Discovery and Expression Analysis of Alternative Splicing Events Conserved among Plant SR Proteins (original) (raw)
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1Department of Biological Sciences, Oakland University
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1Department of Biological Sciences, Oakland University
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1Department of Biological Sciences, Oakland University
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1Department of Biological Sciences, Oakland University
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2Department of Biology, University of Florida
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3Department of Biology and School of Informatics and Computing, Indiana University, Bloomington
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1Department of Biological Sciences, Oakland University
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Published:
19 December 2013
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Hypaitia B. Rauch, Tara L. Patrick, Katarina M. Klusman, Fabia U. Battistuzzi, Wenbin Mei, Volker P. Brendel, Shailesh K. Lal, Discovery and Expression Analysis of Alternative Splicing Events Conserved among Plant SR Proteins, Molecular Biology and Evolution, Volume 31, Issue 3, March 2014, Pages 605–613, https://doi.org/10.1093/molbev/mst238
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Abstract
The high frequency of alternative splicing among the serine/arginine-rich (SR) family of proteins in plants has been linked to important roles in gene regulation during development and in response to environmental stress. In this article, we have searched and manually annotated all the SR proteins in the genomes of maize and sorghum. The experimental validation of gene structure by reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed, with few exceptions, that SR genes produced multiple isoforms of transcripts by alternative splicing. Despite sharing high structural similarity and conserved positions of the introns, the profile of alternative splicing diverged significantly between maize and sorghum for the vast majority of SR genes. These include many transcript isoforms discovered by RT-PCR and not represented in extant expressed sequence tag (EST) collection. However, we report the occurrence of various maize and sorghum SR mRNA isoforms that display evolutionary conservation of splicing events with their homologous SR genes in Arabidopsis and moss. Our data also indicate an important role of both 5′ and 3′ untranslated regions in the regulation of SR gene expression. These observations have potentially important implications for the processes of evolution and adaptation of plants to land.
Introduction
Precise recognition and exclusion of introns are fundamental to the expression of polymerase II-encoded transcripts for the vast majority of eukaryotic genes. This pre-mRNA processing occurs in spliceosomes, a large macromolecular complex composed of more than 170 proteins and five small nuclear ribonucleoproteins (snRNPs; Behzadnia et al. 2007; Kelemen et al. 2013). Frequently, different combinations of alternative splice sites during pre-mRNA processing significantly increase the diversity of transcripts by eukaryotic genes. Four types of alternative splicing events result in a single gene encoding for more than one mRNA isoform (Carvalho et al. 2013). These are alternative 5′ donor splice site, alternative 3′ acceptor splice site, exon skipping, and intron retention. It is becoming evident that alternative splicing has played a major role in the evolution of complex organisms. Serine/arginine-rich (SR) proteins are a family of highly conserved phosphoproteins that play a key role during pre-mRNA processing. They contribute significantly to alternative splicing by influencing the selection of splice sites through their relative abundance and phosphorylation state. Alternatively, spliced isoforms of several SR proteins in plants are tissue specific or developmentally regulated and are a response to environmental stress (Duque 2011).
The characteristic feature of SR proteins is the presence of one or two RNA binding domains (RRM) at their N terminus and a region rich in serine and arginine (RS) at their C terminus (Barta et al. 2010; Manley and Krainer 2010; Reddy and Shad Ali 2011). The RS domain of SR proteins plays an integral role in the employment of splicing factors during the assembly of the spliceosome via protein–protein and protein–RNA interaction (Maniatis and Tasic 2002; Black 2003; Barta et al. 2008). Plant SR genes are grouped into seven subfamilies (RS, RSZ, RS2Z, SC, SCL, SR, and SR45), four of which (RS, RS2Z, SCL, and SR45) are specific to plants (Tanabe et al. 2007; Barta et al. 2008). It remains to be determined whether SR proteins exclusive to plants reflect the intrinsic differences in splicing machinery reported between plants and animals (Barta et al. 1986; van Santen and Spritz 1987; Wiebauer et al. 1988; Pautot et al. 1989; Waigmann and Barta 1992) or are the result of gene duplication events. A majority of plant SR genes encode multiple isoforms of transcripts via alternative splicing. For example, 14 of the 18 SR genes in the Arabidopsis genome display alternative splicing, producing 90 transcript isoforms (Reddy and Shad Ali 2011). Recently, in silico analysis of the available expressed sequence tags (ESTs)/cDNAs in the extant databases of 27 eukaryotic organisms revealed a prodigious occurrence of alternative splicing in SR proteins (Richardson et al. 2011). Intriguingly, these studies revealed a significantly lower number and in some cases absence of alternative splicing of SR genes in the genomes of unicellular eukaryotic species, such as Dictyostelium discoideum, Plasmodium falciparum , and Phytophthora sojae. This could be due to the presence of fewer numbers of introns in their genomes (Richardson et al. 2011). The precise biological relevance of different isoforms of SR proteins remains to be determined. However, their wide prevalence across plant species is very much indicative of their importance in functional regulation of SR transcripts. Furthermore, the profile of alternative spliced transcripts of several SR genes in Arabidopsis display a remarkable alteration during environmental stresses such as heat, salt, and light irradiation (Lazar and Goodman 2000; Palusa et al. 2007; Tanabe et al. 2007; Duque 2011). This observation suggests that a distinct role of SR transcript isoforms in plants is to adapt to different environments. Despite their importance and significant increase in the sequence resources of different plant species, the evolutionary conservation of alternative splicing events as a reflection of their biological significance has not been extensively investigated. The only reports of evolutionary conservation between diverse plant species have been demonstrated in the alternative splicing of an intron spanning the first RRM domain of plant-specific SR proteins belonging to subfamilies RS, RS2Z, and SCL (Iida and Go 2006; Kalyna et al. 2006). The putative protein produced by alternative splicing of this intron is a truncated form lacking an intact RRM1 domain. In Arabidopsis SR gene at-RSZ33, a member of the RS2Z subfamily, an alternative splicing event occurring in this intron demonstrates the regulation of expression for both at-RSZ33 and at-RSp31 (Kalyna et al. 2003, 2006). The lethal impact from the ectopic overexpression of the intronless construct of at-RSZ33 points to the importance of this alternative splicing-mediated autoregulation (Kalyna et al. 2003).
To assess its biological significance, we analyzed the extent of evolutionary conservation of alternative splicing events between different plant species. We searched and manually annotated all the SR genes in the genomes of two economically important and closely related plants, maize and sorghum. The gene structure and alternative splicing events were determined in silico by splice alignment of the available ESTs in the public database with their cognate SR gene. To further test the efficacy of our computational prediction and compare the alternative splicing profile, we performed reverse transcription-polymerase chain reaction (RT-PCR) analysis of each SR gene using total RNA extracted from etiolated maize and sorghum roots and shoots grown under similar conditions. These RT-PCR analyses led to the discovery of many SR alternatively spliced isoforms not represented in the extant EST database. Our data indicates that despite the divergent profiles of alternative splicing of SR genes between maize and sorghum, various transcripts display evolutionary conservation of splicing events in maize, sorghum, Arabidopsis, and moss. In addition, we discovered alternatively spliced SR transcripts with conserved splicing patterns and putative translation products between maize, sorghum, Arabidopsis, and moss. These results point to the important biological significance of alternative isoforms in the regulation of plant gene expression. Intriguingly, we also discovered conservation of alternative spliced introns present in the 5′ and 3′ untranslated region (UTR), which suggests a regulatory function of the UTRs in SR gene expression.
Results
Alternative Splicing Augments the Transcript Diversity of SR Genes
A comprehensive search using PlantGDB led to the identification of 21 maize and 18 sorghum SR genes (table 1), of which only 5 had been previously reported (Gao et al. 2004; Gupta et al. 2005). The SR genes reported here were named following the guidelines proposed for nomenclature of SR proteins in plants (Barta et al. 2010) and are displayed in table 1. To compare the expression of SR genes, total RNA extracted from etiolated roots and shoots of closely related maize and sorghum were subjected to RT-PCR analysis using primers flanking the first and the last exon of the manually predicted gene structure for each SR gene using EST alignment. The sequences of these primers are shown in supplementary table S1, Supplementary Material online, in the supplementary material S1, Supplementary Material online. The resultant RT-PCR products were resolved on agarose gels, purified, cloned, and sequenced in both directions. Here we report a total of 92 SR transcript isoforms in maize, including 19 reported earlier (Gao et al. 2004; Gupta et al. 2005) (table 1). Among these isoforms, 46 are represented in EST collections and 27 are discovered by RT-PCR and not represented in extant EST databases. Similarly, 62 transcript isoforms were identified in sorghum. Of these, 26 are represented in EST collections, whereas 36 are unique to RT-PCR and not represented in EST collections (table 2). All the known forms of alternative splicing events, including the usage of noncanonical splice sites, were detected in both maize and sorghum. As shown in table 3, the usage of an alternative 3′ splice site is the most prevalent in maize, while alternative 5′ splice site usage is the most prevalent in sorghum. The skipping of exons and the retention of unspliced introns were the least abundant alternative splicing events in maize and sorghum, respectively.
Table 1.
SR Homologs in Different Plant Species.
Subfamily | Zea mays | Sorghum bicolor | Arabidopsis thaliana | Physcomitrella patens |
---|---|---|---|---|
SC | zm-SC32 | sb-SC31 | at-SC35 | pp-SC39 |
zm-SC30 | sb-SC30 | pp-SC37 | ||
zm-SC26 | ||||
SCL | zm-SCL30 | sb-SCL31 | at-SCL33 | pp-SCL42 |
zm-SCL28 | sb-SCL28 | at-SCL30 | pp-SCL33 | |
zm-SCL25A | sb-SCL25A | at-SCL30A | ||
zm-SCL25B | sb-SCL25B | at-SCL28 | ||
RS | zm-RS31A | sb-RS34 | at-RS41 | pp-RS27 |
zm-RS31B | sb-RS28 | at-RS40 | ||
at-RS31 | ||||
at-RS31A | ||||
RS2Z | zm-RS2Z39 | sb-RS2Z39 | at-RS2Z33 | pp-RS2Z37 |
zm-RS2Z37A | sb-RS2Z35 | at-RS2Z32 | ||
zm-RS2Z37B | sb-RS2Z34 | |||
zm-RS2Z35 | ||||
RSZ | zm-RSZ28 | sb-RSZ28 | at-RSZ22 | pp-RSZ23 |
zm-RSZ21 | sb-RSZ21 | at-RSZ22A | ||
zm-RSZ20 | sb-RSZ19 | at-RSZ21 | ||
SR | zm-SR32 | sb-SR32 | at-SR34 | pp-SR29 |
zm-SR31 | sb-SR30 | at-SR34A | ||
zm-SR30 | sb-SR27 | at-SR34B | ||
at-SR30 | ||||
SR45 | zm-SR45_1 | sb-SR45 | at-SR45 | pp-SR45 |
zm-SR45_2 | ||||
SC | zm-SC32 | sb-SC31 | at-SC35 | pp-SC39 |
Subfamily | Zea mays | Sorghum bicolor | Arabidopsis thaliana | Physcomitrella patens |
---|---|---|---|---|
SC | zm-SC32 | sb-SC31 | at-SC35 | pp-SC39 |
zm-SC30 | sb-SC30 | pp-SC37 | ||
zm-SC26 | ||||
SCL | zm-SCL30 | sb-SCL31 | at-SCL33 | pp-SCL42 |
zm-SCL28 | sb-SCL28 | at-SCL30 | pp-SCL33 | |
zm-SCL25A | sb-SCL25A | at-SCL30A | ||
zm-SCL25B | sb-SCL25B | at-SCL28 | ||
RS | zm-RS31A | sb-RS34 | at-RS41 | pp-RS27 |
zm-RS31B | sb-RS28 | at-RS40 | ||
at-RS31 | ||||
at-RS31A | ||||
RS2Z | zm-RS2Z39 | sb-RS2Z39 | at-RS2Z33 | pp-RS2Z37 |
zm-RS2Z37A | sb-RS2Z35 | at-RS2Z32 | ||
zm-RS2Z37B | sb-RS2Z34 | |||
zm-RS2Z35 | ||||
RSZ | zm-RSZ28 | sb-RSZ28 | at-RSZ22 | pp-RSZ23 |
zm-RSZ21 | sb-RSZ21 | at-RSZ22A | ||
zm-RSZ20 | sb-RSZ19 | at-RSZ21 | ||
SR | zm-SR32 | sb-SR32 | at-SR34 | pp-SR29 |
zm-SR31 | sb-SR30 | at-SR34A | ||
zm-SR30 | sb-SR27 | at-SR34B | ||
at-SR30 | ||||
SR45 | zm-SR45_1 | sb-SR45 | at-SR45 | pp-SR45 |
zm-SR45_2 | ||||
SC | zm-SC32 | sb-SC31 | at-SC35 | pp-SC39 |
Table 1.
SR Homologs in Different Plant Species.
Subfamily | Zea mays | Sorghum bicolor | Arabidopsis thaliana | Physcomitrella patens |
---|---|---|---|---|
SC | zm-SC32 | sb-SC31 | at-SC35 | pp-SC39 |
zm-SC30 | sb-SC30 | pp-SC37 | ||
zm-SC26 | ||||
SCL | zm-SCL30 | sb-SCL31 | at-SCL33 | pp-SCL42 |
zm-SCL28 | sb-SCL28 | at-SCL30 | pp-SCL33 | |
zm-SCL25A | sb-SCL25A | at-SCL30A | ||
zm-SCL25B | sb-SCL25B | at-SCL28 | ||
RS | zm-RS31A | sb-RS34 | at-RS41 | pp-RS27 |
zm-RS31B | sb-RS28 | at-RS40 | ||
at-RS31 | ||||
at-RS31A | ||||
RS2Z | zm-RS2Z39 | sb-RS2Z39 | at-RS2Z33 | pp-RS2Z37 |
zm-RS2Z37A | sb-RS2Z35 | at-RS2Z32 | ||
zm-RS2Z37B | sb-RS2Z34 | |||
zm-RS2Z35 | ||||
RSZ | zm-RSZ28 | sb-RSZ28 | at-RSZ22 | pp-RSZ23 |
zm-RSZ21 | sb-RSZ21 | at-RSZ22A | ||
zm-RSZ20 | sb-RSZ19 | at-RSZ21 | ||
SR | zm-SR32 | sb-SR32 | at-SR34 | pp-SR29 |
zm-SR31 | sb-SR30 | at-SR34A | ||
zm-SR30 | sb-SR27 | at-SR34B | ||
at-SR30 | ||||
SR45 | zm-SR45_1 | sb-SR45 | at-SR45 | pp-SR45 |
zm-SR45_2 | ||||
SC | zm-SC32 | sb-SC31 | at-SC35 | pp-SC39 |
Subfamily | Zea mays | Sorghum bicolor | Arabidopsis thaliana | Physcomitrella patens |
---|---|---|---|---|
SC | zm-SC32 | sb-SC31 | at-SC35 | pp-SC39 |
zm-SC30 | sb-SC30 | pp-SC37 | ||
zm-SC26 | ||||
SCL | zm-SCL30 | sb-SCL31 | at-SCL33 | pp-SCL42 |
zm-SCL28 | sb-SCL28 | at-SCL30 | pp-SCL33 | |
zm-SCL25A | sb-SCL25A | at-SCL30A | ||
zm-SCL25B | sb-SCL25B | at-SCL28 | ||
RS | zm-RS31A | sb-RS34 | at-RS41 | pp-RS27 |
zm-RS31B | sb-RS28 | at-RS40 | ||
at-RS31 | ||||
at-RS31A | ||||
RS2Z | zm-RS2Z39 | sb-RS2Z39 | at-RS2Z33 | pp-RS2Z37 |
zm-RS2Z37A | sb-RS2Z35 | at-RS2Z32 | ||
zm-RS2Z37B | sb-RS2Z34 | |||
zm-RS2Z35 | ||||
RSZ | zm-RSZ28 | sb-RSZ28 | at-RSZ22 | pp-RSZ23 |
zm-RSZ21 | sb-RSZ21 | at-RSZ22A | ||
zm-RSZ20 | sb-RSZ19 | at-RSZ21 | ||
SR | zm-SR32 | sb-SR32 | at-SR34 | pp-SR29 |
zm-SR31 | sb-SR30 | at-SR34A | ||
zm-SR30 | sb-SR27 | at-SR34B | ||
at-SR30 | ||||
SR45 | zm-SR45_1 | sb-SR45 | at-SR45 | pp-SR45 |
zm-SR45_2 | ||||
SC | zm-SC32 | sb-SC31 | at-SC35 | pp-SC39 |
Table 2.
Overview of SR Genes in Maize and Sorghum.
Zea mays | Sorghum bicolor | |
---|---|---|
SR subfamilies | 7 | 7 |
Total number of SR genes | 21 | 18 |
Total number of SR transcripts | 92 | 62 |
Number of newly identified SR transcripts | 27 | 36 |
Number of alternative spliced SR genes | 20 | 15 |
Number of transcripts with intact RRM domain | 78 | 55 |
Zea mays | Sorghum bicolor | |
---|---|---|
SR subfamilies | 7 | 7 |
Total number of SR genes | 21 | 18 |
Total number of SR transcripts | 92 | 62 |
Number of newly identified SR transcripts | 27 | 36 |
Number of alternative spliced SR genes | 20 | 15 |
Number of transcripts with intact RRM domain | 78 | 55 |
Table 2.
Overview of SR Genes in Maize and Sorghum.
Zea mays | Sorghum bicolor | |
---|---|---|
SR subfamilies | 7 | 7 |
Total number of SR genes | 21 | 18 |
Total number of SR transcripts | 92 | 62 |
Number of newly identified SR transcripts | 27 | 36 |
Number of alternative spliced SR genes | 20 | 15 |
Number of transcripts with intact RRM domain | 78 | 55 |
Zea mays | Sorghum bicolor | |
---|---|---|
SR subfamilies | 7 | 7 |
Total number of SR genes | 21 | 18 |
Total number of SR transcripts | 92 | 62 |
Number of newly identified SR transcripts | 27 | 36 |
Number of alternative spliced SR genes | 20 | 15 |
Number of transcripts with intact RRM domain | 78 | 55 |
Table 3.
Comparison of Alternative Splicing between Maize and Sorghum.
Alternative Splicing Type | Zea mays | Sorghum bicolor |
---|---|---|
Alternative 5′ | 32 | 21 |
Alternative 3′ | 38 | 13 |
Alternative 5′ and 3′ | 25 | 19 |
Exon Skipping | 13 | 16 |
Intron Retention | 16 | 5 |
Non-canonical splice site | 23 | 27 |
Alternative Splicing Type | Zea mays | Sorghum bicolor |
---|---|---|
Alternative 5′ | 32 | 21 |
Alternative 3′ | 38 | 13 |
Alternative 5′ and 3′ | 25 | 19 |
Exon Skipping | 13 | 16 |
Intron Retention | 16 | 5 |
Non-canonical splice site | 23 | 27 |
Table 3.
Comparison of Alternative Splicing between Maize and Sorghum.
Alternative Splicing Type | Zea mays | Sorghum bicolor |
---|---|---|
Alternative 5′ | 32 | 21 |
Alternative 3′ | 38 | 13 |
Alternative 5′ and 3′ | 25 | 19 |
Exon Skipping | 13 | 16 |
Intron Retention | 16 | 5 |
Non-canonical splice site | 23 | 27 |
Alternative Splicing Type | Zea mays | Sorghum bicolor |
---|---|---|
Alternative 5′ | 32 | 21 |
Alternative 3′ | 38 | 13 |
Alternative 5′ and 3′ | 25 | 19 |
Exon Skipping | 13 | 16 |
Intron Retention | 16 | 5 |
Non-canonical splice site | 23 | 27 |
The Vast Majority of SR Transcript Isoforms Are Not Conserved between Maize and Sorghum
Despite sharing high degrees of structural similarity and conserved positions of the introns, the selection of alternatively spliced sites for the majority of SR transcripts is not conserved between maize and sorghum. For example, the gene structure and the expression analysis of maize zm-SC30 gene and its sorghum homolog sb-SC31 in root and shoot tissues is displayed in figure 1. Both these genes belong to the SC subfamily of SR proteins and contain eight exons and seven introns with conserved positions, which span the RRM and RS domains (fig. 1A). The spliced alignment of the ESTs and RT-PCR products with their cognate genes revealed ten and six distinct transcript isoforms of zm-SC30 and sb-SC31, respectively. As displayed in figure 1C and D, these transcripts are generated by differential selection of splice sites during pre-mRNA processing. The pattern of splice site selection is not conserved for any of the alternatively spliced isoforms of SR transcripts between maize and sorghum. Intriguingly, the splicing events that generate the SR transcript isoforms of both zm-SC30 and sb-SC31 genes occur near the 3′ terminus. Thus, the putative proteins for all SR transcript isoforms contain an intact RRM domain and variable arginine/serine-rich region lengths with two exceptions. An alternative acceptor site reduces the length of exon 2 by 7 bp and creates an in-frame stop codon within exon 3 of sb-SC31transcript III. The resulting truncated putative protein product of 71 amino acid residues lacks an intact RRM domain. The sb-SC31 transcript VI uses the donor site of exon 4 and an alternative noncanonical TT acceptor site within exon 8, resulting in the skipping of exons 5, 6, and 7. The truncated putative product of this transcript, 107 amino acids in length, retains an intact RRM domain but completely lacks the carboxyl terminus RS region. Despite intricate differences in splicing profile, we note the creation of an intron in zm-SC30 transcripts IV, V, VI, VII, and VIII through alternate usage of a donor and acceptor site. This intron is located in the 3′ UTR region of the gene. Similar usage of splice sites creates an intron in exon 8 of transcript III localized in the 3′ UTR region of sb-SC31 gene.
Fig. 1.
Gene structure and expression analysis between a maize SR gene zm-SC30 and its sorghum homolog sb-SC31. Panel A displays the gene structure of zm-SC30 and sb-SC31. Boxes and lines exhibit the exons and introns, respectively. Panel B displays the pairwise alignment of the putative translation products of zm-SC30 and sb-SC31. The arrows point to the conserved position of the introns in the alignment. Total RNA was extracted from maize and sorghum roots (R) and shoots (S). The left side of panels C and D displays the RT-PCR products resolved on 1% agarose gel and stained with ethidium bromide. The positions of the molecular weight markers in kilobases are indicated on the left of each gel picture. The right side of panels C and D displays the schematic structures of zm-SC30 and sb-SC31 mRNA isoforms, respectively. Broken lines connect the splice sites used to generate alternatively spliced isoforms while intron retention is indicated by an asterisk. The vertical green and red arrows mark the position of the start and stop codon. The horizontal arrows mark the position of the primers used for PCR amplification. The dinucleotide sequence at the noncanonical splice sites of the alternatively spliced transcripts is indicated. The assigned roman numerals to the alternatively spliced transcripts are shown on the left. The solid diamond marks the RT-PCR transcripts not represented in the EST collection. The RRM domain of each SR gene is highlighted in blue in transcript I.
To further investigate the evolutionary conservation of this process, we performed splice alignment of the available ESTs with their cognate homologous Arabidopsis gene at-SC35 and moss gene pp-SC39. These are displayed in figure 2A and B. As shown, the EST alignment produced an at-SC35 wild-type gene structure with eight exons and seven introns. In addition, the usage of an alternative donor and acceptor splice site in several ESTs created a 190 bp intron in exon 8 localized in the 3′ UTR of transcript II of this gene. This observation indicates the alternative splicing of a 3′ UTR localized intron is conserved between maize, sorghum, and Arabidopsis. Similarly, the homologous moss pp-SC39 gene produced an EST gene structure of nine exons and eight introns. Intriguingly, intron 8 of this gene is 455 bp in length and is located in the 3′ UTR region of this gene corresponding to the conserved retained intron variants of transcripts observed between zm-SC30, sb-SC31, and at-SC35. To analyze the possible alternative splicing of this intron, we performed RT-PCR using primers complementary to exons 1 and 9 and total RNA extracted from moss thallus. The spliced alignment of the resultant PCR product produced two additional alternative spliced isoforms shown in figure 2B. The usage of an alternative donor site in exon 4 and a noncanonical TT acceptor site in exon 9 result in skipping of exons 5, 6, 7, and 8 in transcript II. Also, alternative donor and acceptor sites within exon 9 create two introns of 328 and 177 bp in length. Both these introns are located in the 3′ UTR region of the wild-type gene. Similarly, the usage of an alternative donor in exon 4 and a noncanonical CT acceptor site within exon 9 causes skipping of exons 5, 6, 7, and 8 in transcript III. The putative protein products of transcripts II and III maintain an intact RRM domain but completely lack the RS region. The RT-PCR using nested primers failed to detect alternative splicing of intron 8 (data not presented). Because the distance between the upstream stop codon and the splice junction of an intron is critical in activation of nonsense-mediated decay (NMD) of transcripts, we measured the distance between the authentic stop codon and the exon–exon junction of the 3′ alternatively spliced intron in each case. As displayed in figure 2C, zm-SC30 transcript isoforms IV, V, and VII, sb-SC31 transcript isoform III, at-SC35 transcript isoform II and pp-SC39 transcript isoform II display a distance of more than 55 bp between the stop codon and the exon–exon junction, whereas transcript isoforms VI and VIII of zm-SC30 display a distance of only 41 bp between the stop codon and the exon–exon junction.
Fig. 2.
The retention of the intron (IR) at the 3′ UTR of SC30 subfamily is evolutionarily conserved between different plant species. Panel A displays the scheme of the gene structure for two mRNA isoforms identified by EST alignment to the at-SC35 gene. The total RNA from Physcomitrella patens was subjected to RT-PCR analysis. Panel B shows the resulting products resolved on agarose gel and the spliced alignment of their sequence to pp-SC39 gene. Panel C exhibits the length and distance between the stop codon and the exon–exon junction of the 3′ UTR intron in the mRNA isoforms of SR genes zm-SC35, sb-SC31, at-SC35, and pp-SC39. The multi symbol marks the mRNA isoforms potentially targeted for elimination by NMD.
Transcript Profiles of SR Genes That Display Evolutionary Conservation between Maize, Sorghum, Arabidopsis, and Moss
Between the 92 maize and 62 sorghum isoforms of SR transcripts in this article, only ten pairs displayed conservation covering all the seven subfamilies of plant SR proteins. We searched and performed in silico expression analysis by the homologous SR genes in Arabidopsis and moss (Physcomitrella patens). The moss SR genes that displayed structural similarity and also displayed EST evidence of conserved alternative splicing events were further subjected to RT-PCR analysis to test the efficacy of the computational results. Table 4 exhibits a comprehensive list of evolutionarily conserved alternative splicing events in SR genes discovered between maize, sorghum, Arabidopsis, and moss. In addition to SC, three other members of SR genes belonging to subfamilies RS, RS2Z, and SCL display alternatively spliced isoforms evolutionarily conserved between these four plant species. For example, an alternative donor and an acceptor site create an exon within intron 3 in SR genes belonging to SCL subfamily. This transcript isoform is evolutionarily conserved between SCL genes of maize (zm-SCL25A and zm-SCL25B), sorghum (sb-SCL25A and sb-SCL25B), Arabidopsis (at-SCL30 and at-SCL30a), and moss (pp-SCL29 and pp-SCL31; fig. 3). The formation of an in-frame stop codon within the included exon results in a truncated version of the putative protein with an intact RRM domain but missing the RS region. Similar inclusion of an exon in intron 2 creates a premature termination codon in transcript isoform of SR genes belonging to RS subfamily. This isoform is evolutionarily conserved between maize (zm-RS31A and zmRS31B), sorghum (sb-RS28), Arabidopsis (at-RSp40 and at-RSp41), and moss (pp-RS27). The putative protein of this isoform lacks an intact RRM domain (supplementary fig. S1, Supplementary Material online).
Fig. 3.
Alternative inclusion of an exon in intron 3 of SCL subfamily is under strong evolutionary selection in plants. Panels A and B display maize genes zm-SCL25A and zm-SCL25B and sorghum genes sb-SCL25A and sb-SCL25B, respectively. These panels show RT-PCR products from roots and shoots separated on agarose gels and the spliced alignment of their sequences with their cognate paralogous genes. Panel C displays RT-PCR products from P. patens mRNA and the scheme of their mRNA isoforms corresponding to paralogous genes pp-SCL33 and pp-SCL42. Panel D exhibits the mRNA structures of Arabidopsis paralogous genes at-SCL30 and at-SCL30A derived from EST evidence. The red boxes enclose the exon in intron 3 of the SR transcript isoforms that is conserved.
Table 4.
List of Alternatively Spliced Events Evolutionary Conserved between SR Genes of Different Plant Species.
Subfamily | Type of Alternative Splicing Pattern | Impact on Putative Product | Zea mays | Sorghum bicolor | Arabidopsis thaliana | Physcomitrella patens |
---|---|---|---|---|---|---|
SC | A3SS,a A5SSb | No impact | zm-SC30 | sb-SC31 | at-SC35 | pp-SC39 |
SCL | A3SS, A5SS | Truncated form without RRM domain | zm-SCL25A | sb-SCL25A | at-SCL30 | pp-SCL42 |
zm-SCL25B | sb-SCL25B | at-SCL30a | pp-SCL33 | |||
A3SS, A5SS | Truncated form with incomplete SR region | zm-SCL28 | sb-SCL28 | No evidence | No evidence | |
RS | A3SS, A5SS | Truncated form with incomplete RRM domain | zm-RS31A | sb-RS28 | at-RSp41 | pp-RS27 |
zm-RS31B | at-RSp40 | |||||
RS2Z | A3SS | Truncated form with incomplete RRM domain | zm-RS2Z37A | sb-RS2Z35 | at-RS2Z33 | pp-RS2Z37 |
zm-RS2Z37B | sb-RS2Z34 | at-RS2Z32 | ||||
RSZ | A3SS, A5SS | No impact | zm-RSZ28 | sb-RSZ28 | at-RSZ21 | No evidence |
SR | A3SS, A5SS, A3SS | Truncated form in maize. Extended form in sorghum | zm-SR32 | sb-SR32 | No evidence | No evidence |
A3SS, A5SS, ESc | Extended form in maize. Truncated form in sorghum with incomplete RRM domain | zm-SR31 | sb-SR31 | No evidence | No evidence | |
SR45 | ES, A5SS, A3SS | No significant ORF | zm-SR45_2 | sb-SR45 | No evidence | No evidence |
A3SS, A5SS | No impact | zm-SR45_2 | No evidence | No evidence | pp-SR45 |
Subfamily | Type of Alternative Splicing Pattern | Impact on Putative Product | Zea mays | Sorghum bicolor | Arabidopsis thaliana | Physcomitrella patens |
---|---|---|---|---|---|---|
SC | A3SS,a A5SSb | No impact | zm-SC30 | sb-SC31 | at-SC35 | pp-SC39 |
SCL | A3SS, A5SS | Truncated form without RRM domain | zm-SCL25A | sb-SCL25A | at-SCL30 | pp-SCL42 |
zm-SCL25B | sb-SCL25B | at-SCL30a | pp-SCL33 | |||
A3SS, A5SS | Truncated form with incomplete SR region | zm-SCL28 | sb-SCL28 | No evidence | No evidence | |
RS | A3SS, A5SS | Truncated form with incomplete RRM domain | zm-RS31A | sb-RS28 | at-RSp41 | pp-RS27 |
zm-RS31B | at-RSp40 | |||||
RS2Z | A3SS | Truncated form with incomplete RRM domain | zm-RS2Z37A | sb-RS2Z35 | at-RS2Z33 | pp-RS2Z37 |
zm-RS2Z37B | sb-RS2Z34 | at-RS2Z32 | ||||
RSZ | A3SS, A5SS | No impact | zm-RSZ28 | sb-RSZ28 | at-RSZ21 | No evidence |
SR | A3SS, A5SS, A3SS | Truncated form in maize. Extended form in sorghum | zm-SR32 | sb-SR32 | No evidence | No evidence |
A3SS, A5SS, ESc | Extended form in maize. Truncated form in sorghum with incomplete RRM domain | zm-SR31 | sb-SR31 | No evidence | No evidence | |
SR45 | ES, A5SS, A3SS | No significant ORF | zm-SR45_2 | sb-SR45 | No evidence | No evidence |
A3SS, A5SS | No impact | zm-SR45_2 | No evidence | No evidence | pp-SR45 |
aA3SS, alternative 3′ splice site.
bA5SS, alternative 5′ splice site.
cES, exon skipping.
Table 4.
List of Alternatively Spliced Events Evolutionary Conserved between SR Genes of Different Plant Species.
Subfamily | Type of Alternative Splicing Pattern | Impact on Putative Product | Zea mays | Sorghum bicolor | Arabidopsis thaliana | Physcomitrella patens |
---|---|---|---|---|---|---|
SC | A3SS,a A5SSb | No impact | zm-SC30 | sb-SC31 | at-SC35 | pp-SC39 |
SCL | A3SS, A5SS | Truncated form without RRM domain | zm-SCL25A | sb-SCL25A | at-SCL30 | pp-SCL42 |
zm-SCL25B | sb-SCL25B | at-SCL30a | pp-SCL33 | |||
A3SS, A5SS | Truncated form with incomplete SR region | zm-SCL28 | sb-SCL28 | No evidence | No evidence | |
RS | A3SS, A5SS | Truncated form with incomplete RRM domain | zm-RS31A | sb-RS28 | at-RSp41 | pp-RS27 |
zm-RS31B | at-RSp40 | |||||
RS2Z | A3SS | Truncated form with incomplete RRM domain | zm-RS2Z37A | sb-RS2Z35 | at-RS2Z33 | pp-RS2Z37 |
zm-RS2Z37B | sb-RS2Z34 | at-RS2Z32 | ||||
RSZ | A3SS, A5SS | No impact | zm-RSZ28 | sb-RSZ28 | at-RSZ21 | No evidence |
SR | A3SS, A5SS, A3SS | Truncated form in maize. Extended form in sorghum | zm-SR32 | sb-SR32 | No evidence | No evidence |
A3SS, A5SS, ESc | Extended form in maize. Truncated form in sorghum with incomplete RRM domain | zm-SR31 | sb-SR31 | No evidence | No evidence | |
SR45 | ES, A5SS, A3SS | No significant ORF | zm-SR45_2 | sb-SR45 | No evidence | No evidence |
A3SS, A5SS | No impact | zm-SR45_2 | No evidence | No evidence | pp-SR45 |
Subfamily | Type of Alternative Splicing Pattern | Impact on Putative Product | Zea mays | Sorghum bicolor | Arabidopsis thaliana | Physcomitrella patens |
---|---|---|---|---|---|---|
SC | A3SS,a A5SSb | No impact | zm-SC30 | sb-SC31 | at-SC35 | pp-SC39 |
SCL | A3SS, A5SS | Truncated form without RRM domain | zm-SCL25A | sb-SCL25A | at-SCL30 | pp-SCL42 |
zm-SCL25B | sb-SCL25B | at-SCL30a | pp-SCL33 | |||
A3SS, A5SS | Truncated form with incomplete SR region | zm-SCL28 | sb-SCL28 | No evidence | No evidence | |
RS | A3SS, A5SS | Truncated form with incomplete RRM domain | zm-RS31A | sb-RS28 | at-RSp41 | pp-RS27 |
zm-RS31B | at-RSp40 | |||||
RS2Z | A3SS | Truncated form with incomplete RRM domain | zm-RS2Z37A | sb-RS2Z35 | at-RS2Z33 | pp-RS2Z37 |
zm-RS2Z37B | sb-RS2Z34 | at-RS2Z32 | ||||
RSZ | A3SS, A5SS | No impact | zm-RSZ28 | sb-RSZ28 | at-RSZ21 | No evidence |
SR | A3SS, A5SS, A3SS | Truncated form in maize. Extended form in sorghum | zm-SR32 | sb-SR32 | No evidence | No evidence |
A3SS, A5SS, ESc | Extended form in maize. Truncated form in sorghum with incomplete RRM domain | zm-SR31 | sb-SR31 | No evidence | No evidence | |
SR45 | ES, A5SS, A3SS | No significant ORF | zm-SR45_2 | sb-SR45 | No evidence | No evidence |
A3SS, A5SS | No impact | zm-SR45_2 | No evidence | No evidence | pp-SR45 |
aA3SS, alternative 3′ splice site.
bA5SS, alternative 5′ splice site.
cES, exon skipping.
Maize (zm-RS2Z37A and zm-RS2Z37B), sorghum (sb-RS2Z35 and sb-RS2Z34), Arabidopsis (at-RS2Z32 and at-RS2Z33), and moss (pp-RS2Z37) display evolutionary conservation of a transcript isoform of the RS2Z subfamily, in which alternate usage of an acceptor site in intron 2 increases the length of exon 3 (supplementary fig. S2, Supplementary Material online). This transcript isoform is predicted to produce a truncated protein with an incomplete RRM domain. We also report retention of an intron localized in the 5′- UTR of RSZ subfamily SR transcript isoforms of zm-RSZ28, sb-RSZ28, and at-RSZ21. However, the moss homolog pp-RSZ30 displayed no intron in its 5′ UTR (supplementary fig. S3, Supplementary Material online). Intriguingly, we observed conservation of two transcript isoforms of SR45 subfamily. One isoform conserved only between maize zm-SR45_2 and sorghum sb-SR45 gene displayed skipping of exons 2–8 due to alternative donor and acceptor splice sites within exon 1 and 9, respectively (supplementary fig. S4, Supplementary Material online). The truncated putative product of this isoform lacks the entire RRM domain as well as a major portion of the RS domain. Meanwhile, the second isoform, conserved only between maize zm-SR45_2 and moss pp-SR45, displayed retention of an intron located in the 3′ UTR of the gene (supplementary fig. S5, Supplementary Material online).
The remaining case of splicing conservation observed in SR subfamily was restricted only between maize and sorghum: both maize zm-SR32 and sorghum sb-SR32 genes display a transcript isoform with an alternative inclusion of an exon in intron 11. Also in this transcript, an alternative acceptor site increases the length of exon 11. Despite sharing similarity in splicing profile, the maize transcript encodes a shorter putative product with reduced length of the carboxyl terminus-containing RS domain, whereas the putative product of the sorghum transcript is larger than the wild-type protein (supplementary fig. S6, Supplementary Material online). A transcript isoform displaying alternative inclusion of an exon in intron 2 and skipping of exon 9 is only shared between the maize zm-SR31 and sorghum sb-SR31 genes (supplementary fig. S7, Supplementary Material online). Conversely, the putative protein of this isoform in maize is larger than the wild-type protein, whereas the sorghum isoform is predicted to encode a truncated protein lacking an intact RRM domain.
Discussion
The importance of alternative splicing in the regulation of SR genes in plants is beginning to emerge. However, the biological importance of different transcript isoforms remains largely undetermined. Only a few studies have reported the evolutionary conservation of SR transcript isoforms between different plant species as evidence of their potentially conserved biological role (Iida and Go 2006; Kalyna et al. 2006). Since these studies, a marked increase in both genomic and EST sequences from different plant species have become available in the public databases. These are providing valuable resources to compare gene classification and assess the extent of alternative splicing of SR genes among diverse lineages of eukaryotic species (Richardson et al. 2011). Such high-throughput computation-based analyses provide a good overview of gene content and expression but fall short of accurate annotation of eukaryotic genes (Ashurst and Collins 2003; Schlueter et al. 2005). Usage of numerous alternative and noncanonical splice sites among SR genes makes it a particularly daunting task to correctly annotate gene structure solely by computational methods. Reannotation of computationally predicted gene structure has mostly relied on manual inspection of the gene structure evidence from multiple sources (Foissac and Schiex 2005; Haas et al. 2005). Instead, we used RT-PCR analysis and discovered a number of alternatively spliced isoforms of SR transcripts that are not represented in the available EST database for both maize and sorghum. This result suggests that the depth of the extant EST population is not sufficient to cover all the transcript isoforms of alternatively spliced genes. Recently, our expression analysis of genes captured by Helitron transposons in maize revealed numerous transcript isoforms not present in the EST collection (Barbaglia et al. 2012).
In this article, we used both computational and experimental approaches to accurately annotate the alternative splicing of all genes encoding SR proteins in maize and sorghum. These are the first two closely related representatives of the monocotyledonous plant species with a high-quality complete annotation of their SR proteins.
We previously reported differential splicing of highly similar duplicated SR genes in maize. The two paralogous SR genes, zm-RSp31A and zm-RSp31B, display significant differences in splicing patterns despite sharing a high degree of sequence and structural similarity (Gupta et al. 2005). The polysomal association of the alternative spliced SR isoforms point to their potential biological function. Similarly, recent genome-wide studies in Arabidopsis have reported a significant divergence in the alternative splicing pattern following gene and genome duplication (Zhang et al. 2010). Some of these duplicated gene transcript isoforms are differentially expressed in tissue or in response to particular stress points to their distinctive functional evolution. In contrast, we also reported an alternative splicing of U1-snRNP-specific (U1-70K) protein that is evolutionarily conserved between maize, rice, and Arabidopsis (Gupta et al. 2006). The U1-70K protein plays an important role in both constitutive and alternative splicing of pre-mRNA (Mount et al. 1983). Whether the divergent alternative spliced transcript isoforms between maize and sorghum serve distinct biological function is not apparent.
Despite the presence of introns in the UTRs of a substantial number of eukaryotic genes, they are seldom considered to play biological functions because they are targeted for elimination by NMD. However, recent studies are indicating that introns present in both 5′ and 3′ UTRs play important regulatory roles in vertebrates, including mRNA nuclear export, subcellular localization, and translation (Bicknell et al. 2012). The inclusion and exclusion of introns by alternative splicing in either the 5′ or 3′ UTR has been recently reported to determine whether the transcripts are targeted for NMD decay, thus regulating the transcript levels of genes in Arabidopsis (Kalyna et al. 2012). For example, according to the exon junction complex rule regarding the effect of upstream RNA processing events and RNA stability, a stop codon located more than 50–55 bp upstream to an exon–exon junction triggers the activation of NMD (Chang et al. 2007). In this regard, we find that the distances between the wild-type stop codon and the splice junction of a 3′ UTR alternatively spliced intron in the SC subfamily of maize, sorghum, Arabidopsis, and moss, and the SR45 subfamily of maize and moss generally conform to this rule and targets for NMD. The strong evolutionary selection of this process across these plant species points to a biological importance of this process in potentially regulating the levels of gene expression.
We note that an alternative splicing event that creates an exon in intron 2 has been reported in SR genes of the RS subfamily in Arabidopsis, maize, and rice (Gupta et al. 2005; Kalyna et al. 2006). The usage of an alternative acceptor site in intron 2 that increases the length of exon 3 has been reported in SR genes of the RS2Z subfamily in Arabidopsis and rice (Iida and Go 2006). Multiple splicing events in intron 3 spanning the RRM domain of the SCL subfamily have also been reported in Arabidopsis and rice (Iida and Go 2006; Reddy and Shad Ali 2011; Thomas et al. 2012). Although all seven subfamilies of plant SR genes have introns spanning the RRM domain, the conservation of alternative splicing in these introns is restricted to the RS, RS2Z, and SCL subfamilies. In each case, the highly truncated predicted proteins containing only a part of the RRM domain of these transcripts are likely not translated and might exert their role at the posttranscriptional level by either autoregulating their expression or the expression of the other members of the SR subfamilies (Kalyna et al. 2006). Recently, using splicing assay reporter gene constructs and RNA binding assays, Thomas et al. (2012) demonstrated that Arabidopsis SCL33 protein binds to a conserved region of its intron 3. This points to an autoregulation of alternative splicing in SCL33 (Thomas et al. 2012).
At least 400 My of evolutionary separation between moss and flowering plants represents a period of plant adaptations to land (Rensing et al. 2008). This period is also marked by a dramatic increase in the complexity of the plant genome associated with the loss of genes involved with the aquatic environment and acquisition of genes required for survival in the terrestrial atmosphere (Rensing et al. 2008). Our data demonstrates the presence of these particular alternatively splicing events and intron position in homologous SR genes of subfamilies RS, RS2Z, SC, and SCL across maize, sorghum, Arabidopsis, and moss. This evidence of evolutionary selection of alternatively spliced SR isoforms points to their biological importance in regulation of gene expression during and after plant adaptation to land.
Materials and Methods
Plant Material
The maize-inbred line B73 and Sorghum bicolor seeds were obtained from the Plant Genetic Resource Conservation Unit, USDA, ARS (Griffin, GA). The culture of moss P . patens was a generous gift from Dr Timothy Devarenne, Texas A&M University. The plants were grown in either Percival plant growth incubators or in the greenhouse.
Discovery and Annotation of Plant SR Proteins
We searched the species-specific sequence data sets available at the Plant Genome Database (http://www.plantgdb.org/, last accessed December 16, 2013; Duvick et al. 2008) using known plant SR proteins as queries (Barta et al. 2008, 2010; Richardson et al. 2011). The annotation of the SR genes was implemented using Your Gene structure Annotation Tools for Eukaryotes (yrGATE), a web-based tool for annotation and dissemination of eukaryotic gene structure (http://www.plantgdb.org/yrGATE, last accessed December 16, 2013; Wilkerson et al. 2006). In silico expression analysis was performed by manual inspection of the spliced alignment of the ESTs and their predicted translation products with their corresponding SR genes using the computer software GeneSeqer and SplicePredictor (Usuka and Brendel 2000; Usuka et al. 2000).
RT-PCR Analysis
The total RNA was extracted from 3-day-old dark grown maize (inbred B73) and S . bicolor roots and shoots, and from the moss P . patens using Trizol reagent (Invitrogen). The first strand for RT-PCR analysis was synthesized by oligo dT primers using SuperScript First Strand Synthesis kit (Invitrogen). The gene-specific primers flanking the first and last exons of the annotated SR genes were used for PCR amplification. Supplementary table S1, Supplementary Material online, in the Supplementary Material S1, Supplementary Material online, lists the sequence of primers used for RT-PCR analysis of SR genes. The resultant PCR products were resolved on 1% agarose gel, purified, cloned, and sequenced in both directions by either ABI Prism Dye Terminator sequencing (Applied Biosystem, Foster City, CA) or by the University of Florida Interdisciplinary Center for Biotechnology DNA Sequence Core Laboratory.
Acknowledgments
We thank Dr L Curtis Hannah and Amy Siebert for critically reading the earlier version of this manuscript. This work was supported by the National Science Foundation (grant number DBI 1221984) and by the Research Excellence Fund, Oakland University.
Accession numbers in this manuscript: zm-RSZ28 (KC417028, KC417029, KC417030, KC417031, KC417032, KC417033, KC417034, KC417035), zm-RSZ21 (KC424982, KC424983), zm-RSZ20 (KC424984, KC424985, KC424986, KC424987, KC424988, KC424989), zm-RS2Z39 (KC424990, KC424991, KC424992, KC424993, KC424994, KC424995, KC424996),zm-RS2Z37A (KC424997, KC424998, KC424999), zm-RS2Z37B (KC425000, KC425001, KC425002, KC425003), zm-RS2Z35 (KC425004, KC425005, KC425006), zm-SC32 (KC425007, KC425008, KC425009), zm-SC30 (KC425010, KC425011, KC425012, KC425013, KC425014, KC425015, KC425016, KC425017, KC425018, KC425019), zm-SC26 (KC425020, KC425021, KC425022), zm-SCL30 (KC425023, KC425024, KC425025), zm-SCL28 (KC425026, KC425027, KC425028), zm-SCL25A (KC425029, KC425030, KC425031), zm-SCL25B (KC425032, KC425033, KC425034, KC425035, KC425036, KC425037, KC425038, KC425039, KC425040, KC425041), zm-SR45_1 (KC425042), zm-SR45_2 (KC425043, KC425044, KC425045, KC425046), sb-RS34 (KC425047), sb-RS28 (KC425048, KC425049), sb-RSZ28 (KC425050, KC425051, KC425052, KC425053), sb-RSZ21 (KC425054, KC425055, KC425056, KC425057), sb-RSZ19 (KC425058, KC425059), sb-RS2Z39 (KC425060), sb-RS2Z35 (KC425061, KC425062), sb-RS2Z34 (KC425063, KC425064, KC425065, KC425066, KC425067, KC425068), sb-SC31 (KC425072, KC425073, KC425074, KC425075, KC425076, KC425077), sb-SC30 (KC425069, KC425070, KC425071), sb-SCL31 (KC425078), sb-SCL28 (KC425079, KC425080, KC425081), sb-SCL25A (KC425082, KC425083, KC425084), sb-SCL25B (KC425085, KC425086, KC425087, KC425088), sb-SR32 (KC425089, KC425090), sb-SR30 (KC425091, KC425092, KC425093, KC425094, KC425095, KC425096), sb-SR27 (KC425097, KC425098), sb-SR45 (KC425099, KC425100, KC425101, KC425102, KC425103, KC425104, KC425105, KC425106, KC425107, KC425108), pp-RS27 (KC440857, KC440858), pp-RSZ23 (KC440859, KC440860, KC440861, KC440862), pp-RS2Z37 (KC440863, KC440864), pp-SC39 (KC440865, KC440866, KC440867), pp-SC37 (KC440868, KC440869, KC440870, KC440880), pp-SCL42 (KC440874, KC440875, KC440876, KC440877), pp-SCL33 (KC440871, KC440872, KC440873), pp-SR45 (KC440878, KC440879).
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Author notes
Associate editor: Xun Gu
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July 2021 | 24 |
August 2021 | 17 |
September 2021 | 10 |
October 2021 | 12 |
November 2021 | 28 |
December 2021 | 22 |
January 2022 | 11 |
February 2022 | 14 |
March 2022 | 9 |
April 2022 | 164 |
May 2022 | 21 |
June 2022 | 17 |
July 2022 | 53 |
August 2022 | 8 |
September 2022 | 24 |
October 2022 | 23 |
November 2022 | 19 |
December 2022 | 43 |
January 2023 | 15 |
February 2023 | 14 |
March 2023 | 20 |
April 2023 | 27 |
May 2023 | 34 |
June 2023 | 8 |
July 2023 | 17 |
August 2023 | 10 |
September 2023 | 9 |
October 2023 | 28 |
November 2023 | 18 |
December 2023 | 13 |
January 2024 | 17 |
February 2024 | 20 |
March 2024 | 41 |
April 2024 | 20 |
May 2024 | 62 |
June 2024 | 25 |
July 2024 | 20 |
August 2024 | 17 |
September 2024 | 20 |
October 2024 | 4 |
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