Concerted control of the LaRAV1-LaCDKB1;3 module by temperature during dormancy release and reactivation of larch

Abstract

Dormancy release and reactivation of temperate-zone trees involve the temperature-modulated expression of cell-cycle genes. However, information on the detailed regulatory mechanism is limited. Here, we compared the transcriptomes of the stems of active and dormant larch trees, emphasizing the expression patterns of cell-cycle genes and transcription factors and assessed their relationships and responses to temperatures. Twelve cell-cycle genes and 31 transcription factors were strongly expressed in the active stage. Promoter analysis suggested that these 12 genes might be regulated by transcription factors from 10 families. Altogether, 73 cases of regulation between 16 transcription factors and 12 cell-cycle genes were predicted, while the regulatory interactions between LaMYB20 and LaCYCB1;1, and LaRAV1 and LaCDKB1;3 were confirmed by yeast one-hybrid and dual-luciferase assays. Last, we found that LaRAV1 and LaCDKB1;3 had almost the same expression patterns during dormancy release and reactivation induced naturally or artificially by temperature, indicating that the LaRAV1-LaCDKB1;3 module functions in the temperature-modulated dormancy release and reactivation of larch trees. These results provide new insights into the link between temperature and cell-cycle gene expression, helping to understand the temperature control of tree growth and development in the context of climate change.

Keywords: cambium; cell-cycle gene; dormancy release; larch; reactivation; temperature; transcription factor.

Figure 1.

Photomicrographs of cambium regions in cross-section in the dormant stage (10 March 2013) (A) and the active stage (July 2011) (B) of Larix kaempferi. The image of the cambium region from the active stage was taken in our previous study (Li et al. 2017). P, phloem; X, xylem; C, cambium. Scale bar, 50 μm.

Figure 2.

Summary of the DEUs and their enrichment analysis. (A) Number of DEUs with a given adjusted P-value (Padj). (B) Numbers of DEUs with and without annotation. (C) Numbers of Gene Ontology terms enriched in the annotated DEUs.

Figure 3.

Prediction and verification of transcriptional regulators of 12 differentially expressed cell-cycle genes. (A) Cases of regulation, transcription factors and enriched transcription factors with over-represented targets in the input gene set under the cutoff P-value ≤0.05 predicted using the data from seven plant species: Arabidopsis thaliana, Glycine max, Solanum lycopersicum, Populus trichocarpa, Triticum aestivum, Oryza sativa subsp. japonica and Zea mays, with the PlantRegMap server (http://plantregmap.cbi.pku.edu.cn/regulation_prediction.php; Jin et al. 2017). (B) Summary of the enriched transcription factors from 10 families. (C) Venn analysis of transcription factor families identified by prediction and transcriptomic analysis. (D) Seventy-three cases of regulation (highlighted in green and blue) between 16 transcription factors and 12 expressed cell-cycle genes, both of which were strongly expressed in the active stage of Larix kaempferi, two of which (highlighted in blue) were confirmed by yeast one-hybrid and dual-luciferase assays.

Figure 4.

Analysis of interactions between transcription factors and cell-cycle genes. Yeast one-hybrid assays show that LaMYB20 binds to the promoter of LaCYCB1;1 (ProLaCYCB1;1) (A), and both LaMYB84 (B) and LaRAV1 (C) bind to the promoter of LaCDKB1;3 (ProLaCDKB1;3). Dual-luciferase assays show that LaMYB20 (E) and LaRAV1 (G) increase the promoter activity of LaCYCB1;1 (ProLaCYCB1;1) and LaCDKB1;3 (ProLaCDKB1;3; P ≤ 0.05), respectively, and LaMYB84 (F) does not increase the promoter activity of LaCDKB1;3 (ProLaCDKB1;3). Schematic diagrams of the effector and reporter vector used in dual-luciferase assays is shown in (D) [values are the ratio of firefly luciferase (LUC) to Renilla luciferase (REN) activity]. Data represent the mean ± SD of three biological replications. Error bars represent standard error. Statistical significance was determined using Student’s t-test.

Figure 5.

Analysis of protein interaction. Yeast two-hybrid assays show that LaCDKB1;3 interacts with LaCYCB1;1 (A), and LaMYB84 does not interact with LaRAV1 (B). Bimolecular fluorescence complementation assays further show that LaCDKB1;3 interacts with LaCYCB1;1 in tobacco leaves (C). Scale bars, 20 μm.

Figure 6.

Expression patterns of two cell-cycle genes [LaCDKB1;3 (A) and LaCYCB1;1 (B)] and three transcription factors [LaMYB20 (C), LaMYB84 (D) and LaRAV1 (E)] in the active (9 July 2016) and dormant stages (21 December 2016) of Larix kaempferi seedlings (n ≥ 5) assayed by qRT-PCR with LaSYS as the internal control.

Figure 7.

Expression patterns of two cell-cycle genes (LaCDKB1;3 and LaCYCB1;1) and three transcription factors (LaMYB20, LaMYB84 and LaRAV1) during reactivation naturally induced by temperature. (A) Climatic daily temperatures from 13 February to 20 March 2017. Variation of LaCDKB1;3 (B), LaCYCB1;1 (C), LaMYB20 (D), LaMYB84 (E) and LaRAV1 (F) expression from 13 February to 20 March 2017 during the reactivation of Larix kaempferi seedlings (n ≥ 5) assayed by qRT-PCR with LaSYS as the internal control. The P-value for LaCDKB1;3 was generated between samples harvested on 24 and 28 February 2017, and 6 and 13 March 2017; for LaCYCB1;1 on 6 and 13 March 2017; and for LaRAV1 on 28 February and 6 March 2017.

Figure 8.

Correlations between gene expression and temperature. Pearson correlation coefficients between the expression levels of two cell-cycle genes (LaCDKB1;3 and LaCYCB1;1) and three transcription factors (LaMYB20, LaMYB84 and LaRAV1) and maximum temperature (A), minimum temperature (B) and average temperature (C) from 30 January to 20 March 2017. Pearson correlation coefficient values greater than those indicated by the green and red lines means that the correlations between gene expression and temperature were significant at P ≤ 0.05 and P ≤ 0.01, respectively.

Figure 9.

Expression patterns of two cell-cycle genes (LaCDKB1;3 and LaCYCB1;1) and three transcription factors (LaMYB20, LaMYB84 and LaRAV1) during reactivation artificially induced by temperature. One-year-old pot-grown dormant Larix kaempferi seedlings were transferred from outdoors into a dark room on 21 December 2016. Forty seedlings 19 cm in mean length were used: 10 were kept outdoors and in the dark as controls and 25 were kept in a dark room at 23 °C. Five seedlings were sampled at 2, 4, 6, 8 and 10 days after treatment; for controls five seedlings were sampled at 0, 8 and 10 days. (A) Climatic daily temperatures from 21 to 31 December 2016. Variation of LaCDKB1;3 (B), LaCYCB1;1 (C), LaMYB20 (D), LaMYB84 (E) and LaRAV1 (F) expression assayed by qRT-PCR with LaSYS as the internal control. The P-values for LaCDKB1;3 and LaRAV1 were generated between samples harvested at 2 and 4 days after treatment, at 0 and 10 days for the control seedlings and for LaCYCB1;1 at 4 and 6 days after treatment.

Figure 10.

Expression patterns of two cell-cycle genes (LaCDKB1;3 and LaCYCB1;1) and three transcription factors (LaMYB20, LaMYB84 and LaRAV1) during dormancy release and reactivation naturally or artificially induced by temperature. Forty 2-year-old pot-grown active Larix kaempferi cutting seedlings were transferred from outdoors into a greenhouse on 13 September 2017, when they had not experienced low temperatures. On 30 November 2017, when they had entered the dormant stage, 16 seedlings were transferred outside; after 6 weeks, eight seedlings were sampled on 11 January 2018 and the remaining eight were transferred into the greenhouse and kept for 2 weeks to determine whether dormancy was released; they were sampled on 25 January 2018. The other 24 seedlings were kept in the greenhouse as controls, and eight seedlings were sampled on 30 November 2017 and 11 and 25 January 2018. (A) Climatic daily temperatures from 25 November 2017 to 25 January 2018. Variation of LaCDKB1;3 (B), LaCYCB1;1 (C), LaMYB20 (D), LaMYB84 (E) and LaRAV1 (F) expression assayed by qRT-PCR with LaSYS as the internal control. The P-values for LaCDKB1;3 and LaRAV1 were generated between samples harvested on 11 and 25 January 2018 with natural chilling, and on 25 January 2018 with or without natural chilling.

Figure 11.

Regulatory network model for Larix kaempferi dormancy release and reactivation based on the expression patterns of cell-cycle genes and transcription factors and their relationships and responses to temperature. After fulfillment of the chilling requirement in winter, the increasing spring temperatures induce LaCDKB1;3, LaCYCB1;1, LaRAV1 and LaMYB20 expression; meanwhile, LaRAV1 and LaMYB20 promote LaCDKB1;3 and LaCYCB1;1 expression, respectively, and LaCDKB1;3 interacts with LaCYCB1;1, together promoting the resumption of cell-cycle progression and cambium cell division.