The Inhibitory Effect of AtBES1D on Tomato Seed Germination is Correlated with ABA

Shengke ZHAO Zicheng WANG Sheng LI Sen HE Chengguo JIA

Abstract Brassinosteroids (BRs), a group of polyhydroxylated plant steroid hormones, have fundamental functions in many aspects of plant growth and development. The BRI1-EMS SUPPRESSOR1 (BES1) transcription factor is a positive regulator involved in BRs signaling pathways. We studied the role of AtBES1D (obtained from bes1-D Arabidopsis ) in tomato ( Solanum lycopersicum ) seed germination. Overexpression of AtBES1D in tomato inhibited seed germination compared with wild type Zhongshusihao (ZS4). The expression of abscisic acid (ABA) related genes was enhanced in AtBES1D transgenic tomato seeds during germination. Furthermore, AtBES1D transgenic tomato seeds were hypersensitive to ABA. Our findings suggest that the inhibitory effect of AtBES1D transcription factor on tomato seed germination may be correlated with an enhanced ABA pathway.

Key words ABA; Brassinosteroids (BRs); BRI1-EMS SUPPRESSOR1 (BES1); Seed germination

Received: January 23, 2020Accepted: March 4, 2020

Supported by National Undergraduate Training Program for Innovation and Entrepreneurship (2018A8205).

Shengke ZHAO (1996-), female, P. R. China, undergraduate student, devoted to research about BR signaling pathway.

Corresponding author. E-mail: jiacg@jlu.edu.cn.

Seed germination plays an important role in the production of agronomic and horticultural crops. Germination is a very complex process regulated by plant hormones and other signaling molecules, including abscisic acid (ABA), ethylene, brassinosteroids (BRs), auxins and cytokinins［1］. Generally, ABA is considered as a major player in the regulation of germination. ABA functions as a positive regulator of physiological dormancy and inhibits seed germination［2］. Brassinosteroids (BRs), a class of plant steroidal hormones, were known to play crucial roles in various biological processes, such as seed germination, seedling development, flowering, fruit development, senescence and stress responses［3-4］. Using Arabidopsis as a model system, researchers have established the BR signaling pathway［5］. BRI1-EMS SUPPRESSOR1 (BES1) is a well-characterized basic helix-loop-helix transcription factor in the BR signaling pathway［6-7］.

In this study, we investigated the effects of AtBES1D transcription factor on tomato ( Solanum lycopersicum ) seed germination and determined whether the regulatory function of AtBES1D on tomato seed germination is correlated with ABA biosynthesis and signaling. The results will enable us to gain further insight into the mechanisms of BR signaling in regulating seed germination.

Materials and Methods

Materials and plant growth conditions

Tomato ( Solanum lycopersicum cv. Zhongshusihao; here denoted ZS4) was used in this study. AtBES1D transgenic tomato plants were obtained by Agrobacterium -mediated transformation method with 35S-AtBES1D-GFP constructs. Primary transformed plants (T0) were screened and confirmed by PCR and quantitative real-time PCR (qRT-PCR). T1 generation seeds were obtained by self-pollination of individual transgenic lines. All plants were grown in a standardized greenhouse at Jilin University, with temperature ranging from 22 to 28 ℃. Seeds of homozygous T3 generation were used in the present research.

Seed germination assays

For germination experiments, seeds of ZS4 and AtBES1D transgenic lines were collected at the same time. Harvested seeds were air-dried at room temperature and used for experiments. Seeds were soaked with distilled water at room temperature for 2 h (defined as time 0), and then distributed evenly on one layer of wet filter paper in petri dishes. For ABA sensitivity analysis, seeds of AtBES1D transgenic and ZS4 tomato lines were soaked in ABA (5 μM) solution for 2 h on a room temperature shaker. Seeds were germinated on two layers of filter paper moistened with ABA in 9-cm petri dishes. Four replications were performed, and each petri dish contained 30 seeds. Seeds were germinated in a chamber at 24 ℃ in the dark. The data of seed germination rate were collected at the indicated time-points.

RNA extraction and qRT-PCR analysis

Sample of seeds was collected at each time-point and frozen in liquid nitrogen immediately. Total RNA of each sample was isolated using RNAprep Pure kit (Tiangen, China, Code no DP441). The quantity and quality of all RNA samples were then analyzed using a NanoPhotometer P330 (IMPLEN, Germany) and agarose gel analysis, respectively. The first-strand cDNA synthesis was performed with 1 μg of total RNA using the PrimeScriptTM RT reagent kit (Takara, Code No. RR047A) according to the manufacturer餾 instruction. For qPCR, data were analyzed separately by the 2-ΔΔCT method (Schmittgen and Livak 2008).

Results and Analysis

Overexpression of AtBES1D delays seed germination

In Arabidopsis , BES1 is an important transcription factor involved in the BR signaling pathway［6-7］. However, it was unknown how BES1 affects tomato seed germination. As shown in Fig. 1, there was no significant difference in germination rates observed between ZS4 and AtBES1D transgenic seeds at 12 and 24 h after imbibition. However, the germination percentages of all AtBES1D  transgenic lines were highly decreased compared with ZS4 at 36, 48, 60 and 72 h, indicating that overexpression of AtBES1D  in tomato delays seed germination.

Transcript levels of genes involved in ABA metabolism and signaling

ABA is a major negative regulator of seed germination. We first determined the expression levels ABA biosynthesis and catabolism genes by qRT-PCR during germination. As shown in Fig. 2a , SlNCED1 expression in seeds of BES1D OX #3 was slightly higher than expression in ZS4 at 12, 18 and 24 h, but lower at 36 and 48 h; SlNCED2 was expressed at a much higher level in BES1D OX #3 seeds compared with ZS4, except at 12 h after seed imbibition (Fig. 2b). ABA 8′‐hydroxylases are key enzymes in ABA catabolism, encoded by the cytochrome P450 CYP707A family genes. The transcript level of SlCYP707A1 in ZS4 decreased with the time of seed imbibition, whereas BES1D OX #3 exhibited a highly decreased expression level relative to ZS4 at 0 and 12 h (Fig. 2c). SlCYP707A3 expression in ZS4 was 3.69-fold higher in BES1D OX #3 than in ZS4 at 48 h (Fig. 2d). SlCYP707A4 exhibited similar expression patterns in BES1D OX #3 and ZS4, but its expression was lower in BES1D OX #3 than in ZS4, especially at 12 h (Fig. 2e). In sum, expression of SlCYP707A genes exhibited decreases in BES1D OX #3 compared with expression in ZS4 during seed germination.

In the presence of ABA, ABA receptors (PYR/PYL/RCARs) form a complex and deactivate the negative regulators PP2Cs, which otherwise inactivates the SnRK2s［8］. ABSCISIC ACID INSENSITIVE 3 (ABI3) and ABI5 are key positive regulators of ABA signaling in regulation of seed germination［9］. As expected, expression of SlPP2C2 and SlPP2C3 were decreased, whereas SlABI3 and SlABI5 were up-regulated in BES1D OX #3 seeds compared with in ZS4 (Fig. 2f-Fig. i), indicating that ABA signaling was enhanced in seeds of AtBES1D transgenic lines during germination.

ABA sensitivity analysis of seed germination by AtBES1D -overexpressing and ZS4 plants

The foregoing results suggested that the ABA signaling pathway may be increased in AtBES1D transgenic tomato seeds compared with ZS4. To further investigate this hypothesis, BES1D OX #3, BES1D OX #13 and ZS4 seeds were treated with ABA. As shown in Fig. 3, the seed germination rates of both AtBES1D transgenic lines and ZS4 were severely decreased at 48 to 84 h after ABA imbibition compared with control. ABA treatment rescued the seed germination rate of ZS4 to nearly the same level as the control at 96 to 120 h, whereas the percent of germination of BES1D OX #3 and BES1D OX #13 seeds was only 3.33% and 5% at 120 h, respectively. In sum, seeds of AtBES1D transgenic lines showed enhanced sensitivity to ABA treatment during the process of germination.

Discussion

The positive effect of BRs on seed germination has long been studied extensively with various species. Because of the important function of BR in seed germination, we speculated that overexpression of the AtBES1D transcription factor tomato may improve seed germination. Unexpectedly, we noticed that all AtBES1D transgenic tomato lines were inhibited compared with ZS4 when conducting the germination assay on filter paper (Fig. 1a). This result was opposite to findings for Arabidopsis , but it was reasonable. First, we repeated the germination experiment for at least five times and obtained similar results, even from the T1 to T5 generation. Second, instead of overexpression of AtBES1D , investigators have used the bes1-D mutant in comparable Arabidopsis studies［10-11］. Moreover, seed germination assays were conducted on MS medium for Arabidopsis , whereas we used filter paper instead.

BRs have antagonistic effects on ABA response during seed germination［12-13］. We first examined the expression of several genes involved in ABA biosynthesis and catabolism and ABA accumulation. SlNCED1 was expressed at a lower level compared with SlNCED2 in germinating seeds［14］. It seems that SlNCED1 has a dominant function in ABA synthesis in tomato fruits, whereas SlNCED2 mainly regulates ABA synthesis in tomato seeds. Similar to results reported by Yang et al. ［14］, SlNCED2 also showed the maximum transcript level at 0 h and had a decreasing trend during germination in both BES1D OX #3 and ZS4. We also found SlNCED2 was up-regulated at each time-point during seed germination. ABA catabolism is mediated by ABA 8-hydroxylase encoded by the CYP707A gene family. We found that, during germination of AtBES1D transgenic tomato seeds, the transcript abundances of SlCYP707A1, SlCYP707A3 , and SlCYP707A4 were lower than in ZS4 seeds (Fig. 2c-Fig. 2e). It has been reported that CYP707A3 and CYP707A4 were putative binding targets of BES1 transcription factor in Arabidopsis ［15］. Therefore, AtBES1D may inhibit ABA catabolism by directly regulating CYP707A3 and CYP707A4 transcription, causing increased inhibitory effects on seed germination. In contrast to the result in Arabidopsis, BES1D OX #3 exhibited enhanced sensitivity to ABA, and improved the expression of genes involved in ABA signaling during seed germination. These observations indicate that the regulatory role of AtBES1D in ABA-mediated seed germination depends on plant species and organs.

In conclusion, we proposed the possible mechanism for the inhibitory effect of AtBES1D on tomato seed germination. ABA accumulation in AtBES1D transgenic tomato seeds was improved by enhancing ABA biosynthetic genes and suppressing ABA catabolic genes, resulting in depression of ABA responsive genes. The inhibition of tomato seed germination by AtBES1D may be correlated with enhanced ABA accumulation and signaling pathway.

Shengke ZHAO et al. The Inhibitory Effect of AtBES1D on Tomato Seed Germination is Correlated with ABA

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