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. 2018 Oct 9;9(1):4173.
doi: 10.1038/s41467-018-06696-y.

A Genetic Network Mediating the Control of Bud Break in Hybrid Aspen

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Free PMC article

A Genetic Network Mediating the Control of Bud Break in Hybrid Aspen

Rajesh Kumar Singh et al. Nat Commun. .
Free PMC article

Abstract

In boreal and temperate ecosystems, temperature signal regulates the reactivation of growth (bud break) in perennials in the spring. Molecular basis of temperature-mediated control of bud break is poorly understood. Here we identify a genetic network mediating the control of bud break in hybrid aspen. The key components of this network are transcription factor SHORT VEGETATIVE PHASE-LIKE (SVL), closely related to Arabidopsis floral repressor SHORT VEGETATIVE PHASE, and its downstream target TCP18, a tree homolog of a branching regulator in Arabidopsis. SVL and TCP18 are downregulated by low temperature. Genetic evidence demonstrates their role as negative regulators of bud break. SVL mediates bud break by antagonistically acting on gibberellic acid (GA) and abscisic acid (ABA) pathways, which function as positive and negative regulators of bud break, respectively. Thus, our results reveal the mechanistic basis for temperature-cued seasonal control of a key phenological event in perennial plants.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Delayed and early bud break in plants over- and under-expressing SVL. ad Bud break is earlier in a WT plants than in three independent SVL overexpressing lines, designated SVLoe2 b, SVLoe4 c, and SVLoe8 d. eh In contrast, bud break is later in WT plants h than in independent SVLRNAi lines 2 f, 10 g, and 12 h. I, j Time to bud break relative to wild type controls in SVLoe i and SVLRNAi j lines. Average time taken to bud break ± standard error mean (SEM), with respect to WT considered as 1, is shown with data from 10 plants from each line. Asterisks (*) indicate significant differences (P < 0.05) with respect to WT. k Low temperatures suppress SVL expression. Relative expression of SVL after 10 weeks of SD, followed by 2 and 5 weeks of low temperature (2WC, 5WC at +4 °C) and after 2 weeks subsequent exposure to long days and warmer temperatures (2WLD). SVL expression from three independent biological replicates ± SEM is shown relative to the reference gene UBQ with 10WSD time point set to 1. Different letters A–D over the bars indicate significant differences at P < 0.001. Statistical analysis was done using one way ANNOVA implying Dunnett’s/Tukey’s multiple comparison test
Fig. 2
Fig. 2
SVL negatively regulates expression of FT1 and GA20 oxidases during dormancy release and bud break. Expression patterns of genes encoding FT1, GA20 oxidases (GA20ox_1 and GA20ox_2) in apices of a WT and SVLoe and b WT and SVLRNAi after 10 weeks of SDs(10WSD), at the time of dormancy release (i.e. after 2weeks  of LT, 2WC), and after 2 weeks subsequent exposure to long days and warmer temperatures (2WLD). Expression values of the cited genes shown are averages for three biological replicates ± SEM, relative to the reference gene UBQ and with 10WSD time point set to 1. Asterisks indicate significant (*P < 0.05), very significant (**P < 0.005) and extremely significant (***P < 0.001) differences from corresponding controls, respectively. Statistical analysis was done using multiple t-tests
Fig. 3
Fig. 3
SVL positively regulates expression of the ABA biosynthesis gene NCED3, ABA receptors (RCAR/PYLs), and TCP18/BRC1-like transcription factors during dormancy release and bud break. Expression patterns of NCED3, RCAR/PYL1, RCAR/PYL2, and TCP18 in apices of a WT and SVLoe and b WT and SVLRNAi after 10 weeks of SDs (10WSD), at the time of dormancy release (i.e. after 2weeks of LT, 2WC), and after 2 weeks subsequent exposure to long days and warmer temperatures (2WLD). Expression values of the cited genes shown are averages for three biological replicates ± SEM, relative to the reference gene UBQ and with 10WSD time point set to 1. Asterisks indicate significant (*P < 0.05), very significant (**P < 0.005) and extremely significant (***P < 0.001) differences from corresponding controls, respectively. Statistical analysis was done using multiple t-test
Fig. 4
Fig. 4
SVL binds to FT1, NCED3, and TCP promoters in vivo in chromatin immunoprecipitation (ChIP) assays. a Diagrammatic representation of FT1, NCED3, and TCP18/BRC1 promoters showing the CArG motif and their positions within a 3 kb region. F1–R1 indicates positions of DNA fragments used to assess DNA–protein interactions in ChIP assays, and F2–R2 indicates positions of DNA fragments with no CArG motif used as negative controls in the assays. b Enrichment of the DNA fragments containing the CArG motif quantified by ChIP-q-PCR. Presented values were first normalized by their respective input values, then fold enrichments in WT and Myc-SVL plants relative to negative controls were calculated. Bars show an average values from three independent biological replicates ± SEM. Asterisks indicate significant (*P < 0.05), very significant (**P < 0.005) and extremely significant (***P < 0.001) differences from corresponding controls, respectively. Statistical analysis was done using t-test
Fig. 5
Fig. 5
Overexpression of GA2 oxidase represses early bud break in SVLRNAi lines. ad Bud break is earlier in a WT and b SVLRNAi plants than in c, d two independent lines overexpressing GA2 oxidase in a SVLRNAi background, designated GA2oxoe/SVLRNAi_1 and 6, respectively. e Time to bud break relative to WT controls for SVLRNAi plants, and lines overexpressing GA2 oxidase in a SVLRNAi background. Average time taken to bud break ± SEM, with respect to WT considered as 1, is shown with data from 10 plants from each line. Different letters A–C over the bars indicate significant differences at P < 0.001. Statistical analysis was done using one way ANNOVA implying Tukey’s multiple comparison test
Fig. 6
Fig. 6
RCAR/PYL1 or TCP18/BRC1 overexpression delays bud break in hybrid aspen. Time to bud break relative to WT controls for a RCAR/PYL1oe and TCP18/BRC1oe b plants. Average time taken to bud break ± SEM, with respect to WT considered as 1, is shown with data from 10 plants from each line. Asterisks (*) indicate significant differences (P < 0.01), with respect to WT. Statistical analysis was done using t-test
Fig. 7
Fig. 7
Hypothetical model integrating components involved in bud break. Low temperature reduces ABA levels and suppresses SVL expression, leading to induction of FT1 expression and GA biosynthesis, which promotes bud break. In the absence of low temperatures, high levels of SVL expression induce NCED3 and RCAR/PYL, thereby maintaining high ABA levels and sensitivity in buds, ensuring that they remain dormant. SVL subsequently induces TCP18/BRC1, suppressing bud break. Low temperatures trigger reduction in SVL expression and its suppressive effects, followed by bud break

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