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.