Overexpression of AtAHL20 confers a late-flowering time phenotype in Arabidopsis
Our gain-of-function study showed that overexpression of AtAHL20 confers a late-flowering time phenotype under LDs (Fig. 2). This result is consistent with previous work implicating several Clade-A Arabidopsis AHLs (AtAHL18, AtAHL22, AtAHL27 and AtAHL29) in flowering time regulation (Street et al., 2008; Xiao et al., 2009; Yun et al., 2012). Specifically, transgenic plants overexpressing AtAHL22, AtAHL27 and AtAHL29 displayed a late-flowering phenotype (Street et al., 2008; Xiao et al., 2009; Yun et al., 2012). AtAHL22, AtAHL27 and AtAHL29 single gene knockout mutants did not show any clear flowering-time phenotypes. Only when AtAHL27, AtAHL29, AtAHL22 and AtAHL18 were simultaneously knocked out and/or knocked down, did the quadruple mutant display an early-flowering phenotype (Street et al., 2008; Xiao et al., 2009; Yun et al., 2012). These data suggested that several AHLs may function as part of a complex(es) to regulate gene expression (Tetko et al., 2006; Reeves, 2010; Yun et al., 2012) and that functional redundancy exists between these genes and other Clade-A AHL family members. Indeed, Zhao et al. (2013) proposed a similar model suggesting that various AHLs formed multi-AHL complexes to regulate hypocotyl growth in Arabidopsis. Data from our targeted yeast-two-hybrid assays supports this model by demonstrating that AtAHL20 physically interacted with itself and other Clade-A AHL members; AtAHL19, AtAHL22 and AtAHL29 (Fig. 4). It is, therefore, conceivable that these AHLs regulate flowering time as part of a complex. AtAHL20 did not interact with AtAHL6 (a Clade-B AHL) indicating that not all AHLs interacted with each other. Overall, we have shown that AtAHL20 is the fifth Clade-A AHL to be implicated in flowering time regulation in Arabidopsis.
AtAHL20 is a repressor of FT expression
Gene expression analyses showed that overexpression of AtAHL20 resulted in a depletion of FT transcript levels (Fig. 3). This is not surprising considering that several AHLs, including OsAHL1, AtAHL5, AtAHL10, AtAHL12, AtAHL16, AtAHL20, AtAHL22, AtAHL27 and AtAHL29, have been reported to exhibit promoter binding capabilities or been shown to confer transcriptional repression or activation of downstream target genes (Xu et al., 2013; Xu et al., 2013; Franco-Zorrilla et al., 2014; Franco-Zorrilla and Solano, 2014; Jia et al., 2014; Favero et al., 2016; Zhou et al., 2016; Lee and Seo, 2017; Wong et al., 2019; Favero et al., 2020). In particular, a previous study showed that AtAHL20 is a negative regulator of defenses in Arabidopsis (Lu et al., 2010). We also showed that an AHL20 orthologue in Camelina repressed CsFT expression, which suggests a conserved function across the two species (Figure S2B). It can be hypothesized that several AHLs modulate gene transcription, individually or as part of protein complexes in Arabidopsis and other species (Yun et al., 2012; Zhao et al., 2013; Favero et al., 2016; Lee and Seo, 2017). Our studies did not show a direct biological mechanistic link between AtAHL20 overexpression and repression of FT transcription. However, a close Clade-A AHL family member, AtAHL22, was shown to repress FT expression via a chromatin remodeling process (Yun et al., 2012). This occurs via FT chromatin architecture modification through both H3 acetylation and methylation. In addition, (Favero et al., 2016) also showed that AtAHL29, a Clade-A AHL, directly binds to YUC8 and SAUR19 promoters resulting in gene expression repression. Lee at al. (2017) went further and showed that AtAHL27 and AtAHL29 bind YUC9 promoter and suppress gene expression via chromatin modification activities of SWI2/SNF2-RELATED 1 (SWR1) complex. Recently, Favero et al. (2020) showed that AtAHL29 binds to PIF-targeted loci to reduce binding of PIF to these regions, thereby inhibiting transcriptional activation of growth promoting genes in Arabidopsis petioles. We hypothesize that AtAHL20 may also bind FT promoter elements and suppress its expression, perhaps individually or as part of complex. After all, AtAHL20 has already been shown to have binding affinities for several A/T-containing elements (Franco-Zorrilla et al., 2014; Franco-Zorrilla and Solano, 2014). A definitive answer to the question of the mechanism of gene repression may be provided via future studies that include yeast-one-hybrid and (chromatin immunoprecipitation) ChIP RT-qPCR experiments. Interestingly, 35S:AtAHL20 plants displayed similar seedling hypocotyl growth inhibition and adult plant phenotypes (dwarfism and late flowering) to 35S:AtAHL22 plants (Figs. 2, 5), esc-D/AtAHL27 as well as sob3-D/AtAHL29 (Street et al., 2008; Xiao et al., 2009). Targeted Y2H studies showed that AtAHL20 interacted with AtAHL22, ESC/AtAHL27 and SOB3/AtAHL29 (Fig. 4). It is therefore plausible that these AHLs function redundantly to regulate hypocotyl elongation and flowering time, possibly as part of a complex that includes AtAHL20, AtAHL22, AtAHL27 and AtAHL29, which have all been shown to regulate both biological processes.
Missense mutation in the AT-hook domain abolishes AtAHL20’s overexpression phenotype
We hypothesized that a missense mutation in AtAHL20’s AT-hook domain would abolish function based on similar outcomes in other Clade-A AHL gene family members, AtAHL29 (Street et al., 2008) and AtAHL22 (Yun et al., 2012). Thus, it was not surprising that 35S:AtAHL20m transgenic plants (Fig. 2A-D) lost the late-flowering phenotype typically observed when the wild-type AtAHL20 gene is overexpressed. Our working hypothesis based on the works of (Street et al., 2008; Yun et al., 2012; Zhao et al., 2013)
is that the second arginine residue in the AT-hook domain’s conserved R-G-R core is important for DNA binding, and without it, AtAHL20 is unable to bind AT-rich DNA and recruit chromatin modifying components required to repress FT transcription. This is in line with a deletion mutant study from Lu et al., (2010) who showed that removal of the entire At-hook domain abolished AtAHL20’s suppression function. This raises an interesting question regarding the specific biological importance of the conserved R-G-R amino acid trio found in both type-1 and type-2 At-hook motifs, versus peptide sequences flanking the AT-hook domain, for example. Would the mutation of the second arginine in the R-G-R core motif in all three AHL types (Type-I, -II, -III) also abolish overexpression phenotypes observed in transgenic plants overexpressing these genes? What role does the divergent nature of amino acid sequences flanking the R-G-R core play in Clade-A versus Clade-B AHLs? Studies in mammalian AHL orthologues, HMGA proteins, showed that the different types of AT-hook domains bind DNA with different affinities (Huth et al., 1997; Dragan et al., 2003). HMGA proteins containing a type-1 AT-hook, similar to the one found in Clade-A AHLs (e.g. AtAHL20, AtAHL22, AtAHL27 and AtAHL29) (Zhao et al., 2013), were found to confer the highest affinity to AT-rich DNA due to the nature of the peptide sequence adjacent to the R-G-R core motif. Interestingly, HMGAs containing a type-2 AT-hook, similar to one found in AtAHL6, have decreased DNA-binding affinity to AT-rich DNA (Huth et al., 1997; Dragan et al., 2003). Notably, preliminary data from AtAHL6 gain of function studies showed that transgenic plants overexpressing an aberrant gene carrying an R81->H mutation (35S:AtAHL6m) did not abolish the overexpression phenotype (early-flowering) observed in 35S:AtAHL6 plants. We can thus speculate whether this due to the fact that AtAHL6’s At-hook domain has low DNA-binding affinity to begin with. In the future, it will be important to further investigate the effect of missense mutations in Clade-A versus Clade-B AHLs which contain different At-hook types, and whose conserved R-G-R core is flanked by divergent amino acid sequences.
FT repression by AtAHL20 negatively affects expression of downstream flowering pathway genes
Quantitative PCR data showed that overexpression of AtAHL20 also resulted in repression of TSF, AGL8 and SPL3 expression (Fig. 3B). AGL8 and SPL3function downstream of FT in the flowering pathway (Higgins et al., 2010) whereas TSF acts redundantly with FT as a floral pathway integrator (Yamaguchi et al., 2005). The fact that two redundant floral pathway integrators FT and TSF transcript levels are regulated in a similar manner in 35S:AtAHL20 transgenic plants, raises an interesting question. Does AtAHL20 act directly on these two floral pathway integrators, or act upstream of them Further experiments, including ChIP-Seq, yeast-one-hybrid assays may help identify AtAHL20’s direct targets. Previous work showed that overexpression of AHL29, a Clade-A Type-I gene (just like AtAHL20),also caused delayed flowering in Arabidopsis (Street et al., 2008). Interestingly, preliminary Chip-Seq data from our lab showed that AtAHL29 binds FT. Taken together, these data suggest that AtAHL20 may function in a similar manner, by directly binding to promoters of its downstream targets.
At the same time, it was interesting that overexpression of an aberrant AtAHL20 protein in 35S:AtAHL20m transgenic plants only resulted in the elevation of FT transcript levels but not in downstream flowering pathway genes TSF, AGL8 and SPL3. We speculate that AtAHL20 indirectly affects expression of downstream targets via the direct repression of the main regulatory component of the flowering pathway, FT. Therefore, perhaps the elevation of FT transcript levels in 35S:AtAHL20m transgenic plants is not of enough magnitude to dramatically alter the expression of downstream components.
Pleiotropic AtAHL20 overexpression phenotypes
It was previously shown that AtAHL20 was involved in suppression of plant defenses (Lu et al., 2010). Here we have shown that AtAHL20 is a negative regulator of flowering time (Fig. 2C,D), enhances water stress tolerance (Figure S5) and represses hypocotyl growth (Fig. 5).
The suppression of hypocotyl elongation result is consistent with the phenotype conferred by the sob3-6 dominant negative allele (Street et al., 2008; Zhao et al., 2013). The sob3-6 allele is the basis of the model proposed by Zhao et al. (2013) which hypothesizes that several AHL genes contribute differently, in a quantitative manner, to the suppression of hypocotyl elongation in white light, and that other AHLs with similar functions are also affected by dominant-negative alleles (Street et al., 2008; Zhao et al., 2013). In their study, Zhao et al., (2013) showed that the quadruple mutant sob3-4 esc-8 ahl6 ahl22 conferred a longer hypocotyl phenotype than relevant triple mutant knockout lines and the wild type, but was still shorter than the dominant negative sob3-6 mutant. These results also point to a multifunctional role for AtAHL20 which is synonymous with the concept of moonlighting proteins (Huberts and van der Klei, 2010). Members of this class of multifunctional proteins perform multiple autonomous and often unrelated functions (Huberts and van der Klei, 2010). Previous data showing that AtAHL22, AtAHL27 and AtAHL29 regulate both flowering time and hypocotyl elongation support this hypothesis (Street et al., 2008; Xiao et al., 2009; Yun et al., 2012; Zhao et al., 2013; Favero et al., 2016). Alternatively, AtAHL20 may not display moonlighting characteristics, but indirectly control these processes via its regulation of multiple downstream targets. Ultimately, gain-of-function phenotypes should always be interpreted with caution since overexpression of genes may lead to indiscriminate activation of other genes that are not typically activated by the transcription factor under physiological conditions.