Ethylene signaling transcription factor promote grape growth induced by exogenous carbon

The carbon can be converted into sugar which is not only important for plant growth and development, but also for plant signal transduction, especially in plant hormone response. The objective of this work was to build available genomic and proteomic resource to investigate the molecular mechanisms of exogenous carbon regulating plant growth and development. Grape (Vitis vinifera L. cv. ‘Pinot Noir’) plantlets cultured with exogenous carbon (2% sucrose, 1000 μmol·mol-1 CO2 and with both 2% sucrose and 1000 μmol·mol-1 CO2 were designated as S1, C0 and Cs, respectively). We used S0 (without sucrose, ambient CO2) as CK to analyze the differential expression genes and proteins induced by exogenous carbon. Through the transcriptomic and proteomic analysis, with pooled data for Cs, C0 and S1 compared with CK, 70 differentially expressed genes (DEGs) and 65 differentially expressed proteins (DEPs) were identified. Based on biological functions and physiological characteristics, we identified 8 DEGs and 2 DEPs related to ethylene signaling process. Amongst the DEGs we focussed on ERF TFs, including ERF5 (LOC100244353, LOC100247763, LOC100254616 and LOC100261260), ERF105 (LOC100249507 and LOC100259725), ERF2 (LOC100254640) and CTr (CTr7). Also, there were 2 DEPs related to ethylene metabolism, such as S-adenosylmethionine synthase 5 (SAM synthase 5; XP_002280106.1) and 1-aminocyclopropane-1-carboxylic acid oxidase 2 (ACC oxidase 2; NP_001267871.1) were also identified. The transcriptome and proteome results suggested that exogenous carbon inhibits ethylene biosynthesis through ACC oxidase 2. Additionally, CTr7 and ERF5, which were up-regulated, are related to the ethylene signaling pathway. We

pathways, but which inhibit ethylene biosynthesis.

Conclusions
Exogenous carbon regulates the expression of ethylene biosynthesis and signaling related genes, which may improve plant growth through the ethylene signaling pathway.

Background
Carbon is one of the vital substances of plant cytoskeleton and plays an irreplaceable role in plant growth and development process. The carbon is fixed by photosynthesis and converted into sugar [1]. The sugar plays pivotal roles in plant nutrient balance, optimum carbon to nitrogen ratio can either promote storage reserve mobilization and photosynthesis [2]. It is not only served as fuel supplying plant growth and a necessary compound for the synthesis of other substances, but also a signal which regulating plant growth and development [3,4]. Plants use many sugar sensor proteins, such as Hexokinases (HXK), to interrelate light, and hormone signaling networks for controlling growth and development in response to the changing environment [5,6]. In plants, sugars including sucrose, glucose, fructose, and trehalose, and they have hormone like regulatory activities [7].
Sugar metabolism plays a pivotal role in governing the outcome of various kinds of plantpathogen interactions and defense signaling [8,9]. Sugar is also tightly interconnected with hormonal signaling pathways [1,10]. Gaseous phytohormone ethylene affects many aspects of plant growth. Ethylene is related to the following biological processes: regulation of leaf development, senescence, fruit ripening [11][12][13], stimulation of germination and plant responses to biotic and abiotic [14,15]. Ethylene is a growth inhibitory hormone because ethylene sensitivity is negatively correlated with leaf growth [16]. Ethylene signal transduction is mainly related to ethylene receptor (ETR), constitutive triple response (CTR), ethylene insensitive (EIN) and Ethylene insensitive-like (EIL) [17][18][19][20]. The EIN/EIL proteins bind to upstream regions of ERF transcription factors (ERF TFs) to promote its expression in tissues. ERF TFs had been shown to be involved in various processes of plant development and response to biotic and abiotic stress [11,21].

Ethylene signal in plants is affected by environmental changes. Previous studies have
shown that copper affects ethylene binding growth of ETR1 receptor in Arabidopsis thaliana [22]. Sugars have been proved to act as a signaling molecule to interact with ethylene signal, in regulating plant growth and development. Mutants displaying nonfunctional ethylene receptors (etr1, ein4) or alteration of signal transduction proteins (ein2 and ein3), are hypersensitive to sugar-mediated photosynthesis repression, while constitutive triple response 1 (ctr1), a negative regulator of ethylene signaling, is glucose insensitive [23][24][25]. ERFs, have been classified into AP2/EREBP-TF family, were identified as regulators of genes which related to plant growth [21,26,27].
Molecular connection between ethylene and growth-regulatory pathways has been uncovered, we already know ethylene as inhibitor of leaf growth [16] and ERFs modulate transcription of a wide variety of genes which response to stress [28][29][30]. However, in higher plants the mechanism of exogenous carbon affects ethylene pathway remains unclear and whether exogenous carbon affects plant growth through ethylene pathway is uncertain. Although, the effects of ethylene on grape mostly focused on fruit ripening and postharvest [31,32]. For example, postharvest ethylene treatment affects berry dehydration, polyphenol and anthocyanin conten [33].
While basic models have been suggested for regulatory mechanisms among these pathways, but sugar concentration, localization, or the nature of the sugar signal may differentially affect hormone signals and gene [34]. Therefore, this work aims to investigate the changes in ethylene related genes and proteins under the influence of exogenous carbon. Further study on how does ERFs response to exogenous carbon and regulates plant growth.
We have revealed the effect of high CO 2 concentration on photosynthesis of grape plantlets based on previous analysis [35]. However, the expression of genes, proteins and an understanding of plant growth regulated by exogenous carbon at molecular levels are still undisclosed. In this study, we used 2×2 experimental design in which sucrose and eCO 2 were the main factor, to analyze the differential expression of ethylene-related genes and proteins in grape leaves induced by exogenous carbon through comparing with no carbon treatment, further to reveal the regulation of exogenous carbon on plant growth and development.

Exogenous carbon enhances plant biomass
Grape plantlets in vitro were cultured for 25 days and exogenous carbon is supplied by eCO 2 and sucrose. Those exogenous carbon treatments were compared with no carbon treatments. In agreement with what is known about the effect of exogenous carbon phenotype, our results showed that the leaf area, plantlet height and shoot fresh weight increased significantly in each treatment compared with CK ( Fig. 1). Through the analysis of physiological indicators, exogenous carbon significantly affects plant growth. The fresh weight of the underground part, leaf area and plantlet height of grape plantlet in vitro were 0.07g, 0.01g and 4.63cm 2 , respectively. These data were significantly lower than other treatments. The exogenous carbon significantly affected the fresh weight of aerial and underground part, but only caused changes in the dry weight of the aerial part of Cs.
It had no significant effects on the dry weight of the underground part ( Table 1).

Analysis of transcriptomics
To identify the molecular mechanisms responsible for increased plant growth with supply of exogenous carbon, comparison of gene transcription for plants grown with exogenous carbon and deficiency carbon was performed. A robust data set was collected after data processing, 46.50, 47.05, 46.89 and 47.08 million high-quality reads were obtained at Cs, C0, S1 and CK (Table S2). The bases content were 97.81%, 97.86%, 97.54 and 97.04%, respectively. The GC content were 46.33%, 46.00%, 46.00%, 46.33%, respectively (Table   S2) .

Analysis of proteomics
To better dissect the molecular regulated network in grape plantlets in vitro response to exogenous carbon, we utilized iTRAQ labeling strategy to perform quantitative proteomics and analyze the global protein changes in exogenous carbon supplied plants. From the pooled data for Cs, C0 and S1 compare with CK, 3047 unique proteins were identified.
There were 65 differentially expressed proteins (DEPs) identified from Cs, C0 and S1 compare with CK (P-value <0.05, FC 1.4 or FC <5/7), including 7 UR proteins and 58 DR proteins ( Table 3). The fold change and P-value listed in Table S4.
Among these DEPs, 17 DEPs could not match with the UniParc and RefSeq database. Therefore, the biological functions of these proteins are not clear. The other 48 DEPs matched with proteins of known function to be characterized in the UniProt database, but 7 DEPs functions might still unclear (Fig. 3). Based on biological functions, the 41 DEPs were classified into 7 categories: primary metabolism (25.64%), secondary metabolism (41.03%), energy (2.56%), bio-signaling (7.69%), translation (7.69%) and transport (7.69%) (Fig. 3).
Differential expression of ethylene pathway related genes was confirmed by qRT-PCR and their relative expression level was consistent with FPKM values fold change observed from transcriptional analysis, only 2 genes analyzed by qRT-PCR, i.e., METK5 and ACO2 under S1 treatment were not consistent with our RNA-seq data ( Table 4).

DEGs associated with plant growth
DEGs involved in primary metabolism: There were 9 DEGs related to primary metabolism, 7 up-regulated in Cs, C0 and S1 treatments, 2 down-regulated in exogenous carbon treatments. Those  However, all the proteins associated with primary metabolism were up-regulated in CK and down-regulated in treatments (Fig. 6).
With exogenous carbon supply, proteins that participate in environmental stress are differential expression. Indeed, 16 DEPs were observed up-regulated in CK, these DEPs Interestingly, 1 DEP in glutathione metabolism pathway was up-regulated in exogenous carbon treatment.

Discussion
Exogenous carbon promotes plant growth through ethylene signaling Although SAM synthetase 5 was up-regulated under exogenous carbon treatment, ACC oxidase 2 was down-regulated. The final step in ethylene biosynthesis is catalysised by ACC oxidase [36]. ACC oxidase was referred to as ethylene forming enzyme [37]. However, in sugar-free control, ACC oxidase expression was up-regulated, this change will likely producing additional ethylene, which affected the no normal growth of plants and resulted in plant slower growth (Fig 7). In the absence of exogenous glucose, plant growth is restricted to the seedling stage even after culturing on MS medium [38]. Ethylene is a growth inhibitory hormone [16]. In Arabidopsis, excess ethylene would cause plant dwarfism and slows down growth [39,40]. Therefore, those plantlets which lack of exogenous sugar grows slowly may also be affected by endogenous ethylene.
In the process of ethylene signaling, copper ions likely play a role in ethylene binding and transported by RAN1 [22,41], it serves as a cofactor for ethylene binding and is required for proper biogenesis of the receptors. The results implicate that exogenous carbon, especially eCO 2 could enhance the CTr7 expression. We speculate that eCO 2 may regulate ethylene signal by affecting the transport of copper ions. ERF, which is involved downstream of ethylene signaling, is involved in various processes of plant development [11,21] and different stress responses [42,43]. However, we observed that ERF expression increased with exogenous carbon supply. ERF could promoters of secondary target genes, which contains GCC box, such as chitinase [44].
Therefore, we speculated that exogenous carbon can regulate the expression of other genes through ERF. The ERF transcription factors can be classified as activating-or repressing-transcription factors, with ERF2 and ERF4 being activators and ERF3 being a repressor of transcription [45]. ERF5 is an activator of transcription and interacts with multiple proteins, such as ERF6, ERF8, and SCL13 [46,43]. ERFs belong to the AP2/EREBP transcription factor family [26], which can strongly bind a wide range of cis-regulatory elements, in the promoter of target genes [47,48]. As the final response gene in ethylene signaling pathway, basic endochitinase precursor (NP_001267891.1) changes, proving its relation to ERF [43]. Overexpression of ERF enhances resistance to bacterial and fungal pathogens [49]. Under exogenous carbon treatment, which genes interact with ERF remains to be illustrated. In addition, some ERF enhances the activities of ACC oxidase, thereby promoting ethylene synthesis and signal transduction [50, 51]. However, some ERFs also repress of ACC oxidase activities to prevent ethylene biosynthesis [52][53][54]. In our study, we speculate that ERF5 exhibits an inhibitory effect on ACC oxidase.
After analysis of transcriptome and proteome data, we speculated that exogenous carbon regulates plant growth through ethylene signaling pathways that inhibit ethylene biosynthesis. The expression of ERF5 increased under the action of exogenous carbon may further promoting plant growth. However, mechanisms on how exogenous carbon affects ERF5 and which genes are affected by ERF require further study. ERF TFs likely play a major role in these regulatory pathways. Identification of their direct target genes will be helpful and will improve our understanding of their sometimes contradictory roles in plant growth.

Exogenous carbon affects Primary metabolism
Many DEGs and DEGs are involved in the process of primary metabolism under exogenous carbon treatment. Compared with CK, beta-glucosidase was up-regulated but chitinase was down-regulated. However, the mechanism of tyrosine/DOPA decarboxylase and xyloglucan galactosyl transferase MUR3 needs further study. These DEPs can be categorized into carbohydrate metabolic process, sucrose metabolic process and tricarboxylic acid cycle.
SUS is a sucrose degrading enzyme in plants [2]. SUS produces more energy than INV during metabolism [55]. Probably because of this reason, the expression of SUS was upregulated in control and could produce additional energy to supply plants without sugar.
Additionally, exogenous fructose significantly reduces leaf and root SUS activity [56], so we speculate that exogenous carbon may be converted into fructose in leaves to reduce SUS activity. Supported by exogenous carbon, the leaves were used as the source organs for energy conversion through photosynthesis. However, under sugar free treatment, SUS activity was high and the leaves sank.
Glyceraldehyde-3-phosphate dehydrogenase is a glycolytic enzyme [57]. Citrate synthase is a key enzyme of the citric acid cycle that provides energy for cellular function [58]. Exogenous carbon affects second metabolism through ethylene signaling Plant secondary metabolism and its metabolites are related to plant function and growth [59]. Different environmental conditions regulate the production of secondary metabolites, such as water, flavonoids [60,61] and others. Under exogenous carbon treatment, the secondary metabolism related genes and proteins expressed differently, especially flavonoid synthesis, phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS).
PAL, the important enzyme linking the secondary metabolism to primary metabolism, participates in the biosynthesis of flavonoids, lignins, stilbenes and many other compounds [62]. PAL can be induced by some environmental conditions, such as sunlight, mechanical wounding, methyl jasmonate and salicylic acid [63][64][65][66]. Sugar is also related to PAL activity. El-Awady [67] indicated that sucrose can induce PAL. However, PAL induction is repressed by glucose [68]. Ethylene is involved in the signaling pathways modulating the production of secondary production in plants cells [69]. Under exogenous carbon, ERF up-regulates the expression of PAL and PAL increased. This finding suggests that exogenous carbon affecting secondary metabolism in leaves is associated with ethylene signaling.
CHS is the first enzyme of the flavonoid biosynthesis pathway [70]. CHS can be induced by sugar [71]. The expression of CHS was not significant compared with CK, but the CHS expression was significantly up-regulated under exogenous carbon treatment. The ethylene antagonist 1-MCP can inhibit CHS [72]. Exogenous ethylene can stimulate genes which are related to anthocyanin biosynthesis increase, such as CHS [73]. Ethylene signaling is associated with secondary metabolism [69]. In transcriptome data, the expression of genes related to flavonoid synthesis was up-regulated. This result indicates that exogenous carbon may promote the synthesis of flavonoids in plants by ethylene signaling. The mechanisms by which exogenous carbon cause changes CHS and CHS remain to be further studied.

Conclusions
This study reveals that exogenous carbon may regulates plant growth through ethylene pathway. Exogenous carbon affects plant growth by inhibiting ethylene biosynthesis and ethylene signaling through ACC oxidase 2, CTR and ERF. However, the increased expression of ERF5 under the action of exogenous carbon may promote plant growth.
Without exogenous carbon supplied, the carbon fixed by photosynthesis will further metabolise through glycolysis and tricarboxylic acid cycling. Exogenous carbon can also promote the synthesis of flavonoids in plant.

Plant materials and growth conditions
The 'Pinot Noir' (V. vinifera L.) samples were collected from the main producing area of Gansu Province, northwest China. The plantlets material was propagated from branches of adult mother plants. The voucher specimens of grape were deposited in the Fruit Tree Physiology and Biotechnology Laboratory, College of Horticulture, Gansu Agricultural University. Those plantlets were grown at 26°C, at a 16 h light and 8 h dark cycle. The average photosynthetic photon flux was 120 μmol·m -2 ·s -1 . One climate chamber (PQX-430D-CO 2 ), which have TC-5000 (T) intelligent CO 2 controller to regulate CO 2 concentration at approximately 1000 μmol·mol -1 . The other chamber was maintained with current atmospheric CO 2 . After conventional propagation, nodal segments (average 20 mm in length) with leaves and with two axillary buds were cultured on modified B5 solid medium containing 0.1 mg·L -1 IAA, 50 mL of medium was taken in 150 mL erlenmeyer flasks, which was using gas-permeable membrane sealing. Then put those explants materials into two climate chambers for treatment. Meanwhile, explants were treated by eCO 2 (1000 μmol·mol -1 ) and sucrose after inoculation for 25 days. We use sucrose and CO 2 to provide exogenous carbon. Growth occurred under following four conditions: Cs: modified B5 solid medium containing 0.1 mg·L -1 IAA with 2% sucrose and eCO 2 ; C0: modified B5 solid medium containing 0.1 mg·L -1 IAA without sucrose but with eCO 2 ; S1: modified B5 solid medium containing 0.1 mg·L -1 IAA with 2% sucrose, ambient CO 2 ; S0: modified B5 solid medium containing 0.1 mg·L -1 IAA without sucrose, ambient CO 2 (380 ± 40 μmol·mol -1 ), we use S0 as CK to analyze the differential expression genes and proteins induced by exogenous carbon. Each treatment had three biological replicates with 15 plantlets per replicate.
Plantlet leaves were harvested at 25 days after inoculation. Fully expanded younger leaves (the third and fourth functional leaves) of the cultivars were sampled. Three independent biological replicates were acquired. Each replicate was collected from more than 10 randomly selected plantlets. The leaf samples were transferred immediately to liquid nitrogen and stored at -80°C for transcriptome and iTRAQ analyses.

Growth parameters
The in vitro growth characteristics assessed after 25 days were as follows: fresh weight of aerial parts (g), fresh weight of underground part (g), dry weight of aerial parts (g), dry weight of underground part (g), total dry mass (g), average leaf area (cm 2 ) and plantlet height (cm).

RNA isolation and library preparation for transcriptome analysis
Total RNA was extracted using the mirVana miRNA Isolation Kit (Ambion). Each sample was evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) as as described previously [35].
Analysis of RNA-Sequencing data Raw microarray data was acquired and analyzed as previously described [35]. Raw data (raw reads) were filtered into clean reads using NGS QC Toolkit [74]. Then the clean reads were mapped to reference genome using hisat2 [75]. A differentially expressed gene was defined as a variation in the gene expression test with a P-value < 0.05 and a fold change (FC) >2 or FC< 0.5. Functional gene classification was performed using the UniProtKB/Swiss-Prot database.

Protein extraction, digestion and iTRAQ labeling
Total proteins were extracted from the leaf tissue of grape in vitro as previously described [35]. The protein concentration was quantified by BCA method [76] and the protein purity was detected by SDS-PAGE [77]. Protein digestion was performed according to the FASP procedure [78]. RP chromatography separation and Mass spectrometry analysis iTRAQ labeled peptides were fractionated by RP chromatography separation using the 1100 HPLC System (Agilent). The specific process as described previously [35].

qRT-PCR analysis
The 10 genes related to ethylene pathways were verified by qRT-PCR. Primer sequences used for qRT-PCR are provided in Table S1.

Statistical analysis
Data are expressed as the mean ± SD from three independent biological replicates.
Significance was determined via one-way analysis of variance (ANOVA).

Consent to publish
Not applicable

Availability of data and materials
The datasets generated and analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.        Figure 1 Effects of exogenous carbon on phenotypes of grape plantlets in vitro. The leaf area, plantlet height and shoot increased significantly in exogenous carbon treatment compared with CK.

Figure 2
Distribution and classification of differentially expressed genes (DEGs) at Cs, C0 and S1 compared with CK. UR and DR representing up-regulation and downregulation, respectively.

Figure 3
Distribution and classification of differentially expressed protein (DEPs) at Cs, C0 and S1 compared with CK.

Figure 4
Ethylene metabolism pathway with up-regulated genes and proteins at exogenous carbon versus CK shown in red. The changing DEGs include 7 of ERFs and 1 CTR.

Figure 5
Gene expression heat map shows differential regulation at Cs, C0 and S1 compared with CK based on fragments per kb per million reads (FPKM). Differentially expressed genes have been categorized into primary metabolism; secondary metabolism; cell morphogenesis; bio-signaling; transcription; translation; transport and stress tolerance. Heat map illustrating the relative protein expression at Cs, C0 and S1 compared with CK based on the DEPs fold change (FC). Differentially expressed proteins have been categorized into primary metabolism; secondary metabolism; energy; biosignaling; translation; transport as well as stress tolerance.

Figure 7
Effects of exogenous carbon on genes and proteins expression in grape plantlets in vitro. The DEGs or DEPs of red was up-regulated and blue was down-regulated.

Supplementary Files
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