Untangling complexity: shedding a new light on LEAFY and APETALA1 interactions

by Leonie Verhage and Francois Parcy
Institut de Biosciences et Biotechnologies de Grenoble (France)

Ever since their discovery almost 30 years ago, the transcription factors LEAFY (LFY) and APETALA1 (AP1) (together with its paralog CAULIFLOWER (CAL)) have been extensively studied for their roles in floral transition. Early genetic and molecular experiments indicated that LFY and AP1/CAL were partly redundant and partly complementary in the process of floral initiation, and numerous subsequent studies fit this model (see Denay et al., 2017 and Wils and Kaufmann, 2017 for recent reviews). However, combining a set of new experiments with published datasets, Goslin and colleagues manage to stir up the prevalent view (Goslin et al., 2017).

FrancoisParcyImage2

Scanning electron micrograph of an ap1 cal mutant. Floral meristems are transformed into proliferative inflorescence meristems. This mutant background was used by Goslin and colleagues. (Image courtesy of Marie le Masson and Christine Lancelon Pin)

To unravel the redundancy of LFY and AP1/CAL, the authors utilized a mutant line harboring a 35S:LFY-GR construct in an ap1/cal background (Wagner et al., 1999). With this line, they performed induction experiments and microarray analysis, in the same way as was previously performed with 35S:AP1-GR in the ap1/cal background (Kaufmann et al., 2010), to make the datasets comparable. This allowed them to compare the downstream genes that are regulated by LFY in the absence of AP1/CAL to genes that are regulated by AP1 in the presence of LFY.

Among the many things uncovered by these analyses, a few were expected and many completely unanticipated.

As already reported by Winter et al., 2015, there is a large overlap between the genes that are differentially regulated upon induction of LFY-GR or AP1-GR. It is likely that this represents true redundancy, where LFY and AP1 can regulate genes in the same way, independent of each other. However, due to a lack of experiments where AP1 is induced in the absence of LFY, it cannot be excluded that this set of genes can be regulated by LFY alone, or by LFY and AP1 together.

More surprisingly, many direct targets of LFY were found to be down-regulated, whereas most of the well-known targets are induced (such as the floral organ identity genes or the LATE MERISTEM IDENTITY genes).

Interestingly, a subset of genes showed differential expression in ap1 cal upon AP1 induction but not upon induction of LFY. By comparing these genes with previously published ChIP-seq data of LFY, the authors could identify a set of genes to which LFY is able to bind, but that are not differentially regulated in absence of AP1. This was the case for APETALA3 (AP3) and AGAMOUS (AG), consistent with a previous report showing that AP1 can act on these genes (Ng and Yanofsky, 2001). Hence, for regulation of these B- and C- type floral organ identity genes, LFY and AP1 appear to act interdependently.

The most surprising result, however, was the presence of genes that are differentially expressed upon LFY or AP1 induction, but in different directions. Apparently, besides acting redundantly or interdependently, LFY and AP1 can also act antagonistically. Notably, this turned out to be the case for several genes involved in inflorescence meristem identity, including TERMINAL FLOWER1 (TFL1). Contrary to the longstanding belief that AP1 and LFY are both repressors of TFL1, only AP1 repressed TFL1, whereas LFY actually activates this gene. It is not completely clear why LFY would up-regulate a gene that inhibits floral meristem identity. The authors speculate that it might be a way to better define the floral transition, so that it occurs only when AP1 is expressed high enough to overcome TFL1.

Goslin et al.  paper is a nice example of how to combine new experiments and existing datasets in a time with ever growing amounts of genome-wide data, with a surprising outcome. Two transcription factors that were long thought to function similarly in initiation of flower formation suddenly turn out to have a much more intriguing relationship, posing many new questions. When LFY and AP1 act together, the biochemical basis of their interaction is elusive. They might be part of the same regulatory complex, especially since their binding sites have been reported to be adjacent (Winter et al., 2015), but a direct interaction between the two proteins has not been observed. Analysis by targeted proteomics has uncovered AP1 interactors in floral tissue (Smaczniak et al., 2012), but has never been analyzed in earlier tissue in which LFY is expressed. Another question is how LFY and AP1 sometimes work together, and sometimes do not, sometimes activate and other times repress. One possibility is that there might be spatio-temporal differences in expression of interaction partners of LFY and AP1 (see also the previous Flowering Highlight on Spatially resolved floral transcriptome profiling by Aalt-Jan van Dijk). Altogether, there is still a lot to be understood about these two ‘very well known’ regulators!

References

Denay G, Chahtane H, Tichtinsky G, Parcy F. 2017. A flower is born: an update on Arabidopsis floral meristem formation. Current Opinion in Plant Biology 35, 15–22. https://doi.org/10.1016/j.pbi.2016.09.003

Goslin K, Zheng B, Serrano-Mislata A, et al. 2017. Transcription Factor Interplay between LEAFY and APETALA1/CAULIFLOWER during Floral Initiation. Plant Physiology 174, 1097–1109. https://doi.org/10.1104/pp.17.00098

Kaufmann K, Wellmer F, Muiño JM, et al. 2010. Orchestration of floral initiation by APETALA1. Science 328, 85–89.  https://doi.org/10.1126/science.1185244

Ng M, Yanofsky MF. 2001. Activation of the Arabidopsis B class homeotic genes by APETALA1. The Plant Cell 13, 739–753. https://doi.org/10.1105/tpc.13.4.739

Smaczniak C, Immink RGH, Muiño JM, et al. 2012. Characterization of MADS-domain transcription factor complexes in Arabidopsis flower development. Proceedings of the National Academy of Sciences 109, 1560–1565. https://doi.org/10.1073/pnas.1112871109

Wagner D, Sablowski RW, Meyerowitz EM. 1999. Transcriptional activation of APETALA1 by LEAFY. Science 285, 582–584.  https://doi.org/10.1126/science.285.5427.582

Wils CR, Kaufmann K. 2017. Gene-regulatory networks controlling inflorescence and flower development in Arabidopsis thaliana. BBA – Gene Regulatory Mechanisms 1860, 95–105. https://doi.org/10.1016/j.bbagrm.2016.07.014

Winter CM, Yamaguchi N, Wu M-F, Wagner D. 2015. Transcriptional programs regulated by both LEAFY and APETALA1 at the time of flower formation. Physiologia Plantarum 155, 55–73. https://doi.org/10.1111/ppl.12357

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