Blooming Time for Plant Glycine-Rich Proteins

Abstract

Glycine-rich regions are proposed to be involved in protein-protein and protein-nucleic acid interactions in some mammalian protein families. In plants the occurrence of quasi-repetitive glycine-rich peptides has been reported in different species. They are characterized by having the glycine residues arranged in characteristic repetitive structural motifs, but with distinct primary sequence. The expression of genes encoding glycine-rich proteins (GRP) is developmentally regulated, and also induced, in several plant genera, by physical, chemical and biological factors. The diverse expression pattern of GRP genes, taken together with the distinct sub-cellular localisation of some GRP groups indicate that these proteins are involved in several independent physiological processes. Notwithstanding the absence of a clear definition of the role of GRPs in plant cells, experimental data coming from different research groups have shed some light on the biological function of some GRPs. One of these proteins is AtGRP2, a member of the Cold-Shock Domain protein family in Arabidopsis. AtGRP2 is a nucleo-cytoplasmic protein involved in Arabidopsis development. Expression analysis revealed that the AtGRP2 gene is active in meristematic tissues, being modulated during flower development. Downregulation of AtGRP2 gene using gene silecing techniques resulted in early flowering, altered stamen number and affected seed development.

The glycine-rich protein (GRP) super-family corresponds to a large and complex group of plant proteins that share, as common feature, the presence of glycine-rich domains arranged in (Gly)n-X repeats, which are thought to be involved in protein-protein interactions. Initially isolated from plants, GRP genes encoding proteins with characteristic repetitive glycine stretches have been reported in a wide variety of organisms from cyanobacteria to animals.¹ More than 150 different GRP genes have been identified after a whole-genome analysis of sugarcane,² eucalyptus³ and Arabidopsis (Galvão and Sachetto-Martins et al., unpublished results). Despite the extensive number of reports describing the occurrence of these genes in different species, very little is known about their biological role in plants.

Only recently, experimental data has been generated that shed some light on the biological function of some GRPs. Mayfield and Preuss⁴ demonstrated the involvement of an Arabidopsis oleosin-GRP (AtGRP7/AtOlnB;3/GRP17) in pollen recognition and hydration. GRPs have also been implicated in signal transduction by the characterization of AtGRP3 as the extracellular ligand of the plant receptor kinase WAK1.⁵ Ueki and Citovsky⁶ showed that the expression of a cadmium-induced GRP (cdiGRP) enhances callose deposition and is responsible for inhibiting the long-distance movement of Turnip vein clearing tobamovirus (TVCV) in tobacco plants. The involvement of RNA-binding GRPs in plant cold acclimation has also been observed.^7,8

The diversity of functions and structural domains, together with the different but highly specific expression patterns and distinct sub-cellular localizations of GRPs, indicate that this complex group of proteins is likely to be implicated in numerous independent physiological processes. This diversity led to the concept that GRPs should not be considered as a family of related proteins but as a wide group of proteins that share a common structural domain.¹

Based on their primary structure and functional domains, GRPs can be classified into four major groups.² GRPs from class I are known as the “classic” GRPs. They may contain a signal peptide followed by a glycine-rich region with GGGX repeats. A structural function is attributed to proteins of this class due to their cell wall localization.⁹ The class II GRPs contain a glycine-rich region followed by a cysteine-rich region at their C-terminus. For one member of this family, AtGRP-3, this cysteine-rich domain has been shown to interact with WAKs.⁵ The class III GRPs are proteins with lower glycine content that show a great diversity of structures. The best known proteins from this class are oleosin GRPs, which play a structural role in stabilizing the triacylglycerols of the oil bodies. Previous works demonstrate that several major pollen coat proteins are derived from an endoproteolytic cleavage of oleosin GRPs that originally accumulate within the large cytoplasmic lipid bodies of tapetal cells.¹⁰ GRPs from class IV are RNA-binding GRPs. These GRPs may contain, besides the glycine-rich region, several motifs which include the RNA-recognition motif, the cold-shock domain and zinc fingers.

A subgroup of plant RNA-binding GRPs, which is likely to be involved in cold acclimation, features a cold shock domain (CSD) in the N-terminal half and CCHC retroviral-like zinc fingers in its C-terminal region.¹¹ CDS-containing proteins that function as RNA chaperones have been described in bacteria¹² and, more recently, in plants.¹³

In an attempt to further understand the role of CSD/GRP proteins in plants, we have proceeded to the functional characterization of the AtGRP2 gene, which encodes one of the four members of the CSD protein family in Arabidopsis.

We have shown that the AtGRP2 protein binds ssDNA, dsDNA and RNA homopolymers, and that it has a nucleo-cytoplasmic localization. Investigation of the spatial and temporal expression pattern of the AtGRP2 gene under various conditions demonstrated that AtGRP2 is cold-regulated and preferentially expressed in meristematic and developing tissues, with a tissue-specific expression pattern which is significantly modulated during flower development. We could further demonstrate that silencing of AtGRP2 causes anticipation of flowering, altered stamen number and affected seed development.¹⁴

At the moment, it is not clear how AtGRP2 might act in the flowering time pathway. One hypothesis puts forward that AtGRP2 controls the expression of flowering time regulators at the post-transcriptional level by affecting mRNA processing, stability, or export from the nucleus. It is possible that in the presence of a reduced level of AtGRP2, transcripts of some flowering time regulators become unstable and are rapidly degraded.

Posttranscriptional gene regulation is a critical tool for the regulation of eukaryotic gene expression during growth and development. Many reports highlight the importance of RNA-binding proteins mediating those processes. A KH motif-containing protein, HEN4, is involved in AGAMOUS pre-mRNA processing.¹⁵ HEN4 physically and functionally interacts with HUA1,¹⁶ another RNA-binding protein, and directly binds to AGAMOUS pre-mRNA.¹⁵ CCR2/AtGRP7, a nuclear RNA-binding protein with an RRM motif is a component of a circadian-regulated negative feedback loop in Arabidopsis.¹⁷

The regulation of flowering time by RNA-binding proteins has been observed. FPA, FCA and FLK have been isolated by forward or reverse genetics screenings.^18–21 Those mutants flower late, indicating that the wild-type alleles promote flowering. AtGRP2 seems to operate in the opposite direction, since its downregulation anticipates flowering. These data are in agreement with results derived from AtGRP2-overexpressed transgenic lines, which show a delay in flowering (Bocca, Ramos and Sachetto-Martins, unpublished results).

Floral repression is likely to be the principal mechanism for maintaining vegetative development since genes promoting vegetative growth act by repressing genes required for flower development. Genes with early-flowering mutant phenotype are considered to be floral repressors that inhibit the floral signaling pathways at various levels.²²

It is possible that in the presence of a reduced level of AtGRP2, transcripts of some flowering time regulators become unstable and are rapidly degraded or translated inefficiently. Although additional research is needed in order to gain more insight into the AtGRP2 function, our study demonstrates that AtGRP2 plays an important role during Arabidopsis development with a possible function in cold-response.

Addendum to: Fusaro AF, Bocca SN, Ramos RL, Barrôco RM, Magioli C, Jorge VC, Coutinho TC, Rangel-Lima CM, De Rycke R, Inzé D, Engler G, Sachetto-Martins G. AtGRP2, A Cold-Induced Nucleo-Cytoplasmic RNA-Binding Protein, has a Role in Flower and Seed Development. Planta. 2007;225:1339–1351. doi: 10.1007/s00425-006-0444-4.