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. 2017 Mar;173(3):1709-1723.
doi: 10.1104/pp.16.01933. Epub 2017 Jan 26.

5' to 3' mRNA Decay Contributes to the Regulation of Arabidopsis Seed Germination by Dormancy

Isabelle Basbouss-Serhal  1   2   3 , Stéphanie Pateyron  1   2   3 , Françoise Cochet  1   2   3 , Juliette Leymarie  1   2   3 , Christophe Bailly  4   5   6
Affiliations

5' to 3' mRNA Decay Contributes to the Regulation of Arabidopsis Seed Germination by Dormancy

Isabelle Basbouss-Serhal et al. Plant Physiol. 2017 Mar.

Abstract

The regulation of plant gene expression, necessary for development and adaptive responses, relies not only on RNA transcription but also on messenger RNA (mRNA) fate. To understand whether seed germination relies on the degradation of specific subsets of mRNA, we investigated whether the 5' to 3' RNA decay machinery participated in the regulation of this process. Arabidopsis (Arabidopsis thaliana) seeds of exoribonuclease4 (xrn4) and varicose (vcs) mutants displayed distinct dormancy phenotypes. Transcriptome analysis of xrn4-5 and vcs-8 mutant seeds allowed us to identify genes that are likely to play a role in the control of germination. Study of 5' untranslated region features of these transcripts revealed that specific motifs, secondary energy, and GC content could play a role in their degradation by XRN4 and VCS, and Gene Ontology clustering revealed novel actors of seed dormancy and germination. Several specific transcripts identified as being putative targets of XRN4 and VCS in seeds (PECTIN LYASE-LIKE, ASPARTYL PROTEASE, DWD-HYPERSENSITIVE-TO-ABA3, and YELLOW STRIPE-LIKE5) were further studied by reverse genetics, and their functional roles in the germination process were confirmed by mutant analysis. These findings suggest that completion of germination and its regulation by dormancy also depend on the degradation of specific subsets of mRNA.

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Figures

Figure 1.
Figure 1.
Seeds of Arabidopsis mutants affected in 5′ to 3′ mRNA decay have a dormancy phenotype. A, Plant development 5 weeks after sowing. B to E, Germination of freshly harvested seeds of Col-0 (black circles), vcs-8 (closed orange squares), vcs-9 (open orange squares), xrn4-5 (closed green triangles), and xrn4-3 (open green triangles) in darkness at 15°C (B), at 25°C (C), and at 25°C after 2 weeks (D) and 3 weeks (E) of storage at 20°C and 56% relative humidity (after-ripening). Means ± sd of triplicate experiments are shown.
Figure 2.
Figure 2.
The transcript abundance of genes involved in ABA and GA pathways is affected in mRNA decay mutants. The quantification of transcript abundance is shown for NCED6, NCED9, CYP707A2, ABI5, SLP2, Ga3ox1, Ga20ox4, and Ga2ox2 in seeds of Col-0 (white bars), vcs-8 (striped bars), and xrn4-5 (dotted bars) after 24 h of imbibition at 25°C. a.u., Arbitrary unit (100 value was attributed to Col-0). Means ± sd of triplicate experiments are shown. Letters indicate homogenous groups in a corresponding class (ANOVA test and Newman-Keuls tests, P = 0.05).
Figure 3.
Figure 3.
mRNA decay modifies the seed transcriptome and is related to transcript features. A, Venn diagram showing the number of transcripts displaying significant increases in abundance (after Benjamini and Hochberg correction; ratio > 0.5) in xrn4-5 and vcs-8 mutants compared with wild-type Col-0 after 24 h of imbibition at 25°C. Totals of 973 and 2,312 transcripts more abundant in xrn4-5 and vcs-8 mutants, respectively, were identified. B and C, Relationship between mRNA total length and mRNA abundance in xrn4-5 (B) and vcs-8 (C) mutants. Each point of the scatterplots represents log10 (mRNA total length) on the x axis versus the ratio of change in mRNA log2 expression (mutant to Col-0) on the y axis for transcripts more abundant in mutant seeds. nt, Nucleotides. D and E, Relationship between the length of mRNA 5′ UTRs and mRNA abundance in xrn4-5 (D) and vcs-8 (E) mutants. Each point of the scatterplots represents log2 (5′ UTR length) on the x axis versus the ratio of change in mRNA log2 expression (mutant to Col-0) for transcripts more abundant in mutants on the y axis. F and G, Distribution of transcripts specifically more abundant in xrn4-5 (F) and vcs-8 (G) mutants (striped bars) and of transcripts displaying no significant change in abundance (black bars), compared with Col-0, as a function of the minimal free energy (ΔG) of secondary structure. mRNA classes were clustered based on the free energy of their secondary structure using a 10 kcal mol−1 step. The distribution of up-accumulated or constant transcripts in xrn4-5 and vcs-8 seeds was statistically different, as determined by χ2 test (P < 0.05). H and I, Distribution of transcripts specifically more abundant in xrn4-5 (H) and vcs-8 (I) mutants (striped bars) and of transcripts displaying no significant change in abundance (black bars), compared with Col-0, as a function of their GC content. mRNAs classes were clustered based on the free energy of their secondary structure using a 5% GC content step. The distribution of up-accumulated or constant transcripts in xrn4-5 and vcs-8 seeds was statistically different, as determined by χ2 test (P < 0.05).
Figure 4.
Figure 4.
mRNA decay targets molecular regulators of seed germination and dormancy. GO classification of transcripts specifically more abundant in xrn4-5 (A) and vcs-8 (B) mutants after 24 h of imbibition at 25°C was obtained using REVIGO (http://revigo.irb.hr; Supek et al., 2011), which allows a two-dimensional space representation according to the semantic similarity of GO terms (semantically similar GO terms remain close together in the plot). The size of the circles indicates the frequency of the GO term in the GO database (more general terms are represented by larger circles), and the color of the circles indicates the P value (scale at right; blue and green circles represent GO terms with more significant P values than orange and red circles).
Figure 5.
Figure 5.
The 5′ to 3′ mRNA decay of specific transcripts is associated with dormancy alleviation. A to F, mRNA stability analyzed by the quantification of transcript abundance during seed imbibition with cordycepin (a transcription inhibitor) in Col-0 (black circles), vcs-8 (open orange circles), and xrn4-5 (closed green triangles) for transcripts of MYB33 (At5g06100; A), MYB65 (At3g11440; B), ASP (At1g66180; C), PLY (At3g62110; D), DWA3 (At1g61210; E), and YSL5 (At3g17650; F). Means ± sd of triplicate experiments are shown. Asterisks indicate differences between mutant and Col-0 (ANOVA and Newman-Keuls tests, P = 0.05). G, Effect of the duration of after-ripening treatment at 20°C at 56% relative humidity on subsequent germination percentage after 7 d at 25°C of Arabidopsis seeds. Col-0, asp-1 (SALK_025595), asp-2 (SALK_041567), ply-1 (SALK_017839), ply-2 (SALK_031921), dwa3-1 (SALK_092993), dwa3-2 (SALK_058391), ysl5-1 (SALK_105596), and ysl5-2 (SALK_058656) mutants were assayed during dry storage. The after-ripening requirement for each genotype was estimated by calculating the number of days of after-ripening required to reach 50% germination (DSDS50) and is indicated at right. Values are means of three biological replicates ± sd.
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