GIGANTUS1 is here described to be very important in regulating plant growth development (seed germination, faster growth, flowering time, and biomass accumulation) (Figures 1, 2 and 3). This is the first time that a mutation in GTS1 has been implicated in early germination, growth and development in Arabidopsis thaliana. The molecular mechanism by which GIGANTUS1 regulates plant growth development is still unknown. As a member of WD40 protein family, GTS1 is expected to play central roles in different biological processes including cell division and cytokinesis, flowering, floral development, cytoskeleton dynamics, nuclear export to RNA processing, transcriptional mechanism, and protein-protein interactions . We postulate that GIGANTUS1 might primarily function as a site for protein-protein interaction or mediator of transient interplay among other proteins to regulate different biological processes in plants. The development of protein complexes involves regulatory interactions that are mainly controlled by scaffolding proteins, such as WD40 repeat motifs. These motifs are important features of diverse protein-protein interactions , providing an unbending platform for interactions of proteins with other cellular components and controlling therefore several vital functions of the cell, such as signaling cascades, cellular transport and apoptosis [29–31].
The WD40 domains in GTS1 protein are shown to contain seven or multiples of seven repeats forming a highly stable β-propeller structure (Figure 5A). The 7-fold β-propeller is the most stable β-sheet geometry characterizing the resolved WD40 structures and also used to identify WD40 proteins . However members of this protein family have been also found to contain as high as sixteen repeats . Proteins with less than 7 repeats form an incomplete β-propeller structure and require additional WD-repeats from their neighbors to stabilize themselves, making dimers . There is no apparent folding order for each repeat and the order in which repeats fold might vary among different WD40 proteins, or even within the same protein . These proteins are known be involved in light signaling/photomorphogenesis and flowering , auxin response and cell division , in meristem maintenance , floral development , seed development and flowering , chromatin-based gene silencing and organogenesis , protein turn-over, microtubule dynamics, phospholipid binding and vesicle coating . This justifies the great deal of research interest in the WD40 protein superfamily across plant species. Our data (Figures 2 and 3) suggest that GTS1 belongs to the WD40 protein subfamily regulating auxin response and cell division , meristem maintenance , floral development , seed development and flowering . In this study the Arabidopsis GTS1 ligand-binding domain (main functional domain) lies mainly on the top surface residues, which integrates parts of β-propeller domain (Figure 6B). However, our data revealed that GTS1 WD40 propellers have three distinct surfaces available for interactions: the top region of the propeller, the bottom region (Figure 9), and the circumference [4, 5] (Figures 8 and 9), suggesting the multi-functional properties of GTS1 protein through protein-protein interactions. Indeed, protein-protein and protein-peptide interactions involved the entry site of the central channel of the β-propeller (Figure 6B), where the majority of interaction partners (including small molecules) bind . N- or C-terminal extensions of GTS1 run parallel to the tunnel-like structure, which form the complete 7 WD40 repeat domains, making them accessible for interaction with other partners (Figures 8 and 9). WD40 domains can thus act as large interaction platforms for multiple protein interactions. In comparison to other domains, the proteins containing WD40 motif are components of several interaction pairs [41, 42], and act as scaffolds for larger complex assemblies. This work represents the first time the 3D-structural and molecular features of the GTS1 protein in plants have been examined.
To better understand what area of the GTS1 plays an important role in complex formation for functional conservation across plant species, we identified two fundamentally conserved regions; the first, located on the top rim, constituted by the residues composing blades 4 to 7, including N- and C-terminal arms; and the second large conserved surface is located on the bottom of the propeller and is mainly composed of blades 1, 2 and 3 (Figure 6A). These regions represent potential protein-protein interaction sites .
In general, plant and particularly Arabidopsis-WD repeat proteins are strongly conserved. Most of these proteins are components of basic cellular machinery regulating plant-specific processes. An interesting question arises as to how these proteins evolved into their specific cellular roles. One of the key functional processes of WD proteins is the biogenesis of eukaryotic ribosomes, a highly regulated and dynamic process that begins in the nucleolus with transcription of rRNA precursor (pre-rRNA) and rapidly packaged into the 90S ribonucleoprotein particle containing ribosomal proteins, non-ribosomal proteins, and snoRNA-containing ribonucleoprotein particles (snoRNPs). The 90S pre-RNPs are converted into 43S and 66S ribosome assembly intermediates, which ultimately give rise to mature 40S and 60S ribosomal subunits . It is well known that ribosome biogenesis is driven by a large number of pre-ribosomal factors that associate with and/or dissociate from the pre-ribosomal particles along the maturation pathway. Although there has been much progress to identify ribosome assembly intermediates and their protein and RNA constituents , the information about the architecture of these pre-rRNPs is scarce. It is unclear which proteins are the nearest neighbors within the assembled ribosomes and to what extent neighboring molecules function together.
WD40 protein-protein interaction motifs represent excellent candidates to mediate interactions in the multiprotein subcomplex comprising a neighborhood in assembling ribosomes because of their protein-protein multi-interacting versatility. More than 70 trans-acting factors required for ribosome assembly have been identified , as well as 80 additional assembly factors present in pre-ribosomes . Therefore, such WD40-containing proteins may nucleate the assembly of pre-ribosomes by interacting sequentially or simultaneously with other assembly factors or ribosomal proteins. Among the assembly factors, 17 proteins were found to contain WD40 motifs . Many of the annotated ribosome biogenesis-WD40 repeat proteins were shown to directly interact with, or regulate the levels of other proteins  or to be components of multiprotein subcomplexes. Yeast WD40 protein Ytm1 is a constituent of 66S pre-rRNPs, whose depletion resulted in a deficiency of 60S ribosomal subunits . Its homologue, mammalian WDR12 functions in the maturation of the 60S ribosomal subunit. WDR12 forms a stable complex with a novel member the nucleolar proteins Pes1 and Bop1 (Pe- BoW complex), which are crucial for processing of the 32S precursor ribosomal RNA (rRNA) and cell proliferation . Interestingly, a potential homologous complex of Pes1–Bop1–WDR12 in yeast (Nop7p-Erb1p-Ytm1p) is involved in the control of ribosome biogenesis and S phase entry .
The yeast WD40 repeat protein Mak11 that modulates a p21-activated protein kinase function is an essential factor in nuclear maturation of 60S ribosomal subunits and its depletion led to a cell cycle delay in G1, indicating an early step nucleolar role of Mak11 in ribosome assembly. Another sub-complex, transiently associated with late, nuclear pre-60S precursors, is composed of four proteins and contains Ipi3 as a WD40 repeat member .
In this study, a new interacting counterpart, the Arabidopsis Nop16 protein was identified as a potential ribosome biogenesis factor in plants, which could be implicated in formation of the 60S ribosomal precursor. This process may be regulated by an interaction with GTS1 (Figure 8). This interaction was studied using a docking analysis that showed a stable interaction between GTS1 and Nop16, involving the N-terminal tail and the 4th blade of the first partner, and a cleft formed in the second ribosomal factor by the N-terminal α-helix and the neighboring secondary elements. Another ribosomal protein (L19e protein) was found to interact with GTS1. L19e protein is implicated in the structural stability of ribosome. The interacting area between GTS1 and L19e is very close to that of Nop16 and GTS1 interacting area (Figure 8). This suggests that the interacting mechanism of regulating the biogenesis nucleolar factor Nop16 and the structural ribosome factor L19e may be competitive. Therefore both steps in the 60S ribosomal subunit formation, structural maturation and stabilization may be separate in the time and/or different cellular compartments.
In support of our data are two other examples of WD40 repeat ribosome biogenesis factors, Rrb1 and Sqt1, which interact directly with ribosomal proteins for 60S ribosomal subunit assembly. Rrb1 interacts with the ribosomal protein Rpl3 in the nucleus and regulates its levels , and Sqt1 interacts with Rpl10 in the cytoplasm . Both proteins have a role in the association of the corresponding ribosomal protein with the nascent 60S ribosomal subunits and might regulate the levels of the corresponding ribosomal protein. Other WD40 repeat proteins have been implicated in the formation and stabilization of the small ribosomal subunit 40S. Yeast RACK1 regulates the translation initiation by recruiting PKC to the ribosome [55, 56]. Four RACK1 orthologs identified in Arabidopsis thaliana may have a similar activity . These interactions could provide a mechanism to regulate translation activities of ribosome populations programmed with specific mRNAs .