Introduction
Low temperature stress is the major abiotic stress that is not conducive
to plant growth and yield. Basing the different effects of cold stress
in plants, it can be classified chilling stress and freezing stress
(Jan, Mahboob ul, & Andrabi, 2009). Chilling stress disturbs membrane
processes including opening of ion channels, membrane associated
electron transfer reactions (Ruelland, Vaultier, Zachowski, & Hurry,
2009). The freezing temperature has more negative effects in plants. The
extracellular freezing which means cellular water migrates to this
extracellular ice causing cell dehydration and shrinkage could occur in
freezing stress (Dowgert & Steponkus, 1984). Ultimately, ice can
penetrate the symplast (Gusta & L., 2004), causing a deterioration of
the intracellular structures and death of tissues. In addition, ROS is
accumulated continuously, no matter under chilling stress or freezing
stress, which leads to the degradation of various proteins and protein
complexes in plants and destroys the process of plant biogenesis
(Ruelland et al., 2009). Unraveling the mechanisms of how cold resistant
plant varieties adapt to extremely cold environments could provide
valuable information for enhancing the performance of conventional crops
under freezing stress, via genetic engineering. Plants have evolved
sophisticated mechanisms that limit cold-induced damage. A series of
comprehensive physiological and biochemical events take place during
plants withstanding cold stress (Y. Ding, Shi, & Yang, 2019). At the
physiological level, many substances or protective proteins are
synthesized in plants, such as soluble sugars, proline, and
cold-resistance proteins, of which are involved in osmotic potential,
ice crystal formation, membrane stability and ROS scavenging in plants.
These physiological and biochemical changes in plants are regulated by
different signaling and metabolic pathways (Asif et al., 2014), such as
CBF signaling pathway, sugar metabolism and ROS scavenge system.
Exploring upstream regulation mechanism of these pathways is of great
significance for revealing the molecular basis of plant freezing
tolerance.
CBF signal pathway endows plants cold tolerance by regulating the
expression of downstream cold-resistance proteins (Chinnusamy et al.,
2003a). CBF/DREB1 (C-REPEAT BINDING FACTOR/DEHYDRATION-RESPONSIVE
ELEMENT-BINDING PROTEIN1) genes are involved in cold acclimation of
plants and rapidly induced under cold stress (Q. Liu et al., 1998;
Stockinger, Gilmour, & Thomashow, 1997). Subsequently, COR genes, a
class genes encoding osmolyte and cryprotective proteins, are induced by
CBFs to protect plant from freezing injury (Shi, Ding, & Yang, 2018;
Yamaguchi-Shinozaki & Shinozaki, 1994). Inducer of CBF expression
transcription factor (ICE) is a MYC type bHLH transcription factor,
which is the most characteristic transcription activator of CBF gene
(Chinnusamy et al., 2003b). The ICE1 has been proved to play an
important role in response to low temperature stress in varied plants,
such as rice (Dianjun, Yu, & Kuide, 2007), Arabidopsis (Chinnusamy et
al., 2003b) and tomato (Juan et al., 2015). Not only CBFs gene, ICE1 can
also bind CORs promoter to activate CORs gene expression (Tang et al.,
2020). Due to the importance of ICE1 in CBF signaling pathway, its
upstream regulation mechanism has also been widely studied. Ding et al.
report that ubiquitination, sumo acylation and phosphorylation are
important for ICE1 in Arabidopsis cold tolerance (Yanglin Ding et al.,
2019). ICE1 is ubiquitinated and degraded by E3 ubiquitin ligase HOS1
(high expression of osmotically responsive gene 1), while sumo E3 ligase
SIZ1 (SAP and Miz) mediates sumo acylation of ICE1 and stabilizes ICE1
under low temperature stress (Miura et al., 2007). In addition, protein
kinase OST1 (open stomata 1) mediates ICE1 phosphorylation and cold
tolerance in Arabidopsis. OST1 is cold activated and is negatively
regulated by protein phosphatase EGR2 (clade e growth regulating 2) and
ABI1 (abscisic acid insensitive 1), independent of ABA (Y. Ding et al.,
2019; Y. L. Ding et al., 2015). Cold activated OST1 phosphorylates ICE1
to enhance its stability by destroying its interaction with HOS1 (Y. L.
Ding et al., 2015). Other study also finds that the protein stability
and transcriptional activity of OsICE1 are positively regulated by
OsMPK3 (MAP kinase 3) in rice (Z. Zhang et al., 2017). However, the
upstream regulation mechanism of ICE1 in plant freezing tolerance is
still elusive.
Starch degradation is involved in cold‐induced sugar accumulation
(Ruelland et al., 2009). When suffering cold stress, amylases activity
in plants increases. Subsequently, starch is degraded to glucose and
fructose, which is the substrate for the accumulation of soluble sugar
(Kaplan et al., 2007; Ruelland et al., 2009). The AtBAM3(At4g17090) encoding β-amylase, is induced by low temperature stress
(Kaplan & Guy, 2005; Lundmark, Cavaco, Trevanion, & Hurry, 2006). Thebam3 Arabidopsis mutant is sensitive to cold, and the
accumulation of soluble sugar is significantly reduced(Kaplan & Guy,
2005; Yano & R., 2005). Overexpressing PbrBAM3 in tobacco
(Nicotiana tabacum ) and pear (P.ussuriensis ) can increase
β-amylase activity, promote starch degradation under low temperature
stress, and enhance its cold tolerance (Zhao et al., 2019). ExceptBAM3 , another gene encoding β-amylase, BAM1 , also plays an
important role in starch degradation under chilling and freezing stress
(T. Peng, Zhu, Duan, & Liu, 2014). Moreover, α-glucan hydration
dikinase (GWD) can phosphorylate C-3 and C-6 of α-glucan, this starch
phosphorylation may promote starch degradation by increasing the
water-insoluble glucan to become hydrophilic ensuring better access to
starch degrading enzymes(Hejazi, Fettke, Haebel, Edner, & Ritte, 2010).
And its coding genes SEX1 and SEX4 are induced by low
temperature stress (Berrocal-Lobo et al., 2011; Hejazi et al., 2010).
Based on the importance of starch metabolism in plant response to low
temperature stress, its upstream regulatory mechanism is still to be
explored.
miR399 is an essential regulator of UBC24 (PHO2 )
expression during plant growth and response to stress. The
miR399-UBC24 (PHO2 ) regulation module is conserved in
plants including Arabidopsis (Bari, Datt Pant, Stitt, & Scheible,
2006), rice (Oryza sativa ) (Hu et al., 2011), wheat
(Triticum aestivum ) (J. Wang, Sun, et al., 2013) and maize
(Zea mays ) (Du, Wang, Zou, Xu, & Li, 2018). The
miR399-UBC24 model was first found to play a role in plant
response to Pi deficiency stress (Fujii, Chiou, Lin, Aung, & Zhu,
2005). When Pi is sufficient, UBC24 can degrade the Pi transporter
PHOSPHATE TRANSPORTER 1 (PHT1) by ubiquitination (Huang et al., 2013).
Under Pi-deficient condition, miR399 is strongly induced to downregulate
the expression of UBC24 to increase the level of PHT1 (Aung et
al., 2006; Hu et al., 2011). miR399 has been shown to regulate plant
reproductive development. The miR399-UBC24 module regulates
SEPALLATA MADS box transcription factor genes (SEPs) and ICE1 protein
level to effect floral organ development in Citrus (R. Wang et al.,
2020). The miR399-UBC24 module is also involved in plant other
process such as sugar metabolism (Y. Wang et al., 2017), nutrienrt
starvation responses (Hu et al., 2015), salt, drought and ABA signaling
(Baek et al., 2016). In addition, the results of multi plant species
miRNA sequencing showed that miR399 was induced by low temperature (R.
Hu et al., 2019; Koc, Filiz, & Tombuloglu, 2015). Overexpressing miR399
can improve plant growth in low temperature (Gao, Qiang, Zhai, Min, &
Shi, 2015). However, the mechanism of miR399 in response to freezing
stress is unknown.
Wheat (Triticum aestivum ) is one of the main food crops for the
global population (Rizwan et al., 2019). Freezing stress is one of the
main factors damaging the yield and quality of economic crops such as
wheat. To avoid winterkill it is very important to obtain the wheat
freezing resistance gene (Babben et al., 2018). Dongnongdongmai1 (Dn1)
is the first winter wheat cultivar that can overwinter safely in alpine
region of Heilongjiang province (The minimum temperature in this area
can reach -30℃) and the rate of returning green is greater than 85% (K.
K. Peng et al., 2021; Tian et al., 2021). Winter wheat’s resistance to
cold includes microRNAs (miRNAs)-short, single-stranded, non-coding RNAs
that regulate the posttranscriptional gene expression by targeting mRNAs
for cleavage or repressing translation (Lu, Xu, et al., 2020).
Therefore, Dn1 is valuable germplasm resources for mining freezing
resistance genes for crop variety improvement.
In this work, we systematically studied the interaction of
tae-miR399-TaUBC24 and the expression of
tae-miR399-TaUBC24 in winter wheat using bioinformatics and
research methods in molecular biology, physiology and biochemistry. Then
we determined the physical and genetic interaction of TaUBC24 and a
inducer of CBF expression transcription factor protein, TaICE1, and
reported that in addition to its role in Pi homeostasis and starch
degradation, the mechanism of how tae-miR399-UBC24 module and
ICE1 synergistically regulated plant tolerance to freezing stress, which
providing a broader view on the novel upstream regulating mechanism of
both CBF singnaling pathway and starch degradation response to freezing
stress.