Reduced tolerance to abiotic stress in transgenic Arabidopsis overexpressing a Capsicum annuum multiprotein bridging factor 1

Background The pepper fruit is the second most consumed vegetable worldwide. However, low temperature affects the vegetative development and reproduction of the pepper, resulting in economic losses. To identify cold-related genes regulated by abscisic acid (ABA) in pepper seedlings, cDNA representational difference analysis was previously performed using a suppression subtractive hybridization method. One of the genes cloned from the subtraction was homologous to Solanum tuberosum MBF1 (StMBF1) encoding the coactivator multiprotein bridging factor 1. Here, we have characterized this StMBF1 homolog (named CaMBF1) from Capsicum annuum and investigated its role in abiotic stress tolerance. Results Tissue expression profile analysis using quantitative RT-PCR showed that CaMBF1 was expressed in all tested tissues, and high-level expression was detected in the flowers and seeds. The expression of CaMBF1 in pepper seedlings was dramatically suppressed by exogenously supplied salicylic acid, high salt, osmotic and heavy metal stresses. Constitutive overexpression of CaMBF1 in Arabidopsis aggravated the visible symptoms of leaf damage and the electrolyte leakage of cell damage caused by cold stress in seedlings. Furthermore, the expression of RD29A, ERD15, KIN1, and RD22 in the transgenic plants was lower than that in the wild-type plants. On the other hand, seed germination, cotyledon greening and lateral root formation were more severely influenced by salt stress in transgenic lines compared with wild-type plants, indicating that CaMBF1-overexpressing Arabidopsis plants were hypersensitive to salt stress. Conclusions Overexpression of CaMBF1 in Arabidopsis displayed reduced tolerance to cold and high salt stress during seed germination and post-germination stages. CaMBF1 transgenic Arabidopsis may reduce stress tolerance by downregulating stress-responsive genes to aggravate the leaf damage caused by cold stress. CaMBF1 may be useful for genetic engineering of novel pepper cultivars in the future.


Background
Transcriptional regulatory proteins play a central role in the expression of genomic information during complex biological processes in all organisms. Among these proteins, transcriptional co-activators are key components of eukaryotic gene expression by interacting with both transcription factors and/or other regulatory elements and the basal transcription machinery [1,2]. Multiprotein bridging factor 1 (MBF1), a transcriptional co-activator, enhances transcription of its target genes by bridging the general factor TBP (TATA box Binding Protein) and specific transcription factors bound to their target promoters in eukaryotes such as yeast [3], Drosophila [4] and Arabidopsis [5].
MBF1-type genes (SlER24 and StMBF1) encode functional transcriptional co-activators as demonstrated by their capacity to complement the yeast mbf1 mutant [6,7]. Fusion of tomato SlER24 to EAR (Amphiphilic Repression) in the MicroTom cultivar induced a delay of seed germination, but had no obvious effect on plant growth [6]. Moreover, it was reported that the StMBF1 gene in potato was induced by pathogen attack, oxidative stress, wounding and in response to salicylic acid (SA) treatment [7,8]. Direct evidence of the involvement of MBF1 in plant responses to environmental stresses was obtained by enhancing tolerance to heat and osmotic stresses in transgenic Arabidopsis lines expressing the AtMBF1c gene and more recently AtMBF1a, without growth retardation [9,10]. These data indicate that MBF1-like genes can be associated with a variety of developmental processes in plants such as environmental stress tolerance. To date, there are very few data on the significance of MBF1 in cold stress tolerance.
Pepper (Capsicum annuum L.) is a member of the Solanaceae family, and an important vegetable and spice crop valued for its aroma, taste, pungency and flavor. The pepper fruit is the second most consumed vegetable around the world [11]. Different types of peppers, including chili, mild and sweet peppers are cultivated worldwide. Low temperature is one of the most important abiotic factors limiting the growth, development and geographical distribution of plants [12]. Pepper plants originate from tropical regions and are very sensitive to low temperature, which affects their vegetative development and reproduction, resulting in economic losses [13][14][15]. As part of production and fruit quality improvement, we are interested in investigating plant defense mechanisms to improve resistance to environmental stresses. In our previous report, we showed that exogenous application of ABA increased the tolerance of pepper seedlings to chilling-induced oxidative damage, mainly by enhancing the activity of antioxidant enzymes and expression of related genes [16]. Furthermore, ABAmediated candidate genes associated with chilling stress have been fully characterized in pepper plants using a suppression subtractive hybridization (SSH) method [17]. One of the genes cloned from the reverse subtraction was homologous to Solanum tuberosum MBF1 (StMBF1) encoding the coactivator multiprotein bridging factor 1. Expression of this MBF1 homologue was highly induced by cold stress, whereas ABA-pretreatment decreased its expression in pepper seedlings subjected to cold stress. However, the function of this gene involved in the defense response to chilling stress remains to be elucidated.
In this study, based on the above-mentioned expressed sequence tag (EST) from the reverse SSH library that enriched the up-regulated expressed genes responding to chilling stress, we have functionally characterized the homolog of StMBF1 in pepper (designated as CaMBF1). The results of this study suggest that CaMBF1 transcript in pepper seedlings can be suppressed by SA, salt, osmotic and heavy metal stresses. Overexpression of CaMBF1 in Arabidopsis displayed reduced tolerance to cold and high salt stress.

Results
Isolation of the CaMBF1 cDNA clone and sequence analysis A differential screening of a cold-related pepper seedling cDNA library, using PCR-amplified subtracted and control probes, was performed previously [17]. One of the isolated clones exhibited 80% identity at the nucleotide level to StMBF1 from Solanum tuberosum [8]. A full-length clone of this homologue was obtained by a homology-based candidate gene method, including the complete open reading frame. The gene was named CaMBF1 and submitted to GenBank with the Accession Number JX402927. The size of the CaMBF1 clone was 648 bp, comprising an open reading frame of 420 bp (139 amino acids). The predicted polypeptide was basic, with a pI of 9.86 and a molecular mass of 15.3 kDa. An alignment of the deduced amino acid sequence of CaMBF1 with other homologous sequences is presented in Figure 1. At the amino acid level, CaMBF1 showed a high degree of conservation with known genes of other plant species: Solanum tuberosum (StMBF1, 95% identity) [8], and Arabidopsis thaliana (AtMBF1b, 80% identity; AtMBF1a, 79% identity) [10].
Expression of CaMBF1 in pepper seedlings is severely suppressed by stress and SA treatments A number of MBF1 genes were found to be differentially induced by abiotic stress [10,18,19]. Therefore, we suspected that the CaMBF1 gene may be involved in stress signaling pathways and were interested in its possible function in stress responses. As a first step toward functional analysis, we examined the expression pattern of CaMBF1 in pepper plants using qRT-PCR analysis. This analysis revealed that the CaMBF1 gene was expressed ubiquitously in all developmental stages of plants and in all tested organs, including root, stem, leaf, flower, fruit and seed ( Figure 2). High-level expression was detected in flower and seed, although expression level in root was rather low. As shown in Figure 3, CaMBF1 expression was dramatically decreased by several stress conditions, including 5 mM SA, high salt (300 mM NaCl), osmotic stress (300 mM mannitol), and heavy metal (300 μM Hg). Rapid and robust down-regulation of CaMBF1 transcript was observed at 1 h after salt, osmotic and heavy metal treatments, which decreased to 0.06-fold, 0.03-fold and 0.12-fold, respectively. In contrast, a slight reduction of CaMBF1 transcript was found during 12 h of SA treatment and followed by an increase to the initial level ( Figure 3A).

Reduced tolerance of CaMBF1-overexpressing Arabidopsis plants to cold stress
To test the function of CaMBF1 in Arabidopsis, we generated transgenic plants that constitutively expressed CaMBF1 under the control of the CaMV 35S promoter. Transgenic plants expressing CaMBF1 appeared similar in their growth and development to WT plants. However, as shown in Figure 4, the transgenic plants were larger than the WT plants during the florescence production period; the rosette leaves of transgenic plants were 70% longer and 60% wider than those of WT plants.
To study the response of CaMBF1-expressing plants to abiotic stress, 2-week-old WT and transgenic seedlings were subjected to several stresses, including cold, salinity, and ABA. Firstly, transcript levels of the high homology (AtMBF1a, AtMBF1b or AtMBF1c) modulated by the overexpression of CaMBF1 under normal conditions were determined by qRT-PCR. Compared to WT plants, the expression of the homologous genes was not basically altered in transgenic plants when grown in normal condition ( Figure 5), indicating that overexpression of pepper CaMBF1 gene has no obvious effect on AtMBF1s transcripts in Arabidopsis. The CaMBF1 gene was not detected in WT plants. CaMBF1 transcript in transgenic plants subjected to cold stress, salinity, and ABA was much lower than that detected in transgenic plants under normal conditions ( Figure 6), suggesting that expression of CaMBF1 in Arabidopsis was dramatically decreased by stress treatments such as cold, salinity, and ABA. Furthermore, the visible symptoms of leaf damage in transgenic seedlings were observed to examine the tolerance of CaMBF1-expressing plants to cold stress. As shown in Figure 7, overexpression of the pepper CaMBF1 gene in Arabidopsis aggravated the visible symptoms of leaf damage caused by cold stress in seedlings. Wilting appeared after 6 h of cold stress in transgenic plants and became serious at 24 h, while control leaves only exhibited withering after 48 h of cold stress. Meanwhile, to evaluate the extent of cell damage caused by cold stress in CaMBF1-expressing seedlings, electrolyte leakage was measured. The transgenic plants presented 1.5 folds higher electrolyte leakage than WT, which suggests that the membrane is likely to be impaired in these seedlings subjected to cold stress ( Figure 8). These results suggested that overexpression of CaMBF1 in Arabidopsis could downregulate the expression of genes involved in stress tolerance.
We selected a group of candidate genes and conducted qRT-PCR analysis to test this hypothesis ( Figure 9). Earlier studies have found RD29A, RD22, RAB18, KIN1 and ERD15 to be involved in the response to dehydration and cold/ABA [20][21][22][23]. Compared with normal conditions, cold stress induced RD29A, ERD15 and KIN1 genes expression in both transgenic and WT plants ( Figure 9A, D and E). After cold treatment, the expression of RD29A, ERD15 (except at 48 h) and KIN1 in the transgenic plants was lower than that in the WT plants. Meanwhile, RAB18  and RD22, consistent with the results from leaf chilling injury assays and electrolyte leakage measurement.
The CaMBF1-overexpressing Arabidopsis is hypersensitive to salt stress To further characterize the tolerance of CaMBF1-overexpressing plants to salinity, transgenic seeds were germinated in MS/2 media supplemented with 100 mM NaCl and allowed to grow for 8 days. Transgenic seeds exhibited hypersensitivity to salinity compared with WT seeds ( Figure 10A). On medium containing 100 mM NaCl, 78% of WT seeds germinated within 2 d, whereas the germination percentage for transgenic seeds was only 12% during the same period. In addition, the germination and subsequent growth of transgenic seedlings were comparable to WT plants on normal medium, but were significantly more inhibited by salt stress (Figure 10). The cotyledons of 6-day-old transgenic lines were bleached 7 days after transfer to medium containing 150 mM NaCl and became serious at 9 days, whereas the cotyledons of WT plants were slightly affected ( Figure 10B). On the other hand, the primary root growth of transgenic plants was similar to that of WT plants under salt stress. However, lateral root formation was more severely influenced by salinity in transgenic plants compared with WT plants ( Figure 10B). Similarly, comparative expression analyses of the stress gene markers described above were also performed by qRT-PCR on RNA isolated from 2-week-old plants grown under non-stress and salt stress conditions ( Figure 11). Upon salinity treatment, several gene markers (RD29A, RAB18 and KIN1) were highly induced in both WT and transgenic seedlings ( Figure 11A, B and E). Conversely, RD22 and ERD15 transcripts were dramatically decreased in both transgenic and WT plants subjected to salt stress ( Figure 11C and D). Furthermore, the expression of RD29A, RAB18, KIN1 and ERD15 in the transgenic lines was higher than that in the WT plants under high salt conditions. Therefore, overexpression of CaMBF1 in Arabidopsis appeared to positively regulate the expression of stress-responsive gene markers such as RD29A, RAB18, KIN1 and ERD15, which was not consistent with the results from seed germination and cotyledon greening assays. In some cases, the level of stress gene expression appears to be insufficient to induce tolerance changes [24][25][26].
Altered expression of stress-responsive HSPs in the CaMBF1-overexpressing Arabidopsis To evaluate whether CaMBF1 expression could be correlated with alterations of other stress-responsive genes, classical heat-shock genes, HSP70 and HSP90 were tested in all lines by qRT-PCR ( Figure 12). Compared with control plants, HSP70 and HSP90 transcripts (except at 0 h) were decreased in transgenic plants under normal conditions. After cold treatment, the expression of HSP70 and HSP90 in the transgenic plants was lower than that in the WT plants ( Figure 12A and B); whereas, the expression of these genes in the transgenic lines was higher than that in the WT plants under high salt conditions ( Figure 12C and D), indicating that comparative regulation of HSPs in response to CaMBF1 overexpression could be related to different stresses.

Discussion
Here, we report a putative transcription coactivator from pepper seedlings, the putative amino acid sequence of which was 95% and 80% identical to those of StMBF1 and AtMBF1b, respectively. Therefore, CaMBF1 could be categorized as belonging to the same group as StMBF1 [8]. The deduced amino acid sequences of plant MBF1s revealed the existence of highly conserved amino acid residues in each group [19]. Additionally, tissue-specific expression of CaMBF1 observed here ( Figure 2) suggests that CaMBF1 may be involved in physiological processes of pepper plants. In this regard, the highly homologous StMBF1 also exhibits a ubiquitous tissue distribution [8].
In the present study, CaMBF1 transcript in pepper or Arabidopsis seedlings was dramatically decreased in response to abiotic stresses such as SA, ABA, high salt, osmotic, and heavy metal stress (Figures 3 and 6). Particularly, under cold stress the expression of CaMBF1 was downregulated in Arabidopsis seedlings ( Figure 6). These results indicated that CaMBF1 may be negatively involved in stress signaling pathways. Unlike other MBF1 genes, the expression of AtMBF1c is induced by various stresses, including salinity, drought, heat, H 2 O 2 and ABA, and is not affected by cold stress [19]. Salinity also induced AtMBF1a/b expression [10] and cold stress did not significantly change mRNA accumulation of AtMBF1a and AtMBF1b in Arabidopsis [19].
CaMBF1-overexpressing plants showed extremely large leaf phenotypes (Figure 4). This finding could be explained by similar evidence reported by Tojo et al. [27] who suggested that AtMBF1s play a crucial role in controlling rapid leaf expansion through promotion of cell expansion. The amino acid sequences of MBF1s are widely conserved among plant species. Similarly, transgenic Arabidopsis expressing AtMBF1c were 20% larger than control plants and produced more seeds [9].
The visible symptoms of leaf damage in CaMBF1expressing transgenic Arabidopsis were observed more severely than that in WT plants (Figure 7) and the transgenic plants presented 1.5 folds higher electrolyte leakage than  WT under cold stress (Figure 8), suggesting that the tolerance of transgenic plants to cold stress was reduced. This result was in agreement with the fact that some genes isolated from the reverse SSH library, including a MBF1 homologue, were related to reduction in cold tolerance of plants [17]. Moreover, overexpression of the CaMBF1 gene in Arabidopsis reduced the expression of RD29A, ERD15, KIN1, and RD22 during cold treatment (Figure 9). CaMBF1 may reduce the tolerance of Arabidopsis to cold stress by negatively regulating stress-tolerant gene expression. Suzuki et al. [9] reported that the tolerance of MBF1cexpressing transgenic seedlings to cold stress was similar to that of WT seedlings. On the other hand, CaMBF1expressing transgenic plants showed high susceptibility to salt stress imposed during seed germination ( Figure 10A). In contrast to this result, the triple knock-down mutant (abc-) presented a significant diminution of germination under osmotic stress [28] and MBF1 genes negatively regulated ABA-dependent inhibition of germination [29]. The cotyledons and lateral root formation were more severely influenced by salinity in transgenic plants compared with WT plants ( Figure 10B). Meanwhile, root growth of MBF1a/c-expressing plants adopted to the high or lowsalt condition comparatively better than WT plants [9,10]. Seed germination is controlled by the antagonistic action of gibberellic acid (GA) or ethylene and ABA [30][31][32]. MBF1 may be involved in several hormone signal transduction pathways (ethylene, GA/ABA) during seed germination [6,33]. In addition, the expression of RD29A, RAB18, KIN1 and ERD15 in CaMBF1-expressing transgenic Arabidopsis was higher than that in WT plants under high salt conditions ( Figure 11). Kim et al. [10] also reported that MBF1a-overexpressing transgenic Arabidopsis induced RD29A, ERD15, and KIN2 during the course of salt treatment. The accumulation of a number of defense transcripts was similarly augmented in MBF1c transgenic Arabidopsis in response to heat stress [9]. The expression patterns of the above-mentioned stress gene markers in transgenic plants subjected to cold stress were different from those in transgenic lines under salt stress. This difference could be related to that each stress opens out specific defense mechanisms in young seedlings and the participation of CaMBF1 might be different depending on the stress condition imposed. Since different stresses may disrupt plant growth and development in specific ways, the plant might alleviate damage by different mechanisms. The results of this study, that overexpression of the pepper CaMBF1 gene differently modules the expression of HSPs in Arabidopsis under cold and salt stresses (Figure 12), supported this hypothesis. There were similar reports as follows: constitutive expression of stress-responsive HSP genes was augmented in the abc-mutant, indicating that AtMBF1s may act as negative regulators of HSP in Arabidopsis thaliana seedlings [28]. Suzuki et al. [9] described that transcripts encoding classical HSPs accumulated to a similar level in WT and transgenic plants over-expressing MBF1c; they suggested that the enhanced tolerance of A B Figure 10 Analysis of 35S::CaMBF1 transgenic lines subjected to salt stress. A, Effects of salinity on germination. Complete radicle emergence was used as a marker for germination. 50 seeds were counted at indicated days, and Data represent means standard deviation of three independent experiments. B, Post-germination assay of transgenic seedlings. 6-day-old seedlings were transferred to half-strength Murashige and Skoog (MS/2) medium without (right panel) or with (left panel) 150 mM NaCl. Photographs were taken at 7 d or 9 d after the transfer. these plants to osmotic and heat-shock stress was associated with the expression of other stress-responsive genes rather than with the constitutive expression of HSPs. Finally, our data together with previous evidences support that Capsicum annum CaMBF1 play a different role as Arabidopsis AtMBF1 in response to salt or cold stress. Further studies will be necessary to reveal specific functions for each gene.

Conclusions
This study demonstrates that the manipulation of the CaMBF1 gene from pepper using a transgenic approach can lead to reduced cold-stress and salt-stress tolerance in Arabidopsis. In addition, overexpression of CaMBF1 may reduce stress tolerance by downregulating stressresponsive genes to aggravate the leaf damage caused by cold stress. However, upregulation of such stressresponsive genes appears to be insufficient to induce tolerance of CaMBF1 transgenic plants to salt stress. The CaMBF1 gene could be a candidate gene for future research on abiotic stress signaling pathways and genetic engineering of novel pepper cultivars. The results of this study will be helpful in providing beneficial information to support biotechnology applications and molecular breeding, which clarify the function of a gene involved in abiotic stress in plants.

Plant materials and stress treatments
Pepper (Capsicum annuum L.) cv. P70 seeds were sown at a depth of 1.0 cm into 9-cm-deep plastic pots filled with growth medium consisting of grass charcoal and perlite in a ratio of 3:1 after accelerated germination and grown in a growth chamber using a previously described method [16]. The seedlings at the sixth leaf expansion stage were used to establish the following treatments. ABA and cold treatments were performed as described by Guo et al. [17]. For ABA and cold treatments, seedlings were sprayed with freshly prepared 0.57 mM ABA solution or water (control). At 72 h after foliar application, control and ABA treatment groups were subjected to chilling stress at 6°C. For salt, osmotic, and the heavy metal (Hg) treatments, the seedling roots were immersed in solutions containing 300 mM sodium chloride (NaCl), 300 mM mannitol, or 300 μM Hg and maintained at 25°C for the indicated times. For SA treatment, seedlings were sprayed with 5 mM SA solution and incubated for the indicated times. The treated seedlings were harvested after 0, 1, 3, 6, 12 and 24 h for examination of CaMBF1 expression pattern under various stress conditions. At each time point, two or three upper young leaves from four separate seedlings were collected to form one sample, wrapped with foil, immediately frozen in liquid nitrogen and stored at −80°C. The treatments were arranged in a randomized complete block design with three replicates.

Isolation of CaMBF1 cDNA clone and sequence analysis
The MBF1-homologous EST (GenBank No: JZ198811) characterized from the differential screening of a coldrelated pepper seedling cDNA library was reported by Guo et al. [17]. The full-length open reading frame of the MBF1 homologue was obtained using the cDNA fragment of this homolog as a probe by a homology-based candidate gene method [34]. The full-length forward and reverse primers for CaMBF1 were 5′-GAAGAAAAAAA GCAATGAGTGG-3′ and 5′-GCAGAAACGAATTTA G-GATTTG-3′ respectively. The theoretical molecular weight (Mw) and isoelectric point (pI) were calculated with the ExPASy compute pI/Mw tool [35]. Sequence data were analyzed using Clustal W [36]. Homology searches in database were carried out using the default parameters of the BLAST program on the website http://www.ncbi.nlm.nih.gov:blast [37].

Generation of CaMBF1 transgenic Arabidopsis plants
Full-length forward and reverse primers with an added BamHI site were used to generate a DNA fragment encoding the CaMBF1 gene. The CaMBF1 fragment was inserted into the cloning site of the pMD19 T-vector (Takara, Tokyo, Japan) and then this plasmid DNA was digested using XbaI and BamHI from the pMD19 T-vector. The CaMBF1 DNA fragment was inserted into the XbaI-BamHI site of the pVBG2307 vector under the control of the 35S cauliflower mosaic virus (CaMV) promoter, resulting in the pVBG2307-CaMBF1 construct. The pVBG2307 vector was constructed according to pCAMBIA2300 vector [38]. This construct was confirmed by sequencing and then introduced into Agrobacterium tumefaciens GV3101 using electroporation. Arabidopsis (ecotype Columbia-0, Col-0), chosen for transgenic studies, was grown in a controlled environment chamber at 22°C, with a 14/10 h photoperiod, a light intensity of 120 mmol m −2 s −1 , and 70% relative humidity. Transgenic plants were generated by Agrobacterium-mediated transformation using the floral dip method [39]. CaMBF1-overexpressing transgenic seedlings were confirmed by examining the segregation ratio of the kanamycin selectable marker and by PCR analysis of NPTII and CaMBF1 using the primers NPTII-F/R and CaMBF1-F/R (Additional file 1: Table S1). T2 lines that produced 100% kanamycin -resistant plants in the T3 generation were considered as homozygous transformants.
In each experiment, T2 generations of homozygous transgenic lines (#5, #12 and #21) were selected for further analysis. Similar phenotypes and results used for this study were observed in more than three independent lines of transgenic plants.

Performance of transgenic lines under stress treatments
Two-week-old transgenic seedlings were subjected to various treatments. Cold treatment was conducted in the dark by exposure of plants grown on vermiculite soil at 22°C to 4°C for 48 h, whereas control plants were placed in the dark at 22°C for 48 h. After cold treatment, wild-type (WT) and CaMBF1-overexpressing transgenic plants were visually examined to determine the extent of chilling damage. For high-salinity and ABA treatments, 2-weekold seedlings were submerged in half-strength Murashige and Skoog (MS/2) medium containing 150 mM NaCl or 100 μM ABA solutions, whereas control plants were submerged in a MS/2 medium. Third-fourth rosette leaves were collected from both stress-treated and control plants after 0, 2, 6, 24, and 48 h of cold, salt stress or ABA treatments. At each time point, sample was frozen in liquid nitrogen, stored at −80°C and used for extraction of total RNA. The treatments were arranged in a randomized complete block design with three replicates. Homozygous T2 seeds of the transgenic lines were used for phenotypic analysis. For high-salinity treatment, seeds of WT and transgenic plants were plated on MS/2 agar plates supplemented with 100 mM NaCl, grown in a growth chamber, and assessed for percentage of germination after various times (0, 2, 4, 6 and 8 d). Experiments were done in triplicate for each line (50 seeds each). 6-day-old plants grown on normal MS/2 agar plates were transferred to vertical MS/2 agar plates containing 150 mM NaCl, and grown for another week as previously described [40]. The root growth and cotyledon greening of 24 seedlings were observed.

Measurement of electrolyte leakage
Leaflets from 2-week-old seedlings were transferred to 4°C and incubated for 24 h in the dark in the growth chamber. The conductivity of the suspending solution was measured according to the method of Arce et al. [28]. The electrical conductivity of the solution was measured using an electrical conductivity analyzer (DDS-307; Shanghai Precision Scientific Instrument Co., Ltd., China) before and after autoclaving at 120°C for 30 min to release the total electrolytes. The conductivity was scored at least for 4 plants per line and pretreatment. Electrolyte leakage was expressed as a percentage of total electrolytes.