In this study, we identified eight Alba genes from tomato via genome-wide analysis. The number of Alba gene family members varies across diverse species of the plant kingdom (Fig. 1). Alba genes exist as a multi-gene family in tomato with the average number compared with other plant species, implying that they have biological importance in this plant species.
In agreement with the previous reports [2, 15], in-depth analysis of the phylogenetic relationships among Alba proteins revealed two types. Some of the proteins are relatively large, with an RGG/RG repeat motif or other domain in addition to the generic single Alba domain; most of these proteins belong to the RPP25-like family. The other Alba proteins are relatively small, with only the generic single Alba domain; most of these proteins belong to the RPP20-like family in plants or the archaeal Alba family (Fig. 1). These findings suggest that the emergence of additional motifs and domains in Alba family proteins may have played a significant role in the expansion and diversification of Alba gene family in plant species during the course of evolution.
In agreement with the findings of Verma et al. (2018) [22], our phylogenetic analysis clearly grouped the plant Alba genes into two distinct subclusters: Alba genes from monocots (rice, sorghum, and maize) and those from dicots (tomato, potato, Arabidopsis, chickpea, and grapevine). In addition, the only Alba gene (CreAlba1) identified in the genome of the single-celled green alga Chlamydomonas reinhardtii was clustered together with those of land plant species in the Rpp20-like phylogenetic group, indicating that common Alba genes are shared by chlorophytes and streptophytes, which diverged over one billion years ago [31]. Additionally, Alba genes from the moss Physcomitrella patens, the basal angiosperm Amborella, monocots (sorghum, rice, and maize), and dicots (tomato, potato, grapevine, Arabidopsis, and chickpea) were present within both eukaryotic-specific phylogenetic groups (Fig. 1), suggesting that these two families evolved before bryophytes and angiosperms diverged approximately 450 million years ago [32]. Overall, these findings enabled us to retrace ancient evolutionary transitions in this gene family.
From an evolutionary standpoint, gene duplication events increase the number of genes in a particular gene family, which can help plants adapt to adverse environmental stresses [33, 34]. One duplicated gene pair, SlAlba6/SlAlba7, was predicted in the tomato genome (Figure S3), indicating that the eight SlAlba genes appear to have been derived from an original set of seven ancestral genes. Microsynteny analysis revealed three segmentally duplicated gene pairs between tomato and Arabidopsis, but only a single duplicated gene pair between tomato and rice as well as between Arabidopsis and rice. These results are consistent with the closer evolutionary relationship between tomato and the dicotyledonous model plant Arabidopsis than between tomato and the monocotyledonous model plant rice (Fig. 5).
Analysis of genetic structural diversity is indispensable for the evolutionary analysis of a multi-gene family. Detailed analyses of the conserved motifs and exon-intron structures of the Alba genes revealed that exons and introns as well as conserved motifs were organized in a similar pattern among pylogenetically closely related Alba genes but in a different manner among those from the different clusters. These findings point to the functional redundancy across phylogenetically closely related Alba family genes and present a likely rationale for the functional divergence among the divergent Alba genes during the evolutionary process (Figure S1, Fig. 4).
Predicting the three-dimensional structure of a protein provides valuable information about its possible molecular functions and ligand-binding sites. A previous study uncovered the likely binding affinity of rice Alba proteins to several molecules including DNA and RNA. In the current study, 3D-modeling of SlAlba proteins also predicted possible binding interactions with DNA, RNA, and peptide molecules (Figure S5), supporting their putative functions in transcriptional and translational regulation (Table S9).
The proper transport of a protein to its specific subcellular locations is critical for its optimal activity. A previous subcellular localization experiment revealed that OsAlba1 localizes to the nucleus and cytoplasm [35]. In support of this finding, tomato Alba fusion proteins also showed GFP signals in the nucleus and cytoplasm (Fig. 9). These findings suggest that the diverse Alba proteins are involved in a variety of cellular signaling processes in the cytoplasm and nucleus.
Analyzing the expression patterns of a gene during growth and development and upon exposure to stress stimuli may help determine its functions. In support of previous findings [22], majority of SlAlba genes except SlAlba1 exhibited differential expression profiles across the organs examined, suggesting these genes play distinct regulatory roles in growth and development (Fig. 7). Several of the tomato Alba genes (SlAlba3, SlAlba4, SlAlba6, SlAlba7, and SlAlba8) were predominantly expressed in vegetative organs, but others, including SlAlba1, SlAlba2, and SlAlba5, showed higher transcript levels in reproductive organs such as flowers and fruits. These results suggest that these genes play preferential roles in these organs and developmental phases in tomato.
The initiation and development of a floral bud, a process modulated by multiple floral genes and environmental factors, plays a vital role in fruit set and crop yield [36]. Intriguingly, SlAlba1 was expressed at strikingly higher levels in flower buds, fully bloomed flowers, and senescent flowers compared to root tissues and 1 cm fruits, and no expression was detected in other organs. SlAlba2 was predominantly expressed in flower buds, with its expression level many times higher than that in any other organs (Fig. 7). These findings suggest that SlAlba2 regulates floral bud formation in tomato and that SlAlba1 regulates flower development at all stages via interactions with other regulatory genes.
The roles of Alba genes in fruit development have not previously been studied in any vegetable crop. Tomato is a model organism for the study of climacteric fruits. Therefore, the molecular pathways controlling tomato fruit enlargement (which includes a cell division stage and a cell elongation stage) as well as fruit ripening have been extensively explored [37–39]. Interestingly, SlAlba5 is the only gene that showed higher expression in tomato fruits at all stages of development (except the immature stage), with its peak expression in 1 cm fruits. By contrast, the remaining seven SlAlba genes showed lower expression levels in fruits at all developmental stages compared to the control (leaves; Fig. 7). Our findings suggest that SlAlba5 plays a regulatory role in fruit enlargement and ripening, particularly during the initial stages of fruit development.
Most tomato Alba genes that were highly expressed in vegetative organs had their highest expression levels in stems; only one gene had its highest expression level in leaves, and no gene has its highest expression level in roots. In addition to providing mechanical support to the aerial portions of the plant, the stem facilitates the long-distance translocation of water and nutrients to sustain plant growth under both normal and stressful conditions. The transcript levels of SlAlba3, SlAlba4, SlAlba6, SlAlba7, and SlAlba8 were markedly higher in stems than in any other organ examined, suggesting that they likely function in stem growth, the long-range movement of water and nutrients, and stress tolerance. The higher expression level of SlAlba3 in leaves suggests that it might play a role in leaf development and signaling cascades in leaves (Fig. 7). Taken together, these findings point to the diverse functions of SlAlba family genes in plant development.
Plant responses and adaptation to environmental stresses often involve differential gene expression, which is regulated by a dynamic network of numerous transcription factors and various stress tolerance genes in an ABA-dependent or -independent manner [40–43]. Alba proteins are associated with stress tolerance due to their involvement in genome packaging and organization, transcriptional and translational regulation, post-translational regulation, and RNA metabolism, in addition to responses to different environmental stresses [2, 22, 44]. The possible roles of Alba family genes in plant stress responses were supported by previous studies reporting the stress-induced expression of Alba genes in several plant species, such as cotton, Arabidopsis, and rice [15, 22, 23]. The role of OsAlba1 as a DNA binding protein involved in oxidative stress tolerance was revealed by complementation analysis in yeast. Moreover, the susceptibility of ghAlba4- and ghAlba5-silenced cotton plants to drought and salinity conditions highlighted the possible involvement of Alba genes in plant stress tolerance [23, 35].
In agreement with previous reports, we observed that tomato Alba genes were differentially expressed in response to various abiotic stresses (Fig. 8a-e). SlAlba4 and SlAlba5 expression was significantly induced by heat treatment (Fig. 8a), which is in agreement with the finding that many Alba family genes in Arabidopsis and rice were markedly upregulated in response to mild heat stress (37°C) and moderate heat stress (42°C), respectively [15, 22]. Here we showed that SlAlba6 was sharply upregulated in plants under saline conditions (Fig. 8b), suggesting its possible role in salt tolerance in tomato. This result is in agreement with the recent study finding that Alba genes were expressed at higher levels in cotton upon exposure to salt stress and that ghAlba4 and ghAlba5 cotton plants showed increased sensitivity to salt treatment compared to wild type and control plants [23]. Tomato Alba genes were downregulated under drought stress (Fig. 8d), while several Alba genes in rice and cotton were upregulated by dehydration treatment, pointing to the functional divergence of Alba family genes in different plant species [22, 23]. The expression level of SlAlba8 was considerably elevated following cold stress (Fig. 8c), which is consistent with the finding that a few rice Alba genes were upregulated in response to low temperature stress [22]. The phytohormone ABA is well known for its role in regulating plant acclimation to adverse environmental stresses, including heat, cold, salt, and drought [45–47]. All tomato Alba genes except SlAlba3 were markedly upregulated under ABA treatment (Fig. 8e), which agrees with the finding that multiple Alba genes in rice were induced by ABA treatment [22]. Therefore, SlAlba genes might function in abiotic stress tolerance via their direct roles in ABA signaling.
The regulation of tomato Alba genes under stress conditions was also corroborated by the prevalence of multiple cis-elements related to stress tolerance and hormonal responses in their promoter regions. Such elements might facilitate the regulation of these genes under different abiotic stress conditions (Figure S4, Table S5).
Post-transcriptional regulation of numerous miRNA families plays a vital role in various biological processes, including development and stress tolerance [48, 49]. Several target sites for miRNAs related to stress tolerance and developmental processes were predicted in SlAlba genes (Table S6). This result is consistent with the finding that miRNA target sites are present in the Alba genes of various plant species including rice, Arabidopsis, maize, and sorghum [22].