Analysis of RAVs
RAVs belonged to the AP2/ERF superfamily [27, 28], we took several representative plants in different evolutionary stages as the research objects to analyzed the evolution of RAVs. The RAV family has been identified in many species, such as sweet orange, Vitis vinifera, rice, maize, wheat, soybean, and so on [13, 29–33]. We integrated these results, and we searched and identified RAV family again using the methods described in this article. The result we identified was almost the same with that in other articles. However, 13 RAVs were identified in soybean , several gene which did not contain the AP2 domain should be excluded. In maize, 4 RAVs were screened by Zhou et. al using more kinds of database, which was different with the method we used .
The RAVs are widely associated with the plant growth and development and the regulation against adverse reactions. The overexpression of AtRAV1 and AtRAV2 in cotton can enhance the fiber length differentially under drought stress and delays flowering . The overexpression of CARAV1 which is response to the pathogen infection from pepper increases the tolerance to drought and salt, and enhancing the sensitivity to ABA in transgenic Arabidopsis . A RAV-like protein from soybean is involved in the process of photosynthesis and senescence. Transgenic Nicotiana tabacum (tobacco) plants overexpressing the RAV-like gene will have a slow growth rate, delayed root elongation, delayed flowering time, and a significantly reduced chlorophyll content in the leaves . The RAV family has been identified and studied mainly in eudicots plant species and monocots plant species, especially in Eudicots, but rarely in other plant stages. In Bryophytes and Gymnospermaes, no research related to RAVs is found, while the genome sequences of S. moellendorffii in Lycophytes and A. trichopoda are reported, respectively [36, 37]. Although RAVs have been identified in several other early plants, there has been no further research, either . While we can predict the function of more RAVs using the database of the Phytozome website
We predicted 97 RAVs (Additional file 6) which had no transmembrane domain and signal peptide (same as other transcription factors)  and analyzed B3 and AP2 domains in each protein. Each protein had and only had both B3 and AP2 domains, except CbRAV4 which had an extra domain: MetAP1. Additionally, A complete gene sequence consists of exons and introns, and exons, as part of the coding sequence, take an crucial part in gene function . The RAV members of P. abies had 1–3 exons. PhRAV1 had 3 exons. A few angiosperms’ RAVs and most RAVs of Bryophytes and Lycophytes had two exons, and the most RAV members of the angiosperms pant species had only one exon. According to the analysis result, it could be speculated that some introns have been lost from the Bryophytes with the continuous evolution of plants. This is consistent with the previous conclusion . Because the RAV protein structures and features in Bryophyta did not vary greatly (Fig. 1, Additional file 1b,c), we inferred that the intron losses that occurred during plant evolution was the reason of the number differences of exons between different species. The phenomenon has been confirmed in previous study . However, CbRAV4 had 11 exons which is completely incompatible with the plant evolution condition, and we have not been able to find relevant studies to confirm this phenomenon. Combined with the results of conserved domain analysis, we suggest that CbRAV4 may have evolved into a protein of other families (MetAP family) or formed new functions , so that it has formed a significant difference from the RAV family.
Evolution of RAVs
RAVs are divided into 3 main groups, which is consistent with previous research . Phylogenetic results showed that most non-vascular plants (Bryophytes), A. trichopoda, P. abies and some eudicots plant species were composed of Clade I. Clade II only contained eudicots plant species. The only RAV gene in A. trichopoda was located in Clade I, which could be explained that because in A. trichopoda, which belongs to the Angiosperm and is a sister of flowering plants, six exogenous genomes constructed the mitochondrial genome, one from moss, two from other flowering plants, and three from green algae (Chlorophytes did not contain RAVs) . The RAVs of eudicots plant species may be derived from the Bryophytes, and have formed the proteins belonging to Clade II with special functions during the evolution process. However, the RAV members of the monocots plant species and M. polymorpha belong to Clade III, which indicates that the RAVs in Monocots may also have evolved from Bryophytes and have common ancestors with Eudicots, but the protein functions are differentiated during the evolution process in Angiosperms. Moreover, there are the most RAVs in Gymnosperms, which may mean that there are more obvious evolutions in Gymnosperms than that in Angiosperms. In general, phylogenetic trees suggest that vascular plants may have a common ancestor, but the ancestor of Monocots is inherited from the MpRAV1 protein of Clade III in M. polymorpha, while the ancestor of other seed and non-seed plants may be inherited from the proteins from Bryophytes which are located in Clade I (Fig. 2). With the development of vascular plants, the number of RAV members has increased dramatically (Fig. 3). In addition, due to the use of different protein databases, selection of different species to study, and using a different website/program to analyze, all will affect the analysis results, so the above views need be confirmed by more evidence.
Different groups of RAVs may have different functions in different plant species. The overexpressing of AtRAV1 in Arabidopsis results in a retardation of lateral root and rosette leaf development, and the expression inhibition will lead to an earlier flowering phenotype, indicating that AtRAV1 may function as a negative regulatory component of growth and development . The mRNA level of AtRAV1 from Arabidopsis showed an increase, the highest level, and a decrease in the three leaf stages of late maturity, early senescence and late senescence, which suggested that AtRAV1 has an significant role in regulating leaf senescence . In transgenic tomato plants, with the overexpression and silencing SlRAV2, the expression levels of SlERF5 and PR5 increased and decreased, which enhanced or weakened the BW tolerance, respectively . The BnaRAV-1-HY15 TF in B. napus is highly identical to AtRAV2 in Arabidopsis. The expression of BnaRAV-1-HY15 will be induced by cold, salt and PEG treatments, and is insensitivity to ABA . The more similar the distribution of genes in the same family on the phylogenetic tree, the more similar their functions in different plants . The same class probably means similar characteristic of RAV family. Therefore, we can speculate the function of a RAV gene in any species based on the existing research results, thus providing effective ideas and directions for further research .
Gene duplication events, Ka/Ks ratio, and GO annotation
In all living organisms, gene duplication events are a very common matter, which provides a basis for the formation of new functional genes and the function differentiation of genes in organisms. This also leads to species differences and species specificity [46, 47]. Our finding indicated that gene duplication events appeared only present in Eudicots and Monocots with the evolution of plants. There was no gene duplication in other species because of a small number or none of RAVs. But in P. abies, no gene duplications were identified from 16 RAVs, either. The evolution of RAVs was also related to gene duplications. The increasing of the duplications of RAVs following the evolution of plants, providing evidence for confirming the roles of this family in plant evolution. In addition, this phenomenon that gene duplication events only exist in Eudicots and Monocots also is consistent with the appearance of the function differentiation of RAVs.
The Ka/Ks values calculated by DnaSP software indicated all gene duplication events were suffering purifying selection, which meaning that these plants eliminate deleterious mutations during evolution process, and keeping the protein as it is . We calculated Ka/Ks values in each of the plant species’ RAVs, and found that except for PaRAV13 and PaRAV16 in P. abies with a Ka/Ks value of 2.70, the other genes were all stable and were all under purify selection (Additional file 3). Overall, RAV gene family is relatively stable through the process of plant evolution.
GO annotation is one way to predict the function of genes in terms of BP, MF and CC . Based on these results of GO annotations, about a half of RAVs clustered in the nucleus, the main molecular functions of RAVs were in transcription factor activity and DNA binding, and the main biology process was regulation of transcription and DNA-template (Fig. 4, Additional file 5). While, CbRAV4 in C. rubella was also predicted be related to proteolysis, metal ion binding, metalloexopeptidase activity, and aminopeptidase activity, which was the same as the result of conserved domain and exons analysis, and showed the specificity of the gene CbRAV4.