Molecular Plant Review Article
Growth-Regulating Factors (GRFs): A Small Transcription Factor Family with Important Functions in Plant Biology Mohammad Amin Omidbakhshfard1,2, Sebastian Proost1,2, Ushio Fujikura1 and Bernd Mueller-Roeber1,2,* 1
Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Straße 24–25, Haus 20, 14476 Potsdam-Golm, Germany
2
Max-Planck Institute of Molecular Plant Physiology, Am Mu¨hlenberg 1, 14476 Potsdam-Golm, Germany
*Correspondence: Bernd Mueller-Roeber (
[email protected]) http://dx.doi.org/10.1016/j.molp.2015.01.013
ABSTRACT Growth-regulating factors (GRFs) are plant-specific transcription factors that were originally identified for their roles in stem and leaf development, but recent studies highlight them to be similarly important for other central developmental processes including flower and seed formation, root development, and the coordination of growth processes under adverse environmental conditions. The expression of several GRFs is controlled by microRNA miR396, and the GRF-miRNA396 regulatory module appears to be central to several of these processes. In addition, transcription factors upstream of GRFs and miR396 have been discovered, and gradually downstream target genes of GRFs are being unraveled. Here, we review the current knowledge of the biological functions performed by GRFs and survey available molecular data to illustrate how they exert their roles at the cellular level. Key words: abiotic stress, chromatin remodeling, flower development, growth regulation, leaf development, miRNA Omidbakhshfard M.A., Proost S., Fujikura U., and Mueller-Roeber B. (2015). Growth-Regulating Factors (GRFs): A Small Transcription Factor Family with Important Functions in Plant Biology. Mol. Plant. 8, 998–1010.
INTRODUCTION Transcription factors (TFs) are master regulators of gene expression, and it is estimated that around 4%–7% of the eukaryotic genes encode for TFs; generally, the number of TFs is correlated with the complexity of the organism (Riechmann et al., 2000; Levine and Tjian, 2003; Hughes, 2011). The growth-regulating factor (GRF) family is a small, plant-specific TF family, of which the first member was identified 15 years ago (Van der Knaap et al., 2000). Although in initial studies a function of GRFs was only identified in leaf and stem development (Van der Knaap et al., 2000; Kim et al., 2003; Horiguchi et al., 2005; Kim and Lee, 2006), recent studies have uncovered the functions of GRFs in other aspects of plant biology such as flowering, seed and root development, the control of growth under stress conditions, and the regulation of plant longevity (Hewezi et al., 2012; Kim et al., 2012; Bao et al., 2014; Debernardi et al., 2014; Liang et al., 2014; Liu et al., 2014a; Pajoro et al., 2014). Biochemically, GRFs form a complex with the so-called GRF-interacting factors (GIFs), a group of transcriptional coactivators (Kim and Kende, 2004; Lee et al., 2009; Lee et al., 2014). At the transcript level GRFs are often regulated 998
Molecular Plant 8, 998–1010, July 2015 ª The Author 2015.
by microRNA miR396, and examples are given throughout the review. The GRF family of TFs has been identified experimentally or in silico in many plant species including Arabidopsis thaliana, Brassica napus, Glycine max (soybean), Solanum tuberosum (potato), Oryza sativa (rice), Zea mays (maize), the moss Physcomitrella patens, and all other land plant genomes sequenced to date (Table 1) (Zhang et al., 2008; Yang et al., 2009; Osnato et al., 2010; Kim et al., 2012; Liu et al., 2012; Baloglu, 2014; Filiz et al., 2014; Kuijt et al., 2014; Wang et al., 2014). Typically, 8–20 GRFs are encoded by the land plant genomes, while GIFs are generally present in lower copy numbers, normally in the range of 3–5. GRFs are absent from green algae while GIFs are present (Table 1). Here, we review the current knowledge of the biological functions of GRF TFs (Figure 1), and highlight open questions and important aspects to be addressed in future research.
Published by the Molecular Plant Shanghai Editorial Office in association with Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and IPPE, SIBS, CAS.
Molecular Plant
GRF Transcription Factors Species Eudicots
With WRC
GIFs
9
9
9
3
Arabidopsis lyrata
9
9
9
3
Brassica rapa Thellungiella parvula
9
8
9
3
17
16
17
5
8
8
8
3
Carica papaya
11
9
8
3
Gossypium raimondii
18
18
18
6
Theobroma cacao
10
10
10
3
Citrus sinensis
11
8
9
2
Populus trichocarpa
20
19
19
6
Ricinus communis
14
10
11
2
Manihot esculenta
17
17
17
5
2
2
2
1
Lotus japonicus Medicago truncatula
10
8
10
6
Glycine max
26
24
24
11
Prunus persica
11
10
10
2
Malus domestica
17
12
13
5
Fragaria vesca
10
10
10
2
Citrullus lanatus
8
8
8
2
Cucumis melo
8
7
8
2
Eucalyptus grandis
7
6
7
3
Vitis vinifera
10
8
10
4
Solanum lycopersicum
12
12
12
4
Solanum tuberosum
13
12
12
2
Beta vulgaris
7
6
7
3
Oryza sativa ssp. japonica
13
12
12
2
Oryza sativa ssp. indica*
13
11
12
3
8
7
7
2
12
12
11
3
9
9
9
3
Zea mays
17
14
14
3
Setaria italica*
10
10
10
3
Musa acuminata*
19
17
19
6
Physcomitrella patens
2
2
2
5
Chlamydomonas reinhardtii
0
0
0
1
Ostreococcus lucimarinus
0
0
0
1
Amborella trichopoda
7
6
7
2
Hordeum vulgare* Brachypodium distachyon* Sorghum bicolor
Other
With QLQ
Arabidopsis thaliana
Capsella rubella
Monocots
GRFs
Table 1. Number of GRF and GIF Genes per Species. Data were obtained from PLAZA 3.0 Dicots/Monocots (Proost et al., 2015; species with data from the Monocots version are indicated by an asterisk). As all GRFs/GIFs share a common ancestor, they can be found by looking at the homologous gene families containing any of the GRFs/GIFs. Species are ordered according to their phylogenetic relationship.
STRUCTURAL FEATURES OF GRF TFs The first member of the GRF gene family to be identified was OsGRF1 from rice (O. sativa; Van der Knaap et al., 2000), where it was discovered as a gibberellic acid (GA)-induced gene in intercalary meristem internodes. Ectopic expression of OsGRF1
in Arabidopsis causes impaired stem growth, suggesting that it plays a regulatory role in GA-induced stem elongation. The rice GRF1 protein contains regions that are conserved in homologous proteins from both rice and other plants (with molecular weights mostly in the range of 40–60 kDa). One of them is a Molecular Plant 8, 998–1010, July 2015 ª The Author 2015.
999
Molecular Plant Oil production in seeds BnGRF2
Flower development and flowering time AtGRF1-9, OsGRF1, 6, 10 MADS-box TFs AP1, SEP3 OsGRF6, 10 OsGIFs
AtGRF3 and 5 GIFs
Stem elongation OsGRF1
Coordination of growth with stress responses AtGRF1, 3 and 7
Ear development AtGRF1, 2, 3
such genes are functional and have roles similar to those of characterized GRFs.
Plant longevity
AtGRFs
OsCR4, OsJMJ706
Cell expansion
GRF Transcription Factors
ZmGRF2 and 11 ZmGIF2 and 3
AtGRF7 AtGRF7 under stress
x
DREB2A
DREB2A
Cell proliferation AtGRF1, 2, 3, 4, 5, 9 BrGRF8 and ZmGRF10 Leaf growth GIFs
Adaxial-abaxial patterning
SAM maintenance AtGRF4-6, OsGRF3, 10 and BGRF1 GRFs
AtGRFs and AtGIF1
KNAT2 OsKN2 BKN3
Root growth AtGRF1, 3 Interaction between GRFs and GIFs
Figure 1. GRFs Have Diverse Growth-Related Functions. Graphic summary of the known biological functions reported for GRFs from eudicot and monocot species. References to the processes and genes mentioned are given in the main text. Gene names are shown in italics. Reported interactions between GRFs and GIFs at the protein level (revealed by, e.g., yeast two-hybrid or BiFC studies) or at the genetic level are indicated by a black triangle. Arrow-ending and T-ending lines indicate positive and negative gene regulatory interactions, respectively.
QLQ (glutamine, leucine, glutamine, IPR014978) domain accompanied by mostly bulky aromatic/hydrophobic amino acids (Van der Knaap et al., 2000; Kim et al., 2003; Choi et al., 2004; Zhang et al., 2008). Those conserved regions may be important for QLQ to function as a protein–protein interaction domain (Van der Knaap et al., 2000). The QLQ motif is also present in the SWI2/SNF2 protein from Saccharomyces cerevisiae, where it facilitates the interaction with other proteins to form a complex involved in chromatin remodeling (Treich et al., 1995). The second conserved region is a WRC (tryptophan, arginine, cysteine, IPR014977) amino acid stretch in combination with a C3H motif; this part of the protein is expected to be relevant for DNA binding and targeting of the TF to the nucleus (Raventos et al., 1998; Van der Knaap et al., 2000; Kim et al., 2003; Choi et al., 2004; Zhang et al., 2008). The QLQ and WRC domains are located within the N-terminal part of GRFs. While proteins with a QLQ motif are found in all eukaryotes, WRC represents a plant-specific motif. Another less conserved motif, TQL, is located within the C-terminal region of OsGRF1 (Van der Knaap et al., 2000). Similar to OsGRF1, all nine members in Arabidopsis (AtGRF1 to AtGRF9) harbor the conserved QLQ and WRC domains within the N termini (Kim et al., 2003). Notably, AtGRF9 contains a second WRC domain in its C-terminal part, like BrGRF12 from Chinese cabbage (Brassica rapa; Wang et al., 2014). The vast majority of GRF homologs in other species (derived from PLAZA 3.0; Proost et al., 2015), which groups similar genes, based on the complete amino acid sequence, into gene families) contain both the QLQ and WRC domain. However, some species do have members that lack the QLQ domain, the WRC domain, or even both (Table 1). It is currently unknown if 1000 Molecular Plant 8, 998–1010, July 2015 ª The Author 2015.
In contrast to the conserved N-terminal regions in GRFs, their C-terminal parts are variable and only show low to moderate sequence similarities. In Arabidopsis, the most similar members are AtGRF3 and AtGRF4, with 54% identical amino acids in their C-terminal parts, whereas AtGRF5 and AtGRF6 show the least similarity (16%), similar to AtGRF7 and AtGRF8 (17%), while AtGRF1 and AtGRF2 share 44% identical amino acids in their C-terminal segments. Due to its unique structure with the extra WRC domain in this region, AtGRF9 represents a further, separate member. In addition to the QLQ and WRC domains, two smaller amino acid motifs are present in the C-terminal region of AtGRFs, i.e. TQL and GGPL. TQL is present in AtGRF1 to AtGRF4 and in OsGRF1 to OsGRF5, while GGPL is only found in AtGRF1 to AtGRF4 and in AtGRF7 and AtGRF8, but not in any of the 13 OsGRFs (Van der Knaap et al., 2000; Kim et al., 2003; Choi et al., 2004; PLAZA 3.0). Both motifs also occur in several GRFs from other species. Whether specific functions are associated with them is currently unknown.
REGULATION OF LEAF GROWTH BY GRFs Studies in Arabidopsis using promoter–reporter fusion lines have shown that GRFs are expressed in different parts of roots and shoots, often in growing zones where cell proliferation occurs (Kim et al., 2003; Horiguchi et al., 2005; Kim et al., 2012; Bao et al., 2014; Liang et al., 2014; Pajoro et al., 2014). Furthermore, RNA gel-blot and quantitative RT–PCR analyses showed that AtGRF expression levels decrease as the age of the plant (or the organ) increases (Kim et al., 2003; Rodriguez et al., 2010). Thus, GRFs are generally more expressed in actively growing tissues than in mature ones, which is in accordance with their function in early stages of growth and development in different tissues (Kim et al., 2003; Horiguchi et al., 2005; Rodriguez et al., 2010). Altered expression of AtGRF1 to AtGRF3 causes a change in growth phenotypes, especially in leaves and cotyledons, suggesting that those three genes in particular regulate leaf and cotyledon development (Kim et al., 2003). While single-gene loss-of-function mutants of AtGRF1, AtGRF2, and AtGRF3 did not show a clear developmental phenotype in the initial study, the atgrf1/2/3 triple mutant developed smaller and narrower leaves and shorter petioles compared with wild-type; recently, however, Debernardi et al. (2014) reported a 15% reduced size of the first pair of leaves also for the atgrf3 single-gene mutant. In line with these results, overexpression of individual genes (AtGRF1 and AtGRF2) causes larger leaves than in controls. The altered organ sizes resulting from elevated GRF expression are due to an increase in cell size rather than cell number, suggesting that these GRFs regulate cell expansion in leaves (Kim et al., 2003). However, Kim and Kende. (2004) additionally indicated a role of AtGRF1 to AtGRF3 for regulating cell proliferation during leaf development, which was confirmed for AtGRF3 by Debernardi et al. (2014). AtGRF4 shows some functional redundancy with AtGRF1 to AtGRF3. Similar to the atgrf1/2/3 triple mutant, the atgrf1/2/3/4
GRF Transcription Factors quadruple mutant developed smaller leaf areas. The smaller size of the atgrf multi-gene mutants is due to smaller cells (through atgrf1 and atgrf2) or fewer cells (through atgrf3 and atgrf4) (Kim et al., 2003; Kim and Lee, 2006; Gonzalez et al., 2009; Debernardi et al., 2014). In addition, a high percentage of quadruple mutant plants showed cup-shaped cotyledons, and a significant number of plants also lacked a shoot apical meristem (SAM; similar to the shoot meristemless/stm mutant) (Kim and Lee, 2006). Therefore, it was concluded that AtGRF4 is involved in not only cell proliferation in leaves but also the embryonic development of cotyledons and the SAM (Kim and Lee, 2006). Although AtGRFs seem to have at least partly overlapping roles, the function of AtGRF5 cannot be taken over by other members of the family (Horiguchi et al., 2005; Debernardi et al., 2014). Overexpression lines of AtGRF5 show bigger leaf areas, which, in contrast to AtGRF1 and AtGRF2, is due an increased cell number; and the atgrf5 single-gene mutant develops a narrower leaf compared with wild-type as a result of a decreased cell number (Horiguchi et al., 2005). Notably, AtGRF5 interacts with AN3 (ANGUSTIFOLIA3) to regulate cell proliferation in the leaf primordium. The an3 leaf-shape mutant carries a mutation in the GIF1 (GRF-INTERACTING FACTOR 1) gene; thus, AN3 is identical to GIF1 (Horiguchi et al., 2005). The GIF proteins have recently been reported to associate not only with GRF TFs but also with various chromatin remodeling proteins, suggesting a wider range of transcription regulation function than previously assumed (Debernardi et al., 2014; Vercruyssen et al., 2014). Although AtGRF9 was originally thought to have a minor role in cell proliferation (Horiguchi et al., 2005), more recent studies indicate that GRF9 also contributes to determining final leaf size (Arvidsson et al., 2011; O. Mohammad Amin et al., unpublished results). In Arabidopsis, miR396 shares nearly perfect sequence complementarity with the WRC encoding part of transcripts of seven members of the GRF family, except for AtGRF5 and AtGRF6 (Liu et al., 2009; Rodriguez et al., 2010). Accordingly, miR396a and miR396b play important roles in leaf growth and development by post-transcriptionally repressing GRF gene expression (Liu et al., 2009; Rodriguez et al., 2010). Wang et al. (2011) reported that cell proliferation controlled by miR396targeted AtGRFs is also required for adaxial–abaxial (Ad-Ab) polarity formation during leaf morphogenesis. Similarly to Arabidopsis, expression of GRFs in rice (OsGRFs) is generally high in actively growing tissues including the SAM, leaf primordia, and young leaves. However, while most AtGRFs show comparatively high expression in roots, expression of OsGRFs is low in this organ (Kim et al., 2003; Choi et al., 2004; Bao et al., 2014). In addition, expression of several rice GRFs (OsGRFs 1, 2, 3, 7, 8, 10, and 12) is enhanced by gibberellin (GA3) treatment, while expression of OsGRF9 is reduced (Choi et al., 2004). Also, in Chinese cabbage (B. rapa L. ssp. pekinensis) most GRFs are induced by GA3 treatment (Wang et al., 2014), while in Arabidopsis GRFs are mostly not affected by GA3 (Kim et al., 2003; eFP browser Winter et al., 2007). The available data therefore suggest functional diversification of GRFs, at least with respect to root growth and GA signaling (Van der Knaap et al., 2000; Choi et al., 2004).
Molecular Plant An interesting situation exists in corn (Z. mays), where ZmGRF10 encodes a truncated GRF protein that contains the conserved QLQ and WRC motifs at the N terminus while almost the entire C-terminal part is missing. ZmGRF10 lacks apparent transcription activation function, but retains its ability to physically interact with the transcriptional co-activators ZmGIF1 and ZmGIF2. In contrast to GRFs that positively regulate cell proliferation in leaves, overexpression of ZmGRF10 in maize leads to a decrease in leaf size (mostly leaf length) and plant height alongside an impaired cell proliferation, suggesting that it regulates leaf size by limiting cell proliferation (Wu et al., 2014). Notably, however, yield-related traits were not affected by ZmGRF10 overexpression. Transcriptome profiling by RNA-seq revealed 176 upregulated and 74 down-regulated genes in the third leaf of ZmGRF10 overexpressors compared with non-transgenic siblings. Among the up-regulated transcripts were several from metabolism-related genes (carbohydrate, amino acid, lipid, hormone metabolic pathways) and transcription regulators involved in developmental processes (such as two SCARECROW-like genes, a MADS-box gene and a homeobox gene); the downregulated genes included other growth/development-related transcription regulators and several chromatin-modifying proteins. Thus, ZmGRF10 appears to affect various cellular mechanisms to control growth (Wu et al., 2014). In Chinese cabbage (B. rapa L. ssp. pekinensis), an important vegetable for human food, 17 GRFs have been reported (Wang et al., 2014). With the exception of BrGRF3 and BrGRF14, for which no expression was detected in any tissue, all other BrGRFs were mostly expressed in young leaves and showed lower expression in old leaves, the largest difference in expression between young and old leaves being detected for BrGRF8. Six BrGRFs were more highly expressed in flower buds than in open flowers, while the opposite was observed for seven other members of the gene family. These data suggest that, as in other plants, GRFs in Chinese cabbage have important functions in controlling the growth and development of young leaves and flowers. BrGRF16 was most strongly expressed in roots, indicating a function in this organ. Ectopic overexpression of BrGRF8 in Arabidopsis (CaMV 35S promoter) increases rosette leaf area (leaf length and leaf width) and petiole length due to an increase in cell number but not cell size; silique size and seed number per silique were not affected. The molecular mechanisms triggered by overexpression of BrGRF8 are not known at present (Wang et al., 2014).
FLORAL ORGAN DEVELOPMENT Various studies report a function of GRFs and miR396 in floral organ development. Recently, Pajoro et al. (2014) revealed a role of miR396a for flower formation in Arabidopsis, where it determines sepal-petal identity by regulating GRF transcript levels. Using data from DNA binding and gene expression studies, a stagespecific interaction of the MADS-box TFs APETALA1 (AP1) and SEPALLATA3 (SEP3), which are main controllers of flower specifications, with five (in the case of AP1) or all (in the case of SEP3) members of the GRF family was demonstrated; in addition, AP1 directly controls the expression of SEP3. Of the GRFs expressed in the flower meristem, AtGRF8 appeared to be of particular importance for later floral organ development. Time-course DHS-seq analysis (DNase I hypersensitive sites assay followed Molecular Plant 8, 998–1010, July 2015 ª The Author 2015. 1001
Molecular Plant by deep sequencing) demonstrated that in the case of AtGRF8, the chromatin structure changes through binding of the two MADS-box TFs, which leads to increased accessibility to regulatory elements. Following these initial chromatin changes, AtGRF8 expression increases to take part in flower development. Thus, AP1 and/or SEP3 appear to operate as ‘‘pioneer factors’’ for AtGRF8 expression, which after its induction can exert its flower-related functions. At present, however, the downstream gene regulatory network controlled by AtGRF8 during flower formation in Arabidopsis remains unknown. In addition to sepalpetal identity determination, the miR396/GRF regulatory network is required for proper development of the pistil (Liang et al., 2014). In flowers the gynoecium represents the female reproductive organ, in which ovules develop from dedicated meristems (in Arabidopsis these are located at the floral carpel margins). The AP2-type transcription factor AINTEGUMENTA (ANT) and the transcriptional adaptor protein SEUSS (SEU) are critical for ovule formation such that seu/ant double mutant plants fail to initiate ovule primordia. As single-gene mutants are only partially impaired with respect to ovule primordia formation (ant: 50% of wild-type; seu: almost normal), the two transcription regulators appear to act synergistically in this developmental process. A global transcriptomic study by Wynn et al. (2011) identified 31 genes showing lower expression in the seu/ant double mutant compared with either of the single-gene mutants, suggesting that they play particularly important functions in the establishment of ovule primordia. Notably, half of the genes identified code for TFs of which members of the REPRODUCTIVE MERISTEMS (REM) and GRF families were overrepresented. In situ hybridization confirmed expression of AtGRF5 in wild-type ovule primordia and strongly reduced expression in the seu/ant double mutant in later-stage gynoecia (Wynn et al., 2011). So far, however, the gene regulatory network controlled by AtGRF5 (or other AtGRFs) during ovule formation remains elusive. As GRFs interact with GIF proteins, it is worth mentioning that Lee et al. (2014) recently observed that GIFs play a critical role for the formation of reproductive organs (male and female) in Arabidopsis. More specifically, the atgif1/2/3 triple mutant (with mutations in all three GIF genes in this plant) developed neither a normal carpel margin meristem nor tissues derived from it, namely ovules and the carpel septum, thereby leading to a split gynoecium and the absence of embryo sacs. The triple mutant also failed to develop functional male reproductive organs (anthers); thus, GIFs play a critical role in establishing reproductive competence. In an earlier work, Zhang et al. (2008) suggested that in maize (Z. mays) ZmGRF11 and ZmGIF2, in addition to ZmGRF2 and ZmGIF3, might form functional complexes possibly involved in the growth and development of ears. However, as this conclusion was derived from phenotypes observed in transgenic Arabidopsis plants expressing the maize GRFs/GIFs, which leads to delayed bolting but accelerated growth rate of the main inflorescence stem, the exact functions of the maize proteins remain unknown at present. An involvement of GRFs in floral organ development was also demonstrated for rice, where OsGRF6, besides OsGRF4 and OsGRF10, is highly expressed in young inflorescences (Liu et al., 2014a). Antisense inhibition of OsGRF6 or overexpression of OsmiR396d (which targets OsGRF6 transcripts) led to abnormal 1002 Molecular Plant 8, 998–1010, July 2015 ª The Author 2015.
GRF Transcription Factors floral organ (floret) development (Liu et al., 2014a). Of note, miR396d represents a monocot-specific member of the miR396 family (Jones-Rhoades and Bartel, 2004; Sunkar et al., 2005; Hewezi et al., 2008; Sunkar and Jagadeeswaran, 2008; Liu et al., 2009). Except for OsGRF11, all other OsGRFs have target sequences for OsmiR396d and accordingly its overexpression results in significant down-regulation of most OsGRFs (with the exception of OsGRF9, OsGRF11, and OsGRF12; Li et al., 2010; Liu et al., 2014a). Rice plants overexpressing OsmiR396d rarely exhibit smaller leaf phenotypes (in contrast to miR396 overexpressors in Arabidopsis; Liu et al., 2009), but rather show a greater number of abnormalities and defects in florets compared with wild-type (Liu et al., 2014a). Further analysis revealed that osgrf6 and osgrf10 single-gene mutant plants remained small but did not show obvious abnormalities in the florets, while the osgrf6/osgrf10 double mutant exhibited defects in florets in addition to the small-growth phenotype, suggesting that the two OsGRFs have redundant functions in floret development. Plants overexpressing an miR396d-resistant OsGRF6 variant in the OsmiR396d overexpression background produced normal florets. Furthermore, using yeast twohybrid and bimolecular fluorescence complementation (BiFC) assays, an interaction of OsGRF6 and OsGRF10 with OsGIFs (O. sativa GIFs) was shown. In addition, both GRFs bind to the promoters of OsCR4 (O. sativa Crinkly4 Receptor-like kinase) and OsJMJ706 (O. sativa Jumonji706), which play a role in interlocking palea and lemma maintenance (rice fertility) and flower development, respectively (Sun and Zhou, 2008; Pu et al., 2012; Liu et al., 2014a). Furthermore, the transcription activation ability of OsGRF10 toward the two target genes was enhanced in the presence of OsGIF1. Taken together, it appears that at least one of the mechanisms underlying floret development in rice is controlled through a network involving OsGRFs (most likely OsGRF6), OsGIFs, and miR396d (Liu et al., 2014a). Earlier, Luo et al. (2005) showed that OsGRF1, in addition to its role in regulating leaf growth, controls flowering time. Interestingly, AtGRF1 and AtGRF2 overexpressors in Arabidopsis also showed delayed flowering compared with wild-type (Kim et al., 2003). Considering all evidence, GRFs play an important role in flower development and flowering time control (Van der Knaap et al., 2000; Kim et al., 2003; Luo et al., 2005; Liu et al., 2014a; Pajoro et al., 2014).
A GRF AFFECTING SEED WEIGHT AND OIL CONTENT Liu et al. (2012) reported another interesting aspect about GRFs in B. napus (rapeseed): by comparing two cultivars differing in seed oil content, they discovered two closely related GRF genes, namely BnGRF2a and BnGRF2b, which are expressed at higher levels in ovules of the high-oil seeds than in ovules from the low-oil seeds. Overexpression of the BnGRF2a open reading frame under control of the 35S promoter in transgenic Arabidopsis plants led to pleiotropic growth phenotypes, including cotyledons with longer petioles than those of wildtype, increased leaf area at flowering stage, delayed flowering, and longer flower pistils, which reduced fertility due to insufficient
Molecular Plant
GRF Transcription Factors pollination by the normal stamen in these plants. In addition, more chloroplasts per cell were observed in 35S:BnGRF2a lines, concomitant with an increase in chlorophyll a and b content and a higher photosynthetic efficiency (Liu et al., 2012). The higher chloroplast number might have been caused by elevated expression of the transcription factor GOLDEN2-LIKE2 (GLK2), a key regulator of chloroplast development and maintenance (Waters and Langdale, 2009), in the transgenic plants. Furthermore, seed mass was increased in 35S:BnGRF2a lines (after assisted pollination) and in Napin:BnGRF2a plants, with a >30% increase in weight compared with wild-type (the Napin promoter drives preferential expression in seeds); cotyledons were enlarged without a change in cell size, and seed oil content was elevated by >40%. However, the number of seeds per fruit remained unchanged in Napin-BnGRF2a lines. As seed mass and oil yield were increased relative to the control, these findings are likely to be exploited for the breeding of high-oil crop cultivars in the future. Transcriptome profiling of young leaves revealed expression differences of a large number of genes in 35S:BnGRF2 plants (1601 genes up, 1234 down), many of which encoded TFs. In addition, as may be expected from the enlargedleaf phenotype, several genes encoding cell division and cell cycle control proteins were up-regulated. Furthermore, BnGRF2a triggered elevated expression of chloroplast/photosynthesisrelated genes, in accordance with the higher chlorophyll levels in these plants. Some other genes were classified as being involved in lipid and fatty acid biosynthetic and storage processes (Liu et al., 2012). Of note, overexpression of BnGRF2a resulted in a more pleiotropic phenotype than is normally observed when other GRFs are overexpressed, which may be due to sequence differences between BnGRF2 and GRFs from other species analyzed so far. The fact that BnGRF2 overexpression induces a diverse spectrum of phenotypic alterations indicates that its expression is tightly adjusted to the developmental and metabolic needs of a growing rapeseed plant and during seed formation. It may therefore be highly interesting to unravel the regulatory program that controls BrGRF2 expression in the future.
GRFs AND ROOT DEVELOPMENT In several studies GRFs were found to be expressed in roots of different plant species, suggesting a possible role of GRFs for root development or physiological processes in this organ (Kim et al., 2003; Luo et al., 2005; Hewezi et al., 2012; Hewezi and Baum, 2012; Bazin et al., 2013; Bao et al., 2014). Experimental evidence suggests that a balanced expression of AtGRF1 and AtGRF3, which show the highest expression levels among GRFs in Arabidopsis roots, and miR396 is essential for normal root growth (Hewezi et al., 2012). Thus, mir396a knock-down lines develop longer roots than wild-type, while miR396a and miR396b overexpression lines and plants overexpressing miR396-resistant AtGRF1 or AtGRF3 versions exhibit shorter roots (Hewezi et al., 2012; Bao et al., 2014). Another recently identified target of miR396 is bHLH74, which encodes a basic helix-loop-helix TF (Debernardi et al., 2012); the bhlh74 knock-out line exhibits shorter roots compared with wild-type, while overexpression of bHLH74 leads to longer roots (Bao et al., 2014). It has thus been concluded that a regulatory
network governed by miR396 and its targets, including GRFs and bHLH74, has a central function in normal root growth and development.
GRFs AND LONGEVITY In addition to their functions in different aspects of leaf and flower development, GRFs also play a role in regulating plant senescence. Debernardi et al. (2014) reported that atgrf3 and atgrf5 mutants or transgenic Arabidopsis plants overexpressing miR396 exhibit early senescence, whereas rGRF3 lines that express a miR396-resistant version of GRF3, overexpressors of AtGRF5 and more effectively, rGRF3x35S:GIF1 transgenic lines exhibited a clear delay in leaf senescence. Importantly, these authors showed that the functions of GRF in leaf size determination and senescence are two distinct processes, and that leaf longevity is not simply a result of an alteration of cell proliferation. They showed that the expression of AtGRF3 and AtGRF5 in early leaf development is related to cell proliferation and determination of final leaf size, while expanding their expression during later stages of leaf development increases leaf longevity (Debernardi et al., 2014). At present, however, the molecular mechanisms underlying this longevity extension remain unknown.
GRFs COORDINATE PLANT GROWTH WITH STRESS RESPONSES Unlike most animals that are capable of escaping stressful conditions by moving away, plants are sessile organisms that must cope with upcoming stress directly at the place where their seeds germinated. To this end, plants have evolved sophisticated signaling and defense systems that help them to guard against various types of stresses that often come in combination. Defense responses require significant metabolic input and energy. As a consequence, the growth of plants is often impaired under stress. Numerous studies have demonstrated that activation of stress-related genes, e.g. DREB2A (DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN2A), increases plant tolerance to stress while concurrently inhibiting plant growth (Heidel et al., 2004; Sakuma et al., 2006). Recently, additional studies have shown that GRFs may function in coordinating plant growth with defense signaling and stress responses (Liu et al., 2008; Hewezi et al., 2012; Kim et al., 2012; Casadevall et al., 2013; Casati, 2013; Liu et al., 2014b). During evolution, plants have established mechanisms to repress the expression of stress-related genes under normal conditions, and one such example is represented by AtGRF7 in Arabidopsis, which under non-stress conditions is mainly expressed in developing leaves, some flower tissues, and leaf mid-veins, while expression is rarely detected in cotyledons (Kim et al., 2012). Under normal growth conditions, AtGRF7 overexpression and atgrf7 mutant lines do not show a strong phenotype compared with wild-type, although there is a tendency toward reduced plant size in atgrf7 lines (T-DNA insertion or artificial miRNA inhibition lines). Importantly, however, atgrf7 mutants are more tolerant to salinity and drought stress than are wild-type and AtGRF7 overexpressor lines, a phenotype likely mediated through up-regulation (de-repression) of DREB2A in these lines. DREB2A is a transcription factor whose transcriptional and Molecular Plant 8, 998–1010, July 2015 ª The Author 2015. 1003
Molecular Plant post-translational activation increases osmotic stress tolerance in Arabidopsis (Liu et al., 1998; Kim et al., 2012). Under nonstress conditions, AtGRF7 suppresses DREB2A expression in the wild-type to preserve plant growth rate through direct binding to a TGTCAGG cis element in the DREB2A promoter. Wholegenome expression profiling of the atgrf7-1 T-DNA insertion mutant revealed a high proportion of up-regulated genes annotated for their response to stresses and responsiveness to abscisic acid, including DREB2A. These findings highlight the role of AtGRF7 as a repressor of stress-responsive genes under nonstress conditions (Kim et al., 2012). In addition, GRFs play an important role in biotic stress-related processes, as shown for the plant-parasitic cyst nematode Heterodera schachtii (Hewezi et al., 2012). The parasite employs the plant’s regulatory miR396/GRF module to control the initiation and subsequent maintenance phase of the syncytium (the nematodes’ feeding source created through the fusion of hundreds of root cells) in Arabidopsis. Nematode infection coincides with an initial down-regulation of miR396 expression leading to the up-regulation of its targets, the GRF family members and especially AtGRF1 and AtGRF3, to dedifferentiate root cells to form the syncytium through further redifferentiation. Maintenance and other developmental processes of the syncytium occur concurrent with the subsequent upregulation of miR396, which results in the down-regulation of AtGRF1 and AtGRF3 (and other GRFs) expression to normal levels (Hewezi et al., 2012). Overexpression of miR396a or miR396b in transgenic Arabidopsis plants reduced transcript levels of the GRF targets, as expected, and strongly impaired susceptibility to nematode infection. Notably, susceptibility to H. schachtii was also reduced in the atgrf1/2/3 triple mutant, while no difference to wild-type Arabidopsis was observed in atgrf1 and atgrf3 single-gene mutants, consistent with the view that these GRFs exert redundant functions as previously observed for leaf growth-related phenotypes. To gain insight into the downstream targets affected by AtGRF1 and AtGRF3, Hewezi et al. (2012) determined the transcriptomes of the atgrf1/2/3 triple mutant and plants overexpressing miRNA396-resistant GRF forms, and observed altered expression of a large number of genes (6,385 in total) when compared with wild-type. Notably, of the 7225 genes reported by Szakasits et al. (2009) to change expression during syncytium formation, 44% are altered in the Hewezi et al. (2012) experiment, supporting the important roles of AtGRF1 and AtGRF3 in this process. In addition, a more complex regulatory feedback loop between miR396 and GRFs appears to be involved, not only as the up-regulation of miRNA396 leads to the down-regulation of GRFs, but also because an increased expression of GRFs triggers the down-regulation of miR396 transcript abundance (Hewezi and Baum, 2012). Whether the GRF TFs directly or indirectly control miR396 expression is currently unknown. More recently, a further functional classification of AtGRF1 and AtGRF3 putative downstream targets revealed that most of them are involved in defense responses and processes of disease resistance (Liu et al., 2014b). Thus, considering the growth-related functions of GRFs (Kim et al., 2003; Horiguchi et al., 2005; Kim and Lee, 2006; Kim et al., 2012) and their roles 1004 Molecular Plant 8, 998–1010, July 2015 ª The Author 2015.
GRF Transcription Factors in biotic and abiotic stress responses (Liu et al., 2008; Hewezi et al., 2012; Kim et al., 2012; Casadevall et al., 2013; Casati, 2013), GRF TFs, and more specifically AtGRF1 and AtGRF3, appear to play a central role in the coordination of plant growth with defense signaling (Liu et al., 2014b). In addition, the expression level of miR396 is affected by a wide range of stressful conditions, including low temperature, high salinity, drought, and UV-B radiation (e.g. Liu et al., 2008; Zhou et al., 2012; Casadevall et al., 2013; Casati, 2013; Wang et al., 2013; Liu et al., 2014b). UV-B radiation causes impaired cell proliferation with increased levels of miR396. The inhibitory effect on cell proliferation is dependent on MITOGEN-ACTIVATED PROTEIN KINASE3 (MPK3); however, the molecular basis of this regulation is currently unknown (Casadevall et al., 2013). Interestingly, the change of miR396 expression level in response to stresses is often stronger than that of other miRNA genes (Liu et al., 2008), which is in accordance with the model that the miR396/GRF module plays an important role in the coordination of stress responses with plant growth.
THE miR396/GRF REGULATORY MODULE miRNAs are key regulators of many physiological and developmental processes in plants (e.g. Poethig, 2013; Curaba et al., 2014; Spanudakis and Jackson, 2014). As the GRF gene family is widely present in the plant kingdom, it has been suggested that the miR396/GRF regulatory module (Figure 2) is functionally conserved in plants. In accordance with this model, heterologous expression of Ath-miR396a (from Arabidopsis) in tobacco resulted in a reduction of the expression of three of four tested NtGRF genes, which was accompanied by a severe reduction in leaf size and a narrow-leaf phenotype similar to the Arabidopsis atgrf1/2/3 triple mutant (Yang et al., 2009). Furthermore, flower development was impaired in the AthmiR396a overexpressor lines, which produced more petals and shorter (curved) stamens compared with wild-type, underscoring the role of the miR396/GRF network for floral organ formation. Similarly, ectopic expression of Ptc-miR396c from Populus trichocarpa in tobacco resulted in altered plant growth and flower development with an accompanied reduction of NtGRF transcript abundance (Baucher et al., 2013). In plants there are two major classes (I and II) of the TCP (TEOSINTE-BRANCHED/CYCLOIDEA/PROLIFERATING CELL FACTORS) TFs, which regulate a diverse spectrum of developmental and physiological processes including growth, senescence, and hormone signaling (Martin-Trillo and Cubas, 2010; Uberti Manassero et al., 2013). In snapdragon (Antirrhinum majus), the class II TCP CINCINNATA (CIN) regulates leaf morphogenesis and controls the progression of the mitotic cell cycle arrest front (the border between the cell proliferation and cell expansion zones in growing leaves) (Nath et al., 2003). A close homolog to CIN in Arabidopsis is TCP4. Hyperaccumulation of TCP4 transcript causes an increase in miR396 transcript abundance and a reduction in the transcript levels of all GRFs, including AtGRF5, AtGRF6, and GIF1, which are not the direct targets of miR396, suggesting that TCP4 regulates GRF/GIF expression independent of miR396 (Figure 2)
Molecular Plant
GRF Transcription Factors Low temperature High salinity Drought stress
UV-B miR396
TCPs (TCP4)
G R F s (miR396 targets)
Interaction
(non-miR396 targets)
GIFs
Growth-related processes Figure 2. The miR396/GRF Regulatory Module. MiR396 targets various GRF transcripts, thereby negatively regulating their abundance. The expression of miR396 itself is positively regulated by upstream TCP transcription factors and is enhanced by various types of abiotic stresses, which modulates GRF transcript abundance. Furthermore, GRFs may control miR396 transcript levels (and the expression of other GRFs, see Hewezi and Baum, 2012), although the underlying molecular details are unknown. TCP4 affects the expression levels of some GRFs and GIF1 independent of miRNA396 (Rodriguez et al., 2010). At the protein level, GRFs interact with GIFs to control growthrelated processes (see Figure 1). Note that several, but not all GRFs are miR396 targets.
(Rodriguez et al., 2010). In addition, a recent report by the same group provides experimental evidence for a direct regulation of miR396b by TCP4 (Schommer et al., 2014). Furthermore, it has been suggested that the miR396/GRF regulatory network synergistically interacts with the transacting short interfering RNA (ta-siRNA) biogenesis pathway that requires RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) and DICERLIKE 4 (DCL4) activity during leaf development (Mecchia et al., 2013).
GRFs ARE UPSTREAM REPRESSORS OF KNOX GENES Aerial organs in vascular plants are initiated from the SAM, and proper SAM growth and maintenance determines the growth of lateral organs (leaves). KNOTTED1-LIKE HOMEOBOX (KNOX) genes are among the most important controllers of meristem development and function, and they restrict cell differentiation in the SAM. The role of KNOX genes in SAM formation and maintenance has been shown in many studies; prominent examples are represented by the loss-of-function mutants shoot meristemless (stm) in Arabidopsis and knotted1 (kn1) in maize, which both lack an SAM (Long et al., 1996; Vollbrecht et al., 2000). Normal shoot development occurs when the repression of meristemspecific genes and the activation of organ-specific genes occur in parallel (Lin et al., 2003). One of the best explained pathways for the repression of KNOX expression in the SAM involves the ASYMMETRIC LEAVES1 (AS1) and AS2 proteins in Arabidopsis. According to Guo et al. (2008), an AS1–AS2
repressor complex is formed, which induces the formation of a loop at the KNOX promoter that creates a repressive chromatin state, thereby inhibiting KNOX expression. Recently, Kuijt et al. (2014) reported that OsGRF3 and OsGRF10 interact with the promoter of the KNOX family gene OsKN2 in rice, thereby repressing its expression; their binding to a relevant 34 bp segment of the OsKN2 promoter requires the GRF’s conserved QLQ and WRC domains. Other GRFs containing QLQ and WRC domains, namely AtGRF4, AtGRF5, and AtGRF6 from Arabidopsis, also bind to the same 34 bp rice sequence and activate the HIS3 reporter in a yeast one-hybrid assay (Kuijt et al., 2014). The element is enriched for the CAG motif (and its reverse complementary sequence, CTG), which has previously been shown to be part of the cis-regulatory sequence bound by AtGRF7 (TGTCAGG; Kim et al., 2012). The GRF–KNOX interaction also occurs in other plants; in Arabidopsis, expression of the KNOX gene KNAT2, but not KNAT1, is suppressed by overexpression of AtGRF4, AtGRF5, and AtGRF6 as well as OsGRF10, and this is accompanied by developmental abnormalities as expected from KNOX repression (Kuijt et al., 2014). In barley, BGRF1 (BARLEY GROWTH-REGULATING FACTOR1) binds to an intron-located regulatory element in the BKN3 (BARLEY KNOX3) gene leading to its repression, and OsGRF10 is able to replace BGRF1 as a repressor (Osnato et al., 2010; Kuijt et al., 2014). Collectively, these findings indicate functional conservation of the GRF– KNOX regulatory module across eudicot and monocot species.
GIFs GRF TFs form a complex with the GIFs, a group of transcriptional co-activators. In Arabidopsis, the three proteins GIF1, GIF2, and GIF3 play roles in cell proliferation during leaf development and for maintaining the competence of meristematic cells to proliferate during the development of reproductive organs (Kim and Kende, 2004; Lee et al., 2009, 2014). GIF is a homolog of the human transcription co-activator synovial sarcoma translocation protein (SYT). In Arabidopsis, the GIF1 gene is also known as ANGUSTIFOLIA3 (AN3); the an3 mutant shows a change in the leaf shape (Horiguchi et al., 2005). GIF1 interacts with AtGRF1, AtGRF2, AtGRF4, AtGRF5, and AtGRF9 through their conserved QLQ domains (Kim and Kende, 2004; Horiguchi et al., 2005). While overexpression of GIF1/AN3 leads to bigger leaf areas due to an increase in cell number (Horiguchi et al., 2005), moderate down-regulation of GIF1 expression results in smaller leaves due to a decrease in cell number. Interestingly, severe inhibition of GIF1 expression also led to small leaves with more strongly reduced cell numbers, but cell sizes increased again (Fujikura et al., 2009). This phenomenon, called the ‘‘compensation effect’’, is thus defined as the initiation of cell size enlargement as a result of a severe reduction in cell numbers (Tsukaya, 2002; Kim and Kende, 2004; Horiguchi et al., 2005; Fujikura et al., 2009). Although it is known that GIF1 physically interacts with AtGRF5 and thereby participates in the positive control of cell proliferation during leaf development (Kim and Kende, 2004; Horiguchi et al., 2005), the molecular mechanism of compensation is not known at present. Another unique feature of GIF1 during leaf formation was recently reported by Kawade et al. (2013): GIF1 is expressed in subepidermal leaf tissues, and after the protein is synthesized it Molecular Plant 8, 998–1010, July 2015 ª The Author 2015. 1005
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GRF Transcription Factors Figure 3. Phylogenetic Tree of the GRF Genes in PLAZA 3.0 Dicots. The multiple sequence alignment, edited with partial and outlier genes removed, was obtained for gene family HOM03D000374 from PLAZA 3.0 dicots (Proost et al., 2015) and the tree was constructed using MEGA5 (Tamura et al., 2011). As can be seen, the GRF gene family can be subdivided into six groups which were already present in the ancestor of the flowering plants. By superimposing block duplicates (also derived from PLAZA 3.0 Dicots) on the tree topology, it can be seen that in eudicots groups IV and V expanded through the whole-genome triplication in the ancestor of the eudicots (yellow stars). Similarly, albeit more recently, the GRFs in rice and maize expanded. Within some species (such as poplar and soybean), additional large-scale duplications followed by retention of the duplicates resulted in further expansions. The genes included in each group are listed in Supplemental Table 1. Individual genes, some of which have reported functions, are highlighted. The bar indicates the branch length (over which 0.2 substitutions per site are expected).
moves into epidermal cells and helps control epidermal cell proliferation. Given the importance of the miR396-targeted GRF network for Ad-Ab polarity formation, GIF1 is also required for correct Ad-Ab patterning, which also involves the interaction of GIF1/ AN3 with ASYMMETRIC LEAVES2 (AS2), a nuclear protein important for leaf Ad-Ab patterning (Iwakawa, 2002; Xu et al., 2003; Iwakawa et al., 2007; Xu et al., 2007; Horiguchi et al., 2011). Furthermore, Kanei et al. (2012) demonstrated that GIF1 is essential for the establishment of cotyledon identity in concert with HAN/MONOPOLE/GATA18, a GATA-type transcription factor, through repression of the ectopic expression of PLETHORA1 (PLT1), a master regulator of embryonic apical fates. Strikingly, Debernardi et al. (2014) showed that overexpressing GIF1 alone did not significantly change leaf area (or cell sizes or numbers), in contrast to the report by Horiguchi et al. (2005). One possible explanation for this observation is that final organ size is predominantly controlled by the expression level of GRFs, and GIF1—as a transcriptional co-activator—then supports the gene regulatory activity of the GRF proteins. In agreement with this model, overexpressing GIF1 in an rGRF3 background (i.e. a plant expressing an miR396-resistant version of GRF3) dramatically increases leaf size (Debernardi et al., 2014). Although GIF1 has predominantly been studied in its interaction with GRFs, recent proteomic studies by Debernardi et al. (2014) demonstrated that GIF1 interacts with a number of additional proteins in planta, namely those involved in chromatin remodeling processes, in particular those known to participate in the formation of the SWITCH/SUCROSE NONFERMENTING (SWI/SNF) complex such as different ATPases of the SWI/SNF family (i.e. BRAHMA, SPLAYED, and CHR12). Similar results were obtained by Vercruyssen et al. (2014), who furthermore demonstrated the presence of GIF1/AN3 protein at the 1006 Molecular Plant 8, 998–1010, July 2015 ª The Author 2015.
loci of the AtGRF5 and AtGRF6 genes, and several other transcription control genes such as CYTOKININ RESPONSE FACTOR2 (CRF2), CONSTANS-LIKE5 (COL5), HECATE1 (HEC1), and ARABIDOPSIS RESPONSE REGULATOR4 (ARR4). The comprehensive data provided in the two recent reports provide strong evidence that GIF1/AN3 regulates gene expression through association with chromatin remodelers besides the interaction with GRF TFs.
GRFs EXPANDED THROUGH LARGE-SCALE GENOME DUPLICATION AND GENE RETENTION As land plants have multiple GRFs, comparative genomics can shed some light on when these genes emerged and how they expanded in different organisms. GRFs are lacking in all currently sequenced green algae, and thus appear to be an adaptation specific to land plants (Embryophyta). Two GRF genes can be found in P. patens, a member of the mosses (Bryophyta), while in sequenced flowering plants (Magnoliaphyta) usually eight or more are present (Table 1). A phylogenetic analysis (Figure 3) shows that the GRF family can be subdivided into six large categories, with the moss genes as outgroup. This indicates that the earliest expansion of the gene family occurred after the mosses diverged from flowering plants (over 400 million years ago) (Rensing et al., 2008), but before Amborella trichopoda separated from the other angiosperms (about 130 million years ago). While there is evidence for an angiosperm-wide genome duplication (Amborella Genome Project, 2013), these early expansions in the GRFs cannot be unambiguously linked to it. However, when looking at more recent expansions, retention after a whole-genome duplication (WGD) seems to be the norm; among all GRFs, 170 (out of 331 in total) are derived from
GRF Transcription Factors
Molecular Plant Figure 4. Domain Composition of GRFs. The majority of the 331 GRFs in PLAZA 3.0 have both the characteristic QLQ and WRC domains. A sequence motif was created using the multiple sequence alignment in PLAZA and WebLogo (http://weblogo.berkeley.edu/) to show the core QLQ and WRC motifs along with highly conserved flanking regions. In the extended WRC domain, three cysteine residues in combination with a histidine form a C3H DNA binding domain.
et al., 2014; O. Mohammad Amin et al., unpublished results) are in group II. The extent to which the functions of the proteins in the different groups are conserved in different plant lineages awaits further testing.
a large-scale duplication. Tandem duplications, on the other hand, are rather rare (only eight genes are found to be duplicated in this way). Some early expansions within the six groups can be linked to the whole-genome triplication in the common ancestor of the eudicots (Figure 3, indicated by yellow stars). Based on collinearity data from Vitis vinifera (Jaillon et al., 2007) and Eucalyptus grandis (Myburg et al., 2014), it can be concluded that the two eudicot subgroups of group IV (containing AtGRF7 and AtGRF8) were created during this event. The eudicot subgroups within group V were also created during this event. Similarly, a shared duplication between all cereals lies at the origin of many expansions seen in rice and maize. Further expansion occurred through the several independent WGDs in various plant lineages. The most recent WGD in the ancestor of the Brassicales created AtGRF3 and AtGRF4 in A. thaliana, the two most similar genes in this species. Also, in poplar and soybean (which have 20 and 26 GRF genes, respectively), the higher number of GRFs compared with other eudicots can be explained by gene retention after large-scale duplications. These observations are in line with previous studies that found that genes with a regulatory function are preferentially retained after large-scale duplications (Maere et al., 2005). Unlike single-gene duplications (e.g. tandem duplications), these events create a functional copy of the whole regulatory network, and hence dosage effects no longer have deleterious effects. The QLQ and WRC domains originally identified in the rice and Arabidopsis GRFs are highly conserved in GRFs from all plant species sequenced to date (Figure 4), underlining their functional importance. With respect to the known biological functions, AtGRF1 and AtGRF2, both of which positively affect cell expansion (Kim et al., 2003), are in group I, while AtGRF5 and BrGRF8, which positively regulate cell proliferation (Horiguchi et al., 2005; Wang et al., 2014), are in group V, and AtGRF9 and ZmGRF10, which negatively regulate organ size (Arvidsson et al., 2011; Wu
As GRFs interact with GIFs in several plants, we performed a comparative genomics analysis of GIF sequences across species. Interestingly, while GRFs are only present in land plants, GIFs can also be found in green algae (Table 1). Therefore, GIFs were likely already present in the common ancestor of green algae and land plants, and GRFs are a new gene family in the land plants that expanded fairly rapidly to the six subfamilies we observe today. The fact that GIFs exist in algae without the concurrent presence of GRFs is perhaps not surprising given the recent findings that GIF1/AN3 interacts with several chromatin remodelers to establish an SWI/SNF-AN3 module (Debernardi et al., 2014; Vercruyssen et al., 2014).
CONCLUSIONS AND PERSPECTIVES Research in recent years has uncovered a variety of growthrelated functions of GRFs in A. thaliana and other species, including crops. Despite the significant progress achieved so far, many key issues remain to be addressed in the future; in particular we only now are starting to get a glimpse of how GRFs work at the cellular level. For example, currently we do not know much about the control of GRF expression by upstream transcription regulators (or even further upstream signaling elements); the restricted expression of many GRFs in young growing tissues is intriguing, but not well understood. Similarly, our knowledge about the direct downstream target genes is limited, thus restricting the formulation of mechanistic models of GRF’s functions. However, cis-regulatory elements bound by GRF TFs are beginning to be identified (e.g. Kim et al., 2012), and more direct target genes (besides, e.g., the KNOX genes, or OsCR4 and OsJMJ706 in rice) will soon be discovered by ChIP-seq experiments (in combination with RNA-seq) using, e.g. transgenic plants that express individual GRFs under chemically inducible promoters. A particular challenge in such studies could be the selection of the correct (growing) tissue, for analysis as GRFs may not bind to their target genes in more developed organs when other cellular components (e.g. interacting TFs) are perhaps missing. Over time, we will also better understand how GRFs and, in particular, their gene regulatory networks evolved to affect growthrelated processes. Molecular Plant 8, 998–1010, July 2015 ª The Author 2015. 1007
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GRF Transcription Factors
Another interesting aspect of GRFs is their interaction with GIF proteins; our phylogenetic analysis revealed that while GRF gene copy number ranges from 8 to 20 in most land plant species, the number of GIF genes is considerably smaller (usually 3–5; Table 1). Furthermore, GIFs but not GRFs are present in green algae; thus, GIF1/AN3 may associate with chromatin remodeling proteins in these organisms, as in Embryophyta. The role of algal GIFs has, however, not been investigated thus far. With respect to land plants, it will be exciting to have a closer look at the potential effect of chromatin remodeling— through the SWI/SNF complex—on GRF function.
Casadevall, R., Rodriguez, R., Debernardi, J., Palatnik, J., and Casati, P. (2013). Repression of growth regulating factors by the microRNA396 inhibits cell proliferation by UV-B radiation in Arabidopsis leaves. Plant Cell 25:3570–3583.
At present, evidence is accumulating that GRFs integrate environmental stress signals with growth programs to help balance stress responses against growth. It will be very interesting to unravel the underlying interactions in greater detail and determine to which extent, and through which molecular elements, these two processes can be uncoupled in different environments or in dependence on nutrient availability. Such knowledge may in the future be employed for the development of superior crop varieties that show good growth and biomass accumulation even under stressful conditions.
Debernardi, J.M., Rodriguez, R.E., Mecchia, M.A., and Palatnik, J.F. (2012). Functional specialization of the plant miR396 regulatory network through distinct microRNA-target interactions. PLoS Genet. 8:e1002419.
SUPPLEMENTAL INFORMATION Supplemental Information is available at Molecular Plant Online.
ACKNOWLEDGMENTS M.A.O. and B.M.R. thank the University of Potsdam for financial support. No conflict of interest declared. B.M.R. and S.P. thank the German Research Foundation (DFG) for funding (FOR 948; MU 1199/14–2). We thank the three anonymous reviewers for helpful comments on our manuscript. Received: November 4, 2014 Revised: December 21, 2014 Accepted: January 13, 2015 Published: January 22, 2015
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