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.
Highlights
The survival of plant species largely depends on successful seed production via the formation of flowers; plants have evolved a complex mechanism to ensure successful reproduction (Huijser and Schmid, 2011), including a complicated genetic network that integrates endogenous and environmental cues to modulate the timing of the floral transition
Among the genes that regulate FLOWERING LOCUS C (FLC), the transcript levels of MADS AFFECTING FLOWERING 3 (MAF3), NF-YA2, NF-YA5, and TBP-ASSOCIATED FACTOR 14 (TAF14) increased in upf mutants and among the genes that regulate flowering time in an FLCindependent manner, the transcript levels of Arabidopsis thaliana CENTRORADIALIS homolog (ATC) and B-BOX DOMAIN PROTEIN 19 (BBX19) increased in upf mutants
From our RNA sequencing (RNA-seq) data, we found two floral repressors that showed consistent expression changes in upf mutants: B-BOX DOMAIN PROTEIN 19 (BBX19), which encodes a floral repressor that interacts with CO, thereby preventing CO from inducing FLOWERING LOCUS T (FT) expression (Wang et al, 2014), and ATC, which encodes a floral repressor that interacts with FD to affect the expression levels of the floral meristem identity gene AP1 (Mimida et al, 2001; Huang et al, 2012; Figures 5J,L). Quantitative Real-Time PCR (qPCR) analysis revealed that BBX19 expression was significantly up-regulated with a fold change of ∼2 in upf1-5 and upf3-1 mutants and ∼3-fold in upf1-5 upf3-1 double mutants (Figure 5K)
Summary
The survival of plant species largely depends on successful seed production via the formation of flowers; plants have evolved a complex mechanism to ensure successful reproduction (Huijser and Schmid, 2011), including a complicated genetic network that integrates endogenous and environmental cues to modulate the timing of the floral transition. At least 306 genes and eight genetic pathways affect flowering, including the photoperiod, autonomous, vernalization, ambient temperature, and gibberellic acid-dependent pathways (Bernier and Périlleux, 2005; Bouche et al, 2016). FLC negatively regulates flowering (Michaels and Amasino, 1999) by repressing the expression of FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1; Searle et al, 2006), thereby preventing precocious flowering. MADS AFFECTING FLOWERING 3 (MAF3), a close homolog of FLC, directly interacts with FLC (Gu et al, 2013) and negatively regulates flowering at low temperatures (Suter et al, 2014). FLC is positively regulated by the members of the NUCLEAR FACTOR Y family, including NF-YA2 and NF-YA5 (Xu et al, 2013), which encode CCAAT-binding transcription factors
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