1. Introduction
Plant growth and development require numerous rigorous regulatory processes, and therefore, transcriptional regulation plays an important role in every stage of plant growth and development. TFs bind to their target genes or adjacent regions and control gene expression by turning them on and off as needed [
1,
2], and therefore, they play crucial roles in regulating various plant processes and stress responses. To date, many TF families and their binding sites have been reported [
3]. One such family of TFs is the B3 superfamily, which regulates the expression of various genes. B3 proteins are expressed in various plant tissues, suggesting a role for B3 proteins in different plant processes [
4]. NGA belongs to the RELATED TO ABI3/VP1 (RAV) transcriptional subfamily, forming a subgroup of the B3 superfamily. The RAV subfamily is further divided into two categories: Class-I (proteins with a B3 domain and an AP2 domain) and Class-II (proteins have a B3 domain but no AP2 domain) [
5]. Exceptionally,
Merchantia Polymorpha has two B3 domains in NGA3 [
4]. Although the NGA family has been studied in
A. thaliana and
Brassica napus, it has been poorly explored in other plant species.
In Arabidopsis, four
NGA genes (
AtNGA1,
AtNGA2,
AtNGA3,
AtNGA4) and three
NGA-LIKE genes (
AtNGA-LIKE1,
AtNGA-LIKE2,
AtNGA-LIKE3) have been reported [
6,
7,
8,
9,
10,
11,
12]. NGA TFs are mainly involved in developing pistils; they are also involved in regulating the shape and size of lateral organs such as leaves and petals and the regulation of seed size [
6,
8,
9,
13,
14,
15,
16,
17,
18]. Alvarez et al. [
19] used synthetic miRNAs against
A. thaliana NGA genes, showing that single
NGA mutants exhibited mild phenotypic changes in lateral organs, while quadruple mutants exhibited defective pistils as well as small, broad leaves with the broad perianth [
19]. Later, in 2009, the same group published a detailed report on the
NGA genes,
AtNGA1 and
AtNGA4, expressed in the lateral organs, especially the distal parts [
6,
20]. The gene
STYLISH1 (
AtSTY1), known to be involved in carpel development, activates the NGA gene that indirectly regulates gynoecium development. STY1 and NGA co-regulate the
YUCCA2 (
AtYUC2) and
AtYUC4 genes that promote auxin biosynthesis, thereby regulating the auxin gradient in pistils. In addition, STY1 is known to directly activate
AtYUC4, suggesting a direct link to auxin biosynthesis [
6,
20]. This was further confirmed by Trigueros et al. [
12]. The same group identified a
tower of pisa-1 (
top1) mutant by activation markers in Arabidopsis and observed that this mutant was impaired in silique development, with enlarged patterns and reduced fruit size. The
top1 mutant was found to overexpress
AtNGA3 (due to random T-DNA insertions containing the 4x 35S promoter). As a result, the
TOP1/
NGA3 mutant showed an elongated style [
12]. VIGS mediated silencing of
EcNGA in
Eschscholzia californica resulted in pistils with impaired style and stigma, but the other parts of the flower such as the petals, sepals, and stamens were unaffected, indicating that NGA is redundant in pistil development. Similarly,
NbNGAa and
NbNGAb were downregulated in
Nicotiana benthamiana using VIGS and it was observed that the length of the style was significantly reduced with improperly fused stigma. It has been documented that the
YUCCA (
YUC) genes are involved in auxin biosynthesis and its accumulation in the gynoecium. In Arabidopsis, reduced expression of
YUC genes was reported in
nga mutants, resulting in reduced auxin accumulation in the pistils of
nga mutants [
12]. Furthermore, the expression of
NbYUC2 and
NbYUC6 in the apical portion of the gynoecium was decreased in the silenced
NbNGAa and
NbNGAb lines, suggesting that the role of
NGA genes in activating
YUC genes is involved in promoting auxin gradient across the pistil [
7].
NGA TFs also play critical roles in lateral organ growth and development. Lee et al. [
8] measured the cell proliferation activity in the lateral organs of overexpression lines and loss-of-function mutants of the NGA family in Arabidopsis, and they observed small, narrow leaves in the overexpression lines and large, wide lateral organs in the quadruple mutants. These results suggest that the NGA family negatively controls cell proliferation in lateral organs [
8]. Similar results were observed when
BrNGA1 from Brassica was overexpressed in Arabidopsis, suggesting that
BrNGA1 regulates cell numbers in the lateral organs and roots [
21]. This was further supported by the study of Lee et al. [
9]. They expressed AtNGA1 in the presence of domains such as CLAVATA3 (CLV3), Meristem layer1 (ML1), WUSCHEL (WUS), SHOOTMERISTEMLESS (STM), and AINTEGUMENTA (ANT) in Arabidopsis. Their results showed that NGA expression in meristems incapacitated pluripotent cells, rendering them incapable of cell differentiation, suggesting an important role for NGA TFs as general differentiation and pistil group identity factors [
9].
Despite the limited literature available, NGA has also been involved in various stress responses [
10,
22]. Sato et al. [
10] overexpressed
AtNGA1-GFP under the influence of the 35S promoter, examined the two-week old seedlings subjected to water stress under the confocal microscope and observed increased protein accumulation of
AtNGA1-GFP under drought stress compared to the control conditions. They further showed that NGA1 binds to the G-box of the
AtNCED3 promoter under water-deficit conditions to induce ABA biosynthesis. The study also confirmed that the binding of AtNGA1 to the promoter of
AtNCED3 increased under drought stress. A similar pattern was seen in ABA-deficient mutants of Arabidopsis, where NGA induced
AtNCED3 to synthesize ABA in response to stress. In addition, a study by Guo et al. [
22] showed that the overexpression of
MtNGA1 from
Medicago truncatula in
A. thaliana exhibited increased tolerance to high salt stress. They also exhibited a reduction in the number of branches in the overexpressed lines along with delayed flowering, indicating the importance of NGA as key players in crucial aspects of plant development as well as stress responses. They also examined the reduced shoot branching by analyzing the transcript levels of SMXL genes in the MtNGA1 overexpression lines to observe that the transcript levels of
AtSMXL6,
AtSMXL7, and
AtSMXL8 were downregulated while the expression of
AtMAX1/2,
AtBRC1, and
AtBRC2 were up-regulated. The repressed shoot branching in the transgenic lines provides important evidence that NGA not only influences ABA, but also regulates strigolactones [
22].
To date, phylogenetic analyses of the NGA family of a few plant species such as
A. thaliana,
B. napus,
G. max,
B. distachyon,
O. sativa,
P. patens, and
M. truncatula have been reported in the literature [
10,
21]. Furthermore, Pfannebecker et al. [
23] combined the phylogeny of members of the NGA family of cruciferous, nightshade, and grass families. Their study concluded that each gene family evolved independently through several rounds of gene duplication events.
In this study, we performed a detailed analysis of the NGA family in higher plant species, focusing on Solanaceae and Poaceae. Phylogenetic reconstruction of the gene family was followed by the characterization of the Solanaceae NGA gene family compared to the monocot members of Poaceae. The characterization included gene and protein structure, protein motifs, promoter analysis, Gene Ontology, and quantitative RT-PCR analysis of the NGA genes. Our obtained data provide a comprehensive understanding of the NGA gene family in higher plants and facilitate further research related to crop plant development and new control methods.
3. Discussion
The NGA family belonging to the RAV subfamily of the B3 superfamily is relatively well-characterized in
A. thaliana compared to other plant species [
6,
8,
9,
12,
21]. In Arabidopsis, the NGA family is known to be involved in the development of gynoecium and the regulation of lateral organs. However, functional annotation of the NGA family is still an area of limited knowledge. In this study, we performed phylogenetic reconstruction of the NGA family using several dicots (Solanaceae) and monocots (Poaceae) (
Figure 1).
The NGA phylogenetic tree has a peculiar feature (i.e., the NGA and NGA-LIKE sequences are very well distinguished, suggesting that these genes have evolved separately with well-demarcated evolution in dicots and monocots (
Figure 1)). Furthermore, NGA and NGA-LIKE sequences are defined based on the plant families where members of the Brassicaceae, Solanaceae, and Poaceae are phylogenetically well separated, suggesting that these sequences have resulted from multiple duplication events from the most recent common ancestor. Based on the phylogeny analysis, the NGA sequences from different subfamilies and the number of genes in each species vary. For example, in
B. rapa, ten NGAs and seven NGA-LIKE genes were present, while in
B. vulgaris, only one NGA and one NGA-LIKE gene were identified. The highest number of genes were identified in
C. sativa with 14 NGAs and seven NGA-LIKEs, followed by
T. aestivum with 18 NGAs and eight NGA-LIKEs (
Figure 1;
Figure S2). These results indicate that the NGA genes have evolved due to multiple rounds of duplications leading to the expansion of the gene family. Furthermore, among the monocots, banana forms a distinct clade with respect to both the
NGA and
NGA-LIKE genes, revealing that the genes within this species might have resulted from repeated segmental duplications (
Figure 1 and
Figure S2).
Furthermore, the gene structure analysis gives a framework of gene duplications and the functional relationship among the gene families. The exon–intron structures of the NGA family in our analysis revealed that the numbers of exons and introns were conserved among subfamilies, indicating the conserved function of the genes within subfamilies (
Figure 2). The same trend has been observed among the protein structures where the NGA and NGA-Like proteins share some common motifs; however, few unique motifs are only present within the subfamilies or unique to species. For example, motifs 9, 11, 13, 14, 15, 17, 18, and 19 were acquired during evolution in the NGA and NGA-Like proteins of the Solanaceae species such as
S. lycopersicum,
S. tuberosum,
C. annuum, and
N. tabacum, indicating novel functions of the proteins. Similarly, monocots such as
O. sativa L. japonica,
B. distachyon, and
S. bicolor possess common motifs that are also present in Solanaceae members, suggesting a conserved function of the NGA proteins. Consistent with these results, the three-dimensional structure of the proteins was conserved in these species; however, minor alterations in the amino acid sequences contribute to the functional variations among the NGA proteins (
Figure 4). The presence of protein motif (RLFGV) in the NGA proteins of
A. thaliana,
S. lycopersicum, and
O. sativa implicates that this motif plays an essential role in plant development (
Figure S4). Consistent with this, it has been observed that AtNGA1 possessing the RLFGV motif directly binds to the promoter of
AtNCED3, thereby inducing ABA biosynthesis in Arabidopsis in response to drought stress (
Figure S3) [
10]. The presence of the repressor motif is also reported in
N. benthamiana,
Amborella trichopoda, and
Aquilegia caerulea in their respective NGA protein sequences [
7]. In addition, this repressor motif is reported to be involved in regulating heat stress in the Heat shock factor B family [
7,
24,
25,
27]. These findings indicate the significance of NGA proteins in many aspects of plant development, which is yet to be explored.
The analysis of
cis-elements in the promoter region of the genes would provide clues into the transcriptional regulation of the respective genes. NGA genes are also looked for in the upstream
cis-regulatory elements. It has been observed that light-responsive elements are present in the promoters of the genes, suggesting that light plays an important role in regulating these genes (
Figure 3). Almost half of the genes were observed to be involved in stress-related responses such as drought inducibility and defense, suggesting that these genes play a role in stress response. The NGA genes also possess hormone response elements such as ABA, GA, MeJA, SA, and auxin. ABA and SA are known to participate in plant stress, and the
cis-elements analysis indicates that NGA genes might be involved in defense response [
36,
37,
38]. The presence of auxin-responsive elements in the promoters of the NGA genes is an interesting feature. As discussed above, the NGA family regulates the
AtYUC2 and
AtYUC4 genes involved in auxin biosynthesis, especially in carpel development [
6,
12,
16]. However, the direct link of auxin responsive elements with NGA regulation is yet to be discovered. Furthermore, some of these genes also implicate their role in gibberellin signaling and methyl jasmonate pathways. In addition, phytohormone ABA seems to play a major part in carpel development [
39,
40], and the roles of other hormones such as GA, SA, and MeJA in NGA regulation are still not understood. Among the other
cis-elements, anaerobic induction and meristem development seem to be majorly involved in the regulating of NGA genes.
Gene duplications are the main source of evolution of gene families, predominantly tandem and segmental duplication events [
41]. The synteny analysis of NGA genes of Arabidopsis with tomato, potato, and other species such as
P. trichocapra,
M. truncatula, and
O. Sativa L. japonica showed that most of them have evolved through segmental duplications. However, these duplications are followed by the diversification of gene functions during evolution. In addition, tandem duplications are also not uncommon, as can be seen in the phylogeny with genes or proteins co-existing, resembling their similarities in terms of sequence and functions [
42]. The nucleotide variations are the key to evolution within gene families. The Ka/Ks ratio tells us about the synonymous and non-synonymous changes in the gene sequences acquired during evolution and measures the evolutionary pressure of the nucleotide variations within the sequence of the genes [
43,
44]. The Ka/Ks ratio is assessed in NGA genes. Most of the genes have evolved under negative selection pressure, thereby screening random deleterious mutations, whereas, in
S. bicolor, each gene pair with
SbNGA-LIKE1-2 showed positive Darwinian selection. Our study revealed that the NGA family has evolved under stringent selection pressure, resulting in the conservation of the gene family.
GO analysis revealed the possible roles of NGA genes in Arabidopsis and tomato. Being derived from the B3 superfamily, NGA is primarily involved in gene regulation by sequence-specific DNA binding activity (including
cis-elements) and is predicted to be localized in the nucleus (
Figure 6;
Table 5). As evident from the previous literature on the NGA family in
A. thaliana and
S. lycopersicum, the AtNGAs are involved in regulating leaf morphogenesis and flower development [
6,
9,
12,
21,
45]. In addition,
AtNGA-LIKE1 is thought to be responsive to karrikins, indicating that this gene has a role in Strigolactone signaling. Other functions of
NGA-LIKE genes include negative regulation of transcription, seed growth regulation, leaf shaping, and meristem maintenance. The possible roles of the NGA gene family based on the Gene Ontology results implicate the potential role of the genes in plant growth, development, and defense. Consistent with the Gene Ontology results, the gene expression of the NGA family in Arabidopsis and tomato reflected the importance of the genes in regulating leaf morphogenesis and flower development (
Figure 7). However, the localization of NGA proteins would provide better evidence for protein expression in different cell types rather than gene expression studies. These results correlate with the expression of NGA genes in Arabidopsis and
B. rapa, affecting the development of lateral organs and floral development [
8,
9,
11,
21,
46].