StMAPKK5 responds to heat stress by regulating potato growth, photosynthesis, and antioxidant defenses

Backgrounds As a conserved signaling pathway, mitogen-activated protein kinase (MAPK) cascade regulates cellular signaling in response to abiotic stress. High temperature may contribute to a significant decrease in economic yield. However, research into the expression patterns of StMAPKK family genes under high temperature is limited and lacks experimental validation regarding their role in supporting potato plant growth. Methods To trigger heat stress responses, potato plants were grown at 35°C. qRT-PCR was conducted to analyze the expression pattern of StMAPKK family genes in potato plants. Plant with StMAPKK5 loss-of-function and gain-of-function were developed. Potato growth and morphological features were assessed through measures of plant height, dry weight, and fresh weight. The antioxidant ability of StMAPKK5 was indicated by antioxidant enzyme activity and H2O2 content. Cell membrane integrity and permeability were suggested by relative electrical conductivity (REC), and contents of MDA and proline. Photosynthetic capacity was next determined. Further, mRNA expression of heat stress-responsive genes and antioxidant enzyme genes was examined. Results In reaction to heat stress, the expression profiles of StMAPKK family genes were changed. The StMAPKK5 protein is located to the nucleus, cytoplasm and cytomembrane, playing a role in controlling the height and weight of potato plants under heat stress conditions. StMAPKK5 over-expression promoted photosynthesis and maintained cell membrane integrity, while inhibited transpiration and stomatal conductance under heat stress. Overexpression of StMAPKK5 triggered biochemical defenses in potato plant against heat stress, modulating the levels of H2O2, MDA and proline, as well as the antioxidant activities of CAT, SOD and POD. Overexpression of StMAPKK5 elicited genetic responses in potato plants to heat stress, affecting heat stress-responsive genes and genes encoding antioxidant enzymes. Conclusion StMAPKK5 can improve the resilience of potato plants to heat stress-induced damage, offering a promising approach for engineering potatoes with enhanced adaptability to challenging heat stress conditions.


Introduction
Over the past century, the Earth's average surface temperature has been rising (Hansen et al., 2022;Katlane et al., 2023).Evidence indicates that extreme weather events, including heat waves, are increasing in both frequency and intensity (Walsh et al., 2020).Extreme temperatures confer negative roles in photosynthesis, flower pollination, and fruit setting, which leads to lower crop yields and affects food availability (Beillouin et al., 2020;Dalhaus et al., 2020;Nicholson and Egan, 2020;Wi et al., 2020).As the fourth most important crop in agricultural production, potatoes (Solanum tuberosum L.) are susceptible to high temperature or adverse water supply (Singh et al., 2020).Due to climate change, tuber growth is inhibited above 33°C and high temperature above 25°C induces leaf senescence (Rykaczewska, 2015).We need to better understand how heat stress affects plant growth and development as global temperatures rise in the future.This could offer valuable insights into breeding programs to improve plant heat-stress tolerance.
Protein kinases (PK) constitute a vast and crucial superfamily of enzymes involved in cellular signaling, regulating a wide array of biological processes (Chen et al., 2021a).The mitogen-activated protein kinases (MAPKs) are important and evolutionarily conserved subfamily of protein kinases that play a central role in transducing signals from the cell surface to the nucleus responding to a variety of stimuli, for instance, heat stress (He et al., 2020;Kumar et al., 2020).The hierarchical organization of MAPK signaling cascades, encompassing the MAPKs, MAPK kinases (MAPKKs or MEKs), and MAPKKs kinases (MAPKKKs or MEKKs), forms a crucial framework for transmitting external signals, amplifying stress signaling, and ultimately facilitating communication between the cellular environment and transcriptional responses (Zwerger and Hirt, 2001;Taj et al., 2010;Zhang and Zhang, 2022).Heat stress can indeed trigger various signaling mechanisms within biological systems in three pathways, including Ca 2+ -dependent salt overlay sensitive signaling and osmotic/oxidative stress signaling (Rodriguez et al., 2010).The role of oxidative stress signaling in promoting osmolyte accumulation through the MAPK cascade, involving G-protein receptors, histidine kinases, and protein tyrosine kinases, aligns with known cellular responses to stress (Ara and Sinha, 2014;Röhm et al., 2021).
Genome-wide analyses have progressively identified multiple MAPKKs genes in various plant species.Yin et al. identified 23 MAPKKs in the Gossypiumhirsutum genome and divided into 4 groups.Most of these genes feature the active site motif-D(I/L)K-, which includes two conserved residues, K (lysine) and D (aspartic acid) (Yin et al., 2021).The cucumber genome contains 6 MAPKK genes classified into 4 groups (A-D) (Wang et al., 2015).Ten MAPKK genes have been reported in Arabidopsis genome, which are also classified into 4 groups (A-D) (Danquah et al., 2014;Choi et al., 2017).Bioinformatic analysis of maize genome revealed 9 MAPKK genes and identified 11 subdomains of protein kinases showing serine/threonine specificity (Kong et al., 2013).From the available genome, 5 MAPKK genes were identified in tomato (Wu et al., 2014) and 7 in rice (Kumar et al., 2008), respectively.However, systematic studies of the MAPKK gene family are still required for potato plant.
The MAPKK factors, as the components of the MAPK signaling pathway, exhibit evolutionary conservation across various organisms and regulate a wide array of functions, such as stomatal development (Bergmann et al., 2004), plant architecture (Yu et al., 2014), chlorophyll synthesis (Li et al., 2022), glucose metabolism (Chardin et al., 2017a), apical meristem (Lee et al., 2019), and flower development (Lafleur et al., 2015).However, studies on how StMAPKKs respond to heat stress in potato plants are scarce.This study aimed to describe the expression features of StMAPKKs under heat stress situations.Then, our study analyzed the functional aspects of StMAPKK5, including its roles in potato plant growth, antioxidation, cell membrane integrity, photosynthesis capacity, and mRNA expression of heat stress-responsive genes and antioxidant enzyme genes.The potato cultivar "Atlantic" is a cultivar introduced and popularised in Guangdong Province.As a high-quality and high-yield potato variety, "Atlantic" has been studied rarely under heat stress.Meanwhile, this study aimed to screen heat tolerance genes, which may provide an effective basis for its molecular breeding for heat stress tolerance.

Plant materials and cultivation
The potato (Solanum tuberosum L.) cultivar "Atlantic" was cultivated in vitro using Murashige and Skoog (MS) medium with a pH of 5.8-6.0.The medium was supplemented with 8% sucrose and the plants were cultivated for 4 weeks.Next, four-week seedlings were cultured in the dark for 30 days to induce tuber generation.Approximately 1 g of potato tubers with 1 mm germinated buds were transferred into containers (19 cm ×25 cm ×26 cm) filled with soil-vermiculite mixture (1:1, v/v) and cultivated for 5 weeks in Zhanjiang located at latitude 21°11'43''N and longitude 110°34'56''E.Soil water holding capacity ranged from 75% to 80%, supplemented with 100 mL of nutrient solution (pH: 5.8; 0.20 mmol/L FeSO 4 , 2.57 mmol/L KH 2 PO 4 , 2.08 mmol/L MgSO 4 , 1.29 mmol/L (NH 4 ) 2 SO 4 , and 9.89 mmol/L KNO 3 ) every 7 days.Potato plants in each group exhibiting uniform growth appearance were next cultivated under 35°C.Physiological and photosynthetic indexes were assayed at 0 h, 8 h, 12 h, 24 h, and 48 h after cultivation under heat stress conditions.The control plants were cultivated at 22°C.Each group was prepared with three biological replicates and three technical replicates.
To examine the mRNA expression of MAPKK family genes in various plant tissues, the potted plants were continually cultured.Flower, root, stem, leaf, petiole, stolon, and shoot were collected at the developmental stage, and tuber was collected at the maturity stage.Next, the plants were cultivated under 35°C, and StMAPKKs expression in leaf, stem, and root were examined at 0 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, and 48 h after cultivation under heat stress conditions.

Construction of transgenic plants
The gene encoding the StMAPKK5 protein was cloned into the pBI121-EGFP plasmid using a previously described method (Li et al., 2020), to develop StMAPKK5-overexpressing plants (OE for short).Agrobacterium tumefaciens strains LBA4404 was used for transformation experiments.Potato plants low expressing StMAPKK5 (RNAi or Ri for short) were established with a previously described method (Lu et al., 2019).The primers utilized for plasmid construction are listed in Table 1.

qRT-PCR
TRIzol RNA Extraction kit (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from the collected samples.The firststrand cDNA of the target genes were synthesized using the First-Strand cDNA Synthesis Kit (TransGen Biotech, Beijing, China).qPCR was conducted using a LightCycler 480 II real-time PCR system (Roche, Rotkreuz, Switzerland) with the reaction mixture comprising 0.8 mL of specific primers (0.5 mM), 100 ng of cDNA (Table 1), and 10 mL of SYBR Premix Ex Taq (2 ×) (Takara, Tokyo, Japan).The reactions underwent an initial incubation at 94°C for 3 min, followed by the procedures: 36 cycles of 94°C for 45 s, 59°C for 34 s, and 72°C for 1 min.The relative transcription levels were determined using the 2 -△△Ct method.

Subcellular localization
The expression vector pPBI121-EGFP carrying protein-coding sequence of StMAPKK5 gene was transformed into Agrobacterium tumefaciens GV3101.The primers utilized for plasmid construction are detailed in Table 1.Following the method described by Sparkes et al. (2006), tobacco epidermal cells were infiltrated with the transformed strain.A confocal laser scanning microscope (Olympus, Tokyo, Japan) was used to observe the sample.For the autofluorescence of chlorophyll in chloroplasts, the excitation wavelength and transmission range for emission were set at 640 nm and 675 nm, and 488 nm and 510 nm for the GFP-tagged StMAPKK5 protein.The software Olympus Fluovie was used for imaging processing.

Phylogenetic analysis and amino acid alignment
MEGA 5.05 software was utilized to construct a phylogenetic tree with a neighbor-joining algorithm.DNAman tool (Lynnon Biosoft, San Ramon, CA, USA) was applied to multiple-sequence alignment of the conserved subdomains.

Measurements of plant growth
Non-transgenic (NT) and transgenic plantlets, uniformly measuring 2 cm in height, were planted in MS medium under controlled conditions in an incubator with an 8 h-dark cycle and 16 h-photoperiod at 22°C and 3,000 Lx.To induce heat stress, plants were cultivated under 35°C.Four weeks after heat stress treatment, we measured root fresh/dry weight, plant fresh/dry weight, and plant height.

Measurements of stomatal conductance, transpiration rate, and net photosynthetic rate
Four-week-old potted plants were cultivated under 35°C.After 0 h, 8 h, 16 h, 24 h, and 48 h, we examined stomatal conductance, transpiration rate, and net photosynthetic rate.Stomatal conductance, transpiration rate, and net photosynthetic rate were assessed using a portable LI-6400XT system (Li-COR, Lincoln, NE, USA) between 9:30-11:30.The fourth fully expanded functional leaf was collected from the plant top.The parameters were configured as follows, 50%-70% of relative humidity in leaf chamber, 1,500 mmol•m -2 •s -1 of the photon flux density, and 400 mmol/mol of CO 2 .Four-week-old potted plants were cultivated under 35°C.Following exposure to heat stress for durations of 0 h, 8 h, 16 h, 24 h, and 48 h, the potted plants were assayed for the levels of proline, malondialdehyde (MDA), and hydrogen peroxide (H 2 O 2 ), and activities of peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) activities with methodologies previously established in our research (Zhu et al., 2021).Description of detailed experimental procedures are provided in Supplementary Methods.

Assays for relative electrical conductivity and chlorophyll content
Relative electrical conductivity (REC) and chlorophyll content were assayed according to our previous methods (Zhu et al., 2023).Description of detailed experimental procedures are provided in Supplementary Methods.

Statistical analysis
Statistical analysis was performed using GraphPad Prism Software (GraphPad, San Diego, CA, USA) and IBM SPSS 19.0 Statistical Software (IBM, Chicago, IL, USA).Results are presented as mean ± standard deviation.Histograms and line charts were generated using GraphPad Prism software.For multiple comparisons, 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 were employed.

Phylogenetic analysis and sequence comparison of StMAPKK proteins
After phylogenetic analysis of the amino acid sequences of the reported plant MAPKK homologs, we found StMAPKK5 was homologous with SlMAPKK5, AtMAPKK3 and OsMAPKK3 (Figure 1A).There was a high degree of homology between StMAPKK4 and SlMAPKK4, StMAPKK2 and SlMAPKK2, The phylogenetic relationship and protein sequence alignment of MAPKK proteins with MAPKK proteins.(A) Dendrogram of MAPKK proteins from Solanum tuberosum (St) (StMAPKK1-5), Arabidopsis thaliana (At) (AtMAPKK1-10), Solanum lycopersicum (Sl) (SlMAPKK1-5), and Oryza sativa (Os) (1,3,4,5,6,10-1 and 10-2); Bootstrap values were calculated from 500 resampled datasets with a 50% cut-off; MEGA-X software was used to establish the neighbor-joining phylogenetic tree.(B) Protein sequence of MAPKK proteins were aligned using DNAman software; Identical and similar amnio acids were marked in the same colors.The conserved subdomains were indicated on the bottom, using the Roman numbers (I-XI).The dark line marks the active site motif.Red asterisks indicate substrate specificity, and the black dots above the sequences represent activating sites.The conserved consensus motif GXGXXG is marked as I.  5), and Oryza sativa (Os) (1, 3, 4, 5, 6, 10-1 and 10-2) showed the presence of the highly conserved domains (Figure 1B).StMAPKK5 contains 11 conserved catalytic subdomains typical of MAPKKs and the conserved consensus motif GXGXXG.

Characterization of StMAPKK family genes expression of the various organs of potato
A variety of plant tissues (flower, root, stem, leaf, petiole, stolon, tuber, and shoot) were collected to analyze the expression features of StMAPKK family genes.StMAPKK1 showed the highest level of expression in root (Figure 2A).StMAPKK2 displayed the maximum expression in leaf (Figure 2B), StMAPKK3 in stem and shoot (Figure 2C), StMAPKK4 in root (Figure 2D), stem and leaf, and StMAKK5 in leaf (Figure 2E).These results represented the tissuespecific expression profiles of StMAPKK family genes.We speculated genes within the StMAPKKs family may play a crucial role in regulating the growth and development of different tissues and organs in potato.

Heat stress resulted in differential expression of StMAPKK family genes
Potato leaves, stems and roots were chosen for quantification because the relative expression of StMAPKK5 gene was found to be higher than in other tissues.At 0 h, 1 h, 2 h, 4 h, 8 h, 16 h, 24 h, or 48 h after exposure to heat stress (35°C), potato leaves, stem and roots were obtained.qRT-PCR was then performed to assay the expression features of StMAPKK family genes.The expression of StMAPKK1 increased steadily with the duration of heat stress (p < 0.05) (Figure 3A).However, we observed that the expression of StMAPKK2 (Figure 3B), StMAPKK3 (Figure 3C), and StMAPKK4 (Figure 3D) in leaves, stems and roots changed in a disordered manner in response to heat stress (p < 0.05).Likewise, we noted that heat stress conditions resulted in a consistent increase in the transcript levels of StMAPK5 in potato leaves, stems, and roots (p < 0.05) (Figure 3E).Considering that after treatment, StMAPKK5 transcript levels were up-regulated at a higher rate than StMAPKK1-4 (p < 0.05).Therefore, we speculated that in response to heat stress, StMAPKK5 gene may perform crucial molecular functions.

Impacts of StMAPKK5 on plant morphological phenotypes under heat stress conditions
We generated StMAPKK5 overexpression plants by introducing pBI121-EGFP-StMAPKK5 and create loss-of-function plants through the induction of pART-StMAPKK5-RNAi.In transgenic plants, StMAPKK5 mRNA expression was significantly elevated or decreased relative to the wild-type plant (p < 0.001) (Figures 4A, B).Within the under-expression lines, Ri-2, Ri-3, and Ri-5 were chosen as notable under-expressors, while OE-2, OE-3 and OE-6 were identified as significant over-expressors for subsequent functional analysis under heat stress conditions.In order to better assess the location of StMAPKK5 protein, we examined by tagging StMAPKK5 protein with GFP and dissecting the green fluorescence, with the red autofluorescence of chlorophyll as a location reference.The green fluorescence is evenly distributed in the nucleus, cytoplasm and Frontiers in Plant Science frontiersin.orgcytomembrane (Figure 4C), which was consistent with our previous result (Luo et al., 2024).In comparison with the NT plants, StMAPKK5 overexpression significantly enhanced the thermotolerance of potato plants as evidenced by the increases in plant height, dry weight, dry root weight, fresh weight, and root fresh weight, under normal and heat stress conditions after 4 weeks of cultivation (p < 0.001) (Figures 4D, E).In contrast, under normal or heat stress conditions, RNAi-mediated silencing of StMAPKK5 apparently resulted in the inhibition of plant growth, compared to non-transgenic plants (p < 0.001).

StMAPKK5 overexpression elevated the ability of ROS scavenging under heat stress condition
Heat stresses led to a significant increase in H 2 O 2 in nontransgenic plants, and it continually increased with prolonged duration of heat stress (p < 0.001) (Figure 5A).StMAPKK5 transgenic lines showed significantly reduced H 2 O 2 production at 8 h, 16 h, 24 h, and 48 h following cultivation under heat stress conditions, compared to non-transgenic plants, while RNAimediated silencing of StMAPKK5 reversely increased the accumulation of H 2 O 2 (p < 0.001).MAD generated in non-transgenic plant was found significantly increased as the extension of heat stress (p < 0.001) (Figure 5B).Compared to non-transgenic plants, MAD content was decreased in StMAPKK5 transgenic lines (OE-2, OE-3, and OE-6 after heat stress.In contrast, as compared to non-transgenic plants, knockdown of StMAPKK5 resulted in the accumulation of MDA under heat stress.The level of proline, as a consequence of heat stress, was increased in non-transgenic plants in a time-dependent manner (Figure 5C).The transgenic lines (OE-2, OE-3, and OE-6) accumulated more proline compared to non-transgenic plants under heat stress condition, while StMAPKK5-silencing plants were detected with decreased proline content.Under heat stress conditions, the specific activities of CAT, SOD, and POD were consistently higher in non-transgenic plants (p < 0.001) (Figures 5D-F).After 48 h of heat stress, the activity of CAT, SOD, and POD was found higher in StMAPKK5-transgenic lines compared to non-transgenic plants, respectively.However, compared to non-transgenic plants, StMAPKK5-silencing plants displayed decreased activities of CAT, SOD, and POD, 48 h after heat stress.
In general, chlorophyll synthesis is sensitive to heat stress, serving as an indicator of the extent of heat stress injury.It was observed that heat stress had an adverse effect on chlorophyll in potato plants (Figure 5G).Overexpression of StMAPKK5 may increase the heat stress tolerance of potato plants by maintaining the total chlorophyll content, compared to non-transgenic plants (p < 0.001).StMAPKK5-silencing lines appeared to be more susceptible to heat stress, evidenced by a higher reduction in total chlorophyll content than non-transgenic plants (p < 0.001).REC have commonly been examined to reflect stress-induced injury of cell membrane.Heat stress significantly exacerbated REC with the extension of time (Figure 5H).StMAPKK5 overexpression profoundly inhibited REC while StMAPKK5 silence significantly increased REC, compared to the NT plants (p < 0.001).

Overexpression of StMAPKK5 improved the photosynthetic rate and reduced transpiration responding to heat stresses
Comparisons of measured rates of net photosynthesis suggested that direct suppression of photosynthesis happens at temperatures higher than about 35°C.Cultivation under heat stress contributed to the inhibition of net photosynthetic rate for the NT plants (p < 0.05) (Figure 6A).However, compared to the NT plants, StMAPKK5overexpressing plants showed increased net photosynthetic rates in response to heat stress (p < 0.001).Potato plants lowly expressing StMAPKK5 failed to maintain net photosynthetic rate in comparison with the NT plants (p < 0.001).Transpiration is an essential physiological process, during which course water and mineral nutrients are transferred from soils to plants to dissipate heat.In this study, the transpiration rate of the plants decreased as the duration of thermal incubation increased (Figure 6B).StMAPKK5 overexpression further inhibited transpiration in potato plants, whereas StMAPKK5 low expression promoted transpiration in instead (p < 0.001).Leaf stomata manages plant CO 2 absorption through transpiration and photosynthesis.It was shown that stomatal conductance was decreased responding to increased temperatures (Figure 6C).In transgenic plants, compared to the NT plants, stomatal conductance of StMAPKK5 overexpression plants decreased

StMAPKK5 participated in regulating heatresponsive gene expression
To protect the structure and operation of plant cells from the damaging effects of oxidative stress caused by heat stress, the antioxidant defense system is generated, consisting of antioxidant enzymes, like FeSOD, MnSOD, and CuZnSOD, in chloroplasts, mitochondria, and peroxisomes.Under heat stress conditions, NT plants showed a significant increase in mRNA expression of StFeSOD2, StFeSOD, StMnSOD, StCuZnSOD1, and StCuZnSOD2 (Figures 7A-E).StMAPKK5-transgenic plants (OE-2, OE-3, and OE-6 lines) exhibited increased sensitivity to heat stress, suggesting a higher mRNA expression of StFeSOD2, StFeSOD3, StMnSOD, StCuZnSOD1 and StCuZnSOD2, while Ri-2, Ri-3, and Ri-5 plants exhibited a decreased expressed compared to the NT plants, after 48 h of cultivation under heat stress condition (p < 0.001).However, StMAPKK5-low expressing plants showed decreased gene expression (p < 0.001).Peroxidases, encoded by StPOD66, StPOD47, and StPOD12 are enzymes involved in scavenging ROS, in response to heat stress.The CAT genes encode for catalase, such as StCAT1 and StCAT2, playing a vital role in the antioxidant defense system by scavenging ROS generated under heat stress situations.In the NT plants, StPOD66 (Figure 7F), StPOD47 (Figure 7G), StPOD1 (Figure 7H), StCAT1 (Figure 7I), and StCAT2 (Figure 7J) was highly expressed in response to heat stress (p < 0.001).Compared to the NT plants, the transgenic potato lines (OE-2, OE-3, and OE-6) increased the gene expression, while the RNAi plants profoundly decreased the mRNA expression of peroxidases, in response to heat stress (p < 0.001).The StHSFA3encoded protein belongs to the plant heat shock transcription factor family, orchestrating the transcription of genes that encode heat shock proteins (HSP) assisting the plant in coping with heatinduced damage (Friedrich et al., 2021).After 48 h of cultivation under heat stress condition, the NT plants displayed increased mRNA expression of StHSFA3 (Figure 7K), StHsp20-20 (Figure 7L), StHsp20-33 (Figure 7M), StHsp20-44 (Figure 7N), StHsp70 (Figure 7O), StHsp90.2 (Figure 7P), and StHsp90.4 (Figure 7Q).In contrast to the NT plants, the OE lines showed enhanced transcriptional levels, while the Ri plants exhibited decreased expression of the genes, under heat stress conditions (p < 0.001).The StP5CS gene, which encodes △1-pyrroline-5carboxylate synthetase, plays a critical role in proline biosynthesis, and proline is an important amino acid involved in maintaining cellular osmotic balance and scavenging ROS.Consistently, the NT plants increased the mRNA expression of StP5CS, 48 h after cultivation under heat stress conditions (p < 0.001) (Figure 7R).In comparison with the NT plants, the mRNA expression of StP5CS was evidently enhanced in the OE lines, while reduced in the Ri plants, in response to heat stress.Our results suggested that overexpression of StMAPKK5 triggered genetic responses of potato plant to combat heat stress.

Discussion
The effects of high temperatures are now evident in the reduced yield, nutrient depletion, and diminished quality of potatoes in an aberrant environment (Lee et al., 2020;Singh et al., 2020).The MAPK cascade is a crucial signaling pathway that plays a key module in transducing signals from environmental factors such as heat.The cascade has been extensively investigated and detailed in model plants like Arabidopsis, and crops such as rice owing to their relatively well-established research resources (Kumar et al., 2020;Chen et al., 2021).However, understanding the MAPK cascade in response to heat stress has been more challenging due to the complexity of its genome.Here, transgenic potato plants overexpressing StMAPKK5 displayed an improved heat stress tolerance in comparison to the non-transformed controls.Overexpression of StMAPKK5 triggered antioxidant enzyme genes, such as StFeSOD2, which was then implicated in regulating potato growth and physiology.As a result, StMAPKK5 can be used to create varieties that are more resistant to heat stress.
The modification of MAPKK protein expression under abiotic and biotic stress conditions appear to be a plant strategy for adapting and defending against challenging survival environments.Yang et al. revealed that in response to heat stress, the transcription level of OsMKK1 in Xanthomonas oryzae was apparently up-regulated (Yang et al., 2024).As for the abiotic stress, Kumar et al. OsMAPKK1,OsMAPKK3,OsMAPKK4,OsMAPKK6, and OsMAPKK10-2 expression levels are differentially regulated by heat, cold, drought, and salinity stresses (Kumar et al., 2008).In Prunus mume under exposure to cold, the expression of 4 PmMKK genes (PmMKK5, PmMKK6, PmMKK20, and PmMKK3) is reduced with prolonged treatment (Wen et al., 2023).The expression patterns of MKK genes were determined in different tissues of poplar by Wang et al., and the expression of MKK2a was altered after cultivation under salt tress (Wang et al., 2022).However, limited studies have explored the expression patterns of StMAPKKs under heat stress conditions.Considering that plant MAPKKs have crucial functions responding to abiotic stresses, and their expressions are regulated by heat stress (Kumar et al., 2008;Kumar and Sinha, 2013), we firstly investigated StMAPKKs expression distribution in different organs of potato plant and expression profile under heat stress conditions.The expression of StMAPKK1, StMAPKK2, StMAPKK3, StMAPKK4, and StMAPKK5 was distinctly distributed in flower, root, stem, leaf, petiole, stolon, tuber, and shoot.Furthermore, mRNA expression of the five genes in leaf, stem and root were affected to different extent.It is noteworthy that the increase in StMAPKK5 expression was relatively high and stable.Subsequently, we further constructed StMAPKK5 overexpression and low-expression plants to analyze its biological functions.
It's concerning to see the visible impact of aberrant environmental conditions on potato production, nutrient levels, and quality.Transgenic technology has been explored in creating varieties with traits like resistance to tolerance to environmental stressors like high temperature.Chen et al. concluded that the interaction between SaMKK2 and SaMAPK4/7 positively induces the expression of the downstream genes (SLD2, OPR2, and CBFs), thereby endowing potatoes with innate cold resistance (Chen et al., 2022).StMKK1 is involved in potato defense against the potato late-blight pathogen Phytophthora infestans, the gray-mold fungal pathogen Botrytis cinerea, and the bacterial wilt pathogen Ralstonia solanacearum (Chen et al., 2021b).However, it is still unknown whether StMAPKK5 mediates the growth of potato plant under heat stress.Our results showed that in response to heat stress, StMAPKK5 overexpression maintained or even fostered the plant growth, while StMAPKK5 down-regulation even decreased the growth compared to the non-transgenic plants.Duan et al. suggested a potential relationship between OsMKK4 and grain size or growth (Duan et al., 2014).GhMKK3 overexpression facilitates root growth in transgenic N. benthamiana (Wang et al., 2016).These findings are consistent with our results, providing a rationale for the involvement of MAPKK genes in plant growth.
Plants activate various defense mechanisms to alleviate the harmful impacts of oxidative stress or consequential injury to cells or tissues in response to abiotic stresses (drought, cold, heat, and salinity stresses) (Sachdev et al., 2021).These defense mechanisms include various nonenzymatic antioxidants and antioxidant enzymes (SOD, CAT, and POD) to neutralize ROS and maintain cellular homeostasis (Ahmad et al., 2009;Caverzan et al., 2016).High temperature affects multiple cellular processes, including photosynthesis, respiration and protein stability, and the disruption of these normal physiological activities leads to an overproduction of ROS and causes oxidative stress (Kumar, 2012;Posch et al., 2019;Zahra et al., 2023).Previous work has reported that in response to heat stress, MAPK cascade is involved in signal transduction, which affects plant physiological processes, such as photosynthesis, respiration, transpiration, nutrient uptake, tropisms, and senescence (Lee et al., 2016;Chardin et al., 2017b;Gouda et al., 2020;Liu et al., 2020).In this study, there was a reduction in contents of H 2 O 2 and MDA, and an increase in antioxidant activities, induced by StMAPKK5 overexpression under heat stress conditions.Chlorophyll content and REC are regarded as the physiological parameters mostly affected by heat and drought stress (Rehman et al., 2016).From our findings, it can be concluded that StMAPKK5 overexpression can alleviate the adverse effects on photosynthetic efficiency and cell membrane integrity.SOD functions in the primary defense against oxidative damage in cells, and participates in the detoxification, neutralization, scavenging, and converting of superoxide radicals (Alscher et al., 2002;Mishra and Sharma, 2019).There are three main types of SOD, each containing a different metal cofactor, including FeSOD, MnSOD, and CuZnSOD (Mishra and Sharma, 2019).Catalase detoxifies hydrogen peroxide and maintains redox homeostasis responding to heat stress (Dat et al., 1998).Peroxidase prevents the accumulation of hydrogen peroxide and reduce oxidative stress, working in coordination with other antioxidants like SOD and catalase (Veal and Day, 2011).StMAPKK5 overexpression significantly enhances the mRNA expression of SOD, catalase and peroxidase responding to heat stress.HSFA protein activate HSP expression, which are molecular chaperones that assist in protein refolding and prevent protein denaturation under heat stress conditions (Bourgine and Guihur, 2021;Khan et al., 2021).From our results, the increase in StHSFA3 mRNA expression and the induction of StHsp70,StHsp90.2,and StHsp90.4 seems to be related to StMAPKK5 overexpression.P5CS gene is involved in the biosynthesis of proline that serves as an osmoprotectant, maintaining osmotic balance during heat stress (Liu and Wang, 2020).StMAPKK5 overexpression-induced heat stress resistance may be through the regulation of StP5CS gene.Thus, StMAPKK5 overexpression modulated relative expression of heatstress responsive genes in response to heat stress.

Conclusions
Extreme high temperature leads to lower crop yields and confers negative roles in food availability.Here, we found the constructed StMAPKK5-transgenic potato plants exhibited an intensified heat stress tolerance compared to the non-transgenic plants.However, StMAPKK5 deficiency showed relatively poor thermal stress resistance.The adverse effects of heat stress on antioxidant activities POD, CAT, and SOD, photosynthetic efficiency and cell membrane integrity were alleviated by StMAPKK5 overexpression.The enhanced heat stress resistance in plants due to StMAPKK5 overexpression may be attributed to alterations in the relative expression of antioxidant enzyme genes and heat-stress responsive genes.Scientific Institution Basal Research Fund for Chinese Academy of Tropical Agricultural Sciences (No. 1630062024005).
FIGURE 2 mRNA expression of StMAPKK family genes of the various tissues of potato.Relative expression of (A) StMAPKK1, (B) StMAPKK2, (C) StMAPKK3, (D) StMAPKK4, and (E) StMAPKK5 at mRNA levels, in flower, root, stem, leaf, petiole, stolon, tuber, and shoot.Data were the means ± standard deviation.Different letters indicated significant difference between two groups (p-value less than 0.05, calculated by one-way ANOVA, followed by LSD and Duncan or Dunnett's T3).
FIGURE 3Expression profiles of StMAPKK family genes at mRNA level in potato leaves, stem and roots in response to heat stress.Relative mRNA levels of (A) StMAPK1, (B) StMAPKK2, (C) StMAPKK3, (D) StMAPKK4, and (E) StMAPKK5 in leaves, stem, and roots.Four-week-old normally grown plants were exposed to heat stress (35°C) for 0 h, 1 h, 2 h, 4 h, 8 h, 16 h, 24 h, or 48 h.Data were the means ± standard deviation.Different letters indicated significant difference between two groups (p-value less than 0.05, calculated by one-way ANOV, followed by LSD and Duncan or Dunnett's T3).

TABLE 1
List of specific primers for qRT-PCR and plasmid construction.Assays for levels of proline, MDA, and H 2 O 2 , and activities of POD, SOD, and CAT