AgGMP encoding GDP-D-mannose pyrophosphorylase from celery enhanced the accumulation of ascorbic acid and resistance to drought stress in Arabidopsis

Introduction

GDP-D-mannose pyrophosphorylase (GMPase) is a rate-limiting enzyme in the L-galactose pathway, a key biosynthetic pathway for L-ascorbic acid (AsA) in plants. In this pathway, the initial step is the formation of GDP-D-mannose, which is catalyzed by GMPase (Wang et al., 2011). AsA is a water-soluble antioxygenic organic micromolecule widely found in plants. It’s essential for cardiovascular function, immune cell development, connective tissue health, and iron utilization. As a key enzyme in AsA biosynthesis, GMPase in higher plants plays important roles in removing reactive oxygen species (ROS) generated by adverse environmental conditions (Xue et al., 2018), such as high temperature, low temperature (Li et al., 2018a; Liu et al., 2019; Huang et al., 2016), and salt stress (Zhang et al., 2012). In addition, the resistance of abiotic stress in plants could be improved through modulating GMP gene expression to increase the AsA content. Until now, GMP genes have been isolated from a number of higher plants, including Arabidopsis thaliana (Wang, Guo & Tang, 2011), Camellia sinensis (Xiao et al., 2015), Solanum esculentum (Wang et al., 2011), and Dendrobium huoshanense (He, Yu & Tei, 2017).

Plant resistance against abiotic stress was associated with the changes of antioxidant defense system, which consisted of enzymatic and non-enzymatic antioxidants (Garg, Varshney & Jain, 2014; Hasanuzzaman et al., 2017; Li et al., 2018a; Liu et al., 2021). AsA belongs to non-enzymatic antioxidants (Liu et al., 2019), drought stress affects AsA content by controlling the activities of enzymes involved in AsA metabolism (e.g. GMP, GalDH, APX, DHAR, MDHAR) (Huang et al., 2016; Liu et al., 2019). AsA can directly remove ROS (reactive oxygen species) produced by stress, and also indirectly remove H₂O₂ through the AsA-GSH cycle to protect tissues from harmful oxidative products and to keep certain enzymes in their required reduced forms (Liebler, Kling & Reed, 1986; Padh, 1990; Barth, Gouzd & Steele, 2009), as well as improve the ability of plants to resist abiotic stress.

Under low and high temperature stresses, the relative expression of SlGMP, GMPase activity, contents of AsA and dehydroascorbic acid (DHA) were increased, and the content of malondialdehyde (MDA) was decreased in transgenic tomato plants (Wang et al., 2011). The overexpression of SlGMP also delayed the senescence of potato (Lin et al., 2011). OsGMP gene affected AsA synthesis and GMPase activity in rice (Oryza sativa), and overexpression of OsGMP reduced the inhibitory effect of NH₄⁺ on root growth in A. thaliana (Li et al., 2010). The OsGMP gene also was reported to play a key role in the rice during its nutritional and reproductive stages under salt stress (Kempinski, Haffar & Carina, 2011; Qin et al., 2016a, 2016b). The GmGMP1 gene can be induced to express under high temperature, low temperature, drought and salt stresses. The overexpression of GmGMP1 gene in A. thaliana and soybean (Glycine Max) increases GMPase activity and AsA content, and enhances the ability for eliminating ROS (Xue et al., 2018). The tobacco (Nicotiana Tabacum) harboring PpGMP showed stronger resistance to salt or drought stresses by increasing the AsA content (Ai, Liao & Li, 2016). AtGMP positively regulates the synthesis of AsA. AsA content in transgenic lettuce (Lactuca sativa) overexpressing AtGMP gene increased to 2.5-fold of that in control (Wang, Guo & Tang, 2011).

Celery (Apium graveolens) growth is influenced by multiple environmental factors. In previous studies, it is reported that GMP gene participated in plant response to stress. We speculated that AgGMP may be involved in celery resisting abiotic stress through modulating the AsA accumulation. However, the characteristics and transcription regulation mechanism of AgGMP under drought stress in celery remain unclear. In this study, AgGMP, a gene encoding GDP-D-mannose pyrophosphorylase, was cloned from celery, and then its expression patterns were detected in celery under abiotic stress treatments. The transgenic A. thaliana plants overexpressed AgGMP were obtained to examine the AsA level and compared their root growth with wild-type under drought stress. This study further clarified the roles of AgGMP in abiotic stress, and provided a theoretical basis for stress response of celery.

Materials and Methods

Results

Analysis of AgGMP sequence

The full lengths cDNA sequence of AgGMP gene was obtained from ‘Jinnan Shiqin’ (GenBank No. OL757646), which contained an open reading frame (ORF) with the length of 1,086 bp, encoding 361 amino acids (Fig. 1A). The secondary structure was mainly composed of 30.77% α-helixes, 29.09% extended strand, 9.07% β-turn and 37.67% random coils. Prediction results indicated that the protein tertiary structure of AgGMP protein was mainly composed of three α-helixes and certain random coils (Fig. 1B). AgGMP protein has no signal peptide according to the prediction by Signal P software. The amino acid sequence of AgGMP were aligned with homologous sequences from Daucus carota (carrot, AQM57027.1), Lycopersicon esculentum (tomato, accession ID DQ449030), Solanum tuberosum (potato, NP_001275205.1), Camellia sinensis (tea plant, AGI78460.1), Arabidopsis thaliana (AT4G30570), Oryza sativa (rice, LOC4327472), Malpighia glabra (acerola, ABB53473.1), Brassica rapa subsp. chinensis (non-heading Chinese cabbage, AET14212.1), Nicotiana tabacum (tobacco, BAB62108.1) (Fig. 2). GMP proteins were highly conserved (90.80% of consistency) among different species. Phylogenetic analysis demonstrated that AgGMP protein had the closest evolutionary relationship with carrot (Fig. 3).

Expression profiles of AgGMP under abiotic stress in celery

As shown in Fig. 4, the relative expression levels of AgGMP gene in ‘Jinnan Shiqin’ were up-regulated significantly under low temperature, high temperature, salt and drought treatments. The relative expression levels of AgGMP gene were obviously increased at 1 h after low temperature, high temperature and salt treatments, respectively (Figs. 4A–4C). In salt treatment, the relative expression levels of AgGMP gene were remarkably elevated at 8 h and 24 h than that at 0 h, which were 2.52 and 2.79-folds of that at 0 h, respectively. The highest expression of AgGMP gene were occurred at 2 h, 4 h, 1 h and 2 h of low temperature, high temperature, salt and drought treatments, which were 2.70, 2.43, 1.54, and 4.2-folds of the 0 h, respectively. Under drought stress, the expression profiles of AgGMP exhibited an increase at 1 h, 2 h, 4 h, which were 1.11, 1.54 and 1.47, and then decreased at 8 h, 24 h, which were 0.82 and 0.85, respectively (Fig. 4D). The results indicated that AgGMP involved in the response to abiotic stress in celery.

Subcellular localization analysis of AgGMP

The vector AgGMP-GFP and pA7-GFP were transferred into the onion epidermis cell to detect the subcellular localization of AgGMP. The onion epidermal cell with AgGMP-GFP and pA7-GFP displayed bright fluorescence throughout the entire cell, suggesting that the AgGMP protein was located in cytoplasm (Fig. 5A).

Overexpression of AgGMP up-regulated the AsA content in Arabidopsis

There are no obvious differences observed in phenotype among three OE lines and WT plants (Fig. 6A). According to the AsA content determination, the transgenic A. thaliana lines contained higher AsA accumulation compared with WT (Figs. 6B and 6C). The AsA contents in WT, AgGMP-1, AgGMP-2 and AgGMP-5 lines were 42.25, 48.04, 51.98, and 52.73 mg/100g FW (fresh weight), respectively. The results indicated that overexpression of AgGMP up-regulated the AsA level in transgenic A. thaliana.

The change of seedling root lengths in A. thaliana under 300 mM mannitol treatment

The seeds of three A. thaliana OE lines and WT were germinated and grown on MS medium containing different mannitol concentrations. The root lengths of 7-day-old (day after germinating) A. thaliana plants were measured. On MS medium without mannitol, no significant difference in root lengths was observed between WT and transgenic A. thaliana lines hosting AgGMP gene. The root lengths of WT, AgGMP-1, AgGMP-2, and AgGMP-5 lines, were 2.98, 2.55, 3.31 and 3.00 cm, respectively (Fig. 7A). Whereas, the roots lengths were inhibited in WT and three transgenic A. thaliana lines treated with 300 mM mannitol, which were 1.71, 2.08, 2.23, and 1.81 cm, respectively (Fig. 7B). On MS medium without mannitol, the root lengths of A. thaliana were longer, more lateral roots and root hairs, as well as better root growth. The root lengths of transgenic plants were longer than that of WT on MS medium containing 300 mM mannitol. In particular, the root lengths of AgGMP-2 were significantly increased compared with WT.

Discussion

Celery is one of important leafy vegetables with rich nutrients (Kooti & Daraei, 2017; Li et al., 2018b), such as anthocyanin (Feng et al., 2018b, 2021), apigenin (Tan et al., 2017; Yan et al., 2019; Wang et al., 2021), carotenoids (Li et al., 2019; Yin et al., 2020; Ding et al., 2021), AsA (Liu et al., 2021), and dietary fiber (Duan et al., 2020). AsA is not only is one of important nutrients, but also a key mediator that triggers plant response to various abiotic stress (Barth, Gouzd & Steele, 2009). AsA accumulation is affected by abiotic stress, such as salt (Zhang et al., 2012), low temperature, high temperature (Wang et al., 2011), drought stress (Padh, 1990), light (Li et al., 2020a), and a variety of nutrient elements (Li et al., 2020b), which is speculated to accomplished by increasing the activity of related enzymes involved in the AsA biosynthesis pathway in plants.

In plants, there are four pathways for AsA biosynthesis, including L-galactose pathway, myo-inositol pathway, L-gulose pathway, and D-galacturonate pathway. The GMP gene, encoding GMPase, plays an essential role in the L-galactose pathway. GMPase catalyzes the initial steps of AsA biosynthesis to form GDP-D-mannose, the precursor of AsA. Thus, GMP gene could affect AsA content by controlling the activities of GMPase. Our present result indicated that AgGMP involved in the response to abiotic stress in celery. The identified function of AgGMP is similar to that of soybean GmGMP1, GMP genes expression were both significantly induced by heat, cold and salt stresses. Under abiotic stress treatments, the expression level of GmGMP1 peak at 1 h and then decreased (Xue et al., 2018). Under drought stress, the expression levels of the AgAPX1 and AgGMP of celery were also similar, which significantly higher than that of the control at 2 h, and peaked at 4 h followed by a decrease (Liu et al., 2019).

It is acknowledged that drought is an important factor affecting agricultural production, improving plant drought resistance also is an ongoing hot topic. Considering evidences have suggested the relationship between AsA and stress resistance (Wang et al., 2011; Zhang et al., 2012). One of the consequences of abiotic stress is that it triggers an oxidative burst due to formation of reactive oxygen species. AsA could directly remove ROS produced by stress, and also indirectly remove H₂O₂ through the AsA-GSH cycle to protect tissues from harmful oxidative products, as well as keep certain enzymes in their required reduced forms. GMPase is a key rate limiting enzymes in AsA biosynthesis. Declined expression of GMP gene, encoded GMPase, usually was followed by a decreased resistance to drought stress in plants. Overexpression of the GMP gene of soybean enhanced the plant drought resistance in transgenic plants (Xue et al., 2018). In this study, we found that the relative expression levels of AgGMP gene were up-regulated significantly under low temperature, high temperature, salt and drought treatments, which reached the peak at 2 h, 4 h, 1 h, and 2 h, respectively. The transcript of AgGMP in celery were induced by drought stress, the expression of AgGMP in celery was significantly up-regulated of AgGMP under drought stress at 2 h and reach peak at 4 h and then declined.

To further investigate the function of AgGMP gene in response to drought stress, the AsA content and root lengths of three A. thaliana OE lines and WT were measured. Compare with the WT, overexpression of AgGMP up-regulated the AsA content in three transgenic A. thaliana lines, the root lengths were also longer when subjected to 300 mM mannitol. It is possible that overexpression of AgGMP can improve the activity of GMPase that involved in AsA biosynthesis pathway in A. thaliana, and increase the AsA accumulation to neutralize part of the stress effects. AsA could remove ROS and H₂O₂ produced by drought stress, protect tissues from harmful oxidative products and keep certain enzymes in their required reduced forms (Wang et al., 2011; Zhang et al., 2012). The three transgenic A. thaliana lines showed reduced root damage caused by mannitol and presented greater tolerance to drought stress. We speculated that AgGMP was participated in the process of celery resisting drought stress and overexpression of AgGMP can induce an increased resistance to drought stress in transgenic plants by enhancing the accumulation of AsA.

Conclusion

GMPase was a rate-limiting enzyme in the L-galactose pathway, a key biosynthetic pathway for L-ascorbic acid (AsA) in plants. Here, the gene AgGMP encoding the AgGMPase was identified and characterized. The AgGMP gene contained an ORF of 1,086 bp, encoding 361 amino acids. Sequence alignment suggested that AgGMP protein was highly conserved among different plant species. The transcript of AgGMP was induced by abiotic stress in celery, the heterologous overexpression of AgGMP in A. thaliana proved the role of AgGMP in regulating AsA accumulation and modifying drought stress resistance. These findings suggested that AgGMP acted a regulator in AsA accumulation and response to abiotic stress in celery. In the future, we hope to achieve its homologous over-expression through transgenic technology and knock it out using the CRISPR/Cas9 method in celery.

Supplemental Information