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AgMYB5, an MYB transcription factor from celery, enhanced β-carotene synthesis and promoted drought tolerance in transgenic Arabidopsis

BMC Plant Biology volume 23, Article number: 151 (2023) Cite this article

Abstract

Background

Water shortage caused by global warming seriously affects the yield and quality of vegetable crops. β-carotene, the lipid-soluble natural product with important pharmacological value, is abundant in celery. Transcription factor MYB family extensively disperses in plants and plays regulatory roles in carotenoid metabolism and water scarcity response.

Results

Here, the AgMYB5 gene encoding 196 amino acids was amplified from celery cv. ‘Jinnanshiqin’. In celery, the expression of AgMYB5 exhibited transactivation activity, tissue specificity, and drought-condition responsiveness. Further analysis proved that ectopic expression of AgMYB5 increased β-carotene content and promoted drought tolerance in transgenic Arabidopsis thaliana. Moreover, AgMYB5 expression promoted β-carotene biosynthesis by triggering the expression of AtCRTISO and AtLCYB, which in turn increased antioxidant enzyme activities, and led to the decreased contents of H2O2 and MDA, and the inhibition of O2− generation. Meanwhile, β-carotene accumulation promoted endogenous ABA biosynthesis of transgenic Arabidopsis, which resulted in ABA-induced stomatal closing and delayed water loss. In addition, ectopic expression of AgMYB5 increased expression levels of AtERD1, AtP5CS1, AtRD22, and AtRD29.

Conclusions

The findings indicated that AgMYB5 up-regulated β-carotene biosynthesis and drought tolerance of Arabidopsis.

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Introduction

Celery (Apium graveolens L.) is one of the world's most important vegetable crops in the Apiaceae family [1]. Traditionally, celery leaf blades and petioles have long been appreciated for their nutritional and pharmacological properties [2, 3]. In contrast to other vegetables, celery is rich in a range of nutrients, including carotenoids, ascorbic acid, apigenin, and flavonoids [4, 5]. At the same time, celery contains a vast array of secondary metabolites with properties of anti-oxidative damage, anti-inflammation, and anti-diabetes. Due to global warming and irregular rainfall, both celery production and quality are negatively affected by drought stress [6].

Transcription factors (TFs) have attracted attention because of their vital role as upstream regulators of structural genes. MYBs [7], NACs [8], bHLHs [9], and WRKYs [10] have been proven the potential capacity of regulating plant drought tolerance. Moreover, by interacting directly with abscisic acid (ABA), TFs played positive regulatory roles in the development and drought stress of plants. In addition, TFs from Vitis vinifera [11], Oryza sativa [12], and Zea mays [13], were found to be the negative regulators of plant drought tolerance.

In plants, MYBs are composed of one of the most important families of TFs [14]. As well as regulating organ development, MYBs widely participate in tissue formation and is important for response mechanisms to multiple environmental stresses [15]. In recent years, MYBs from Triticum aestivum [16], Sesamum indicum [17], and Lycopersicon esculentum [18], have been proven the function of regulating drought tolerance. There is evidence that MYBs respond to drought stress by coordinating the stomatal opening and closing [19], influencing flower development [20], regulating phytohormone signal transduction [21], and affecting cuticular wax accumulation [22].

In addition, MYBs act as key regulators of promoting and repressing carotenoid metabolism in plants [23]. As the major source of retinol in humans, carotenoids have the efficacies of anti-oxidative, anti-cancer, immunomodulatory, and delaying senescence. However, the human body is unable to synthesize carotenoids, therefore, we must obtain them through plant-based foods. β-carotene, belonging to the branch of carotenoids, contributes to the treatment of eye diseases, cardiovascular disorders, and neoplasms [24]. In plants, β-carotene is biosynthesized in plastids through the metabolism of terpenoids and polyketides, and mediated by various enzymes, including phytoene desaturase (PDS), ζ-carotene desaturase (ZDS), carotenoid isomerase (CRTISO), and lycopene β-cyclase (LCYB) [25]. Recent studies suggested that in Vitis vinifera [26], Capsicum annuum [27], and Ulva prolifera [28], overexpression of MYBs promoted or repressed β-carotene accumulation. However, the regulatory role of MYBs in abiotic resistance and carotenoid accumulation in celery still lacks a thorough understanding.

To obtain new insights into the biological function of the MYB family in celery, based on our published celery genome information [2, 4] and the transcriptomic data of celery under drought stress [29, 30], the gene encoding the transcription factor AgMYB5 was amplified from celery cultivar ‘Jinnanshiqin’ and heterologously expressed in A. thaliana. Physiological traits of wild type (WT) and transgenic Arabidopsis, including the contents of total carotenoid, β-carotene, malondialdehyde (MDA), hydrogen peroxide (H2O2), and superoxide anion-radicals (O2−), as well as the activities of superoxide dismutase (SOD), peroxide (POD), catalase (CAT), were detected and analyzed. Moreover, water loss rate, ABA content, stomatal aperture, and relative expression profiles of drought-stress-responsive genes were also measured and analyzed. Our work demonstrated that AgMYB5 played potentially vital roles in β-carotene accumulation and drought tolerance of plants.

Materials and methods

Plant material, culture conditions, and abiotic stress treatment

The seeds of celery (A. graveolens L., cv. ‘Jinnanshiqin’) and Arabidopsis thaliana (‘Columbia-0’) were kindly provided by the State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University (32°04′N, 118°85′E). Celery was incubated in the plant cultivation laboratory of Yancheng Teachers University (33°38’N, 120°20’E). The culturing temperature and day length was set at 24 °C for 16 h of day-time and 18 °C for 8 h of night-time. To gain insights into the expression pattern of AgMYB5 in celery under abiotic stress, two-month-old healthy celery plants were cultured under four kinds of abiotic stress conditions, heat (38 °C treatment), cold (-7 °C treatment), salt (200 mM NaCl treatment), and drought (200 g/L PEG 6000 treatment) stress. Celery samples were collected at 6 h, 12 h, and 24 h after stress treatments, and quickly frozen in liquid nitrogen.

A. thaliana (ecotype ‘Columbia-0’) was used for AgMYB5 transformation. In the artificial climate chamber, climatic conditions were 24 °C, 70% relative humidity, and long photoperiod of 15 / 9 (light/dark). Here, WT and AgMYB5 transgenic Arabidopsis seeds were sterilized with 75% ethanol and 10% sodium hypochlorite, germinated on Murashige and Skoog (MS) solid medium, transferred to soil in pots (nine seedlings per pot) and grown under normal conditions for three weeks. One week later, all Arabidopsis plants were treated with drought stress (200 g/L PEG 6000 treatment) for 7 d. Then, A. thaliana growth conditions were observed and analyzed, while its physiological and molecular indexes were assayed.

AgMYB5 amplification and bioinformatic analysis

Total RNA of celery and A. thaliana was extracted using CTAB isolating buffer (Catalog No. R21004, purchased from Shanghai Yuanye Biotechnology Co., Ltd), and cDNA was synthesized with EasyScript® Reverse Transcriptase (Catalog No. AE101-02, purchased from Beijing Transgene Biotechnology Co., Ltd). In PCR amplifications with AgMYB5-specific primers, cDNA was used as a template. In addition, the upstream and downstream primers were designed with DNAMAN 9.0 and synthesized by Nanjing GenScript Biotechnology Co., Ltd. Nanjing GenScript Biotechnology Co., Ltd. also undertook the task of PCR product sequencing.

ExPASy Translate Tool (https://web.expasy.org/translate/) was used to translate nucleic acid sequences of AgMYB5 into amino acid sequences. ExPASy-Protparam tool (https://web.expasy.org/protparam/) was used to calculate the physiochemical properties of AgMYB5. NCBI BLAST tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was used for the search of MYB5 in the other plant species. The secondary and tertiary structure prediction of AgMYB5 was conducted by SOPMA (https://npsa-prabi.ibcp.fr/) and Swiss-Model (http://swissmodel.Expasy.org/), respectively. Multiple amino acid sequence alignment of MYB5s was performed using DNAMAN 9.0, and the NJ phylogenetic tree was built by MEGA 7.0.

A. thaliana transformation and assay for drought tolerance evaluation

The ORF product of AgMYB5 was obtained with primers (Forward primer: 5’-TTTACAATTACCATGGGGAGAAGCCCGTGTTGTTCCA-3’, reverse primer: 5’-GTTTCGACTGATTGCTTCTGCCT-3’) and digested with Bam HI and Sac I. After that, AgMYB5 ORF was introduced into Agrobacterium GV3101 by the pCAMBIA-1301 vector, and the floral dip method [31] was utilized to transform A. thaliana. Transgenic A. thaliana were screened using hygromycin (50 mg/L, w/v) and continuously cultured to obtain homozygous T3 transgenic A. thaliana lines.

The copy number of exogenous AgMYB5 in transgenic Arabidopsis was measured with a previous method [32]. Briefly, genomic DNA was extracted from the leaves of AgMYB5 transgenic Arabidopsis lines and diluted into five-fold. Through qRT-PCR, the Cq values of At4-HPPD and AgMYB5 were detected and the standard curves of At4-HPPD and AgMYB5 were conducted. Then, the copy number of AgMYB5 was calculated according to the formula [33].

In vitro evaluation for drought tolerance of A. thaliana was conducted according to the method [34]. A. thaliana seeds were germinated on solid MS medium supplement with PEG 6000 (w/v, 200 g/L) for 14 d. Afterward, the growth characteristics of these A. thaliana seedlings were observed and analyzed. Besides, in vivo evaluation was determined by the method [35]. Briefly, four-week-old WT and AgMYB5 transgenic A. thaliana seedlings in pots were treated with PEG 6000 solution for 7 d. Then, the stomatal aperture [36] of Arabidopsis abaxial epidermal peels and the water loss rate [37] of Arabidopsis samples were determined.

Determination of total carotenoid and β-carotene

The total carotenoid content was measured according to the acetone-hexane method [38]. Briefly, 2.0 g of A. thaliana samples were frozen and ground in liquid nitrogen. The powder was extracted with an acetone-hexane mixture and cold stood for 10 min. After decanting and running the solution through a 0.45 mm filter, the extract’s absorbance was measured reflecting the total carotenoid content.β-carotene content was determined by the method [39]. The powder, described above, was incubated at 50 °C for 1 h. After filtration with 0.45 μm, 20 μL of the extract was injected into Vanquish Flex system for ultra-high performance liquid chromatography (UPLC) analysis. Acetonitrile and methanol (1:9, v/v) were applied as the eluants with 0.8 mL/min of the flowing rate. After that, the effluent was monitored simultaneously at 450 nm by a diode array detector.

Measurement of ABA, MDA, H2O2, O2 −, and antioxidant enzymes

An assay of MDA content, SOD and POD activities was performed based on the measurement [40]. The generation rate of O2− was measured following the previous method [41]. ABA content was measured with a Plant ABA ELISA kit (Catalog No. H251-1). CAT activity was assayed with a Plant CAT ELISA Kit (Catalog No. A007-1). H2O2 content was measured based on the Plant H2O2 ELISA Kit (Catalog No. A064-1). In addition, these ELISA kits were acquired from Nanjing Jiancheng Biological Engineering Co., Ltd.

Quantitative real-time PCR (qRT-PCR)

In this experiment, A. graveolens gene expression levels were quantified and normalized to AgGAPDH [42], while A. thaliana gene expression in WT and transgenic lines were normalized with AtActin3. AgMYB5, ABA synthesis-related genes (AtABA1, AtNCED6, AtABA2, AtAAO3), β-carotene synthesis-related genes (AtPDS3, AtZDS, AtCRTISO, AtLCYB), and drought response-related genes (AtERD1, AtP5CS1, AtRD22, AtRD29), whose relative expression levels were determined by qRT-PCR (Primer information in Table 1). The qRT-PCR reaction was conducted by TransStart® Top Green qPCR SuperMix (Catalog number: AQ131-01, purchased from Beijing Transgene Biotechnology Co., Ltd.). Besides, the CT method [43] was used to calculate gene expression relative to the references.

Table 1 Nucleotide sequences and characteristics of primers
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Transcriptional activity assay by Y2H

The ORF that encodes AgMYB5 was inserted into the pGBKT7 plasmid and shuttled into the Y2H vector (Catalog number: PG1029, purchased from Wuhan Protein Interaction Biotechnology Co., Ltd.). Meanwhile, Y2H vectors with pCL1 and empty pGBKT7 plasmids were set as positive and negative controls, respectively. Over two days, the solid medium was placed in a stationary position upside down to initiate cultivation at 30 °C. In the YPDA liquid medium, positive clones were cultivated overnight to turbidity. After centrifuging at 5000 rpm for 5 min, the sediment was diluted with ddH2O and inoculated on SD/-His/-Ade solid medium (X-gal).

Statistical analysis

To determine whether there were any significant differences in physiological indexes and gene expression profiles between WT and AgMYB5 transgenic Arabidopsis lines, the data were statistically analyzed with point t-test, one-way and two-way ANOVA and graphed with GraphPad 7.0. A significant difference was defined as *P < 0.05 and **P < 0.01. All plant lines were compared by Tukey’s multiple range test (P < 0.05). In addition, each experiment was repeated three times and included three biological replicates.

Results

Bioinformatic analysis of AgMYB5

Bioinformatic analysis showed that the AgMYB5 gene (NCBI accession No. OP272489) contained 588 nucleotide base pairs and 196 amino acids in the ORF (Fig. 1A), with the predicted molecular weight and theoretical pI value of 22.65 kDa and10.44, respectively. Multiple amino acid sequence alignment revealed a reasonable degree of sequence similarity between AgMYB5 and other MYB5s from A. thaliana (NP_187963.1), Camellia sinensis (QSV39846.1), Dimocarpus longan (QRV61369.1), Lotus corniculatus (QXT50540.1), and Nicotiana tomentosiformis (XP_009601374.1) (Fig. 1B). Among them, AgMYB5 shared the highest similarity with CsMYB5 (32.58%). The NJ phylogenetic tree constructed by MEGA 7.0 also revealed close evolutionary relationships between AgMYB5 and CsMYB5 (Fig. 1C). Besides, protein structure prediction suggested that AgMYB5 protein was composed of the alpha helix (31.63%), extended strand (6.63%), beta-turn (6.12%), and random coil (55.61%) (Fig. 1D).

Fig. 1
figure 1

Sequence analysis of AgMYB5 gene. A Nucleotide sequence and predicted amino acid sequence of AgMYB5. B Multiple amino acid sequence alignment of AgMYB5 (OP272489), AtMYB5 (NP_187963.1), CsMYB5 (QSV39846.1), DlMYB5 (QRV61369.1), LcMYB5 (QXT50540.1), and NtMYB5 (XP_009601374.1). C The NJ phylogenetic tree of AgMYB5, NtMYB5, SlMYB5, OsMYB5, and MeMYB5. D Protein tertiary structure prediction of AgMYB5

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Transcriptional activity assay and function prediction of AgMYB5

The vector of pGBKT7-AgMYB5 was constructed to verify the transcriptional activity of the AgMYB5 gene. As shown in Fig. 2A, on SD/-His/-Ade solid medium, positive control of pCL1 grew normally and appeared blue when incubated in X-gal. As the negative control, pGBKT7 plasmid was found to grow normally on SD/-Trp/-Leu solid medium but was inviable on SD/-His/-Ade solid medium. Moreover, the pGBKT7-AgMYB5 plasmid was found to grow normally on SD/-Trp/-Leu and SD/-His/-Ade solid mediums and appeared blue on SD/-His/-Ade medium containing X-gal. These results demonstrated that the AgMYB5 protein could activate the transcription of reporter genes His3 and Laz, suggesting that AgMYB5 was capable of transcriptional activation.

Fig. 2
figure 2

Bioinformatic analysis and expression characteristics of AgMYB5. A Verification of transcript activation ability by Y2H. B The relative expression level of AgMYB5 in different tissues of ‘Jinnanshiqin’. The asterisk indicates a statistically significant difference (*, P < 0.05; **, P < 0.01). C Conserved domain analysis of AgMYB5. D The relative expression level of AgMYB5 in different tissues of ‘Jinnanshiqin’ under heat (38 °C), cold (-7 °C), salt (200 mM NaCl), and drought (200 g/L PEG 6000) conditions. Significant differences are indicated by different lettered columns (P < 0.05)

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Using qRT-PCR, a comparison of AgMYB5 expression profiles among different tissues was presented in Fig. 2B. AgMYB5 exhibited differentiated expression patterns in various tissues of celery (cv. Jinnanshiqin). Compared with that of the petiole (P < 0.01) and root (P = 0.03), the highest expression of AgMYB5 was obtained in the leaf. Besides, conserved domain analysis revealed that the AgMYB5 gene contained an MYB5 domain (Fig. 2C), which was related to abiotic stress response [44]. Therefore, the qRT-PCR analysis demonstrated that AgMYB5 exhibited specific expression patterns under various abiotic stresses (Fig. 2D). Notably, with drought treatment for 24 h, compared with that of 6 h treatment, the relative expression level of AgMYB5 increased by 4.97 folds in the leaf, 5.56 folds in the petiole, and 58.51 folds in the root, respectively. The above results demonstrated that AgMYB5 was highly responsive to various abiotic stresses, especially drought stress.

Promoted β-carotene biosynthesis of AgMYB5 transgenic Arabidopsis

The contents of total carotenoid and β-carotene of celery (cv. Jinnanshiqin) samples at three developmental stages, including stage 1 (S1, 35 d post-planting), stage 2 (S2, 50 d post-planting), and stage 3 (S3, 65 d post-planting), were assayed. At the same time, the AgMYB5 expression level was also determined. The results showed that total carotenoid content increased from 0.052 mg/g at S1 to 0.625 mg/g at S3, and β-carotene content increased from 0.42 mg/g at S1 to 0.51 mg/g at S3 (Fig. 3A). Meanwhile, the expression profiles of AgMYB5 increased by 4.17 folds (S2) and 10.33 folds (S3) as compared to that of S1 (Fig. 3B). These results implied that AgMYB5 contributed to β-carotene accumulation.

Fig. 3
figure 3

Total carotenoid and β-carotene accumulation and AgMYB5 expression in different developmental stages of ‘Jinnanshiqin’. Three developmental stages of celery included S1 (35 d post-planting), S2 (50 d post-planting), and S3 (65 d post-planting). A The total carotenoid and β-carotene contents and (B) relative expression level of AgMYB5 during different developmental stages. The asterisk indicates a statistically significant difference (*, P < 0.05; **, P < 0.01)

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To further verify the role of AgMYB5 in β-carotene biosynthesis, we obtained homozygous AgMYB5 transgenic Arabidopsis (OE1 and OE4) through self-pollination. The copy number of exogenous AgMYB5 was about one in OE1 and OE4 (Supplementary Figure S1), and qRT-PCR results showed that the expression level of AgMYB5 was slightly higher in the independent transgenic line of OE4 (Fig. 4A). The β-carotene content assay showed that under normal conditions, OE1 and OE4 accumulated slightly more β-carotene than that of WT, and drought stress led to significant accumulation of β-carotene in transgenic Arabidopsis lines (Fig. 4B), especially between the group OE4 vs WT (P < 0.001). Furthermore, the qRT-PCR analysis demonstrated that no significant changes in the expression of AtPDS and AtZDS were detected under normal and drought conditions, while under drought stress, AtCRTISO (P = 0.009) and AtLCYB (P = 0.001) in OE4 showed significantly higher expression levels in comparison with that in WT (Fig. 4C). These results indicated that ectopic expression of AgMYB5 significantly enhanced β-carotene biosynthesis of transgenic A. thaliana by regulating the expression of AtCRTISO and AtLCYB.

Fig. 4
figure 4

Validation of the positive AgMYB5 transgenic Arabidopsis lines. A The transcript levels of AgMYB5 in transgenic Arabidopsis lines (OE1 and OE4). B β-carotene content and (C) relative expression levels of β-carotene biosynthesis-related genes in WT and transgenic Arabidopsis lines (OE1 and OE4). The asterisk indicates a statistically significant difference (*, P < 0.05; **, P < 0.01)

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Enhanced drought tolerance of AgMYB5 transgenic Arabidopsis

To investigate the regulatory roles of AgMYB5 under drought conditions, Arabidopsis seeds were germinated on MS solid medium and MS medium containing PEG 6000 (200 g/L). Under normal conditions, a similar growth phenotype was observed in WT and AgMYB5 transgenic Arabidopsis lines. However, under drought stress, OE1 and OE4 lines presented a better growth situation (Fig. 5A). The data showed that under drought stress, compared to WT, the average root length (Fig. 5B) and fresh weight (Fig. 5C) of OE4 increased by 30.06% (P = 0.004) and 48.85% (P < 0.001), respectively.

Fig. 5
figure 5

Drought tolerance of WT and transgenic Arabidopsis lines (OE1 and OE4) germinated on MS-agar plates. A The Phenotype of WT and transgenic lines under normal and drought conditions. B Average Root length and (C) fresh weight of WT and transgenic lines under normal and drought conditions. The asterisk indicates a statistically significant difference (*, P < 0.05; **, P < 0.01)

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In vitro drought tolerance evaluation showed that four-week-old WT and AgMYB5 transgenic A. thaliana seedlings in pots (nine plants with the same age per pot) displayed similar developmental patterns. Meanwhile, under drought stress, all WT seedlings wilted and lost water significantly, while AgMYB5 transgenic lines displayed mild wilting. With the extension of the PEG 6000 treatment, the water loss rate of transgenic line OE4 was the lowest, while that of WT was the highest (Fig. 6A). After drought treatment, transgenic line OE1 and OE4 had a significantly higher survival rate, which increased by 2.03-fold (OE1) and 2.66-fold (OE4) than WT, respectively (Fig. 6B). Because stomatal closure may be the first response of plants to drought stress, as well as observing the state of stomata, we also measured the stomata aperture of WT and transgenic lines by ImageJ software (Fig. 6C). Under normal conditions, the stomata aperture of transgenic line OE4 was slightly lower as compared to that of WT and transgenic line OE1. Under drought stress, all Arabidopsis plants exhibited decreased stomata aperture in varying degrees. Among these, the stomata aperture of OE4 was the lowest (0.74 μm), followed by OE1 (1.12 μm) and then WT (1.44 μm) (Fig. 6D). These results demonstrated that AgMYB5 expression significantly enhanced drought tolerance of transgenic A. thaliana by inducing stomatal closure.

Fig. 6
figure 6

Drought tolerance of WT and transgenic Arabidopsis lines planted in soil. A The water loss rate (percent water loss per hour) and (B) the survival rate of WT and transgenic lines (OE1 and OE4). C The extent of stomatal closure and (D) stomata aperture of WT and transgenic lines under normal and drought conditions. The asterisk indicates a statistically significant difference (*, P < 0.05; **, P < 0.01)

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Promoted ABA biosynthesis of AgMYB5 transgenic Arabidopsis

Since ABA is derived from β-carotene and can cause stomatal closure, a measurement of endogenous ABA content in Arabidopsis was conducted. Under normal conditions, no significant differences in endogenous ABA content were assayable between WT and AgMYB5 transgenic A. thaliana lines (OE1 and OE4). However, under drought stress, endogenous ABA content of OE1 and OE4 increased by 3.05 folds and 2.91 folds, respectively, whereas that of WT increased by only 1.61 folds (Fig. 7A). The qRT-PCR analysis further examined the expression of ABA synthesis-related genes, including AtABA1, AtNCED6, AtABA2, and AtAAO3. The data exhibited that in transgenic lines under drought conditions, four genes encoding ABA biosynthesis were up-regulated. Particularly AtNCED6, encoding the rate-limiting enzyme in ABA biosynthesis, whose expression in transgenic line OE4 was 2.71 times greater than that in WT (Fig. 7B). These findings indicated that the expression of AgMYB5 in transgenic A. thaliana activated the endogenous ABA synthesis pathway.

Fig. 7
figure 7

Effects of AgMYB5 expression on endogenous ABA biosynthesis pathway. A Endogenous ABA contents and (B) relative expression levels of ABA synthesis-related genes (AtABA1, AtNCED6, AtABA2, and AtAAO3) of WT and transgenic lines under normal and drought conditions. The asterisk indicates a statistically significant difference (*, P < 0.05; **, P < 0.01)

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Protected ROS scavenging system of AgMYB5 transgenic Arabidopsis

Under drought stress, plants respond to excessive accumulation of ROS by promoting antioxidant enzyme activities and protecting the ROS scavenging system [45]. Thus, we analyzed the antioxidative enzymatic activities and ROS contents of A. thaliana under normal and drought conditions. Compared to WT, AgMYB5 transgenic A. thaliana showed increased SOD, POD, and CAT activities by 152.17% (OE1) and 169.57% (OE4), 48.65% (OE1) and 62.16% (OE4), and 84.02% (OE1) and 116.11% (OE4), respectively (Fig. 8A). At the same time, comparing AgMYB5 transgenic lines with WT, we observed decreases in MDA content, H2O2 content, and O2− generation of 26.32% (OE1) and 34.21% (OE4), 24.02% (OE1) and 36.27% (OE4), and 21.05% (OE1) and 31.58% (OE4) (Fig. 8B). These results demonstrated that AgMYB5 expression in transgenic A. thaliana significantly promoted antioxidant enzymatic activities and inhibited the accumulation of ROS induced by drought stress.

Fig. 8
figure 8

Antioxidant capacity of WT and transgenic lines under normal and drought conditions. A Activities of SOD, POD, and CAT enzyme and (B) contents of MDA, H2O2, and O2− generation of WT and transgenic lines (OE1 and OE4). The asterisk indicates a statistically significant difference (*, P < 0.05; **, P < 0.01)

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Up-regulated drought-responsive genes of AgMYB5 transgenic Arabidopsis

Four drought-responsive genes, including AtERD1, AtP5CS1, AtRD22, and AtRD29, were screened out to determine the effects of AgMYB5 ectopic expression on transgenic A. thaliana. We observed relatively similar gene expression patterns in WT and transgenic Arabidopsis plants under normal conditions. Under drought conditions, relative expression levels of AtERD1 (Fig. 9A), AtP5CS1 (Fig. 9B), AtRD22 (Fig. 9C), and AtRD29 (Fig. 9D) were significantly higher in transgenic lines (especially OE4) than that in WT. The data of AtERD1 was chosen as an example to illustrate the expression pattern. Under drought stress, compared to that of WT, the relative expression level of AtERD1 increased by 1.79-fold (P < 0.0001) in OE1 and 2.11-fold (P < 0.0001) in OE4. These results indicated that AgMYB5 enhanced drought tolerance of transgenic Arabidopsis by up-regulating the expression profiles of drought-response genes.

Fig. 9
figure 9

Relative expression levels of drought-response genes, including (A) AtERD1, (B) AtP5CS1, (C) AtRD22, and (D) AtRD29, in WT and transgenic lines (OE1 and OE4) under normal and drought conditions. The asterisk indicates a statistically significant difference (*, P < 0.05; **, P < 0.01)

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Discussion

In plants, TFs usually exhibit differential expression patterns in different tissues and under various abiotic stresses. Kundan et al. identified 99 R2R3-MYBs from Cannabis sativa, and these TFs were found to have tissue-specific expression [46]. Here, we found that under various abiotic stress conditions, AgMYB5 showed different expression patterns in celery, as well as tissue specificity. Moreover, under drought stress, AgMYB5 expression was significantly higher in the root, which might be because when soils become dry, roots sense water scarcity earlier than the other tissues and continuously release root-source signals [47]. MYBs, MADSs, NACs, and WRKYs are responsible for carotenoid biosynthesis and degradation [48]. Among them, MYBs played a key role in regulating β-carotene production. In Liriodendron chinense, based on genome-wide association, HPLC, and qRT-PCR, four MYBs were identified to have the function of regulating β-carotene accumulation in petals [49]. In the green mutant of Citrus reticulata, CrMYB68 suppressed α- and β-carotene accumulation by down-regulating BCH2 and NCED5 [50]. RNA-Seq analysis revealed that SlMYB72 affected β-carotene synthesis by regulating the expression of SlPDS, SlZDS, and SlLCYB, while RNA interference with SlMYB72 inhibited lycopene and β-carotene accumulation [51]. In the present study, it was found the changes in AgMYB5 expression and β-carotene content were closely related in celery, and ectopic expression of AgMYB5 in A. thaliana promoted β-carotene accumulation and up-regulated β-carotene synthesis-related genes in a water-deprived environment, which confirmed the involvement of AgMYB5 in β-carotene biosynthesis under drought stress. At the same time, β-carotene, an excellent natural antioxidant, confers stress resistance to plants by scavenging excess ROS [52]. In Dioscorea esculenta, the over-production of β-carotene led to a stronger DPPH radical-scavenging capacity and a higher photosynthetic efficiency [53]. Our results showed that drought stress induced the activities of SOD, POD, and CAT, and increased the contents of MDA, H2O2, and O2− at various levels. Moreover, compared with that in WT, increased antioxidant enzymatic activities and decreased ROS contents were found in transgenic lines, which indicated that AgMYB5 expression protected ROS scavenging system by enhancing β-carotene production.

It has been reported that in the ABA biosynthesis of plants, NCED is the key enzyme, and β-carotene acts as the precursor [54, 55]. In transgenic Oryza sativa, OsZDS overexpression enhanced β-carotene biosynthesis, which also promoted OsNCED1 expression and induced ABA accumulation [56]. In A. thaliana, ABA treatment induced AtD27 (a gene encoding β-carotene isomerase) expression, and the AtD27 mutant exhibited lower ABA content [57]. In the present study, compared to WT, ABA content and expression profiles of ABA biosynthesis-related genes were higher in transgenic lines, revealing that under drought stress, AgMYB5 promoted endogenous ABA biosynthesis by enhancing β-carotene accumulation. In drought-stressed plants, ABA is a major phytohormone involved in regulating stomatal aperture [58]. According to the reports from Populus euphratica [59], Oryza sativa [60] and Solanum lycopersicum [61], plants increased drought tolerance by modulating stomatal closing. Our results demonstrated that compared to WT, AgMYB5 inhibited water loss by regulating stomatal closure, thereby enhancing drought tolerance of transgenic Arabidopsis.

Recently studies have shown that TFs respond to biotic and abiotic stress by cooperating with stress-response genes. In Gossypium hirsutum, GhWRKY17 overexpression significantly reduced drought and salt tolerance by down-regulating the transcript levels of GhERD and GhLEA [62]. Compared to WT, VvNAC17 transgenic Arabidopsis showed stronger drought tolerance and enhanced expression of AtCOR47, AtP5CS, AtRD22, and AtRD29A [63]. Our study found that under drought stress, the expression of AtERD1, AtP5CS1, AtRD22, and AtRD29 were significantly enhanced in AgMYB5 transgenic lines as compared to that in WT, indicating that through activating the expression of drought stress-response genes, AgMYB5 enhanced drought tolerance of transgenic Arabidopsis. Previous reports showed that AcMYB3R, obtained from Actinidia chinensis, activated the expression of AtRD22, AtCOR15A, and AtRD29, which led to enhanced drought tolerance of transgenic Arabidopsis [64]. A similar study also reported that overexpression of TaSIM, an R2R3-MYB gene of Triticum aestivum, boosted the expression of AtRD22 and AtRD29, which revealed the function of TaSIM in the drought response mechanism [65].

In conclusion, we isolated a transcription factor AgMYB5 from the celery cultivar ‘Jinnanshiqin’. AgMYB5 presented the properties of transcriptional auto-activation and tissue specificity under drought stress, as well as a correlation with carotenoid accumulation. In transgenic Arabidopsis, AgMYB5 ectopic expression enhanced β-carotene biosynthesis and increased β-carotene content under drought stress, which in turn protected transgenic Arabidopsis against ROS-mediated peroxidation. Meanwhile, β-carotene accumulation induced ABA biosynthesis, which triggered stomatal closure and reduced water loss of transgenic Arabidopsis under drought stress. In addition, drought-response genes were significantly up-regulated by AgMYB5 ectopic expression (Fig. 10). Interestingly, unlike that under normal conditions, β-carotene and ABA accumulation, antioxidant enzyme activity, and stomatal aperture in transgenic Arabidopsis showed significant differences under drought stress, implying that AgMYB5 was barely expressed under normal conditions but induced by drought stress. Altogether, our findings suggest that AgMYB5 promotes β-carotene accumulation and plays a pivotal role in the drought tolerance of Arabidopsis. However, the anti-drought ability validation of AtMYB5 from A. thaliana is still required, which will give a more powerful reference for MYB5 function investigation. Meanwhile, AtMYB5 function validation will be an excellent subject for us to study the molecular regulation mechanism of MYBs in plant drought resistance.

Fig. 10
figure 10

The potential regulation model of AgMYB5 in β-carotene biosynthesis and drought tolerance of transgenic Arabidopsis

Full size image

Availability of data and materials

AgMYB5 sequence data from ‘Jinnanshiqin’ in this study has been submitted to the NCBI database with accession No. OP272489. Sequence data used in this article can be found in the GenBank database (http://www.ncbi.nlm.nih.gov/Genbank) under the following accession numbers: AtMYB5 (NP_187963.1); CsMYB5 (QSV39846.1); DlMYB5 (QRV61369.1); LcMYB5 (QXT50540.1) and NtMYB5 (XP_009601374.1). The data sets supporting the conclusions of this article are included within the article. Celery (A. graveolens L., cv. ‘Jinnanshiqin’) and Arabidopsis thaliana (‘Columbia-0’) were deposited at the State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University (32°04′N, 118°85′E).

References

  1. Wang XJ, Luo Q, Li T, Meng PH, Pu YT, Liu JX, Zhang JX, Liu H, Tan GF, Xiong AS. Origin, evolution, breeding, and omics of Apiaceae: a family of vegetables and medicinal plants. Hortic Res. 2022;9:uhac076. https://doi.org/10.1093/hr/uhac076.

    Article  Google Scholar 

  2. Li MY, Feng K, Hou XL, Jiang Q, Xu ZS, Wang GL, Liu JX, Wang F, Xiong AS. The genome sequence of celery (Apium graveolens L.), an important leaf vegetable crop rich in apigenin in the Apiaceae family. Hortic Res. 2020;7:9. https://doi.org/10.1038/s41438-019-0235-2.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Li MY, Hou XL, Wang F, Tan GF, Xu ZS, Xiong AS. Advances in the research of celery, an important Apiaceae vegetable crop. Crit Rev Biotechnol. 2018;38(2):172–83. https://doi.org/10.1080/07388551.2017.1312275.

    Article  CAS  PubMed  Google Scholar 

  4. Feng K, Hou XL, Li MY, Jiang Q, Xu ZS, Liu JX, Xiong AS. CeleryDB: a genomic database for celery. Database (Oxford). 2018;2018:bay070. https://doi.org/10.1093/database/bay070.

    Article  CAS  PubMed  Google Scholar 

  5. Feng K, Xing G, Liu J, Wang H, Tan G, Wang G, Xu Z, Xiong A. AgMYB1, an R2R3-MYB factor, plays a role in anthocyanin production and enhancement of antioxidant capacity in celery. Vegetable Res. 2021;1(1):1–12. https://doi.org/10.48130/VR-2021-0002.

    Article  Google Scholar 

  6. Feng K, Liu JX, Xing GM, Sun S, Li S, Duan AQ, Wang F, Li MY, Xu ZS, Xiong AS. Selection of appropriate reference genes for RT-qPCR analysis under abiotic stress and hormone treatment in celery. PeerJ. 2019;7:e7925. https://doi.org/10.7717/peerj.7925.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Baldoni E, Genga A, Cominelli E. Plant MYB Transcription Factors: Their Role in Drought Response Mechanisms. Int J Mol Sci. 2015;16(7):15811–51. https://doi.org/10.3390/ijms160715811.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Manna M, Thakur T, Chirom O, Mandlik R, Deshmukh R, Salvi P. Transcription factors as key molecular target to strengthen the drought stress tolerance in plants. Physiol Plant. 2021;172(2):847–68. https://doi.org/10.1111/ppl.13268.

    Article  CAS  PubMed  Google Scholar 

  9. Castilhos G, Lazzarotto F, Spagnolo-Fonini L, Bodanese-Zanettini MH, Margis-Pinheiro M. Possible roles of basic helix-loop-helix transcription factors in adaptation to drought. Plant Sci. 2014;223:1–7. https://doi.org/10.1016/j.plantsci.2014.02.010.

    Article  CAS  PubMed  Google Scholar 

  10. Tripathi P, Rabara RC, Rushton PJ. A systems biology perspective on the role of WRKY transcription factors in drought responses in plants. Planta. 2014;239(2):255–66. https://doi.org/10.1007/s00425-013-1985-y.

    Article  CAS  PubMed  Google Scholar 

  11. Hou L, Fan X, Hao J, Liu G, Zhang Z, Liu X. Negative regulation by transcription factor VvWRKY13 in drought stress of Vitis vinifera L. Plant Physiol Biochem. 2020;148:114–21. https://doi.org/10.1016/j.plaphy.2020.01.008.

    Article  CAS  PubMed  Google Scholar 

  12. Lim C, Kang K, Shim Y, Yoo SC, Paek NC. Inactivating transcription factor OsWRKY5 enhances drought tolerance through abscisic acid signaling pathways. Plant Physiol. 2022;188(4):1900–16. https://doi.org/10.1093/plphys/kiab492.

    Article  CAS  PubMed  Google Scholar 

  13. Mao Y, Xu J, Wang Q, Li G, Tang X, Liu T, Feng X, Wu F, Li M, Xie W, et al. A natural antisense transcript acts as a negative regulator for the maize drought stress response gene ZmNAC48. J Exp Bot. 2021;72(7):2790–806. https://doi.org/10.1093/jxb/erab023.

    Article  CAS  PubMed  Google Scholar 

  14. Dubos C, Stracke R, Grotewold E, Weisshaar B, Martin C, Lepiniec L. MYB transcription factors in Arabidopsis. Trends Plant Sci. 2010;15(10):573–81. https://doi.org/10.1016/j.tplants.2010.06.005.

    Article  CAS  PubMed  Google Scholar 

  15. Li J, Han G, Sun C, Sui N. Research advances of MYB transcription factors in plant stress resistance and breeding. Plant Signal Behav. 2019;14(8):1613131. https://doi.org/10.1080/15592324.2019.1613131.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Li X, Tang Y, Li H, Luo W, Zhou C, Zhang L, Lv J. A wheat R2R3 MYB gene TaMpc1-D4 negatively regulates drought tolerance in transgenic Arabidopsis and wheat. Plant Sci. 2020;299:110613. https://doi.org/10.1016/j.plantsci.2020.110613.

    Article  CAS  PubMed  Google Scholar 

  17. Dossa K, Mmadi MA, Zhou R, Liu A, Yang Y, Diouf D, You J, Zhang X. Ectopic expression of the sesame MYB transcription factor SiMYB305 promotes root growth and modulates ABA-mediated tolerance to drought and salt stresses in Arabidopsis. AoB Plants. 2020;12(1):081. https://doi.org/10.1093/aobpla/plz081.

    Article  CAS  Google Scholar 

  18. Cui J, Jiang N, Zhou X, Hou X, Yang G, Meng J, Luan Y. Tomato MYB49 enhances resistance to Phytophthora infestans and tolerance to water deficit and salt stress. Planta. 2018;248(6):1487–503. https://doi.org/10.1007/s00425-018-2987-6.

    Article  CAS  PubMed  Google Scholar 

  19. Li J, Zhao S, Yu X, Du W, Li H, Sun Y, Sun H, Ruan C. Role of Xanthoceras sorbifolium MYB44 in tolerance to combined drought and heat stress via modulation of stomatal closure and ROS homeostasis. Plant Physiol Biochem. 2021;162:410–20. https://doi.org/10.1016/j.plaphy.2021.03.007.

    Article  CAS  PubMed  Google Scholar 

  20. Su Z, Ma X, Guo H, Sukiran NL, Guo B, Assmann SM, Ma H. Flower development under drought stress: morphological and transcriptomic analyses reveal acute responses and long-term acclimation in Arabidopsis. Plant Cell. 2013;25(10):3785–807. https://doi.org/10.1105/tpc.113.115428.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Singh D, Laxmi A. Transcriptional regulation of drought response: a tortuous network of transcriptional factors. Front Plant Sci. 2015;6:895. https://doi.org/10.3389/fpls.2015.00895.

    Article  PubMed  PubMed Central  Google Scholar 

  22. Song Q, Kong L, Yang X, Jiao B, Hu J, Zhang Z, Xu C, Luo K. PtoMYB142, a poplar R2R3 MYB transcription factor, contributes to drought tolerance by regulating wax biosynthesis. Tree Physiol. 2022;42(10):2133–47. https://doi.org/10.1093/treephys/tpac060.

    Article  PubMed  Google Scholar 

  23. Allan AC, Espley RV. MYBs Drive Novel Consumer Traits in Fruits and Vegetables. Trends Plant Sci. 2018;23(8):693–705. https://doi.org/10.1016/j.tplants.2018.06.001.

    Article  CAS  PubMed  Google Scholar 

  24. Milani A, Basirnejad M, Shahbazi S, Bolhassani A. Carotenoids: biochemistry, pharmacology and treatment. Br J Pharmacol. 2017;174(11):1290–324. https://doi.org/10.1111/bph.13625.

    Article  CAS  PubMed  Google Scholar 

  25. Hirschberg J. Carotenoid biosynthesis in flowering plants. Curr Opin Plant Biol. 2001;4(3):210–8. https://doi.org/10.1016/S1369-5266(00)00163-1.

    Article  CAS  PubMed  Google Scholar 

  26. Mahjoub A, Hernould M, Joubès J, Decendit A, Mars M, Barrieu F, Hamdi S, Delrot S. Overexpression of a grapevine R2R3-MYB factor in tomato affects vegetative development, flower morphology and flavonoid and terpenoid metabolism. Plant Physiol Biochem. 2009;47(7):551–61. https://doi.org/10.1016/j.plaphy.2009.02.015.

    Article  CAS  PubMed  Google Scholar 

  27. Ma X, Yu Y, Jia J, Li Q, Gong Z. The pepper MYB transcription factor CaMYB306 accelerates fruit coloration and negatively regulates cold resistance. Hortic. 2022;295:110892.

    Article  CAS  Google Scholar 

  28. He Y, Li M, Wang Y, Shen S. The R2R3-MYB transcription factor MYB44 modulates carotenoid biosynthesis in Ulva prolifera. Algal Res. 2022;62:102578. https://doi.org/10.1016/j.algal.2021.102578.

    Article  Google Scholar 

  29. Jiang Q, Wang F, Li MY, Tan HW, Ma J, Xiong AS. High-throughput analysis of small RNAs and characterization of novel microRNAs affected by abiotic stress in a local celery cultivar. Sci Hortic. 2014;169:36–43. https://doi.org/10.1016/j.scienta.2014.02.007.

    Article  CAS  Google Scholar 

  30. Li MY, Wang F, Jiang Q, Ma J, Xiong AS. Identification of SSRs and differentially expressed genes in two cultivars of celery (Apium graveolens L.) by deep transcriptome sequencing. Hortic Res. 2014;1:10. https://doi.org/10.1038/hortres.2014.10.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Zhang X, Henriques R, Lin SS, Niu QW, Chua NH. Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nat Protoc. 2006;1(2):641–6. https://doi.org/10.1038/nprot.2006.97.

    Article  CAS  PubMed  Google Scholar 

  32. Li T, Liu JX, Deng YJ, Xu ZS, Xiong AS. Overexpression of a carrot BCH gene, DcBCH1, improves tolerance to drought in Arabidopsis thaliana. BMC Plant Biol. 2021;21(1):475. https://doi.org/10.1186/s12870-021-03236-7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Weng H, Pan A, Yang L, Zhang C, Liu Z, Zhang D. Estimating number of transgene copies in transgenic rapeseed by real-time PCR assay withHMG I/Y as an endogenous reference gene. Plant Mol Biol Rep. 2004;22(3):289–300. https://doi.org/10.1007/BF02773139.

    Article  CAS  Google Scholar 

  34. Noman M, Jameel A, Qiang WD, Ahmad N, Liu WC, Wang FW, Li HY. Overexpression of GmCAMTA12 Enhanced Drought Tolerance in Arabidopsis and Soybean. Int J Mol Sci. 2019;20(19):4849. https://doi.org/10.3390/ijms20194849.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Liu JX, Feng K, Duan AQ, Li H, Yang QQ, Xu ZS, Xiong AS. Isolation, purification and characterization of an ascorbate peroxidase from celery and overexpression of the AgAPX1 gene enhanced ascorbate content and drought tolerance in Arabidopsis. BMC Plant Biol. 2019;19(1):488. https://doi.org/10.1186/s12870-019-2095-1.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Galbiati M, Matus JT, Francia P, Rusconi F, Cañón P, Medina C, Conti L, Cominelli E, Tonelli C, Arce-Johnson P. The grapevine guard cell-related VvMYB60 transcription factor is involved in the regulation of stomatal activity and is differentially expressed in response to ABA and osmotic stress. BMC Plant Biol. 2011;11(1):142. https://doi.org/10.1186/1471-2229-11-142.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Ré DA, Capella M, Bonaventure G, Chan RL. Arabidopsis AtHB7 and AtHB12evolved divergently to fine tune processes associated with growth and responses to water stress. BMC Plant Biol. 2014;14(1):150. https://doi.org/10.1186/1471-2229-14-150.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Liu Y, Shi Y, Su D, Lu W, Li Z. SlGRAS4 accelerates fruit ripening by regulating ethylene biosynthesis genes and SlMADS1 in tomato. Hortic Res. 2021;8(1):3. https://doi.org/10.1038/s41438-020-00431-9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Ding X, Liu JX, Li T, Duan AQ, Yin L, Wang H, Jia LL, Liu YH, Liu H, Tao JP, Xiong AS. AgZDS, a gene encoding ζ-carotene desaturase, increases lutein and β-carotene contents in transgenic Arabidopsis and celery. Plant Sci. 2021;312:111043. https://doi.org/10.1016/j.plantsci.2021.111043.

    Article  CAS  PubMed  Google Scholar 

  40. Sun M, Feng XX, Gao JJ, Peng RH, Yao QH, Wang LJ. VvMYBA6 in the promotion of anthocyanin biosynthesis and salt tolerance in transgenic Arabidopsis. Plant Biotechnol Reports. 2017;11(5):299–314. https://doi.org/10.1007/s11816-017-0452-9.

    Article  Google Scholar 

  41. Zhu J, Wang WS, Ma D, Zhang LY, Ren F, Yuan TT. A role for CK2 β subunit 4 in the regulation of plant growth, cadmium accumulation and H2O2 content under cadmium stress in Arabidopsis thaliana. Plant Physiol Biochem. 2016;109:240–7. https://doi.org/10.1016/j.plaphy.2016.10.004.

    Article  CAS  PubMed  Google Scholar 

  42. Li MY, Wang F, Jiang Q, Wang GL, Tian C, Xiong AS. Validation and Comparison of Reference Genes for qPCR Normalization of Celery (Apium graveolens) at Different Development Stages. Front Plant Sci. 2016;7:313. https://doi.org/10.3389/fpls.2016.00313.

    Article  PubMed  PubMed Central  Google Scholar 

  43. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc. 2008;3(6):1101–8. https://doi.org/10.1038/nprot.2008.73.

    Article  CAS  PubMed  Google Scholar 

  44. Jacob P, Brisou G, Dalmais M, Thévenin J, van der Wal F, Latrasse D, Suresh Devani R, Benhamed M, Dubreucq B, Boualem A. The Seed Development Factors TT2 and MYB5 Regulate Heat Stress Response in Arabidopsis. Genes (Basel). 2021;12(5):146. https://doi.org/10.3390/genes12050746.

    Article  CAS  Google Scholar 

  45. Choudhury FK, Rivero RM, Blumwald E, Mittler R. Reactive oxygen species, abiotic stress and stress combination. Plant J. 2017;90(5):856–67. https://doi.org/10.1111/tpj.13299.

    Article  CAS  PubMed  Google Scholar 

  46. Kundan M, Gani U, Fayaz M, Angmo T, Kesari R, Rahul VP, Gairola S, Misra P. Two R2R3-MYB transcription factors, CsMYB33 and CsMYB78 are involved in the regulation of anthocyanin biosynthesis in Cannabis sativa L. Industrial Crops and Products. 2022;188:115546. https://doi.org/10.1016/j.indcrop.2022.115546.

    Article  CAS  Google Scholar 

  47. Schachtman DP, Goodger JQD. Chemical root to shoot signaling under drought. Trends Plant Sci. 2008;13(6):281–7. https://doi.org/10.1016/j.tplants.2008.04.003.

    Article  CAS  PubMed  Google Scholar 

  48. Sun T, Li L. Toward the ‘golden’ era: The status in uncovering the regulatory control of carotenoid accumulation in plants. Plant Sci. 2020;290:110331. https://doi.org/10.1016/j.plantsci.2019.110331.

    Article  CAS  PubMed  Google Scholar 

  49. Yang L, Liu H, Hao Z, Zong Y, Xia H, Shen Y, Li H. Genome-Wide Identification and Expression Analysis of R2R3-MYB Family Genes Associated with Petal Pigment Synthesis in Liriodendron. Int J Mol Sci. 2021;22(20):11291. https://doi.org/10.3390/ijms222011291.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Zhu F, Luo T, Liu C, Wang Y, Yang H, Yang W, Zheng L, Xiao X, Zhang M, Xu R, Xu JG, Zeng YL, Xu J, Xu Q, Guo WW, Larkin RM, Deng XX, Cheng YJ. An R2R3-MYB transcription factor represses the transformation of α- and β-branch carotenoids by negatively regulating expression of CrBCH2 and CrNCED5 in flavedo of Citrus reticulate. New Phytol. 2017;216(1):178–92. https://doi.org/10.1111/nph.14684.

    Article  CAS  PubMed  Google Scholar 

  51. Wu M, Xu X, Hu X, Liu Y, Cao H, Chan H, Gong Z, Yuan Y, Luo Y, Feng B, Li ZG, Deng W. SlMYB72 Regulates the Metabolism of Chlorophylls, Carotenoids, and Flavonoids in Tomato Fruit. Plant Physiol. 2020;183(3):854–68. https://doi.org/10.1104/pp.20.00156.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Morales M, Roach DA, Quarles BM, Cotado A, Salguero-Gómez R, Dwyer J, Munné-Bosch S. Validity of photo-oxidative stress markers and stress-related phytohormones as predictive proxies of mortality risk in the perennial herb Plantago lanceolata. Environ. Exp Bot. 2021;191:104598. https://doi.org/10.1016/j.envexpbot.2021.104598.

    Article  CAS  Google Scholar 

  53. Kang L, Ji CY, Kim SH, Ke QB, Park SC, Kim HS, Lee HU, Lee JS, Park WS, Ahn MJ. Suppression of the β-carotene hydroxylase gene increases β-carotene content and tolerance to abiotic stress in transgenic sweetpotato plants. Plant Physiol Biochem. 2017;117:24–33. https://doi.org/10.1016/j.plaphy.2017.05.017.

    Article  CAS  PubMed  Google Scholar 

  54. Moreno JC, Mi J, Alagoz Y, Al-Babili S. Plant apocarotenoids: from retrograde signaling to interspecific communication. Plant J. 2021;105(2):351–75. https://doi.org/10.1111/tpj.15102.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Cowan AK, Rose PD. Abscisic Acid Metabolism in Salt-Stressed Cells of Dunaliella salina: Possible Interrelationship with beta-Carotene Accumulation. Plant Physiol. 1991;97(2):798–803. https://doi.org/10.1104/pp.97.2.798.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Liu X, Hu Q, Yan J, Sun K, Liang Y, Jia M, Meng X, Fang S, Wang Y, Jing Y. ζ-Carotene Isomerase Suppresses Tillering in Rice through the Coordinated Biosynthesis of Strigolactone and Abscisic Acid. Mol Plant. 2020;13(12):1784–801. https://doi.org/10.1016/j.molp.2020.10.001.

    Article  CAS  PubMed  Google Scholar 

  57. Abuauf H, Haider I, Jia K-P, Ablazov A, Mi J, Blilou I, Al-Babili S. The Arabidopsis DWARF27 gene encodes an all-trans-/9-cis-β-carotene isomerase and is induced by auxin, abscisic acid and phosphate deficiency. Plant Sci. 2018;277:33–42. https://doi.org/10.1016/j.plantsci.2018.06.024.

    Article  CAS  PubMed  Google Scholar 

  58. Hsu PK, Dubeaux G, Takahashi Y, Schroeder JI. Signaling mechanisms in abscisic acid-mediated stomatal closure. Plant J. 2021;105(2):307–21. https://doi.org/10.1111/tpj.15067.

    Article  CAS  PubMed  Google Scholar 

  59. Zhang Y, Sun Y, Liu X, Deng J, Yao J, Zhang Y, Deng S, Zhang H, Zhao N, Li J. Populus euphratica apyrases increase drought tolerance by modulating stomatal aperture in arabidopsis. Int J Mol Sci. 2021;22(18):9892. https://doi.org/10.3390/ijms22189892.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Zhao J, Zhang W, da Silva JAT, Liu X, Duan J. Rice histone deacetylase HDA704 positively regulates drought and salt tolerance by controlling stomatal aperture and density. Planta. 2021;254(4):79. https://doi.org/10.1007/s00425-021-03729-7.

    Article  CAS  PubMed  Google Scholar 

  61. Xu BQ, Wang JJ, Peng Y, Huang H, Sun LL, Yang R, Suo LN, Wang SH, Zhao WC. SlMYC2 mediates stomatal movement in response to drought stress by repressing SlCHS1 expression. Front Plant Sci. 2022;13:952758. https://doi.org/10.3389/fpls.2022.952758.

    Article  PubMed  PubMed Central  Google Scholar 

  62. Yan H, Jia H, Chen X, Hao L, An H, Guo X. The cotton WRKY transcription factor GhWRKY17 functions in drought and salt stress in transgenic Nicotiana benthamiana through ABA signaling and the modulation of reactive oxygen species production. Plant Cell Physiol. 2014;55(12):2060–76. https://doi.org/10.1093/pcp/pcu133.

    Article  CAS  PubMed  Google Scholar 

  63. Ju YL, Yue XF, Min Z, Wang XH, Fang YL, Zhang JX. VvNAC17, a novel stress-responsive grapevine (Vitis vinifera L.) NAC transcription factor, increases sensitivity to abscisic acid and enhances salinity, freezing, and drought tolerance in transgenic Arabidopsis. Plant Physiol Biochem. 2020;146:98–111. https://doi.org/10.1016/j.plaphy.2019.11.002.

    Article  CAS  PubMed  Google Scholar 

  64. Zhang YB, Tang W, Wang LH, Hu YW, Liu XW, Liu YS. Kiwifruit (Actinidia chinensis) R1R2R3-MYB transcription factor AcMYB3R enhances drought and salinity tolerance in Arabidopsis thaliana. J Integr Agr. 2019;18(2):417–27. https://doi.org/10.1016/S2095-3119(18)62127-6.

    Article  CAS  Google Scholar 

  65. Yu Y, Bi C, Wang Q, Ni Z. Overexpression of TaSIM provides increased drought stress tolerance in transgenic Arabidopsis. Biochem Biophys Res Commun. 2019;512(1):66–71. https://doi.org/10.1016/j.bbrc.2019.03.007.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

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Funding

This research was supported by Natural Science Foundation for Higher Education Institutions in Jiangsu Province (21KJB210007), Key Research and Development Program of Jiangsu (BE2022386), and Priority Academic Program Development of Jiangsu Higher Education Institutions Project (PAPD).

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  1. College of Marine and Biological Engineering, Yancheng Teachers University, Yancheng, 224002, Jiangsu, China

    Miao Sun, Qin-Yi Xu, Zhi-Peng Zhu, Jian-Xiang Yu, Yao-Xian Guo & Shu Tang

  2. State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Horticulture, Nanjing Agricultural University, Nanjing, 210095, Jiangsu, China

    Miao Sun, Pei-Zhuo Liu & Ai-Sheng Xiong

  3. College of Food Science and Technology, Nanjing Agricultural University, Jiangsu, 210095, China

    Miao Sun & Zhi-Fang Yu

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Miao Sun: Original writing, funding acquisition. Qin-Yi Xu: Data analysis. Zhi-Peng Zhu, Pei-Zhuo Liu, Jian-Xiang Yu, Yao-Xian Guo, and Shu Tang: Experiment. Zhi-Fang Yu: Manuscript revision. Ai-Sheng Xiong: Funding acquisition, manuscript revision. All authors have read and approved the final manuscript.

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Correspondence to Ai-Sheng Xiong.

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The authors confirm that all the experimental methods complied with.relevant institutional, national, and international guidelines and legislation. Wealso specify that all the permissions or licenses regarding the seed collectionwere obtained from Nanjing Agricultural University and Yancheng Teachers University for only research purpose.

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Additional file 1:

Figure S1. Copy number of AgMYB5 in transgenic Arabidopsis lines (OE1 and OE4).

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Sun, M., Xu, QY., Zhu, ZP. et al. AgMYB5, an MYB transcription factor from celery, enhanced β-carotene synthesis and promoted drought tolerance in transgenic Arabidopsis. BMC Plant Biol 23, 151 (2023). https://doi.org/10.1186/s12870-023-04157-3

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Keywords

BMC Plant Biology

ISSN: 1471-2229

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