Potato (Solanum tuberosum L.) cultivar “Atlantic” was used in this study, which is mainly due to its high yields of tubers and its resistance to common scab, latent and mild mosaics. Potato plants were in vitro planted in Murashige and Skoog (MS) medium with pH adjusted among 5.8-6.0. The medium was supplemented with 3% sucrose for seedling growth. To induce tuber generation, 8% sucrose was utilized. The cultivation was conducted in a controlled biotron, with the following conditions: 22°C/15°C (day/night), 16-h/8-h light/dark photoperiod (2,800 Lux), and 50% air humidity. Potato tubers with a sprouted bud of 1 mm in height were transferred into soil and vermiculite (1:1, v/v) pots measuring 18 cm × 26 cm × 27 cm and maintained for 5 weeks. To induce heat stress, the plants were next fostered under mild (30°C) and acute (35°C) conditions. Potato leaves were collected for detecting transcriptional patterns of StGATA family genes, 0 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, and 48 h after heat treatment. For the quantitation of StGATAs genes expression, there were 144 seedlings (1 line × 2 heat treatments × 8 different time periods × 3 replications × 3 pots); For the quantitation of heat-responsive genes, there were 630 seedlings (7 lines × 2 heat treatments × 5 different time periods × 3 replications × 3 pots); For the examination of physiological and photosynthetic indexes, there were 630 seedlings (7 lines × 2 heat treatments × 5 different time periods × 3 replications × 3 pots).

To generate potato plants highly expressing StGATA2 gene (OE for short), StGATA2 protein-encoding gene (GenBank Accession No. XM_006347854.2) was amplified using the specific primers (forward, 5’-CTCGAGATGGATGTCTACGGCGTGCACTCT-3’ and reverse 5’-GTCGACGCAGACCCGAAAGTGATGTCCGTACATTC-3’) (Bioeditas, Shaanxi, China). The PCR products were cloned into pBI121-EGFP plasmid according to a previous method (Li et al., 2020). The constructed plasmid was introduced into Agrobacterium tumefaciens strains LBA4404, followed by infecting the potato tuber slide (2 mm) according to Si’s method (Si et al., 2003). Potato plants with StGATA2 knocked down (Ri for short) were constructed with a previous method (Lu et al., 2019). The sense cDNA sequence was amplified using forward primer (Kpn I) and reverse primer (EcoR I) and inserted as an Kpn I-EcoR I fragment into pHANNIBAL (pHAN-StMAPK1-F); The anti-sense cDNA sequence was amplified using forward primer (Hind III) and reverse primer (BamH I), and inserted as a Hind III-BamH I fragment into pHANNIBAL (pHAN-StGATA2-R). The pHAN-StGATA2-RF were subcloned at Sac I and Spe I sites into pART vector (pART-StGATA2-RNAi). pART-StGATA2-RNAi was introduced into LBA4404, which was used to infect potato tuber slides.

After agrobacterium-mediated transformation, the tuber slides were cultivated on a solid medium in the dark at 28°C for 48 h. Then plants were continually cultivated on differentiation media in the dark at 22°C with 16-h/8-h light/dark photoperiod (2,400 Lux). The medium was changed every 12 days and the plants were cultured for about 3 to 4 weeks for shoot differentiation. Plant shoots were cut after growing to 1.5 cm and the shoots were transferred to the rooting medium for screening with 75% mg/mL of kanamycin. After 3 weeks of cultivation on MS medium, the leaves were collected for detecting StGATA2 by PCR using the specific primers for RNAi (forward: 5’-ATGGATGTCTACGG CGTGCACTCT-3’ and reverse: 5’- ATTAGCCGGAAAATTACTAAATGAAT-3’) and NPT II (forward: 5’-CTCACCTTGCTCCTGCCGAGA-3’ and reverse: 5’-CGCCTTGAGCCTGG CGAACAG-3’).

mRNA expression of StGATA family genes was examined 0 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, and 48 h after heat stress that has been described as abovementioned. For StGATA2 mRNA expression in transgenic plants, the leaves were collected 3 weeks after cultivation on MS medium. To examine the expression of heat stress genes (StSOD, StCAT, StPOD and StP5CS), plants leaves were collected 0 h, 8 h, 12 h, 24 h and 48 h after heat treatments. TRIzol RNA Extraction Kit (Invitrogen, Carlsbad, CA, USA). The first-strand cDNA was synthesized using the First-Strand cDNA Synthesis Kit (TransGen Biotech, Beijing, China). qPCR was performed using 100 ng of cDNA, 10 μL of SYBR Premix Ex Taq (2 ×) (Takara, Tokyo, Japan), and 0.8 μL of specific primers (0.5 μM) on ABI3000 System (Applied Biosystems, Foster City, CA, USA). The reaction procedures were: one cycle at 94°C for 3 min and 36 cycles of amplification at 94°C for 45 s, 59°C for 34 s, and 72°C for 1 min. The relative mRNA expression was calculated using the formula 2^-△△Ct. Solanum tuberosum translation elongation factor 1α gene (StEF1α) served as an internal control. The specific primers were listed in Table 1 .

Protein-coding sequence of StGATA2 was amplified and ligated to the expression vector pPBI121-EGFP, using the following primers: forward, 5’-CTCGAGATGGATGTCTACGGC GTGCACTCT-3’ and reverse, 5’-GTCGACGCAGACCCGAAAGTGATGTCCGTACATTC-3’. The constructed plasmid was transformed into Agrobacterium tumefaciens GV3101. The transformed strain infiltrated tobacco epidermal cells in accordance with a previous method reported by Sparkes et al. (2006). The green fluorescence was detected 48 h after infiltration under a Leica TCA confocal scanning laser microscopy (Leica, Weztlar, Germany).

Phylogenetic tress for GATA domain-containing homologs with highly similarity was constructed using MEGA 5.05 Software. Multiple-sequence alignment of GATA domain-containing amino acid sequence was analyzed using DNAman tool (Lynnon Biosoft, San Ramon, CA, USA)

Potato plants were imaged 2 days after heat stress. Potato plants cultivated at 35°C were transferred to 22°C conditions and maintained for 7 days. CK plants were cultivated at 22°C for 2 days. The plants were measured for plant height, plant fresh weight (FW), plant dry weight (DW), root FW and root DW.

Stomatal apertures were examined according to a previous method reported by Tricker et al. (2005). After 35 days of cultivation, transgenic or non-transgenic plants were subjected to heat stress treatment for 24 h or 48 h. The comparable fully-expanded leaves of transgenic or non-transgenic plants were selected between 9:30-11:00 am for measuring stomatal aperture. Clear nail polish was applied to the third fully expanded fully functional leaf located at the top of the plant, followed by covering with scotch tape. The leaves were mounted on glass slides and imaged using a Zeiss-Axiolmager M2 microscope (Zeiss, Germany). Three biological replicates were performed for each condition separately.

The third leaf from the plant top was collected when it fully expands during 9:30-11:30. Net photosynthetic rate, transpiration rate and stomatal conductance were examined using a portable photosynthetic LI-6400XT system (Li-COR, Lincoln, NE, USA). The photon flux density was set as 1,500 μmol·m^-2·s^-1. The relative humidity in leaf chamber was 50%-70%. CO₂ concentration was 400 μmol/mol.

To assess physiological and biochemical changes in plants under heat stress, 5-week-old StGATA2 transgenic plants and non-transgenic control plants grown under greenhouse conditions were subjected to heat stress treatments at 30°C and 35°C. Contents of H₂O₂, proline, and malondialdehyde (MDA) and activities of catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) were determined according to our previous methods (Zhu et al., 2021).

All experiments were carried out with three biological replicates and three technological replicates. Data are shown as the mean ± standard deviation. Statistical analysis was done with IBM SPSS 19.0 Statistical Software (IBM, Chicago, IL, USA) and GraphPad Prism Software (GraphPad, San Diego, CA, USA). Multiple comparisons were analyzed by one-way ANOVA with Tukey test or Dunnett’s T3 for post-hoc analysis or two-way ANOVA corrected by Sidak’s multiple comparisons test. Histograms were computed with GraphPad Prism software, heatmap by the pheatmap function in R version 4.2.3.

To provide an overview of expression patterns of GATA family genes, we cultivated potato plants under heat stresses. qRT-PCR results showed that mRNA expression of the 57 members from GATA family genes were distinctly altered during mild (30°C) or acute (35°C) heat stresses (0-48 h), which was presented in heatmaps ( Figures 1A, B ). Importantly, under heat stress conditions, StGATA2 expression was induced 1-48 h after heat stress (30°C and 35°C) treatment and peaked at 48 h with 7.5-fold and 8.4-fold increases in mRNA expression, respectively ( Figure 1 ). Notably, StGATA2 transcript levels were maintained at higher levels (expression increased more than 7 folds) at 8 h, 12 h, 24 h and 48 h after heat stress treatment (P < 0.05). The results indicated that heat stress at 30°C and 35°C induced a sustained and stable high expression of StGATA2. Consequently, we speculated that StGATA2 gene may play molecular functions in response to heat stresses.

Phylogenetic tree of GATA factors was generated, including Solanum tuberosum GATA2 (StGATA2), Solanum lycopersicum GATA2 (SlGATA2), Nicotiana tabacum GATA4 (NtGATA4), Capsicum annuum GATA4 (CaGATA4), Capsicum baccatum GATA2 (CbGATA2), Arabidopsis thaliana GATA2 (AtGATA2), Oryza sativa GATA12 (OsGATA12) ( Figure 2A ). Solanum tuberosum GATA2 contains CTHC-X18-CNAC motif ( Figure 2B ). To examine StGATA2 function, we obtained StGATA2 overexpression plants by expressing pBI121-EGFP-StGATA2 and generated a loss-of-function plants by introducing pART-StGATA2-RNAi. The relative mRNA expression of StGATA2 in transgenic plants were shown in Figures 2C, D , which significantly increased or knocked down when compared to the wild type (p<0.001). Amongst the overexpression lines, OE-1, OE-2 and OE-5 were selected as the significant over-expressors, and Ri-1, Ri-4 and Ri-6 as the significant under-expressors used for the functional analysis in response to heat stresses. StGATA2 protein obviously located in the nucleus ( Figure 3 ). The subcellular localization of GATA2 to the nucleus is the structural premise for DNA binding and transcription activation.

Next, we cultivated the 5-week-old transgenic or non-transgenic plants under heat stresses for 2 days, and investigated whether StGATA2 gene is involved in regulating plant morphology or growth such as plant height, plant weight and root weight. The morphological features of the plants were imaged 2 days after cultivation at 22°C (CK), 30°C, or 35°C. Figure 4A showed that neither StGATA2 over-expression nor under-expression affected plant morphological structure, compared to wild-type plants at 22°C. However, the height of OE plants in Figure 4B or Ri plants in Figure 4C was significantly different from the non-transgenic plants.

The statistical analysis results revealed that under mild heat stress (30°C), OE plants were characterized by increased plant height ( Figure 4E ), while plant fresh weight ( Figure 4F ), plant dry weight ( Figure 4G ), root fresh weight ( Figure 4H ) or root dry weight ( Figure 4I ) were unchanged (p>0.05) relative to NT plants (p<0.05). In contrast, the height of Ri plants was not significantly different from NT plants (p>0.05) ( Figure 4E ), while StMAPK1 under-expression reduced plant fresh weight ( Figure 4F ), plant dry weight ( Figure 4G ), root fresh weight ( Figure 4H ) or root dry weight ( Figure 4I ) (p<0.05) 2 days after growth at 30°C. As for the acute heat stress (35°C), OE plants displayed significant increases in plant height, plant fresh weight, plant dry weight, root fresh weight, and root dry weight, which were significantly decreased in Ri plants compared to non-transgenic plants (p<0.05).

Further, we compared the morphological structures of transgenic plants to non-transgenic plants 7 days after the plants receiving heat stresses were cultivated at 22°C. Figure 4D showed that compared to the non-transgenic plants, OE plants showed an increased plant height and more apical branches. However, Ri plants grows slowly, with a shorter height compared to NT plants. These results indicated that StGATA2 is implicated in the growth of potato plants in response to heat stresses, especially acute heat stress.

Measurements revealed that average ratio of stomal width to length was significantly decreased in StGATA2 overexpressing plants compared with NT plants, 24 or 48 h after mild heat stresses (p<0.05, p<0.01) ( Figures 5A, B ). StGATA2 under-expression resulted in a significant increase in stomatal aperture (p<0.05), 24 h or 48 h after mild or acute heat stresses. Under heat stress conditions, the net photosynthetic rate was increasingly enhanced in OE plants, while decreased in Ri plants (p<0.05) ( Figures 5C, D ). Additionally, there were evidence of StGATA2 overexpression inhibiting transpiration rate ( Figures 5E, F ) and decreasing stomatal conductance ( Figures 5G, H ) (p<0.05, p<0.01). Comparing to the NT plants, 30°C or 35°C heat stresses resulted in significant increases in transpiration rate and stomatal conductance of StGATA2 under-expression plants (p<0.05, p<0.01, p<0.001). All of these results confirmed that StGATA2 is closely correlated with heat stress-induced modifications in photosynthesis and transpiration rate.

Biochemical changes in potato plants were assayed to evaluate the capacity of StGATA2 to trigger protective mechanisms against heat stress. There is a significantly increase in contents of H₂O₂, MDA, and proline, as well as the activity of CAT, SOD and POD, following mild and acute heat stress treatment (p<0.05, p<0.01) ( Figure 6 ). Compared to the NT plants, StGATA2 overexpression significantly restrained the accumulation of H₂O₂ and MDA, while further induced the generation of proline ( Figures 6A–F ). Under heat stress conditions, StGATA2 overexpression enhanced the activity of CAT, SOD and POD compared to the non-transgenic plants ( Figures 6G–L ). However, there was an opposite trend toward the contents of H₂O₂, MDA and proline, as well as the activity of CAT, SOD, and POD in the Ri plants following heat stress, compared with OE plants. These data suggested that StGATA2 overexpression induced biochemical responses of potato plant against heat stress.

Besides, under heat stress conditions, mRNA expression of heat stress responsive genes was increased in non-transgenic, including StSOD ( Figures 7A, B ), StCAT ( Figures 7C, D ), StPOD ( Figures 7E, F ), StP5CS ( Figures 7G, H ) (p<0.05, p<0.01, p<0.001). However, StGATA2 over-expression further elevated the transcription of these 4 heat stress-responsive genes following heat stress treatment, compared to non-transgenic plants. In contrast, Ri plants showed the opposite results. This analysis indicated that StGATA2 overexpression induced genetic responses of potato plant against heat stress.