Introduction
Apple (Malus × domestica Borkh.) is a widely cultivated and economically important fruit crop in temperate regions worldwide owing to its high nutritional value, good storage, and lengthy supply period. Fuji apple is the main cultivar in China, but there are cultivation and production problems, including flowering difficulties and severe alternate bearing (Fan, Zhang, Lei, Chen, Xing, Ma, Zhao & Han, 2016, Guitton, Kelner, Velasco, Gardiner, Chagné & Costes, 2012). However, with global warming, an increase in the average temperature in winter will result in earlier apple flowering (Romanovskaja & Bakšiene, 2009, 刘璐, 郭梁, 李曼华, 傅玮东 & 栾青, 2020), and if there is cold weather in early spring, then significant flower and fruit losses will result. Additionally, at present, extreme hot weather occurs frequently in summer, causing other problems, such as growth impairment and production decline (Yao, Song, Wang, Song, Jiao, Wang & Zheng, 2020, ZHOU, SUN, LIU, JIN, ZHANG & WEI, 2016), which have seriously affected the development of the apple industry in China.
Floral induction pathways have been extensively studied, and six signalling pathways, photoperiodic, vernalization, autonomic, gibberellin, temperature-sensitive, and age, regulate flowering in the model plant Arabidopsis thaliana (Bäurle & Dean, 2006, Komeda, 2004, Teotia & Tang, 2015). In apple, the functions of some key flowering-related genes have been well studied in recent years, such asAPETALA1 (AP1 ), LEAFY (LFY ), FLOWERING LOCUS T (FT) , and TERMINAL FLOWER 1 (TFL1 ). For instance, overexpression of MdMADS5 , a putative homolog ofAP1 , leads to significant early flowering in Arabidopsis (Kotoda, Wada, Kusaba, Kano-Murakami, Masuda & Soejima, 2002). AFL1 andAFL2 , two orthologues of LFY , have been isolated from apple buds, and their overexpression lines in Arabidopsis flower significantly earlier than wild type (WT), with the overexpression ofAFL2 leading to a more obvious early-flowering phenotype than the overexpression of AFL1 (Wada, Cao, Kotoda, Soejima & Masuda, 2002). Apple anti-TERMINAL FLOWER 1 transgenic lines flower significantly earlier than the WT, with the earliest flowering at 8 months, while the WT did not flower for 6 years (Kotoda, Iwanami, Takahashi & Abe, 2006). The overexpression of the apple FT gene in Arabidopsis, poplar, and apple results in significantly earlier flowering in these plants, and transgenic poplar and apple flower during in vitro cultivation (Trankner, Lehmann, Hoenicka, Hanke, Fladung, Lenhardt, Dunemann, Gau, Schlangen, Malnoy & Flachowsky, 2010). The overexpression of MdFT1 and MdFT2 independently in Arabidopsis results in significantly earlier flowering under both long- and short-day conditions (Li, Tao, Yao, Hao & You, 2010). Through transcriptome analyses, the induction of apple flower buds was found to be regulated by sugar and hormone signalling pathways (Xing, Zhang, Li, Shen, Zhao, Ma, An & Han, 2015). Other omics studies have revealed the molecular mechanisms involved in responses to exogenous treatments, such as sugar (Liu, Feng, Pan, Zhong, Chen, Yuan & Li, 2016), 6-benzylaminopurine (Li, Zhang, An, Fan, Zuo, Zhang, Zhang, Gao, Han & Xing, 2019), and gibberellins (Zhang, Gottschalk & van Nocker, 2019), and their effects on the flowering of apples. However, research on apple flowering is still relatively limited.
A nuclear pore complex (NPC) is composed of a class of nucleoporins (Nups ) located in the nuclear pore (Tamura, Fukao, Iwamoto, Haraguchi & Hara-Nishimura, 2010). More than 30 Nups have been identified in Arabidopsis and 38 members have been identified in apple (Tamuraet al. , 2010, Zhang, An, Jia, Zhang, Liang, Zhang, Zhou, Ma, Han, Xing & Ren, 2020). Some Nups interact and form three subcomplexes:Nup62 , Nup93 , and Nup107–160 (Tamura et al. , 2010, Zhu, Wang, Tang, Hsu, Xie, Du H, Yang, Tao & Zhu, 2017).Nups control the transport of substances, such as RNA and proteins, between the nucleus and cytoplasm (Parry, 2013, Zhang, Wang, Kim, Yan, Yan, Pang & Hua, 2020). Proteins rely on importin α, importin β, and Ran-GTP complexes to pass through the NPC and enter the nucleus (Gorlich, Seewald & Ribbeck, 2003, Hill, 2009, Takizawa, Weis & Morgan, 1999). Importin β1 interacts with importin α, Ran-GTP, andNup62 directly in Arabidopsis, which further illustrates that plants and humans share a similar nuclear transport mechanism (Luo, Wang, Ji, Fang, Wang, Tian & Li, 2013). Nups play important roles in regulating plant growth and development, as well as biotic and abiotic stresses (Parry, 2013, Xu & Meier, 2008, Yang, Wang, Chu, Zhu & Zhang, 2017). For example, HOS1 , Nup96 , Nup54 ,Nup58 , Nup62 , Nup136 , and Nup160 are important for plant flowering (Cheng, Zhang, Huang, Huang, Zhu, Chen, Miao, Liu, Fu & Wang, 2020, Jung, Park, Lee, To, Kim, Seki & Park, 2013, Lazaro, Mouriz, Piñeiro & Jarillo, 2015, Parry, 2014, Tamuraet al. , 2010). HOS1 , Nup85 , Nup96 , andNup133 participate in abiotic stress pathways (Dong, Agarwal, Zhang, Xie & Zhu, 2006, Dong, Hu, Tang, Zheng, Kim, Lee & Zhu, 2006, Ishitani, Xiong, Lee, Stevenson & Zhu, 1998, Zhang et al. , 2020, Zhu et al. , 2017). MOS7 , Nup96 , Nup160 , andSec1 play important roles in plant immunity (Cheng, Germain, Wiermer, Bi, Xu, García, Wirthmueller, Després, Parker, Zhang & Li, 2009, Roth & Wiermer, 2012, Zhang & Li, 2005), and Nup96 ,Nup160 , and TPR affect hormone signalling pathways (Jacob, Mongkolsiriwatana, Veley, Kim & Michaels, 2007, Parry, Ward, Cernac, Dharmasiri & Estelle, 2006, Robles, Deslauriers, Alvarez & Larsen, 2012, Wiermer, Cheng, Imkampe, Li, Wang, Lipka & Li, 2012, Xu, Rose, Muthuswamy, Jeong, Venkatakrishnan, Zhao & Meier, 2007).
Heat shock factors (HSFs) are important components of signal transduction and play important roles in diverse stress pathways (Scharfa, Berberich, Ebersberger & Nover, 2012). The HSF family in plants has more members (21 HSFs in Arabidopsis) and more complex regulatory mechanisms (Nover, Bharti, Doring, Mishra, Ganguli & Scharf, 2001, Wang, Liu, Yu, Guo, Chen, Jiang, Xu, Fang, Wang, Zhang & Chen, 2020) than in vertebrates (4 HSFs ) or Drosophila (only 1HSF ). The structures of plant HSFs are relatively consistent from the N- to C-termini. The N-terminus has a DNA-binding domain, followed by heptad hydrophobic repeats involved in oligomerization, and a nuclear localization signal. The C-terminus contains a nuclear export signal and short peptide motifs. HSFs may be divided into three classes, A, B, and C, on the basis of their structural differences (Nover et al. , 2001). Class A has the C-terminal short peptide AHA domain, which has an activator function, while the B and C classes lack this domain (Kotak, Port, Ganguli, Bicker & von Koskull-Döring, 2004). HSFs specifically identify and bind heat shock elements (HSEs), which contain nGAAnnTTCn or nTTCnnGAAn in the downstream target genes’ promoters (Littlefield & Nelson, 1999). Class A members (HSFA1a , HSFA1b ,HSFA1d , HSFA1e , HSFA2 , and HSFA3 ) positively regulate plant heat tolerance (Charng, Liu, Liu, Chi, Wang, Chang & Wang, 2007, Nishizawa-Yokoi, Nosaka, Hayashi, Tainaka, Maruta, Tamoi, Ikeda, Ohme-Takagi, Yoshimura, Yabuta & Shigeoka, 2011, Qian, Chen, Liu, Yang, Li & Zhang, 2014, Schramm, Larkindale, Kiehlmann, Ganguli, Englich, Vierling & Von Koskull-Döring, 2008, Tian, Wang, Zhao, Lan, Yu, Zhang, Qin, Hu, Yao, Ni, Sun, Rossi, Peng & Xin, 2020), while, in contrast, Class B HSFs (HSFB1 and HSFB2b ) negatively regulate heat-induced HSFs and plant heat tolerance (Ikeda, Mitsuda & Ohme-Takagi, 2011). In addition to responding to heat stress, plant HSFs also participate in other stress pathways. For instance,HSFA1a -overexpression (OE) plants have enhanced tolerance under low/high pH levels and to hydrogen peroxide treatments (Qian et al. , 2014). HsfA2 knockout (KO) mutants (hsfa2 ) and double mutants (hsfa1a/hsfa1b ) both lose their heat-dependent adaptability to hypoxia (Banti, Mafessoni, Loreti, Alpi & Perata, 2010). Under high-light stress, the photosystem II activity of KO-hsfa1d/a1e mutants decreases, while that of WT plants remains high (Nishizawa-Yokoi et al. , 2011). The drought and salt tolerance levels of Oryza sativa OE-HSFBb transgenic lines significantly decrease, but they are significantly enhanced inOsHSFBb -RNAi lines (Xiang, Ran, Zou, Zhou, Liu, Zhang, Peng, Tang, Luo & Chen, 2013). Apple HSFA8a regulates the synthesis of flavonoids, thus enhancing the drought tolerance of apple (Wang et al. , 2020). In addition, some HSFs (HSFA2 , HSFA1E , andHSFA4C ) appear to be involved in plant flowering pathways (Chen, Zhao, Ge, Han, Ning, Luo, Wang, Liu, Zhang & Wang, 2018, Liu, Feng, Gu, Deng, Qiu, Li, Zhang, Wang, Deng, Wang, He, Bäurle, Li, Cao & He, 2019). Thus, HSFs may play important roles in plant development and stress tolerance.
Currently, there are no reported functional studies of Nups in apple.Nup62 is a member of the Nup62 subcomplex in the central core of the nuclear pore (Tamura et al. , 2010, Zhang et al. , 2020), and nup62 A. thaliana mutants have been reported to flower early, indicating Nup62 ’s involvement in flowering pathways (Parry, 2014). In this study, we characterized appleNup62 , which showed a high transcription level at the flower bud developmental stage and was responed to high temperature. The overexpression of MdNup62 in Arabidopsis resulted in earlier flowering compared with WT. Moreover, The overexpression ofMdNup62 in Arabidopsis and tomato both reduced heat resistance. Further, we performed a yeast two-hybrid (Y2H) sieve library experiment to screen for proteins that interact with MdNup62 , and the interactions between MdNup62 and the MdHSFs were confirmed. And the overexpression of MdHSFA1d and MdHSFA9bindependently in Arabidopsis resulted in earlier flowering and enhancing heat resistance. Thus, MdNup62 and the MdHSFs regulate flowering and respond to temperature changes. These results provide a theoretical reference for managing the impact of global warming on the apple industry.