Heat stress affects various components of photosynthetic machinery of which Rubisco activation inhibition due to heat sensitive Rubisco activase (RCA) is the most prominent. Detailed comparison of RCA coding genes identified a tandem duplication event in the grass family lineage where the duplicated genes showed very different evolutionary pattern. One of the two genes showed high level of sequence conservation whereas the second copy, although present only 1.5kb away, was highly variable among various plant species because of loss of introns, alternative splicing and loss of the last exon coding redox regulated C-terminal extension domain. Gene specific expression analysis, both at the transcription as well as the protein level, showed very different expression pattern of the two RCA copies. Expression of the highly conserved copy was higher under normal plant growing conditions that decreased many folds under heat stress with substantial genotypic variation, but the variable copy showed much higher expression under heat stress conditions across all grass species. The cultivated rice has only one functional gene as the second copy became nonfunctional due to multiple deletions but Oryza brachyantha and Oryza australiensisstill have two functional Rca genes. Detailed analysis of the promoter region of the two copies among various plant species showed insertion of several transposable elements harboring heat responsive elements in the heat inducible copy of the gene. The conserved RCA copy of wheat didn’t have any transposable insertions whereas in that of maize has one heat shock element and sorghum had two. It would be interesting to study if the higher level of heat stress tolerance observed in sorghum and maize is associated with the differences observed for RCA.
Research Article
Tandemly Duplicated Rubisco Activase Genes of Cereals Show Differential Evolution and Response to Heat Stress
https://doi.org/10.21203/rs.3.rs-4676428/v1
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Rubisco Activase
Cereal Crops
Gene Duplication
Heat Stress
Photosynthesis
Molecular Evolution
and Regulation of Gene Expression
The optimal temperature for photosynthesis varies among crops, from 25-30°C in wheat to 30-35°C in rice (Nagai and Makino, 2009). Every degree-Celsius increase over the optimal temperature may result in up to 6% reduction in grain yield of wheat and 3.2% of rice (Zhao et al., 2017). Although many plant metabolic pathways are affected by heat stress, the effect on photosynthesis is critical as any constraint on the process will directly impact development, growth, biomass production, and crop yield. Plant photosynthetic machinery involves complex components and processes, many of which including Rubisco activation, enzymes and proteins of photosystems, membrane permeability and ATP generation are vulnerable to high temperature (Allakhverdiev et al. 2008). The critical temperature and its duration affecting each photosynthetic process often differ among species (Feller et al., 1998). Plants exposed to temperatures above 40°C for extended periods show irreversible damage to the permeability of thylakoid membranes and electron transport chain, resulting in photosynthesis inhibition. The photosystems and electron transport are usually not affected until 40°C, whereas for many species including some important crop plants including wheat the Rubisco activation level and photosynthetic rate (Pn) start to decline even at moderately elevated temperatures (Feller et al., 1998).
The functional state of Rubisco is maintained by Rubisco activase (RCA) that removes inhibitory sugar phosphates from the active sites of Rubisco with energy derived from ATP hydrolysis (Salvucci et al., 1985). RCA is a nuclear encoded, cytosol synthesized chloroplast protein, generally consisted of two immunologically related polypeptides called α isoform (45 to 46 kDa) and β isoform (41 to 43 kDa). The size difference between the two isoforms is due to the presence of a redox regulated C-terminal extension (CTE) domain that is present in the α isoform but is lacking in the β form. In most plants, both α and β isoforms of RCA are capable of activating Rubisco (Werneke et al., 1989; Rundle and Zielinski, 1991; To et al., 1999; Salvucci et al., 2003). Shown in Arabidopsis, the two cysteine (Cys) residues located in the CTE domain of RCA α isoform make a disulfide bond under oxidizing conditions. This significantly increases its sensitivity to physiological level of ADP/ATP ratio and affects both ATP hydrolysis and Rubisco activation (Zhang and Portis, 1999; Zhang et al., 2002). The major function of larger isoform is to regulate the activity of smaller RCA isoforms under low light/dark conditions in order to conserve energy. The equimolar concentrations of the larger and the smaller isoforms, completely inhibited the activity of smaller isoform under higher ADP/ATP ratio in light conditions (Zhang et al., 2001). Interestingly, tobacco, which expresses only the β isoform, is also sensitive to physiological levels of ADP/ATP ratio and responds similar to species with both isoforms (Carmo-Silva and Salvucci, 2013). Selection for genotypes with higher Rca expression levels at normal conditions in soybean, maize, and some other crops showed a positive correlation with Rubisco activity, photosynthetic rate, and seed yield (Morales et al., 1999; Yin et al., 2010, 2014).
In-vitro analysis has shown that while the RUBISCO enzyme is stable even at 50oC, RCA is very sensitive to heat stress and its activity start to decline at temperatures over 35oC (Crafts-Brandner and Salvucci, 2000). As a result, at higher temperatures RCA is unable to keep pace with faster Rubisco deactivation thus constrains photosynthesis (Crafts-Brandner and Salvucci, 2000). In crops including cotton and wheat, even a moderate level of heat stress decreases the Rubisco activation levels, which correlated with reduced net photosynthesis, increased RuBP level, and decreased levels of 3-phosphoglycerate in leaves (Law and Crafts-Brandner, 1999). Independent studies on various crops have shown that photosynthetic heat acclimation is directly associated with the acclimation of heat sensitive RCA to high temperature (Law and Crafts-Brandner, 1999; Yamori et al., 2006). This conclusion was further supported by the studies showing increase in the thermal tolerance of Arabidopsis photosynthesis by improving RCA thermostability (Kurek et al., 2007; Kumar et al., 2009).
Previously, we showed that the Rca gene structure is highly conserved in higher plants, and the common grass ancestor Rca, similar to Arabidopsis AtRca1, contained seven exons and six introns, enabling expression of the RCA α isoform with redox regulated CTE domain coded from the seventh exon (Nagarajan and Gill, 2018). The alternatively spliced mRNA of the same gene contains a premature stop codon and produces a shorter redox insensitive RCA β isoform (Werneke et al., 1989). We also reported that the Rca gene is tandemly duplicated (Rca1 and Rca2) among grass species and the duplication event probably occurred in the common grass ancestor, but before the divergence of subfamilies (Nagarajan and Gill, 2018). Heat stress induced changes in the RCA expression pattern at protein level were reported earlier in rice (Scafaro et al., 2010; Wang et al., 2010), wheat (Feller et al., 1998; Law and Crafts-Brandner, 2001; Ristic et al., 2009), and maize (Crafts-Brandner and Salvucci, 2002; Ristic et al., 2009). High temperature stress altered the ratio of α and β isoform levels in rice (Wang et al., 2010), but induced new RCA isoform expression in wheat and maize (Law and Crafts-Brandner, 2001; Crafts-Brandner and Salvucci, 2002; Ristic et al., 2009). A recent study in wheat showed that heat stress altered the expression pattern of Rca genes (Scafaro et al., 2019). Later, (Degen et al., 2020) showed a single amino acid substitution (M159I) in TaRCA2-β isoform could increase the thermal stability of the enzymatic activity by up to 5°C. However, the heat stress induced expression pattern of RCA isoforms in important cereal crops and key evolutionary changes causing the differential gene expression are still not well studied. Earlier, we proposed an evolutionary model for tandemly duplicated Rca genes of the grass species (Nagarajan and Gill, 2018). We also showed that after the duplication event, one of the gene copies, Rca1, have lost the third, the fifth and the sixth introns, which also abolished the alternative splicing mechanism of the Rca transcripts. Other changes observed in Rca1 among various grass species included nonfunctional Rca1 in some Oryzoideae species; 2) loss of Rca1 sequences coding the redox regulated CTE domain in Pooideae including that in Brachypodium, barley, and wheat and the loss of various introns (Ragarajan and Gill, 2018). 3) loss of Rca2 sequences coding for the redox regulated CTE domain in Panicoideae species (maize, sorghum and foxtail millet), and 4) loss of various introns in both Rca1 and Rca2 of these crops. Whereas, the second gene copy, Rca2, did not amass any major changes in its gene structure although loss of some introns and sequences coding for the redox regulated CTE domain in Panicoideae species including maize, sorghum and foxtail millet was observed.
In the present study, first, we studied the origin of the tandem duplication of Rca genes. Specifically, we tracked the evolution of the rice OsRca1 by comparing the sequences of various Oryzoideae species. Second, we studied the effect of heat stress on the expression pattern of tandemly duplicated cereal Rca gene copies both at the mRNA as well as at the protein level and investigated possible causes (i.e. promoter elements) of the variation. Finally, we performed a phylogenetic study to analyze the rate of amino acid substitution and the evolutionary relationship of different monocot RCA isoforms. The information was then used to understand the role of tandemly duplicated Rca genes in Rubisco activation, and photosynthetic acclimation to heat stress in important cereal crops.
Tandem duplication of Rca gene occurred after the divergence of Poales families
With the objective to accurately determine origin of the tandem duplication in the RCA gene, we evaluated the Rca gene structure in three distantly related monocot species: banana (Musa acuminata), date palm (Phoenix dactylifera) and pineapple (Ananus comosus; family: Bromeliaceae; order Poales). Unlike the grass family, no tandem duplication of the Rca gene was observed in any of these species. Instead, these species contained three Rca gene copies on separate chromosomes (table S1). The Rca gene copies in banana were located on chromosomes 2, 10 and 11, whereas in date palm, the copies were present on two different unplaced scaffolds NW_008246537.1 (1.6 Mbp) and NW_008246711.1 (0.506 Mbp) and were not tandemly duplicated as was seen in grasses. The gene copies in pineapple were located in the linkage groups (LG) LG14, LG18, and LG23. Otherwise, the structure of the gene copies was similar to those of other higher plants. In all three species, the exon sizes and relative order were highly conserved among the three copies as was seen in other plant species. Major variation was however observed for intron length (Fig. 2b). Based on these observations, we concluded that the gene duplication event occurred after the divergence of the Poales families and is probably specific to the Poaceae.
Rca gene structure among cereals
After evaluating Rca gene structure among various grass family members, we identified and sequence confirmed Rca gene copies from seven (O. brachyantha, O. australiensis, O. sativa, T. aestivum, Z. mays, S. bicolor, and Setaria italica) species. Since rice showed major variation for the Rca gene structure, we also obtained sequence of seven cultivated rice accessions and two wild rice species (Oryza australiensis and Oryza brachyantha). For each accession, both genomic and cDNA copies were cloned and sequenced. While the Rca2 sequence from one of the accessions was reported earlier (To et al., 1999; Scafaro et al., 2016), we cloned and sequenced the Rca1 gene from O. australiensis (GenBank Acc. MH115971), and O. brachyantha (GenBank Acc. MH220418), and Rca2 gene from O. brachyantha (GenBank Acc. MH660917).
The structural comparison of Rca1 gene copy across various cultivated and wild rice species revealed presence of large deletions in the exons 2, 3, 4, 5, 6 and 7 which appeared to have caused frameshift mutation in the Rca1 of cultivated rice species (Fig. 3). In other species, between 11 to 13 independent deletions were detected in all four exons, resulting in 157 to a total loss of 218 nucleotides. These Rca1 deletions appear to have occurred multiple times, but in different time periods during the evolution. For example, O. barthi and O. glumipatula have a 15 bp deletion on the third exon, whereas O. nivara, O. sativa, and O. rufipogon showed a 39 bp deletion (Fig. 3; Box1). Similarly, the fourth exon of Rca1 of O. nivara, O. sativa, and O. rufipogon contains two additional deletions of 13 and 24 nucleotides (Fig. 3; Box 2), which were absent in other wild species. Additionally, species specific indels in the Rca1 gene were also identified. The six bp addition in the second exon of O. punctata Rca1, a 25 bp deletion in the fourth exon of O. nivara, and a nine bp instead of the 14 bp deletion in the second exon of O. glumipatula Rca1 occurred probably after the divergence of the species. However, it is not clear how the 24 bp deletion is absent in O. sativa ssp. indica but present in most other species including O. sativa ssp. japonica. Interestingly, the intron lengths of Rca1 were determined before the major Oryza species divergence and maintained thereafter (Fig. 3). Alignment of RCA2 amino acid sequences of these species showed 100% similarity, except for O. brachyantha and O. punctata, which had only 95.5% and 97.4% similarities with the other Oryza species respectively.
Rca1 gene structure in Oryzoideae subfamily
From the comparative analysis of Rca genes in Poaceae, it was evident that both Pooideae and Panicoideae subfamilies have two functional Rca gene copies present in tandem (Fig. 1). The OsRca1 copy contained multiple small deletions that seem to have occurred after the divergence of these sub-families. Comparison of the two wild and seven cultivated rice species revealed the presence of a putative Rca1 gene in all these species. Structurally, the Rca1 gene in O. australiensis, O. brachyantha, and O. punctata contained four exons with slight size variation for the three introns (Fig. 3). The remaining five species including the two accessions of cultivated rice showed length of the three introns were remarkably conserved. The exons of these five species showed 11 to 13 deletions (Fig. 3). Nine of these deletions were common to all five species and the remaining were specific to one or more species. At the predicted protein level, the largest protein among these three species was predicted to be 466aa in O. punctata. The predicted protein in O. australiensis is 465aa as compared to 458 in O. brachyantha. Unlike the RCA1 of Pooideae members, these predicted peptides also contain the light sensitive CTE domain as identified in the RCA1 of the Panicoideae members.
Thermostability conferring amino acids in RCA1 isoform of cereals
Previous studies showed that Arabidopsis transgenic plants expressing RCA variants with certain amino acid substitutions were heat tolerant, and the modified Arabidopsis RCA showed up to 10°C increase in stability and Rubisco activation activity (Kurek et al., 2007). One such substitution T274R (Threonine 274 Arginine) in the variant 183H12, which alone could provide the maximal heat stability and activity, was identified in all three copies of TaRCA1 and ZmRCA2 (Fig. 6). Other natural variants observed in the same position were glutamine (Q) in SbRCA1, SbRCA2, and ZmRCA1, and lysine (K) in ObRCA1 and ObRCA2 of O. brachyantha, and in all of the RCA2 isoforms of rice and wheat. Based on these results, we hypothesize that the isoform expressed during heat stress may have better thermal stability than the isoforms expressed at normal conditions because of the putative thermal tolerant amino acid substitutions. The role of these natural RCA variants in thermal tolerance is not known, and further experiments are needed to identify the significance of these sequence changes in providing thermal tolerance.
Phylogenetic analysis of monocot RCA
Phylogenetic analysis performed with the alignment of RCA protein from distantly related monocots, using the MEGA7 maximum likelihood method, revealed different clusters of RCAs, based on the sequence similarity and evolutionary pattern (Fig. 7). In deep nodes, the phylogenetic relationship is unclear, and the bootstrap (BS) values are low (< 70%). The comparative analysis showed how the duplicated Rca genes evolved in cereals. Thus, we did not collapse the deep nodes with lower statistical support. Based on the phylogenetic analysis, RCA isoforms of banana, date palm and pineapple, and the cereal isoforms, formed distinct clades, with cereals further comprising of RCA1 and RCA2 from the tandemly duplicated genes. Although the RCA isoforms of banana (MaRCA1, 2 and 3), date palm (PdRCA1 and 2), and pineapple (AcRCA1, 2 and 3) were clustered together, the subgroup pattern within the cluster suggests that the genes coding these isoforms were duplicated or triplicated after the divergence of monocot orders. Similarly, the RCA1 and RCA2 of cereals formed different clades, but within each clade, the isoforms of closely related species, or species from a shared family, showed less divergence with high BS values. The branch and individual leaf lengths show the rate of substitution, the RCA1 isoforms of wild rice and Panicoideae species showed a higher rate of changes than did the RCA1 of Pooideae. Together, these results suggest that after the divergence for a common monocot ancestor, the Rca gene copy number increased either through polyploidization or tandem duplication and evolved at different rates in the studied species.
Expression pattern of RCA isoforms in cereals
Detailed analysis of 302 ESTs from banana (Musa acuminata) and 331 from pineapple (Ananus comosus) showed that in both banana and pineapple, only one of the three copies codes for RCA-α, whereas the other two copies code for only the β isoform (Supplementary Table S1). EST sequences showed no evidence of alternative splicing in the RCA-α coding copy, suggesting a possible loss of the splice junction during evolution. In comparison, alternate splicing was observed in date palm (Phoenix dactylifera) based on the analysis of 305 ESTs. One of the two Rca copies, PdRca1, codes for both RCA-α and RCA-β isoforms via alternative splicing, whereas the other gene copy (PdRca2) codes only for the RCA-α isoform.
Evaluating the EST data, MaRca1-α, MaRca2-β, and MaRca3-β of Banana has 101, 101 and 100 ESTs respectively; PdRca1-α, PdRca1-β and PdRca2-α of date palm has 103, 102 and 100 ESTs respectively and AcRca1-α, AcRca2-β and AcRca3-β of pineapple has 110, 115 and 106 ESTs respectively. Based on this data, the three splice variants appear to be equally expressed in these three plants. With the objective to compare expression of the gene(s) at the RNA level with that at the protein level, and to identify the relative contribution of the two tandemly duplicated Rca gene copies, we performed gene specific expression analyses using quantitative Real Time PCR (qRT-PCR) for both Rca1 and Rca2 genes of O. brachyantha, O. australiensis, O. sativa, T. aestivum, Z. mays, and S. bicolor at normal as well as at high temperature conditions (Fig. 5). In cultivated rice (O. sativa), either under heat stress or normal plant growth conditions, no expression was observed for the Rca1 copy in either of the two cultivars that were used for the analysis (Fig. 5a). O. australiensis showed some expression of Rca1 under normal growth conditions and the expression reduced both under 35oC and 45oC treatment. O. brachyantha on the other hand showed a different expression pattern for Rca1 where its expression was very low under normal plant growth conditions but increased under the heat stress conditions. Rca1 expression was however significantly less than that for Rca2. The Rca2 copy in cultivated rice showed very high expression level under normal plant growth conditions and its expression reduced dramatically under heat stress conditions. There was significant difference between the two cultivated rice lines for the expression of Rca2 copy. The two rice wild relatives showed dramatically higher expression for Rca2 under heat stress. Even between the two wild relatives, O. brachyantha showed significantly higher expression at 45oC treatment as compared to O. australiensis.
Heat stress also affected the alternative splicing mechanism, as the reduction in the levels of transcripts coding RCA2α varied during heat stress (Fig. 4). In rice, OsRca2 gene expresses OsRca2α and OsRca2β transcripts via alternate splicing mechanism. At normal conditions, the level of OsRca2β transcripts was many hundred-folds higher than that of the OsRca2α transcripts, but at 42°C, the OsRca2α transcript level was either equal or higher than that of the OsRca2β transcripts. Interestingly, ObRca2α transcripts were stable at all studied temperatures, however, ObRca2β showed more than two-fold reduction at higher temperatures. These results suggest that the temperature increase, at least among various rice species, favors transcripts coding RCA2α isoform. Even though the transcripts encoding OsRCA2α and OsRCA2β showed a significant reduction at 45°C in rice, the quantity of both the isoforms from the heat stressed leaves is comparable or higher to their control leaves (Fig. 4).
The expression pattern of the Rca gene copies in wheat was very different from that observed in rice. Rca1 showed essentially no expression at normal plant growth conditions, but its expression increased dramatically under heat stress conditions (Fig. 4b). The expression pattern was very similar among the three cultivars (Chinese Spring, Giza168 and PBW343) although significant differences were observed among cultivars. In all three cultivars, transcript level for Rca2 copy was significant at all three temperature conditions although its level at 37oC was significantly higher than either of the other two conditions. Genotypic differences were significant among the three genotypes at each temperature condition. Between the two isoforms of Rca2, transcript level of Rca2α isoform was much lower than the Rca2β form and stayed about the same over the three temperature treatments (Fig. 4b). The Rca2β form showed significant increase at 37oC treatment but reduction at 42oC.
Both in maize and sorghum, Rca1 copy made only the alpha isoform of the gene whereas the Rca2 copy made the beta form (Fig. 4c and d). In both of these species, the beta form was made at all three temperatures although significant differences were observed both among temperatures as well as among genotypes. The alpha form showed essentially no accumulation at 25oC, slight increase at 35oC treatment and dramatic increase at 45oC. In general, the genotypic differences for relative expression of the two isoforms were much higher than that observed among the wheat genotypes.
Using the same plant samples as that used for real-time expression analysis, RCA protein level was also studied using immunoblot analysis with an RCA specific antibody (Fig. 5). Except for wheat, all of the studied cereals expressed a 42 kDa RCAβ isoform at normal as well as under heat stress conditions whereas the 46 kDa RCAα isoform was expressed only during the heat stress conditions (Fig. 5). In comparison to Rubisco, the expression level of wheat RCAβ was significantly higher at the normal temperature as compared to that under heat stress. Relative to RCAβ, expression of the RCAα is much lower at normal conditions but is undetectable after the heat treatment. Although it is difficult to accurately quantify, expression levels of both Rubisco, as well as RCA of rice, maize, and sorghum, increased after 45°C treatment as compared to that at the normal temperature. Wheat, on the other hand did not show the 46 kDa RCAα isoform under heat stress conditions along with a significant reduction in the RCAβ level. With the current analysis, it was not possible to determine if the 42 kDa RCAβ isoform of wheat is expressed from TaRca1 or the TaRca2 copy.
Regulatory elements in the Rca genes of cereals
As the two Rca gene copies of cereals showed very different expression pattern, we looked for differences in expression control elements between promoters of the two copies. We evaluated regulatory elements in ~ 1.5 kb region upstream of the transcription start site of the two Rca gene copies of the studies species along with the intronic sequences, with a focus on heat regulated elements (material and methods). Along with the core promoter elements, this analysis showed various putative cis-elements including that for circadian rhythm, light, hormone and biotic and abiotic stress responsives (Table S2). The Rca1 copy showed insertion of heat shock elements (HSE) in its promoter with wild rice (O. brachyantha) having three HSEs. These HSEs were missing in the Rca1 promoter of cultivated rice (O. sativa). Interestingly, Rca2 of O. sativa have HSEs in its promoter and in the 5th intron. Transposable element (TE) of size 366bp, 347bp and 320 bp were present in the promoter of Rca1, promoter of Rca2 and in the 5th intron of Rca2, respectively (Fig. 1). In wheat, Rca1 promoter of the A and the B genome showed one HSEs and the D genome has two. There were no HSEs present in the promoter of Rca2 of wheat but promoter of D genome showed insertion of 162 bp TE. In maize (Z. mays) and sorghum (S. bicolor), both Rca1 and Rca2 had insertion of HSEs with sorghum having maximum of 5 HSEs in the promoter of Rca1. Promoter of Rca2 had two HSEs in sorghum and one in maize. TE were also present in the promoter of Rca1 in both maize and sorghum (Fig. 1)
Adaptation of organisms to the changing environmental conditions via modifications in the basic biological processes is a common feature of evolution. For better adaptation, an organism often relies on new gene mutations which may develop neo-functions, however, that could also cause detrimental effects if the mutated gene is essential for the organism’s survival. Gene and genome duplications provide the base genetic material for the origin of novel genes or trait development during evolution.
Tandem duplication of Rca genes was first reported in barley (Rundle and Zielinski, 1991) and later in wheat (Carmo-Silva et al., 2015). In rice, DNA gel blot analysis with cDNA coding RCA as a probe showed the presence of more than one Rca gene in the genome (Zhang and Komatsu, 2000). We (Nagarajan and Gill, 2018) showed that the tandem duplication event occurred in the common grass ancestor and the structural changes then accumulated after the divergence of the Poaceae families. In this study we have shown that this tandem duplication of Rca gene is Poaceae-specific and probably occurred after the divergence of Poales order as one (Bromeliaceae) of the 16 families did not have the duplication. It would be possible to pinpoint the exact timeline of the duplication event once the genome sequence information for the other 14 families is available.
Phylogenetic and fossil data analyses have suggested that the grass sub-families probably diverged from a common ancestor between 50-70 million years ago (MYA) (Kellogg, 2001; Salse et al., 2008). The tandem duplication event of Rca genes might have occurred before the divergence of the grass sub-families as the Oryzoideae, Pooideae, and Panicoideae members have the duplicated genes (Fig. 2). The conserved heat induced expression pattern of Rca1 in all three sub-families suggests that the promoter elements required for heat responsive expression might have integrated into the Rca1 promoter of the grass ancestor. Later, species specific TE insertions added additional HSEs in the promoter region and further altered the gene expression. Effect of TE insertions on the neighboring gene expression is a well-documented phenomenon in plants and other organisms (see reviews, Feschotte, 2008; Lisch, 2013; De Souza et al., 2013). Similarly, integration of biotic and abiotic responsive cis-regulatory elements in the promoter regions of genes by TE was also reported in plants (Beguiristain et al., 2001; Naito et al., 2009), including HSEs (Ito et al., 2013). The insertions of TEs with HSE elements in duplicated Rca gene regions of cereals could be the source for evolutionary adaptation through altered gene expression. Heat stress induced changes in the RCA expression pattern were reported earlier in rice (Scafaro et al., 2010; Wang et al., 2010), wheat (Feller et al., 1998; Law and Crafts-Brandner, 2001; Ristic et al., 2009), and maize (Crafts-Brandner and Salvucci, 2002; Ristic et al., 2009). Our copy specific Rca transcript quantification along with immunoblot analysis have specifically revealed expression pattern of each of the Rca copies and of its splice varients. We have also shown differences and similarlities among Rca of various species for a differential response to heat stress. In comparison to maize and sorghum, the wheat Rca showed almost an opposite accumulation pattern of different Rca isoforms and splice varients (Figures 4&5). Role of these differences in explaining differences in heat stress tolerance among wheat, maize and sorghum needs to be validated.
One of the important findings of this study is the identification of a functional Rca1 gene copy among wild rice species. The genus Oryza is composed of 23 recognized species, including two cultivated (O. sativa and O. glaberrima) and 21 wild rice species with 10 different genomes (AA, BB, CC, BBCC, CCDD, EE, FF, GG, HHJJ, and HHKK) (Ge et al., 1999; Vaughan et al., 2003). Our analysis on the non-functionalization of Rca1 showed that the species O. sativa, O. glaberrima, O. barthii, O. glumipatula, O. nivara, and O. rufipogon with AA genome have multiple deletions in the exonic sequences thus have non-functional Rca1. Interestingly, O. punctata with BB genome, O. australiensis with EE, and O. brachyantha with FF genome contain a complete and functional Rca1 gene. These results suggest that the non-functionalization of Rca1 in the AA genome species occurred after the divergence of Oryza species and also specifically in the AA genome lineage. Recent research on thermal tolerance of wild rice species O. australiensis showed unaffected net photosynthetic and leaf elongation rates, even at 45°C, whereas O. meridionalis and cultivated rice O. sativa showed intermediate tolerance to > 50% decline in net photosynthesis and leaf growth rates (Scafaro et al., 2016). Rubisco activation levels of these species at high temperature correlated with the net photosynthesis and leaf elongation rate. Interestingly, the thermal stability of recombinant OaRCA2 was similar to the OsRCA2 of cultivated rice in the in vitro assays, but the leaf extracted RCA of O. australiensis showed no Rubisco activation inhibition at temperatures as high as 42°C, whereas rice RCA inhibition occurred at temperatures above 34°C. The authors characterized the OaRca2 of O. australiensis and compared its thermal stability with that of rice OsRca2. They speculated that some unknown factor(s) in the leaf extract could contribute to its higher thermal activity and stability (Scafaro et al., 2016). Based on our Rca1 comparative analysis of Oryza sp. and heat stress induced expression of OaRca1 of O. australiensis, we propose that the presence of OaRCA1 could be one of the reasons for increased thermal stability of Rubisco activation and unaffected net photosynthetic observed in the above-mentioned study. However, further experiments are needed to characterize the OaRca1 and utilize this gene in rice breeding through alien introgression to improve photosynthetic thermal tolerance of rice cultivars.
Various heat shock elements (HSEs) present in the sequences upstream of the transcription start sites (TSS) were identified in ObRca1 of O. brachyantha, OsRca2 of rice, all three copies of wheat TaRca1, both ZmRca1 and ZmRca2 of maize, and both SbRca1 and SbRca2 of sorghum (Fig. 1). The number of HSEs also varied among species with the promoter of SbRca1 containing up to five, ObRca1 containing three, TaRca1-D and SbRca2 containing two, and the remaining ones showing only a single HSE in the upstream region. It is tempting to speculate a correlation of higher level of heat stress tolerance in sorghum and wild rice with higher number of HSE’s but additional experimentation is needed to confirm.
Previous studies have shown that certain transposable elements are preferentially inserted into the regulatory sequences of various genes and play an important role in the expression control of the neighboring genes (Makarevitch et al., 2015). In some cases, heat stress induced gene expression was acquired from TE insertion events (Ito et al., 2011). The duplicated Rca genes of closely related yet evolutionarily diverged cereals, and their heat stress induced expression pattern may serve as an ideal model for understanding different TE insertions and their role in the stress regulated expression. To identify TEs in the genic region, we used the duplicated gene sequences, along with 2Kb sequence flanking on both sides, as the query to search the crop specific TE database. We identified independent TE insertion events in the promoter sequence of all six Rca1 copies of the studied species (Table 2). Similarly, there were TE insertions in the promoter and the fifth intron of rice OsRca2, and in the promoter of TaRca2-D copy (Fig. 1). Interestingly, many of these TEs contain HSEs; these TEs inserted in different time periods of evolution served as a potential source for the HSEs and may have caused heat stress induced expression pattern in the Rca1 genes. A detailed list of the identified TEs, their sizes, and locations relative to the genes is given in Table 2.
In conclusion, the comparative and expression analyses of RCA coding genes of important cereal crops revealed a tandem gene duplication, which occurred in the common grass ancestor subsequently led to neo-functionalization and evolved to respond differently during heat stress. One of the two copies of the tandem duplication maintained a very high level of conservation whereas the second copy showed tremendous divergence to evolve species specific function of the gene. We identified the insertion of various transposable elements harboring heat responsive elements in the promoter region. These results suggest that one of the duplicated Rca genes showing divergence, which could be responsible for the heat induced expression pattern and thermal stability of these heat tolerant cereal crops. Understanding these evolutionary changes are important as this knowledge can be used to engineer future plants with better and wider adaptability.
Gene model prediction and validation
The genomic regions comprising Rca genes of banana, date palm, and pineapple were searched using the well characterized 474 aa sequence of AtRCA1 (AT2G39730.1) (https://www.arabidopsis.org/) as the query in Phytozome 11 (https://phytozome.jgi.doe.gov) and NCBI database. We selected banana, date palm, and pineapple because of its position in the monocot evolution with sequenced genome at the time of analysis. Gene models were developed using the predicted transcripts for each of these genes from NCBI database (Table S1). The Rca gene models for rice (cv. Nipponbare), wheat (cv. Chinese spring), maize (inbred B73), and sorghum (inbred BTx623) were adopted from our previous study (Nagarajan and Gill, 2018). To identify the Rca genes of wild rice species, AtRCA1 was used as a query sequence to search 11 Oryza species (Jacquemin et al., 2013) of Ensembl plants DNA database with default settings (http://plants.ensembl.org/index.html). For the Oryza Rca1 structural analysis, we did not include O. meridionalis, O. glaberrima, and O. longistaminata, because the sequences for the region was not complete in these three species. The criteria used for searching true orthologs in this study were described elsewhere (Dhaliwal et al., 2014). For identification of cis-regulatory elements in the promoter region, we used the 1.5 Kb upstream sequence from ATG start codon for each gene to search in PlantCARE database (Lescot et al., 2002). The transposable elements for Oryza species were identified from RiTE-DB (http://www.genome.arizona.edu/cgi-bin/rite/index.cgi); wheat from cereal repeat database (http://wheat.pw.usda.gov/ggpages/Repeats/), maize from MaizeGDB (http://www.maizegdb.org), and sorghum from TREP database (http://botserv2.uzh.ch/kelldata/trep-db/index.html). For validation of the gene models, cDNA sequences coding the mature isoform of Rca were PCR amplified using attB sites flanking transcript specific primers (Supplementary table S3) with PrimeSTAR GXL polymerase (Clontech-Takara, USA) and cloned in pDONR201 vector using BP Clonase-II enzyme (Invitrogen, USA) as per the manufacturer’s recommendation. Multiple clones per construct were sequenced and the data was validated with the previously predicted gene models. Rca gene from banana, date palm, and pineapple was included in the analysis due to their position in the monocot evolution. Gene models were developed using the predicted transcripts for each of these genes from NCBI database (Table S1).
To identify the Rca genes of wild rice species, AtRCA1 was used as a query sequence to search 11 Oryza species (Jacquemin et al., 2013) of Ensembl plants DNA database with default settings (http://plants.ensembl.org/index.html). For the Oryza Rca1 structural analysis, we did not include O. meridionalis, O. glaberrima, and O. longistaminata, because the sequences for the region was not complete in these three species.
Plant growth condition and heat treatment
Rice cultivars Nipponbare (GSOR 100) and N22 (PI 389102), maize inbred lines B73 (PI 550473) and Mo22 (PI 558534), and sorghum genotypes BTx623 (PI 564163), SC1019 (PI 656071), SC1047 (PI 656072) and B35 (PI 147224) were received from Germplasm Resources Information Network (https://www.ars-grin.gov). The wild rice species Oryza brachyantha (IRGC 101232) and Oryza australiensis (IRGC 101397) used in this study were received from Gerald E. Edwards, Washington State University, Pullman, WA, USA. The wheat cultivars Chinese Spring, Giza168, and PBW343 were used in the experiment. All plant materials were grown in Conviron PGR15 growth chamber equipped with high-intensity discharge lamps capable of Photosynthetically Active Radiation (PAR) of > 600 µmol/m2/sec before treating at high temperatures. Except for the rice and wild rice species, all plants were planted in 4 x 9-inch-deep pots using Sunshine LC1 potting mixture (SunGro Horticulture, Bellevue, WA, USA) supplemented with 14g Osmocote 14-14-14 slow releasing fertilizer. Cultivated and wild rice species were grown as mentioned in Giuliani et al. (2013). The wheat plants were grown under controlled temperature of 24/18°C with 16/8 hours of the day/night cycle. For rice maize, and sorghum genotypes the temperature was 25/22°C with 12/12 hours day/night cycle. Heat shock treatments were given to plants with fully expanded leaves (four-five weeks old). The chamber temperature was gradually increased (5°C/hour) from morning to reach the target temperatures and maintained for four hours before collecting leaf samples. To match the circadian rhythm, samples from control sets were also collected at the same time period. Leaf samples were frozen immediately in liquid nitrogen and stored in -80°C freezers for subsequent RNA and protein extractions.
Immunoblot analysis
Protein extraction and immunoblot analyses were performed as described in Yamori and von Caemmerer (2011) with some modifications. Protein concentrations were measured using Bradford reagent (Bio-Rad). For Fig. 4, 2ul of the total protein was loaded per well on 1D-SDS-PAGE. Immunoblots were probed first with cotton anti-Rubisco activase (Agrisera, Catalog # AS10-700) antibody and later with horseradish peroxidase-conjugated secondary antibody as per the manufacturers’ recommendation. Immunological bindings were detected using Pierce ECL Western Blotting Substrate (Thermo Scientific) with Kodak X-ray film developing system.
RNA extraction, cDNA preparation, and Real Time (RT) PCR analysis
Total RNA from the normal and heat stress treated leaf samples were extracted using the hot phenol RNA extraction method. In brief, 100–200 mg frozen tissue samples were ground in liquid nitrogen and 0.75 ml of preheated (80°C) extraction buffer containing a mix of 100 mM Tris-HCl (pH 8.0), 100 mM Lithium Chloride (LiCl), 20 mM Ethylenediaminetetraacetic acid (EDTA), 1% Sodium dodecyl sulfate (SDS) prepared in Diethyl pyrocarbonate (DEPC) treated water and equal volume of phenol (pH 4.8) was added. Before centrifugation, the homogenates were vortexed, chloroform (0.35 ml) was added and vortexed till the mix turn whitish green. The resulted supernatant was separated, precipitated with 8M LiCl in ice for two hours and spun at 4°C for 30 min. The RNA pellets were washed with 2M LiCl and 70% ethanol and air dried. The pellets were dissolved in DEPC treated water and DNAse digestion was performed using Turbo™ DNAse kit (Thermo Fisher Scientific; Catalog # AM2238) as per the manufacturer’s recommendation. Purified RNA was loaded on a 1% agarose gel to check the quality and quantity of the sample. First strand cDNA was synthesized from the purified RNA samples using M-MLV Reverse transcriptase (Promega; Catalog # M1701) with anchored-oligo (dT)18 primer as per the manufacturer’s recommendation. The cDNA samples were diluted 50 times and used in the RT-PCR reactions. All the RT-PCR analysis were performed using the KAPA SYBR® FAST qPCR master mix (Kapa Biosystems Inc; Catalog # KK4609) on LightCycler® 480 (Roche Diagnostics), as per the manufacturer’s instructions. The sequences of all the PCR primers used for the RT-PCR are given in Table S3. The relative expression for each sample was calculated using 2− ΔCT, where ΔCT = (CT gene of interest – CT internal control) (Schmittgen and Livak 2008) .
Phylogenetic analysis
Phylogenetic studies were performed using the putative RCA amino acid sequences retrieved from the annotated gene models. The complete list of the sequences used is listed in the SI. The analysis was carried out using the Maximum likelihood method in the Molecular Evolutionary Genetics Analysis version 7 (MEGA7) (Kumar et al., 2016). Alignment for tree construction was done using MUSCLE with the Gonnet scoring matrix. Reliability of the obtained trees was tested using 1000 bootstrap replicates.
Conflict of interest statement
The authors declare no conflict of interest.
Author contributions
RN and KSG designed the research, RN and KSK performed the experiments and data analysis and wrote the initial draft of manuscript. KSK, AM and KSG critically revised and prepared the final draft of the manuscript.
Funding
This work was supported by the USDA National Institute of Food and Agriculture, Hatch (project number WNP00449) and United States Agency for International Development Feed the Future Innovation Lab-Climate Resilient Wheat (Grant number AID-OAA-A-13-00008).
Author contributions:
RN and KSG designed the research, RN and KSK performed the experiments and data analysis and wrote the initial draft of manuscript. KSK, AM and KSG critically revised and prepared the final draft of the manuscript.
Acknowledgements
We sincerely thank Dr. G.N. Mohan Kumar, Department of Horticulture, Washington State University for providing advice on protein extraction and immunoblot analyses, Sheila Fitzgerald for proofreading the manuscript, and Dr. Gerald Edwards and Dr. Rita Giuliani, School of Biological Sciences, Washington State University for providing wild rice species and protocol for growing rice plants under control conditions.
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Table 2. List of transposable elements identified in the grass Rca genic region
|
Gene |
TE type |
Size (bp) |
Region |
E value |
No. of HSEs (sequence) |
|
ObRca1 |
Mutator-like (MULE) |
336 |
Promoter |
0 |
2 (aaaaaattta; caaatttttt) |
|
OsRca2 |
Mutator-like (MULE) |
347 |
Promoter |
0 |
1 (aaaaaatttg) |
|
OsRca2 |
MuDR-like (MITE) |
320 |
Intron (5) |
0 |
1 (gtaatttttt) |
|
TaRca1-A |
Gypsy (LTR) |
319 |
Promoter |
3e-43 |
0 |
|
TaRca1-B |
Mariner (MITE) |
239 |
Promoter |
1e-18 |
0 |
|
TaRca1-D |
Copia (LTR) |
967 |
Promoter |
0 |
0 |
|
TaRca2-D |
Mariner (MITE) |
162 |
Promoter |
5e-21 |
0 |
|
ZmRca1 |
Class II/III TE |
368 |
Promoter |
0 |
1 (agataattcg) |
|
SbRca1 |
CACTA-consensus |
584 |
Promoter |
2e-97 |
1 (acaaaatttc) |