GROWTH-REGULATING FACTORs (GRFs) are sequence-specific DNA-binding transcription factors that regulate various aspects of plant growth and development. GRF proteins interact with a transcription cofactor, GRF-INTERACTING FACTOR (GIF), to form a functional transcriptional complex. For its activities, the GRF-GIF duo requires the SWITCH2/SUCROSE NONFERMENTING2 chromatin remodeling complex. One of the most conspicuous roles of the duo is conferring the meristematic potential on the proliferative and formative cells during organogenesis. GRF expression is post-transcriptionally down-regulated by microRNA396 (miR396), thus constructing the GRF-GIF-miR396 module and fine-tuning the duo’s action. Since the last comprehensive review articles were published over three years ago, many studies have added further insight into its action and elucidated new biological roles. The current review highlights recent advances in our understanding of how the GRF-GIF-miR396 module regulates plant growth and development. In addition, I revise the previous view on the evolutionary origin of the
Transcription factors control gene expression and thus regulate the patterns of plant growth and development. The number of transcription factors in
It has been more than three years since the last comprehensive review articles were published on the GRF-GIF-miR396 module (11–13). During that time, many reports have been published, elucidating its new biological roles and identifying its downstream and upstream genes as well as target
GRF proteins contain two highly conserved QLQ and WRC domains in the N-terminal half (6–8). The QLQ domain consists of highly conserved Gln-Leu-Gln (QX3LX2Q) and neighboring residues. The QLQ domain provides an interface for interacting with GIFs (9, 10). The WRC domain consists of the conserved spacing of three Cys and one His residues (CX9CX10CX2H, simply the C3H motif), which acts as a DNA-binding domain (DBD) (6, 7, 16–18). The C-terminal regions of GRF proteins are highly variable in length and composition of amino acid residues, and they function as a transactivation domain (7–10). AtGRFs with truncated C-termini have been shown to lose their transactivation activities, while OsGRF10 (rice) and ZmGRF10 (maize) with short C-termini have also exhibited no activities (9, 19, 20).
GIFs were identified by their capability interacting with GRFs and characterized as transcription cofactors with no DBD (9, 10). The interacting partnership between almost all members of the two protein families has been demonstrated in all plants tested (19–25). GIF proteins have the highly conserved SNH domain in the N-terminus that directly interacts with the GRF QLQ domain. The C-terminal regions of GIFs exert transactivation activities and are rich in Gln (Q) and Gly (G), and are thus called the QG domain.
As transcription factors with the WRC DBD, GRFs are expected to regulate the expression of downstream target genes and bind to specific regulatory
Aside from GTEs, chromatin immunoprecipitation assays (ChIP) revealed that AtGIF1/AN3 proteins were strongly associated with the G-box and GAGA elements in the Arabidopsis genome, and that these elements were found to reside in the promoters of some target genes, including
As mentioned above, ChIP assays revealed that AtGIF1/AN3 and maize GIF1/AN3 (ZmGIF1/ZmAN3) proteins were associated with the promoter regions of certain target genes (23, 25). The assays also showed that these associations were not limited to those known target genes, but detected widely over the whole genome of Arabidopsis, suggesting that AtGIF1/AN3 may be a key transcription cofactor acting together with GRFs and/or other transcription factors. Consistently with the notion, a series of tandem affinity purification (TAP) and co-immunoprecipitation (co-IP) approaches revealed that GIF1/AN3 proteins of Arabidopsis and maize were co-purified with the components of SWI2/SNF2 chromatin-remodeling complexes, including the core SWI2/SNF2 chromatin-remodeling ATPases, such as BRAHMA (BRM) and SPLAYED (SYD) (21, 23, 24). Upregulation of AtGIF1/AN3 target genes also required intact activities of BRM. These results give rise to the notion that GIF1/AN3 transcription cofactors may recruit both SWI2/SNF2 complexes and GRFs to GTEs, thus activating or repressing target genes (Fig. 1).
The results and notion are consistent with the fact that the human GIF homolog, SYT, directly interacts with the human BRM and its homolog (31, 32), as well as the fact that TAP experiments using the human SYT as bait also retrieved the components of human SWI2/SNF2 chromatin-remodeling complexes (33). The result suggests that the interaction between GIF1/AN3 and the SWI2/SNF2 complex may be mediated via direct interaction between GIF1/AN3 and plant BRM homologs, and that the interaction between GIF1/AN3 and the SWI2/SNF2 complex is not only evolutionarily conserved in metazoans and plants, but also essential for transcriptional regulation, despite the fact that metazoans lack GRFs.
Some additional interesting features of the GRF-GIF action are that they activate their own transcription through a positive feedback loop in Arabidopsis, rice, and maize, likely by forming the GRF-GIF-SWI2/SNF2 complex at the promoter sites of target GRFs and GIFs (Fig. 1; for detailed information, see 11, 25); and that Arabidopsis and rice GIF1 proteins translocate between different cell layers through plasmodesmata, thus coordinating the cell proliferation activities of different cell layers (34, 35).
In Arabidopsis, miR396 species, products of
The function of the GRF-miR396 module holds up for other eudicot plants as well. The overexpression of Arabidopsis and
It has been shown that the AtGIF family controls both the rate and duration of cell division (37, 44). An increase in the
The movement of the cell cycle arrest front (AF) from the distal to proximal regions of Arabidopsis leaf primordia during the early stages of leaf growth has been well documented (45).
A pea transcriptional complex consisting of BIGGER ORGANS (BIO) and ELEPHANT EAR LIKE LEAF1 (ELE1) negatively regulates leaf growth and interacts with a WUSCHEL-related transcription factor, LATHYDROIDES (LATH) (50). LATH has been shown to directly bind to a promoter region of a pea
CINCINNATA-like TCP (CIN-TCPs) transcription factors control the transition from cell proliferation to expansion during leaf morphogenesis and act as growth repressors (for review, see 52). The overexpression of Arabidopsis
It should be noted that not all Arabidopsis GRFs seem to act as positive regulators of leaf growth. The loss-of-function
It appears that monocot GRFs and GIFs primarily act as positive regulators of cell proliferation in leaves.
The overexpression of
It should be noted that, like loss-of-function mutant leaves of Arabidopsis
It has been reported that increases in the
Recently, an Arabidopsis gain-of-function mutant,
It was recently demonstrated that AtGRFs are required for the transition of stem cells into transit-amplifying cells in the root meristem (62). Briefly, the abolishment of
AtGIF1/AN3 also plays crucial roles in QC organization, which are, interestingly, independent of
Regarding monocot root growth, the roots of
Since GRFs and GIFs form a functional unit for transcriptional regulation, the deactivation of
The rod-shaped gynoecium phenotypes of those mutants were exacerbated by the
Rice GIF1/MKB3 has been shown to be involved in floral organ development, as spikelets of the
The deactivation of
Both the rice
Based on results derived from the deactivation of monocot
The Arabidopsis inflorescence stem showed a bi-phasic growth pattern in response to different dosages of
In monocot plants, the activities of the GRF-GIF-miR396 module affected the grain size and architecture of panicles, such as the length and number of branches as well as spikelet numbers. The up-regulation of
Evidence indicates a role of eudicot GRFs in determining seed size.
Syncytium formation occurring in Arabidopsis roots by an infective cyst nematode (
Genes involved in the regulation of defense responses and disease resistance were found to be enriched in the potential target candidates of AtGRF1 and AtGRF3 (79). In support of that, Arabidopsis plants expressing
In Arabidopsis leaves, UV-B light induced the accumulation of miR396 and thus reduced the abundance of
It has been described in the last review articles that GRFs are land plant-specific genes, since genomic and transcriptomic resources available then revealed their presence only in plants (land plants or embryophytes) but not in metazoans, fungi, and protists, including ‘green algae’ (11, 12). The ‘green algae’ are members of chlorophytes and charophytes that are paraphyletic to land plants (Fig. 2). However, a recent paper reported the presence of a single GRF gene in the genome sequence of a charophyte,
How could the GRF gene have been invented in an ancestral charophyte? This question may remain unanswered for years. One may speculate that an ancient QLQ domain derived from the N-terminus of the SWI2/SNF2 chromatin-emodeling ATPases (BRM and its homologs) have acquired the WRC domain, resulting in an ancestral GRF gene (9, 11). SWI2/SNF2 ATPases are universally present in eukaryotes, including viridiplantae, and they play essential roles in the chromatin remodeling process (11, 87, 88). According to the Pfam profile, QLQ domains exist in 66 different architectures with 2303 entries (http://pfam.xfam.org, PF08880). Half of the entry proteins have the QLQ domain together with WRC as GRF proteins; roughly the other half together with the SNF2_N domain of the SWI2/SNF2 ATPases; and only a few entries are together with other kinds of domains. These combinatorial structures with QLQ are compatible with the notion that the SWI2/SNF2 ATPase QLQ domain might be co-opted into an ancient GRF gene.
The Pfam profile also reveals that the WRC domain, which contains the DNA-binding C3H motif, is present in streptophytes and Mamiellophyceae, but is not present in Chlorophyceae and Trebouxiophyceae or any other organisms (Fig. 2; http://pfam.xfam.org, PF08879). WRC domains exist in 26 different architectures with 1984 entries: more than half of the entry proteins have the WRC domain together with the QLQ domain as GRFs, a quarter are mostly uncharacterized proteins with a single WRC domain but with no associated known domains, and the rest have single or multiple WRC domains associated with other kinds of known domains. Interestingly, the GRFs of
The origin of GIF genes is much more ancient than that of GRFs, as they exist in most eukaryotes, such as virideplantae, and metazoans, and not in fungi and protists other than ‘green algae’ (11, 12). GIFs are present in the genomes of a charophyte (
Both genomic and cDNA sequences were available for some of those algal GIFs (
In summary, the GRF-GIF-miR396 module plays essential roles in the growth and development of angiosperms. It regulates the meristematic potential of primordial cells during leaf growth, determining the final size and shape of the leaf organ. The GRF-GIF duo is a prerequisite for floral organ development, and thus enables the production of the formative cells, such as CMMs and egg cells as well as microsporangia and sperm cells. It is also involved in the regulation of leaf longevity and photosynthetic efficiency in mature leaves. Importantly, the monocot GRF-GIF duo also promoted the yield traits, such as grain size and panicle architecture, warranting crop productivity. Finally, the GRF gene has a charophycean origin, so studies on GRFs of basalmost land plants and charophytes could shed light on their significance in the evolution-developmental history of a main lineage of life, the streptophyte.
This work was supported by the Korea Research Foundation Grants, 2015R1D1A1A01059934 and 2018R1D1A1B07050016. I apologize to all colleagues whose work could not be cited due to space constraints.
The authors have no conflicting interests.
|Proteins||Target genes||Transcriptional regulation|
aNucleotide sequences read from the 5′ to 3′ direction.
bMinus and plus symbolize up- and down-regulation of target gene expression, respectively.
cS indicates G and C; M, A and C; R, A and G.