Possible Role of MADS AFFECTING FLOWERING 3 and B-BOX DOMAIN PROTEIN 19 in Flowering Time Regulation of Arabidopsis Mutants with Defects in Nonsense-Mediated mRNA Decay

Abstract

Eukaryotic cells use nonsense-mediated mRNA decay (NMD) to clear aberrant mRNAs from the cell, thus preventing the accumulation of truncated proteins. In Arabidopsis, two UP-Frameshift (UPF) proteins, UPF1 and UPF3, play a critical role in NMD. Although deficiency of UPF1 and UPF3 leads to various developmental defects, little is known about the mechanism underlying the regulation of flowering time by NMD. Here, we showed that the upf1-5 and upf3-1 mutants had a late-flowering phenotype under long-day conditions and the upf1-5 upf3-1 double mutants had an additive effect in delaying flowering time. RNA sequencing of the upf mutants revealed that UPF3 exerted a stronger effect than UPF1 in the UPF-mediated regulation of flowering time. Among genes known to regulate flowering time, FLOWERING LOCUS C (FLC) mRNA levels increased (up to 8-fold) in upf mutants, as confirmed by qPCR. The upf1-5, upf3-1, and upf1-5 upf3-1 mutants responded to vernalization, suggesting a role of FLC in delayed flowering of upf mutants. Consistent with the high FLC transcript levels and delayed flowering in upf mutants, levels of FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) mRNAs were reduced in the upf mutants. However, RNA-seq did not identify an aberrant FLC transcript containing a premature termination codon (PTC), suggesting that FLC is not a direct target in the regulation of flowering time by NMD. Among flowering time regulators that act in an FLC-dependent manner, we found that MAF3, NF-YA2, NF-YA5, and TAF14 showed increased transcript levels in upf mutants. We also found that BBX19 and ATC, which act in an FLC-independent manner, showed increased transcript levels in upf mutants. An aberrant transcript containing a PTC was identified from MAF3 and BBX19 and the levels of the aberrant transcripts increased in upf mutants. Taking these results together, we propose that the late-flowering phenotype of upf mutants is mediated by at least two different pathways, namely, by MAF3 in an FLC-dependent manner and by BBX19 in an FLC-independent manner.

Keywords: Arabidopsis; NMD; UPF1; UPF3; flowering.

Figure 1

Late flowering of upf1-5, upf3-1, and upf1-5 upf3-1 mutants under LD conditions. (A) Late-flowering phenotypes of upf1-5 and upf3-1 mutants grown at 23°C under LD conditions. Scale bar = 2 cm (B) Total leaf numbers of upf1-5 and upf3-1 mutants presented as a box plot (see Section Materials and Methods for further information on box plots). (C) The severely bushy phenotype of upf1-5 upf3-1 double mutants with no well-defined leaves in the vegetative phase (middle) and multiple inflorescences generated from the axillary meristems in the reproductive phase (right). Scale bar = 2 cm (due to the late flowering of upf1-5 upf3-1 mutants, pictures of wild-type Col-0 and double mutants were taken at different times. Col-0 plants were photographed at 5 weeks old and upf1-5 upf3-1 double mutants were photographed at 8 weeks old). (D) Days to flowering of upf1-5, upf3-1, and upf1-5 upf3-1 mutants presented as a box plot. A t-test was used to assess the statistical significance of flowering time between wild-type and upf mutants (p-values are mentioned for each group).

Figure 2

Differentially expressed genes in upf1-5, upf3-1, and upf1-5 upf3-1 mutants. (A) A heatmap showing differentially expressed genes among upf1-5, upf3-1, upf1-5 upf3-1, and wild-type plants. Based on the differential gene expression, upf1-5 mutants and wild-type plants grouped together, whereas upf3-1 and upf1-5 upf3-1 double mutants grouped together, indicating the high similarity between them. Upright triangle: up-regulation; inverted triangle: down-regulation. (B) Venn diagram of common and unique DEGs that were up-regulated in upf1-5, upf3-1, and upf1-5 upf3-1 mutants as compared to wild-type plants. (C) Venn diagram of common and unique DEGs that were down-regulated in upf1-5, upf3-1, and upf1-5 upf3-1 mutants as compared to wild-type plants. Note that the number of up-regulated genes was higher than down-regulated genes, suggesting the possible accumulation of NMD substrates in upf mutants.

Figure 3

An increase in FLC mRNA levels and a decrease of FT and SOC1 mRNA levels in upf mutants. (A,B) FPKM levels determined by RNA-seq (A) and relative expression levels determined by qPCR (B) of FLC in upf1-5, upf3-1, upf1-5 upf3-1, and wild-type plants. (C,D) FPKM levels determined by RNA-seq (C) and relative expression levels determined by qPCR (D) of FT in upf1-5, upf3-1, upf1-5 upf3-1, and wild-type plants. (E,F) FPKM levels determined by RNA-seq (E) and relative expression levels determined by qPCR (F) of SOC1 in upf1-5, upf3-1, upf1-5 upf3-1, and wild-type plants.

Figure 4

Vernalization response of upf1-5, upf3-1, and upf1-5 upf3-1 mutants. (A,B) Acceleration of flowering by vernalization in upf1-5 (A) and upf3-1 mutants (B). NV: non-vernalized, V: vernalized. Scale bar = 2 cm (C) Box plot showing total leaf numbers of vernalized/non-vernalized upf1-5 and upf3-1 mutants. Both upf1-5 and upf3-1 mutants flowered earlier in response to vernalization treatment. (D) Vernalization response of upf1-5 upf3-1 double mutants. NV: non-vernalized, V: vernalized. (E) Box plot showing days to flowering of vernalized/non-vernalized upf1-5, upf3-1, and upf1-5 upf3-1 mutants. A t-test was used to assess the statistical significance of flowering time difference in response to vernalization (p-value s mentioned for each group).

Figure 5

Expression of FLC and its regulators in upf mutants. (A) Heatmap shows the expression of 58 genes that possibly regulate FLC expression. MAF3, TAF14, NF-YA2, and NF-YA5, which showed expression patterns similar to that of FLC, are shown separately. (B,C) FPKM levels determined by RNA-seq (B) and relative expression levels determined by qPCR (C) of MAF3 in upf1-5, upf3-1, upf1-5 upf3-1, and wild-type plants. (D,E) FPKM levels determined by RNA-seq (D) and relative expression levels determined by qPCR (E) of TAF14 in upf mutants. TAF14 mRNA levels increased in upf single and double mutants, as detected by RNA-seq (D) and confirmed by qPCR (E). (F,G) FPKM levels determined by RNA-seq (F) and relative expression levels determined by qPCR (G) of NF-YA2 in upf mutants. (H,I) FPKM levels determined by RNA-seq (H) and relative expression levels determined by qPCR (I) of NF-YA5 in upf mutants. (J,K) FPKM levels determined by RNA-seq (J) and relative expression levels determined by qPCR (K) of BBX19 in upf mutants. (L,M) FPKM levels determined by RNA-seq (L) and relative expression levels determined by qPCR (M) of ATC in upf mutants.

Figure 6

Production of aberrant MAF3 and BBX19 transcripts containing PTCs in upf mutants. (A,B) Comparison of the structures of a normal transcript (AT5G65060.1) and an aberrant transcript (AT5G65060.3) from MAF3 (A) and comparison of the structures of a normal transcript (AT4G38950.3) and an aberrant transcript (AT4G38950.4) that we identified from BBX19 (B) from our RNA-seq analysis. Each bottom panel shows deduced amino acid sequences from the normal transcript and the aberrant transcript containing a PTC, which is indicated by an asterisk. Sequences in blue indicate the cDNA fragment that is absent in the aberrant transcript. Red arrows indicate the binding sites of primers used to detect aberrant transcripts from MAF3 and BBX19. Gray boxes indicate coding regions. (C,D) FPKM levels of the aberrant transcript of MAF3 (AT5G65060.3) (C) and BBX19 (AT4G38960.4) (D) in upf mutants. (E) Detection of AT5G65060.3 and AT4G38960.4 transcripts via RT-PCR. Note that an amplicon with the same size was amplified from genomic DNA (gDNA) from MAF3 (asterisk). PP2AA3 was used as an internal control.

Figure 7

Model explaining the late-flowering phenotype of NMD-deficient mutants. Two independent pathways likely participate in the UPF-mediated regulation of flowering time. In the FLC-dependent pathway, MAF3 seems to be the direct target of NMD and the up-regulation of MAF3 in NMD-deficient mutants causes up-regulation of FLC, which in turn represses FT and SOC1 to delay flowering. In the FLC-independent pathway, BBX19 seems to be the direct target of NMD and its up-regulation suppresses FT expression, which eventually inhibits flowering.