**1. Introduction**

Pepper (*Capsicum annuum* L.) is an important member of the Solanaceae family and is one of the most important spice and vegetable crops in many countries [1]. It is rich in capsaicin, capsanthin, and vitamins, which can improve appetite and health [2]. Pepper grows well in warm climates but is sensitive to high temperatures, with the suitable temperature range for growth and development being 20–30 ◦C [3]. When the temperature exceeds 35 ◦C, the plant will suffer from heat stress (HS) and show symptoms of high temperature injury in the whole growth stage, which will adversely affect the plant morphology, physiological and biochemical metabolic processes, and other aspects [3,4]. With the intensification of the greenhouse effect, global temperatures have risen, impacting the growth and development of crops and presenting a severe challenge for many agricultural regions in the world, and leading to a drastic reduction in economic yields and quality [5]. Therefore, investigating the molecular mechanisms underlying the response of pepper to HS is imperative for developing varieties that are better adapted to more hostile conditions.

HS affects plant cell structure, protein denaturation, and lipid transport, resulting in the destruction of the plasma membrane structure and the death of specific cells or tissues. HS causes plant transpiration water loss, decreased photosynthetic rate, and abnormal metabolism, which affect the growth and development of plants [4]. Photosynthesis is a

**Citation:** Wang, F.; Yin, Y.; Yu, C.; Li, N.; Shen, S.; Liu, Y.; Gao, S.; Jiao, C.; Yao, M. Transcriptomics Analysis of Heat Stress-Induced Genes in Pepper (*Capsicum annuum* L.) Seedlings. *Horticulturae* **2021**, *7*, 339. https:// doi.org/10.3390/horticulturae7100339

Academic Editor: Yuyang Zhang

Received: 24 August 2021 Accepted: 21 September 2021 Published: 24 September 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

very heat-sensitive physiological process and is easily inhibited by HS, affecting almost all photosynthetic processes, including photosystem II, photosystem I, electron transport chain, adenosine triphosphate (ATP) synthesis, and carbon fixation [6,7]. In addition to the decrease in net photosynthetic efficiency and photosystem activity, reactive oxygen species (ROS) accumulate, resulting in the destruction of D1 protein and antenna pigment in serious cases, and thus reducing the ability of plants to absorb and utilize light energy and sequester carbon [8,9]. Additionally, ROS accumulation caused by HS in plants results in oxidative damage to cells. High temperatures cause metabolic imbalances and production of ROS in plants, which aggravate lipid peroxidation and protein denaturation of the cell membrane, thus affecting the structure and function of biofilms; severe cases can lead to cell damage and plant death [10]. High temperatures also greatly effect plant metabolism; for example, most of the genes in the anthocyanin biosynthesis pathway of eggplant are induced and downregulated under high temperatures, resulting in a decrease in anthocyanin accumulation [11]. Many abiotic stresses, including HS, directly or indirectly affect the synthesis, concentration, metabolism, transport, and storage of sugars. As a potential signal molecule, soluble sugars interact with light, nitrogen, and abiotic stresses to regulate plant growth and development [12–14].

Thermal signal perception and transduction are important parts of plant stress resistance, involving a number of signal transduction pathways, including calcium-dependent protein kinases (CDPKs) and mitogen-activated protein kinases (MAPK/MPKs), signal molecules (such as ROS), and plant hormones, which play important roles in various cellular signaling networks, by transmitting extracellular stimuli to generate intracellular responses. Thermal signal perception and transduction actively regulate gene expression and protein function under various stresses and ultimately cause adaptation to environmental stresses [15–19]. For example, CRISPR/Cas9-mediated *SlMAPK3* tomato mutants were more heat-tolerant than wild-type plants, showing less plant wilting and membrane damage, a lower ROS content, higher antioxidant enzyme activities, and higher transcriptional levels [20]. The heat-induced 47 kD MBP-phosphorylated protein SlMPK1 negatively regulates the heat tolerance of tomato by mediating antioxidant protection and redox metabolism; SlSPRH1, a protein homolog rich in serine and proline, is the target protein of SlMPK1 and can be phosphorylated by SlMPK1. Overexpression of SlSPRH1 reduces the heat tolerance and antioxidant capacity of plants and is related to SlSPRH1 phosphorylation. The SlMPK1-SlSPRH1 module negatively regulates the high-temperature signal in the high-temperature response process and cooperates with the antioxidant stress system [21]. Evidence shows that HS is accompanied by a certain degree of oxidative stress, and there is a crosstalk between the signals of heat and oxidative stresses. A study showed that H2O2 erupts after a short period of time under HS, owing to the activity of NADPH oxidase [22]. This outbreak was related to the induction of HS response genes [23]. H2O2 or menadione pretreatment can also improve heat tolerance in plants [24]. BZR1, the key regulator of brassinoid (BR) response, regulates the HS response of tomato through RBOH1-dependent ROS signaling; at least in part through the regulation of FER2 and FER3 [25].

Plant heat shock transcription factors (Hsfs) are important regulatory factors of signal transduction, which mediate the transcription of heat shock factors (HSPs) and other HSinduced genes [26]. *HsfA1a* regulates the initial response, and *HsfA1a* and *HsfB1* are constitutively expressed at a steady-state low abundance of mRNA. Under HS, the accumulation of *HsfA2* mRNA and protein is strongly induced, and *HsfA2* becomes the most abundant Hsf, regulating heat tolerance during recovery or after repeated HSs [27–29]. Under non-stress conditions, overexpression of *HsfB1* stimulates the co-activation of *HsfB1*, which promotes the accumulation of HS-related proteins and enhances heat tolerance [30,31]. Hsps are regulated by Hsfs, including *Hsp100*/*ClpB*, *Hsp90*/*HtpG*, *Hsp70*/*DnaK*, *Hsp60*/*GroEL*, and small *Hsp* (*sHsp*), which are generally considered to be important molecular chaperones for maintaining and/or restoring protein homeostasis, which plays a vital role in plant survival under HS [32,33]. In addition to Hsfs, other large families of transcription factors in plants are also involved in HS responses, such as WRKY, bZIP, MYB, and NAC. As a

downstream negative regulator of the H2O2-mediated HS response, *CaWRKY27* prevents improper responses during HS and recovery [3]. *CabZIP63*, a member of the bZIP family in pepper, directly or indirectly regulates the expression of *CaWRKY40* at the transcriptional and post-transcriptional levels and forms a positive feedback loop with *CaWRKY40* during the response of pepper to ralstonia solanacearum inoculation or high temperature–high humidity [34]. Overexpression of *SlAN2* induced the upregulation of the expression of several structural genes in the anthocyanin biosynthesis pathway and caused anthocyanin accumulation in tomato, which enhanced the tolerance to HS [35].

In nature, when plants are subjected to HS, their ability to recover is important, as the stronger the ability to recover, the faster the plant can restore their metabolic balance and maintain their normal growth. However, the regulatory molecular mechanisms and networks of pepper have not yet been reported. Therefore, in this study, we performed transcriptome analysis of the heat-tolerant variety 17-03 and the heat-sensitive variety H1023 during HS recovery, to identify candidate genes that had altered transcription levels in the pepper leaves. Collectively, our findings provide a theoretical basis for the cultivation of high-quality, heat-resistant varieties.
