This is the first comprehensive study of HVA22 genes in a solanaceous crop species. We identified and comprehensively characterized 15 non-redundant SlHVA22 genes at a genome-wide scale. As in other flowering plant species, the existence of HVA22 genes as a multiple gene family in tomato (Fig. 1) suggested their important biological role in this model fruit crop. TB2/DP1/HVA22 family proteins have been identified in eukaryotes but not in prokaryotes, indicating that they might have evolved after the divergence of prokaryotes and eukaryotes, playing a vital role in eukaryote evolution.
We performed a phylogenetic analysis to explore the evolutionary relationship among HVA22 homologs from diverse species in the plant kingdom. The phylogenetic tree depicted a major clustering of proteins from dicots (Arabidopsis, citrus, tomato and potato) and monocots (rice, maize and sorghum) along with lower plants (Fig. 1). The corresponding proteins from the moss Physcomitrella patens and the single-celled green alga Chlamydomonas reinhardtii only occurred in phylogenetic groups II and III, revealing that the HVA22 proteins from these two phylogenetic groups (II and III) were more primitive than proteins in other groups (I and IV) and may have evolved from the common ancestor of chlorophytes and streptophytes, which diverged over one billion years ago . The prevalence of only the HVA22 homologs from angiosperms in groups I and VI indicated that the homologs from these phylogenetic groups may have evolved before the divergence of monocots and dicots (~ 200 million years ago) .
All HVA22 genes in the Citrus species contain TMDs (Ferreira et al., 2019); however, SlHVA22 genes varied in terms of absence or presence of different numbers of TMDs (Fig. 2). This domain organization may play a role in the structural and functional divergence of HVA22 genes in tomato. In addition, SlHVA22 genes contained other types of domains, such as zinc-finger domains identified in DNA- and RNA-binding proteins (C2H2-type zinc-finger domain, U1-type zinc-finger domain) and RVT_3 domains in a few tomato genes. The domains may have also enhanced the diversification of the tomato HVA22 gene family. The conserved motifs, exons and introns identified in SlHVA22 gene family members were arranged in a similar manner across evolutionarily closely related SlHVA22 genes but in a dissimilar arrangement from those of different phylogenetic groups (Figs. S1 and S2). This could explain the functional similarity among evolutionarily closely related SlHVA22 genes and the functional dissimilarity across divergent ones over the course of evolution.
Three segmentally duplicated gene pairs (SlHVA22a/SlHVA22m, SlHVA22e/SlHVA22n and SlHVA22g/SlHVA22o) were predicted in tomato (Fig. S5 and Table 2). Therefore, HVA22 family members might have evolved from an original set of 12 progenitor genes in tomato. The duplicated genes of each pair resided on different chromosomes, one of which contained only one HVA22 gene, whereas the others had three or four HVA22 genes (Fig. S3), suggesting that gene duplication increased not only the number of HVA22 genes in the tomato genome but also the number of chromosomes carrying them in tomato. This result indicates that more chromosomes in tomato may have needed to harbor HVA22 genes during evolution to boost important biological functions in tomato cells such as adaptation to unfavorable environmental conditions.
A comparative microsyntenic map constructed to explore the evolutionary relationship among HVA22 orthologs from Arabidopsis, tomato and rice revealed four pairs of orthologous genes between Arabidopsis and tomato but none between rice and Arabidopsis or tomato (Fig. 4). These results are well correlated with the closer evolutionary connection of tomato with Arabidopsis than with rice and also suggest that four SlHVA22 genes might have derived from Arabidopsis during species divergence.
Cis-acting elements in the promoter sequences of genes can act like circuit breakers to switch on and off the transcription of their genes upon exposure to different environmental stimuli [27, 28]. The presence of several cis-regulatory elements related to hormonal and abiotic stress responses upstream of tomato HVA22 genes highlighted their probable roles in tomato abiotic stress tolerance (Fig. S6). This result is corroborated by the prevalence of hormonal and stress-related cis elements, which can interact with diverse trans-acting genes, including stress-related transcription factors, in the promoter regions of stress-induced HVA22 genes in other plant species [ 1, 3, 6, 13, 17].
To determine whether the abiotic stress responses of SlHVA22 genes could also be related to miRNAs, we analyzed the miRNA target sites in SlHVA22 genes. Eleven out of fifteen SlHVA22 gene family members were targeted by tomato miRNAs that regulate abiotic stress tolerance in tomato, such as Sly-MIR159b, Sly-MIR166c-5p, Sly-MIR1917, Sly-MIR395a, Sly-MIR396a, Sly-MIR396a-5p, Sly-MIR482a, Sly-MIR5302a, Sly-MIR5303, Sly-MIR6023, Sly-MIR6024, Sly-MIR9470-5p, Sly-MIR9474-5p, Sly-MIR9479 and sly-MIR9479-3p [29–32] (Table S6). This indicates that many SlHVA22 genes might be linked to the post-transcriptional regulation of miRNAs in tomato abiotic stress tolerance.
Protein phosphorylation, a crucial post-translational modification regulated by kinases and phosphatases, is crucial in signaling pathways and stress responses in plants [33, 34]. Prediction of phosphorylation sites using the NetPhos 3.1 Server revealed that many putative phosphorylation sites were prevalent in all tomato HVA22 proteins (Table S7), which is consistent with the distribution of phosphorylation sites in HVA22 homologs in other species [3, 4, 6].
The 3D structure of a protein can provide useful clues to predict its possible interaction with other molecules and its biological functions. Thus, we analyzed 3D models of tomato HVA22 proteins to gain a better understanding of their molecular structural conformations and putative functions. Most HVA22 proteins had similar numbers of α-helixes, β-sheets and coils (Table S8), suggesting structural conservation in most HVA22 family members during evolution. SlHVA22 proteins were predicted to harbor ligand-binding sites that interact with various molecules, such as ions, intracellular messengers or receptor molecules, to initiate a change in cell function. SlHVA22 proteins were also predicted to have various molecular functions, including the ability to bind to a variety of ligands, transporter activity and transferase activity based on the GO terms (Fig. 5 and Table S11). These results indicate that SlHVA22 proteins might have several important biological functions in tomato.
The determination of protein subcellular localization can indicate putative functions. Subcellular localization analysis revealed that tomato HVA22 proteins were predominantly localized to the ER (Fig. 8). This finding was consistent with the localization of HVA22 homologs from barley and yeast in the ER, suggesting that HVA22 homologs from diverse species might have a conserved function, such as vesicular trafficking in abiotic stress responses [1, 8].
A qRT-PCR assay revealed varied expression patterns of SlHVA22 genes in the different organs tested, suggesting that they might have distinct regulatory functions in the growth and development of tomato (Fig. 9). Of the 15 tomato HVA22 genes, 8 genes (SlHVA22c, SlHVA22f, SlHVA22g, SlHVA22i, SlHVA22k, SlHVA22l, SlHVA22n and SlHVA22o) had high transcript levels in reproductive organs (flowers or fruits), whereas 7 genes (SlHVA22a, SlHVA22b, SlHVA22d, SlHVA22e, SlHVA22h, SlHVA22j and SlHVA22m) had high transcript levels in vegetative organs, such as leaves, roots, or stems, hinting at their preferential roles in these organs and developmental stages in tomato.
The expression of SlHVA22d was highest in leaves, IM fruit, MG fruit and B5 fruit, pointing to its possible role in the growth of leaves and fruit. The roots play a vital role in the uptake of water and minerals for growth and development of plants. The mRNA transcript levels of SlHVA22a and SlHVA22m were highest in roots, suggesting their probable involvement in root growth and uptake of nutrients and water. The stem is the main organ that provides mechanical strength to the aerial parts of the plant and transports water and nutrients to promote plant growth and development under normal and unfavorable environmental conditions. The higher mRNA transcript accumulation of SlHVA22b, SlHVA22e, SlHVA22h and SlHVA22j in stems compared to in other organs hints at their likely role in stem development, long-range translocation of water and minerals as well as stress adaptation (Fig. 9).
Of the eight SlHVA22 genes that were highly expressed in reproductive organs, two genes (SlHVA22f and SlHVA22o) were predominantly expressed in flowers, suggesting their probable role in flower development. Tomato, as a model fleshy fruit crop, has been widely studied to understand the regulatory mechanisms governing the growth and ripening of climacteric fruits [35–37]. The expression of SlHVA22k and SlHVA22l was higher in fruit at all fruit developmental stages, except at the 1-cm fruit stage, compared with in other organs. This result suggests that SlHVA22k and SlHVA22l may actively function throughout the developmental stages of fruit starting from the cell expansion phase. The mRNA transcript levels of SlHVA22n were higher in IM and MG fruit than in fruit at other developmental stages, suggesting its active role in the cell expansion phase of fruit development. SlHVA22c is the only gene whose expression peaked in IM fruit, indicating that it may influence the early cell expansion phase of tomato. The higher expression levels of SlHVA22g in B and B5 fruit suggested its possible role in tomato fruit ripening. The transcript levels of SlHVA22i were high in B fruit (> 80-fold higher than in the control) and highest in B5 fruit (~ 300-fold higher than in the control). This finding indicates that SlHVA22i may be a novel gene implicated in the regulation of fruit ripening. The predominant expression of the duplicated gene pair (SlHVA22a/SlHVA22m) in the same organ (root) suggested functional conservation, whereas the differential expression levels of other duplicated gene pairs (SlHVA22e/SlHVA22n and SlHVA22g/SlHVA22o) in different organs suggested functional diversification after gene duplication (Fig. 9).
Plant adaptation to diverse environmental stresses is regulated by gene networks, including transcription factors and downstream stress-related genes in ABA-dependent or -independent manners [38–41]. Previous studies have reported ABA- and stress-induced differential expression of HVA22 gene family members in various plant species [3, 17, 18]; interactions between the cis elements located in the promoter regions of HVA22 homologs with several ABA- and stress-related genes; and the exploitation of the HVA22 promoter as a stress-inducible promoter of stress-related genes in transgenic plants [12–15, 19]. Therefore, it is likely that SlHVA22 genes function in tomato abiotic stress tolerance.
In the current study, tomato HVA22 genes displayed differential transcript levels upon exposure to abiotic stress stimuli (Fig. 10a and b). Most SlHVA22 genes were significantly downregulated, while several genes were dramatically upregulated, and only one gene (SlHVA22j) was not responsive under cold treatment. This result agrees with a previous study reporting the cold-induced expression of barley HVA22 gene and the differential responses of Arabidopsis HVA22 homologs under cold stress conditions [13, 17]. The expression of SlHVA22b, SlHVA22i and SlHVA22n was highly induced following cold stress (Fig. 10a), suggesting their potential role in cold stress tolerance in tomato.
In contrast to cold stress, many SlHVA22 genes were upregulated, whereas few genes (SlHVA22a, SlHVA22f, SlHVA22h and SlHVA22n) were downregulated by heat treatment. In response to heat stress, 11 of the 15 SlHVA22 genes were upregulated, and SlHVA22i showed the highest expression (Fig. 10a). These genes may be crucial in tomato heat tolerance. These observations are corroborated by a previous study that determined that a mutation in YOP1, the yeast (Saccharomyces cerevisiae) HVA22 homolog, results in a growth defect in yop1 mutants under mild temperature stress (37°C) .
Drought treatment upregulated half of the tomato HVA22 genes and downregulated the other half. SlHVA22i had the highest expression level in response to drought stress, followed by SlHVA22n (Fig. 10a). This suggests that SlHVA22i and SlHVA22n might actively function in the tomato drought response. These findings are consistent with a previous report in which the expression of AtHVA22 homologs was differentially regulated by drought and another report in which CcHVA22d-overexpressing transgenic tobacco exhibited a lower dehydration rate and buildup of H2O2 than the WT [6, 17].
Under salt stress conditions, the transcript levels of most SlHVA22 genes increased, but those of a few genes (SlHVA22d, SlHVA22h and SlHVA22o) decreased. Eleven SlHVA22 genes were upregulated, with SlHVA22m having the greatest expression, followed by SlHVA22i, SlHVA22b and SlHVA22n (Fig. 10b). This indicates the potential involvement of these genes in the salt response of tomato. This is in agreement with previous studies reporting the salt-responsive expression of HVA22 homologs in yeast and several plant species, and elevated mRNA transcripts of the tomato HVA22 homolog (SlHVA22n in this study) in the salt-tolerant tomato LeERF1 and LeERF2 transgenic lines [3, 18, 20].
ABA is a well-studied phytohormone that regulates a variety of stress-related genes to promote plant tolerance to unfavorable environmental conditions such as cold, heat, drought and salt stress [42–44]. Except for SlHVA22d, the expression of all SlHVA22 genes was altered by ABA treatment, with SlHVA22i exhibiting the highest expression, followed by SlHVA22f and SlHVA22n (Fig. 10b). This finding agrees with previous work reporting the responses of HVA22 homologs from barley and Arabidopsis upon exposure to ABA, hinting at their functional role in abiotic stress adaptation in tomato in an ABA-dependent manner [4–17].
We performed a co-expression network analysis of SlHVA22 genes using RNA sequencing data to further understand their putative functions in tomato. The genes co-expressed with SlHVA22 genes were involved in diverse biological pathways including abiotic stress responses and development (Figs. 6 and 7, Fig. S7 and Table S12), suggesting the important biological role of SlHVA22 genes in tomato. Multiple abiotic stress-responsive genes, such as SlyHSF-24 (Solyc09g009100), SlDEAD22 (Solyc07g042010) and SlDEAD29 (Solyc09g090740) [45–47], were co-expressed with SlHVA22a, which is in agreement with our finding that SlHVA22a responded to abiotic stress treatment. In addition, SlPIP1;5 (Solyc08g081190), which was highly expressed in roots and under salt treatment, was co-expressed with SlHVA22a, whose expression peaked in roots and on exposure to salt stress , suggesting that these genes might interact with each other in the root development and salt tolerance of tomato.
We also identified several genes in the co-expression networks that were related to abiotic stress responses and/or expressed in fruits, such as SPS1 (Solyc07g007790), SlPDI7-2 (Solyc11g019920), SlMC8 (Solyc10g081300), SlWRKY1 (Solyc07g047960), SlWRKY3 (Solyc08g081610), Solyc07g064820 (Mitogen-activated protein kinase kinase 2-like), Solyc07g040960 (Salt responsive protein 2) and SlGPAT6 (Solyc09g014350) in the co-expression network of SlHVA22g [49–55]; SlRabGAP9a (Solyc07g049580), SlRabGAP21a (Solyc12g009610), SlGT-33 (Solyc12g043090), SlFdAT1 (Solyc12g088170), Solyc11g045120 (Translation initiation factor SUI1) and Solyc11g044910 (β-D-xylosidase) in that of SlHVA22k [56–62]; and Solyc10g078600 (phosphate transporter 1–1), C2H2 zinc finger (C2H2-ZF) (Solyc11g017140), Solyc11g045120 (Translation initiation factor SUI1), Solyc11g044910 (β-D-xylosidase), SlRabGAP9b (Solyc12g005930), SlRabGAP21a (Solyc12g009610) and SlFdAT1 (Solyc12g088170) in that of SlHVA22l [56, 57, 59, 61, 63, 64] (Fig. 6 and Table S12). These findings suggested that these three genes, whose transcript levels were high in fruit and induced by abiotic stresses, likely function together with the co-expressed genes in the fruit development and abiotic stress response of tomato.