a regulatory effect on flower development, mainly regulating the growth of stamens and the division of anthers, which significantly influences plant fertility [13]. There are few intron insertions in GRAS genes during the evolutionary process, among which the DELLA family has a more straightforward gene structure, which is more conserved, and the function of DELLA proteins has been widely studied in recent years, but ZmGRAS46 in the DELLA subfamily has not been investigated, so this gene was selected in this study to investigate its regulatory role on flowering.
In recent years, global warming and an increase in temperature have affected the flowering period of plants; Krista et al. showed that the change in the flowering period had an impact on the nectar secretion of flowers, disrupting plant–pollinator interactions, thus affecting the entire ecosystem [14]. Late frost damage severely impacts the early flowering species Siberian plum, reducing yield [15]. Sorghum is a short-day plant with a solid photoperiodic response, so it becomes essential to develop the cultivation of photoperiod-insensitive cereals in temperate regions [16]. These studies have shown that changes in flowering time can significantly affect plant growth and development, so to solve the problem of early and late flowering for plant flowering, related research becomes essential.
In this study, we successfully cloned ZmGRAS46 and obtained positive Arabidopsis plants by genetic transformation. Through expression pattern analysis and phenotypic observation, we demonstrated that ZmGRAS46 delayed plant flowering by repressing CO, FT, SOC1, and LFY. This regulatory effect was dependent on the GA signaling pathway. In addition, this study provides new insights for the subsequent analysis of the molecular mechanism of the GRAS gene regulation of plant flowering.

2. Materials and Methods

2.1. Plant Materials and Treatments

All Arabidopsis thaliana seedlings used in this study were in Columbia-0 wild-type (Col-0 WT) background. The CDS region of ZmGRAS46 was cloned, and the gene was ligated into the pCAMBIA3301 vector by seamless cloning. Arabidopsis was genetically transformed by the Agrobacterium-mediated method, and the overexpression-positive Arabidopsis plants, OE6 and OE8, which had the highest expression of ZmGRAS46, were selected for the present experiment.
In this experiment, the WT, OE6, and OE8 seeds were vernalized in dark culture at 4 °C for 3 days. Then, the vernalized seeds were planted in nutrient soil containing vermiculite using a pipette gun and placed in an artificial climate chamber (22 °C, monochromatic white light, 400–700 nm continuous spectrum, 16 h light/8 h dark, 130 μmol/(m2·s), 65%RH) to wait for their germination. Arabidopsis was sprayed with 200 μmol/L GA3 (Shanghai yuanye Bio-Technology Co., Ltd., Shanghai, China) every three days starting. On the 15th day of cultivation, the growth of the treated Arabidopsis was observed and recorded.

2.2. Generation of Constructs and Transgenic Plants

For creating ZmGRAS46 overexpression constructs, the protein-coding sequence of ZmGRAS46 was PCR-amplified with primers and then cloned into the pCAMBIA3301 vector driven by a Cauliflower mosaic virus (CaMV) 35 S promote [17]. The recombinant plasmid was transformed into Agrobacterium tumefaciens GV3101, and Arabidopsis thaliana was dip-flower infested by the Agrobacterium-mediated method [18]. The T3 plants of positive lines were selected with Glyphosate (5 mg/L, Sigma, Kawasaki-shi, Japan), and the viable Arabidopsis thaliana plants were identified by PCR for the subsequent experiments. All primers used in this study are listed in Supplementary Table S1.

2.3. RNA Extraction, Reverse Transcription, and Gene Expression Quantification

The treated samples were prepared by freezing in liquid nitrogen and stored in a refrigerator at −80 °C for RNA extraction in subsequent experiments. RNA extraction by Trizol method, and cDNA was synthesized using the TransScript All-in-one First-Strand cDNA Synthesis SuperMix for qPCR (TRANS, Xiamen, China) following the manufacturer’s instructions. Gene expression levels were quantified by quantitative real-time PCR (qRT-PCR) using ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China) with a Thermal Cycler Dice Real-Time System TP800 (TaKaRa, San Jose, CA, USA) following the manufacturer instructions. AtActin2 was used as a reference. The expression value was calculated using the comparative CT method [19]. Three replications were performed for each sample and each experiment.

2.4. Measurement of Physiological and Biochemical Indicators

On the 15th of cultivation, 200 μmol/L GA3 was sprayed on WT, OE6, and OE8 Arabidopsis plants once every three days. SOD, POD, Pro, H2O2, O2−, and chlorophyll contents were measured in treated and untreated leaves when the plants entered flowering stage. The antioxidant enzyme activities of SOD and POD were measured according to Xiang’s method [20]. The proline content was analyzed using the acid ninhydrin method [21]. The content of H2O2 and O2− was calculated by using the acetone method, and the specific method was referred to as Wang’s method [22]. The absorbance of chlorophyll solution was measured at 649 nm and 665 nm, and the chlorophyll content was measured by Fitter’s method [23].

2.5. Yeast Two-Hybrid (Y2H) Assay

Relatively high-scoring ZmMADS62 proteins were obtained by screening through STRING online software (Version: 11.0) analysis. In order to verify whether ZmGRAS46 and ZmMADS62 indeed interacted with each other, we constructed the pGBKT7-ZmGRAS46 decoy vector and the ZmMADS62-AD prey vector. After proving that the pGBKT7-ZmGRAS46 prey vector did not have toxicity and self-activating activity (Figures S1 and S2), ZmGRAS46-BK vector plasmid and ZmMADS62-AD vector plasmid were transformed into the same AH109 yeast sensory state at the same time. The transformed bacterial fluids were coated on SD/-Trp-Leu two-deficient solid medium. Single colonies were picked and put into the YPDA liquid medium at 29 degrees Celsius for two days. The cultured bacterial liquid, positive bacterial liquid, and negative bacterial solution were spotted on SD/-Trp-Leu two-deficient solid medium and SD/-Trp-Leu-Ade-His, SD/-Trp-Leu-Ade-His-X-α-Gal four-deficient solid yeast medium at 29 degrees Celsius for two days to observe the growth [24].

2.6. Statistical Analysis

All results in this study were performed in more than three replicates. SPSS 19.0 software (SPSS Inc., Chicago, IL, USA) was used for statistical analysis of experimental measurement data; Students’ t-tests were used to confirm the variability of results between treatments, respectively. p < 0.05 (*) and p < 0.01 (**).

3. Results

3.1. Overexpression of ZmGRAS46 Inhibition Flowering

Zhou et al. found that transcription factors can control plants’ structure and flowering time [25]. In order to investigate the regulatory effects of ZmGRAS46 on flowering, we transfected Arabidopsis thaliana with ZmGRAS46 using an Agrobacterium-mediated method in this experiment. We obtained two overexpression plants (OE6 and OE8) with high expressions of ZmGRAS46, which were used in the subsequent experiments (Figure S3). In this study, we observed the phenotypes of overexpression plants OE6 and OE8. We found that the flowering time of the overexpression plants was significantly later than that of the wild-type plants (Figure 1A,B). The wild-type plants (WT) flowered about seven days earlier than the overexpression plants. The number of rosette leaves at flowering was about 10 in the wild-type Arabidopsis thaliana. However, the number of rosette leaves at flowering was around 26 in the positive Arabidopsis thaliana (Figure 1C,D). These results suggest that the overexpression of ZmGRAS46, in its early stages, focuses on promoting rosette leaf growth in Arabidopsis while delaying flowering in Arabidopsis.

3.2. ZmGRAS46 Promotes Flower Bud Formation and Inhibits Floral Branch Elongation

During Arabidopsis thaliana flowering, plants OE6 and OE8 had approximately 12 flower buds, while wild-type Arabidopsis had around 7 buds. The number of buds of the positive plants was significantly more than that of the wild-type Arabidopsis plants (Figure 2A,B). The length of the main branches of wild-type Arabidopsis plants was significantly longer than that of OE6 and OE8 by about eight centimeters (Figure 2C,D). The results showed that the overexpression of ZmGRAS46 promoted the formation of flower buds and inhibited the elongation of main branches.

3.3. Determination of Physiological and Biochemical Indices in T3 Generation of Arabidopsis thaliana Plants Transgenic for ZmGRAS46 after GA3 Treatment

The GA20ox and GA3ox genes catalyze the formation of active GA, and the GA2ox gene can render gibberellins irreversibly inactive [26,27,28]. The experiment involved spraying gibberellin onto wild-type and overexpressed Arabidopsis thaliana OE6 and OE8 until they entered the flowering stage. Then, Arabidopsis thaliana flower buds and blossoms were sampled. A fluorescence qPCR revealed that the expressions of GA2ox2, GA3ox2, GA20ox1, and GA20ox2 were decreased in positive Arabidopsis thaliana OE6 and OE8 and that the expression of GA3ox1 was elevated in flower buds and decreased in blossoms (Figure 3A,B). It suggests that exogenous gibberellin spraying inhibited endogenous gibberellin synthesis, altering the changes of gibberellin in plants.
Bao S et al. showed that ubiquitination degraded DELLA when bound to GA [7]. In order to verify whether the expression of ZmGRAS46 was changed, this experiment sampled treated Arabidopsis leaves, flowers, and buds and found that the expression of ZmGRAS46 was reduced (Figure 3C), indicating that the application of GA3 would reduce the expression of ZmGRAS46, and in order to explore what would happen to Arabidopsis positive for the reduction of the ZmGRAS46 content, a later stage of the Arabidopsis was treated and the phenotypes were observed and recorded.
The application of gibberellins affects plants’ physiological and biochemical indices, and excessive concentrations affect the peroxide scavenging capacity of plants [29]. In order to verify whether the application of 200 μmol/L GA3 gibberellin has any adverse effect on the growth condition of plants, we measured the physiological and biochemical indexes of Arabidopsis thaliana. In this experiment, Arabidopsis leaves just entering the flowering stage were sampled. Arabidopsis leaves without GA3 treatment were the control group. In contrast, GA3-treated Arabidopsis leaves were the experimental group. The results of the measurements showed that spraying with GA3 could increase POD, SOD, Pro, and total chlorophyll contents in wild-type and overexpressed Arabidopsis thaliana OE6 and OE8. They would reduce H202 and O2− in wild-type Arabidopsis thaliana and OE6 and OE8 plants (Figure 4). The results showed that spraying 200 μmol/L GA3 was beneficial to plant growth and that it could be possible to spray 200 μmol/L GA3 for the subsequent experiments.

3.4. ZmGRAS46 Is Dependent on the GA Pathway to Regulate Flowering

Wild-type plants and OE6 and OE8 plants were planted in nutrient soil containing vermiculite, and 200 μmol/L GA3 was sprayed on Arabidopsis thaliana starting at 15 days of plant growth and every three days, and the growth of Arabidopsis thaliana after spraying was observed. Observations on the 30th of cultivation showed that untreated wild-type Arabidopsis had formed flower buds, but OE6 and OE8 had not yet formed flower buds, whereas GA3-treated WT, OE6, and OE8 plants had elongated main branches (Figure 5A). We found that there was no significant difference in flowering time between GA3-treated wild-type plants, and both OE6 and OE8 plants flowered at about 26 days of growth (Figure 5B). This suggests that spraying GA3 can change the flowering time of ZmGRAS46 overexpression and advance OE6 and OE8, which originally delayed flowering, to a similar flowering time as the wild type. When the main branches were elongated, we recorded and observed the length of the main branches and found that the length of the main branches of the overexpression plants was significantly elongated after spraying GA3 with no significant difference compared with the wild type, indicating that the spraying of GA3 could change the length of the main branches with the overexpression of ZmGRAS46 so that the length of the main branches, which was shorter than that of the wild type, was similar to the length of the main branches of the wild-type plants (Figure 5C,D). It was shown that ZmGRAS46 was dependent on the GA pathway to regulate flowering, and spraying gibberellin reduced the expression of ZmGRAS46, restoring the phenotype of the overexpression of ZmGRAS46 that delayed flowering as well as inhibited floral branch elongation.

3.5. ZmGRAS46 Regulates Diverse Genes That Are Involved in Flowering Regulation

To investigate the role of ZmGRAS46 in regulating the flowering pathway, this experiment sampled flower buds and flowers of GA3-treated and untreated Arabidopsis thaliana. The results by fluorescence quantification showed that the expression of CO, FT, SOC1, and LFY was less than that of WT plants in OE6 and OE8, indicating that the overexpression of ZmGRAS46 inhibited the formation of CO, FT, SOC1, and LFY to delay flowering. Our results indicated the possibility that ZmGRAS46 represses flowering through the CO-FT-SOC1-mediated photoperiodic flowering pathway. CO, FT, SOC1, and LFY expressions were elevated in overexpressed OE6 and OE8 plants after GA3 treatment. This indicates that spraying GA3 can reduce the expression of ZmGRAS46 and promote the elevated expression of flowering-related genes CO, FT, SOC1, and LFY, which contributed to the early flowering of Arabidopsis thaliana, demonstrating that ZmGRAS46 is dependent on the GA pathway for the regulation of flowering (Figure 6).

3.6. Interaction of ZmGRAS46 and ZmMADS62 Proteins

Delayed flowering affects fruit yield. Li et al. showed that MADS-box regulates fruit ripening time and quality [30,31]. In order to investigate the effect of ZmGRAS46 on fruit quality, this experiment was conducted by screening and validation to find ZmMADS62 in the MADS-box family. ZmGRAS46-BK vector plasmid and ZmMADS62-AD vector plasmid were simultaneously transformed into the same AH109 yeast sensory state. After the transformation, the cultured, positive, and negative bacterial spots were incubated in an SD/-Trp-Leu two-deficient solid medium and SD/-Trp-Leu-Ade-His and SD/-Trp-Leu-Ade-His-X-α-Gal four-deficient solid yeast medium for two days at 29 °C, respectively. The results showed that the pGADT7-T + pGBKT7-53 positive control and ZmGRAS46-BK + ZmMADS62-AD experimental group grew colonies on both two- and four-deficient media, and the colonies turned blue on four-deficient media coated with X-α-gal. In contrast, the pGADT7-T + pGBKT7-lam negative control had colonies on two-deficient media growth growing colonies on all four-deficient media (Figure 7), suggesting that the ZmGRAS46-ZmMADS62 module interaction may play a role in regulating plant yield.

4. Discussion

A complex regulatory network synergistically regulates plant flowering. In this study, we showed that ZmGRAS46 in the GRAS family can affect the flowering phenotype of Arabidopsis thaliana and repress the expression of CO, FT, SOC1, and LFY. The treatment by GA3 demonstrated that this gene mediates the GA pathway to regulate plant flowering.
BrLAS is a GRAS transcription factor in kale-type oilseed rape that alters the phenotype of Arabidopsis, delays the flowering time of the plant, and can respond to it under drought stress [32]. The GAI1-like gene in the GRAS family delays plant flowering and reduces plant height in Rhus chinensis [33]. SlGRAS26, a GRAS transcription factor in Solanum lycopersicum, responds to abiotic stress and alters plant height, affecting the initiation of inflorescence meristematic organization [25]. The above studies showed that the GRAS family can affect the flowering time of plants, which is consistent with the results of the present study that show ZmGRAS46 delayed plant flowering and altered plant phenotypes (Figure 1 and Figure 2), which is inconsistent with the above studies, although the present study did not investigate as to whether ZmGRAS46 responded to abiotic stresses. In the study on upland cotton, GhGRAS55 of the GRAS family was found to affect plant germination and promote early plant maturation [34]. For the GRAS family, the effect of early and late plant maturity in this study is consistent with the function of this study. However, inconsistently, ZmGRAS46 did not affect plant germination.
Many members of the GRAS family regulate flowering through the GA pathway, and in studies on kale, it was found that BraRGL1 repressed the transcriptional activation of the BraLFY gene with BraSOC1. However, the presence of GA3 enhanced the activation of BraSOC1, suggesting that the BraRGL1-BraSOC1 module regulates mossing and flowering in cabbage through GA signaling [35]. Plants overexpressing SlGARS7 (SlGRAS7-OE) in tomato exhibited various phenotypes associated with many behaviors, including plant height, root and shoot length, and flowering time. It was observed that many genes related to growth hormone and gibberellin (GA) were down-regulated, and sensitivity to GA3/IAA was altered in SlGRAS7-OE seedlings [36]. The overexpression of a tomato miR171 target gene SlGRAS24 impacts multiple agronomical traits via regulating gibberellin and auxin homeostasis [37]. Consistent with these, this study found that ZmGRAS46 depends on the GA signaling pathway to regulate flowering (Figure 5). This study did not investigate other hormonal pathways, such as growth hormone, which provides a new direction for subsequent research.
CONSTANS (CO), a zinc fiber protein, is a crucial regulator for plant flowering, and light signaling has a stabilizing effect on CO proteins, which play a central role in regulating photoperiodic flowering [38,39]. In this study, the expression of flowering-related genes was determined, and the overexpression of ZmGRAS46 suppressed CO expression and reduced CO expression in Arabidopsis (Figure 6). This suggests ZmGRAS46 may mediate the photoperiodic pathway to regulate flowering in Arabidopsis, but the role of ZmGRAS46 in the photoperiodic pathway regulation of flowering was not investigated in this study. In follow-up experiments, we will change the photoperiod and perform different light treatments to investigate whether ZmGRAS46 mediates the photoperiodic pathway to regulate plant flowering. Li et al. showed that SOC1- and FT-overexpressed repressors integrate the gibberellin (GA) signaling pathway and the FLOWERING LOCUS C (FLC)-mediated vernalization pathway in regulating flowering time. In this study, the overexpression of ZmGRAS46 altered the expression of SOC1 and FT but not FLC, and follow-up experiments are needed to investigate whether ZmGRAS46 mediates the vernalization pathway [40].
Crops need to have high yields. Gautam et al. showed that early flowering mutants were obtained by the mutagenesis of rice with gamma rays. EMS can affect rice yield, suggesting that crops’ early maturity is of some research significance [41]. Wheat yield is affected by environmental, management, and genotypic factors. To cope with different climatic changes, farmers must have different varieties, e.g., early, mid–late, and late flowering varieties, and sow according to seasonal conditions [42,43]. The publication of related studies indicates the importance of research on the effect of flowering time on yield. Li et al. showed that MADS-box transcription factors regulate fruit ripening, that SOC1 in the MADS-box mediates plant flowering, and that this family also has a role in influencing yield [25,44]. The K-domain of a blueberry’s SUPPRESSOR of CONSTITUTIVE EXPRESSION OF CONSTANS 1 (VcSOC1K) has similarities to five MADS-box genes in maize. The constitutive expression of VcSOC1K has been demonstrated to be very effective in improving maize grain yield [45]. The expression of the MADS-box transcription factor gene, Zmm28, resulted in increased vegetative growth, photosynthetic capacity, and nitrogen utilization in maize plants, and these positive changes led to a significant increase in grain yields [46]. Since the MADS-box family impacts plant yield, ZmMADS62, a family member, was chosen as the prey vector in this study. The experimental results showed that ZmGRAS46 could interact with ZmMADS62 (Figure 7). However, it was not investigated whether the ZmGRAS46-ZmMADS62 module regulated yield, and the effect of ZmGRAS46 on yield will be investigated in subsequent experiments.

5. Conclusions

In this study, we showed that ZmGRAS46 negatively regulated flowering in Arabidopsis thaliana by inhibiting the expression of CO, FT, SOC1, and LFY. The overexpression of ZmGRAS46 affected the plant’s phenotype, which promoted rosette leaf growth, facilitated bud formation, and inhibited the elongation of the main stem. The application of GA3 rescued the delayed flowering phenotype of Arabidopsis. It promoted the elongation of flowering shoots, demonstrating that ZmGRAS46 regulates flowering in Arabidopsis by mediating the GA pathway. In recent years, global warming and the increase in temperature have changed the flowering period of plants, affecting their growth and development, and it has become necessary to study their flowering period. This study elucidated the mechanism by which ZmGRAS46 is mediated in the GA pathway to regulate plant flowering, and it provided a theoretical basis for solving the problem of early and late flowering.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy14010155/s1, Figure S1: Toxicity validation of pGBKT7-ZmGRAS46 prey vector; Figure S2: Validation of pGBKT7-ZmGRAS46 prey vector self-activation; Figure S3: Analysis of relative expression of ZmGRAS46 in eight T3 generation positive overexpressing Arabidopsis thaliana plants; Table S1: All primer sequences used in this study.

Author Contributions

Conceptualization, S.G., B.G., Y.Z. and Y.M.; methodology, H.Z. and Z.J.; writing—original draft preparation, H.Z. and S.L.; writing—review and editing H.Z., P.J. and S.G. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Jilin Province Science and Technology Development Plan Project [20220202008NC, 20230202003NC].

Data Availability Statement

All data generated or analyzed during this study are available within the article or upon request from the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Flowering phenotypes of Arabidopsis seedlings with different ZmGRAS46 backgrounds. (A,B) The flowering phenotypes of Col-0 wild-type (WT) and ZmGRAS46-overexpressing (OE6, OE8) lines under long-day (16 h/8 h light/dark) conditions. (C,D) Phenotypes of rosette leaves of Col-0 wild-type (WT) and ZmGRAS46-overexpressing (OE6, OE8) lines under long-day (16 h/8 h light/dark) conditions. Using Student’s t-test, asterisks indicate statistically significant differences (** p < 0.01). Data are shown as mean ± SD from three independent experiments.
Figure 1. Flowering phenotypes of Arabidopsis seedlings with different ZmGRAS46 backgrounds. (A,B) The flowering phenotypes of Col-0 wild-type (WT) and ZmGRAS46-overexpressing (OE6, OE8) lines under long-day (16 h/8 h light/dark) conditions. (C,D) Phenotypes of rosette leaves of Col-0 wild-type (WT) and ZmGRAS46-overexpressing (OE6, OE8) lines under long-day (16 h/8 h light/dark) conditions. Using Student’s t-test, asterisks indicate statistically significant differences (** p < 0.01). Data are shown as mean ± SD from three independent experiments.
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Figure 2. Phenotypic observation of Arabidopsis with different ZmGRAS46 backgrounds. (A,B) Observation on bud of Col-0 wild-type (WT) and ZmGRAS46-overexpressing (OE6, OE8) lines under long-day (16 h/8 h light/dark) conditions. (C,D) Length of main branches of Col-0 wild-type (WT) and ZmGRAS46-overexpressing (OE6, OE8) lines under long-day (16 h/8 h light/dark) conditions. Using Student’s t-test, asterisks indicate statistically significant differences (** p < 0.01). Data are shown as mean ± SD from three independent experiments.
Figure 2. Phenotypic observation of Arabidopsis with different ZmGRAS46 backgrounds. (A,B) Observation on bud of Col-0 wild-type (WT) and ZmGRAS46-overexpressing (OE6, OE8) lines under long-day (16 h/8 h light/dark) conditions. (C,D) Length of main branches of Col-0 wild-type (WT) and ZmGRAS46-overexpressing (OE6, OE8) lines under long-day (16 h/8 h light/dark) conditions. Using Student’s t-test, asterisks indicate statistically significant differences (** p < 0.01). Data are shown as mean ± SD from three independent experiments.
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Figure 3. Differential expression of genes related to GA3 synthesis and metabolism in Arabidopsis seedlings with different ZmGRAS46 backgrounds. (A) Differential expression of GA3 anabolic genes in Arabidopsis buds. (B) Differential expression of GA3 anabolic genes in Arabidopsis blossom. (C) Expression analysis of ZmGRAS46 in different conditions. Using Student’s t-test, asterisks indicate statistically significant differences (* p < 0.05; ** p < 0.01; ns: not significant). Data are shown as mean ± SD from three independent experiments.
Figure 3. Differential expression of genes related to GA3 synthesis and metabolism in Arabidopsis seedlings with different ZmGRAS46 backgrounds. (A) Differential expression of GA3 anabolic genes in Arabidopsis buds. (B) Differential expression of GA3 anabolic genes in Arabidopsis blossom. (C) Expression analysis of ZmGRAS46 in different conditions. Using Student’s t-test, asterisks indicate statistically significant differences (* p < 0.05; ** p < 0.01; ns: not significant). Data are shown as mean ± SD from three independent experiments.
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Figure 4. Determination of physiological and biochemical indexes of Arabidopsis with different ZmGRAS46 backgrounds. (A) Analysis of POD activity. (B) Analysis of Pro activity. (C) Analysis of H2O2 activity. (D) Analysis of SOD activity. (E) Analysis of O2− activity. (F) Analysis of total chlorophyll content. Using Student’s t-test, asterisks indicate statistically significant differences (* p < 0.05; ** p < 0.01; ns: not significant). Data are shown as mean ± SD from three independent experiments.
Figure 4. Determination of physiological and biochemical indexes of Arabidopsis with different ZmGRAS46 backgrounds. (A) Analysis of POD activity. (B) Analysis of Pro activity. (C) Analysis of H2O2 activity. (D) Analysis of SOD activity. (E) Analysis of O2− activity. (F) Analysis of total chlorophyll content. Using Student’s t-test, asterisks indicate statistically significant differences (* p < 0.05; ** p < 0.01; ns: not significant). Data are shown as mean ± SD from three independent experiments.
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Figure 5. Flowering phenotypes of Arabidopsis seedlings with different ZmGRAS46 backgrounds after GA3 treatment. (A,B) Flowering phenotypes of Col-0 wild-type (WT) and ZmGRAS46 overexpressing (OE6, OE8) strains after GA3 treatment under long-daylight (16 h/8 h light/dark) conditions. (C,D) Primary branch length of Col-0 wild-type (WT) and ZmGRAS46 gene overexpressing (OE6, OE8) strains under long daylight (16 h/8 h light/dark) conditions after GA3 treatment condition. Using Student’s t-test, asterisks indicate statistically significant differences (ns: not significant). Data are shown as mean ± SD from three independent experiments.
Figure 5. Flowering phenotypes of Arabidopsis seedlings with different ZmGRAS46 backgrounds after GA3 treatment. (A,B) Flowering phenotypes of Col-0 wild-type (WT) and ZmGRAS46 overexpressing (OE6, OE8) strains after GA3 treatment under long-daylight (16 h/8 h light/dark) conditions. (C,D) Primary branch length of Col-0 wild-type (WT) and ZmGRAS46 gene overexpressing (OE6, OE8) strains under long daylight (16 h/8 h light/dark) conditions after GA3 treatment condition. Using Student’s t-test, asterisks indicate statistically significant differences (ns: not significant). Data are shown as mean ± SD from three independent experiments.
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Figure 6. Differential expression of flowering-related genes in Arabidopsis seedlings with different ZmGRAS46 backgrounds. (A) Differential expression of flowering-related genes CO, FT, LFY, and SOC1 in Arabidopsis flower buds. (B) Differential expression of flowering-related genes CO, FT, LFY, and SOC1 in Arabidopsis blossom condition. Using Student’s t-test, asterisks indicate statistically significant differences (* p < 0.05; ** p < 0.01; ns: not significant). Data are shown as mean ± SD from three independent experiments.
Figure 6. Differential expression of flowering-related genes in Arabidopsis seedlings with different ZmGRAS46 backgrounds. (A) Differential expression of flowering-related genes CO, FT, LFY, and SOC1 in Arabidopsis flower buds. (B) Differential expression of flowering-related genes CO, FT, LFY, and SOC1 in Arabidopsis blossom condition. Using Student’s t-test, asterisks indicate statistically significant differences (* p < 0.05; ** p < 0.01; ns: not significant). Data are shown as mean ± SD from three independent experiments.
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Figure 7. Interaction validation of ZmGRAS46-ZmMADS62. Positive control: pGADT7-T + pGBKT7-53; experimental group: ZmGRAS46-BK + ZmMADS62-AD; negative control: pGADT7-T + pGBKT7-Lam.
Figure 7. Interaction validation of ZmGRAS46-ZmMADS62. Positive control: pGADT7-T + pGBKT7-53; experimental group: ZmGRAS46-BK + ZmMADS62-AD; negative control: pGADT7-T + pGBKT7-Lam.
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MDPI and ACS Style

Zhang, H.; Jiang, Z.; Jiao, P.; Zhao, Y.; Gao, B.; Liu, S.; Guan, S.; Ma, Y. ZmGRAS46 Negatively Regulates Flowering Time in Arabidopsis thaliana. Agronomy 2024, 14, 155. https://doi.org/10.3390/agronomy14010155

AMA Style

Zhang H, Jiang Z, Jiao P, Zhao Y, Gao B, Liu S, Guan S, Ma Y. ZmGRAS46 Negatively Regulates Flowering Time in Arabidopsis thaliana. Agronomy. 2024; 14(1):155. https://doi.org/10.3390/agronomy14010155

Chicago/Turabian Style

Zhang, Honglin, Zhenzhong Jiang, Peng Jiao, Yang Zhao, Bai Gao, Siyan Liu, Shuyan Guan, and Yiyong Ma. 2024. "ZmGRAS46 Negatively Regulates Flowering Time in Arabidopsis thaliana" Agronomy 14, no. 1: 155. https://doi.org/10.3390/agronomy14010155

APA Style

Zhang, H., Jiang, Z., Jiao, P., Zhao, Y., Gao, B., Liu, S., Guan, S., & Ma, Y. (2024). ZmGRAS46 Negatively Regulates Flowering Time in Arabidopsis thaliana. Agronomy, 14(1), 155. https://doi.org/10.3390/agronomy14010155

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