Cloning and assessment of cold resistance in the transcription factor PhIPT5 from Populus hopeiensis

doi:10.21203/rs.3.rs-4943804/v1

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Cloning and assessment of cold resistance in the transcription factor PhIPT5 from Populus hopeiensis

https://doi.org/10.21203/rs.3.rs-4943804/v1

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Background:

Populus hopeiensis, a significant afforestation species, faces substantial growth constraints due to cold stress. The IPT gene, a pivotal rate-limiting enzyme in cytokinin synthesis, plays a crucial role in controlling plant reactions to both biotic and abiotic pressures. In this study, we isolated the PhIPT5 gene from Populus hopeiensis and analyzed its biological characteristics and cold tolerance with the aim of providing guidance for the production of cold-resistant poplars.

Results:

The coding sequence (CDS) of the PhIPT5 gene spans 981 bp, encoding 333 amino acid residues with a molecular weight of 37.07 kDa. The PhIPT5 protein has alkaline stability and hydrophilicity. Phylogenetic analysis revealed that Populus hopeiensis IPT5 is closely related to Populus alba. Subcellular localization studies revealed the chloroplastic localization of PhIPT5. We constructed an overexpression vector for PhIPT5 and transformed it into Populus hopeiensis, resulting in improved cold tolerance in transgenic seedlings. Analysis of cytokinin metabolites revealed significantly greater levels in leaves harboring the PhIPT5 gene than in those harboring the CK gene even after exposure to cold. Furthermore, our findings suggest that the PhIPT5 gene primarily regulates the isoamyl pyrophosphate cytokinin metabolism pathway, leading to the synthesis of tZ, iP, and DZ cytokinins.

Conclusion:

Our isolation of PhIPT5 from Populus hopeiensis demonstrated that its overexpression enhances resistance to cold stress in transgenic plants. This work provides a foundation for further elucidating the function of IPT genes and has significant implications for advancing research on enhancing cold tolerance in Populus hopeiensis.

Populus hopeiensis

PhIPT5

Cytokinin

Cold stress

Plant endogenous hormones are organic compounds synthesized in plants and primarily include auxin, cytokinin, gibberellin, abscisic acid, ethylene and brassinosterol [1]. Throughout the process of plant growth, these various hormones collectively regulate plant growth and development by either promoting or inhibiting one another [2]. IPT gene research has shown that cytokinins (CTKs) can stimulate plant cell division and expansion, participate in bud differentiation and apical dominance, and delay leaf senescence [3, 4]. In addition to influencing root elongation, they also affect various other plant growth and development processes. Furthermore, their involvement is crucial in how plants respond to stress [5].

Isopentenyl transferases (IPTs) function as the primary rate-limiting enzymes in cytokinin (CTK) synthesis, and their activity is regulated by this process [6]. Initially, discovered in Agrobacterium tumefaciens, this gene family includes ATP/ADP-IPTs and tRNA-IPTs. The former enzymes utilize ATP or ADP as their primary substrates, leading to the biosynthesis of isopentenyladenine (iP)-type and trans-zeatin (tZ)-type cytokinins (CTKs). In contrast, tRNA-IPTs are responsible for synthesizing cis-zeatin (cZ)-type CTKs by transferring the isopentenyl group to the N6 atom of adenine in tRNA [7–9].

Under stress conditions, activation of the isopentenyltransferase gene leads to the production of CTK, which effectively scavenges free radicals, enhances SOD and CAT enzyme activities, reduces lipid peroxidation, and minimizes MDA accumulation. These actions collectively mitigate stress-induced damage to plant growth and development [10, 11]. The IPT gene family has been identified in Arabidopsis thaliana, Oryza sativa, Solanum lycopersicum, and Malus domestica, and similar homologous IPT genes have been cloned in Humulus lupulus, Glycino max, Zea mays, and Malus hupehensis, among other cultivated plants. Furthermore, it has been established that the expression levels of this gene closely correlate with plant cytokinin content, leaf aging processes, increased yield potential, and enhanced stress resistance mechanisms within these plant species[12–21]. Zhang et al. utilized a biolistic transformation method to introduce the IPT gene into Festuca elata and reported enhanced cold resistance as well as delayed senescence in transgenic plants [22]. Yu et al. demonstrated that overexpression of the IPT gene in transgenic rice maintained normal physiological activities and high root vitality under cold stress conditions while reducing cold-induced damage to plants [23]. These findings collectively suggest that the regulation of CTK levels mediated by IPT genes can modulate plant resistance mechanisms. Currently, research on IPT genes has focused predominantly on herbaceous species, with limited exploration within woody poplar species, particularly with respect to stress resistance.

Populus hopeiensis, a member of the Salicaceae family, is a hybrid species resulting from the crossbreeding of Populus tomentosa and Populus davidiana. Known for its tall, straight, and aesthetically pleasing appearance, characterized by smooth bark and a large round crown, this tree has emerged as an exceptional choice for afforestation and landscaping in the Loess Plateau and sandstorm-prone areas of Northwest China and North China. Its well-developed root system, robust growth potential, rapid maturation, and high adaptability make it uniquely suited to these challenging environments [24]. In this study, we successfully cloned the coding region sequence of the isovalenyltransferase gene PhIPT5 from Populus hopeiensis. Through bioinformatics methods, including sequence analysis, subcellular localization, tissue-specific expression analysis, and assessment of the low-temperature stress response at the gene level, our findings lay a solid foundation for deeper insights into the biological functions of IPT genes in poplar growth development as well as their stress responses. This work aims to provide valuable reference data for future research in poplar breeding.

Materials

The plant material utilized comprised Populus hebeiensis tissue culture seedlings. The strains used included E. coli DH5α and Agrobacterium tumefaciens GV3101. Vector construction involved the utilization of the plasmids pMD19-T and pCAMBIA1302. These materials were housed in the Forest Tree Genetics and Breeding Laboratory at Inner Mongolia Agricultural University.

RT‒qPCR

The RNA extraction process involved the utilization of a specialized kit (TIANGEN's RNAprep Pure Plant Plus Kit) for isolating total RNA from leaves, followed by quality assessment through gel electrophoresis. Reverse transcription was subsequently employed to synthesize first-strand cDNA. Transcriptome data were utilized for primer design to screen the IPT gene and isolate PhIPT5 cDNA. A specific primer set, IPT5 F/R, was designed on the basis of the cDNA sequence via version 5.0 of a program. The target gene was then amplified from genomic DNA via these primers, resulting in the isolation of the full-length PhIPT5 gene via PCR under specific cycling conditions. A gel recovery kit was used for product retrieval following electrophoretic detection, as per the kit instructions. The recovered DNA fragments were ligated into the pMD19-T vector according to Table 4 specifications and transformed into E. coli DH5α cells for resistance screening on LB solid media supplemented with 50 mg·L^− 1 kanamycin. Positive monoclonal colonies were selected for colony PCR with the reaction system detailed in (Table 1), and the bacterial mixture exhibiting the correct bands was subjected to sequencing analysis.

To investigate the impact of cold stress and different levels of overexpression on the expression of the PhIPT5 gene, RNA was isolated from leaves and converted into cDNA via reverse transcriptase. The PhIPT5 gene was then amplified via qRT‒PCR via specific primers (Table 1) and SYBR Green Master Mix (Roche). Three independent biological experiments were conducted with Actin as an internal reference. The fold change in expression level was determined via the ΔΔCT method (fold change = 2^−[ΔΔCT]). The real-time fluorescence quantitative RT‒PCRs involved an initial incubation at 95°C for 3 min, followed by 35 cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min; finally, the reaction was stored at 72°C for 7 min before being cooled to 4°C.

Table 1

Cloning and quantitative primers for the *PhIPT5* gene
Uses	Primer name	Primer sequences
Gene clone	IPT5-F	5’-3’GGAGAGAACACGGGGGACTTTGCAAC
Gene clone	IPT5-R	5’-3’ ATGACCATGAGGCTTTCTTTGACCG
qRT‒PCR	IPT5-RT-F	5’-3’TTCAGGCACCATGCATCACT
qRT‒PCR	IPT5-RT-R	5’-3’GATCGACCCGCTCTGATACG
Reference genes	Actin-F	5’-3’ACCCTCCAATCCAGACACTG
Reference genes	Actin-R	5’-3’TTGCTGACCGTATGAGCAAG

Sequence analysis of PhIPT5

NCBI (https://www.ncbi.nlm.nih.gov/genbank/) was used to deduce the coding sequence of PhIPT5 on the NCBI website and explore homologous genes of PhIPT5 in the database. ProtParam (https://web.ExPASy.org/protparam/) was used for analysis of physical and chemical properties, the Prot Scale (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=nasp_sopma.html) was used for hydrophobicity analysis, and SOPMA (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page

=nasp_sopma.html) and SWISS-MODEL (https://swissmodel.ExPASy.org/) for the prediction of secondary and tertiary structures, respectively, and MEGA software was used to construct a phylogenetic tree with bootstrap values of 1000.

Construction of the overexpression vector of the GFP fusion subcellular localization vector

The overexpression vector pBWA(V)HS-ccdB-EGFP was obtained by linking PhIPT5 to pBWA(V) HS-CCDB-EGFP with Biorun 2× EasyClone Mix recombinase. The overexpression vector was transformed into DH5α receptor cells, and the positive clones were identified via colony PCR and sequenced for further identification. The correct plasmid transformed into Agrobacterium GV3101 receptive cells was detected, pBWA(V)HS-ccdB-EGFP was used as a negative control, positive clones were selected and propagated, Agrobacterium infection solution was prepared, and the OD₆₀₀ was adjusted to approximately 0.60. The lower epidermis of tobacco leaves was injected with a syringe, cultured in darkness for 48 h, and then observed and photographed under a confocal laser microscope.

Genetic transformation and identification of transgenic strains of Populus hebeiensis

The Agrobacterium GV3101 strain, transformed with the overexpression vector, was initially streaked onto LB solid media supplemented with 50 mg·L^− 1 kanamycin and 50 mg·L^− 1 rifampicin. Following incubation in the dark at 28°C for approximately 48 h, a single colony was selected and inoculated into 5 mL of LB liquid medium supplemented with 50 mg·L^− 1 kanamycin and 50 mg·L^− 1 rifampicin. The culture was then agitated at a constant temperature of 180 r for 12–18 h before being transferred to a larger volume (100 mL) of LB liquid medium containing the same concentrations of antibiotics for an additional period of 5–6 h, aiming for OD₆₀₀ values between 0.4–0.6. The mixture was subsequently centrifuged at 3500 r for 15 min to separate the supernatant from the pellet, which was subsequently resuspended in lysosomes for subsequent infection experiments.

Multiple vials were selected, each containing robust Populus hebeiensis tissue culture seedlings with healthy, flat leaves. After young leaves were excised and 3‒6 vertical incisions were made along the main vein via a blade, the leaves were placed on coculture media for precultivation at 28°C in darkness for 1 d. The precultivated leaves were subsequently immersed in a previously prepared infection solution for 15 min. After removal from the solution, excess moisture was absorbed via sterile filter paper before placing the leaves back onto the coculture medium for further cocultivation at 28°C in darkness for 3 days.

After the cocultivation process, the transgenic leaves were transferred to differentiation screening media, which were subsequently replaced approximately 15 days later. Upon reaching a length of approximately 2 cm, the kanamycin-resistant shoots were excised and transferred to rooting screening media. The resistant shoots successfully developed roots after approximately 12 d, and within 30 d, they had regenerated into complete plants, indicating the initial acquisition of the transgenic strains.

Cold stress treatment and determination of the physiological indices of the transgenic plants

On the basis of previous research on the response of the PhIPT5 gene to low temperatures, it can be inferred that the expression level of the PhIPT5 gene is particularly sensitive when subjected to stress at 5°C for 24 h. Consequently, two transgenic strains with a growth period of 60 days and high expression levels, along with nontransgenic control seedlings, were selected and exposed to a temperature of 5°C in an incubator for 24 hours. The CK strains treated at 25°C were used as the control group. All other conditions remained consistent except for the temperature variation, and each group included 3 biological replicates. Samples were collected immediately after stress treatment from segments spaced 3–6 at the apex of morphology to determine each index. Peroxidase (POD) and superoxide dismutase (SOD) activities, malondialdehyde (MDA) content, and conductivity were assessed. POD activity was measured through quantitative guaiacol oxidation; SOD activity inhibition of the photochemical reduction of nitrobenzene tetrazole was determined via spectrophotometry; the free MDA content was determined via the thiobarbituric acid method; the relative conductivity was determined via the immersion method; and the chlorophyll content was determined via ethanol extraction.

Determination of cytokinin metabolites

The 60-day-old CK and transgenic plants were provided by Wuhan Metville Biotechnology Co., LTD. The cytokinin metabolites of Hebei Poplar with the PhIPT5 gene and wild-type control lines were analyzed using the AB SciexQTRAP LC‒MS/MS platform, with 3 biological replications per sample. Differential gene expression analysis was conducted using the KEGG database (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC102409/) to enrich biochemical, metabolic, and signal transduction pathways associated with the differentially expressed genes .

Cloning the PhIPT5 gene

The RNAprep Pure Plant Plus Kit from TIANGEN was used for the extraction of total RNA from tissue-cultured seedlings of Populus tomentosa leaves, followed by reverse transcription of the extracted total RNA via the TaKaRa PrimeScript RT reagent Kit to generate cDNA. The Populus hopeiensis cDNA was used as a template for PCR amplification of its coding sequence. The PCR mixture comprised 10 µL of Easy Taq Mix, 1 µL each of upstream and downstream primers (10 µmol/L), 2 µL of template, and 6 µL of deionized water. The PCR program consisted of initial denaturation at 95°C for 3 min; one cycle at 95°C for 30 s, 58°C for 30 s, and 72°C for 1 min; a final extension step at 72°C for 7 min; and storage at 4°C. The sequences of primers used are detailed in Table 2. Agarose gel electrophoresis was employed to detect the amplified products.

The relative expression of the PhIPT5 gene in young leaves was assessed via qRT‒PCR, while the expression of PhIPT5 under different temperature stresses (exposed to 25°C, 10°C, 5°C, and 0°C for 3 h) was analyzed. The RT‒PCR results indicated that at 25°C, the relative expression of PhIPT5 in young leaves remained stable over time; however, as the temperature decreased, the relative expression of PhIPT5 decreased to approximately 1/4 (Fig. 1). These findings suggest that varying cold stress conditions influence the regulation of the PhIPT5 gene in young leaves.

Bioinformatics analysis of the PhIPT5 gene

The full predictive open reading frame (ORF) of PhIPT5 spans 981 base pairs and codes for a total of 333 amino acids(Fig. 2). The expected protein has a molecular weight of 37.07 kDa, an isoelectric point of 7.64 (alkaline), an instability coefficient of 36.86, and a hydrophobicity value of -0.223. These results strongly indicate that the PhIPT5 protein can be classified as a stable alkaline hydrophilic protein.

Upon analysis, it was determined that among the 327 amino acids constituting the PhIPT5 protein, approximately 49.24% formed alpha helices, while approximately 11.62% formed extended strand structures; the remaining portion (39.14%) adopted random coil configurations (Fig. 3A). Additionally, the tertiary structure model reveals a tightly folded core region alongside a more loosely arranged area, which may play a crucial role in maintaining the overall structural integrity and functionality of the protein(Fig. 3B).

The amino acid sequences of PhIPT5 were compared with those of 15 species showing high homology in the NCBI database, including Populus alba, Populus nigra, Populus trichocarpa, Populus euphratica, Salix koriyanagi, Salix suchowensis, Ricinus communis, and Tripterygium wilfordii.Hevea brasiliensis, Hibiscus trionum, Gossypium hirsutum, Manihot esculenta, Hibiscus syriacus and Euphorbia lathyris. MEGA 6.0 software was used to construct the phylogenetic tree (Fig. 4). The resulting tree revealed three major branches: Euphorbia lathyris formed a single branch within Euphorbiaceae; Ricinus communis, Hevea brasiliensis and Manihot esculenta grouped into another branch; and the remaining species clustered into the third branch. The genetic and evolutionary relationships between the PhIPT5 protein from Populus hopeiensis and that from Populus alba are closely related.

Subcellular localization of GFP-PhIPT5 in transiently transformed tobacco

To determine the precise subcellular localization of the PhIPT5 protein, this investigation developed the fusion expression vector p35S::PhiPT5-GFP, which enables the expression of the fusion protein in tobacco leaves through Agrobacterium tumefaciens-mediated delivery. The findings depicted in (Fig. 5) revealed that the fluorescence signal of p35S::PhIPT5 coincided with that of the endoplasmic reticulum localization marker HDEL (His-Asp-Glu-Leu)-Mcherry, suggesting the potential localization of PhIPT5 within chloroplasts.

Generation of a transgenic Populus hopeiensis strain

The plant overexpression vector pCAMBIA1302::PhIPT5 was constructed through double enzyme digestion and T4 DNA ligase. The DH5α strain of Escherichia coli was then transformed, and Kana-resistant LB medium was used for positive screening. After correct sequencing of a single colony, the plasmid was isolated and subsequently introduced into Agrobacterium GV3101 in its competent state for screening resistance to rifampicin. Following the shaking of a single colony, colony PCR was employed to verify the successful transfer of the target gene and vector to Agrobacterium.

During the coculture process, when white Agrobacterium appeared around the leaves, they were transferred to differentiation medium for screening. Some transgenic leaves produced resistant differentiated buds, whereas others withered and died. Each resistant bud was separated and numbered. When the resistant bud reached approximately 2 cm in size, it was cut off and transferred to rooting screening medium for further screening. After approximately 10 days, transgenic resistant buds developed adventitious roots and regenerated into whole plants; any false positive meristem buds that failed to take root were discarded. The rooted transgenic tissue culture-generated seedlings were cultivated at room temperature before being transplanted into small pots; after acclimatization in an artificial climate chamber, they were moved into larger pots before being finally transferred into a greenhouse (Fig. 6).

Analysis of the cold tolerance of transgenic Populus hopeiensis overexpressing PhIPT5

The activity of the antioxidant enzymes SOD and POD, as well as the soluble protein content in plants, serve as indicators of the degree of stress-induced damage to plants. Following a 3h exposure to 0°C stress, both SOD and POD activities, along with soluble protein levels, increased in CK and transgenic Populus hebeiensis compared with those at 25°C. Notably, the activities of SOD, POD, and soluble protein were significantly greater in the transgenic strains than in the CK strains(Fig. 7).

The MDA content and conductivity are indicators of cell membrane integrity. After 3 h of exposure to 0°C stress, the MDA content and conductivity increased in both CK and transgenic Populus hopeiensis compared with those at 25°C; however, the MDA content and conductivity were lower in the transgenic strain than in the CK strain(Fig. 7).

At 25°C, the chlorophyll and carotenoid contents were notably greater in transgenic strain No. 1 than in the CK control or transgenic strain No. 2. As the temperature decreased, both the chlorophyll and carotenoid contents decreased to varying degrees for both the transgenic and CK strains. Nevertheless, at 5°C, these contents remained greater for the transgenic strains than for the CK strains(Fig. 7).

Under cold stress conditions, the levels of soluble protein, POD, SOD, and chlorophyll were significantly greater for transgenics than for CKs; moreover, the increase in MDA content and conductivity was markedly lower. These results indicate that, under cold stress conditions, transgenics exhibit greater resistance than their CK counterparts.

Determination of cytokinin metabolites in transgenic Populus hebeiensis under cold stress

The cytokinin metabolites of the transgenic and nontransgenic strains of Populus hebeiensis were analyzed via ultrahigh-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). A total of 36 cytokinin metabolites were identified (Table 2).

Table 2

Cytokinins in transgenic *Populus hopeiensis* under cold stress
Serial number	Full name of the substance	English Abbreviations	CAS number
1	2-METHYLTHIO-cis-ZEATIN RIBOSIDE	2MeScZR	52049-48-6
2	6-(2-hydroxybenzylamino)-9-beta-D-ribofuranosylpurine	oTR	50868-58-1
3	PARA-TOPOLIN	pT	80054-30-4
4	meta-TOPOLIN-9-GLUCOSIDE	mT9G	179528-30-4
5	Kinetin Riboside	KR	4338-47-0
6	trans-Zeatin-riboside	tZR	6025-53-2
7	6-Furfurylaminopurine	K	525-79-1
8	ortho-TOPOLIN-9-GLUCOSIDE	oT9G	160299-96-7
9	DL-DIHYDROZEATIN RIBOSIDE	DHZR	22663-55-4
10	2-CHLORO-trans-ZEATIN (2ClZ)	2CltZ	29736-30-9
11	2-METHYLTHIO-cis-ZEATIN (2MeScZ)	2MeScZ	52020-11-8
12	2-METHYLTHIO-N6-ISOPENTENYLADENINE (2MeS-iP)	2MeSiP	20758-33-2
13	2-methylthio-N-6-isopentenyladenosine	2MeSiPR	20859-00-1
14	N6-BENZYLADENOSINE	BAPR	4294-16-0
15	KINETIN-9-GLUCOSIDE	K9G	98177-43-6
16	para-TOPOLIN RIBOSIDE	pTR	23666-24-2
17	DL-DIHYDROZEATIN	DZ	14894-18-9
18	6-BENZYLAMINOPURINE 9-(BETA-D-GLUCOSIDE)	BAP9G	4294-17-1
19	N6-BENZYLADENINE-7-GLUCOSIDE	BAP7G	56159-42-3
20	DIHYDROZEATIN-7-GLUCOSIDE	DHZ7G	91599-03-0
21	IHYDROZEATIN-O-GLUCOSIDE RIBOSIDE	DHZROG	62512-95-2
22	N6-(delta 2-Isopentenyl)-adenine	IP	2365-40-4
23	N6-ISOPENTENYLADENOSINE-D6	IPR	7724-76-7
24	6-[4-HYDROXY-3-METHYL-CIS-2-BUTENYLAMINO]PURINE	cZ	32771-64-5
25	cis-ZEATIN-9-GLUCOSIDE	cZ9G	169565-72-4
26	cis-ZEATIN RIBOSIDE	cZR	15896-46-5
27	cis-ZEATIN-O-GLUCOSIDE RIBOSIDE	cZROG	125225-72-1
28	N6-ISOPENTENYLADENINE-7-GLUCOSIDE	iP7G	59384-58-6
29	N6-ISOPENTENYLADENINE-9-GLUCOSIDE	iP9G	83087-94-9
30	ortho-TOPOLIN	oT	20366-83-0
31	4-[[(9-beta-D-Glucopyranosyl-9H-purin-6-yl)amino]methyl]phenol	pT9G	1046433-04-8
32	trans-Zeatin	tZ	1637-39-4
33	TRANS-ZEATIN GLUCOSIDE	tZOG	56329-06-7
34	6-Benzylaminopurine	BAP	1214-39-7
35	Meta-Topolin	mT	75737-38-1
36	meta-TOPOLIN RIBOSIDE	mTR	110505-76-5

Cluster analysis was employed to examine the differential expression of multiple cytokinins in various control groups, aiming to ascertain the alterations in cytokinin metabolites in Populus hebeiensis leaves under cold stress (Fig. 8).

At 25°C, the expression of cytokinins in the transgenic lines was significantly greater than that in the CK lines. When the transgenic and nontransgenic lines were compared at 25°C and 5°C, respectively, the expression of cytokinins in both types of lines was downregulated to varying degrees due to cold stress. However, under cold stress at 5°C, most cytokinins in the transgenic lines were significantly upregulated, with some even surpassing the levels in the CK lines. These findings suggest that the expression of cytokinins is greater in the transgenic lines than in the CK lines and that cold stress can inhibit their expression to a greater extent in the CK lines.

Analysis of the endogenous cytokinin metabolic pathway in transgenic Populus hebeiensis under cold stress

Owing to the interactions of metabolites within organisms, various pathways are formed. The metabolites were enriched and annotated via the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, revealing that all the comparison groups were enriched in the zein synthesis pathway (Fig. 9). Zeosine is involved in two pathways: isovaleryl pyrophosphate and mevalonate. The isovaleryl pyrophosphate pathway begins with the combination of DMAPP with ATP, ADP, and AMP, followed by catalysis by IPT to produce isovaleryl triphosphate, isovaleryl diphosphate, and isovaleryl adenosine monophosphate sequentially. CYP735A catalyzes the formation of trans-zeanoside adenosine triphosphate, trans-zeanoside diphosphate, and trans-zeanoside adenosine monophosphate, subsequently leading to further synthesis into isovalenyl-adenosine, which then forms isovalenyl-adenine. Trans-zeanoside adenosine monophosphate can generate trans-zeanoside, which can synthesize trans-zeanoside tZ or dihydrozeanoside adenosine monophosphate, leading to dihydrozeanoside DZ synthesis. Mevalonate has two pathways: DMAPP combines with tRNA catalyzed by IPT to form isovalenyldeoxyribonucleic acid, which further generates cis-isovalenyldeoxyribonucleic acid, resulting in cis-zeosine monophosphate and subsequently producing cis-zeosine nucleosides, leading to cis-zeosin cZ.

Analysis of cytokinin levels regulated by the PhIPT5 gene in various strains under cold stress

The alterations in the levels of the four types of cell kinins primarily facilitated by the PhIPT5 gene under cold stress were examined (Fig. 10), and the levels of all four kinins in the transgenic lines were notably greater than those in the CK lines at 25°C. Following exposure to a temperature of 5°C, the levels of these cytokinins decreased to varying extents in both the transgenic and CK lines; however, the reduction was significantly less pronounced in the transgenic lines than in their CK counterparts. Compared with those of the CK strains, the iP content of the transgenic strains remained relatively stable after cold treatment but decreased significantly in the CK strains. Furthermore, the tZ and DZ contents decreased substantially under cold treatment in both the transgenic and nontransgenic lines; nevertheless, the hormone content remained considerably greater in the transgenic lines. The cZ content was nearly six times greater in transgenics than in CK at 25°C; following cold treatment, there was a significant decrease observed only within the former group but still maintained a level almost twice as high as that found within the latter.

Cloning and bioinformatics analysis of the PhIPT5 gene

The IPT gene is a pivotal enzyme that catalyzes the initial step in the cytokinin biosynthesis pathway[25]. This gene is closely associated with various aspects of plant growth and development, including delaying leaf senescence, enhancing stress resistance, increasing crop yield, and increasing the fruit setting rate through its encoding of isopentyltransferase. On the basis of their amino acid sequence structure, IPT enzymes can be categorized into ATP/ADP-IPTs and tRNA-IPTs[26]. In this investigation, the PhIPT5 gene from Populus hopeiensis was cloned via PCR and subjected to bioinformatic analysis to elucidate both the structural and functional characteristics of the gene and its encoded protein.

The CDS length of the cloned gene was determined to be 981 bp, which could encode 333 amino acid residues. Amino acids serve as fundamental components of proteins with diverse functions. Protein hydrophilicity refers to the ability of a protein to attract and bind water molecules within the protein structure. Hydrophilic proteins possess hydrogen bond networks on their hydrophobic surfaces that attract water molecules, thereby maintaining protein stability and promoting organismal metabolic processes. The presence or absence of signal peptides can determine a protein's self-transport ability[26].

The subcellular localization prediction for the PhIPT5 protein primarily indicates chloroplast localization. Research by Huynh et al. revealed that IPT genes can modulate CTK biosynthesis, leading to increased chlorophyll content and improved plant photosynthetic efficiency[27], thus suggesting the potential involvement of this protein in leaf photosynthesis. Gene cis-acting elements play roles in regulating gene expression; notably, multiple elements related to the light response, hormone response, and cold response were identified in the PhIPT5 gene.

Physiological and biochemical changes in populus hebeiensis seedlings harboring the PhIPT5 gene under cold stress

In plants subjected to low-temperature stress, membrane permeability increases, leading to electrolyte imbalance and resulting in cell metabolism disorders. The conductivity method is commonly used to assess the extent of membrane damage, providing a more accurate reflection of plant cold tolerance[28]. The measurement of relative conductivity revealed an increase in each strain following low-temperature treatment; however, compared with the nontransgenic strain, the transgenic strain presented significantly lower conductivity, indicating less severe membrane damage.

Cold tolerance results in the accumulation of a substantial amount of free radicals in plants, thereby intensifying membrane lipid peroxidation and leading to an increase in the malondialdehyde content. The higher the content is, the higher the cold tolerance of plants[29]. The change in the MDA content paralleled that in the relative conductivity. Following exposure to low-temperature stress, the MDA content increased across all the strains; however, the MDA content of the transgenic strains significantly decreased compared with that of the nontransgenic controls, indicating reduced levels of free radicals and decreased membrane lipid peroxidation in the transgenic strains.

Under cold tolerance, there is an increase in reactive oxygen species (ROS) levels within plants. To mitigate ROS-induced damage, alterations in antioxidant enzyme activities facilitate ROS clearance. Investigations into POD, SOD, and CAT levels during eucalyptus and walnut exposure to cold stress revealed varying changes in enzyme activities over time [30, 31]. Additionally, osmoregulatory substances serve as indicators of plant resilience to cold temperatures; heightened soluble protein levels contribute to increased bound water content while maintaining intracellular osmotic pressure. Notably, a fluctuating trend [32] in soluble protein (SP) levels was observed among poplar clones subjected to cold stress. Furthermore, exposure to cold tolerance resulted in elevated concentrations of soluble proteins along with increased POD and SOD activities; notably, higher POD and SOD activities were detected in the transgenic strains than in their CK counterparts, indicating robust cold tolerance within these genetically modified variants.

The process of photosynthesis is highly sensitive to low temperatures, particularly those affecting chloroplasts, which serve as the primary sites for this biological function. Low temperatures not only disrupt the structure and function of chloroplasts but also impede the synthesis of chlorophyll, consequently diminishing the overall photosynthetic capacity of plants[33]. Research indicates that brief exposure to low temperatures can reduce the leaf chlorophyll content, enabling plants to adapt to adverse conditions[34]. In line with the results of our study, both the chlorophyll a and b levels decreased in the transgenic strains following low-temperature treatment; however, these levels remained higher than those observed in the nontransgenic strains. These findings suggest that low-temperature treatment had a more pronounced inhibitory effect on CK.

Zhang et al. utilized the gene gun method to introduce the IPT gene into tall fescue and assessed SOD, POD, and MDA levels in select strains during the late growth phase. These findings indicated that the transgenic strains Li4 and Li69 exhibited favorable performance across all three indices, resulting in significantly increased cold resistance and delayed plant senescence[22]. Chen evaluated various physiological parameters, including relative electrical conductivity, chlorophyll content, and root activity, in IPT transgenic rice at different growth stages. Compared with wild-type controls, transgenic lines presented reduced changes in relative electrical conductivity and lower degrees of chloroplast damage under low-temperature treatment during the booting stage and flowering stage [35]. Additionally, other studies have demonstrated notable inhibition of photosynthesis in tobacco and Jatropha under cold stress[36, 37].

Analysis of endogenous cytokinin metabolites in Populus hebeiensis with PhIPT5 gene transfer at low temperature

The endogenous hormones of plants are closely associated with the entire growth process, and various hormones collaborate to regulate diverse life activities of plants. They also play a role in the response of plants to nonbiotic stress [38]. Studies indicate that under low-temperature stress, plants adjust the expression of their endogenous cell division hormones to adapt to adverse conditions. Low temperature triggers the synthesis of ABA, while CTK exerts an antagonistic effect on it, leading to a decrease in its content. It has been demonstrated that CTK can modulate plant aging under low-temperature stress, thereby indirectly enhancing cold tolerance [39]. Multiple types of endogenous cell division hormones with two synthesis pathways exist: de novo synthesis and tRNA degradation. Research suggests that de novo synthesis is the primary source of cell division hormones in plants and is catalyzed by isopentenyl transferase [40]. In this study, qualitative and quantitative detection was carried out for cell division hormone-like metabolites in the leaves of transgenic and nontransgenic poplar stocks subjected to low-temperature treatment, resulting in the identification of 36 metabolites. Differentially abundant metabolite analysis revealed that most metabolites exhibited specific responses to low temperature. The expression level of cell division hormone-like metabolites was significantly greater in the transgenic stocks than in the nontransgenic controls after low-temperature treatment. Although the expression levels of metabolites were suppressed in both the transgenic and nontransgenic cultivars following low-temperature treatment, those in the transgenic cultivar remained higher than those in the nontransgenic cultivar. These findings indicate that the activity of cell division hormone metabolites was greater in the transgenic cultivar than in the WT cultivar and was less affected by suppression due to low temperatures.

Several studies have investigated the levels of endogenous hormones in 5 different Cynodon dactylon strains under low-temperature stress. The findings revealed that as the stress temperature decreased, the ABA content initially increased but then decreased across all the breeds, whereas the CTK content tended to decrease. However, breeds with high cold tolerance presented the lowest decrease in CTK content[39]. Zeng Guanghui conducted an analysis of endogenous hormones in tea tree leaves under natural low temperatures and reported a significant decrease in cytokinin levels[38].

In this study, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was used to identify the zeatin bioanabolic pathway. Pathway analysis revealed that iP, tZ, DZ, and cZ were the primary types of endogenous cytokinins in the plants. The combination of DAMPP and ATP/ADP/AMP is catalyzed by ATP/ADP-IPTs to produce iP, tZ, and DZ. Moreover, the combination of DAMPP and tRNA catalyzed the production of cZ by TRNA-IPTS. Following low-temperature treatment, the levels of iP, tZ, DZ, and cZ in the transgenic lines were significantly greater than those in the nontransgenic lines. While the levels of iP, tZ, and DZ remained relatively stable in the transgenic lines after low PhIPT5-temperature treatment compared with the significant decrease observed in the nontransgenic lines, there was a notable decrease in the cZ content within the transgenic lines but no significant change within the nontransgenic lines after treatment. These results suggest that transfer of the gene significantly increased the cytokinin content in Hebei Yang leaf slices; however, low temperatures inhibited their expression, leading to decreased content. Under identical treatment conditions, tZ presented the highest content among all four metabolites, followed by iP and Dz, with cX displaying the lowest concentration. Combined with bioinformatics analysis, these findings suggest that PhIPT5 encodes ATP/ADP-IPTs, further indicating its role in regulating the isopentylpyrophosphate cytokinin metabolism pathway, which primarily involves the synthesis of tX.

Analysis of the response of the PhIPT5 gene to low temperature

The isopentyltransferase encoded by the IPT gene exhibits specific responses to both biological and abiotic stresses. The endogenous cytokinin content directly correlates with the expression of the IPT gene, indicating its involvement in plant stress resistance through cytokinin regulation[41]. Wang et al. reported increased endogenous cytokinin levels and enhanced cold resistance in wheat expressing the TaIPT8 gene under drought stress compared with wild-type strains[42]. Similarly, transfer of the IPT gene in Arabidopsis thaliana led to elevated endogenous cytokinin levels and improved plant resistance[43]. Low-temperature stress increases the abscisic acid (ABA) content while gradually decreasing the CTK content, negatively regulating IPT gene expression[44].

In this study, the PhIPT5 gene was overexpressed in Populus hebeiensis via an overexpression vector. Cold tolerance indices and cytokinin metabolites were assessed in transgenic Populus hebeiensis under low-temperature stress. The results indicated the downregulation of differential cytokinin metabolites following low-temperature treatment. Specifically, the iP, tZ, DZ and cZ contents decreased significantly after low-temperature treatment, suggesting a decrease in PhIPT5 gene expression and a subsequent reduction in cytokinin content. Notably, PhIPT5 gene expression was positively correlated with the expression of cytokinins but was negatively regulated by low temperatures.

ABA（Abscisic Acid）

CDS(Coding sequence)

CKXs（Cytokininoxidase/dehydrogenase）

CTK（Cytokinine）

d（day）

h（hour）

KEGG（Kyoto Encyclopedia of Genes and Genomes）

LB（Luria-Bertanimedium）

MDA（Malondialdehyde）

min（minute）

MS（Murashige & Skoog medium）

PCR（Polymerase Chain Reaction）

RT-PCR（Real-time Quantitative PCR）

POD（Peroxidase）

SOD（Superoxide dismutase）

Acknowledgements

Not application.

Authors’ contributions

ZQ analyzes the data, edits and writes the manuscript. TT and WN conducted the experiment. JL edited the manuscript. YE designed the experiment and edited the manuscript. All authors read and approved the manuscript.

Funding

Supported by the National Science and Technology Major Project (2018ZX08020002-005-005). These funding agencies have no role in research design, sample collection, data analysis or interpretation, and manuscript writing.

Availability of data and materials

All data and materials are presented in the main paper and additional supporting file.

Ethics approval and consent to participate

There are no ethical issues involved.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Author details

¹Inner Mongolia Agricultural University, Hohhot, China.

²Forestry and Grassland Bureau of Zhuozi County, Ulanqab City, Inner Mongolia, China

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Table 1 and 2 are available in the Supplementary Files section.

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Cloning and assessment of cold resistance in the transcription factor PhIPT5 from Populus hopeiensis

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method

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RT‒qPCR

Construction of the overexpression vector of the GFP fusion subcellular localization vector

Cold stress treatment and determination of the physiological indices of the transgenic plants

Determination of cytokinin metabolites

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