Discussion
Plant flowering has always been an important topic in crop and horticultural sciences, and issues with apple flowering have long hindered the development of the apple industry in China (Fan et al. , 2016, Guitton et al. , 2012). The Nups control protein transport between the nucleus and cytoplasm, and they participate in a variety of biological processes, including flowering (Parry, 2013, Zhang et al. , 2020).HOS1 regulates the binding of some nuclear genes toFLOWERING LOCUS C (FLC ) chromatin at low temperatures and weakens the transcriptional inhibition of FLC by HDA6 (Junget al. , 2013). In A. thaliana , Nup96 promotes the stability of HOS1 , and HOS1 conjugates and degradesCO , then promotes FLC expression, leading to delayed flowering. In addition, HOS1 increases the stability ofNup96 and thus maintains this regulatory pathway to control the flowering time (Cheng et al. , 2020, Lazaro et al. , 2015). Mutations in Nup54 , Nup58 , Nup62 , Nup136 , and Nup160 have resulted in a prominent earlier flowering phenotype compared with WT (Parry, 2014, Tamura et al. , 2010). In the present study, MdNup62 maintained a high expression level during flower development. To verify the flowering function ofMdNup62 , we determined the flowering phenotypes of OE-MdNup62 A. thaliana lines. Interestingly, the phenotypes of the overexpression lines were consistent with Arabidopsis deletion mutants and showed obvious early flowering. Previous studies found that both Nup62 deletion mutants and overexpression strains of Arabidopsis have increased the sensitivities to auxin, indicating that the overexpression does not result in a functional gain, but rather a functional loss, like the mutant (Boeglin, Fuglsang, Luu, Sentenac, Gaillard & Cherel, 2016). Therefore, the overexpression ofMdNup62 in this study may also result in a functional loss. However, MdNup62 is involved in the flowering pathway.
With global warming, extreme high-temperatures will occur more frequently, which will seriously affect the normal growth and development of plants (Yao et al. , 2020, ZHOU et al. , 2016). And Nups are involved in temperature-stress responses. HOS1 is an important negative regulator of cold-signal transduction in plant cells, and hos1-1 has poor cold resistance compared with WT (Ishitaniet al. , 1998). HOS1 specifically binds to and degradesICE1 , thereby reducing cold resistance (Dong et al. , 2006). The expression levels of cold-resistance-related genes, such asCBF, in atnup160 mutants were impaired and seedlings grew slowly, especially at low temperatures (Dong et al. , 2006). Nup85 and Nup133 control mRNA output only under warm conditions and are more sensitive to transcription factor localization at warm temperatures (Zhang et al. , 2020). In this study, MdNup62 responded to high-temperature stress in apple. However, OE-MdNup62 lines had reduced high-temperature resistance in both Arabidopsis and tomato. By analysing the relative expression levels of HSPs (HSP101 , HSP22-ER , HSP21.0 , and HSP70T-2 ) in transgenic plants, we found no obvious correlations between OE-MdNup62 lines and WT at a normal growth temperature, but OE-MdNup62 lines had significantly lower HSP expression levels than WT under high-temperature conditions.
In plants, Nup-interacting proteins have been studied (Cheng et al. , 2020, Zhang et al. , 2020, Zhu et al. , 2017), and some potential Nup85 -interacting proteins have been identified by immunoprecipitation and subsequent mass spectrometry in Arabidopsis, such as the Nup107–160 subcomplex (Nup160 , Nup133 ,Nup43 , Nup96 , Nup107 , Seh1 , andSec13 ), several mediator subunits (MED16 , MED14 , and MED18 ), HOS1 , and Sec13A . The interactions between Nup85 and three proteins, HOS1 , Sec13A , andMED18 , have been confirmed (Zhu et al. , 2017). Additionally, a direct interaction between Nup96 and HOS1in Arabidopsis has also been reported (Cheng et al. , 2020). In our previous study, the interaction between MdNup54 andMdNup62 was confirmed in apple (Zhang et al. , 2020). However, there are no reports of direct interactions between transcription factors and Nups in plants. We previously identified an interaction between apple MdNup54 and MdKNAT4/6 using a yeast double-hybridization test, but further verification is needed (Zhang et al. , 2020). In this study, we verified direct interactions between MdNup62 and MdHSF s, indicating that the Nups may directly recognize related transcription factors and thus regulate their transport. This provides a new direction of study for Nups.
Because of the early flowering of OE-MdNup62 Arabidopsis lines, MdHSF s that interact withMdNup62 may be also involved in the flowering pathway. Consistent with this conjecture, some HSFs are associated with flowering (Chenet al. , 2018, Liu et al. , 2019). HSFA1E andHSFA4C directly target and positively regulate the flowering geneSOC1 in lettuce (Chen et al. , 2018). ArabidopsisHSFA2 directly targets and promotes the expression ofREF6 , and the REF6–HSFA2 loop directly targets and activates HTT5 , which coordinates early flowering (Liu et al. , 2019). In this study, we found that MdHSFA9b andMdHSFA1d maintained high expression levels during flower bud induction. Additionally, OE-MdHSFA9b and OE-MdHSFA1dArabidopsis lines flower significantly earlier than WT. This suggests that MdHSFA9b and MdHSFA1d promote plant flowering.MdNup62 , MdHSFA9b , and MdHSFA1d share the same flowering phenotype, possibly because the overexpression ofMdNup62 fosters HSF accumulation in the nucleus, promoting the expression of downstream flowering-related genes and advancing flowering.
HSFs play important roles in regulating plant resistance to high temperatures. HSFA1 positively regulates the heat tolerance of tomato, the expression of HSFA2 is dependent on HsfA1 , and the thermotolerance of the posttranscriptional silencing of theHsfA1 gene in protoplasts can be restored by plasmid-borneHsfA2 (Mishra, 2002). In Arabidopsis, HSFA3 directly up-regulates the expression of Hsp18.1-Cl and Hsp26.5-MII , and both HSFA3 mutants and RNAi significantly reduce high temperature resistance (Schramm et al. , 2008). The overexpression of HsfA1a increases Hsp18.2 and Hsp70 expression levels, as well as heat-shock tolerance (Qian et al. , 2014).HSFA1b regulates high-temperature resistance through OPR3and the jasmonate signalling pathway (Tian et al. , 2020).HSFA1d and HSFA1e activate HsfA2 transcription, and a double knockout of HSFA1d and HSFA1e impairs tolerance to heat-shock stress (Nishizawa-Yokoi et al. , 2011). In Arabidopsis, HsfA2 acts as a heat-induced transactivator to maintain the expression levels of HSPs and prolong the duration of acquired thermotolerance (Charng et al. , 2007). InMedicago truncatula , HSFA9 plays important roles in thermotolerance (Zinsmeister, Berriri, Basso, Ly Vu, Dang, Lalanne, Da Silva, Leprince & Buitink, 2020). In the current study, we obtained similar results for MdHSFA9b and MdHSFA1d . The expression levels of HSPs in the two overexpression Arabidopsis lines were significantly greater than in WT, and both lines had enhanced high-temperature resistance levels. Like the flowering and auxin phenotypes (Boeglin et al. , 2016), the opposite phenotypes between OE-MdNup62 and OE-MdHSFA9b, OE-MdHSFA1dindicates that the overexpression of MdNup62 may also result in a lack of function under heat-stress conditions. Similar to the results of this study, Zhang et al. (2020) found that nup85 andnup133 increase the ubiquitous protoplast (nucleus and cytosol) signals of IAA17 and PIF4 at 28°C compared with at 22°C. Furthermore, the nup96 and hos1 mutants show significant increases in the ubiquitous localizations of IAA17 andPIF4 signals at 28°C (72% and 66%, respectively) compared with 22°C (40% and 49%, respectively)(Zhang et al. , 2020, 北京爱琴海乐之技术有限公司, 2005)(北京爱琴海乐之技术有限公司, 2005; Zhang et al. , 2020)(Zhang et al. , 2020). Thus, the nuclear accumulations of the IAA17 and PIF4 proteins innup85 , nup96 , nup133 , and hos1 are reduced compared with WT, and the defects are more severe at 28°C. Therefore, we hypothesized that the transport of MdHSFA9b , MdHSFA1d , and other MdHSF s is inhibited in OE-MdNup62 lines at high temperatures, resulting in the inhibition of the transcription of downstream HSP s, which further reduces high-temperature resistance.
On the basis of these findings, we constructed a hypothetical model ofMdNup62- related pathways involved in high-temperature resistance (Figure 11). At normal temperature, apple MdHSFs were not induced, and not much transported into nucleus that cannot lead to up-regulate expression of MdHSPs in WT and OE-MdNup62 . However, at high temperature, apple MdHSFs were significantly induced, and then transported into the nucleus through NPC channels to promote the expression of MdHSPs in WT, in which enhanced high-temperature resistance. But for OE-MdNup62 lines, the structure of the apple NPC changed, and blocked the transport of high temperature induced MdHSFs into the nucleus that cannot induce much MdHSPs expression causing heat injuring (Figure 11). Additionally, OE-MdNup62 , OE-MdHSFA9b and OE-MdHSFA1d lines showed significant early flowering phenotype compared with WT (Figure 3, 9; Figure S3).
In conclusion, temperature is an important factor affecting flowering. With global warming, apple flowering will occur earlier, increasing the risk of chilling-related injury. Moreover, extreme hot weather is also occurring frequently. Both climatic conditions seriously affect the development of the apple industry. MdNup62 interacts withMdHSFs to regulate flowering and heat-resistance pathways in plants. Thus, both MdNup62 and the MdHSFs regulate flowering and respond to temperature changes. This research provides a theoretical reference for managing the impact of global warming on the apple industry.