**6. Conclusions**

212 Gene Duplication

The existence of multiple genes for each subunit in the plant genome indicates that the specificity of subunit interaction may be determined by preferential protein–protein interaction, tissue or cell-specific expression of each gene or a combination of both (Yazawa and Kamada, 2007). The large number of possible combinations has hindered the analysis of plant NF-Y complexes and suggests that they might act in a more intricate system than in vertebrates and yeast, which have only one gene that encodes each HAP subunit (Yazawa and Kamada, 2007). Additionally, the multiple copies for each NF-Y subunit raises a question if a specific NF-Y subunit interacts with any other two NF-Y subunits or if the NF-Y subunit interacts with only specific member(s) of the other two subunits (Thirumurugan *et* 

Although the presence of many genes encoding NF-Y subunits suggests a high degree of genetic redundancy in plants, the analysis of mutants in single NF-Y genes in Arabidopsis has been associated with defects in development and enhanced stress sensitivity, suggesting a specialized function for each member (Lotan *et al.*, 1998; Kwong *et al.*, 2003; Lee *et al.*, 2003; Zanetti *et al.*, 2010). This could indicate that duplicated genes have passed through a

Some proteins may require several key substitutions before acquiring a new function, while others may be more mutationally labile. An example includes the terpene synthase gene family in Norway spruce (*Picea abies*). These genes appear to have undergone repeated rounds of neofunctionalization (Figure 2b) (Keeling *et al.*, 2008) and a small number of key amino acid substitutions among paralogs was sufficient to alter the substrate specificity and terpenoid product profiles (Flagel and Wendel, 2009). Another example of neofunctionalization (Figure 2b) in plants is observed in Arabidopsis, where a specific amino acid residue identified in LEC1 and LEC1-LIKE (L1L) is responsible for differentiating their functions (seed development) from those of other NF-YB members (Kwong *et al.*, 2003; Lee *et al.*, 2003; Yamamoto *et al.*, 2009). In addition, the analysis of amino acid substitution rates in plants has been appointed for the asymmetric evolution of certain duplicates of NF-YB and NF-YC subunits, which appears to be coupled with the asymmetric

With respect to expression patterns, the Arabidopsis NF-Y gene family presents some members that are ubiquitously expressed and others that are tissue specific or induced only after the switch to reproductive growth in flowers and siliques (Gusmaroli *et al.*, 2001; 2002; Yazawa and Kamada, 2007). The difference observed in the expression pattern of these genes could represent an example of *cis*-regulatory divergence (Figure 3c), where the *cis*element of gene evolves independently from the other members of gene family, and

Because genes that harbor NF-Y binding domains include genes that are constitutive, inducible, and cell-cycle-dependent, the regulation of the expression of these genes cannot be exclusively due to NF-Y binding to DNA. In this scenario, the interaction with other transcription factors, either functionally or physically, will contribute to the NF-Y action (Matuoka and Chen, 2002). In addition, the independent evolution of protein-binding domains present in duplicated gene architecture can contribute to protein network divergence (Figure 3b2), increasing the numbers of possible interacting partners of NF-Y

When compared with other forms of mutation, a notable feature of duplication is that it creates genetic redundancy. This redundancy fosters evolutionary innovation, creating the opportunity for duplicates to explore new evolutionary terrain (Flagel and Wendel, 2009).

divergence in gene function (Yang *et al.*, 2005; Yamamoto *et al.*, 2009).

becomes regulated by different stimuli and/or *trans*-activators.

*al.*, 2008).

genes.

neofunctionalization process (Figure 2b).

Whereas various classes of structural and metabolic genes preferentially return to a single copy state following whole-genome duplication (Paterson *et al.*, 2010), transcription factors tend to be preferentially retained among the duplicated genes in *A. thaliana* (Flagel and Wendel, 2009). Our findings support the hypotheses that this preference seems to be true for all plant species, based on the number of genes identified for each NF-Y subunit. Certainly, further studies encompassing functional assays are required to ascertain the role of these genes in plant metabolism.

The number of interacting partners in a molecular network (connectivity) of a particular gene also influences the probability of duplication gene retention (Flagel and Wendel, 2009). In this scenario, the high number of genes coding for the three subunits of NF-Y transcription factor in higher plants leads to numerous interaction possibilities among different genes of each subunit and among these genes and other transcription factors what could contribute to gene retention of the NF-Y transcription factor family in plants.
