The characterization of the LEAFY COTYLEDON 2 activation domains reveals its conserved dual mode of action in flowering plants

Abstract

Seed development in Arabidopsis thaliana is largely controlled by a set of transcription factors (TFs) called LAFL, including LEAFY COTYLEDON 2 (LEC2). In this study, we investigated the structure/function relationships of the protein LEC2 outside the well-described B3 DNA-binding domain. The results presented here unveil the presence of transcription activation domains (ADs) within the unstructured ends of the protein that are conserved in eudicots. Expression in both yeast and moss protoplasts of deleted and mutated versions of LEC2 confirmed the transcriptional activity of these ADs. Surprisingly, the expression of LEC2 variants lacking their ADs restored a wild-type seed phenotype in lec2 mutant, showing that these ADs are not essential for LEC2 function in seed development. Moreover, ZmAFL2/ZmABI19, a maize B3 factor related to LEC2 but deprived of N-ter AD, can also complement lec2 seed phenotype and induce abnormal vegetative development when overexpressed in Arabidopsis, supporting this observation. This work suggests that LEC2 can act both as a classical transcriptional activator or without transactivation activity, probably through its interaction with the pioneer factor LEC1. Taken together, the results provide important insights into the function of the LAFL master regulators during seed development, from cell differentiation to storage accumulation in seed.

Keywords: LAFL; LEC2; activation domain; epigenetic control; seed; transcriptional regulation.

Figure 1

Transcriptional ADs are predicted within N‐ and C‐ter parts of AtLEC2 and appear to be highly conserved. (A) The blue curve represents the probability of the presence of a transcriptional AD, obtained with ADpred (Erijman et al., 2020), on the AtLEC2 sequence, represented as the x‐axis. (B) The red curve represents the AD prediction of 60 AtLEC2 orthologous genes. The prediction has been averaged and normalized on the AtLEC2 sequence. The round and triangular landmarks correspond to the reference points in the B2 and at the end of the B3 domain respectively, used to normalize the predictions.

Figure 2

The transactivation domains predicted by ADpred are functional in yeast. Left panel: schematic representation of different variants of LEC2 cloned in pDEST32. Right panel: transactivation assay in yeast. SD–leu allows the selection of the positive clones, SD–leu–his allows the selection of clones with a histidine autotrophy reflecting the transcriptional activity. (A) Different deletions of LEC2 have been tested according to the AD predictions. (B) Putative ADs have been synthetically mutagenized and the whole proteins as well as different fragments have been tested. The stars represent mutations in AD sequences.

Figure 3

The transcriptional activity of LEC2 is strongly affected when the transcriptional ADs are deleted or mutated. (A) Schematic representation of LEC2 variants in relation to ADs predicted by ADpred (Erijman et al., 2020). The predicted activation domains have been (i) deleted or (ii) synthetically mutated according to the predictions. (iii) ZmAFL2, a B3‐containing Zea mays TF. The stars represent mutations in AD sequences. (B) Transcriptional activity of different LEC2 variants or ZmABI19/ZmAFL2 on proOLE1::GFP measured in moss protoplasts. Data normalized to the transcriptional activity of AtLEC2 on proOLE1::GFP. The standard error is represented. Significant groups, indicated by lowercase letters, were obtained using a Kruskal–Wallis test with risk α = 0.05, followed by a post hoc Dunn's test to adjust the P‐values with the Benjamini–Hochberg (BH) correction.

Figure 4

LEC2 with no AD as well as ZmAFL2/ZmABI19 can restore AtLEC2 functions. (A) Complementation of lec2‐1 mutant with different versions of LEC2 and ZmAFL2 under proLEC2 control. (B) Overexpression of AtLEC2 and ZmAFL2 leads to an abnormal vegetative development of the plantlets. Scale bars: 200 μm.

Figure 5

LEC2 is proposed to have a dual function as a pioneer factor and as a transcription factor. As a pioneer factor (A) LEC2 function would not require its ADs. In a complex with LEC1, it would bind to its targets and induce chromatin remodeling, making early development‐related genes accessible. This pioneer activity would induce a change in cell fate, leading to the expression of ABI3 and FUS3, whose expression controls seed maturation. As a TF (B), LEC2 would induce the expression of seed maturation‐related genes in conjunction with ABI3 and FUS3.