Trehalose-6-phosphate phosphatase (TPP) participates in cold acclimatization of Actinidia arguta depending on the ethylene signal transduction pathway

doi:10.21203/rs.3.rs-3700943/v1

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Trehalose-6-phosphate phosphatase (TPP) participates in cold acclimatization of Actinidia arguta depending on the ethylene signal transduction pathway

https://doi.org/10.21203/rs.3.rs-3700943/v1

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Actinidia arguta (kiwiberry) is a perennial deciduous vine that has a very strong overwintering ability. We speculated that trehalose metabolism, which plays a pivotal role in the stress tolerance of plants, may be involved in the cold acclimatization of A. arguta. Transcriptome analysis showed that the expression of AaTPPA, encoding a trehalose-6-phosphate phosphatase (TPP), was upregulated in response to low temperature. AaTPPA expression levels were much higher in lateral buds, roots, and stem cambium than in leaves in autumn. In AaTPPA-overexpressing (OE) Arabidopsis thaliana, trehalose levels were 8 ~ 11 times higher than that of the wild type (WT) and showed different phenotypic characteristics from WT or overexpression lines of OtsB, the E. coli TPP gene. Surprisingly, AaTPPA-OE A. thaliana had significantly higher freezing tolerance than WT and OtsB-OE lines. Transient overexpression of AaTPPA in A. arguta leaves increased ROS scavenging capacity and the contents of soluble sugars and proline. AaERF64, an ethylene-responsive transcription factor, was induced by ethylene treatment, and bound to the GCC-box of the AaTPPA promoter to activate its expression. AaTPPA expression was also induced by abscisic acid (ABA). Overall, the temperature decrease in autumn induces AaERF64 expression through the ethylene signal transduction pathway, which in turn upregulates AaTPPA expression, leading to the accumulation of osmotic protectants including soluble sugars and proline in the overwintering tissues of A. arguta.

trehalose

ethylene-responsive transcription factor

cold tolerance

freezing stress

winter acclimatization

Environmental and biological factors that are unfavorable or detrimental to the normal growth and development of plants are referred to as stresses. Drought, waterlogging, heat, cold, salinity, and invasion by pathogens are the six main stresses that plants face inevitably during their survival (VanWallendael et al. 2019). Among them, cold stress is one of the major abiotic stresses that affect plant growth in early spring and late autumn, which is categorized into two terms: chilling stress (0–20°C) and freezing stress (< 0°C) (Guo et al. 2018). To cope with cold stress, most plants have evolved two strategies including cold tolerance and cold acclimatization. Cold tolerance is one of the strategies for plants to respond to temporary low temperatures, during which the contents of osmotic protectants and the activities of antioxidant enzymes increase, and stress-signaling phytohormones including gibberellin (GA), abscisic acid (ABA), ethylene, and jasmonic acid (JA) are also produced (Shinozaki and Yamaguchi-Shinozaki 2000). Cold acclimatization is another strategy for plants to cope with long seasonal weather changes, which is triggered upon prior exposure to non-freezing chilling stress. During this process, freezing-protective substances, such as soluble sugars and anti-freezing proteins, accumulate in overwintering tissues of plants to protect biological membranes and biomolecules from damage, and at the same time, morphological changes occur to minimize energy consumption (Ding et al. 2020; Kaplan et al. 2007).

Recently, it has been reported that trehalose (α-D-glucopyranosyl-1,1-α-D-glucopyranoside) metabolism plays a distinct role in plant tolerance to stresses, such as drought, salt, cold, and so on (Hassan et al. 2022). Trehalose is a non-reducing disaccharide that exists in most organisms except vertebrates (Avonce et al. 2006). Generally, in plants, trehalose-6-phosphate (Tre6P) is first synthesized from UDP-glucose and glucose-6-phosphate by trehalose-6-phosphate synthase (TPS), which is in turn dephosphorylated to produce trehalose by trehalose-6-phosphate phosphatase (TPP) (Cabib and Leloir 1958). Finally, it is hydrolyzed into two glucose molecules by trehalase (TRE) (Phan et al. 2022). In plant genomes, TPSs and TPPs are a large family of genes. For example, Arabidopsis thaliana alone has 11 TPS genes and 10 TPP genes (Lunn 2007). The plant TPS genes are further divided into two classes; the class I genes have TPS enzyme activity, and the class II genes have no enzymatic activity (Blazquez et al. 1998; Paul et al. 2008). Regrettably, the reason why plants have so many TPS and TPP genes has not been fully uncovered. Perhaps they might be related to the critical biological roles of the trehalose metabolites. Here, trehalose itself is an effective osmotic protectant, but its levels in most plants are too low to provide osmotic protection. Instead, trehalose and Tre6P act as signaling molecules that induce a set of factors related to stress tolerance and sugar metabolism in plants (Iturriaga et al. 2009). However, so far, only SUCROSE-NON-FERMENTING1-RELATED KINASE1 (SnRK1) has been identified as a target of Tre6P, and no other direct targets of Tre6P and trehalose have been found (Tsai and Gazzarrini 2014). Tre6P binds directly to SnRK1 and inhibits its activity, thereby controlling the expression of over 1,000 genes regulated by SnRK1 (Baena-Gonzalez et al. 2007).

Kiwiberry (Actinidia arguta (Sieb. et Zucc.) Planch ex Miq) is a perennial deciduous vine tree belonging to the genus Actinidia of the Actinidiaceae family. A. arguta is known for its fruit having uniquely delicious taste and rich nutrients and is also one of the valuable cold-tolerant fruit tree resources that do not freeze to death even under − 34°C in winter (Calugaru-Spataru et al. 2013; Latocha 2017). However, its sprouting buds are very sensitive to frost damage in spring. Recently, due to the "false vernalization" phenomenon caused by global warming, the production of kiwiberries has decreased significantly, causing great economic loss to fruit farming in China.

From the transcriptome data of A. arguta under chilling stress, we found that the expression level of a TPP gene (AaTPPA) increased in response to low temperature. AaTPPA overexpression in A. thaliana and transient overexpression in A. arguta leaves enhanced the cold tolerance of these plants by accumulating soluble sugars and proline and increasing the scavenging activity of reactive oxygen species (ROS). Furthermore, we found that AaTPPA was transcriptionally activated by AaERF64 which was induced by ethylene treatment, and that their expression levels increased in overwintering tissues, such as lateral buds, stem cambium, and roots, in response to the temperature decrease in autumn.

Plant materials and treatments

Two A. arguta species, A. arguta cv. Kuilv (KL) (cold resistant variety) and A. arguta cv. Longcheng No.2 (L2) (cold susceptible variety), were used in this study. A. arguta (KL) seedlings were supplied from the Horticulture Branch of Heilongjiang Province, and A. arguta (L2) seedlings were grown in pots from hardwood cuttings. A. arguta (KL) plants at different growth stages (cuttings, 2-month-old seedlings, 5-month-old plantlets) were treated under different low temperatures (4, 9, 15°C) for different times (0, 2, 4, 6 h), respectively, and sprouting buds, young leaves, and mature leaves were collected for RNA extraction. 5-month-old A. arguta (L2) plantlets grown in pots were sprayed with ethephon (ethylene precursor) (10 µmol/L), abscisic acid (ABA) (100 µmol/L), or jasmonic acid methyl ester (MeJA) (10 µmol/L), respectively, once a day for 15 d. After spraying, each pot was covered with a transparent plastic bell to prevent leakage of the phytohormones. Mature leaves, lateral buds, stem cambium, and fine roots were collected for RNA extraction and biochemical analysis. All samples included three biological replicates.

Transcriptome analysis

A. arguta (KL) plantlets grown in pots for 5 months were set outside at 4–10°C on 9 May 2020 (Harbin, China) for cold treatment. Leaves were collected after 0, 2, 4, and 6 h treatment, and were used for RNA extraction. Total plant RNA was extracted according to the instructions of RNAprep Pure Plant Kit (TIANGEN, Beijing, China). cDNA was synthesized according to the method of Clontech SMARTer PCR cDNA Synthesis Kit and sequenced on the Illumina HiSeq X sequencing platform. RNA-seq was set up with three biological replicates. Reads of RNA-seq data were mapped to the reference genome sequence of Actinidia chinensis cv. Hongyang using HISAT2v2.0.5. The values of gene fragments per kilo million bases were generated using Perl script. Differential expression analysis was performed using the DESeq R package (1.18.0). The resulting P-values were adjusted using Benjamini and Hochberg's approach (Love et al. 2014).

Gene cloning and sequence analysis

The open reading frames (ORF) of AaTPPA and AaERF64 were amplified from the cDNA of A. arguta (KL) leaves using 2 × Phanta Max Master Mix (Vazyme), OtsB were amplified from the genome DNA of E. coli DH5α (primers are listed in Table S3). PCR amplified products were purified using Gel Extraction kit (TIANGEN), ligated with a linear pMD18-T vector (Takara), and then transformed into E. coli DH5a. Transformants were confirmed by PCR test with M13-F/R primers, and sent for sequence analysis. The protein-coding sequences (CDS) of AaTPPA, OtsB, and AaERF64 were cloned into the binary vector GV1300 between KpnⅠ and BamHⅠ restriction sites, respectively, under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Constructed GV1300-AaTPPA, GV1300-OtsB, and GV1300-AaERF64 vectors were used for subcellular localization assay and A. thaliana transformation. The CDSs of AaTPPA and OtsB were also cloned into the E. coli expression vector pET-14b for procaryotic expression and purification (Fig. S3).

Quantitative real-time PCR (RT-qPCR) analysis

Samples were ground in liquid nitrogen, and then total RNA was extracted using RNAprep Pure Plant Kit (TIANGEN, Beijing, China). cDNA was synthesized according to the instruction of PrimeScript Reverse Transcriptase kit (Takara, Japan). 18S rRNA was used as the internal reference for A. arguta, and AtActin was used as the internal reference for Arabidopsis thaliana. qPCR was carried out using Premix Ex TaqTM (Takara) mix on Lightcycler480 system (Roche, USA). Three biological replicates were used for each sample. All data were analyzed by the relative quantification (2^−ΔΔCt) method (Livak and Schmittgen 2001). The gene-specific primers are listed in Table S4.

Bioinformatic analysis

Multiple alignments were performed using the ClustalW BLOSUM protocol of MEGA7. Phylogenetic trees were constructed using the Neighbor-Joining (NJ) method and enriched using iTOL (https://itol.embl.de/tree/). The nuclear localization signal peptides were predicted using the online website INSP (http://www.csbio.sjtu.edu.cn/cgi-bin/INSP/INSP.py). The protein 3D-structures were predicted using the online website Phyre² (http://www.sbg.bio.ic.ac.uk/phyre2/). Cis-acting elements of the promoter region were analyzed using the online website PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

Recombinant protein expression and purification.

To determine TPP enzyme activity, CDSs of AaTPPA and OtsB were inserted into pET-14b E. coli expression vector at NdeI and BamHI, respectively. The recombinant vectors were transformed into E. coli Rosetta (DE3), and the positive colonies were verified by PCR test. The transformants were incubated at 37°C until OD₆₀₀ reached 0.6 ~ 1.0, and after the addition of IPTG to 0.5 mmol/L, were incubated at 28°C for the induction of the recombinant proteins (Fig. S2a, c). His6-tagged AaTPPA and OtsB were purified by the Ni-affinity chromatography according to the instruction of His-tag Protein Purification Kit (Beyotime Biotech Inc., China) (Fig. S2b, d).

Subcellular localization

GV1300-AaTPPA, GV1300-OtsB, and GV1300-AaERF64 plasmids were transferred into Agrobacterium tumefaciens strain GV3101. GV1300 empty vector was used as the control plasmid. Tobacco (Nicotiana benthamiana) leaves were infiltrated with the transformed Agrobacterium suspensions (OD₆₀₀ = 0.6-1.0) and were kept in a dark chamber for 1 d, then under normal conditions for 2 d, after which the transformed leaves were observed under a fluorescence microscope. These vectors were also transfected into onion epidermal cells. The onion epidermis was spread flat on Murashige and Skoog 1962 (MS) culture media comprising 8 g/L agar and 30 g/L sucrose (pH 5.8), and co-cultured with the transformed Agrobacterium for 24 h in the dark and 48 h in the light, and then observed under a fluorescence microscope. DAPI was used as a nuclear labeling dye.

A. thaliana transformation

Agrobacterium tumefaciens strain GV3101 harboring the purpose plasmids were used to transform A. thaliana (Col-0) by the floral dip method (Davis et al. 2009). T₀ seeds were screened on MS media containing hygromycin (Hyg) (50 mg/L). Selected T₁ plants were verified by genotyping with primers used for cloning the corresponding genes, and then at T₂ generation, the positive transgenic lines were tested for the expression levels of the corresponding genes. Two T₃ transgenic lines with high expression levels of the transgenes were chosen for further gene functional analysis.

Morphological analysis of the transgenic A. thaliana

T₃ seeds of AaTPPA and OtsB-OE lines were observed for morphology, and the seed size was calculated using ImageJ software. Seeds of AaTPPA and OtsB-OE lines were inoculated on MS media (each plate contained 76 seeds), and the germinated seeds were counted after 10 d cultivation. Seedlings of AaTPPA and OtsB-OE lines grown on MS media for 12 d were measured for size and primary root length. All seedlings grown on MS media for 12 d were transplanted into pots. AaTPPA and OtsB-OE plants grown for 28 d were observed and measured for leaf size and chlorophyll content. Vivid rosette leaves of the plants grown for 45 d were counted, and then the ratio of surviving versus total leaves was calculated to evaluate the degree of senescence. Adult plants were measured for branch numbers, height, and silique counts.

Cold tolerance verification of the transgenic A. thaliana

T₃ seeds of AaTPPA and OtsB-OE lines were inoculated on MS media without Hyg antibiotics. WT seeds were also inoculated on the same plate, and each plate contained 4 × 19 seeds. All plates were cultured in an incubator for 14 d, and then set in a refrigerator at -10°C for 0.5, 1, 2, and 4 h, respectively. After freezing treatments, 3 plates of each group were used for ion leakage assay, and others continued to culture under normal conditions for 2 d, and then the survival rate was measured. AaTPPA and OtsB-OE plants grown for 21 d were subjected to freezing stress at -10°C for 0, 1, 2, and 4 h, respectively, and then continued to grow under normal conditions for 2 d, after which the leaf wilting rate was calculated. After culturing 4 h-treated plants for 7 d, the reviving rate was calculated. For chlorophyll fluorescence, NBT, and DAB assay, 21-day-old plants were exposed to sunny outside of -2 ~ -4°C for 0, 1, and 2 h in winter.

Construction of the plant expression vector with DsRed marker

GV1300 vector was digested by EcoRI and PmeI restriction enzymes and then ligated with chemically synthesized loxP fragment using Exnase II recombinase to construct the GVloxP vector. Next, the GVloxP vector was cut at KpnI and SpeI restriction sites within the loxP construct and then recombined with DsRed expression cassette cloned from MtPT4-p3 vector (purchased from Addgene (Timoneda et al. 2021)) to construct the GVloxP/DsRed vector (Fig. S5). The CDSs of AaTPPA and AaERF64 were introduced into the GVloxP/DsRed vector at SmaI and PspXI restriction sites under enhanced CaMV 35S promoter, respectively, and these recombinant vectors (GVloxP/DsRed-AaTPPA and GVloxP/DsRed-AaERF64) were transfected into A. arguta leaves.

Transient transformation of A. arguta leaves

GVloxP/DsRed-AaTPPA and GVloxP/DsRed-AaERF64 plasmids were transformed into A. tumefaciens strain GV3101(pSoup). GVloxP/DsRed vector was used as the mock plasmid. Young leaves of A. arguta (L2) plantlets grown in pots for 2 months were infiltrated with the transformed Agrobacterium suspensions (OD₆₀₀ = 0.6-1.0) and then were kept in a dark chamber for 1 d. The treated plantlets continued to grow under normal conditions for 5 d, after which the infiltrated leaves at attached state were observed under a fluorescence stereo-microscope (Nikon SMZ25, Japan). The plantlets with red fluorescent leaves were set in a refrigerator at -4°C for 2 h for cold stress treatment, and then the fluorescent leaves were collected for further analysis.

Analysis of physiological characteristics

Conductivity was determined by DDS-ⅡA conductivity meter for E₀ (water control), E₁ (sample was soaked into water and shaken at RT for 1 h), and E₂ (sample was boiled for 15 min). Ion leakage was calculated as the formula: REL (%) = 100×(E₁-E₀)/(E₂-E₀) (Xu et al. 2021). For chlorophyll fluorescence assay, plants were placed in a dark room for 30 min, and then whole plants were imaged using a PHOTON SYSTEMS INSTRUMENTS (PSI Ltd.). The optimal/maximal rate of the quantum yield of photosystem II (Fv/Fm) was measured with FluorCam7 software. The histochemical analyses of H₂O₂ and (O₂⁻) were visualized by staining with 3,3-diaminobenzidine (DAB) and nitro blue tetrazolium chloride (NBT), respectively (Daudi and O'Brien 2012; Kumar et al. 2014). The leaves of A. thaliana and A. arguta were immersed in NBT (0.1 g/mL NBT, 50 mmol/L KH₂PO₄, pH 6.4) and DAB (0.05 g/mL DAB, 50 mmol/L Tris-HCl, pH 5.6) staining solutions after cold stress, and incubated for 3 h at 37°C in dark, after which chlorophyll was discarded in Carnoy's solution (ethanol: lactic acid: glycerol = 3:1:1). The relative average intensity of NBT and DAB staining was analyzed using ImageJ software. Malondialdehyde (MDA), a product of membrane lipid peroxidation, was determined by the thiobarbituric acid method as described previously (Soleimanzadeh et al. 2010). 0.3 g of leaves were ground in liquid nitrogen, and placed in 4 mL of 50 mmol/L potassium phosphate buffer (pH 7.8) in a centrifuge tube. The supernatant was taken, and mixed with 2 mL of 0.5% thiobarbituric acid (TBA), and the mixture was heated in a boiling water bath for 15 min. The absorbance was read at 450 nm, 532 nm, and 600 nm, respectively.

Metabolites and enzyme activities assay

To measure trehalose and sucrose contents, samples were prepared according to the method of (Wang et al. 2020). 21-day-old A. thaliana leaves (100 mg) were harvested and rapidly frozen in liquid nitrogen. Each sample was grounded, and evenly mixed with 500 µL chloroform:acetonitrile (3:7, v/v) and incubated in an ice-bath with occasional shaking. The organic materials were extracted twice with 400 µL water at 4°C. The supernatants were dried in a vacuum dryer. The dry samples were quantified by gas chromatography coupled with mass spectrometry (GC-MS), according to the method of (Garg and Singla 2016). For the determination of Tre6P, samples were prepared as above, and then analyzed using an ultra-performance liquid chromatograph tandem quadrupole mass spectrometer (UPLC-MS/MS) as described previously (Lin et al. 2023).

The content of soluble sugars was measured by the anthrone method. 0.5 g samples were grounded with 2 mL PBS, and 0.1 mL of the supernatant was mixed with 3.5 mL of anthrone, and placed in a water bath at 90°C for 10 min. After cooling, the OD₆₂₀ was measured. The proline content was measured as follows: 0.5 g samples were ground with 2 mL PBS, and 1 mL of the supernatant was mixed with 1.5 mL of ninhydrin and 1 mL of glacial acetic acid, placed in a boiling water bath for 30 min, and then the OD₅₂₀ was measured.

TPP (E.C.3.1.3.12) enzyme activity was assayed by monitoring phosphate ion (Pi) release from Tre6P (Garg and Pandey 2016). The reaction volume was set to 0.3 ml containing 25 mM Tris-HCl (pH 7.0), 10 mM MgCl₂, and 1 mM Tre6P with 0.05 ml of enzyme solution. Pi concentration was measured by the zinc acetate method as follows: 0.3 mL reaction solution was mixed with 0.9 mL of a solution containing 100 mM zinc acetate and 15 mM ammonium molybdate (pH 5.0), and incubated in ice for 1 min. The OD₃₅₀ was immediately measured. Enzyme activities were expressed per mg of protein. SOD, POD, and CAT activities were assayed according to the method of (Huang et al. 2022a).

Cloning of the AaTPPA Promoter and its activity assay by GUS staining

First two sections (1685 bp) upstream to AaTPPA CDS were cloned by conventional PCR amplification with forward primers designed based on the conserved sequences between the TPPA genes of four Actinidia species (A. chinensis cv. Hongyang, A. chinensis cv. Red5, A. eriantha cv. White, A. rufa cv. Fuchu) (Fig. S8a, b), the remaining five sections (2453 bp) were cloned by TAIL-PCR (thermal asymmetric interlaced PCR) according to the manual of Genome Walking Kit (Takara) (Fig. S8c-g). Primers used for cloning the promoter are listed in Table S5. For the promoter activity assay, the CaMV 35S promoter of the GUS expression cassette in pCAMBIA1303 vector was substituted with four segments of the promoter region and 5'-UTR of AaTPPA gene at NcoI and HindIII restriction sites, respectively. These recombinant vectors were transformed into A. thaliana using A. tumefaciens-mediated transformation (refer to A. thaliana transformation method). GUS staining was conducted on AaTPPAproX::GUS transgenic A. thaliana. Seedlings and various organs of the plants were stained with X-Gluc solution in darkness at 37°C overnight. Subsequently, Carnoy's solution was used to remove chlorophyll before taking photographs.

cDNA library screen by Yeast-one-hybrid (Y1H) method

The bait sequences were inserted into the pAbAi vector at SalI and HindIII restriction sites to construct pAbAi-bait plasmids. The pAbAi-bait plasmids were single-digested with BbsI to excise the URA3 gene, and then 1 µg of the linearized plasmid was transformed into Y1HGold yeast competent cells, and determined for the minimum inhibition concentration (MIC) of aureobasidin A (AbA) required to inhibit the bait strain at background levels. The competent cells of the bait strain were co-transformed with A. arguta cDNA library inserted into pGADT7 vector, and screened on SD/-Ura/-Leu media containing the MIC of AbA. Normally growing yeast colonies were selected for PCR test and sequencing.

Yeast-two-hybrid (Y2H) assay for self-transcriptional activity

The full CDS, N-terminal (1–69 aa), and C-terminal domain (70–169 aa) sequences of AaERF64 were cloned into the pGBKT7 (BD) vector at NdeI and EcoRI restriction sites, and transformed into Y2HGold yeast competent cells, respectively. BD-Lam was used as a negative control, and BD-53 was used as a positive control. The transformed yeast cells were screened on SD/-Leu media, and the self-activation test was performed on SD/-Leu/-Trp/-His/-Ade and SD/-Leu/-Trp/-His/-Ade/+X-α-Gal media at different dilutions.

Dual luciferase assay

Dual luciferase assay was performed as described previously (Duan et al. 2019). The CDS of AaERF64 was cloned into the pGreenII-62-SK vector to prepare the effector vector, and the AaTPPAproS3 sequence was cloned into the pGreenII-0800 vector to prepare the reporter vector (Fig. 7e). The constructed vectors were transformed into A. tumefaciens GV3101 (pSoup), respectively, and then co-infiltrated to tobacco (N. benthamiana) leaves. The leaves were treated with a solution containing 0.1 mmol/L potassium D-fluorescein and 0.01% TritonX-100 and then observed using a Tanon 5200 fully automated chemiluminescence image analysis system. Firefly luciferase (Fluc) and renal luciferase (Rluc) activities were determined using a GloMax® 20 light detector according to the instruction for Dual-Luciferase® Reporter Assay System (Promega, Beijing). 20 µL of cell lysates of infiltrated sections were added to a light tube containing 100 µL of LAR II to measure Fluc activity, and subsequently, 100 µL of Stop & Glo® Reagent was added to measure Rluc activity.

Electrophoretic mobility shift assay (EMSA)

The CDS of AaERF64 was cloned into pGEX-4T-1 expression vector and transformed into E. coli host cells to express the recombinant protein GST-AaERF64, which was purified by GST-tag affinity chromatography (Fig. S11). Biotin-labeled DNA probes were synthesized by AZENTA LIFE SCIENCES (Beijing, China). The biotin-tagged hot and mut-probes were mixed with the GST-AaERF64 protein in buffer (0.25 mol/L HEPES, 0.5 mol/L KCl, 20 mmol/L MgSO₄, respectively, and 10 mmol/L DTT) and incubated at 25°C for 30 min. 6.6% PAGE was performed at 80 V for 30 min, and then the EMSA gel was scanned using the Typhoon FLA7000 system (USA).

Statistical analysis

All data in this study were analyzed and plotted using GraphPad Prism 6.0 and IBM SPSS 25.0 (Chicago, IL), and statistical significance was tested using Student's t-test with differences considered significant at *P < 0.05, **P < 0.01, and ***P < 0.001. Duncan's multiple comparisons were used at significance levels of P < 0.05.

Identification of the cold-responsive TPP gene of A. arguta and its subcellular localization

From the transcriptome data of A. arguta cv. Kuilv (KL), we found 39 trehalose-metabolism-related genes including 21 TPSs, 17 TPPs, and one TRE. Comparative analysis of their expression levels between normal (CK) and low temperature (LT) conditions showed that among these genes, TPP#3, #5, #8, and #9 expressed differentially in response to cold stress (Fig. 1a). Since A. arguta (KL) is a tetraploid plant (2n = 4×=116) (Liu et al. 2011), these cold-responsive TPPs might be alleles. Therefore, we cloned all cDNAs of TPP#3, #5, #8, and #9, and found more than 22 different sequences (Fig. S1a). Multiple alignment and phylogenetic tree of these sequences showed that the cloned TPP genes were largely divided into three groups regardless of the primers used for PCR amplification (Fig. S1b). The identities between groups were over 84.6% (Fig. S1c), suggesting that these cold-responsive TPP genes of each group were alleles or duplicate genes, as expected. Among these genes, we selected the Group I TPP gene as the purpose cold-responsive TPP gene, which has the highest expression difference between CK and LT conditions (TPP#5; Appendix S1). This cold-responsive TPP gene of A. arguta had the highest homology with TPPA (trehalose-6-phosphate phosphatase A) of Populus trichocarpa and Arabidopsis thaliana (Fig. 1b). From this, we termed the cold-responsive A. arguta TPP gene as AaTPPA. The similarity of AaTPPA with AtTPPA, PtrTPPA, and OtsB (the E. coli TPP gene) was 66.8%, 73.3%, and 21.4%, respectively (Fig. S2).

The TPP gene family belongs to the HAD (L-2-haloacid dehalogenase) superfamily of phosphatases and other hydrolases, and the proteins of this superfamily are characterized by having three conserved motifs (HAD motif I, II, and III) that form the catalytic active site (Fieulaine et al. 2005). AaTPPA also had all the HAD motifs, considering that it could have the enzymatic activity of trehalose-6-phosphate phosphatase (Fig. S2). To determine its enzymatic activity, the His6-tag-linked AaTPPA protein was expressed in E. coli, and purified by Ni-affinity chromatography (Fig. S3). The His6-tag-linked OtsB protein was used as a positive control. The recombinant AaTPPA protein was mainly expressed in the form of an inclusion body in the E. coli host cells, while the recombinant OtsB protein was present in the cytoplasmic soluble fraction, suggesting that AaTPPA is largely different from OtsB in physiochemical properties. The enzymatic activity of AaTPPA was more than twice that of OtsB at 9–25°C and decreased largely above 35°C (Fig. 1d).

AaTPPA was predicted to have a monopartite nuclear localization signal (NLS) of RKKAK(73–77) (Fig. S2a). In addition, the predicted 3D structure of AaTPPA showed that it has a catalytic domain similar to OtsB, but AaTPPA has an additional N-terminal domain of an unknown function which is common in plant TPP genes (Fichtner and Lunn 2021) (Fig. 1c). The analysis of subcellular localization in onion epidermal cells and tobacco leaf epidermal cells showed that both AaTPPA and OtsB were present in the cell nucleus and the whole cytoplasm (Fig. 1e, f). This result is consistent with a previous report that AtTPPA, AtTPPB, AtTPPC, AtTPPF, and AtTPPH are present in the cytosol and nucleus (Krasensky et al. 2014).

AaTPPA expression is induced in response to mild low-temperature

A. arguta acquires cold acclimatization as the weather temperature gradually decreases in autumn (Van Labeke et al. 2015). Thus, it was necessary to determine at what temperature the expression of the cold-responsive AaTPPA was induced. As a result, the expression level of AaTPPA in mature leaves gradually increased under the mild low-temperature of 15°C, and it was 3.5 times higher at 6 h than before the low-temperature treatment. AaTPPA expression decreased gradually over time at 4°C and 9°C (Fig. 2a).

A. arguta has a strong overwintering ability, but the developing young tissues are very susceptible to frost damage in spring (Marosz 2009). So, we investigated the low-temperature responsiveness of AaTPPA at different leaf growth stages. At the sprouting bud stage, AaTPPA did not respond to mild low temperature, and at the young leaf stage, its expression level increased about twice only after 6 h at 15°C (Fig. 2b). In addition, AaTPPA expression was not induced at all in the sprouting buds and the young leaves under 4°C and 9°C treatments.

Based on the above results, we investigated changes in AaTPPA expression levels in mature leaves of A. arguta grown in a garden as the weather temperature dropped in autumn. Consistent with the indoor experiment, the expression levels of AaTPPA in mature leaves gradually increased until August 23, 2022 (Harbin, China), when the daily low-temperature elapsed 13°C, and then decreased as the temperature dropped below 10°C (Fig. 2d). The weather temperatures below 10^oC suppressed the growth of shoot apical meristems, and accelerated leaf senescence (Fig. 2c). Leaves began to fall on September 25, the deadline for sampling. This result indicated that AaTPPA was transiently induced in mature leaves in response to temporary mild low temperature, and its expression was not maintained at the elevated level during the entire autumn, suggesting that temporary expression of AaTPPA in leaf tissues might not make a critical contribution to the acquisition of overwintering ability of A. arguta.

Therefore, to determine the main acting sites of AaTPPA for acquiring cold acclimatization of A. arguta, the plant's roots, stem cambium, lateral buds, and mature leaves were collected on September 25 in 2022, and AaTPPA expression levels were analyzed (Fig. 2e). The result showed that AaTPPA expression levels in lateral buds, roots, and stem cambium were much higher than that in leaves. Since lateral buds, roots, and stem cambium are dormant tissues in winter, the high expression levels of AaTPPA in these tissues implied that this gene might play an essential role in the cold acclimatization of A. arguta.

Developmental and morphological characteristics of AaTPPA-overexpressing Arabidopsis

Among the transgenic A. thaliana plants, #4 and #7 lines with high expression levels of AaTPPA were selected for AaTPPA-overexpressing (OE) lines (Fig. S4a). We also constructed A. thaliana transformed with OtsB which only has a catalytic domain, unlike the plant TPP enzymes, and OtsB-OE #6 and #7 lines were selected as positive controls (Fig. S4b). Seeds of the selected OE lines were all hygromycin-resistant at T₃ generation, confirming that these lines were homozygous.

First, we measured trehalose and Tre6P levels in AaTPPA and OtsB-OE A. thaliana lines. Surprisingly, trehalose levels in AaTPPA-OE lines were 8 ~ 11 times higher than that of the wild type (WT), whereas no significant changes were observed in OtsB-OE lines (Fig. 3a). T6P levels in A. thaliana were too low to distinguish significant differences between WT and the OE lines, but the result showed a tendency that T6P levels of OtsB-OE lines were lower than those of WT and AaTPPA-OE lines (Fig. S4c). To assay changes in Tre6P levels, we further investigated the expression levels of several SnRK1 marker genes (Fig. S4e). As a result, changes in the expression levels of SnRK1 marker genes in OtsB-OE lines were consistent with the previous report (Zhang et al. 2009), but in AaTPPA-OE lines, there was no significant difference from WT except for the expression of AtDXP. This suggested that Tre6P levels of AaTPPA-OE A. thaliana did not change significantly enough to affect the SnRK1 activity, unlike that of OtsB-OE lines.

Next, we compared the developmental and morphological characteristics between AaTPPA and OtsB-OE A. thaliana. The seeds of all the OE lines were smaller and misshapen compared to WT (Fig. 3b, c). Especially, the seeds of AaTPPA-OE lines were smaller even than those of OtsB-OE lines. However, the small seed size did not affect the germination rate, suggesting that overexpression of the TPP genes only affected seed filling but not embryogenesis (Fig. S5a). Seedlings of the OE lines grown in MS media were also smaller in size and had short primary root lengths at first (Fig. S5b, c; Fig. 3d, e), but afterward, there was no significant difference in growth compared to WT (Fig. S5d), demonstrating that the initial growth disturbance was only due to a lack of nutrients in the seeds of the TPP-OE lines. AaTPPA overexpression rather seemed to enhance vegetative growth in the later developmental stages (Fig. 3f).

AaTPPA-OE lines had elongated rosette leaves like WT, but OtsB-OE lines had broad-shaped leaves (Fig. S5d; Fig. 3g). In addition, chlorophyll contents of rosette leaves were significantly lower in OtsB-OE lines, but there was no difference between AaTPPA-OE lines and WT (Fig. S5e). As in previous studies, bolting periods of all the OE lines were delayed than WT (Fig. 3f), and even at the time when all the rosette leaves of WT had wilted, the rosette leaves of the OE lines remained vivid (Fig. 3h). Interestingly, AaTPPA-OE lines had more branches and siliques than WT, but OtsB-OE lines had significantly fewer branches and siliques (Fig. 3i; Fig. S5g). No significant difference was observed in heights between the OE lines and WT (Fig. S5f).

Overall, AaTPPA-OE A. thaliana had much higher trehalose levels and showed distinct developmental and morphological characteristics. Like OtsB-OE lines, AaTPPA-OE lines had misshapen seeds and delayed reproductive growth and senescence. However, unlike OtsB, AaTPPA overexpression did not affect the leaf shape and chlorophyll content. Furthermore, AaTPPA-OE lines had more branches and siliques compared to WT and OtsB-OE lines.

Heterogeneous overexpression of AaTPPA enhances the freezing tolerance of Arabidopsis

First, we investigated the freezing tolerance of the OE A. thaliana lines at the seedling stage. The survival rate of AaTPPA-OE #4 line was significantly higher than that of WT after freezing treatment at -10°C for 0.5 and 1 h. On the contrary, OtsB-OE lines showed a relatively low survival rate compared to WT (Fig. S6a, b). Meanwhile, the ion leakages of both AaTPPA-OE and OtsB-OE seedlings were lower than that of WT after 1 h of freezing treatment, suggesting that the TPP overexpression had a protective effect on biological membranes (Fig. S6c).

Next, A. thaliana plants grown in pots were subjected to freezing stress at -10°C. The leaf wilting rates of AaTPPA-OE lines of 4 h treatment group were significantly lower than that of other lines (Fig. 4a, b). Surprisingly, after normal cultivation of this group for 7 d, the reviving rate of AaTPPA-OE #4 line was twice that of WT, while the reviving rates of OtsB-OE lines were significantly lower than that of WT (Fig. 4c).

Freezing stress-treated A. thaliana lines were stained with NBT to assay the levels of superoxide anion (O₂⁻) (Fig. S6d, e). Under normal conditions, O₂⁻ levels of all the OE lines were significantly lower than that of WT, suggesting that the TPP overexpression increased ROS scavenging capacity. Under freezing conditions, O₂⁻ levels of all the lines decreased as freezing time increased, and its levels were maintained higher in all the OE lines than in WT. This result could be interpreted that photosystem I (PSI), the generator of O₂⁻ in chloroplasts, was destructed under extreme freezing conditions, but the TPP overexpression prevented PSI from freezing destruction. The fact that the plant photosystems were protected in the OE lines was also proved by chlorophyll fluorescence assay. After cold stress at -2°C for 2h, Fv/Fm values remained the highest in AaTPPA-OE lines, and the values of OtsB-OE lines were also higher than that of WT (Fig. 4d, e).

We also evaluated the degree of hydrogen peroxide (H₂O₂) accumulation using DAB staining (Fig. S6f, g). AaTPPA-OE lines tended to accumulate less H₂O₂ than WT and OtsB-OE lines under normal and freezing stress, but no significant difference was recognized. Moreover, the activities of SOD, POD, and CAT increased more in AaTPPA-OE lines than in WT and OtsB-OE lines under normal and cold stress conditions (Fig. S6h, i, j), and the MDA contents also increased more in AaTPPA-OE lines after cold stress (Fig. 4f), indicating that AaTPPA overexpression plays a positive role in activating the plant antioxidant system.

The above results implied that AaTPPA overexpression could have a protective effect on biological membranes and biomolecules by accumulating osmotic protectants. For proof of concept, the contents of sucrose, soluble sugars, and proline, which are the main osmotic protectants of plants, were measured, showing that under normal and cold stress conditions more sucrose and soluble sugars were accumulated in both AaTPPA and OtsB-OE lines than those in WT, and proline contents were higher only in AaTPPA-OE lines (Fig. 4g, h, i).

In summary, AaTPPA overexpression enhanced the cold tolerance of A. thaliana, but OtsB overexpression did not affect the cold tolerance obviously and was rather negative in some aspects.

Transient overexpression of AaTPPA in A. arguta leaves improves cold tolerance indices

Through the Agrobacterium tumefaciens-mediated transformation, target genes can be transiently overexpressed in plant leaf tissues. However, this method has low reproducibility, because the expression efficiency of the target gene is different each time depending on the growth state of the infiltrated leaves and the experimenter's technique. To improve the reproducibility of this transient transformation method, we created a new plant expression vector (GVloxP/DsRed) which contains a red fluorescence protein (DsRed) as a marker, allowing to monitor the degree of transfection in the infiltrated leaves by a non-invasive manner (Fig. S7). The overexpression vector, GVloxP/DsRed-AaTPPA, was prepared by replacing the CDS of the hygromycin resistance gene (HygR) of this vector with AaTPPA CDS (Fig. 5a).

Young leaves of A. arguta cv. Longcheng No.2 (L2) plantlets grown in pots were infiltrated with Agrobacterium containing GVloxP/DsRed-AaTPPA vector, and after 3 d, they were observed under a fluorescence stereomicroscope, and leaves with obvious red fluorescence were collected to determine the gene expression level (Fig. 5b, c). The result demonstrated that the AaTPPA expression level was incomparably higher in AaTPPA-OE group than in EV (the mock group only infiltrated with GVloxP/DsRed empty vector) and CK (the control group not transfected).

Next, A. arguta (L2) plantlets with red-fluorescent leaves that were transformed as described above were subjected to cold stress at -4°C for 2 h, and then several cold tolerance-related indices, such as ROS accumulation, ion leakage, MDA, soluble sugars, and proline contents, were analyzed. Interestingly, DAB staining showed that H₂O₂ accumulation in EV group was particularly higher, explaining that the leaf tissues might be damaged by the transfection process, or that the overexpression of marker genes, such as DsRed and HygR, might be harmful to the physiology of the plant (Fig. 5d, e). Nevertheless, H₂O₂ level of OE group was significantly lower than that of CK group after cold stress. In addition, after cold stress, the ion leakage and MDA contents of AaTPPA-OE leaves were significantly lower than those of CK and EV groups (Fig. 5f). Soluble sugars and proline contents were higher in AaTPPA-OE group even under normal conditions. Cold stress increased the contents of these osmotic protectants in all groups, but AaTPPA-OE group still maintained the highest level (Fig. 5f).

Cloning of the AaTPPA promoter and verification of its transcriptional activity

Since the genome sequence of A. arguta has not been published yet, the promoter sequence of AaTPPA had to be cloned to seek upstream transcription factors that affect the AaTPPA expression in response to low temperature. According to the genome data of A. chinensis cv. Red5, the homologous TPPA gene had an exceptionally long 5'-UTR (3,162 bp) upstream of the ATG translational start codon (Fig. S8h). Therefore, to obtain the promoter sequence of AaTPPA, we had to clone a region of at least 4,000 bp upstream of the ATG codon. By combining the conventional PCR and TAIL-PCR (thermal asymmetric interlaced PCR), a total region of 4,138 bp upstream of the ATG codon was cloned and sequenced (Fig. S8, Appendix S2). Alignment with the TPPA gene sequence of A. chinensis cv. Red5 showed that among the whole cloned sequence, the promoter region was approximately 1185 bp, and the remaining section (2953 bp) belonged to 5'-UTR (Fig. S9a).

In the previous study on the promoters of TPP genes, ~ 2000 bp sequence upstream to CDS was considered to be the promoter region (Wang et al. 2022). So, to determine whether the long 5'-UTR of AaTPPA affects the gene expression, the CaMV 35S promoter of the GUS gene in pCAMBIA1303 vector was substituted by four sections of the cloned 5'-UTR plus promoter region to construct the promoter activity checking vector (Fig. S9a, b). The AaTPPApro2 section contained a 1685 bp sequence from the ATG codon to the middle of intron 1, the AaTPPApro3 section contained a 2688 bp sequence including the almost intron 1, and the AaTPPApro9 included the whole 5'-UTR and promoter region of AaTPPA (4138 bp). The AaTPPApro8 section contained only the predicted promoter region (Fig. S9a). The GUS expression cassettes driven by the above four different sections were transformed into A. thaliana, and the screened transgenic T₁ plants were verified by PCR test on the corresponding sections (Fig. S9c-j).

GUS gene driven by AaTPPApro9 was expressed in almost all tissues including roots, vascular tissues, mesophyll, stems and branches, sepals and petals, siliques, and developing seeds (Fig. 6a). In comparison, GUS driven by AaTPPApro8 had the same expression pattern as AaTPPApro9, except for young leaves and developing seeds. On the contrary, AaTPPApro2 and AaTPPApro3-driven GUS were not expressed in roots and were mainly expressed in vascular tissues. AaTPPApro3::GUS was also specifically expressed in developing seeds. The above results showed that the AaTPPApro8 sequence had sufficient promoter activity in almost all tissues except for developing seeds and young leaves.

Next, to determine whether the AaTPPApro8 section has cold-stress responsive characteristics, we analyzed the expression levels of the AaTPPApro8-driven GUS gene under normal and low temperature (9°C) conditions (Fig. 6b, c). The result showed that GUS staining was more intense in the low-temperature treatment group than in the normal group, indicating that the AaTPPApro8 sequence has a low-temperature responsive cis-acting element.

Taken together, it can be concluded that AaTPPA has a long 5'-UTR of about 3000 bp and that the AaTPPApro8 sequence of 1274 bp has a low-temperature responsive characteristic as well as constitutive promoter activity.

AaERF64 binds to the AaTPPA promoter and activates its transcription

We searched for cis-acting regulatory elements in the promoter region of 1185 bp upstream from the predicted transcriptional start site using the online software PlantCARE (Fig. 7a, Table S1). Based on the motif analysis, we adopted three sections which are clustered with functional motifs, and each fragment was inserted into pAbAi vector, respectively, to prepare bait vectors. Among the Y1HGold strains transformed with these bait vectors, only the AaTPPAproS3 introduced strain was completely suppressed on 300 ng/mL aureobasidin A (AbA) containing medium, and the others were insensitive to AbA because of their constitutive promoter activity. Therefore, proteins interacting with the AaTPPAproS3 section were screened from the A. arguta cDNA library using the yeast-one-hybridization (Y1H) method. As a result, three ethylene-responsive transcription factors (ERF) were identified as transcription factors. From the transcriptome data of A. arguta, the ERF gene (gene28680) which was responsive to low temperature was selected for further study (Table S2). Its interaction with the AaTPPA promoter was reconfirmed by using the Y1H assay (Fig. 7b). This ERF gene had the highest homology with ERF64 of Actinidia deliciosa (AdERF64) in NCBI's nucleotide database (Fig. S10a), so we called it AaERF64 (Appendix S3).

In plants, ERF genes constitute a large family of transcription factors which is a part of the AP2/ERF (APETALA2/ETHYLENE RESPONSE FACTOR) superfamily (Riechmann et al. 2000). Generally, proteins of AP2/ERF superfamily have an AP2 DNA-binding domain of approximately 60–70 amino acids (Sakuma et al. 2002). AP2/ERF superfamily is divided into 12 phylogenetic groups in A. thaliana, and some of them are further divided into several subgroups (Nakano et al. 2006). The phylogenetic tree with 122 ERF family members of A. thaliana showed that AaERF64 belongs to the V group of the ERF family, and has the closest relation to ERF#003 (At5g25190.1) of A. thaliana (Fig. S10a). This group is characterized to have one CMV-2 motif (Nakano et al. 2006). Bioinformatic analysis showed that both the AP2 DNA-binding domain and CMV-2 motif are well conserved in the AaERF64 protein (Fig. S10c). In addition, the N-terminal contains NLS peptide, indicating that this protein could be localized in the cell nucleus. The predicted 3D structure showed that the AaERF64 protein is composed of two domains including the AP2 DNA-binding domain (4–69 aa) and a poorly predicted C-terminal domain (70–169 aa). The C-terminal domain was thought to be a transcriptional activation domain due to containing the CMV-2 motif (Fig. S10b).

Subcellular localization assay of AaERF64 in tobacco leaf epidermal cells showed that AaERF64 was localized in the cell nucleus, satisfying the first requirement for acting as a transcription factor (Fig. 7c). Its transcriptional activation ability was examined by the Y2H self-transactivation test (Fig. 7d). The result demonstrated that AaERF64 had a transcriptional activation function and that the C-terminal domain (70–169 aa) was a transcriptional activation domain, as expected. In addition, the dual luciferase assay also demonstrated that AaERF64 could interact with an element of the AaTPPA promoter to activate the transcription of the downstream gene in plant cells (Fig. 9e). Furthermore, we transiently transfected A. arguta leaves with the GVloxP/DsRed-AaERF64 vector and proved that AaERF64 overexpression upregulates the expression of the endogenous AaTPPA in A. arguta genome (Fig. 8a). These results revealed that AaERF64 acts as a transcriptional activator of AaTPPA in A. arguta.

Next, we searched for the AaERF64-binding cis-element within the AaTPPA promoter sequence. All the ERF family proteins have the conservative AP2 DNA-binding domain, which has been known to specifically bind to GCC-box (Ohme-Takagi and Shinshi 1995). We found three GCC-box motifs in the AaTPPAproS3 section, which was used as a bait sequence for the AaERF64 screen (Fig. 7f). Three DNA fragments (EM1 ~ 3) containing each GCC-box motif were introduced into the pAbAi vector, respectively, and their binding potential with AaERF64 was examined by employing the Y1H assay. The result revealed that the EM2 fragment (22 bp) interacted with AaERF64. Furthermore, electrophoretic mobility shift assay (EMSA) proved that AaERF64 directly binds to the GCC-box motif (GCCGAC) of the EM2 sequence (Fig. 7g).

AaERF64 expression is induced through the ethylene signal transduction pathway

As shown above, AaTPPA expression levels were particularly high in overwintering tissues of A. arguta in autumn (Fig. 2e). This time, we investigated changes in the expression levels of AaTPPA and AaERF64 in these tissues according to the temperature decrease in autumn (Fig. 8b). Based on the previous experimental data, we selected three sampling times; the first time was the day when the weather temperature was the highest in summer, 2023, the second time was the day when the daily low-temperature dropped below 15°C, and the last time was the day when the daily low-temperature dropped below 10°C. At the last time, the shoot apical buds began to degenerate.

In mature leaves, stem cambium, and lateral buds, the expression levels of AaTPPA were positively correlated to those of AaERF64, supporting the prior result that AaERF64 overexpression induced upregulation of AaTPPA expression (Fig. 10a). Unexpectedly, in roots, no significant changes were observed in the expression levels of AaERF64 depending on the period, but the expression levels of AaTPPA gradually increased as the temperature became colder. This suggested that AaERF64 has no action in roots and that AaTPPA has a high expression level specifically in roots, and may be regulated by other pathways.

In mature leaves, consistent with the results in 2022 (Fig. 2d), the expression levels of AaERF64 and AaTPPA were the highest at the mild low temperature (daily high-temperature 24°C, daily low-temperature 14°C), and afterward, the expression of both genes decreased. However, AaERF64 expression in lateral buds and stem cambium continued to increase until the last day (September 15, 2023), and so did AaTPPA expression. This result suggested that under excessively low temperature conditions, leaves become withered, leading to the reduction of overall gene expression, whereas in lateral buds and stem cambium, expression of genes, especially the genes related to cold stress, continue to be activated even in late autumn.

Next, we conducted research to identify the signal transduction pathway that induces the expression of the AaERF64 transcription factor. According to previous studies, plant TPP genes were induced by several phytohormones, such as abscisic acid (ABA), jasmonic acid methyl ester (MeJA), and gibberellin (GA) (Ge et al. 2008; Lin et al. 2020; Huang et al. 2022b), and the cis-acting element analysis of the AaTPPA promoter predicted that there exists MeJA and ABA-responsive motifs (Fig. 7a, Table S1). For AaERF64 as an ethylene-responsive factor, we selected ethephon (the precursor of ethylene), MeJA, and ABA as the putative inducible phytohormones (Fig. S12c). In A. arguta leaves, AaTPPA was induced by ethylene and ABA, and AaERF64 was induced only by ethylene treatment. Induction of AaERF64 and AaTPPA expression by ethylene occurred at 1–2 d of treatment, whereas induction of AaTPPA expression by ABA peaked at 12 h and then decreased. Interestingly, ABA treatment rather suppressed AaERF64 expression. This suggested that the induction of AaTPPA expression by ABA occurs through a pathway different from AaERF64 and that the induction by ABA occurs transiently in leaves, which is reversible when the ABA signal disappears.

We further investigated changes in the expression levels of AaTPPA and AaER64 in the overwintering tissues of A. arguta by ethylene treatment (Fig. 8c). In the lateral buds and stem cambium, the expression levels of AaTPPA and AaER64 continued to increase until 15 d of treatment, reaching over 10-fold higher than before treatment. However, their expression levels in leaves did not increase further after 5 d of treatment. Soluble sugars and proline are the main osmotic protectants in plants (Ding et al. 2020). Undoubtedly, lateral buds, stem cambium, and roots of A. arguta also contained much soluble sugars and proline in late autumn (Fig. S12a, b). Ethylene treatment also increased the contents of soluble sugars and proline in the lateral buds and stem cambium more than in the leaves (Fig. 8c). However, in roots, AaTPPA and AaER64 expression did not respond to ethylene treatment, and there were no significant changes in the contents of soluble sugars and proline. This suggested that the increase in AaTPPA expression and the accumulation of soluble sugars and proline in roots in autumn might occur depending on other signaling pathways, maybe by ABA (Fig. 8b).

AaTPPA overexpression dramatically increases trehalose levels of Arabidopsis

Schluepmann et al. reported that trehalose levels of OtsB-OE Arabidopsis plants were below the detection limit of 2 nmol/g FW, which were not significantly different from WT (Schluepmann et al. 2003). Meanwhile, trehalose levels in AtTPPF-OE A. thaliana seedlings were ~ 7 µg/g FW, not different from WT, but the levels in AtTPPI-OE seedlings were twice that of WT (Lin et al. 2019; Lin et al. 2023). In our experiments, AaTPPA had a substantially higher TPP enzymatic activity than OtsB (Fig. 1d). OtsB overexpression did not affect trehalose levels of A. thaliana, consistent with the previous study, but AaTPPA overexpression resulted in 8 ~ 11 times higher trehalose levels compared to WT (Fig. 3a). This suggests that TPP enzyme activities vary depending on plant species and types of TPP genes.

We also investigated the effect of AaTPPA overexpression on the levels of Tre6P, the substrate of the TPP enzyme reaction. The quantitative assay of Tre6P and changes in the expression levels of SnRK1 marker genes indicated that Tre6P levels were reduced in OtsB-OE lines, but there was no such trend in AaTPPA-OE lines (Fig. S4c, e). This result is similar to the previous reports that OtsB overexpression lowered Tre6P levels by more than 5-fold in A. thaliana, while AtTPPI overexpression only lowered them by about 20% compared to WT (Schluepmann et al. 2003; Lin et al. 2023).

The difference in the change of Tre6P levels was reflected in the developmental and morphological characteristics of AaTPPA and OtsB-OE A. thaliana lines. Schluepmann et al. reported that mature leaves of OtsB-OE Arabidopsis were lighter green and larger than those of WT, and these differences in leaf phenotypes were caused by the change in Tre6P levels, not by trehalose (Schluepmann et al. 2003). In our study, rosette leaves of AaTPPA-OE lines showed similar phenotypes to WT, unlike OtsB-OE lines (Fig. 3g, Fig. S4d, e). This result was also regarded as indirect evidence that Tre6P levels were not significantly changed in rosette leaves of AaTPPA-OE lines.

However, AaTPPA-OE lines showed OtsB-like phenotypes in other organs; both had small and misshapen seeds, late bolting, and delayed senescence (Fig. 3b, c, f, h). Previous studies reported that OtsB-OE Arabidopsis seeds often had an asymmetrical shape and lower oil content than WT (Zhai et al. 2021). When OtsB was expressed in peas under an embryo-specific promoter, Tre6P levels and starch accumulation were reduced, resulting in the formation of wrinkled seeds (Meitzel et al. 2020). Previous studies also indicated that OtsB-OE Arabidopsis bolted up to 3 weeks later than WT, and had a pronounced apical dominance and plenty of seed set (Schluepmann et al. 2003). JcTPPJ-OE Arabidopsis also bolted 2–3 d later and had more rosette leaves compared to WT (Zhao et al. 2019). The reason why AaTPPA overexpression did not affect the leaf morphology while affecting the reproductive growth and seed formation may be because the modes of trehalose metabolism are different between these tissues. In Arabidopsis leaves, AtTPS1 participates in trehalose metabolism, but during embryogenesis in seeds, other types of TPSs, AtTPS2 and AtTPS4, are expressed instead of AtTPS1 (Fichtner and Lunn 2021). These different TPS genes may have different interactions with AaTPPA.

Although the bolting period was delayed, AaTPPA-OE lines had more branches and siliques, unlike WT and OtsB-OE lines (Fig. 4i, Fig. S5g). In plant leaf cells, Tre6P plays a role as an indicator of sucrose levels. Sucrose accumulation in mesophyll cells increases Tre6P levels which serve as a signal to activate sucrose transport channels (SWEETs) to export more sucrose from the mesophyll to sink tissues (Fichtner and Lunn 2021). From this point of view, we can assume that, unlike OtsB, AaTPPA overexpression does not significantly reduce Tre6P levels in leaf cells, so excessive sucrose accumulation in mature leaves finally increases Tre6P levels to reach a threshold, triggering the activation of transporters to export the accumulated sucrose to phloem, which results in vigorous sprouting of branches.

Taking the above results together, we suppose that AaTPPA overexpression dramatically increases trehalose levels, but does not significantly reduce Tre6P levels at least in leaves. On the contrary, OtsB overexpression greatly reduces Tre6P levels but does not significantly change trehalose levels. Previous studies suggested that the reason why OtsB overexpression did not change trehalose levels was because the trehalase activity of plant cells was too high to degrade the synthesized trehalose immediately, preventing its accumulation (Van Houtte et al. 2013). Moreover, there was a report that trehalase activity was increased in yeast TPS and TPP fusion gene (ScTPS1-ScTPS2) transgenic plants than in non-transgenic plants (Romero-Reyes et al. 2023). In this regard, it might be thought that the rate of trehalose synthesis in AaTPPA-OE Arabidopsis leaves was high enough to exceed the capacity of trehalase.

More interestingly, despite the high enzymatic activity of AaTPPA, Tre6P levels in leaves of AaTPPA-OE lines seemed not to be decreased significantly, indicating that the TPS enzyme activity was also increased. To determine whether AaTPPA overexpression might have affected the expression of AtTPS1, the major TPS gene in Arabidopsis, we measured its transcription levels, but no significant difference was observed between WT and the OE lines (Fig. S5d). This suggested that TPS activity in AaTPPA-OE plant leaves was post-translationally regulated. In other words, AaTPPA might interact directly or indirectly with the AtTPS1 protein to regulate its enzymatic activity. Therefore, we measured the TPS, TPP, and TRE enzyme activities, but no difference was found between WT and the OE lines. Perhaps the regulation of TPS and TRE activities under in vitro conditions would be different from the in vivo environment. Previous studies have suggested that genes of trehalose metabolism might interact with each other. Some class II TPS proteins perform regulatory functions by forming complexes with class I TPS in rice (Zang et al. 2011). In addition, it has been claimed that some TPP enzymes exhibit the “moonlight effect”, performing unknown regulatory functions rather than catalytic activity (Kretzschmar et al. 2015; Claeys et al. 2019). The fact that the biochemical and morphological characteristics of TPS-TPP fusion gene transgenic plants are consistent with AaTPPA-OE plants also supports the evidence of AaTPPA and AtTPS1 interaction. In rice overexpressing fusion of OtsA and OtsB, the TPS and TPP genes of E. coli, trehalose levels increased to almost 0.1% of fresh weight without significant change in Tre6P levels, and there were no noticeable abnormal phenotypes, such as growth retardation and changes in root morphology, that were observed in rice overexpressing OtsA or OtsB, respectively (Jang et al. 2003). In addition, when the fusion of ScTPS1 and ScTPS2, the yeast TPS and TPP genes, were overexpressed in Arabidopsis, trehalose levels increased more than 4 times compared to WT, and no differences in morphology and development were recognized, while only ScTPS1-OE lines showed abnormal phenotypes (Miranda et al. 2007). Considering the above data, it is believed that the accumulation of trehalose in AaTPPA-OE plants might be the result of the interaction of AaTPPA with AtTPS1, thus increasing Tre6P synthesis, the rate-limiting step of trehalose metabolism.

Protein 3D-structure prediction showed that OtsB has only a catalytic domain, whereas AaTPPA has an additional N-terminal domain of unknown function (Fig. 1c). From this, unlike OtsB, the regulatory action of AaTPPA is believed to be due to its N-terminal domain. In the future, it is necessary to verify the role of this N-terminal domain by investigating transgenic lines that are introduced with N-terminal truncated AaTPPA. In detail, much attention should be paid to elucidating the interaction between the N-terminal domain of TPPA and the N-terminal regulatory domain or C-terminal TPP-like domain of TPS1 whose functions are still unknown (Fichtner et al. 2020).

AaTPPA plays a role in enhancing the plant freezing tolerance

There were several examples showing that the introduction of a plant TPP gene could increase the tolerance of plants to cold stress. AtTPPI overexpression in Arabidopsis enhanced cold tolerance, and OsTPP1 overexpression in rice increased tolerance to salt and cold stress (Lin et al. 2023; Ge et al. 2008). However, there have been no published reports on the effect of OtsB on plant stress tolerance. Therefore, we studied the effect of AaTPPA on the freezing tolerance of A. thaliana in contrast with OtsB-OE lines. From the results, we concluded that AaTPPA overexpression significantly enhanced the freezing tolerance of A. thaliana, but OtsB did not contribute to the freezing tolerance (Fig. 4a, b, c). In addition, among AaTPPA-OE plants, the #4 line, which had the highest AaTPPA expression and trehalose levels, was superior in all physiological and biochemical indices related to cold tolerance, suggesting that trehalose levels are positively related to the cold tolerance of the plants. Previous studies also showed that there was a certain correlation between trehalose levels and the stress tolerance of plants. In Arabidopsis, rice, and wheat, overexpression of TPS-TPP fusion genes of E. coli or yeast origin increased trehalose levels as well as the tolerance to salt, drought, cold, and heat stresses (Garg et al. 2002; Jang et al. 2003; Miranda et al. 2007; Romero-Reyes et al. 2023). In addition, exogenous applications of trehalose to plants could increase the tolerance to various stresses (Luo et al. 2023; Islam et al. 2023; Zhang et al. 2022; Yuan et al. 2022; Yang et al. 2022). The above data suggest that trehalose plays a more decisive role than Tre6P in improving the stress tolerance of plants.

However, the impact of Tre6P on the stress tolerance of plants cannot be completely ignored. In plants, there exists the sucrose:Tre6P nexus model: sucrose accumulation increases Tre6P levels, which conversely inhibits the sucrose biosynthesis, thereby maintaining the constant of sucrose versus Tre6P ratio (generally 1000:1) in plant cells (Figueroa and Lunn 2016). According to this theory, in plant leaf cells, the decrease in Tre6P levels enhances the sucrose biosynthesis, and suppresses its export to phloem, resulting in the accumulation of sucrose. Sucrose is one of the osmotic protectants and directly contributes to the stress tolerance of plants. Indeed, our results showed that in both AaTPPA and OtsB-OE lines, several stress tolerance-related indices, such as ion leakage, ROS scavenging, Fv/Fm value, and the content of soluble sugars, were superior to WT, which could be explained in relation to the sucrose accumulation due to the reduction in Tre6P levels by TPP overexpression. Nevertheless, the overall freezing tolerance of OtsB-OE lines was inferior to WT. It might be because the excessive reduction of Tre6P levels caused by uncontrollable TPP enzyme activity had a negative impact on the growth and development of the plants.

AaTPPA overexpression can increase the tolerance of A. arguta to freezing stress. Because tissue culture-dependent plant transformation system has not been established yet for A. arguta, we could not conduct the freezing tolerance verification by using AaTPPA-OE A. arguta plants. Instead, we conducted assays for several cold tolerance-related indices of AaTPPA transiently overexpressing A. arguta leaves (Fig. 5). The results demonstrated that ROS scavenging activity and the content of osmotic protectants such as soluble sugars and proline were higher than that of control and mock groups. These results were consistent with previous studies showing that the introduction of TPP genes activated the expression of stress-related genes participating in the antioxidant system and the production of osmotic protectants in other plants (Lin et al. 2019; Huang et al. 2022b; Lin et al. 2023).

The results of the transient overexpression of AaTPPA in A. arguta leaves and the fact that its expression levels increased in the overwintering organs in response to low temperature promise that this gene will greatly contribute to the freezing tolerance of A. arguta as in A. thaliana. However, to increase the cold tolerance of A. arguta by introducing the TPP gene, a technology that can spatiotemporally control the gene expression must be employed, because the indiscriminate overexpression of TPP genes may have negative effects on plant growth and development in some aspects.

AaTPPA participates in the cold acclimatization of A. arguta in autumn

There are dozens of TPP genes in a plant genome, and these genes are ascribed to have been formed and evolved through gene duplication. The Arabidopsis genome has 10 TPP genes, while the rice genome has 13 TPP genes, and every TPP gene is expressed differently depending on tissues, development stages, and environmental conditions (Vandesteene et al. 2012; Du et al. 2022). Through transcriptome analysis, we found that there were at least 17 TPP genes in the A. arguta genome, and among them, we selected AaTPPA as the research target, whose expression level increased in response to low temperature (Fig. 1a).

The expression level of AaTPPA increased in response to mild low temperatures around 15°C, and its induction by low temperature was more sensitive in mature tissues than in young tissues (Fig. 2a, b). In the fields, the period meeting these conditions is at the beginning of autumn when the weather temperature begins to decrease. Indeed, the expression analysis of AaTPPA from midsummer to late autumn showed that its expression began to increase after the daily low-temperature began to fall below 15°C. When the temperature decreased further, the expression levels of AaTPPA continued to increase in the overwintering tissues, such as lateral buds, roots, and stem cambium, even as the shoot apical meristems and young tissues withered and degenerated (Fig. 2c, d, e, 8b). These results strongly implied that AaTPPA might be involved in the overwintering preparation of A. arguta in autumn.

For plants to overcome cold winter safely, osmotic protectants such as inorganic and organic ions, soluble sugars, and soluble proteins that prevent cell damage from freezing must be sufficiently accumulated in overwintering tissues (Ding et al. 2020). Trehalose metabolism could be involved in the accumulation of these osmotic protectants. In A. thaliana plants and A. arguta leaves overexpressing AaTPPA, the osmotic protectants, such as soluble sugars and proline, were accumulated along with a marked increase in trehalose levels. As a result, the cell membranes were protected from freezing, reducing ion leakage, and the antioxidant enzyme activities were preserved, increasing ROS scavenging capacity (Fig. 4, 5, Fig. S6). Changes in Tre6P levels might also have an impact on the accumulation of osmotic protectants in overwintering tissues. According to previous studies, a decrease in Tre6P levels in sink tissues serves as a sugar shortage signal, which activates sucrose transporters and promotes the decomposition of stored starch. As a result, the sink tissues import and produce more sucrose (Paul et al. 2018). According to this theory, upregulation of AaTPPA expression in the overwintering tissues would decrease Tre6P levels, leading to enhancing sucrose import and starch decomposition, which would supply more nutrients necessary to synthesize the osmotic protectants.

We identified the AaERF64 transcription factor that binds to the AaTPPA promoter. AaERF64 binds to the GCC-box of the AaTPPA promoter to activate its transcription and showed the same expression pattern as AaTPPA in response to the temperature decrease in autumn (Fig. 7, 8a, b). Furthermore, we demonstrated that AaERF64 was induced by ethylene treatment (Fig. 8c, Fig. S12c). In plants, ethylene acts as a stress-induced signaling molecule, and its biosynthesis is generally enhanced under low temperature conditions (Murakami et al. 2014). The ethylene signal transduction pathway includes ethylene receptors, CTR1 (constitutive triple response 1), EIN2, EIN3 (ethylene insensitive 2 and 3), EIL (EIN3-like), ERF proteins, and ethylene-responsive genes (Huang et al. 2018). There were reports that ethylene biosynthesis was enhanced by low temperature in Actinidia plants and that the ripening of fruits was promoted by internally synthesized ethylene or its external application (Mworia et al. 2010; Wang et al. 2015). The above reports and our results support the idea that ethylene is the most upstream signaling molecule that increases AaTPPA expression in response to the temperature decrease in autumn. In addition, the expression level of AaTPPA was transiently increased by ABA treatment, and it is considered that AaTPPA expression is induced in response to temporary stress depending on the ABA signaling pathway, especially in roots (Fig. S12c). The induction of AaTPPA expression by ABA was reversible, whereas its induction by ethylene was irreversible because ethylene can feedback activate its endogenous biosynthesis (Mworia et al. 2010).

Combining the above results, we propose a model of the cold acclimatization mechanism of A. arguta (Fig. 9). The temperature decrease in autumn enhances ethylene biosynthesis in A. arguta plants. The synthesized ethylene induces the expression of the AaERF64 transcription activator through its signal transduction pathway in overwintering tissues, such as lateral buds and stem cambium. AaERF64 binds to the AaTPPA promoter, increasing its expression level. AaTPPA expression is also induced by ABA in roots. AaTPPA catalyzes the conversion reaction from Tre6P to trehalose, increasing trehalose levels and decreasing Tre6P levels in the corresponding tissue cells. Trehalose serves as a signaling molecule that activates the expression of stress-related genes involved in the production of osmotic protectants. Meanwhile, the reduction of Tre6P levels in overwintering tissues would enhance the sucrose import from source leaves, and accelerate the decomposition of stored starch, resulting in the production of more osmotic protectants, such as soluble sugars and proline. As a result, A. arguta plants acquire the overwintering ability to survive even under extreme freezing conditions.

Author contribution

TE and PL conceived the research project and set up the experiments. JP, NE, and YX conducted metabolites and biochemical analysis. LM and JL performed the data analysis. TE and JP wrote the manuscript. JL and HK edited the manuscript. FM and PL organized and supervised the overall project. FM and GJ critically revised the manuscript for publication. All authors have read and agreed to the published version of the manuscript.

Funding

The authors would like to acknowledge the technical support from Analysis and Testing Center of Northeast Forestry University. This research was supported by the Fundamental Research Funds for the Central Universities (No.2572022DS13).

Declaration of Competing Interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

All relevant data can be found within the manuscript and its supporting materials.

Avonce N, Mendoza-Vargas A, Morett E, Iturriaga G (2006) Insights on the evolution of trehalose biosynthesis. BMC Evol Biol 6. 10.1186/1471-2148-6-109
Baena-Gonzalez E, Rolland F, Thevelein JM, Sheen J (2007) A central integrator of transcription networks in plant stress and energy signalling. Nature 448(7156):938–U910. 10.1038/nature06069
Blazquez MA, Santos E, Flores CL, Martinez-Zapater JM, Salinas J, Gancedo C (1998) Isolation and molecular characterization of the Arabidopsis TPS1 gene, encoding trehalose-6-phosphate synthase. The Plant journal: for cell and molecular biology 13(5):685–689. 10.1046/j.1365-313X.1998.00063.x
Cabib E, Leloir LF (1958) THE BIOSYNTHESIS OF TREHALOSE PHOSPHATE. J Biol Chem 231(1):259
Calugaru-Spataru T, Ivanova R, Dascaliuc A (2013) In vitro multiplication and cultivation of Actinidia arguta in the Republic of Moldova. BIOLOGIJA 59(3):301–308
Claeys H, Vi SL, Xu X, Satoh-Nagasawa N, Eveland AL, Goldshmidt A, Feil R, Beggs GA, Sakai H, Brennan RG, Lunn JE, Jackson D (2019) Control of meristem determinacy by trehalose 6-phosphate phosphatases is uncoupled from enzymatic activity. Nat Plants 5(4):352–357. 10.1038/s41477-019-0394-z
Daudi A, O'Brien JA (2012) Detection of Hydrogen Peroxide by DAB Staining in Arabidopsis Leaves. Bio-protocol 2 (18)
Davis AM, Hall A, Millar AJ, Darrah C, Davis SJ (2009) Protocol: Streamlined sub-protocols for floral-dip transformation and selection of transformants in Arabidopsis thaliana. Plant Methods 5:3. 10.1186/1746-4811-5-3
Ding YL, Shi YT, Yang SH (2020) Molecular Regulation of Plant Responses to Environmental Temperatures. Mol Plant 13(4):544–564. 10.1016/j.molp.2020.02.004
Du LY, Li SM, Ding L, Cheng XX, Kang ZS, Mao HD (2022) Genome-wide analysis of trehalose-6-phosphate phosphatases (TPP) gene family in wheat indicates their roles in plant development and stress response. BMC Plant Biol 22(1):20. 10.1186/s12870-022-03504-0
Duan E, Wang Y, Li X, Lin Q, Zhang T, Wang Y, Zhou C, Zhang H, Jiang L, Wang J, Lei C, Zhang X, Guo X, Wang H, Wan J (2019) OsSHI1 Regulates Plant Architecture Through Modulating the Transcriptional Activity of IPA1 in Rice. Plant Cell 31(5):1026–1042. 10.1105/tpc.19.00023%J. The Plant Cell
Fichtner F, Lunn JE (2021) The Role of Trehalose 6-Phosphate (Tre6P) in Plant Metabolism and Development. In: Merchant SS (ed) Annual Review of Plant Biology, Vol 72, 2021, vol 72. Annual Review of Plant Biology. pp 737–760. 10.1146/annurev-arplant-050718-095929
Fichtner F, Olas JJ, Feil R, Watanabe M, Krause U, Hoefgen R, Stitt M, Lunn JE (2020) Functional Features of TREHALOSE-6-PHOSPHATE SYNTHASE1, an Essential Enzyme in Arabidopsis. Plant Cell 32(6):1949–1972. 10.1105/tpc.19.00837
Fieulaine S, Lunn JE, Borel F, Ferrer JL (2005) The structure of a cyanobacterial sucrose-phosphatase reveals the sugar tongs that release free sucrose in the cell. Plant Cell 17(7):2049–2058. 10.1105/tpc.105.031229
Figueroa CM, Lunn JE (2016) A Tale of Two Sugars: Trehalose 6-Phosphate and Sucrose. Plant Physiol 172(1):7–27. 10.1104/pp.16.00417
Garg AK, Kim J-K, Owens TG, Ranwala AP, Choi YD, Kochian LV, Wu RJ (2002) Trehalose accumulation in rice plants confers high tolerance levels to different abiotic stresses. Proc Natl Acad Sci USA 99(25):15898–15903. 10.1073/pnas.252637799
Garg N, Pandey R (2016) High effectiveness of exotic arbuscular mycorrhizal fungi is reflected in improved rhizobial symbiosis and trehalose turnover in Cajanus cajan genotypes grown under salinity stress. Fungal Ecol 21:57–67. 10.1016/j.funeco.2016.04.001
Garg N, Singla P (2016) Stimulation of nitrogen fixation and trehalose biosynthesis by naringenin (Nar) and arbuscular mycorrhiza (AM) in chickpea under salinity stress. Plant Growth Regul 80(1):5–22. 10.1007/s10725-016-0146-2
Ge LF, Chao DY, Shi M, Zhu MZ, Gao JP, Lin HX (2008) Overexpression of the trehalose-6-phosphate phosphatase gene OsTPP1 confers stress tolerance in rice and results in the activation of stress responsive genes. Planta 228(1):191–201. 10.1007/s00425-008-0729-x
Guo XY, Liu DF, Chong K (2018) Cold signaling in plants: Insights into mechanisms and regulation. J Integr Plant Biol 60(9):745–756. 10.1111/jipb.12706
Hassan MU, Nawaz M, Shah AN, Raza A, Barbanti L, Skalicky M, Hashem M, Brestic M, Pandey S, Alamri S, Mostafa YS, Sabagh AEL, Qari SH (2022) Trehalose: A Key Player in Plant Growth Regulation and Tolerance to Abiotic Stresses. J Plant Growth Regul. 10.1007/s00344-022-10851-7
Huang F, Jiang Y, Zhang S, Liu S, Eh T-J, Meng F, Lei P (2022a) A Comparative Analysis on Morphological and Physiological Characteristics between Castor Varieties (Ricinus communis L.) under Salt Stress. Sustainability 14(16). 10.3390/su141610032
Huang GH, Qu Y, Li T, Yuan H, Wang AD, Tan DM (2018) Comparative Transcriptome Analysis of Actinidia arguta Fruits Reveals the Involvement of Various Transcription Factors in Ripening. Hortic Plant J 4(1):35–42. 10.1016/j.hpj.2018.01.002
Huang Xy L, Tj YZ, Wy L, Yn F, Wang L, Ma Yc L Xf (2022b) Overexpression of genes encoding enzymes involved in trehalose synthesis from Caragana korshinskii enhances drought tolerance of transgenic plants. Biol Plant 66:207–218. 10.32615/bp.2022.023
Islam S, Mohammad F, Siddiqui MH, Kalaji HM (2023) Salicylic acid and trehalose attenuate salt toxicity in Brassica juncea L. by activating the stress defense mechanism. Environ Pollut 326. 10.1016/j.envpol.2023.121467
Iturriaga G, Suarez R, Nova-Franco B (2009) Trehalose Metabolism: From Osmoprotection to Signaling. Int J Mol Sci 10(9):3793–3810. 10.3390/ijms10093793
Jang IC, Oh SJ, Seo JS, Choi WB, Song SI, Kim CH, Kim YS, Seo HS, Do Choi Y, Nahm BH, Kim JK (2003) Expression of a bifunctional fusion of the Escherichia coli genes for trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase in transgenic rice plants increases trehalose accumulation and abiotic stress tolerance without stunting growth. Plant Physiol 131(2):516–524. 10.1104/pp.007237
Kaplan F, Kopka J, Sung DY, Zhao W, Popp M, Porat R, Guy CL (2007) Transcript and metabolite profiling during cold acclimation of Arabidopsis reveals an intricate relationship of cold-regulated gene expression with modifications in metabolite content. Plant J 50(6):967–981. 10.1111/j.1365-313X.2007.03100.x
Krasensky J, Broyart C, Rabanal FA, Jonak C (2014) The Redox-Sensitive Chloroplast Trehalose-6-Phosphate Phosphatase AtTPPD Regulates Salt Stress Tolerance. Antioxid Redox Signal 21(9):1289–1304. 10.1089/ars.2013.5693
Kretzschmar T, Pelayo MAF, Trijatmiko KR, Gabunada LFM, Alam R, Jimenez R, Mendioro MS, Slamet-Loedin IH, Sreenivasulu N, Bailey-Serres J, Ismail AM, Mackill DJ, Septiningsih EM (2015) A trehalose-6-phosphate phosphatase enhances anaerobic germination tolerance in rice. Nat Plants 1(9). 10.1038/nplants.2015.124
Kumar D, Yusuf MA, Singh P, Sardar M, Sarin NB (2014) Histochemical Detection of Superoxide and H2O2 Accumulation in Brassica juncea Seedlings. Bio-protocol 4(8):e1108. 10.21769/BioProtoc.1108
Latocha P (2017) The Nutritional and Health Benefits of Kiwiberry (Actinidia arguta) - a Review. Plant Foods Hum Nutr 72(4):325–334. 10.1007/s11130-017-0637-y
Lin Q, Wang J, Gong J, Zhang Z, Wang S, Sun J, Li Q, Gu X, Jiang J, Qi S (2023) The Arabidopsis thaliana trehalose-6-phosphate phosphatase gene AtTPPI improve chilling tolerance through accumulating soluble sugar and JA. Environmental and Experimental Botany 205. 10.1016/j.envexpbot.2022.105117
Lin QF, Wang S, Dao YH, Wang JY, Wang K (2020) Arabidopsis thaliana trehalose-6-phosphate phosphatase gene TPPI enhances drought tolerance by regulating stomatal apertures. J Exp Bot 71(14):4285–4297. 10.1093/jxb/eraa173
Lin QF, Yang J, Wang QL, Zhu H, Chen ZY, Dao YH, Wang K (2019) Overexpression of the trehalose-6-phosphate phosphatase family gene AtTPPF improves the drought tolerance of Arabidopsis thaliana. BMC Plant Biol 19(1):15. 10.1186/s12870-019-1986-5
Liu C, Sun X, Dai H, Zhang Z (2011) In vitro induction of octoploid plants from tetraploid Actinidia arguta. Acta Hort 913:185–190. 10.17660/ActaHortic.2011.913.23
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25(4):402–408. 10.1006/meth.2001.1262
Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15(12):550. 10.1186/s13059-014-0550-8
Lunn JE (2007) Gene families and evolution of trehalose metabolism in plants. Funct Plant Biol 34(6):550–563. 10.1071/fp06315
Luo Y, Sun M, Gao Y, Lang S, Wang Y (2023) EXOGENOUS TREHALOSE PROTECTS PHOTOSYSTEM II IN HEAT-STRESSED WHEAT. Bot Sci 101:186–196. 10.17129/botsci.3038
Marosz A (2009) Comparison of winter hardiness and growth of Actinidia arguta and A. kolomikta cultivars grown in central Poland. Acta Agrobotanica 62:179–188. 10.5586/aa.2009.040
Meitzel T, Radchuk R, McAdam EL, Thormählen I, Feil R, Munz E, Hilo A, Geigenberger P, Ross JJ, Lunn JE, Borisjuk L (2020) Trehalose 6-phosphate promotes seed filling by activating auxin biosynthesis. New Phytol 229(3):1553–1565. 10.1111/nph.16956
Miranda JA, Avonce N, Suarez R, Thevelein JM, Van Dijck P, Iturriaga G (2007) A bifunctional TPS-TPP enzyme from yeast confers tolerance to multiple and extreme abiotic-stress conditions in transgenic Arabidopsis. Planta 226(6):1411–1421. 10.1007/s00425-007-0579-y
Murakami S, Ikoma Y, Yano M (2014) Low Temperature Increases Ethylene Sensitivity in Actinidia chinensis 'Rainbow Red' Kiwifruit. J Japanese Soc Hortic Sci 83(4):322–326. 10.2503/jjshs1.CH-104
Mworia EG, Yoshikawa T, Yokotani N, Fukuda T, Suezawa K, Ushijima K, Nakano R, Kubo Y (2010) Characterization of ethylene biosynthesis and its regulation during fruit ripening in kiwifruit, Actinidia chinensis 'Sanuki Gold'. Postharvest Biol Technol 55(2):108–113. 10.1016/j.postharvbio.2009.08.007
Nakano T, Suzuki K, Fujimura T, Shinshi H (2006) Genome-wide analysis of the ERF gene family in Arabidopsis and rice. Plant Physiol 140(2):411–432. 10.1104/pp.105.073783
Ohme-Takagi M, Shinshi H (1995) Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element. Plant Cell 7(2):173–182. 10.1105/tpc.7.2.173
Paul MJ, Gonzalez-Uriarte A, Griffiths CA, Hassani-Pak K (2018) The Role of Trehalose 6-Phosphate in Crop Yield and Resilience. Plant Physiol 177(1):12–23. 10.1104/pp.17.01634
Paul MJ, Primavesi LF, Jhurreea D, Zhang YH (2008) Trehalose metabolism and signaling. Annu Rev Plant Biol 59:417–441. 10.1146/annurev.arplant.59.032607.092945
Phan T, Crepin N, Rolland F, Van Dijck P (2022) Two trehalase isoforms, produced from a single transcript, regulate drought stress tolerance in Arabidopsis thaliana. Plant Mol Biol 108(6):531–547. 10.1007/s11103-022-01243-2
Riechmann JL, Heard J, Martin G, Reuber L, Jiang C, Keddie J, Adam L, Pineda O, Ratcliffe OJ, Samaha RR, Creelman R, Pilgrim M, Broun P, Zhang JZ, Ghandehari D, Sherman BK, Yu G (2000) Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes. Sci (New York NY) 290(5499):2105–2110. 10.1126/science.290.5499.2105
Romero-Reyes A, Valenzuela-Avendano JP, Figueroa-Soto CG, Mascorro-Gallardo JO, Iturriaga G, Castellanos-Villegas A, Rivera-Dominguez M, Valenzuela-Soto EM (2023) Wheat Transformation with ScTPS1-TPS2 Bifunctional Enzyme for Trehalose Biosynthesis Protects Photosynthesis during Drought Stress. Appl Sciences-Basel 13(12). 10.3390/app13127267
Sakuma Y, Liu Q, Dubouzet JG, Abe H, Shinozaki K, Yamaguchi-Shinozaki K (2002) DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration- and cold-inducible gene expression. Biochem Biophys Res Commun 290(3):998–1009. 10.1006/bbrc.2001.6299
Schluepmann H, Pellny T, van Dijken A, Smeekens S, Paul M (2003) Trehalose 6-phosphate is indispensable for carbohydrate utilization and growth in Arabidopsis thaliana. Proc Natl Acad Sci USA 100(11):6849–6854. 10.1073/pnas.1132018100
Shinozaki K, Yamaguchi-Shinozaki K (2000) Molecular responses to dehydration and low temperature: differences and cross-talk between two stress signaling pathways. Curr Opin Plant Biol 3(3):217–223
Soleimanzadeh H, Habibi D, Ardakani MR, Paknejad F, Rejali F (2010) Effect of Potassium Levels on Antioxidant Enzymes and Malondialdehyde Content under Drought Stress in Sunflower (Helianthus annuus L). Am J Agricultural Biol Sci 5(1). 10.3844/ajabssp.2010.56.61
Timoneda A, Yunusov T, Quan C, Gavrin A, Brockington SF, Schornack S (2021) MycoRed: Betalain pigments enable in vivo real-time visualisation of arbuscular mycorrhizal colonisation. PLoS Biol 19(7). 10.1371/journal.pbio.3001326
Tsai AYL, Gazzarrini S (2014) Trehalose-6-phosphate and SnRK1 kinases in plant development and signaling: the emerging picture. Front Plant Sci 5:11. 10.3389/fpls.2014.00119
Van Houtte H, Vandesteene L, Lopez-Galvis L, Lemmens L, Kissel E, Carpentier S, Feil R, Avonce N, Beeckman T, Lunn JE, Van Dijck P (2013) Overexpression of the Trehalase Gene AtTRE1 Leads to Increased Drought Stress Tolerance in Arabidopsis and Is Involved in Abscisic Acid-Induced Stomatal Closure. Plant Physiol 161(3):1158–1171. 10.1104/pp.112.211391
Van Labeke MC, Soleme J, Debersaques FJAH (2015) Cold acclimation patterns of Actinidia arguta cultivars in a temperate climate zone. (1096):467–470
Vandesteene L, Lopez-Galvis L, Vanneste K, Feil R, Maere S, Lammens W, Rolland F, Lunn JE, Avonce N, Beeckman T, Van Dijck P (2012) Expansive Evolution of the TREHALOSE-6-PHOSPHATE PHOSPHATASE Gene Family in Arabidopsis. Plant Physiol 160(2):884–896. 10.1104/pp.112.201400
VanWallendael A, Soltani A, Emery NC, Peixoto MM, Olsen J, Lowry DB (2019) A Molecular View of Plant Local Adaptation: Incorporating Stress-Response Networks. In: Merchant SS (ed) Annual Review of Plant Biology, Vol 70, vol 70. Annual Review of Plant Biology. pp 559–583. 10.1146/annurev-arplant-050718-100114
Wang W, Chen Q, Xu S, Liu WC, Zhu X, Song CP (2020) Trehalose-6‐phosphate phosphatase E modulates ABA‐controlled root growth and stomatal movement in Arabidopsis. J Integr Plant Biol 62(10):1518–1534. 10.1111/jipb.12925
Wang W, Cui H, Xiao X, Wu B, Sun J, Zhang Y, Yang Q, Zhao Y, Liu G, Qin T (2022) Genome-Wide Identification of Cotton (Gossypium spp.) Trehalose-6-Phosphate Phosphatase (TPP) Gene Family Members and the Role of GhTPP22 in the Response to Drought Stress. Plants-Basel 11(8). 10.3390/plants11081079
Wang YH, Xu FX, Feng XQ, MacArthur RL (2015) Modulation of Actinidia arguta fruit ripening by three ethylene biosynthesis inhibitors. Food Chem 173:405–413. 10.1016/j.foodchem.2014.10.044
Xu L, Hu Y, Jin G, Lei P, Sang L, Luo Q, Liu Z, Guan F, Meng F, Zhao X (2021) Physiological and Proteomic Responses to Drought in Leaves of Amygdalus mira (Koehne) Yu et Lu. Front Plant Sci 12:620499. 10.3389/fpls.2021.620499
Yang Y, Yao Y, Li J, Zhang J, Zhang X, Hu L, Ding D, Bakpa EP, Xie J (2022) Trehalose Alleviated Salt Stress in Tomato by Regulating ROS Metabolism, Photosynthesis, Osmolyte Synthesis, and Trehalose Metabolic Pathways. Front Plant Sci 13. 10.3389/fpls.2022.772948
Yuan G, Sun D, An G, Li W, Si W, Liu J, Zhu Y (2022) Transcriptomic and Metabolomic Analysis of the Effects of Exogenous Trehalose on Salt Tolerance in Watermelon (Citrullus lanatus). Cells 11(15). 10.3390/cells11152338
Zang BS, Li HW, Li WJ, Deng XW, Wang XP (2011) Analysis of trehalose-6-phosphate synthase (TPS) gene family suggests the formation of TPS complexes in rice. Plant Mol Biol 76(6):507–522. 10.1007/s11103-011-9781-1
Zhai ZY, Keereetaweep J, Liu H, Feil R, Lunn JE, Shanklin J (2021) Expression of a Bacterial Trehalose-6-phosphate Synthase otsA Increases Oil Accumulation in Plant Seeds and Vegetative Tissues. Front Plant Sci 12. 10.3389/fpls.2021.656962
Zhang Y, Primavesi LF, Jhurreea D, Andralojc PJ, Mitchell RAC, Powers SJ, Schluepmann H, Delatte T, Wingler A, Paul MJ (2009) Inhibition of SNF1-Related Protein Kinase1 Activity and Regulation of Metabolic Pathways by Trehalose-6-Phosphate. Plant Physiol 149(4):1860–1871. 10.1104/pp.108.133934
Zhang Z, Sun M, Gao Y, Luo Y (2022) Exogenous trehalose differently improves photosynthetic carbon assimilation capacities in maize and wheat under heat stress. J Plant Interact 17(1):361–370. 10.1080/17429145.2022.2041119
Zhao ML, Ni J, Chen MS, Xu ZF (2019) Ectopic Expression of Jatropha curcas TREHALOSE-6-PHOSPHATE PHOSPHATASE J Causes Late-Flowering and Heterostylous Phenotypes in Arabidopsis but not in Jatropha. Int J Mol Sci 20(9). 10.3390/ijms20092165

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04 Dec, 2023

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Trehalose-6-phosphate phosphatase (TPP) participates in cold acclimatization of Actinidia arguta depending on the ethylene signal transduction pathway

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Plant materials and treatments

Gene cloning and sequence analysis

Quantitative real-time PCR (RT-qPCR) analysis

Bioinformatic analysis

Subcellular localization

Construction of the plant expression vector with DsRed marker

Analysis of physiological characteristics

Metabolites and enzyme activities assay

cDNA library screen by Yeast-one-hybrid (Y1H) method

Yeast-two-hybrid (Y2H) assay for self-transcriptional activity

Dual luciferase assay

Electrophoretic mobility shift assay (EMSA)

Statistical analysis

Results

Discussion

Declarations

Author contribution

Funding

Declaration of Competing Interests

Data availability

References

Supplementary Files

Status:

Version 1