REVIEW PAPER The AFL subfamily of B3 transcription factors: evolution and function in angiosperm seeds

Pilar Carbonero, Raquel Iglesias-Fernández and Jesús Vicente-Carbajosa Centro de Biotecnología y Genómica de Plantas (UPM-INIA), and E.T.S.I. Agrónomos, Campus de Montegancedo, Universidad Politécnica de Madrid, Pozuelo de Alarcón, 28223-Madrid, Spain

Seed development follows zygotic embryogenesis; during the maturation phase reserves accumulate and desiccation tolerance is acquired. This is tightly regulated at the transcriptional level and the AFL (ABI3/FUS3/LEC2) subfamily of B3 transcription factors (TFs) play a central role. They alter hormone biosynthesis, mainly in regards to abscisic acid and gibberellins, and also regulate the expression of other TFs and/or modulate their downstream activity via protein- protein interactions. This review deals with the origin of AFL TFs, which can be traced back to non-vascular plants such as Physcomitrella patens and achieves foremost expansion in the angiosperms. In green algae, like the unicellu- lar Chlamydomonas reinhardtii or the pluricellular Klebsormidium flaccidum, a single B3 gene and four B3 paralogous genes are annotated, respectively. However, none of them present with the structural features of the AFL subfamily, with the exception of the B3 DNA-binding domain. Phylogenetic analysis groups the AFL TFs into four Major Clusters of Ortologous Genes (MCOGs). The origin and function of these genes is discussed in view of their expression pat- terns and in the context of major regulatory interactions in seeds of monocotyledonous and dicotyledonous species.

Key words: AFL subfamily (ABI3/VP1, FUS3, LEC2), B3 transcription factors, desiccation tolerance, germination, green algae, seed development, vascular plants.

Introduction The seed represents a key adaptation in the evolutionary suc- environmental conditions. The acquisition of desiccation tol- cess and diversification of land plants, mainly due to the des- erance is present both in non-vascular plants, namely bryo- iccation tolerance associated with the entrance of the embryo phytes, as well as in vascular plants. The vegetative tissues of into a quiescent state (Vicente-Carbajosa and Carbonero, non-vascular plants can commonly withstand strong dehy- 2005; Soppe and Bentsink, 2016). As such, seeds facili- dration. In contrast, in vascular plants the seeds tend to be tate plant dispersal and growth resumption under optimal the only organs to resist severe water-deficits for long periods

Abbreviations: A, activation domain; ABI3, ABA-INSENSITIVE 3; AIP2, ABI3-Interacting Protein; AFL, ABI3/VP1, FUS3, LEC2; ARF10, Auxin Response Factor 10; At2S1, 2S albumin (At2S1); AtGA3ox2, Gibberellin3-oxidase2; CHD, chromodomain helicase DNA-binding domain; CRU3, cruciferin; dog1, Delay of Germination 1; DFUS3, Dicot-FUS3; ER, endosperm rupture; FUS3, FUSCA3; GD1, GERMINATION DEFECTIVE 1; H3K27me3, H3 lysine 27 trimethyltranferase; HSI2, High-level expression of Sugar-Inducible gene 2; HSPs, heat shock proteins; LAFL, AFL genes and LEC1; LEA, late embryogenesis abundant; LEC2, LEAFY COTYLEDON 2; MCOGs, Major Clusters of Ortologous Genes; MFUS3, Monocot-FUS3; MW, molecular weight; mya, million of years ago; NLS, nuclear localization signal; PHD, plant homeodomain; PHS, pre-harvest sprouting; PIL5, phytochrome interacting factor-like 5; PRC, Polycomb group Repressive Complex; PKL, PICKLE; SCR, seed coat rupture; SOM, SOMNUS; SSPs, seed storage proteins; TIPs, Tonoplast Intrisic Proteins; TF, transcription factor; TT2, Transparent Testa 2; TTG1, Transparent Testa Glabra1; VAL, VIVIPAROUS1/ABI3-Like; VP1, Viviparous-1; WRI1, WRINKLED1; YUC2/4, flavin monooxygenase-like proteins 2 or 4 of time, with the exception of the desiccation tolerant vegeta- non-vascular green eukaryotes allows tracing back the ori- tive tissues in the polyphyletic group of resurrection plants gins and ancestral function of the AFL network. (Bartels and Salamini, 2001; Farrant and Moore, 2011; Bewley et al., 2013). In the context of the seed, extensive stud- ies have been done on the regulatory networks involved in The AFL genes from Physcomitrella patens seed maturation, desiccation tolerance and germination, with to vascular plants members of the B3 TF family identified as master regulators in these processes. A prominent feature of AFL proteins is the presence of a The Viviparous-1 (VP1) gene of maize (Zea mays) was the B3 DNA-binding domain. Since B3 TF genes have not been first plant gene identified that encodes a B3 type transcrip- found in the yeast Saccharomyces cerevisiae, the nematode tion factor (TF). Maize vp1 mutant seeds are characterized Caenorhabditis elegans, the insect Drosophila melanogaster, by their incapacity to enter dormancy at the end of the matu- the zebrafishDanio rerio, and the mammalians Mus musculus ration phase and by their display of premature features dur- and Homo sapiens, they are considered to be specific to pho- ing the subsequent germination and post-germination events. tosynthetic eukaryotes (Yamasaki et al., 2013). Consequently, This eventually leads to a precocious germination of the seeds sequences of AFL genes were investigated in plant genome while still in the corn cob, referred to as pre-harvest sprout- databases (see Supplementary Table S1 at JXB online), ing (PHS). VP1 and its orthologous genes in other cereals focusing on the following selected species: the green unicel- have been described as key components of the abscisic acid lular alga Chlamydomonas reinhardtii, the pluricellular alga (ABA) signalling pathway during seed maturation. They are Klebsormidium flaccidum, the moss Physcomitrella patens, the associated with the activation of genes encoding seed stor- fern Selaginella moellendorffii, the gymnosperm Picea abies, age proteins (SSPs), late embryogenesis abundant (LEA) the basal angiosperm Amborella trichopoda, the dicotyledon- enzymes, and anthocyanin biosynthesis enzymes, as well as ous angiosperms Arabidopsis thaliana, Solanum lycopersicum, with the repression of post-germination genes for reserve Ricinus communis and Populus trichocarpa, and the mono- mobilization i.e. α-amylases, proteases, etc (McCarty et al., cotyledonous angiosperms Oryza sativa, Brachypodium dis- 1991; Suzuki et al., 2003; Gubler et al., 2005). The control tachyon and Hordeum vulgare. The AFL genes from Zea mays of antagonistic gene expression programs upon seed matura- are not included here, since excellent articles on these genes tion and germination has been likewise reported for barley have been published recently (Suzuki and McCarty, 2008; (Hordeum vulgare) HvVP1 and supports the central role of Grimault et al., 2015). VP1 as a gene expression switch at key stages of seed develop- A phylogenetic dendrogram has been constructed with ment (Abraham et al., 2016). the forty-four predicted, non-redundant B3 AFL deduced The orthologous gene to VP1 in Arabidopsis thaliana is proteins, which have molecular weights (MW) between 25.6 ABA-INSENSITIVE 3 (ABI3) (Suzuki et al., 2001). Similarly and 79.5 KDa, and isoelectric points (Ip) from 4.73–8.51. to monocot vp1 mutants, abi3 seeds are unable to complete The CLUSTAL Omega tool (McWilliam et al., 2013) and maturation and do not acquire a quiescent state. ABI3 and MEGA6 software (Tamura et al., 2013) were used to con- VP1 belong to the AFL subfamily of B3 transcription fac- struct the dendrogram, using the maximum likelihood tors, which is named after three members within A. thali- method, a bootstrap analysis with 1,000 replicates, complete ana: ABA Insensitive 3 (ABI3: At3g24650), FUSCA3 deletion and the Jones Taylor Thornton matrix, as settings. (FUS3: At3g26790) and LEAFY COTYLEDON 2 (LEC2: The MEME programme software, version 4.11.2 (Bailey et At1g28300). These three members together with LEC1, an al., 2009) was used to identify conserved motives (Fig. 1; ortholog of the NF-YB subunit of the CCAAT-binding Supplementary Table S1, Table S2). The tree topology indi- TF, form a TF regulatory network that controls seed devel- cates that the majority of AFL proteins could be grouped opment from embryogenesis to maturation and desiccation into four Major Clusters of Orthologous Genes (MCOGs): (Vicente-Carbajosa and Carbonero, 2005; Santos-Mendoza ABI3/VP1, LEC2, Monocot-FUS3 (MFUS3) and Dicot- et al., 2008; Suzuki and McCarty, 2008, Fatihi et al., 2016). FUS3 (DFUS3). PaAFL1 and PaAFL2 of P. abies and ABI3 has been also associated with other processes such as AmtrAFL1 of A. trichopoda cannot be included in any of embryo degreening, seedling growth, plastid development, these clusters. The B3 TFs of the green algae C. reinhardtii axillary meristem dormancy and onset of flowering (Delmas and K. flaccidum are used to root the tree. A single B3 gene, et al., 2013, and references therein). ChrB3I, has been found in the Chlamydomonas genome and Notably, the developmental transition from seed to seed- four well-annotated B3 genes in the draft genome draft of ling is controlled by AFL members and their antagonistic Klebsormidium, KfB3.1–4 (Hori et al., 2014). These members VAL (VIVIPAROUS1/ABI3-Like) repressors, VAL1/2/3, are included in the green algae cluster (Fig. 1), suggesting which are implicated in shutting down the AFL network dur- that the B3 TF gene family evolved from the Chlorophyta ing the vegetative phases of development (Suzuki et al., 2007; division 1200–725 million of years ago (mya), exclusive Wang and Perry, 2013; Jia et al., 2014; Schneider et al., 2016). to photosynthetic organisms (Finet et al., 2010). These Here, we present an updated overview of the AFL net- data indicate that B3 genes originated from a single com- work and the connections to other regulatory components mon ancestor in C. reinhardtii, which after gene duplication that influence its effect in the seeds of monocot and dicot and subsequent divergent evolution, gave rise to the differ- species. The identification of homologous AFL members in ent subfamilies, including the AFL B3 subfamily. The AFL Fig. 1. Phylogenetic dendrogram with deduced protein sequences of the AFL (ABI3/FUS3/LEC1) subfamily of B3 transcription factors from Physcomitrella patens (Pt), Selaginella moellendorffii (Sm), Picea abies (Pa), Amborella trichopoda (AmTr), Arabidopsis thaliana (At), Solanum lycopersicum (Sl), Ricinus communis (Rc), Populus trichocarpa (Pt), Oryza sativa (Os), Brachypodium distachyon (Bd) and Hordeum vulgare (Hv). The B3 proteins from Chlamydomonas reinhardtii (Chr) and Klebsormidium flaccidum (Kf) are also included. Bootstrapping values with values higher than 25% are indicated in the branches. Distribution of the conserved motifs among the deduced protein sequences, identified via MEME analysis, are represented by numbers in coloured boxes. A, B1, B2, and B3 correspond to conserved domains, as described by Nakamura and Toyama (2001). Exons are represented with arrows and introns with black lines. Distribution of the B3 DNA-binding domain is depicted in red. The number of exons, the gene size (bp) and the chromosome where the corresponding locus appears (ChrLoc) are indicated, as well as the total number of chromosomes in the species (Chrt). members proper appear for the first time in the bryophytes FUS3 (PtFUS3.1–2) and two LEC2 (PtLEC2.1–2) genes can and are represented in P. patens by three AFL genes closely be annotated. While in the monocotyledonous H. vulgare and similar to ABI3/VP1 (PpVP1-A, PpVP1-B, PpVP1-C). In B. distachyon, the AFL subfamily is represented by four mem- the fern S. moellendorffii, the genes SmVP1.1–3 have been bers: one VP1 gene (HvVP1 and BdVP1, respectively), one also identified as clear members of the AFL family. While FUS3 gene (HvVP1 and BdFUS3, respectively) and two LEC2 VP1 orthologs can be identified inP. patens and S. moellen- genes (HvLEC2.1–2, BdLEC2.1–2, respectively). In O. sativa dorffii; FUS3 and LEC2 orthologs are not found in these besides VP1 (OsVP1) and FUS3 (OsFUS3), three LEC2 genes species (Khandelwal et al., 2010). This indicates that the (OsLEC2.1, OsLEC2.1´, OsLEC2.2) are found. With the AFL subfamily and specifically theABI3/VP1 orthologs exception of R. communis (RcLEC2), all the other dicot and arose over the course of evolution with the appearance of monocot species explored have two or more LEC2 proteins. terrestrial plants. The topology of the tree is supported by the bootstrap In A. thaliana, as in many of the Brassicaceae sequenced values, by the exon-intron structure and by the MEME so far (Elahi et al., 2015; Willing et al., 2015), and in the cas- analysis (Fig. 1; Supplementary Table S2). According to the tor bean (R. communis), the AFL subfamily is represented MEME analysis, protein sequences in the ABI3/VP1 clus- by the classical three members: AtABI3, AtFUS3, AtLEC2, ter share motifs 1, 2, 3, 4, 5, 6 and 7. The B3 domain con- and RcABI3, RcFUS3, RcLEC2, respectively. However the S. taining the DNA-binding sequence spans motifs 1–4 and 7, lycopersicum genome contains two ABI3 genes (SlABI3.1–2) motif 5 forms the B1 domain and motif 6 constitutes the B2 and one FUS3 gene (SlFUS3), while a LEC2 ortholog could domain that contains the nuclear localization signal (NLS) not be detected. In P. trichocrapa, one ABI3 (PtABI3), two (Supplementary Table S2). The VP1 protein A, B1, B2 and B3 Fig. 2. Proposed model of the transcriptional regulation of seed genes mediated by ABI3/LEC2/FUS3 transcription factors during embryogenesis, maturation, dormancy and germination of Arabidopsis thaliana (A) and Hordeum vulgare (B) seeds. AIP, ABI3 Interacting Protein; Amy6.4, Amylase 6.4; BPBF, a DOF TF; BLZ2/1, bZIP TFs; GAMYB, a MYB-R2R3 TF; HSPs, Heat Shock Proteins; Hor2, Hordein 2; LEAs, Late Embryogenesis Abundant proteins; MR1, a MYB-R1 TF; PA, proanthocyanidins; SAD, a DOF TF; SSPs, Seed Storage Proteins; TIPs, Tonoplast Intrisic Proteins; TTG1, Transparent Testa Glabra 1; TT2, Transparent Testa 2; WRI1, WRINKLED1; YUC2/4, flavin monooxygenase-like proteins 2 or 4. domains are well-defined in the angiosperms explored (Fig. 1), The LEC2 cluster comprises protein sequences that share both in the dicotyledonous species and the Gramineae species motifs 1, 2, 3, and 4, with the exception of P. thicocarpa such as O. sativa, B. distachyon and H. vulgare. The B1, B2 PtLEC2.2 that lacks motif 3. Protein sequences included and B3 domains are highly conserved among the ABI3/VP1 in the Monocot-LEC2 subcluster share motifs 1, 2, 3, 4, proteins, including those from P. patens (Marella et al., 2006). 7, and 12. However O. sativa OsLEC2.2 and A. thricopoda In A. thaliana, the B2 domain together with N-terminal acti- AmtrLEC2 do not have motif 12 and O. sativa OsLEC2.1 is vation (A) domain of ABI3 have been described as critical for devoid of motif 7. full transactivation of genes encoding LEA and SSP proteins, Protein sequences in the Monocot-FUS3 cluster share namely AtEm1, AtEm6, At2S1 and At2S2 (Bies-Etheve et al., motifs 1, 2 3, and 4 as well as the activation domain, motif 8, 1999; Mönke et al., 2004), while its B1 domain is necessary which is located within the C-terminal region and not the for the interaction with the bZIP TF ABI5 (Nakamura et al., N-terminal region as occurs for the ABI3/VP1 genes. This 2001). In the Arabidopsis mutant abi3-8, substitution of the probably arose due to exon shuffling. The Dicot-FUS3 cluster leucine (L) residue at position 298 with phenylalanine (F) in shares motifs 1, 2, and 4. All members in the Monocot-FUS3 the B1 domain affects the capacity of ABI3 to interact with and Dicot-FUS3 clusters have a predictive/canonical NLS ABI5 (Nambara et al., 2002). In rice, TRAB1, the ortholog (see Supplementary Table S1 at JXB online). of Arabidopsis ABI5, interacts with OsVP1 and mediates The AFL gene structure is heterogeneous, comprising ABA induced transcription upon seed maturation and ger- genomic structures with 4–11 exons. However, the ABI3/VP1 mination (Hobo et al., 1999). ABI3, FUS3 and LEC2 bind cluster contains genes with a more homogeneous organiza- specifically to the RYcis -elements (5´-CATGCATG-3´) that tion: 5–7 exons and 3–5 introns with conserved positions. The are present in many seed-specific gene promoters from spe- ABI3/VP1 first exon is longer than the others, and the B3 cies as distantly related as A. thaliana, Ginkgo biloba, Zamia domain is placed between exons 2 to 5, with the exception of furfuracea and Vicia faba (Schallau et al., 2008). PpVP1-B and PpVP1-A, where it is included between exons 1 to 2 and RcABI3 and PtABI3, where the B3 domain spans Moreno-Risueño et al., 2008; Tiedemann et al., 2008). During exons 3 and 6. In the Monocot-LEC2 subcluster, genes have embryogenesis, FUS3 and LEC2 transcriptionally repress the 8–11 exons, and the position of the introns is conserved. Their Gibberellin3-oxidase2 (AtGA3ox2) gene that encodes a criti- B3 domain spans exons 5 to 8, however in AmtrLEC2 the B3 cal enzyme for the biosynthesis of active gibberellins (GA) domain is placed between exons 4 to 7. The FUS3 genes have (Curaba et al., 2004; Baud et al., 2016). LEC2 mediates 5–7 exons and the position of the B3 domain varies between auxin responses by inducing the expression of genes YUC2 exons 2 to 5 or 3 to 6. HvFUS3, BdFUS3, OsFUS3, PtFUS3.1, and YUC4, which encode YUC flavin monooxygenases, thus PtFUS3.2 and RcFUS3 conserve a long first intron (Fig. 1). promoting early cell differentiation in developing embryos To determine if the function of the AFL genes is con- (Stone et al., 2008). served in the seeds of monocotyledonous and dicotyledon- ous species, the expression patterns of the AFL genes from Protein and lipid storage accumulation during seed A. thaliana, H. vulgare and O. sativa were explored using the maturation Bio-Analytic Resource for plant biology (http://bar.utoronto. ca/; Supplementary Fig. S1). Expression profiles forABI3/ In A. thaliana, LEC2 is also important in the embryo upon VP1 in the three species analyzed are similar and the tran- maturation as it induces ABI3 and FUS3 transcription, scripts accumulate through seed development, peaking at the which encode TFs necessary for the accumulation of SSPs maturation stage. While HvFUS3 and AtFUS3 gene expres- and lipids (Braybrook and Harada, 2008). Mutations in sion is abundant at middle to late stages of seed develop- LEC2 diminish seed oil and protein content (Ángeles-Núñez ment, OsFUS3 is almost undetectable. AtLEC2, HvLEC2.1 and Tiessen, 2011). The ectopic expression of the ABI3 and and OsLEC2.2 are more abundant upon early to middle FUS3 genes under the control of a constitutive promoter seed developmental stages, while HvLEC2.2, OsLEC2.1 and (35S) stimulates the transcription of 2S albumin (At2S1) and OsLEC2.1´ genes are faintly expressed during this period. cruciferin (CRU3) genes in vegetative tissues. Moreover, co- However, OsLEC2.1´ transcripts are highly expressed in expression of ABI3 with AtbZIP10 or AtbZIP25 results in a young and mature leaves. Since new gene functions are estab- remarkable increase in the activation capacity of these bZIP lished by gene duplication events that promote the appearance TFs over the At2S1 gene. The RY cis-element that is bound of gain of function (neofunctionalization), loss of function by ABI3, FUS3 and LEC2 is overrepresented in the promot- (pseudogenization), and redundant function (subfunctionali- ers of seed maturation genes and the G-box like sequences, zation) genes, the emergence of HvLEC2.2, OsLEC2.1 and which are bound by bZIP TFs, are located in its vicinity - OsLEC2.1´ over the course of evolution could reflect neo- both are essential for full transcriptional activation (Reidt functionalization and pseudogenization phenomena (Lynch et al., 2000; Lara et al., 2003; Kagaya et al., 2005; Braybrook and Force, 2000). et al., 2006; Moreno-Risueño et al., 2008; Guerriero et al., 2009; Wang and Perry, 2013; Roscoe et al., 2015; Baud et al., 2016). Similarly, in Brassica napus, the BnABI3 protein tran- AFL TFs in Eudicotyledonous seeds scriptionally activates the napin napA promoter through a In A. thaliana, AFL TFs are key regulators of seed embryo- RY/G complex, containing RY repeat elements and a G-box genesis, maturation, the dormancy-germination transition, (Ezcurra et al., 2000). post-germination storage reserve mobilization and ABA sig- In A. thaliana, the gene encoding the TF Transparent nalling. ABI3 and FUS3 expression is significantly reduced Testa Glabra1 (TTG1), which is involved in suppressing the in the lec2 mutant, and the normal phenotype is rescued accumulation of seed oil reserves, is negatively regulated by when ABI3 or FUS3 are overexpressed in the mutant back- FUS3 during embryogenesis and seed maturation (Tsuchiya ground. ABI3 and FUS3 positively control the expression of et al., 2004; Chen et al., 2015). However, Transparent Testa each other, indicating that ABI3, FUS3 and LEC2 config- 2 (TT2), an R2R3 MYB TF, regulates proanthocyanidins ure a hierarchical and redundant genetic and molecular net- biosynthesis in the Arabidopsis seed coat, binding directly work. This occurs not only in Arabidopsis but also in other to the FUS3 gene promoter (Wang et al., 2014). A synergis- dicotyledoneous seeds (Vicente-Carbajosa and Carbonero, tic interaction of ABI3, LEC2 and LEC1 in the activation 2005; To et al., 2006; Santos-Mendoza et al., 2008; Verdier of the OLEOSIN1 gene promoter in the developing embryo and Thompson, 2008; Baud et al., 2016; Fatihi et al., 2016). has been described (Baud et al., 2016). LEC2 transcription- The following is a concise description of the major impact ally regulates the expression of WRINKLED1, an AP2 TF of of AFL activity at different stages of seed development, par- the utmost importance during fatty acid biosynthesis upon ticularly regarding its connection to other known regulators. seed maturation (Baud et al., 2007). These genetic interac- tions could be conserved in oilseed species, since constitu- Zygotic embryogenesis and morphogenesis tive overexpression in A. thaliana of the LEC2 orthologous gene from R. communis promotes the transcription of FUS3, In A. thaliana, LEC2 and FUS3 have central roles in seed ABI3, WRINKLED1 and OLEOSIN genes in itsleaves (Kim embryogenesis and morphogenesis, their transcripts being HU et al., 2013). abundantly expressed at these stages. Mutations at these loci The chromatin remodelling factor PICKLE (PKL), a typically result in the appearance of trichomes and antho- chromodomain helicase DNA-binding domain (CHD) pro- cyanin accumulation in cotyledons (Gazzarrini et al., 2004; tein, represses LEC1, LEC2 and FUS3 expression during post-embryonic growth (Dean-Rider et al., 2003). The AFL Germination sensu stricto and reserve mobilization genes and LEC1 (LAFL) are repressed epigenetically by the E3 H2A monoubiquitin ligase activity of Polycomb group A. thaliana seeds germinate in two sequential steps, where Repressive Complex (PRC) 1 and by the histone H3 lysine seed coat rupture (SCR) is followed by the endosperm rup- 27 trimethyltranferase (H3K27me3) activity of PRC2. It has ture (ER), thus allowing radicle emergence i.e. germination been proposed that VAL proteins and AtBMI1-mediated sensu stricto; reserve mobilization is considered a post-ger- H2Aub marks initially repress seed maturation genes, and mination event (Pritchard et al., 2002; Iglesias-Fernández this repression is maintained by H3K27me3 activity medi- et al., 2011; Iglesias-Fernández et al., 2013). While DELLA ated by PRC2 and PRC1 (Berger et al., 2011; Yang et al., factors, which are negative regulators of GA signalling, affect 2013; Merini and Calonje, 2015). The High-level expression both steps, ER is blocked by ABA through the transcrip- of Sugar-Inducible gene 2 (HSI2)/VAL1 plant homeodomain tion factors ABI3 and ABI5 (Piskurewicz et al., 2009). ABI3 (PHD)-like domain has been described as responsible for this interacting with ABI5 and DELLAs, or with the bHLH type H3K27me3 repressing mechanism (Veerappan et al., 2014). TF PIL5 (phytochrome interacting factor-like 5), activates VAL1 target genes belong to a subset of seed maturation the expression of SOMNUS (SOM) which encodes a C3H- genes and the 39% of the FUS3 regulon is derepressed in the type zinc finger protein that regulates the expression of genes val1 mutant (Schneider et al., 2016). involved in the synthesis of ABA and in the catabolism of GA, thus inhibiting seed germination (Park et al., 2011; Lim et al., 2013). In S. lycopersicum, SlABI3 stimulates transcript Acquisition of desiccation tolerance and embryo accumulation of SlABI5 and confers hypersensitivity to ABA growth arrest during seed germination (Bassel et al., 2006; Gao et al., 2013). In addition to their role in embryo formation and seed It has been also proposed to have a seed germination repres- maturation, AFL genes are master regulators in the acqui- sor role for FUS3 at high temperatures (Chiu et al., 2012). sition of desiccation tolerance and in repressing the tran- A genetic interaction between FUS3, ABI5 and the ERF/ sition from embryonic to vegetative development. During AP2 TF ABI4, whichregulates lipid mobilization, has been late seed maturation, FUS3 and ABI3 control the expres- reported to be involved in controlling seed pigment accumu- sion of dehydration related genes, such as those encoding lation, viviparism and reserve mobilization during post-ger- LEAs and heat shock proteins (HSPs) (Kotak et al., 2007). mination (Brocard-Gifford et al., 2003; Penfield et al., 2006). The A. thaliana abi3 and fus3 mutants produce seeds with ABI4 also plays a role in seed dormancy through the regula- a low degree of dormancy and desiccation tolerance. In tion of ABA and GA homeostasis (Shu et al., 2013). this respect, the interaction of AFL members with other ABI3 gene expression is activated by the homeodomain regulators is critical, as demonstrated in numerous reports TFs BLH1 and KNAT3 (Kim D et al., 2013). The RAV1 TF (Graeber et al., 2010; Dekkers et al., 2016). For instance, binds to and represses ABI3 expression upon seed imbibi- the double mutant abi3 and dog1 (Delay of Germination 1) tion (Feng et al., 2014). Interestingly, BEST1/TPL/HDA19, (Bentsink et al., 2006) has non-dormant green seeds that an epigenetic repressor complex, provokes histone deacety- are highly ABA insensitive, thus enhancing the phenotype lation of ABI3 chromatin in response to brassinosteroids, of the abi3-1 single mutant. It has also been reported that thus attenuating the ABA-mediated arrest of early seedling auxins enhance seed dormancy by inducing ABI3 activation development (Ryu et al., 2014). ABI3 is degraded during seed mediated by the ARF10 (Auxin Response Factor 10) TF germination in a process mediated by the 26S proteasome (Liu et al., 2013). The WRKY41 TF is also an important pathway, and is polyubiquitinated by the ABI3-Interacting regulator of ABI3 expression during the establishment of Protein (AIP2) that contains a RING motif (Zhang et al., seed dormancy (Ding et al., 2014). Interestingly, ABI3 tran- 2005). AIPs interact with the C-terminal region of the ABI3 scriptionally controls the expression of two seed-specific protein i.e. the B2 and B3 domains. They therefore function vacuolar aquaporin genes, TIP3-1 and TIP3-2, involved in the transcriptional network of regulators that control seed in maintaining seed longevity (Mao and Sun, 2015). In a development (Zeng et al., 2013). recent report, González-Morales et al. (2016), identified key regulators of desiccation tolerance associated with the AFL AFL TFs in the monocotyledonous seeds network and demonstrated a rescue tolerance phenotype by overexpressing the corresponding genes in the abi3 mutant. Although the AFL genes have been identified in several cereal Similarly, the importance of interactions has been reported species, such as maize, wheat, oat, and barley (Nakamura for FUS3 in the establishment of seed dormancy, where it and Toyama, 2001), and their orthologs of ABI3 and FUS3 prevents seed germination through its interaction with the have essentially similar roles as in dicots, no clear function SnRK1 kinase AKIN10 (Tiedemann et al., 2008; Tsai and has yet been reported for LEC2 in monocots regarding Gazzarrini, 2012). In P. trichocarpa, PtABI3 not only plays zygotic embryogenesis or embryo morphogenesis. Results a role in seed maturation and dormancy but its expression from a recent study based exclusively on protein sequence is also needed for growth cessation of buds during winter data (Devic and Roscoe, 2016) propose a different classifica- (Rohde et al., 2002). In Populus tremula, PtFUS3.2 is ubiq- tion of AFL members in monocotyledonous species, where uitously expressed, with high levels of transcripts in buds, LEC2 homologs could not be clearly identified. This sug- wood and roots (Supplementary Fig. S2). gests that a different regulatory network, compared to dicots, could control seed development in monocots (Sreenivasulu (McCarty et al., 1991; Gubler et al., 2005). In barley, the tran- and Wobus, 2013). Further functional investigations will clar- scriptional repressor activity of HvVP1 on the α-amylase gene ify the true orthology relationships of AFL members between Amy6.4, activated by GAMYB, has been also demonstrated monocot and dicot species. during post-germination (Abraham et al., 2016). However in Sorghum bicolour, SbVP1 gene expression could not be cor- Storage accumulation and acquisition of desiccation related with PHS (Carrari et al., 2003). In T. aestivum, sev- tolerance during seed maturation eral alternatively spliced TaVP1 transcripts are found. Since most of them do not encode the full length protein, it has The endosperm is the major tissue where monocot seeds been proposed that these TaVP1 misspliced forms contribute accumulate storage compounds, such as proteins and starch, to the high susceptibility of modern wheat cultivars to PHS during the maturation phase. This process is mainly regulated (McKibbin et al., 2002; Yang et al., 2007). No alternative at the transcriptional level, where the AFL TFs are actively splicing forms in barley or maize have been reported. involved. In H. vulgare, HvVP1 and HvFUS3 transcripts are abundant in the endosperm and in the embryo during seed development. Moreover, HvVP1 and HvFUS3 TFs, through AFL genes beyond the seed: desiccation binding to RY cis-elements and interacting with other TFs tolerance in vegetative tissues such as the Opaque2-like bZIP, BLZ1/BLZ2, and the MYB- R2R3, GAMYB, control the expression of seed matura- Desiccation tolerance is the ability of a living organism to tion specific genes likeHor2 , which encodes a B-hordein. survive strong dehydration. This is mediated by the ABA Interestingly, the loss-of-function mutant fus3 in A. thali- hormone and by the ABI3 gene expression network (Alpert, ana can be complemented by the barley HvFUS3 gene, thus 2005; Khandelwal et al., 2010). Desiccation tolerance is a restoring normal expression of the albumin At2S3 gene key feature of plants that have conquered terrestrial habitats (Vicente-Carbajosa et al., 1998; Gubler et al., 1999; Oñate and can already be seen in the bryophytes (Khandelwal et al., et al., 1999; Díaz et al., 2002; Moreno-Risueño et al., 2008; 2010). In angiosperms, the expression of AFL members and Abraham et al., 2016). The wheat ortholog, TaVP1, activates particularly of ABI3/VP1 is essentially restricted to the seed. the expression of the LEA protein encoding gene, Em, in the However, remarkably in maize, VP1 is not only expressed in caryopsis Triticum aestivum L. Maize VP1 controls the C1 the embryo and aleurone cells of developing seeds, but is also gene that encodes a MYB-like TF and is a key regulator of induced in phloem cells of leaves and stems upon drought the anthocyanin biosynthesis pathway in maize (Paz-Ares stress (Cao et al., 2007). Similarly, Bedi et al. (2016) recently et al., 1986; Hattori et al., 1992). reported the importance of ABI3 for the expression of dehy- Although the maize AFL members have not been consid- dration inducible genes in vegetative tissues. By contrast, ered in the present phylogenetic analyses, functional infor- ancestral members of the AFL network, such as PpABI3A- mation about fiveZmAFL genes - ZmAFL2, orthologous B-C in P. patens, are constitutively expressed in vegetative tis- to FUS3, ZmAFL3/ZmVp1, and three orthologs to LEC2 sues. PpABI3A partially restores the ABA sensitivity of the ZmAFL4, ZmAFL5 and ZmAFL6 - has been recently reported A. thaliana abi3-6 mutant during seed germination and its (Grimault et al., 2015). ZmAFL4 expression is restricted to vegetative tissues overcome desiccation following a signalling the pollen and to the developing endosperm, and ZmAFL4 pathway similar to that found in seeds, where ABA and ABI3 loss-of-function mutants have reduced starch content in the activate downstream genes such as those encoding LEA pro- endosperm upon seed maturation. The transient co-expres- teins (Marella et al., 2006). The PpABI3A TF transactivates sion of ZmAFL4 and ZmAFL3/ZmVp1 in P. patens transcrip- the wheat Em gene promoter in P. patens protonemal tissue. tionally activates a maize oleosin promoter (Grimault et al., Altogether, these data support conserved transcriptional reg- 2015). In rice, the B3 transcription factor GERMINATION ulation by ABI3/VP1 through plant evolution, from mosses DEFECTIVE 1 (GD1), represses seed germination and seed- to vascular plants (Sakata et al., 2010; Yotsui et al., 2013). ling growth by regulating GA and carbohydrate metabolism. Furthermore these data suggest that an ancestral AFL net- Moreover, GD1 activates the expression of the gene OsLFL1 work appeared to control dehydration tolerance mechanisms (OsFUS3 in our phylogenetic analysis) by directly binding to associated with the ability of plants to conquer land habitats. a RY-element present in its promoter, thus causing the upreg- The observation that germin genes, expressed in fern spores, ulation of the expression of a subset of seed maturation genes contain RY-elements in their promoters, and can be regulated (Guo et al., 2013). by AFL members (Shutov et al., 1999) is also in agreement with the proposed ancient function of the AFL network. Germination and post-germination Elucidation of the molecular mechanisms by which the expression of this network is restricted to seed tissues in sper- VP1 is the major AFL TF associated with the repression matophytes is an open and stimulating question. Interestingly, of genes encoding hydrolytic enzymes i.e. α-amylase and it has been reported that ABI3/VP1 does not mediate the proteases, involved in the storage mobilization of cereal acquired tolerance to dehydration of the polyphyletic group seeds during post-germination. This repression was first of resurrection plants, such as Craterostigma plantagineum, described in Z. mays and later in T. aestivum, Avena fatua and suggesting that an ABI3 independent pathway leads this pro- O. sativa, and has been associated with the avoidance of PHS cess (Chandler and Bartels, 1997; Farrant and Moore, 2011). In this respect, the work by González-Morales et al. (2016) Bentsink L, Jowett J, Hanhart CJ, Koornneef M. 2006. Cloning of DOG1, a quantitative trait locus controlling seed dormancy in Arabidopsis. indicates that different mechanisms leading to desiccation tol- Proceedings of the National Academy of Sciences of the United States of erance in vegetative tissues could arise in those plants by the America 103, 17042‒17047. selective induction of AFL subnetworks. This would avoid Berger N, Dubreucq B, Roudier F, Dubos C, Lepiniec L. 2011. the detrimental effects derived from the unrelated functions Transcriptional regulation of Arabidopsis LEAFY COTYLEDON2 involves RLE, a cis-element that regulates trimethylation of histone H3 at lysine-27. of ABI3/VP1 in photosynthesis and other important physi- The Plant Cell 23, 4065–4078. ological processes. Bewley JD, Bradford KJ, Hilhorst HWM, Nonogaki H. 2013. Seeds: physiology of development, germination and dormancy. New York, NY: Springer. 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Expression patterns upon seed Sciences of the United States of America 103, 3468‒3473. development of the AFL genes of Arabidopsis thaliana (A), Brocard-Gifford IM, Lynch TJ, Finkelstein RR. 2003. Regulatory networks Hordeum vulgare (B) and Oryza sativa (C). in seeds integrating developmental, abscisic acid, sugar, and light Supplementary Fig. S2. Expression patterns of AFL genes signaling. Plant Physiology 131, 78–92.1 Cao X, Costa LM, Biderre-Petit C, Kbhaya B, Dey N, Perez P, McCarty in the vegetative and reproductive organs of Populus tremula. DR, Gutierrez-Marcos JF, Becraft PW. 2007. Abscisic acid and stress signals induce Viviparous1 expression in seed and vegetative tissues of maize. Plant Physiology 143, 720–731. Acknowledgments Carrari F, Benech-Arnold R, Osuna-Fernandez R, Hopp E, Sanchez R, Iusem N, Lljavetzky D. 2003. Genetic mapping of the Sorghum bicolor vp1 We thank Prof. Ángel J. Matilla for critical review of the manuscript. 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