Search
Submit Manuscript
1.9CiteScore
Advanced Search
MAP  >  Fruit Research
2024 Volume 4
Article Contents
Abstract
Introduction
Materials and methods
Plant materials, growth conditions, and stress treatment
Vector construction
Chlorophyll and anthocyanin content determination
Physiological analysis
RNA isolation and qRT-PCR analysis
Results
Screening and characterization of lncRNAs
Generation of lncRNA overexpression lines
Effect of lncRNA overexpression on heat tolerance in Arabidopsis
Effect of lncRNA overexpression on the expression quantity of genes related to high-temperature response in Arabidopsis
Effect of lncRNA overexpression on drought tolerance in Arabidopsis
Discussion
Conclusions
Author contributions
Data availability
Acknowledgments
Conflict of interest
Supplementary information
Rights and permissions
References
About this article
Next Previous
ARTICLE   Open Access    

Genome-wide identification and expression profiling of the SWEET family in Actinidia polygama (Sieb. & Zucc.) Maxim.

  • 1.

    College of Horticulture, Jilin Agricultural University, Changchun, Jilin 130118, China

  • 2.

    Key Laboratory of Forest Silviculture and Conservation of the Ministry of Education, College of Forestry, Beijing Forestry University, Beijing 100083, China

  • 3.

    Institute of Agricultural Biotechnology, Jilin Academy of Agricultural Sciences, Changchun 130033, China

  • # Authors contributed equally: Li Chen, Hui-Fang Song, Jia-Xin Liu

More Information
  • Received: 11 December 2023
    Revised: 10 February 2024
    Accepted: 17 February 2024
    Published online: 06 May 2024
    Fruit Research  4,  Article number: e017 (2024)  |  Cite this article
  • Sugar was transported from photosynthetic source cells to sink cells, sugar efflux transporter protein (sugars will eventually be exported to transporters, SWEETs) play an important role in the process. Although SWEET family members had been identified in many plants, transcriptome or genomics analysis of Actinidia polygama SWEET genes remains uncharacterized. In this study, 14 SWEET genes of Actinidia polygama were identified by protein Blast. The structural characteristics of SWEET genes showed that the number of amino acids encoded by the gene family was between 233 and 304, the relative molecular weight was between 25,918.83 and 33,192.12, the isoelectric point was within the range of 6.96 to 9.71, 14 ApSWEET from Actinidia polygama and the known grape and Arabidopsis SWEETs were divided into four clades (I, II, III, and IV) according to the phylogenetic relationships. The gene structure analysis showed that most of ApSWEET genes have six exons and five introns except ApSWEET5 and ApSWEET14. All ApSWEET proteins also contained P-loop, MtN3-slv, and transmembrane domain. Expression patterns of 14 ApSWEET in different organs and at different fruit developmental stages were analyzed. ApSWEET1 and ApSWEET5 exhibited tissue-specific expression, whereas other genes were more ubiquitously expressed. ApSWEET1, ApSWEET10, and ApSWEET11 exhibited higher expression in fruit. The results of this study provide insights into the characteristics of the SWEET genes in Actinidia polygama and may serve as a basis for further functional studies of such genes.

  • Introduction

    Plants are continuously subjected to unpredictable environmental conditions and encounter a multitude of stressors throughout their growth and development, posing a significant challenge to global crop production and food security[1]. Heat and drought are undoubtedly the two most important stresses that have a huge impact on crops. Both elicit a wide array of biochemical, molecular, and physiological alterations and responses, impacting diverse cellular processes and ultimately influencing crop yield and quality[2].

    A primary physiological consequence of both stresses is the diminished photosynthetic capacity, partially resulting from the degradation of chlorophyll due to leaf senescence under stress conditions. Chlorophyll accumulation was diminished in numerous plants subjected to drought or heat stress conditions[3,4]. Various environmental stresses prompt excessive generation of reactive oxygen species (ROS), initiating oxidative damage that compromises lipids, and proteins, and poses a serious threat to cellular functions[2]. To mitigate oxidative stress and minimize damage, plants have developed various protective mechanisms to neutralize ROS. Several antioxidant enzymes, such as SOD, POD, and CAT, are integral to cellular antioxidative defense mechanisms. Additionally, antioxidants such as anthocyanins and proline serve as crucial ROS scavengers[5,6]. The elevation in temperature typically induces the transient synthesis of heat shock proteins (Hsps), which function as molecular chaperones in protecting proteins from denaturation and aggregation, with their activity primarily regulated at the transcriptional level by heat shock factors (Hsfs)[7]. The significance of Hsps and Hsfs in all organisms, including plants, has been assessed in various stress conditions that could disrupt cellular homeostasis and result in protein dysfunction[7]. Drought stress can also trigger the transcription of a suite of marker genes, including RD29A, RD29B, NCED3, AREB1, Rab18, etc., which assist plants in mitigating cellular damage during dehydration and bolstering their resilience to stress[8−10].

    Previous research efforts focusing on the regulatory control of stress-related genes have largely centered around protein-coding genes. In recent years, non-protein-coding transcripts have emerged as important regulatory factors in gene expression. Among them, long non-coding RNAs (lncRNAs) lncRNAs have been identified as implicated in various abiotic stresses[11,12]. LncRNAs are a class of non-coding RNAs (ncRNAs) exceeding 200 nucleotides in length. They possess minimal or no protein-coding potential[13]. In plants, lncRNAs are specifically transcribed by RNA polymerases Pol IV, Pol V, Pol II, and Pol III[14,15]. LncRNAs exhibit low abundance and display strong tissue and cellular expression specificity relative to mRNAs. Moreover, sequence conservation of lncRNAs is was very poor across different plant species[13,16,17]. The widespread adoption of high-throughput RNA sequencing technology has revealed lncRNAs as potential regulators of plant development and environmental responses. In cucumber, RNA-seq analysis has predicted 2,085 lncRNAs to be heat-responsive, with some potentially acting as competitive endogenous RNAs (ceRNAs) to execute their functions[18]. In radish, a strand-specific RNA-seq (ssRNA-seq) technique identified 169 lncRNAs that were differentially expressed following heat treatment[19]. In Arabidopsis, asHSFB2a, the natural antisense transcript of HSFB2a was massively induced upon heat stress and exhibited a counteracted expression trend relative to HSFB2a. Overexpression of asHSFB2a entirely suppressed the expression of HSFB2a and impacted the plant's response to heat stress[20]. For drought stress resistance, 244 lncRNAs were predicted in tomatoes to be drought responsive probably by interacting with miRNAs and mRNAs[21]. Under drought stress and rehydration, 477 and 706 lncRNAs were differentially expressed in drought-tolerant Brassica napus Q2 compared to drought-sensitive B. napus, respectively[22]. In foxtail millet and maize, 19 and 644 lncRNAs, respectively, were identified as drought-responsive[23,24]. Despite the identification of numerous lncRNAs by high-throughput sequencing, which suggests their potential involvement in various abiotic stress processes, only a minority have been experimentally validated for function.

    In our previous study, we characterized 1,229 differentially expressed (DE) lncRNAs in Chinese cabbage as heat-responsive, and subsequent bioinformatics analysis reduced this number to 81, which are more likely associated with heat resistance[25]. lnc000283 and lnc012465 were selected from among them for further functional investigation. The findings indicated that both lnc000283 and lnc012465 could be promptly induced by heat shock (HS). Overexpression of either lnc000283 or lnc012465 in Arabidopsis plants enhanced their capacity to tolerate heat stress. Additionally, both lnc000283 and lnc012465 conferred drought tolerance to transgenic Arabidopsis.

    Materials and methods

    Plant materials, growth conditions, and stress treatment

    The lncRNA sequences examined in this study were from Chiifu-401-42 Chinese cabbage and all Arabidopsis plants were of the Col-0 background. Transgenic plants expressing lnc000283 and lnc012465 were generated using the Agrobacterium tumefaciens-mediated floral dip method[26]. Single-copy and homozygous T3 plants were identified through genetic segregation on an agar medium supplemented with kanamycin. The T3 generation plants, or their homozygous progeny, were utilized in the experiments.

    For phenotypic assessment, Arabidopsis seeds were initially sown on filter paper moistened with ddH2O and placed in a 4 °C freezer for 2 d. Subsequently, they were evenly planted in nutrient-rich soil and transferred to a growth chamber operating a 16-h day/8-h night cycle, with day/night temperatures of 22 °C/18 °C and a light intensity of 250 μmol·m−2·s−1. After 10 d of growth, Arabidopsis plants with uniform growth were transferred to 50-hole plates. Arabidopsis plants grown in Petri dishes were firstly seed-sterilized and then sown on 1/2 MS medium supplemented with 10 g·L−1 sucrose. The seeds were then placed in a 4 °C refrigerator for 2 d in the dark before transferring them to a light incubator. The day/night duration was set to 16 h/8 h, the day/night temperature to 21 °C/18 °C, and the light intensity to 100 μmol·m−2·s−1.

    For heat treatment, 3-week-old seedlings were subjected to 38 °C for 4 d within a light incubator, subsequently transferred to their original growth conditions under the same light/dark cycles. For drought treatment, 3-week-old Arabidopsis seedlings were deprived of water for 10 d, followed by rehydration to facilitate a 2-d recovery period. Plants were photographed and surveyed both before and after treatment.

    Vector construction

    The lncRNA sequences (lnc000283 and lnc012465) were chemically synthesized based on RNA-seq data, with restriction sites for BamH1 and Kpn1 engineered upstream and downstream. The resultant lncRNA constructs were subcloned into the pCambia2301 binary vector, incorporating a cauliflower mosaic virus (CaMV) 35S promoter. The recombinant vectors were transformed into Escherichia coli TOP10 competent cells (Clontech), incubated at 37 °C overnight, after which single clones were selected for PCR verification, and the confirmed positive colonies were submitted for sequencing. Following verification, the correct plasmids were introduced into A. tumefaciens strain GV3101 using the freeze-thaw method and subsequently transformed into Arabidopsis wild-type (Col) plants.

    Chlorophyll and anthocyanin content determination

    To quantify the chlorophyll content, the aerial portions of wild-type and transgenic Arabidopsis plants, grown in Petri dishes were weighed, minced, and then subjected to boiling in 95% ethanol until fully decolorized. Aliquots of 200 μL from the extract were transferred to a 96-well plate and the absorbance at 663 nm and 645 nm was measured via spectrophotometry by a microplate reader (Multiskan GO, Thermo Scientific, Waltham, MA, USA). Three biological replicates were analyzed for WT and each transgenic line. Chlorophyll content was determined according to the formula of the Arnon method[27]: Chlorophyll a = (12.72A663 − 2.59A645) v/w, Chlorophyll b = (22.88A645 − 4.67A663) v/w, Total chlorophyll = (20.29A645 + 8.05A663) v/w.

    The quantification of anthocyanin was performed as follows: aerial parts of wild-type and transgenic Arabidopsis plants, cultivated in Petri dishes, were weighed and ground to powder in liquid nitrogen. Subsequently, the samples were incubated in 600 μL of acidified methanol (containing 1% HCl) at 70 °C for 1 h. Following this, 1 mL of chloroform was added, and the mixture was vigorously shaken to remove chlorophyll. The mixture was then centrifuged at 12,000 rpm for 5 min, after which the absorbance of the aqueous phase was determined at 535 nm using a spectrophotometer (Shimadzu, Kyoto, Japan). Three biological replicates were analyzed for WT and each transgenic line. The relative anthocyanin content was calculated according to anthocyanin concentration and extraction solution volume. One anthocyanin unit is defined as an absorption unit at a wavelength of 535 nm in 1 mL of extract solution. In the end, the quantity was normalized to the fresh weight of each sample.

    Physiological analysis

    Three-week-old transgenic and WT A. thaliana plants, subjected to normal conditions or varying durations of heat or drought stress, were utilized for subsequent physiological assessments. All assays were performed in accordance with the method described by Chen & Zhang[28]. In brief, 0.1 g of fresh leaf tissue was homogenized in 500 μL of 100 mM PBS (pH 7.8) while chilled on ice. The homogenate was then centrifuged at 4 °C, and the resultant supernatant was employed for further analysis. For the determination of MDA content, 100 μL of the supernatant was combined with 500 μL of a 0.25% thiobarbituric acid (TBA) solution (which was prepared by dissolving 0.125 g of TBA in 5 mL of 1 mol·L−1 NaOH before being added to 45 mL of 10% TCA) and boiled for 15 min. Following a 5 min cooling period on ice, the absorbance was measured at 532 nm and 600 nm. The activity of POD was determined as follows: initially, 28 μL of 0.2% guaiacol and 19 μL of 30% H2O2 were sequentially added to 50 mL of 10mM PBS (pH 7.0), after thorough heating and mixing, 1 mL was transferred into a cuvette, then 50 μL of the supernatant was added to the cuvette and the absorbance at 470 nm was monitored every 15 s for 1 min. To determine the proline content, a reaction solution was prepared by mixing 3% sulfosalicylic acid, acetic acid, and 2.5% acidic ninhydrin in a ratio of 1:1:2, then 50 μL of the supernatant was added to 1 mL of the reaction solution, which was then subjected to a boiling water bath for 15 min (the solution turned red after the boiling water bath). Following cooling on ice, the absorbance at 520 nm was recorded. For the quantification of proline, an L-proline standard curve was prepared by dissolving 0, 5, 10, 15, 20, 25, and 30 μg of L-proline in 0.5 mL of ddH2O, followed by the addition of 1 mL of the reaction solution and measuring the absorbance at 520 nm. The proline content in the samples was then determined based on the L-proline standard curve.

    RNA isolation and qRT-PCR analysis

    Total RNA was isolated from the aerial parts of Arabidopsis using the TaKaRa MiniBEST Plant RNA Extraction Kit, followed by purification and reverse transcription using the PrimeScript RT reagent Kit with gDNA Eraser (Takara). The cDNA product was diluted 10 times and real-time PCR was conducted in triplicate for each biological replicate using SYBR PCR Master Mix (Applied Biosystems) on the ABI 7500 system under the following conditions: 98 °C for 3 min, followed by 40 cycles of 98 °C for 2 s and 60 °C for 30 s. The relative expression levels of each gene were normalized against the transcript abundance of the endogenous control UBC30 (At5g56150) and calculated using the 2−ΔCᴛ method. The specific primers employed for qRT-PCR are detailed in Supplemental Table S1.

    Results

    Screening and characterization of lncRNAs

    In our prior investigation, dozens of lncRNAs associated with the heat stress response in Chinese cabbage were identified through informatics analysis. Two lncRNAs (lnc000283 and lnc012465) were chosen for genetic transformation in Arabidopsis to elucidate their functions comprehensively. Transcriptome data analysis indicated that the expression of lnc000283 and lnc012465 in Chinese cabbage were both induced by HS. To verify the accuracy, the expression patterns of lnc000283 and lnc012465 were confirmed through quantitative real-time PCR (qRT-PCR), and the results from qRT-PCR were consistent with those obtained from RNA-seq (Fig. 1a). The corresponding homologous genes in Arabidopsis were identified as CNT2088434 and CNT2088742, exhibiting sequence similarities of 88% and 87%, respectively (Supplemental Fig. S1). Subcellular localization predictions using the lnclocator database (www.csbio.sjtu.edu.cn/bioinf/lncLocator) suggested that both lncRNAs are localized within the nucleus (Supplemental Table S2). Bioinformatics analysis was conducted using the CPC tool (http://cpc.cbi.pku.edu.cn/) indicated that lnc000283 and lnc012465 are noncoding sequences, with coding probabilities of 0.0466805 and 0.0432148, respectively comparable to the well-characterized lncRNAs COLDAIR and Xist, but significantly lower than those of the protein-coding genes UBC10 and ACT2 (Fig. 1b).

    Figure 1.  Characteristics of lnc000283 and lnc012465. (a) Expression level of lnc000283 and lnc012465 in Chinese cabbage leaves treated at 38 °C at different time points, as determined by qRT-PCR and RNA-seq. CK is a representative plant before heating, and T1, T4, T8, and T12 denote plants that were subjected to 38 °C for 1, 4, 8, and 12 h, respectively. The expression levels were normalized to the expression level of Actin. (b) Analysis of coding potential for lnc000283 and lnc012465. The coding potential scores were calculated using the CPC program. UBC10 (At5g53300) and ACT2 (At3g18780) are positive controls that encode proteins. COLDAIR (HG975388) and Xist (L04961) serve as negative controls, exhibiting minimal protein-coding potential.
    DownLoad: Full-Size Img PowerPoint

    Generation of lncRNA overexpression lines

    To elucidate the role of lnc000283 and lnc012465 in response to abiotic stress, overexpression vectors were constructed for these lncRNAs, driven by the CaMV 35S promoter, and they were introduced into Arabidopsis thaliana (Col-0 ecotype). Through PCR identification and generational antibiotic screening, two homozygous positive lines for lnc012465 and lnc000283 were obtained. The relative expression levels of these lncRNAs were assessed using qRT-PCR (Fig. 2a). When plants were grown in 1/2 MS medium, with the consumption of nutrients, and reduction of water, the leaves of WT began to turn yellow, but the lnc000283 and lnc012465 overexpression lines developed a deep purple color of leaf veins (Fig. 2b). Examination of chlorophyll and anthocyanin contents in the plants revealed that both overexpression lines had higher levels of chlorophyll and anthocyanin compared to the WT, suggesting that the transgenic plants might possess enhanced resistance to nutritional or water stress (Fig. 2c, d).

    Figure 2.  Arabidopsis plants overexpressing lnc000283 and lnc012465 had higher anthocyanins and chlorophyll content. (a) The relative expression level of lnc000283 and lnc012465 in WT and different transgenic lines. UBC10 (At5g53300) was used as an internal control. Each value is mean ± sd (n = 3). (b) The phenotype of WT and Arabidopsis overexpressing lnc000283 or lnc012465 grown on 1/2 MS medium 50 d after sowing. The (c) anthocyanin and (d) chlorophyll content of WT and transgenic Arabidopsis overexpressing lnc000283 or lnc012465. The asterisks above the bars indicate statistical significance using Student's t-test (*, p < 0.05; **, p < 0.01).
    DownLoad: Full-Size Img PowerPoint

    Effect of lncRNA overexpression on heat tolerance in Arabidopsis

    Given that lnc000283 and lnc012465 were highly induced by heat, the thermotolerance of the overexpressing (OE) plants were compared to that of the wild type. Arabidopsis plants were initially exposed to a an HS treatment at 38 °C for 4 d, followed by recovery at room temperature. The death caused by HS was processive. Post-severe HS challenge for 4 d, OE plants initially appeared similar to WT, but upon recovery, their leaves started to fold or curl, followed by a transition to yellow, white, and eventually drying out (Fig. 3a). OE lnc000283 and OE lnc012465 plants exhibited enhanced thermotolerance compared to WT, with lnc012465 showing particularly strong tolerance (Fig. 3a; Supplemental Fig. S2a). After 5 d of recovery, leaf coloration indicated that transgenic plants maintained a significantly higher percentage of green leaves and a lower percentage of bleached leaves compared to WT (Fig. 3b; Supplemental Fig. S2b). Under non-heat-stress conditions, WT and OE plants possessed comparable water content. However, following heat stress, the fresh-to-dry weight ratio of OE lnc000283 and lnc012465 lines was significantly greater than that of WT (Fig. 3c; Supplemental Fig. S2c). Abiotic stresses frequently trigger the production of excessive reactive oxygen species (ROS), which are believed to cause lipid peroxidation of membrane lipids, leading to damage to macromolecules. Leaf MDA content is commonly used as an indicator of lipid peroxidation under stress conditions; therefore, the MDA content in both transgenic and WT plants was assessed. Figure 3d shows that the MDA content in WT plants progressively increased after heat treatment, whereas in the two lines overexpressing lnc012465, the MDA content increased only slightly and remained significantly lower than that in WT at all time points. In plants overexpressing lnc000283, the MDA content did not significantly differ from that of WT before heat stress. However, after 4 d of heat treatment, the MDA content was significantly lower compared to WT (Supplemental Fig. S2d). The results suggested that the expression of both lnc012465 and lnc000283 can mitigate injury caused by membrane lipid peroxidation under heat-stress conditions. Peroxidase (POD) is a crucial antioxidant enzyme involved in ROS scavenging. Figure 3e and Supplemental Fig. S2e demonstrate that POD activity increased in both transgenic and WT plants after heat treatment. However, the increase in WT plants was modest, whereas OE lnc000283 and OE lnc012465 plants exhibited consistently higher POD activity. As anticipated, proline levels were induced in response to stress in all studied plants (Fig. 3f; Supplemental Fig. S2f). However, under normal conditions and 2 d post-heat stress treatment, the proline content in OE lnc000283 and OE lnc012465 plants did not exhibit significant changes compared to WT (Fig. 3f; Supplemental Fig. S2f). Moreover, after 4 d of heat stress, the proline content in OE lnc012465 lines was significantly lower than in WT, and the OE lnc000283 transgenic line 12-6 also showed a marked decrease in proline content compared to WT (Fig. 3f; Supplemental Fig. S2f). The results indicated that the thermotolerance of plants overexpressing either lnc000283 or lnc012465 was independent of proline accumulation.

    Figure 3.  Overexpressing lnc012465 lines are more tolerant to heat stress. (a) Phenotypes of WT and OE lnc012465 plants were assessed before and after exposure to heat stress. The heat treatment was applied to 25-day-old Arabidopsis plants. (b) The percentage of leaves with different colors in Arabidopsis after heat treatment and recovery for 5 d. (c) The fresh-to-dry weight ratio of Arabidopsis leaves was measured before and after 38 °C heat treatment. (d)−(f) depict the MDA content, POD activity, and proline content in Arabidopsis leaves at varying durations of heat stress. The asterisks above the bars indicate statistical significance using Student's t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).
    DownLoad: Full-Size Img PowerPoint

    Effect of lncRNA overexpression on the expression quantity of genes related to high-temperature response in Arabidopsis

    To elucidate the molecular mechanisms by which lncRNAs enhance thermotolerance in Arabidopsis, the expression of the Hsf gene HsfA7a and three Hsps (Hsp25.3, Hsa32, and Hsp18.1-CI) in OE lnc000283, OE lnc012465, and WT Arabidopsis plants were investigated at various time points following heat treatment. As shown in Fig. 4 and Supplemental Fig. S3, both Hsf and Hsps exhibited a rapid response to heat stress with strong induction. Notably, the transcripts of HsfA7a and Hsp25.3 were significantly upregulated at 1 h after heat exposure, then experienced a sharp decrease. Hsa32 and Hsp18.1-CI were highly induced at 1 h and, unlike the other proteins, sustained high expression levels at 3 h (Fig. 4; Supplemental Fig. S3). At 1 h post-heat treatment, the transcript levels of Hsa32 and HsfA7a in OE lnc000283 did not significantly differ from those in WT. However, by 3 h, Hsa32 expression was roughly 50% of the WT level, while HsfA7a expression was approximately double that of WT (Supplemental Fig. S3). The overexpression of lnc000283 did not significantly affect the transcript level of Hsp25.3 at any of the tested time points. Notably, Hsp18.1-CI expression in both lines overexpressing lnc000283 was significantly induced at all three detection points post-heat treatment, reaching approximately 4-9-fold higher levels than in the WT (Supplemental Fig. S3). In Arabidopsis plants with elevated expression of lnc012465, the expression patterns of all Hsp and Hsf genes were similar to those in plants overexpressing lnc000283, with the notable exception of Hsa32. Unlike the WT, Hsa32 did not show a trend of down-regulation at 3 h post-heat treatment (Fig. 4). The findings suggest that the substantial induction of Hsp18.1-CI may play a role in enhancing the thermotolerance of Arabidopsis plants overexpressing lnc000283 and lnc012465.

    Figure 4.  The expression of HSF and HSP genes in lnc012465 overexpressing lines before and after different heat treatment times. Gene expression levels were quantified using RT-qPCR and normalized to UBC10 (At5g53300). Each value represents the mean ± standard deviation (n = 3). The asterisks above the bars indicate statistical significance using Student's t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).
    DownLoad: Full-Size Img PowerPoint

    Effect of lncRNA overexpression on drought tolerance in Arabidopsis

    Prior research has implicated a significant proportion of genes in conferring resistance to various abiotic stresses. To elucidate the functions of lnc000283 and lnc012465 more thoroughly, WT and transgenic plants were subjected to drought stress by depriving them of water for 9 d. It was noted that the majority of leaves in WT plants withered and dried, whereas the OE lnc000283 and OE lnc012465 plants exhibited reduced withering, with only a minority displaying dryness (Fig. 5a; Supplemental Fig. S4a). Eight days post-rewatering, a negligible fraction of WT seedlings exhibited recovery, whereas the overwhelming majority of transgenic plants regained vigorous growth (Fig. 5a; Supplemental Fig. S4a). The transgenic plants demonstrated a significantly higher survival rate compared to the WT plants. Following 9 d of water deficit treatment, less than 40% of the WT plants survived, whereas the OE 012465 lines 8-7 and 9-1 exhibited survival rates of 100% and 95%, respectively, and the OE 000283 lines 11-10 and 12-6 had survival rates of 87% each. (Fig. 5b; Supplemental Fig. S4b). Water loss serves as a critical metric for assessing plant drought tolerance, hence the fresh-to-dry weight ratio of excised leaves was assessed via desiccation analysis. Following 4 d of drought treatment, the fresh-to-dry weight ratio for WT plants was reduced to 43%, whereas for OE lnc000283 lines 11-10 and 12-6, it was reduced to 73% and 75%, respectively. For OE 012465 lines 8-7 and 9-1, the ratios were reduced to 67% and 62%, respectively (Fig. 5c; Supplemental Fig. S4c). The findings indicated that lnc000283 and lnc012465 endow the transgenic plants with drought tolerance.

    Figure 5.  Overexpressing lnc012465 lines are more tolerant to drought stress. (a) Phenotype of WT and OE lnc012465 plants before and after subjecting to drought stress. Drought treatment was carried out on 20-day-old Arabidopsis plants. (b) The percentage of leaves with different colors in Arabidopsis after heat treatment and recovery for 5 d. (c) The fresh weight to dry weight ratio of Arabidopsis leaves before and after undergoing 38 °C heat treatment. (d)−(f) MDA content, POD activity, and proline content in Arabidopsis leaves under different times of heat stress. The asterisks above the bars indicate statistical significance using Student's t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001)
    DownLoad: Full-Size Img PowerPoint

    MDA content in leaves is a standard biomarker for assessing the extent of drought stress-induced damage. Prior to drought stress exposure, MDA levels in WT and transgenic plants were comparable. However, following 7 and 9 d of water deficit, the MDA content in the transgenic plants was markedly reduced compared to the WT, suggesting a less severe degree of membrane lipid peroxidation in the transgenic plants (Fig. 5d; Supplemental Fig. S4d). Oxidative stress frequently coincides with drought stress, hence the activity of POD was assessed to evaluate the ROS scavenging ability. The findings indicated that as the duration of drought treatment increased, POD activity progressively rose. Before drought exposure, the POD activity in lines 11-10 and 12-6 of OE 000283 was 2.4-fold and 2.2-fold higher than that of the WT, respectively (refer to Supplemental Fig. S4e). Following drought treatment, the POD activity in the transgenic lines remained significantly elevated compared to the wild type, although the enhancement was less pronounced than before the treatment (Supplemental Fig. S4e). In the OE 012465 plants, the POD activity in lines 8-7 and 9-1 significantly surpassed that of the wild type, with the discrepancy being more pronounced during drought stress (Fig. 5e). The proline content in WT and OE 000283 plants exhibited no significant differences before and after 7 d of treatment. However, after 9 d of drought, the proline content in OE 000283 plants was significantly lower compared to that in the WT (Supplemental Fig. S4f). OE 000465 plants showed no significant difference from the wild type before and after drought treatment (Fig. 5f). The findings were consistent with those under heat stress, indicating that the enhanced stress resistance due to the overexpression of lnc000283 and lnc012465 in Arabidopsis is not reliant on proline accumulation.

    Following drought stress treatment, the expression levels of drought-related genes such as RD29A, RD29B, NCED3, AREB1, and Rab18 were significantly elevated in plants overexpressing lnc000283 and lnc012465 compared to WT plants. These findings suggest that lnc000283 and lnc012465 modulate Arabidopsis drought tolerance by regulating the expression of genes associated with the drought stress response (Fig. 6; Supplemental Fig. S5).

    Figure 6.  The expression of drought-responsive genes in lnc012465 overexpressing lines before and after different drought treatment time. Gene expression levels were determined by qRT-PCR normalized against UBC10 (At5g53300). Each value is mean ± sd (n = 3). The asterisks above the bars indicate statistical significance using Student's t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).
    DownLoad: Full-Size Img PowerPoint

    Discussion

    The integrity of global food security is under threat due to the confluence of rapid population expansion and profound climatic shifts[29]. Amidst the shifting climatic landscape, heat and drought stress have emerged as primary limitations to crop yield and global food security. Understanding how plants detect stress cues and acclimate to challenging conditions is a pivotal biological inquiry. Moreover, enhancing plant resilience to stress is essential for maintaining agricultural productivity and fostering environmental sustainability[2]. Concurrently, the advancement of next-generation sequencing (NGS) technology has led to the identification of a substantial number of lncRNAs that participate in diverse stress responses, with functional analyses having been conducted on several of these molecules.[30] For instance, in the case of potatoes, the lncRNA StFLORE has been identified to modulate water loss through its interaction with the homologous gene StCDF1[31]. LncRNA TCONS_00021861 can activate the IAA biosynthetic pathway, thereby endowing rice with resistance to drought stress[32]. In wheat, the expression of TalnRNA27 and TalnRNA5 was upregulated in response to heat stress[33]. Our prior investigation identified a total of 81 lncRNAs in Chinese cabbage that engage in intricate interactions with their respective mRNA targets across various phases of heat treatment[25]. Two lncRNAs, lnc000283 and lnc012465, were chosen for subsequent functional analysis. Findings confirmed that these lncRNAs endow transgenic Arabidopsis plants with enhanced tolerance to both heat and drought, thereby offering novel resources for enhancing stress resistance through genetic engineering.

    Abiotic stresses frequently trigger the synthesis of anthocyanins, serving as natural antioxidants that mitigate oxidative damage by neutralizing surplus reactive oxygen species (ROS), thereby protecting plants from growth inhibition and cell death, allowing plants to adapt to abiotic stresses[34,35]. For instance, during chilling stress, the accumulation of anthocyanins within leaves can mitigate oxidative damage, thereby enhancing the photosynthetic rate[36]. Consequently, the level of abiotic stress tolerance can be inferred from the concentration of anthocyanins. The reduction of photosynthetic ability is one of the key physiological phenomena of stresses, which is partly due to the degradation of chlorophyll caused by leaf senescence during stress. The reduced accumulation of chlorophyll in the plants was seen in many plants when exposed to drought or heat stress conditions. The current investigation revealed that lncRNA-overexpressing plants cultivated in Petri dishes exhibited increased accumulation of both chlorophyll and anthocyanins in advanced growth phases, indicating that these transgenic plants, overexpressing lnc000283 and lnc012465, demonstrated enhanced stress tolerance and superior growth performance relative to WT (Fig. 2c, d).

    Upon exposure to heat stress, there is a marked induction of transcription for numerous genes that encode molecular chaperones in plants, with the vast majority of these genes contributing to the prevention of protein denaturation-related damage and the augmentation of thermotolerance[37−39]. The present investigation identified multiple heat-inducible genes in plants overexpressing lnc000283 and lnc012465, as well as in WT (Fig. 4; Supplemental Fig. S3). The findings indicated that of the four HSP or HSF genes examined, Hsp18.1-CI exhibited a significantly greater abundance in both OE lnc000283 and OE lnc012465 plants compared to the WT following heat treatment for several days. Hsp18.1-CI, formerly referred to as Hsp18.2 has been the subject of investigation since 1989.[40] Following the fusion of the 5' region of Hsp18.2 in frame with the uidA gene of Escherichia coli, the activity of GUS, serving as the driver gene was observed to increase upon exposure to HS[40]. The Arabidopsis hsfA2 mutant exhibited diminished thermotolerance after heat acclimation, with the transcript levels of Hsp18.1-CI being substantially reduced compared to those in wild-type plants following a 4-h recovery period[41]. The findings revealed that the upregulation of Hsp18.1-CI protein is a critical mechanism by which plants achieve enhanced protection against heat stress in adverse environmental conditions, thereby bolstering their thermotolerance.

    Plants cultivated in natural settings are often subjected to concurrent multiple abiotic stresses, which can exacerbate threats to their routine physiological functions, growth, and developmental processes[42,43]. Elucidating the molecular mechanisms underlying plant responses to abiotic stress is crucial for the development of new crop varieties with enhanced tolerance to multiple abiotic stresses. Previous research has indicated that the overexpression of certain protein-coding genes can endow plants with resistance to a variety of abiotic stresses. For instance, tomatoes with robust expression of ShCML44 demonstrated significantly enhanced tolerance to drought, cold, and salinity stresses[44]. Overexpression of PeCBF4a in poplar plants confers enhanced tolerance to a range of abiotic stresses[45]. With respect to lncRNAs, transgenic Arabidopsis plants that overexpress lncRNA-DRIR displayed marked increased tolerance to salt and drought stresses compared to the wild-type[46]. In the present study, both overexpression lines of lnc000283 and lnc012465 exhibited resistance to heat and drought stresses, thereby contributing to the enhancement of plant resilience against multiple stresses (Figs 3, 5; Supplemental Figs S2, S4).

    The number of genes implicated in plant drought resistance is regulated by both ABA-dependent and ABA-independent pathways[47,48]. It is well established that the expression of RD29A exhibits a high level of responsiveness to drought stress, operating through both ABA-dependent and ABA-independent mechanisms[49]. RD29B, AREB1, and RAB18 are governed by an ABA-dependent regulatory pathway[10,49,50]. NCED3 is involved in ABA biosynthesis[51]. In the present study, the transcript levels of RD29A, RD29B, NCED3, AREB1, and RAB18 were significantly elevated in OE lnc000283 and OE lnc012465 plants compared to those in the WT plants (Fig. 6; Supplemental Fig. S5). The findings indicated that the drought tolerance imparted by OE lnc000283 and OE lnc012465 plants is contingent upon an ABA-dependent mechanism.

    Prior research has indicated that certain long non-coding RNAs (lncRNAs) can assume analogous roles across diverse biological contexts. For example, the lncRNA bra-miR156HG has been shown to modulate leaf morphology and flowering time in both B. campestris and Arabidopsis[52]. Heterogeneous expression of MSL-lncRNAs in Arabidopsis has been associated with the promotion of maleness, and similarly, it is implicated in the sexual lability observed in female poplars[53]. In the present study, lnc000283 and lnc012465 were induced by heat in Chinese cabbage, and their heterologous expression was found to confer heat tolerance in Arabidopsis. Additionally, sequences homologous to lnc000283 and lnc012465 were identified in Arabidopsis (Supplemental Fig. S1). The data suggest that these sequences may share a comparable function to that of heat-inducible sequences, potentially accounting for the conservation of lnc000283 and lnc012465'os functionality across various species.

    Conclusions

    In conclusion, the functions of two heat-inducible lncRNAs, lnc000283 and lnc012465 have been elucidated. Transgenic Arabidopsis lines overexpressing these lncRNAs accumulated higher levels of anthocyanins and chlorophyll at a later stage of growth compared to the WT when grown on Petri dishes. Furthermore, under heat and drought stress conditions, these OE plants exhibited enhanced stress tolerance, with several genes related to the stress resistance pathway being significantly upregulated. Collectively, these findings offer novel insights for the development of new varieties with tolerance to multiple stresses.

    Author contributions

    The authors confirm contribution to the paper as follows: study conception and supervision: Li N, Song X; experiment performing: Wang Y, Sun S; manuscript preparation and revision: Wang Y, Feng X, Li N. All authors reviewed the results and approved the final version of the manuscript.

    Data availability

    All data generated or analyzed during this study are included in this published article and its supplementary information files.

    Acknowledgments

    This work was supported by the National Natural Science Foundation of China (32172583), the Natural Science Foundation of Hebei (C2021209019), the Natural Science Foundation for Distinguished Young Scholars of Hebei (C2022209010), and the Basic Research Program of Tangshan (22130231H).

  • The authors declare that they have no conflict of interest.

  • Supplemental Table S1 The protein sequences of SWEET genes from A. polygama.
    Supplemental Table S2 The protein sequences of AtSWEETs.
    Supplemental Table S3 The protein sequences of VvSWEETs.
    Supplemental Table S4 The primer sequences of ApSWEET genes for qRT-PCR.
  • [1]

    Patrick JW. 1997. Phloem unloading: sieve element unloading and post-sieve element transport. Annual Review of Plant Biology 48:191−222

    doi: 10.1146/annurev.arplant.48.1.191

    CrossRef   Google Scholar

    [2]

    Hall AJ, Minchin PEH, Gould N, Clearwater MJ. 2017. A biophysical model of fruit development with distinct apoplasmic and symplasmic pathways. Acta Horticulturae 1160:367−374

    Google Scholar

    [3]

    Xiao W, Sheen J, Jang JC. 2000. The role of hexokinase in plant sugar signal transduction and growth and development. Plant Molecular Biology 44:451−61

    doi: 10.1023/A:1026501430422

    CrossRef   Google Scholar

    [4]

    Kühn C, Grof CPL. 2010. Sucrose transporters of higher plants. Current Opinion in Plant Biology 13:288−98

    doi: 10.1016/j.pbi.2010.02.001

    CrossRef   Google Scholar

    [5]

    White PJ, Ding G. 2023. Long-distance transport in the xylem and phloem. In Marschner's Mineral Nutrition of Higher Plants, 4th edition, eds. Rengel Z, Cakmak I, White PJ. Academic Press. pp. 73−104. https://doi.org/10.1016/B978-0-12-819773-8.00002-2
    [6]

    Li Y, Liang G, Nai G, Lu S, Ma W, et al. 2023. VaSUS2 confers cold tolerance in transgenic tomato and Arabidopsis by regulation of sucrose metabolism and ROS homeostasis. Plant Cell Reports 42:505−20

    doi: 10.1007/s00299-022-02972-w

    CrossRef   Google Scholar

    [7]

    Balparda M, Bouzid M, Martinez MDP, Zheng K, Schwarzländer M, et al. 2023. Regulation of plant carbon assimilation metabolism by post-translational modifications. The Plant Journal 114:1059−79

    doi: 10.1111/tpj.16240

    CrossRef   Google Scholar

    [8]

    Garg V, Kühn C. 2022. Subcellular dynamics and protein-protein interactions of plant sucrose transporters. Journal of Plant Physiology 273:153696

    doi: 10.1016/j.jplph.2022.153696

    CrossRef   Google Scholar

    [9]

    Chen LQ, Qu XQ, Hou BH, Sosso D, Osorio S, et al. 2011. Sucrose efflux mediated by SWEET proteins as a key step for phloem transport. Science 335:207−11

    doi: 10.1126/science.1213351

    CrossRef   Google Scholar

    [10]

    Bai Q, Chen X, Zheng Z, Feng J, Zhang Y, et al. 2023. Vacuolar Phosphate Transporter1 (VPT1) may transport sugar in response to soluble sugar status of grape fruits. Horticulture Research 10:uhac260

    doi: 10.1093/hr/uhac260

    CrossRef   Google Scholar

    [11]

    Hedrich R, Sauer N, Neuhaus HE. 2015. Sugar transport across the plant vacuolar membrane: nature and regulation of carrier proteins. Current Opinion in Plant Biology 25:63−70

    doi: 10.1016/j.pbi.2015.04.008

    CrossRef   Google Scholar

    [12]

    Gautam T, Dutta M, Jaiswal V, Zinta G, Gahlaut V, et al. 2022. Emerging roles of SWEET sugar transporters in plant development and abiotic stress responses. Cells 11:1303

    doi: 10.3390/cells11081303

    CrossRef   Google Scholar

    [13]

    Salvi P, Agarrwal R, Kaja, Gandass N, Manna M, et al. 2022. Sugar transporters and their molecular tradeoffs during abiotic stress responses in plants. Physiologia Plantarum 174:e13652

    doi: 10.1111/ppl.13652

    CrossRef   Google Scholar

    [14]

    Yang C, Zhao X, Luo Z, Wang L, Liu M. 2023. Genome-wide identification and expression profile analysis of SWEET genes in Chinese jujube. Peer J 11:e14704

    doi: 10.7717/peerj.14704

    CrossRef   Google Scholar

    [15]

    Gao Y, Wang ZY, Kumar V, Xu XF, Yuan DP, et al. 2018. Genome-wide identification of the SWEET gene family in wheat. Gene 642:284−92

    doi: 10.1016/j.gene.2017.11.044

    CrossRef   Google Scholar

    [16]

    Kumawat S, Sharma Y, Vats S, Sudhakaran S, Sharma S, et al. 2022. Understanding the role of SWEET genes in fruit development and abiotic stress in pomegranate (Punica granatum L.). Molecular Biology Reports 49:1329−39

    doi: 10.1007/s11033-021-06961-2

    CrossRef   Google Scholar

    [17]

    Hir RL, Spinner L, Klemens PAW, Chakraborti D, de Marco F, et al. 2015. Disruption of the sugar transporters AtSWEET11 and AtSWEET12 affects vascular development and freezing tolerance in Arabidopsis. Molecular Plant 8:1687−90

    doi: 10.1016/j.molp.2015.08.007

    CrossRef   Google Scholar

    [18]

    Hu W, Hua X, Zhang Q, Wang J, Shen Q, et al. 2018. New insights into the evolution and functional divergence of the SWEET family in Saccharum based on comparative genomics. BMC Plant Biology 18:270

    doi: 10.1186/s12870-018-1495-y

    CrossRef   Google Scholar

    [19]

    Liu HT, Lyu WY, Tian SH, Zou XH, Zhang LQ, et al. 2019. The SWEET family genes in strawberry: identification and expression profiling during fruit development. South African Journal of Botany 125:176−87

    doi: 10.1016/j.sajb.2019.07.002

    CrossRef   Google Scholar

    [20]

    Miao H, Sun P, Liu Q, Miao Y, Liu J, et al. 2017. Genome-wide analyses of SWEET family proteins reveal involvement in fruit development and abiotic/biotic stress responses in banana. Scientific Reports 7:3536

    doi: 10.1038/s41598-017-03872-w

    CrossRef   Google Scholar

    [21]

    Li Y, Feng S, Ma S, Sui X, Zhang Z. 2017. Spatiotemporal expression and substrate specificity analysis of the cucumber SWEET gene family. Frontiers in Plant Science 8:1855

    doi: 10.3389/fpls.2017.01855

    CrossRef   Google Scholar

    [22]

    Feng CY, Han JX, Han XX, Jiang J. 2015. Genome-wide identification, phylogeny, and expression analysis of the SWEET gene family in tomato. Gene 573:261−72

    doi: 10.1016/j.gene.2015.07.055

    CrossRef   Google Scholar

    [23]

    Wei X, Liu F, Chen C, Ma F, Li M. 2014. The Malus domestica sugar transporter gene family: identifications based on genome and expression profiling related to the accumulation of fruit sugars. Frontiers in Plant Science 5:569

    doi: 10.3389/fpls.2014.00569

    CrossRef   Google Scholar

    [24]

    Eckardt NA. 2021. Sweeter than SWEET: a single-cell leaf vasculature transcriptome atlas. The Plant Cell 33:445−46

    doi: 10.1093/plcell/koaa059

    CrossRef   Google Scholar

    [25]

    Chen LQ. 2014. SWEET sugar transporters for phloem transport and pathogen nutrition. New Phytologist 201:1150−55

    doi: 10.1111/nph.12445

    CrossRef   Google Scholar

    [26]

    Chandran D. 2015. Co-option of developmentally regulated plant SWEET transporters for pathogen nutrition and abiotic stress tolerance. IUBMB Life 67:461−71

    doi: 10.1002/iub.1394

    CrossRef   Google Scholar

    [27]

    Guan Y, Huang X, Zhu J, Gao J, Zhang H, et al. 2008. RUPTURED POLLEN GRAIN1, a member of the MtN3/saliva gene family, is crucial for exine pattern formation and cell integrity of microspores in Arabidopsis. Plant Physiology 147:852−63

    doi: 10.1104/pp.108.118026

    CrossRef   Google Scholar

    [28]

    Chong J, Piron MC, Meyer S, et al. 2014. The SWEET family of sugar transporters in grapevine: VvSWEET4 is involved in the interaction with Botrytis cinerea. Journal of Experimental Botany 65:6589−601

    doi: 10.1093/jxb/eru375

    CrossRef   Google Scholar

    [29]

    Breia R, Conde A, Pimentel D, Conde C, Fortes AM, et al. 2020. VvSWEET7 is a mono-and disaccharide transporter up-regulated in response to botrytis cinerea infection in grape berries. Frontiers in Plant Science 10:1753

    doi: 10.3389/fpls.2019.01753

    CrossRef   Google Scholar

    [30]

    Klemens PAW, Patzke K, Deitmer J, Spinner L, Le Hir R, et al. 2013. Overexpression of the vacuolar sugar carrier AtSWEET16 modifies germination, growth, and stress tolerance in Arabidopsis. Plant Physiology 163:1338−52

    doi: 10.1104/pp.113.224972

    CrossRef   Google Scholar

    [31]

    Seo PJ, Park JM, Kang SK, Kim SG, Park CM. 2011. An Arabidopsis senescence-associated protein SAG29 regulates cell viability under high salinity. Planta 233:189−200

    doi: 10.1007/s00425-010-1293-8

    CrossRef   Google Scholar

    [32]

    Julius BT, Leach KA, Tran TM, Mertz RA, Braun DM. 2017. Sugar transporters in plants: new insights and discoveries. Plant and Cell Physiology 58:1442−60

    doi: 10.1093/pcp/pcx090

    CrossRef   Google Scholar

    [33]

    Durand M, Porcheron B, Hennion N, Maurousset L, Lemoine R, et al. 2016. Water deficit enhances C export to the roots in Arabidopsis thaliana plants with contribution of sucrose transporters in both shoot and roots. Plant Physiology 170:1460−79

    doi: 10.1104/pp.15.01926

    CrossRef   Google Scholar

    [34]

    Huang D, Chen Y, Liu X, Ni D, Bai L, et al. 2022. Genome-wide identification and expression analysis of the SWEET gene family in daylily (Hemerocallis fulva) and functional analysis of HfSWEET17 in response to cold stress. BMC Plant Biology 22:211

    doi: 10.1186/s12870-022-03609-6

    CrossRef   Google Scholar

    [35]

    Nie P, Xu G, Yu B, Lyu D, Xue X, et al. 2022. Genome-wide identification and expression profiling reveal the potential functions of the SWEET gene family during the sink organ development period in apple (Malus × domestica Borkh.). Agronomy 12:1747

    doi: 10.3390/agronomy12081747

    CrossRef   Google Scholar

    [36]

    Xie H, Wang D, Qin Y, Ma A, Fu J, et al. 2019. Genome-wide identification and expression analysis of SWEET gene family in Litchi chinensis reveal the involvement of LcSWEET2a/3b in early seed development. BMC Plant Biology 19:499

    doi: 10.1186/s12870-019-2120-4

    CrossRef   Google Scholar

    [37]

    Wang J, Xue X, Zeng H, Li J, Chen L. 2022. Sucrose rather than GA transported by AtSWEET13 and AtSWEET14 supports pollen fitness at late anther development stages. New Phytologist 236:525−37

    doi: 10.1111/nph.18368

    CrossRef   Google Scholar

    [38]

    Schmitt AJ, Roy R, Klinkenberg PM, Jia M, Carter CJ. 2018. The octadecanoid pathway, but not COI1, is required for nectar secretion in Arabidopsis thaliana. Frontiers in Plant Science 9:1060

    doi: 10.3389/fpls.2018.01060

    CrossRef   Google Scholar

    [39]

    Lin IW, Sosso D, Chen LQ, Gase K, Kim SG, et al. 2014. Nectar secretion requires sucrose phosphate synthases and the sugar transporter SWEET9. Nature 508:546−49

    doi: 10.1038/nature13082

    CrossRef   Google Scholar

    [40]

    Fei H, Yang Z, Lu Q, Wen X, Zhang Y, et al. 2021. OsSWEET14 cooperates with OsSWEET11 to contribute to grain filling in rice. Plant Science 306:110851

    doi: 10.1016/j.plantsci.2021.110851

    CrossRef   Google Scholar

    [41]

    Xiao Q, Zhen L, Wang Y, Hou X, Wei X, et al. 2022. Genome-wide identification, expression and functional analysis of sugar transporters in sorghum (Sorghum bicolor L.). Journal of Integrative Agriculture 21:2848−64

    doi: 10.1016/j.jia.2022.07.034

    CrossRef   Google Scholar

    [42]

    Eom JS, Chen LQ, Sosso D, Julius BT, Lin IW, et al. 2015. SWEETs, transporters for intracellular and intercellular sugar translocation. Current Opinion in Plant Biology 25:53−62

    doi: 10.1016/j.pbi.2015.04.005

    CrossRef   Google Scholar

    [43]

    Zhang X, Feng C, Wang M, Li T, Liu X, et al. 2021. Plasma membrane-localized SlSWEET7a and SlSWEET14 regulate sugar transport and storage in tomato fruits. Horticulture Research 8:186

    doi: 10.1038/s41438-021-00624-w

    CrossRef   Google Scholar

    [44]

    Yu M, Chen L, Liu D, Sun D, Shi G, et al. 2022. Enhancement of photosynthetic capacity in spongy mesophyll cells in white leaves of Actinidia kolomikta. Frontiers in Plant Science 13:856732

    doi: 10.3389/fpls.2022.856732

    CrossRef   Google Scholar

    [45]

    Zhang X, Wen B, Zhang Y, Li Y, Yu C, Peng Z, et al. 2022. Transcriptomic and biochemical analysis reveal differential regulatory mechanisms of photosynthetic pigment and characteristic secondary metabolites between high amino acids green-leaf and albino tea cultivars. Scientia Horticulturae 295:110823

    doi: 10.1016/j.scienta.2021.110823

    CrossRef   Google Scholar

  • Cite this article

    Chen L, Song HF, Liu JX, Jiang XX, Ai J, et al. 2024. Genome-wide identification and expression profiling of the SWEET family in Actinidia polygama (Sieb. & Zucc.) Maxim.. Fruit Research 4: e017 doi: 10.48130/frures-0024-0010
    Chen L, Song HF, Liu JX, Jiang XX, Ai J, et al. 2024. Genome-wide identification and expression profiling of the SWEET family in Actinidia polygama (Sieb. & Zucc.) Maxim.. Fruit Research 4: e017 doi: 10.48130/frures-0024-0010
    shu

Figures(11)  /  Tables(2)

Download PDF

Article Metrics

Article views(2911) PDF downloads(307)

Other Articles By Authors

ARTICLE   Open Access    

Genome-wide identification and expression profiling of the SWEET family in Actinidia polygama (Sieb. & Zucc.) Maxim.

  • 1.

    College of Horticulture, Jilin Agricultural University, Changchun, Jilin 130118, China

  • 2.

    Key Laboratory of Forest Silviculture and Conservation of the Ministry of Education, College of Forestry, Beijing Forestry University, Beijing 100083, China

  • 3.

    Institute of Agricultural Biotechnology, Jilin Academy of Agricultural Sciences, Changchun 130033, China

  • Received: 11 December 2023
  • Revised: 10 February 2024
  • Accepted: 17 February 2024
  • Published online: 06 May 2024
Fruit Research  4,  Article number: e017  (2024)  |  Cite this article

Abstract: Sugar was transported from photosynthetic source cells to sink cells, sugar efflux transporter protein (sugars will eventually be exported to transporters, SWEETs) play an important role in the process. Although SWEET family members had been identified in many plants, transcriptome or genomics analysis of Actinidia polygama SWEET genes remains uncharacterized. In this study, 14 SWEET genes of Actinidia polygama were identified by protein Blast. The structural characteristics of SWEET genes showed that the number of amino acids encoded by the gene family was between 233 and 304, the relative molecular weight was between 25,918.83 and 33,192.12, the isoelectric point was within the range of 6.96 to 9.71, 14 ApSWEET from Actinidia polygama and the known grape and Arabidopsis SWEETs were divided into four clades (I, II, III, and IV) according to the phylogenetic relationships. The gene structure analysis showed that most of ApSWEET genes have six exons and five introns except ApSWEET5 and ApSWEET14. All ApSWEET proteins also contained P-loop, MtN3-slv, and transmembrane domain. Expression patterns of 14 ApSWEET in different organs and at different fruit developmental stages were analyzed. ApSWEET1 and ApSWEET5 exhibited tissue-specific expression, whereas other genes were more ubiquitously expressed. ApSWEET1, ApSWEET10, and ApSWEET11 exhibited higher expression in fruit. The results of this study provide insights into the characteristics of the SWEET genes in Actinidia polygama and may serve as a basis for further functional studies of such genes.

    HTML

    Introduction

    • Sugars as the main soluble solid component are important nutrients and a key factor influencing the flavor quality of fruits[1]. They also play a crucial role in regulating the expression of fruit-related genes, as well as plant growth and development[2], stress responses[3], and other developmental processes. In leaves, sugars are produced as important photoassimilates and are loaded into the phloem system through the symplasm pathway. They are then unloaded in sink organs, such as fruits and flower, through the apoplast pathway[4]. Sugar transporters that play an indispensable role in phloem loading, nectar secretion, and reproductive tissue development. Many studies have explored the mechanism of sucrose transport from source cells to sink cells, which involves the synergistic effects of multiple transporters[4−8]. At present, sugar transporters in plants are classified into two types: Sugars Will Eventually be Exported Transporters (SWEETs) and major facilitator superfamily (MFS) transporters. Sugars Will Eventually be Exported Transporters (SWEETs) have been newly identified in plants in recent years[9]. Sugar transporters of MFS are further classified into MSTs and SUTs[10], which primarily facilitate sugar influx into the cytosol. However, some MSTs, namely the tonoplast sugar transporter (TST) and the vacuolar glucose transporter (VGT) involved in transporting sugars from the cytosol to vacuoles and act as H+/sugar antiporters[11]. Both MSTs and SUTs consist of 12 transmembrane α-helices and mediate membrane transport of different sugars[12,13].

      SWEET transporters differ from the classic 12 transmembrane structural domains of the major facilitator superfamily. The typical characteristics of SWEET genes include seven TM domains, including two MtN3_saliva domains, which are connected to a low conserved single TM, forming a 3-1-3 symmetric structure[14]. Phylogenetic analysis shows that members of SWEET can be divided into four clades. Clades I, Clades II and Clades IV are mainly hexose transporters, while Clade III is mainly a sucrose transporter[15]. There is substantial evidence suggesting that SWEET protein in Clade I and II may transport glucose, most of the Clade III SWEET proteins are sucrose transporters, and Clade IV evolved from other SWEET clades to primarily act as vacuolar transporters, regulating fructose transport[14]. Members of the SWEET family are widely distributed, and SWEET genes are found not only in plants but also in prokaryotes and animals[16]. With the development of plant genome research, the identification and functional study of SWEET family genes have been carried out in many plant species, and the number of SWEET family members varies significantly among different plants. For example, the SWEET family consists of 17 members in Arabidopsis, 22 in Saccharum spontaneum, 20 in strawberry, 15 in pomegranate, 19 in jujube, 29 in tomato, and 33 in apple[17−23]. Previous studies have reported that members of the SWEET gene family are involved in many important physiological processes of plant growth and development, including nectar production, seed, and pollen development, and the regulation of phloem loading, phloem transport, phloem unloading, abiotic stress, and pathogen interaction by regulating carbohydrate compounds[18, 24−26]. SWEET proteins are also associated with flower, fruit, and seed development. For example, AtSWEET8 mainly affected fertility during early inflorescence development, and AtSWEET13 mainly affected fertility during late inflorescence development[27]. The SWEET gene family was also involved in plant interactions with pathogens, such as VvSWEET4, which shows strong up-regulation of expression with infection of Botrytis cinerea[28, 29].

      Furthermore, SWEET proteins are key factor in regulating the distribution of soluble sugars, which is closely related to plant stress resistance[26]. Overexpression of two homologues AtSWET16 and AtSWEET17 in Arabidopsis thaliana can improve the cold resistance of transgenic plants[26, 30]. Overexpression of AtSWEET15 show more sensitivity to salt stress, and loss of function mutations in AtSWEET15 show higher salt tolerance[31]. Drought stress affects the redistribution of carbohydrates in plants[32]. Under water deficiency conditions, the expression of AtSWEET11 and AtSWEET12 in leaves and roots increased, accompanying an increase in the transportation capacity of sucrose from leaves to roots, indicating that plants regulate the redistribution of carbohydrates by regulating the expression of SWEETs under water deficiency conditions[33].

      Actinidia polygama (Sieb. & Zucc.) Maxim. is a perennial vine plant, its fruit contains many unique and interesting flavors and many nutrients, including organic acids, amino acids, flavonoids, dietary fiber, and vitamins C and E. In addition, the leaves, fruits, stems, and roots also were exploited as nutraceuticals or medicine. Therefore, A. polygama as a valuable wild resource has very high nutritional and medicinal value. However, the current domestication of A. polygama is not complete, particularly in terms of improving fruit quality. During the ripening process of A. polygama fruit, sugar accumulation is closely related to fruit quality and edible ability. In this study, we identified the SWEET gene family members of A. polygama, and analyzed the physical and chemical properties, phylogenetic relationship, gene structure, and promoter elements of SWEET family proteins. We also studied the expression of SWEET gene family members in different organs of A. polygama, which laid a foundation for further study on the structure and function of the SWEET gene family.

    Results

      Identification and physical and chemical analysis of SWEET

    • By searching the SWEET gene on the website (ID: PRJDB13926), a total of 23 SWEET gene family members of A. polygama were screened out, and nine SWEET subfamily members were eliminated because of lack of the conserved structures. According to the physicochemical property table (Table 1), the amino acid quantity of the SWEET gene family ranged from 233 to 304. The molecular weight ranges from 25,895.58 to 33,192.12. The isoelectric point is between 6.96 and 9.71, and the stability coefficient of ApSWEET4, ApSWEET5 and ApSWEET14 proteins was greater than the remaining 11 proteins. The aliphatic index was 94.76~118.24, which was a stable protein. Furthermore, most of the SWEET genes were found to be present on the cell membrane, rarely on the chloroplast and Golgi apparatus.

      Table 1.  Physicochemical properties of SWEET gene family proteins.

      Gene IDNumber of amino acidsMolecular weightpIAsp + GluArg + LysInstability indexPredicted location
      ApSWEET127531,0938.17171937.35 (stable)Cell membrane
      ApSWEET224427,049.866.86161635.79 (stable)Cell membrane
      ApSWEET330433,192.129.49193036.79 (stable)Chloroplast
      ApSWEET425429,132.97.61192046.56 (unstable)Cell membrane
      ApSWEET525928,602.29.71132445.53 (unstable)Cell membrane
      ApSWEET623425,895.588.48141635.70 (stable)Cell membrane
      ApSWEET723826,891.19.18131936.22 (stable)Cell membrane
      ApSWEET823726,653.718.87131734.25 (stable)Cell membrane
      ApSWEET925327,530.99.5122329.02 (stable)Cell membrane
      Golgi apparatus
      ApSWEET1025227,529.89.49122025.31 (stable)Cell membrane
      ApSWEET1123625,918.839.2681436.71 (stable)Cell membrane
      ApSWEET1223425,977.879.0391436.26 (stable)Cell membrane
      ApSWEET1323826,391.359.0191438.49 (stable)Cell membrane
      ApSWEET1423326,641.99.36152443.15 (unstable)Cell membrane

      Phylogenetic and structural analysis of SWEET gene family members in A. polygama

    • To study the phylogenetic relationships among SWEET genes in A. polygama and other plant species, a neighbor-joining phylogenetic tree was constructed by aligning 14 ApSWEET sequences, 17 AtSWEET sequences, and 14 VvSWEET sequences (Supplemental Tables S1–S3). Apparently, 45 proteins were clustered into four different groups (Fig. 1). In detail, six ApSWEETs (ApSWEET9, 10, 11, 12, 13, 14) showed high homology with three AtSWEETs (AtSWEET1–3) and three VvSWEETs (VvSWEET2a, 2b, 3) in group I. In group II, three ApSWEETs (ApSWEET5, 7, 8) were clustered with five AtSWEETs (AtSWEET4–8) and three VvSWEETs (VvSWEET5a, 5b, 7). ApSWEET1/4 were homologous to seven AtSWEETs (9–15) and five VvSWEETs (VvSWEET9, 10, 11, 12, 15) in group III. Three ApSWEET (ApSWEET2, 3, 6), two AtSWEETs (AtSWEET16, 17) and three VvSWEETs (VvSWEET17a, 17b, 17d) were included in group IV. The exon-intron structural evolution showed that most of the ApSWEET contained six exons, except for ApSWEET5 and ApSWEET14 which contained five exons (Fig. 2). ApSWEET2 and ApSWEET4 had the shortest and longest sequence, respectively. ApSWEET11, ApSWEET12, and ApSWEET13 had similar exon-intron structures. In addition, ApSWEET1 and ApSWEET4, ApSWEET3, and ApSWEET6 also showed similar structures, these genes belong to the same group. These results suggested that ApSWEETs in the same group shared similar exon-intron organizations.

      Figure 1. 

      Phylogenetic analysis of the ApSWEETs from A. polygama, Arabidopsis thaliana, and Vitis vinifera. The Neighbor-joining tree was drawn using MEGA7.0 with 1,000 bootstraps. The roman numbers (I–IV) labeled with various colors indicate different clades: green – Clade I, orange – Clade II, red – Clade III, blue – Clade IV.

      Figure 2. 

      ApSWEET gene structure of A. polygama.

      Analysis of conserved motif of ApSWEET gene family

    • The Motif1~Motif10 conserved motifs were found in the SWEET gene of A. polygama. (Fig. 3), whereas motifs 1, 2, 4, and 5 were observed in all ApSWEET members. In addition, Motif3 was observed in 13 ApSWEET members except ApSWEET4. Six genes lacked Motif6, and five genes lacked Motif7. Only ApSWEET13, ApSWEET12 and ApSWEET2 genes contained Motif8, ApSWEET10, ApSWEET5 and ApSWEET4 contained Motif9, ApSWEET1 and ApSWEET14 contained Motif10. Most of the conserved motifs had a relatively consistent relationship with the evolutionary tree with the same order of number, suggesting that these genes had strong conserved structures and similar gene functions. We confirmed that the ApSWEET proteins also contained P-loop, MtN3-slv, and transmembrane domain (Fig. 4). The typical structure of plant SWEET proteins consists of seven predicted transmembrane (7-TM) helices forming two MtN3_slv domains (triple-helix bundles, THB) connected by a linker transmembrane helix (TM4). All ApSWEET genes comprise two sugar transporter domains for intercellular exchange.

      Figure 3. 

      The conserved motif analyses of ApSWEETs proteins.

      Figure 4. 

      Conserved structural domains of ApSWEETs.

      Secondary and tertiary structure analysis of ApSWEET gene family members

    • SOPMA was used to analyze the secondary structure of the SWEET gene family in A. polygama, including Alpha helix, chain extension structure, Beta turn, and Random coil (Table 2). These results showed that Alpha helix and Random coil were significantly higher than the chain extension structure and Beta turn. In addition, the Alpha helix proportion of ApSWEET6 was the highest, and ApSWEET9 was the lowest. Meanwhile, the proportion of chain extension structure in ApSWEET9 was the highest, and ApSWEET3 was the lowest. The beta turn of ApSWEET8 was the highest, and ApSWEET4 was the lowest. A random coil of ApSWEET4 was the highest, but ApSWEET6 was the lowest.

      Table 2.  Secondary structure analysis of ApSWEET family members.

      Alpha
      helix      		 Extended
      strand      		Beta turnRandom
      coil
      ApSWEET145.09%16.73%2.18%36.00%
      ApSWEET241.80%22.54%2.87%32.79%
      ApSWEET349.01%15.46%5.26%30.26%
      ApSWEET437.80%18.11%0.79%43.31%
      ApSWEET535.91%21.24%4.63%38.22%
      ApSWEET650.00%21.79%3.85%24.36%
      ApSWEET740.34%21.85%4.20%33.61%
      ApSWEET840.51%21.94%5.91%31.65%
      ApSWEET933.99%25.30%4.35%36.36%
      ApSWEET1046.83%17.06%3.97%32.14%
      ApSWEET1142.37%19.49%5.08%33.05%
      ApSWEET1241.88%20.94%4.27%32.91%
      ApSWEET1346.22%19.33%2.52%31.93%
      ApSWEET1436.48%21.89%3.86%37.77%

      SWISS-MODEL was used for homology modeling analysis (Fig. 5), and it was found that the three-dimensional structure of ApSWEET proteins could be roughly divided into two categories. ApSWEET1, ApSWEET2, ApSWEET4, ApSWEET5, ApSWEET6, ApSWEET7 and ApSWEET8 was clustered into one group, and ApSWEET3, ApSWEET9, ApSWEET10, ApSWEET11, ApSWEET12, ApSWEET13 and ApSWEET14 was clustered into one group.

      Figure 5. 

      Single protein structure of ApSWEETs.

      Chromosomal localization and collinear analysis of the ApSWEET gene

    • Chromosome analysis of genes (Fig. 6) showed that except for chromosome ApChr19, other genes were evenly distributed on 11 chromosomes. Three ApSWEETs genes were distributed in clusters on chromosomes ApChr19, ApSWEET12, and ApSWEET13 may be due to gene replication. According to the collinearity analysis diagram (Fig. 7), ApSWEET2 and ApSWEET6, ApSWEET4 and ApSWEET7, ApSWEET11 and ApSWEET3/12/13 exist collinearity, which may be obtained by chromosome fragment replication.

      Figure 6. 

      Chromosome location of ApSWEET gene family.

      Figure 7. 

      Collinearity analysis of the ApSWEET gene family.

      Promoter analysis of the ApSWEET gene family

    • In order to study the potential regulatory factors of the ApSWEET gene, the 2,000 bp promoter region of this family was analyzed (Fig. 8), and 88 elements in promoter regions of all ApSWEETs genes were predicted. The results showed that response elements such as low temperature, light, and hormone appeared in most gene promoter regions, indicating that genes may be affected by low temperature, light, and hormone levels. They were classified into three groups based on their functional associations: stresses (ARE, DRE, STRE, LTR, MBS, and MYC), hormones (ABRE, TATC-box, CGTCA motif/TGACG motif, HD-Zip1, P-Box, GARE-motif, GA-motif, ERE, and TCA-element) and light (GT1-motif, TCCC-motif, TCT-motif, G-Box, Gap-box, LAMP-element). Among these elements, six elements were responsive to stress, ten elements were responsive to hormones, and six elements were responsive to light. Four development-related elements are responsive to meristem expression (CAT-box), cis-regulatory element involved in endosperm expression (GCN4), involved in endosperm-specific negative expression (AACA), and seed-specific regulation (RY). These findings indicated that ApSWEETs may respond to hormones or be involved in plant growth and stress resistance.

      Figure 8. 

      The cis-elements in the promoter sequences of ApSWEETs gene in A. polygama.

      Expression analysis of ApSWEET gene

    • Real-time fluorescence PCR was used to detect the expression of ApSWEET members in leaves, stems, flowers, roots, mature fruits (Fig. 9) and fruits of different developmental stages (Fig. 10). These results showed that the expression level of ApSWEET2, ApSWEET3, ApSWEET10, ApSWEET11 and ApSWEET13 was higher in leaves, the expression level of ApSWEET2, ApSWEET3, ApSWEET10, ApSWEET11, ApSWEET9, and ApSWEET13 was higher in stem, the expression level of ApSWEET5 and ApSWEET11 was higher in flower, the expression level of ApSWEET3, ApSWEET10, and ApSWEET11 was higher in the root, the expression level of ApSWEET1, ApSWEET10 and ApSWEET11 was higher in fruit. Most of the SWEET genes were found to be ubiquitously expressed in all tissues except for ApSWEET1 and ApSWEET5, the two genes were specifically expressed in the fruit and flower.

      Figure 9. 

      ApSWEET expression of different tissues.

      Figure 10. 

      ApSWEET expression of different fruit development stage.

      During fruit development, only ApSWEET5 had higher expression at an early stage of fruit development, ApSWEET1, ApSWEET2, ApSWEET10, and ApSWEET11 had higher expression at the mid and late stages of fruit development (Fig. 10). In addition, the glucose content in A. polygama is higher than the fructose and sucrose content from the initial measurement on June 11th 2022 (Fig. 10). In the final measurement on September 27th 2022, the glucose content is 2.7 times and 5.4 times higher than the fructose content and sucrose content. Therefore, glucose content was the highest, followed by fructose, and sucrose content was the lowest during A. polygama development.

      After the mature fruits are harvested, only ApSWEET1 shows strong expression at different storage periods. ApSWEET10 showed strong expression only on the first day after harvest, but the expression levels of ApSWEET11 gradually decrease with prolonged fruit storage time (Fig. 11).

      Figure 11. 

      ApSWEET expression of different storage periods.

    Discussion

    • Although SWEET genes have been extensively studied in various species, such as Arabidopsis thaliana[17], Hemerocallis fulva[34], Chinese jujube[14], Malus[35] and Litchi chinensis[36], the presence of SWEET gene family in A. polygama has not been reported. In this study, we identified 14 members of the SWEET gene family in A. polygama. The number is less than three in Arabidopsis and seven in rice[17, 18]. Gene duplication has been shown to contribute to the expansion of SWEET genes in soybean and potato, enabling them to adapt to environmental changes. This could explain why these species have more SWEET proteins than others. Moreover, different predictions of their physical and chemical properties suggest that SWEET genes may have diverse functions in plants[17]. Eleven of the identified genes were located on different chromosomes, consistent with findings from Arabidopsis. It is speculated that different members of the gene family may perform specific biological functions in different plant tissues. However, further research is needed to elucidate their exact roles in plant growth and development.

      The structure and number of conserved motifs among members of the SWEET gene family were similar, and there was also a relative relationship between the evolution tree and gene structure. For example, ApSWEET11, ApSWEET12 and ApSWEET13 exhibited similar gene structures and belonged to the same clade. However, some gene structures displayed noticeable differences, suggesting the occurrence of expansion, reduction, or mutation during the evolution of the SWEET gene family. These variations may be associated with the diverse functions of the genes. The SWEET family members generally consist of five to six exons in their structures. It is speculated that the diversity of gene function may arise from the loss or addition of exons during the evolutionary process of these family genes. Additionally, all the members of SWEET gene family in A. polygama contained four highly conserved motifs: Motif1, Motif2, Motif4 and Motif5. These conserved motifs may play a key role in the biological function of the SWEET protein of A. polygama.

      The expression patterns in different organs are closely correlated with gene function and serve as a predictor of biological functions. Numerous studies have reported the involvement of SWEET genes in various physiological processes, which usually were associated with specific tissue expression patterns. Our results also revealed that some SWEET genes were ubiquitously expressed in the flower, fruit tissues, root, leaf and stem. For example, ApSWEET5 and ApSWEET11 were highly expressed in flower. Some studies have reported that SWEET genes may play an important role in reproductive development. For example, AtSWEET13 and AtSWEET14 were found to be expressed in the anther wall, responsible for facilitating sucrose efflux into locules to support pollen development and maturation[37], and mutations in AtSWEET9 had been shown to impair nectar secretion[38,39]. These genes were specifically expressed in pollen. Similarly, OsSWEET11 and SbSWEET9-3 were highly expressed in the panicle[40, 41], suggesting that these genes may be essential for reproduction. Additionally, ApSWEET1 and ApSWEET5 exhibited specific expression in fruit. Similarly, high expression of VvSWEET transporters in flowers and berries highlighted a putative important role in sugar partitioning during flower and fruit development[4]. Furthermore, ApSWEET3 and ApSWEET10 displayed high expression in the roots (Fig. 5), while ApSWEET2, ApSWEET9, and ApSWEET10 were relatively highly expressed in the leaves and stem, similar to the function of AtSWEET17 as fructose transporter[42], these genes were proposed to participate in the phloem loading of photoassimilate in leaves of A. polygama. Our results suggested these genes may be involved in flower development, as well as the short and long-distance transportation and distribution of sugars. However, the regulatory mechanisms underlying the expression of these genes require further clarification.

      In fruit, sugar (such as glucose, sucrose and fructose) is an important index that determines the quality of fruit. Many studies have reported functions of SWEET genes in both sink and source organs, particularly in fruits like tomato, grape, apple and Chinese jujube. The different expression patterns of the SWEET gene during fruit development are closely related to their function and can be used to predict biological functions. For instance, in apples, the expression of SWEET genes at young (MdSWEET1.1/2, MdSWEET2.4 and MdSWEET3.5) and ripe fruit development stages were different[23]. In grape, the expression of VvSWEET10, VvSWEET12, and VvSWEET15 was higher in young fruit, but VvSWEET15 was more abundant in mature fruit[28]. Furthermore, SlSWEET7a, SlSWEET14 and SlSWEET15 in tomato fruit are responsible the for transportation of glucose, fructose and sucrose[43], and VvSWEET4 acted as a glucose transporter in grape[28]. In our study, seven ApSWEET genes were expressed at five different fruit development stage. Among them, three ApSWEET genes exhibited high expression at the early stage of fruit development, and two ApSWEET genes showed high expression in ripe fruit. Moreover, there is a certain correlation between gene expression and homology, with some genes belonging to the same clade as ApSWEET1, VvSWEET10, VvSWEET11 and VvSWEET15[4, 23]. On the basis of sugar content during fruit development, it was also speculated that ApSWEET1 and two ApSWEET (ApSWEET2 and ApSWEET3) may be involved in the transportation and distribution of sucrose and fructose during fruit ripening, while ApSWEET10 and ApSWEET11 may play a role in transportation and distribution of glucose during fruit ripening.

    Materials and methods

      Plant materials

    • Sampled plants were grown outdoors on the campus of Jilin Agricultural University, Jilin, China (43°48'48'' N, 125°24'15'' E). The annual precipitation is 867 mm, and the annual highest and lowest temperatures are 35 and −40 °C, respectively. Each field plot was divided into three subplots, and the seedlings were planted in each plot (3 m × 4 m). The phosphorous (4.12 ± 0.31 g/Kg), nitrogen (32.17 ± 1.98 g/Kg) and potassium (3.51 ± 0.19 g/Kg) concentrations were sufficient. Seedlings in all experiments were of uniform size. The expression of ApSWEET genes was detected in A. polygama has different tissues and fruits at different development stages. Leaf, flower, root, stem, and fruit were collected. Fruit collected at 10, 40, 60, 80, and 85 d after full bloom respectively. After the mature fruits are harvested, fruit were stored for 1, 2, 3, 4, and 5 d. All fresh plant samples were collected with three independent replicates and immediately frozen in liquid nitrogen, then stored at −80 °C.

      Identification of gene family members

    • Actinidia polygama SWEET gene family was identified by protein Blast of the 17 Arabidopsis SWEET proteins against the Actinidia polygama genome database (https://figshare.com/s/f46aea0009a54a6a0528).

      The NCBI CDD (www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and PFAM (http://pfam.sanger.ac.uk/) website were used to search for the conserved domains of the candidate members.

      Physicochemical properties and structural analysis

    • For the protein sequences encoded by the gene family members obtained above, Expasy (http://web.expasy.org/) was used to predict their molecular weight, isoelectric point, stability and other physicochemical properties, respectively. WoLFPSORT (www.genscript.com/wolf-psort.html) was used to predict protein subcellular localization, and TMHMM (https://services.healthtech.dtu.dk/service.php?TMHMM-2.0) used to predict transmembrane structure. The gene structure of SWEET gene family members of Actinidia polygama was analyzed according to the location information of introns and exons on chromosomes. MEME was used to predict the motif of the protein-conserved domain. The analysis results were visualized using TBtools and modified by AI. The online software SOPMA (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html) was used to predict the secondary structure of SWEET protein, including random curling, chain extension structure, alpha helix and beta-turn. Using the SWISS-MODEL to analyze homology modeling (www.swissmodel.expasy.org).

      Construction of molecular evolutionary tree

    • The MUSCLE was used for sequence comparison of the gene family members and was beautified with GENEDOC. The comparison results were clipped with trim Al and analyzed with IQ-TREE evolutionary tree. Finally, the exhibited (http://tree.bio.ed.ac.uk/software/figtree/) for the beautification of the illustration.

      Chromosome localization and collinearity analysis of gene family

    • Based on the whole genome and location information downloaded from the Actinidia polygama genome database (https://figshare.com/s/f46aea0009a54a6a0528), TBtools was used to conduct chromosome localization, collinearity, and gene tandem repeat event analysis for all SWEET gene family members of Actinidia polygama, and the results were visualized. MCScan was used to compare the whole genome sequence of Actinidia polygama, and the collinearity relationship was obtained. The homologous gene map was drawn with TB tools.

      Gene family promoter analysis

    • The 2 kb nucleotide sequences upstream of the transcription starting points of 14 genes of the gene family were predicted using PlantCARE, and TBtools software was used for visualization.

      RNA extraction and real-time PCR

    • To validate the reliability of RNA-Seq, qRT-PCR for transcripts was carried out as described by Yu et al.[44]. Total RNA was extracted from leaves, flowers, root, stem, and fruit sampled simultaneously. For each experiment, 1 µg of clean RNA was converted to cDNA using the PrimeScript™ RT reagent Kit (TaKaRa Bio., Dalian, China) according to the manufacturer's protocol. Gene-specific primers were designed using Primer5.0 software (Premier Biosoft). Gene expression was performed using the SYBR Green Real-time PCR kit (TaKaRa Bio). ACTIN was used as a housekeeping gene after examining its constitutive expression pattern from the RNA-seq results. Relative gene expression levels were calculated with the 2−ΔΔCᴛ method[45]. The sequences of the primers used for qRT-PCR are listed in Supplemental Table S4.

    Author contributions

    • The authors confirm contribution to the paper as follows: study conception and design: Wang ZX, Wang YP; performing the research: Chen L, Song HF, Liu JX, Jiang XX, Ai J. All authors reviewed the results and approved the final version of the manuscript.

    Data availability

    • All data generated or analyzed during this study are included in this published article and its supplementary information files.

      Acknowledgments

      • This study was supported by the Natural Science Foundation of China (to ZXW, GLS and JA, 31870673) and Jilin Province Development and Reform Commission, Grant/Award (Number: 2022C037-1); Department of Science and Technology of Jilin Province, Grant/Award (Numbers: 20210204083YY, 202101013697JC). This work complies with Chinese law. We thank Professor Ya-dong Li from the Jilin Agricultural University, Changchun, for providing recommendations for data collection.

      Conflict of interest

      • The authors declare that they have no conflict of interest.

      • # Authors contributed equally: Li Chen, Hui-Fang Song, Jia-Xin Liu

      Supplementary information
      Rights and permissions
      • Copyright: © 2024 by the author(s). Published by Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (11)  Table (2) References (45)
  • About this article
    Cite this article
    Chen L, Song HF, Liu JX, Jiang XX, Ai J, et al. 2024. Genome-wide identification and expression profiling of the SWEET family in Actinidia polygama (Sieb. & Zucc.) Maxim.. Fruit Research 4: e017 doi: 10.48130/frures-0024-0010
    Chen L, Song HF, Liu JX, Jiang XX, Ai J, et al. 2024. Genome-wide identification and expression profiling of the SWEET family in Actinidia polygama (Sieb. & Zucc.) Maxim.. Fruit Research 4: e017 doi: 10.48130/frures-0024-0010
    shu

Catalog

  • HTML

  • Introduction
  • Results

  • Identification and physical and chemical analysis of SWEET

  • Phylogenetic and structural analysis of SWEET gene family members in A. polygama

  • Analysis of conserved motif of ApSWEET gene family

  • Secondary and tertiary structure analysis of ApSWEET gene family members

  • Chromosomal localization and collinear analysis of the ApSWEET gene

  • Promoter analysis of the ApSWEET gene family

  • Expression analysis of ApSWEET gene

  • Discussion
  • Materials and methods

  • Plant materials

  • Identification of gene family members

  • Physicochemical properties and structural analysis

  • Construction of molecular evolutionary tree

  • Chromosome localization and collinearity analysis of gene family

  • Gene family promoter analysis

  • RNA extraction and real-time PCR

  • Author contributions
  • Data availability
  • Acknowledgments
  • Conflict of interest
  • Supplementary information
  • Rights and permissions
  • About this article
logo

Contents

Policies

Services

Partnerships

© 2025 Maximum Academic Press, except Open Access articles. MAP is a member of ALPSP, COPE, CrossRef, cOAlition S, OASPA and STM.

Privacy Policy  |  Site Map  |  Terms and Conditions

/

DownLoad:  Full-Size Img  PowerPoint
Return
Return