Figure legends
Fig. 1 Bioinformatic analysis of tae-miR399 and TaUBC24. (A) Base conservation analysis of miR399. (B) Sequence alignment analysis of miR399. (C) RNA secondary structure analysis of pre-tae-miR399, the straight line represented the mature sequence. (D) Cis-acting element analysis of upstream promoter sequence of tae-miR399 and its targetsTaUBC24 . The mature miR399 sequences were of monocots (A. tauschii , B. distachyom ,O. sativa , H. vulgare , S. bicolor , P. vulgaris , T. aestivum and Z. mays ) and eudicots (A. thaliana, G. max, G. raimondii, N. tabacum, M. truncatula, P. trichocarpa, S. tuberosum , G. hirsutum , M. domestica , C. melo , M. esculenta , C. cardunculus ,L. usitatissimum , P. persica , S. lycopersicum ,F. vesca , P. abies , C. sativa , A. officinalis , B. napus , V. vinifera , A. caerulea ,R. communis , A. lyrate , V. unguiculate , T. cacao and V. vinifera ) were obtained from the miRbase database.
Fig. 2 The expression levels of tae-miR399 and its targetsTaUBC24 under low temperature stress. (A) Expression of Dn1 tae-miR399 in field under low temperature. (B) Expression of Dn1TaUBC24 in field under low temperature. (C) Expression of Dn1 tae-miR399 in green house under low temperature. (D) Expression of Dn1TaUBC24 in green house under low temperature. All values in the figure are mean ± SD (n = 3), P values (the field samples versus 5℃, the indoor samples versus 22℃) were calculated with one-way ANOVA, *P <0.05, ** P <0.01, ***P <0.001, **** P <0.0001.
Fig. 3 tae-miR399 targets TaUBC24 . (A) Alignment between tae-miR399 and six MREs in the 5’ UTR of TaUBC24 . (B) The model of cDNA structure and mRNA cleavage site of TaUBC24 determined by 5ʹ RACE. tae-miR399 complementary sites (red lines) with the nucleotide positions of TaUBC24 indicated. The RNA sequence of each complementary site from 5ʹ to 3ʹ and the miRNA sequence from 3ʹ to 5ʹ are shown in the expanded regions. The black arrow indicated a cleavage site verified by RLM 5′-RACE, with the frequency of cloned PCR products shown above the alignment. (a) The cleavage site of TaUBC24 in wheat. (b) The cleavage site of pTaUBC24 in N. benthamiana . (C) β-Glucuronidase (GUS) phenotype observed by histochemical staining. (a) pBI121- GUS (b) pBI121-tae-miR399 (c) pBI121-pTaUBC24 (d) the mixture of pBI121-tae-miR399 and pBI121-pTaUBC24
Fig. 4 Phenotype and survival rate of Arabidopsis lines under freezing stress. (A) Phenotypes of Arabidopsis lines under freezing stress. (B) Root phenotype of Arabidopsis seedlings. (C) Survival rate of Arabidopsis lines under freezing stress. The values are mean ± SD (n = 3), P values (versus WT) were calculated with one-way ANOVA, *P <0.05, ** P <0.01, ***P <0.001, **** P <0.0001. (D) Electronic conductivity of leaves in Arabidopsis lines under freezing stress. (E) MDA content of Arabidopsis lines under freezing stress. The values are mean ± SD (n = 3), P values (versus 24℃) were calculated with two-way ANOVA, * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.
Fig. 5 The gene expression level of miR399 and AtUBC24in Arabidopsis lines under freezing stress. (A) The expression of miR399 in Arabidopsis lines under freezing stress. (B) The expression ofAtUBC24 in Arabidopsis lines under freezing stress. All values in the figure are mean ± SD (n = 3), P values (versus 24℃) were calculated with two-way ANOVA, * P <0.05, **P <0.01, *** P <0.001, ****P <0.0001.
Fig. 6 The gene expression level of CBF signal pathway in Arabidopsis lines under freezing stress. (A) The expression ofAtCBF1 in Arabidopsis lines under freezing stress. (B) The expression of AtCBF2 in Arabidopsis lines under freezing stress. (C) The expression of AtCBF3 in Arabidopsis lines under freezing stress. (D) The expression of AtCOR15A in Arabidopsis lines under freezing stress. (E) The expression of AtCOR15B in Arabidopsis lines under freezing stress. (F) The expression of AtCOR47 in Arabidopsis lines under freezing stress. (G) The expression ofAtCOR413IM in Arabidopsis lines under freezing stress. All values in the figure are mean ± SD (n = 3), P values (versus 24℃) were calculated with two-way ANOVA, * P <0.05, **P <0.01, *** P <0.001, ****P <0.0001.
Fig. 7 The protein level and transcription level of AtICE1 in Arabidopsis lines under freezing stress. (A) Protein level of AtICE1 in Arabidopsis lines under freezing stress. (B) Labeled AtICE1 bands were quantified using Q9 Aliance software. (C) Transcription level of AtICE1 in Arabidopsis lines under freezing stress. All values in the figure are mean ± SD (n = 3),P values (protein content versus WT, expression of AtICE1versus 24℃) were calculated with one-way or two-way ANOVA, *P <0.05, ** P <0.01, ***P <0.001, **** P <0.0001.
Fig. 8 Phosphorus content and transcript levels of selected PSI genes in Arabidopsis lines under freezing stress. (A) Total phosphorus content of Arabidopsis lines under freezing stress. (B) The content of inorganic phosphorus in Arabidopsis under freezing stress. (C) The expression of AtPHO1 in Arabidopsis lines under freezing stress. (D) The expression of AtPHT1;1 in Arabidopsis lines under freezing stress. (E) The expression of AtPHT1;2 in Arabidopsis lines under freezing stress. (F) The expression of AtPHT1;4 in Arabidopsis lines under freezing stress.
Fig. 9 Contents of starch and soluble sugar in Arabidopsis lines under freezing stress. (A) Soluble sugar content in Arabidopsis under freezing stress. (B) Starch content of Arabidopsis lines under freezing stress. All values in the figure are mean ± SD (n = 3),P values (versus 24℃) were calculated with two-way ANOVA, *P <0.05, ** P <0.01, ***P <0.001, **** P <0.0001.
Fig. 10 Expression and activity of starch hydrolase in Arabidopsis lines under freezing stress. (A) Activity of α-amylase in Arabidopsis under freezing stress. (B) Activity of β-amylase in Arabidopsis under freezing stress. (C) Expression of AtAPL1 in Arabidopsis lines under freezing stress. (D) Expression of AtAPL3 in Arabidopsis under freezing stress. (E) Expression of AtBAM1 in Arabidopsis under freezing stress. (F) Expression of AtBAM3 in Arabidopsis under freezing stress. (G) Expression of AtSEX1 in Arabidopsis lines under freezing stress. (H) Expression of AtSEX4 in Arabidopsis under freezing stress. All values in the figure are mean ± SD (n = 3), P values (versus 24℃) were calculated with two-way ANOVA, *P <0.05, ** P <0.01, ***P <0.001, **** P <0.0001.
Fig. 11 ROS content in Arabidopsis lines under freezing stress. (A) DAB staining of Arabidopsis leaves under freezing stress. (B) NBT staining of Arabidopsis leaves under freezing stress. (C) H2O2 content of Arabidopsis lines under freezing stress. (D) O2- content of Arabidopsis lines under freezing stress. All values in the figure are mean ± SD (n = 3), P values (versus 24℃) were calculated with two-way ANOVA, * P <0.05, ** P <0.01, ***P <0.001, **** P <0.0001.
Fig. 12 Expression and activity of SOD, POD and CAT in Arabidopsis lines under freezing stress. (A) Activity of SOD in Arabidopsis under freezing stress. (B) Expression of AtSOD1 in Arabidopsis lines under freezing stress. (C) Expression of AtSOD2in Arabidopsis under freezing stress. (D) Expression of AtSOD3 in Arabidopsis under freezing stress. (E) Activity of POD in Arabidopsis under freezing stress. (F) Expression of AtPER3 in Arabidopsis lines under freezing stress. (G) Activity of CAT in Arabidopsis under freezing stress. (H) Expression of AtCAT2 in Arabidopsis lines under freezing stress. (I) Expression of AtCAT3 in Arabidopsis lines under freezing stress. All values in the figure are mean ± SD (n = 3), P values (versus 24℃) were calculated with two-way ANOVA, *P <0.05, ** P <0.01, ***P <0.001, **** P <0.0001.
Fig. 13 TaUBC24 physically interacts with TaICE1 protein. (A) Yeast two-hybrid (Y2H) assays to determine interactions between TaUBC24 and TaICE1. (B) Bimolecular fluorescence complementation (BiFC) assays for determine the interactions of TaUBC24 with TaICE1.
Fig. 14 Proposed working model for the underlying mechanism used by tae-miR399-UBC24 model to regulate plant response to freezing stress.