B-box containing protein 1 from Malus domestica (MdBBX1) is involved in the abiotic stress response

Plant growth conditions and treatments

For organ-specific expression analyses, different apple organs, including roots, stems, leaves, flowers and fruits, were sampled from 6-year-old apple trees growing at the experimental station of Shandong Agricultural University (Tai’an, Shandong, China).

Apple (golden delicious) seedlings were cultivated under greenhouse conditions (relative humidity of 60–75%) at 22 ± 1 °C with a 16 h light/8 h dark photoperiod for approximately 1 year. Then, the uniformly growing seedlings were selected for stress treatments. For salt and drought stress treatments, the apple seedlings were watered with solutions of 250 mM NaCl or 25% (w/v) polyethylene glycol-6000 (PEG-6000), and control seedling received the same amount of water only. For ABA treatment, 100 µM ABA solutions were directly sprayed on the seedlings. For cold stress, the apple seedlings were subjected to 4 °C condition, while seedlings growing at room temperature (25 °C) were used as the controls. Samples were collected from three different kinds of seedlings at 0, 3, 6, 9 and 12 h after treatment, as was done in a previous study (Yuan et al., 2013). Then, the collected samples were immediately frozen in liquid nitrogen and stored at −70 °C till used. Subsequently, the total RNA was extracted from the collected samples using an improved cetyl-trimethylammonium bromide (CTAB) procedure (Gasic, Hernandez & Korban, 2004).

The seeds of WT (Col-0) and transgenic Arabidopsis were disinfected and sown on 1/2 Murashige and Skoog (MS) media. After culturing at 4 °C for 3 days to undergo vernalization, the seedlings were transferred to a greenhouse condition, which included a 22 ± 1 °C temperature with a 16 h light/8 h photoperiod. Then, 3-week-old WT and transgenic plants were treated with 250 mM NaCl or 25% (w/v) PEG-6000 as described by Liu et al. (2019a), and the control seedlings were treated with water only. Plant growth status was observed and the survival rates were determined daily. Each treatment was performed at least three times.

Quantitative real-time PCR (qRT-PCR) analysis

QRT-PCR is a commonly used approach for the quantitative detection of gene expression in real time (Deepak et al., 2007). All the primers used in this investigation were designed according to the target gene sequences via the Beacon Designers software and were shown in Table S1. qRT-PCR was carried out using a SYBR® PrimeScript™ RT-PCR Kit (TaKaRa, Dalian, China) and run on a CFX96TM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The Arabidopsis Actin8 and apple actin genes were used as reference genes (Wang et al., 2016). The thermal cycling parameters were as follows: 40 cycles of 95 °C denaturation for 15 s, 55 °C annealing for 20 s and 70 °C extension for 15 s. The qRT-PCR data were analyzed by the 2^−ΔΔCt method (Livak & Schmittgen, 2001). The relative expression of MdBBX1 in the treated samples was compared with that in the nontreated samples at each treatment time point with significant differences (P < 0.05) determined based on Tukey’s multiple test.

Subcellular localization of MdBBX1

The full-length coding sequence of MdBBX1 was amplified from apple and inserted into the pROKII vector containing a GFP gene and the CaMV35S promoter. Cells of Agrobacterium tumefaciens GV3101 with the recombinant plasmid were cultured overnight, resuspended in osmotic solution (10 mM, Mole MgCl₂, 10 mM 2-[N-morpholino] ethanesulfonic acid (MES) and 150 mM acetosyringone), and then injected into the leaves of 1-month-old Nicotiana benthamiana plants. The fluorescent signal of MdBBX1-GFP was detected via a confocal microscope (LSM 510 META, Carl Zeiss, Jena, Germany) after 2–3 days. The nuclei were subsequently stained with 100 g/mL 4′, 6 -diamidino-2-phenylindole (DAPI) (Solarbio, Beijing, China) for 10 min. Leaves overexpressing 35S-GFP were used as controls (Wang et al., 2018).

Construction of expression plasmids

The cDNA sequence of MdBBX1 was inserted into the polyclonal sites of pET-30a (+) (Novagen), which contained His-tagged sequences. Then, the recombinant vector was transformed into Escherichia coli BL21 cells. The recombinant sequences in the plasmids were sequenced by Sangon Biotechnology Company (Shanghai, China).

Survival test of Escherichia under different abiotic stresses

Survival analysis of Escherichia coli under salt and drought stress was conducted as described by Du et al. (2014). The cells were cultured in Luria-Bertani (LB) liquid media until the OD₆₀₀ reached 0.4–0.6, and then the expression of the recombinant protein was induced for 2 h using isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37 °C. All the bacterial cultures were first diluted to an OD₆₀₀ of 0.6 and then diluted 10⁻³, 10⁻⁴ and 10⁻⁵ times. For the survival test on solid media, 10 µL cultures of each dilution were spotted onto solid LB media that included 500 mM KCl, 500 mM NaCl or 600 mM mannitol and incubated for 12 h at 37 °C. Then, the colony numbers in each dish for the culture diluted to 10⁻⁵ were counted. Each experiment was performed at least three times.

For the survival test in liquid media, the cultures were first diluted to an OD₆₀₀ of 0.6, after which 200 µL of the cultures were put into 20 mL of LB solution that included 500 mM NaCl, 500 mM KCl or 600 mM mannitol and incubated at 37 °C on a rotary shaker (150 rpm). Then, the bacterial suspension was collected every 2 h for 24 h, after which the OD₆₀₀ of the culture was measured. Each experiment was repeated at least 3 times.

Generation of transgenic plants

The pROKII-MdBBX1 recombinant plasmids were transformed into Arabidopsis in accordance with the floral-dip method via Agrobacterium tumefaciens (GV3101)-mediated transformation. Subsequently, the MdBBX1 overexpression seedlings were screened on MS agar media supplemented with 50 µg/mL kanamycin and were further identified via PCR using MdBBX1 and GFP primers. The specific primers used are shown in the Table S1.

Analysis of germination status under different abiotic stresses

Fifty seeds of WT or overexpression (OE) lines were sown onto 1/2-strength MS agar media supplemented with different concentrations of mannitol (300 or 400 mM), NaCl (150 or 200 mM) or ABA (0.2 or 0.6 µM). Seed germination was observed and measured every 12 h. For root length analysis, the seeds were grown vertically on 1/2-strength MS media as described above. The root length of 20 seedlings was measured after 10 days, and each treatment was performed at least three times.

Measurements of proline (PRO), malondialdehyde (MDA), and reactive oxygen species (ROS) contents and antioxidant enzyme activity

For physiological index measurements, the free PRO content was measured using a spectrophotometric PRO kit (Solarbio Life Sciences, Beijing, China). The MDA content was measured using a thiobarbituric acid reactive substances assay (Aguilar Díaz de León & Borges, 2020; Hodges, Delong & Prange, 1999) and the contents of hydrogen peroxide (H₂O₂) and superoxide anions (O₂^.−) were measured using O₂^.− and H₂O₂ kits, respectively (Nanjing Jiancheng Bioengineering Institute, China). Similarly, the total protein contents were determined using a BCA Protein Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), and the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) were measured based on the protocols of the corresponding kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) (Bai et al., 2020; Li et al., 2019; Ma et al., 2019).

Statistical analysis

All experiments were conducted at least three times. The data presented are the means ± standard deviations of three replications. Statistical significance was analyzed using SPSS software (version 17.0), and Turkey’s multiple range comparison tests were performed to determine the significance of differences between samples (P < 0.05 or P < 0.01).

Organ-specific expression pattern analysis and subcellular localization of MdBBX1

The organ-specific expression pattern of MdBBX1 was analyzed via qRT-PCR. The results revealed that the transcript level of MdBBX1 was obviously higher in the leaves than in other organs (Fig. 1A). To identify the subcellular localization of MdBBX1, an MdBBX1-GFP construct and empty GFP plasmid were introduced into epidermal cells of tobacco leaves, after which the nuclei were stained by DAPI. The fluorescence signal and DAPI staining were predominantly distributed in the nucleus (Fig. 1B), which indicated that MdBBX1 was localized there.

The MdBBX1 promoter contains elements related to the abiotic stress response

Some BBX members were found to respond positively to abiotic stresses in a previous study (An et al., 2020; Crocco & Botto, 2013; Liu et al., 2018; Shalmani et al., 2018). To explore the potential functions of MdBBX1 in response to a variety of abiotic stresses, the DNA sequence within 2000 bp upstream of MdBBX1 (the promoter sequence) was scanned via PlantCARE software (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). The results showed that many cis-acting elements that may be involved in responses to abiotic stress, light and other signals were present in the MdBBX1 promoter region (Table 1). For instance, MBS (MYB-binding site) elements are involved in the response to drought stress, and ABA-responsive elements (ABREs) function in response to exogenous ABA. LTRs are found to participate in low-temperature responsiveness. In addition, some cis-acting elements such as TC-rich repeats and TCA elements are involved in responses to defense and stress or to salicylic acid. Taken together, these results indicated that MdBBX1 may participate in the response to abiotic stress.

To further investigate whether MdBBX1 is expressed in response to abiotic stress, apple seedlings were treated with solutions of 100 µM ABA, 25% polyethylene glycol (PEG), or 250 mM NaCl or 4 °C for different durations. As shown in Fig. 2, the transcript levels of MdBBX1 were upregulated in both the leaves and the roots under exogenous ABA, salt and PEG treatment, MdBBX1 expression was maximized after 6 h of stimulation by ABA, PEG or NaCl compared with the control in the roots and increased by approximately 120-, 4- and 2-fold, respectively. In addition, the transcript levels of MdBBX1 were upregulated by 12-, 20- and 10-fold after treatment with ABA, PEG or NaCl in the leaves. Notably, the expression of MdBBX1 was significantly upregulated under cold conditions only in the roots. Taken together, the above results showed that the expression of MdBBX1 was induced in response to different abiotic stresses, which suggested that MdBBX1 may be involved in the response to abiotic stress.

Ectopic expression of MdBBX1 in Escherichia improved cell tolerance to abiotic stress

To determine the stress resistance function of MdBBX1, heterogeneous expression of MdBBX1 was induced in Escherichia coli growing on solid media under different stress conditions. Survival tests of Escherichia coli cells were carried out, with empty vectors used as controls. As shown in Figs. 3A–3B, the growth of the cells in nonstress media showed slight significant difference between those harboring MdBBX1 and those harboring the empty vector. However, when the same concentration of cultures was inoculated onto plates with stress media, the number of Escherichia coli colonies expressing MdBBX1 was significantly higher than that of the control colonies harboring the empty vector. To further confirm the function of MdBBX1, a growth curve of Escherichia coli in liquid media was constructed. As shown in Fig. 3C, under nonstress conditions, few differences were observed among the growth curves of cells with and without MdBBX1 expression. However, under different stress conditions, the growth rate of Escherichia coli expressing MdBBX1 was significantly faster than that of the control cells carrying the empty vector. The results suggested that MdBBX1 provided strong abiotic stress tolerance.

Overexpression of MdBBX1 increased resistance to abiotic stress in Arabidopsis

To determine the role of MdBBX1 in abiotic stress resistance in plants, three transgenic lines (OE1, OE2 and OE3) with similar expression levels of MdBBX1 were obtained and subjected to salt and drought treatment (Fig. S1). As shown in Fig. 4A, on normal media, the germination rate and growth status of seedlings exhibited no obvious differences between the WT and transgenic lines. However, under salt stress, the OE seeds presented a significantly higher germination rate than did the WT seeds. On the stress media that included 200 mM NaCl, the germination rate of the OE seeds reached 60% compared with 20% for WT seeds after treatment for 48 h. In addition, the root length of OE lines was obviously longer than that of the WT plants on 150 mM NaCl media (Fig. 4B). When the 3-week-old seedlings were watered with 250 mM NaCl for 10 days, the leaves of WT began to turn yellow, but few yellow leaves were observed on the OE seedlings. After treatment for 15 days, more wilted and chlorotic leaves were observed on the WT than in the OE lines. After treatment with salt for 20 days, the OE plants presented a significantly higher survival rate (approximately 90%) than did the WT plants (approximately 40%) (Fig. 5A). In addition, under salt stress, the PRO accumulation in the OE plants was obviously higher than that in the WT (Fig. 5B), while the levels of MDA were obviously lower in the OE lines than in the WT (Fig. 5C).

Similarly, after mannitol treatment, the OE lines also displayed significantly higher germination rates and longer root lengths than did the WT line (Figs. 6A–6B). Moreover, the transgenic plants presented a higher survival rate than did the WT plants when treated with 25% PEG-6000 (Fig. 6C). When treated for 25 days, nearly 60% of WT plants wilted and died, while the leaves of OE plants yellowed slightly. The survival rate of the OE plants was approximately 90%, which was significantly higher than the survival rate of the WT plants. Taken together, these results indicated that overexpression of MdBBX1 enhanced the abiotic stress resistance of the transgenic plants during germination and vegetative stage.

Overexpression of MdBBX1 in Arabidopsis decreased sensitivity to ABA

ABA plays a critical role in the physiological regulation of plant development in seed germination and in abiotic stress responses (Vishwakarma et al., 2017). To determine the potential function of MdBBX1 in response to ABA, seeds of the OE and WT lines were plated on MS media either without or with ABA. Under MS media without ABA, the WT and OE lines displayed similar germination rates. However, under ABA treatment, the germination rates of the OE lines were significantly higher than those of the WT (Fig. 7A). Moreover, after the seedlings grew on MS media with ABA for 10 days, the primary root length of the OE seedlings was obviously greater than that of the WT seedlings (Fig. 7B). To determine the effect on root growth, the seeds of WT and OE were sown on MS media for 2 days first and then transferred to plates with media that included 0.6 μM ABA. The root length of the OE seedlings was still longer than that of the WT seedlings (Fig. S2). The above results suggested that overexpression of MdBBX1 decreased ABA sensitivity in Arabidopsis during the germination and seedling stages.

In the ABA signaling pathway, HY5 and ABI5 are crucial for seed germination and seedling development (Chen & Xiong, 2008; Finkelstein, Gampala & Rock, 2002; Finkelstein & Lynch, 2000b). To determine whether the development of MdBBX1-overexpressing seedlings under abiotic stresses was related to HY5 or ABI5, the transcript levels of ABI5 and HY5 were measured after ABA treatment. The results revealed that the expression of ABI5 was markedly reduced in the OE plants compared with the WT plants, while HY5 changed only slightly (Fig. 7C). Another OE line overexpressing a different MdBBX family member (MdBBX10), which is ABA sensitive, was also evaluated under the same treatment (Liu et al., 2018). As shown in Fig. 7C, the changes in ABI5 expression levels were opposite between MdBBX1 and MdBBX10, which was reasonably expected in terms of a response to ABA treatment.

Overexpression of MdBBX1 reduced ROS accumulation in transgenic plants

Various abiotic stresses often lead to the accumulation of excessive amounts of ROS, particularly H₂O₂ and O₂^•−, which has an important impact on plant growth and development (Mittler et al., 2004). To analyze whether MdBBX1 responds to abiotic stress through the regulation of ROS levels, the accumulation of O₂^•− in WT and OE plants was assessed via nitro blue tetrazolium (NBT) staining. No obvious difference was found between the WT and OE lines. However, under NaCl and PEG conditions, the OE lines accumulated lower levels of O₂^•− than the WT did (Fig. 8A). Furthermore, the contents of H₂O₂ and O₂^•− were measured, and the results showed that their contents in the OE lines were significantly lower than those in the WT (Figs. 8B, 8C). Similarly, under normal conditions, the activities of SOD, POD and APX were not notably different between the OE and WT lines. However, after salt treatment, the activities of SOD, POD and APX in the OE lines significantly increased compared with those in the WT lines (Fig. 9). Together, these results suggested that overexpression of MdBBX1 could decrease the accumulation of ROS in transgenic plants by mediating the activities of ROS-scavenging enzymes.