tae-miR399-UBC24 module regulates Arabidopsis CBF
signaling pathway in response to freezing stress
In this study, we analyzed the expression pattern and biological
function of Dn1 tae-miR399 and its target gene TaUBC24 under low
temperature stress, and provided evidence for tae-miR399 mediatedTaUBC24 expression as a new regulatory mechanism in winter wheat
response to low temperature stress. Due to the limitation of
experimental conditions, it is very difficult to carry out tissue
transfection in wheat. Therefore, Arabidopsis thaliana was used
for functional gain analysis. The results showed that in transgenic
Arabidopsis plants, OEmiR399 lines had a cold tolerance phenotype, and
OEmiR399 Arabidopsis plants had a higher survival rate, lower ion
permeability and membrane lipid damage than WT (Fig. 4). These results
indicates that tae-miR399 has a certain biological function of cold
resistance.
Although a large number of cold responsive miRNAs have been identified,
further functional analysis is needed to determine their roles in plant
cold response, especially resistance to freezing stress. It has found
that overexpressing miR394 transgenic Arabidopsis plants show stronger
cold tolerance, and the expression of multiple genes in CBF signaling
pathway are higher than those of WT, indicating that miR394 is a
positive regulator of plant response to low temperature stress (Song,
Li, Cao, & Qi, 2019). Overexpressing miR319 in rice has a better
survival rate than that of wild-type, the expressions of CBFsincrease, and the content of ROS is also significantly lower than that
of wild-type, which indicates that miR319 is involved in plant cold
tolerance (S. T. Wang et al., 2014). At present, the mechanism of miRNA
regulating wheat response to low temperature stress is still limited,
and the specific mechanism of wheat miRNAs mediated CBF signaling
pathway is still unclear. ICE1, as a MYC bHLH protein, also plays a role
in CBF signaling pathway by regulating downstream target genes (Deng,
Ye, Fan, Pu, & Yan, 2017). The previous study showed that CsUBC24 and
CsICE1 interact in citrus, and CsUBC24 mediate degradation of CsICE1 but
not regulate the transcription level of CsICE1 (R. Wang et al.,
2020). Therefore, we tested the transcription level of AtICE1 in
OEmiR399 Arabidopsis plants and found that only at 4°C, the expression
level of AtICE1 in OEmiR399 Arabidopsis was higher than that of
WT (Fig. 7C). However, the ICE1 protein level of OEmiR399 Arabidopsis
plants was always higher than that of WT (Fig. 7A and B). These results
suggests that overexpressing tae-miR399 can increase the protein content
of AtICE1 by down-regulating the expression of AtUBC24 . In
Arabidopsis, ICE1 plays a key role in the development of leaf stomata
and the response to chilling and freezing stress, and can combine with
the CBFs promoter sequence to promote its expression (Miura et
al., 2007). Recent studies have shown that except binding to the
promoter sequence of CBFs, ICE1 can also directly initiate the
expression of most genes in the CBF signaling pathway, such asCOR47 , COR413IM , MPK and HOS (Tang et al.,
2020). In this study, the expression of CORs detected in all
Arabidopsis lines were up-regulated in different degrees under low
temperature. It is worth noting that only the expression levels ofAtCOR47 and AtCOR413IM in OEmiR399 Arabidopsis plants were
significantly higher than those in WT under cold acclimation and
freezing stress (Fig. 6F and G). The expression of AtCBFs in
OEmiR399 Arabidopsis plants were significantly higher than that in WT
under freezing stress (Fig. 6A, B and C). The results indicate that
AtICE1 with high protein level promotes the expression of its downstream
target genes (AtCBFs, AtCOR47 and AtCOR413IM ) by binding
to the promoter region under freezing stress. Some studies have shown
that plants can activate the expression of CBFs in the early
stage of cold acclimation, and when the content of CBFs protein reaches
a certain level in the later stage, the expression of CBFs is
inhibited to prevent plants from over chilling defense (Chinnusamy et
al., 2003a; Novillo, Alonso, Ecker, & Salinas, 2004). For example, when
Arabidopsis was treated at 4℃ for 24 h, its AtCBFs peaked at 3 h
and then gradually decreased(Joachim Kilian et al., 2007). In this
study, AtCBFs also showed similar expression changes. At 3 days
of cold acclimation, the expression of AtCBFs in WT had returned
to the level before untreated, while the expression of AtCBFs in
OEmiR399 Arabidopsis plants was significantly lower than that at 24℃
(Fig. 6A, B and C). It implies that during cold acclimation, the AtCBFs
protein content of OEmiR399 Arabidopsis plants is significantly higher
than that of WT. In addition, we found that TaUBC24 interacts with
TaICE1 through Y2H and BiFC experiments (Fig. 13). These results
suggested that the interaction between UBC24 and ICE1 may be conserved
in plants. Also in wheat, tae-miR399-TaUBC24 module may mediate
the CBF signaling pathway in response to freezing stress by inhibiting
the degradation of TaICE1 protein.
tae-miR399-UBC24 module regulates Pihomeostasis and phosphorus
utilization efficiency in
Arabidopsis in response to freezing stress
PHO2 down-regulates the expression of PSI-related genes (such as PHTs)
by promoting the degradation of PHO1, and negatively regulates Pi uptake
and translocation (Chiou & Lin, 2011; Liang et al., 2014). Under
phosphorus deficiency, plants adapt complex strategies to enhance the
acquisition of phosphorus, for example, the remodeling of the root
structure and the reactivation of phosphorus in plants (Chiou & Lin,
2011; Liang et al., 2014). In this study, at 24℃, the Pi content of
OEmiR399 Arabidopsis plants was significantly higher than that of WT,
and the primary roots of OEmiR399 Arabidopsis plants were elongated,
which was similar to the phenotype of overexpressing miR399 Arabidopsis
(Chiou et al., 2006), rice (Hu et al., 2011) and maize(Du et al., 2018).
These results imply that overexpressing tae-miR399 may promote the
absorption and transport of Pi in Arabidopsis. During the cold
acclimation, the total phosphorus content and the expression ofAtPHT1; 1 and AtPHT1; 4 in OEmiR399 Arabidopsis plants
were significantly higher than that in WT (Fig. 8D and F). However, the
Pi content of OEmiR399 Arabidopsis plants was significantly lower than
that of WT (Fig. 8B). These results may imply that during cold
acclimation, the phosphorous transporter in OEmiR399 Arabidopsis plants
is active, and the transported Pi has been rapidly converted to
organophosphorus. Previous reports have reported that at 5°C, the Pi and
organophosphates of pho2 Arabidopsis mutants are significantly
higher than those of WT, and the many phosphatases and other metabolic
enzymes activities of pho2 increase (Hurry, Strand, Furbank, &
Stitt, 2000), which is consistent with our research results. Therefore,
we speculate that overexpressing tae-miR399 may increase Pi absorption
and phosphorus utilization efficiency of Arabidopsis during cold
acclimation. The Pi content and the expression of AtPHTs in
OEmiR399 Arabidopsis plants under freezing stress were similar to cold
acclimation, indicating that under freezing stress, the phosphorus
utilization efficiency of OEmiR399 Arabidopsis plants is higher than
that of WT. These results demonstrate that the function of tae-miR399 in
response to Pi signal in Arabidopsis is conserved, and that Pi signal
plays an important and positive role in response to low temperature
stress in Arabidopsis. In addition, the expression of AtPHO1 of
OEmiR399 Arabidopsis plants has been at a low level under low
temperature in this study, which may be a feedback regulation of
increasing AtPHO1 protein content caused by down-regulatingAtUBC24 expression (Fig. 8C).