Genetic analysis of SnRK1β3 subunit of peach and the functional identification of overexpression transformed tomato

The sucrose non-fermentation-related kinase 1 (SnRK1) protein complex in plants plays an important role in energy metabolism, anabolism, growth, and stress resistance. This protein complex typically consists of an α subunit, three β subunits, one or two βγ subunits, and a γ subunit. Studies on plant SnRK1 have primarily focused on the functional α subunit, with the β regulatory subunit remaining relatively unexplored. The present study aimed to elucidate the evolutionary relationship, structural prediction, and interaction with the core α subunit of peach SnRK1β3 (PpSnRK1) subunit. In addition, we produced transgenic tomato plants overexpressing PpSnRK1 (OEPpSnRK1). We mainly tested the growth index and drought resistance of transgenic tomato plants.The results showed that PpSnRK1 has a 354 bp encoded protein sequence (cds), which is mainly located in the nucleus and cytoplasm. Phylogenetic tree analysis showed that PpSnRK1β3 has similar domains to other woody plants. Transcriptome analysis of OEPpSnRK1β3 showed that PpSnRK1β3 is widely involved in plant physiological regulation processes and stress response. Functional analyses of these transgenic plants revealed prolonged growth periods, enhanced growth potential, improved photosynthetic activity and superior drought stress tolerance.

Methods
Phylogenetic tree analysis

Protein sequences of SnRK1β3 were gathered from the NCBI database for 14 plant species ([https://www.ncbi.nlm.nih.gov/](https://www.ncbi.nlm.nih.gov/)), which included *Arabidopsis thaliana*,* Populus trichocarpa*,* Prunus persica*,* Vitis vinifera*,* Malus domestica*,* Prunus avium*,* Ananas comosus*,* Nicotiana attenuata*,* Carya illinoinensis*,* Durio zibethinus*,* Mangifera indica*,* Pyrus x bretschneideri*,* Triticum aestivum*, and *Gossypium hirsutum*. These sequences were analyzed using MEGA7 software to construct an evolutionary tree using the neighbor-joining method with 1000 bootstrap copies [1]. Structural domains were analyzed using Pfam ([http://pfam-legacy.xfam.org/](http://pfam-legacy.xfam.org/)) [2]. Protein sequence alignment was performed using DNAMAN software [3].

Yeast two-hybrid (Y2H) assay

The PpSnRK1α cds was cloned into the PGBKT7 vector, and the cds of PpSnRK1β3 was cloned into the PGADT7 vector (Primers: Table S2). These two vectors were transformed together into Y2H gold yeast strain and cultured on SD/−T-L (−Leu/−Trp) and SD/−T-L-H-A (−Leu/−Trp/-His/−Ade) selective medium and incubated at 28°C for two days. The strong interaction genes PGBKT7-53 and PGADT7-T were transformed as positive controls, and PGBKT7-α and PGADT7 empty vectors, PGADT7-β3 and PGBKT7 empty vectors and two empty vectors were used as negative controls [4]. The yeast spots on the two-defect medium were diluted with ddH2O by 10−1, 10−2, 10−3 fold to the four-defect medium to observe the growth of the yeast.

Bimolecular fluorescence complementation (BiFC) assay

The cds of PpSnRK1α was cloned into the YC vector and PpSnRK1β3 was cloned into the YN vector (Primers: Table S2). The two vectors were mixed, transferred to Agrobacterium GV3101, and injected into the leaves of *Nicotiana benthamiana*. The PpSnRK1α-YC with YN empty vectors and PpSnRK1β3-YN with YC empty vectors were used as negative controls [5]. Fluorescence within the tobacco cells was observed using laser confocal microscopy (LSM880, Zeiss, Germany) after two days of dark treatment. DAPI indicated the location of the nucleus, and the mixed field was composed of bright field, YFP fluorescence field, and DAPI field.

Dual luciferase assay

The coding sequences of PpSnRK1α and PpSnRK1β3 were cloned into the pGreenII 0800-nLUC vector and the pGreenII 0800-cLUC vector (Primers: Table S2). Negative controls included the PpSnRK1β3-CLUC and NLUC empty vectors, as well as the PpSnRK1α-NLUC and CLUC empty vectors, along with the empty NLUC and CLUC vectors. These vectors were introduced into Agrobacterium GV3101 and delivered into the leaves of Nicotiana benthamiana. Following a 3-day incubation period, fluorescence was visualized using a fluorescence microscope (AXIO, Zeiss, Germany) [4].

Acquisition and experimental treatment of overexpressed tomato material

The cds of PpSnRK1β3 was cloned into the Pri101 vector and transformed into *Agrobacterium* GV3101. Based on the method of *Goel D et al. *[6], the stem segments of tomato were infected with *Agrobacterium*, and the transgenic T0 generation plants were obtained in symbiotic medium, differentiation medium and rooting medium . After two generations of screening and typing, the T2 generation of overexpressing PpSnRK1β3-1, PpSnRK1β3-2 and PpSnRK1β3-3 strains was obtained.

In 2024, the experiments were conducted at Shandong Agricultural University's experimental base in Tai’an City, Shandong Province, China (36◦170 745900 N,117◦160 771200 E). Each T2 generation tomato was planted in a plastic black square 8.5 cm× 6 cm × 8 cm pot, mixed with substrate at a peat soil:vermiculite =ratio of 1:1, and cultivated in the tissue culture laboratory under light:darkness = 16 h:8 h conditions.

Transgenic and wild-type tomatoes with consistent growth at the seedling stage were selected for transcriptome sequencing and the determination of physiological indicators. Transgenic and wild-type tomatoes were treated with 4% PEG-6000 to simulate a drought environment for 14 days, and the tomatoes without drought treatment were used as the control [7]. Physiological indicators of the tomatoes under stress in each group were measured.

Determination of chlorophyll content

Samples of fresh, clean peach leaves (0.2 g) were extracted for 24 h in a 95% ethanol solution. The extract was analyzed using a Pharma-Spec UC-2450 ultraviolet spectrophotometer from Shimadzu (Kyoto, Japan) at OD665, OD649, and OD470. These measurements were used to calculate the chlorophyll content of the leaves [8].

Determination of photosynthetic parameters

The net photosynthetic rate (Pn) was recorded using a CIRAS-3 portable photosynthetic system (PP Systems, Massachusetts, USA) under light conditions [69]. A SPAD chlorophyll instrument (spad-502, Hanshatech, China) was used to determine the leaf SPAD value [9]. The leaves were darkened for 30 min and then the Fv/Fm ratio was determined using a hand-held leaf fluorometer (Handy PEA, Hanshatech, China) [10].

Measurement of MDA, H2O2, O2− ,relative electrolyte leakage

The malondialdehyde (MDA) content of the tomatoes was measured using the thiobarbituric acid (TBA) method [11]. The relative electrolyte leakage of the tomatoes was assessed using a DDS-12 conductometer (Hangzhou Wanda Instrument Factory, Hangzhou, China) [12].In addition, the hydrogen peroxide content in the tomato leaves was determined by the trichloroacetic acid (TCA) method, the superoxide anion content of the tomato leaves was measured using the sulfonamide colorimetric method [13].

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