A SNP associated with alternative splicing of RPT5b causes unequal redundancy between RPT5a and RPT5b among Arabidopsis thaliananatural variation
© Guyon-Debast et al; licensee BioMed Central Ltd. 2010
Received: 25 March 2010
Accepted: 3 August 2010
Published: 3 August 2010
The proteasome subunit RPT5, which is essential for gametophyte development, is encoded by two genes in Arabidopsis thaliana; RPT5a and RPT5b. We showed previously that RPT5a and RPT5b are fully redundant in the Columbia (Col-0) accession, whereas in the Wassilewskia accession (Ws-4), RPT5b does not complement the effect of a strong rpt5a mutation in the male gametophyte, and only partially complements rpt5a mutation in the sporophyte. RPT5b Col-0 and RPT5b Ws-4 differ by only two SNPs, one located in the promoter and the other in the seventh intron of the gene.
By exploiting natural variation at RPT5b we determined that the SNP located in RPT5b intron seven, rather than the promoter SNP, is the sole basis of this lack of redundancy. In Ws-4 this SNP is predicted to create a new splicing branchpoint sequence that induces a partial mis-splicing of the pre-mRNA, leading to the introduction of a Premature Termination Codon. We characterized 5 accessions carrying this A-to-T substitution in intron seven and observed a complete correlation between this SNP and both a 10 to 20% level of the RPT5b pre-mRNA mis-splicing and the lack of ability to complement an rpt5a mutant phenotype.
The accession-dependent unequal redundancy between RPT5a and RPT5b genes illustrates an example of evolutionary drifting between duplicated genes through alternative splicing.
Gene duplications contribute to evolution . They arose mostly through polyploidy (i.e. genome duplication) that has been a widespread phenomenon among Angiosperms . Arabidopsis thaliana has undergone several rounds of genome duplication and up to 23% of its proteome is encoded by duplicated genes . The evolutionary outcomes of gene duplication were first theorized by Ohno , who predicted that although duplicated genes are redundant at first, they soon evolve and one of the duplicates either accumulates detrimental mutations and is lost, or gains new function(s). As a consequence, over time the duplicated genes become partially or no longer redundant. In some cases, both genes may have retained unequal redundancy: a mutation in one of the two duplicated genes causes a mutant phenotype while a mutation in the other does not; in the double mutant, this phenotype is strongly enhanced  At its utmost, independent evolution of duplicated genes among close accessions is the basis of incompatibility known as the Dobzhansky-Muller type syndrome [6, 7].
Gene evolution can be assessed by studying natural variation in a single species. A recent survey highlighted that nucleotide sequence polymorphism appears in all gene regions, both coding and non-coding . In particular, alternative splicing, that creates multiple mRNA from the same gene unit, has been described recently as a source of variation among natural accessions. For example, this process alters FLC function in the Arabidopsis accession Bur-0  as well as in some Brassica rapa accessions . Although evolution through alternative splicing may contribute to the creation of new functions through exon sliding , it has mainly been described as inducing Premature Termination Codon (PTC). Transcripts affected by PTC are often targets of Nonsense-Mediated Decay (NMD), leading to their degradation [12, 13]. Interestingly, it has been shown that alternative splicing is often conserved between species as diverse as Rice and Arabidopsis, suggesting that this process has an important role in gene function . However, the extent of variation in alternative splicing among accessions in a single species is not known.
In previous work we showed that two Arabidopsis genes, RPT5a and RPT5b, are necessary for the development of both gametophytes . These genes encode an AAA-ATPase subunit that acts as a component of the 26S proteasome machinery. Loss-of-function for both genes is male and female gametophyte lethal. In the Col-0 accession, both RPT5a and RPT5b genes act in a redundant manner. However, in the Ws-4 accession, RPT5b does not complement for rpt5a mutant phenotype in male gametophyte (and to a lesser extent in the sporophyte). Sequencing the 4800 bp RPT5b locus identified only two SNPs that differed between the RPT5b Col-0 and RPT5b Ws-4 genes, one located in the promoter (named SNP1) and the other at the end of the seventh intron (named SNP2). Those polymorphisms will be referred to as SNP1Ws (or SNP1Col) and SNP2Ws (or SNP2Col). These findings led us to suggest that inactivation of the RPT5b Ws-4 allele could be a result of either diminished RPT5b expression related to SNP1Ws, or else due to a mis-splicing event occurring at intron 7 related to SNP2Ws . Here we address this question by looking among 487 Arabidopsis accessions for RPT5b alleles with polymorphisms similar to those detected in the Ws-4 accession. Redundancy between RPT5a and RPT5b genes was then tested regarding each of these two SNPs. This approach allowed us to demonstrate that SNP2 is responsible for RPT5b inactivation through pre-mRNA mis-splicing, leading to PTC. This A-to-T variation in intron 7, which is shared by 5 unrelated accessions, is predicted to create a new branchpoint sequence upstream from RPT5b intron seven 3' splice site, leading to its mis-splicing.
Screening of Arabidopsis accessions for variation at the RPT5blocus
Our previous work focussed on two accessions for which Arabidopsis insertional mutant collections are available, Col-0 and Ws-4. We presumed that the two SNPs highlighted between the RPT5b Col-0 and RPT5b Ws-4 alleles were responsible for their different abilities to complement the male gametophyte lethal phenotype in a rpt5a background.
We previously showed that a strong rpt5a-1 male gametophyte mutation was complemented by a transgene harbouring a 4 kb RPT5b Col construct . Likewise, 3 constructs were transformed into rpt5a-1/RPT5a plants, each harbouring the same 4 kb of RPT5b with different combinations of Col/Ws SNP1 and SNP2. It was tested whether those constructs would allow the transmission of rpt5a-1 through the male gametophyte (Additional file 1: Figure S1). Surprisingly, all 4 RPT5b constructs (SNP1Col SNP2Col, SNP1Ws SNP2Ws, SNP1Col SNP2Ws and SNP1Ws SNP2Col) did complement the rpt5a-1 mutation. The empty binary vector used as a control did not. It has to be noted that those transformed plants expressed the endogenous RPT5b Ws gene as well as the RPT5b transgene. It is very likely that the additive expression of the endogenous RPT5b together with the transgene is sufficient to complement the rpt5a-1 mutation and is therefore masking the partial loss of function of the RPT5b Ws allele.
List of Arabidopsis accessions selected for this study
VNAT reference number
Country of origin
For practical matter relative to plant crosses, we retained 4 accessions for each haplotype displaying about the same flowering time as Col-0 and Ws-4. Those selected accessions are: Bu-11, Cal-0, Da-0 and Wu-0 for the SNP1Ws/SNP2Ws haplotype, and Akita, Bsch-0, Sn5(1) and Uk-4 for the SNP1Ws/SNP2Col haplotype.
The lack of complementation of rpt5a mutant phenotype by RPT5b gene correlates with SNP2Ws but not with SNP1Ws
F1 progenies from crosses of rpt5a-4 mutant with Akita, Bsch-0, Sn5(1) and Uk-4 were found to display almost no dead pollen (Fig. 2D). This means that RPT5b Akita , RPT5b Bsch-0 , RPT5b Sn5(1) , and RPT5b Uk-4 alleles complement the rpt5a-4 mutation, in contrast to RPT5b Ws-4 . As these accessions have a SNP1Ws/SNP2Col haplotype, it can be concluded that the SNP1Ws is not involved in the process that impairs the RPT5b Ws-4 allele from complementing the rpt5a-4 mutation. This is consistent with previous data showing that the promoter SNP1 did not seem to modify the GUS gene expression pattern when a 1200 bp promoter, either from Col-0 or from Ws-4, was fused to the GUS gene reporter .
By contrast, in F1 progeny from crosses of rpt5a-4 mutant with Bu-11, Cal-0 or Wu-0, approximately 25% non-viable pollen was observed, as for rpt5a-4/RPT5a; RPT5b Col-0 /RPT5b Ws-4 plants (Fig. 2C and 2D). This pollen lethality shows that RPT5b Bu-11 , RPT5b Cal-O and RPT5bWu-0alleles do not complement the rpt5a-4 mutation. As these accessions have a SNP1Ws/SNP2Ws haplotype, and because SNP1 was shown not to be involved, we conclude that the SNP2Ws is responsible for the lack of complementation of rpt5a mutation. Likewise, rpt5a-4/RPT5a; RPT5b Col-0 /RPT5b Da-0 plants displayed 42% pollen lethality, while only 28% dead pollen was observed in a wild-type RPT5a background, which is consistent with an additive effect of the Col X Da-0 incompatibility described above and a rpt5a-4; RPT5b Da-0 male gametophyte lethality. Taken together, these data strongly suggest that the SNP2Ws, located in intron 7, is responsible for the lack of complementation of the rpt5a mutation by RPT5b loci.
SNP2Ws generates a new putative branchpoint consensus sequence in RPT5bintron 7
SNP2Ws correlates with RPT5bmis-splicing in the sporophyte
Previously, a test was developed to quantify the amount of RPT5b intron 7 mis-splicing among mRNA ( and Fig. 3C and 3D). Fragments of RPT5b cDNA surrounding intron 7 were cloned before analysis to make sure that the level of mis-splicing could be accurately assessed (Fig. 3E). However no significant difference could be detected in the level of RPT5b intron 7 mis-splicing between Col-0 and Ws-4 inflorescence . One explanation would be that the mis-splicing occurring only in male gametophyte cells or mother cells would be diluted by splicing occurring in surrounding tissues, making it very difficult to analyse at this stage.
This demonstrates that SNP2Ws, located upstream of the 3'intron site consensus, is very likely responsible for a significant amount of mis-splicing that causes the lack of complementation of rpt5a mutation by RPT5b alleles carrying this SNP2Ws.
In the present study, we confirm that RPT5a and RPT5b display unequal redundancy in some Arabidopsis accessions and we characterized 4 new accessions in which the situation is similar. Moreover, by investigating Arabidopsis natural variation, we show that one SNP, located in RPT5b intron seven, causes this unequal redundancy between RPT5a and RPT5b genes. The A to T substitution in position -8 to the intron 3' splice site correlates with a 25% mis-splicing rate in Ws-4 and in 4 other accessions (Bu-11, Cal-0, Da-0 and Wu-0). Sequence analyses reveal that the SNP2Ws generates a new putative branchpoint sequence that probably competes with the original branchpoint. As a result, another 3' splice site is selected downstream, resulting in a 10 bp deletion in the RPT5b mRNA. However, how this competition occurs between both branchpoint sequences, and consequently both 3' splice sites is unclear. In mammalian genes, despite the steric hindrance caused by the closeness between the branchpoint and the next downstream AG dinucleotide, AG sequences located as close as 4 nt downstream of the branchpoint would still be used at some extent . Data suggest that 3' splicing site scanning occurs in plants as in mammals [17, 21], explaining why even in the presence of SNP2Ws, the first AG remains the main 3' splicing site. In the 5 accessions carrying SNP2Ws, only a fraction of RPT5b pre-mRNA is mis-spliced (between 12 and 25%), though our assay might underestimate this percentage if some of the mis-spliced form is degraded through NMD, as it is frequently the case with mRNA displaying PTC [13, 22]. However, semi-quantitative RT-PCR shows that RPT5b mRNAs accumulate similarly in all accessions we tested (Fig. 5A). The subtle defect in RPT5b pre-mRNA splicing may also explain why transgenes expressing various RPT5b constructs all complement a rpt5a strong male gametophyte defect: the additive expression of both the transgenic and endogenous RPT5b expression is probably sufficient to overcome the lack of RPT5a by providing enough functional RPT5b.
Presumably, this SNP2, through causing a "mild" effect on splicing, is likely to have a stronger effect on RPT5b protein accumulation with deleterious consequences on both male gametophyte and sporophyte development. Indeed, F2 plants that segregate with a rpt5a-4/rpt5a-4; RPT5b Col-0 /RPT5b Bu-11 genotype displayed a short root that is typical of mutants partially affected in the proteasome machinery such as rpt2a, rpn10 and rpn12 as well as weaker alleles of rpt5a [15, 23, 24] (Data not shown). This suggests that the unequal redundancy between RPT5a and the other RPT5b alleles bearing the SNP2Ws extend to sporophyte development.
We previously pointed that while the lack of redundancy between RPT5a and RPT5b Ws-4 has a dramatic effect on male gametophyte development (leading to pollen lethality) it has no or very little effect on the female gametophyte development . Several studies point out that alternative splicing can be regulated by hormones, stresses or developmental cues (i.e. the pattern of alternative splicing can be organ-specific) [25–27]. In this regard, it is interesting to note a recently described set of mutants that impair female gametophyte development: LACHESIS, CHLOTO/GAMETOPHYTIC FACTOR1 and ATROPOS are essential for female gamete cell fate and all three genes encode spliceosomal components [28, 29]. The authors speculate that splicing regulation could be required for maturing specific gametophytic factors mRNA. Likewise, it could be that male gametophytes also have a specific spliceosome machinery that would make them more susceptible to SNP2 Ws related-alternative splicing while the female machinery would not. It will be interesting to see whether mutations affecting male-gametophyte development will be discovered by screening for more mutations of the spliceosome machinery.
The RPT5b alternatively-spliced mRNA described here leads to PTC that is supposed to induce NMD , i.e. degradation of the mRNA to prevent the translation of an incomplete protein. Although alternative splicing has been proposed as a way to generate novel proteins and therefore enhance transcriptomic/proteomic plasticity, there are no indications that this is the case here: the present alternate splicing leads to a RPT5b devoid of the conserved AAA-ATPase domains H and K [30, 31] that is likely to be a non-functional protein as is the C-term deleted RPT2a protein in the hlr-2 mutant . Besides, PTC caused by alternative splicing has been demonstrated to act as a way to regulate gene function by the RUST mechanism (Regulated Unproductive Splicing and Translation) : it triggers the production of mRNAs encoding non functional proteins and those mRNAs are the targets of NMD. However, we found that the level of RPT5b Ws-4 mis-splicing was similar in seedlings in both wild-type RPT5a background and in rpt5a-2 or rpt5a-3 mutant background (Fig. 4B), suggesting that there is no feedback on RPT5b mis-splicing based on RPT5 protein content.
Alternative splicing has been extensively assessed in Arabidopsis  showing that up to 42% of intron-containing genes are alternatively spliced. The majority of those alternate transcripts lead to PTC, suggesting it has an important role for gene expression regulation. New sequencing methods as High Throughput Sequencing [33–35] offer the opportunity to assess how alternative splicing varies between accessions and to see how it can contribute to transcriptomic/proteomic diversification among natural variation.
RPT5a and RPT5b both encode the RPT5 subunit, a component of the Regulatory Particle of the 26S proteasome, the complex that degrades ubiquitin-labelled targets as part of the Ubiquitin proteasome System (UPS) . The 26S/UPS is an essential pathway involved in development and regulatory processes in eukaryotes, including plants [37, 38]. In Arabidopsis, most of the 26S proteasome components are encoded by duplicated genes  and it is not clear whether these duplications are the basis of a mere redundancy or of a proteasome plasticity. In this regard, assessing the redundancy between RPT5a and RPT5b among natural variation in Arabidopsis thaliana brings new insights by showing that these genes offer another example of unequal redundancy  through alternative splicing in some unrelated accessions, without any apparent gain of functions. A recent study carried out in Arabidopsis shows that alternative splicing plays an important role for divergence between duplicated genes . It would be interesting to test whether other proteasome subunits display such incompatibilities and therefore determine whether some of those other duplicated genes are drifting away or not.
Plant Materials and Growth Conditions
Arabidopsis thaliana Columbia (Col-0) and Wassilewskija (Ws-4) accessions were used as reference plants. The rpt5a-4 T-DNA insertion mutant allele has been already described . It was isolated in the SALK collection as line S046321 in Col-0 accession (SIGnAL; http://signal.salk.edu/cgi-bin/tdnaexpress). As previously, the rpt5a-4 mutant allele was genotyped with the S46321-L and LBSalk2 primers while the wild-type allele was scored with S46321-U and S46321-L. All primers are described in Additional file 2: Table S1.
The 487-accessions collection (Additional file 3: Table S2.) was provided by the Resource Centre of INRA Versailles http://dbsgap.versailles.inra.fr/vnat. All Col (Col-0 and Col-7) and Ws accessions (Ws, Ws-1, Ws-2, Ws-3 and Ws-4) were found to present the same RPT5b haplotype; therefore only Col-0 and Ws-4 was used in this study. For growth on either plates or soil, seeds were stratified for 2 days at 4°C and grown at 18 to 20°C, with 16 h light (fluorescent lights: 100 μmol photons m-2 s-1) and 8 h dark cycles. Immature flowers were emasculated and manually cross-pollinated for crosses.
PCR-Genotyping of RPT5bSNPs
Genomic DNA of the 487 accessions was extracted in 96-well plates from seedlings, using a SDS-based buffer . SNP1 and SNP2 were detected as previously reported using dCAPS markers . For the SNP1 located in the RPT5b promoter, a 266 bp fragment was amplified with primers JLV096 and JLV097 and digested with VspI. Only the fragment originating from the Ws allele was restricted into 249 and 17 bp fragments. For the SNP2, located in the RPT5b intron seven, a 280 bp fragment was amplified with primers RPT5b-3369 and RPT5b-Hind3 and digested with HindIII. Only the fragment originating from the Col allele was restricted into 250 and 30 bp fragments.
Pollen viability was assessed using Alexander staining  and observed with a PL Fluotar X25 dry objective on a Leitz Diaplan microscope.
Total RNA was extracted using TRIZOL-reagent (Invitrogen) from 100 mg of tissue (6 day-old plantlets). Contaminating DNA was removed by DnaseI treatment with RNase Free DNase set (Qiagen) using spin columns of the Rneasy Plant mini kit (Qiagen).
RT-PCR was performed with RevertAid H Minus M-MuLV Reverse Transcriptase (Fermentas) on 500 ng RNA according to the supplier's instructions. RPT5b and APT cDNAs were amplified with specific primers: e5 and e10 for RPT5b, APT5' and APT3' for APT.
RPT5b e5-e10 cDNA fragments were amplified by 25 cycles of PCR with e5 and e10 primers. The PCR products were then cloned into pTOPOII plasmid (Invitrogen) and the resulting constructs transformed into DH10B competent cells. A hundred recombinant clones were analysed by PCR using N and e10 primers for normal cDNA detection and M and e10 primers for mis-spliced cDNA.
We thank Christine Camilleri for her very helpful advice on both the project and the manuscript. We are also very thankful to Raphaël Mercier for his advice that helped to improve the manuscript and to Matthieu Simon for his help with the accessions and the use of MSAT markers. Finally, we thank Denis Headon (Roslin Institute, Edinburgh, UK) for editing the manuscript.
- Moore RC, Purugganan MD: The evolutionary dynamics of plant duplicate genes. Curr Opin Plant Biol. 2005, 8 (2): 122-128. 10.1016/j.pbi.2004.12.001.PubMedView ArticleGoogle Scholar
- Bowers JE, Chapman BA, Rong J, Paterson AH: Unravelling angiosperm genome evolution by phylogenetic analysis of chromosomal duplication events. Nature. 2003, 422 (6930): 433-438. 10.1038/nature01521.PubMedView ArticleGoogle Scholar
- Blanc G, Hokamp K, Wolfe KH: A recent polyploidy superimposed on older large-scale duplications in the Arabidopsis genome. Genome Res. 2003, 13 (2): 137-144. 10.1101/gr.751803.PubMedPubMed CentralView ArticleGoogle Scholar
- Ohno S: Evolution by Gene Duplication. London: George Allen and Unwin 1970,View ArticleGoogle Scholar
- Briggs GC, Osmont KS, Shindo C, Sibout R, Hardtke CS: Unequal genetic redundancies in Arabidopsis--a neglected phenomenon?. Trends Plant Sci. 2006, 11 (10): 492-498. 10.1016/j.tplants.2006.08.005.PubMedView ArticleGoogle Scholar
- Bikard D, Patel D, Le Mette C, Giorgi V, Camilleri C, Bennett MJ, Loudet O: Divergent evolution of duplicate genes leads to genetic incompatibilities within A. thaliana. Science. 2009, 323 (5914): 623-626. 10.1126/science.1165917.PubMedView ArticleGoogle Scholar
- Bomblies K, Lempe J, Epple P, Warthmann N, Lanz C, Dangl JL, Weigel D: Autoimmune response as a mechanism for a Dobzhansky-Muller-type incompatibility syndrome in plants. PLoS Biol. 2007, 5 (9): e236-10.1371/journal.pbio.0050236.PubMedPubMed CentralView ArticleGoogle Scholar
- Alonso-Blanco C, Aarts MG, Bentsink L, Keurentjes JJ, Reymond M, Vreugdenhil D, Koornneef M: What has natural variation taught us about plant development, physiology, and adaptation?. Plant Cell. 2009, 21 (7): 1877-1896. 10.1105/tpc.109.068114.PubMedPubMed CentralView ArticleGoogle Scholar
- Werner JD, Borevitz JO, Uhlenhaut NH, Ecker JR, Chory J, Weigel D: FRIGIDA-independent variation in flowering time of natural Arabidopsis thaliana accessions. Genetics. 2005, 170 (3): 1197-1207. 10.1534/genetics.104.036533.PubMedPubMed CentralView ArticleGoogle Scholar
- Yuan YX, Wu J, Sun RF, Zhang XW, Xu DH, Bonnema G, Wang XW: A naturally occurring splicing site mutation in the Brassica rapa FLC1 gene is associated with variation in flowering time. J Exp Bot. 2009, 60 (4): 1299-1308. 10.1093/jxb/erp010.PubMedPubMed CentralView ArticleGoogle Scholar
- Tarrio R, Ayala FJ, Rodriguez-Trelles F: Alternative splicing: a missing piece in the puzzle of intron gain. Proc Natl Acad Sci USA. 2008, 105 (20): 7223-7228. 10.1073/pnas.0802941105.PubMedPubMed CentralView ArticleGoogle Scholar
- Barbazuk WB, Fu Y, McGinnis KM: Genome-wide analyses of alternative splicing in plants: opportunities and challenges. Genome Res. 2008, 18 (9): 1381-1392. 10.1101/gr.053678.106.PubMedView ArticleGoogle Scholar
- Maquat LE: Nonsense-mediated mRNA decay: splicing, translation and mRNP dynamics. Nat Rev Mol Cell Biol. 2004, 5 (2): 89-99. 10.1038/nrm1310.PubMedView ArticleGoogle Scholar
- Wang BB, Brendel V: Genomewide comparative analysis of alternative splicing in plants. Proc Natl Acad Sci USA. 2006, 103 (18): 7175-7180. 10.1073/pnas.0602039103.PubMedPubMed CentralView ArticleGoogle Scholar
- Gallois JL, Guyon-Debast A, Lecureuil A, Vezon D, Carpentier V, Bonhomme S, Guerche P: The Arabidopsis proteasome RPT5 subunits are essential for gametophyte development and show accession-dependent redundancy. Plant Cell. 2009, 21 (2): 442-459. 10.1105/tpc.108.062372.PubMedPubMed CentralView ArticleGoogle Scholar
- Brown JW, Simpson CG: Splice site selection in plant pre-mRNA splicing. Annu Rev Plant Physiol Plant Mol Biol. 1998, 49: 77-95. 10.1146/annurev.arplant.49.1.77.PubMedView ArticleGoogle Scholar
- Simpson CG, Clark G, Davidson D, Smith P, Brown JW: Mutation of putative branchpoint consensus sequences in plant introns reduces splicing efficiency. Plant J. 1996, 9 (3): 369-380. 10.1046/j.1365-313X.1996.09030369.x.PubMedView ArticleGoogle Scholar
- Liu HX, Filipowicz W: Mapping of branchpoint nucleotides in mutant pre-mRNAs expressed in plant cells. Plant J. 1996, 9 (3): 381-389. 10.1046/j.1365-313X.1996.09030381.x.PubMedView ArticleGoogle Scholar
- Smith CW, Porro EB, Patton JG, Nadal-Ginard B: Scanning from an independently specified branch point defines the 3' splice site of mammalian introns. Nature. 1989, 342 (6247): 243-247. 10.1038/342243a0.PubMedView ArticleGoogle Scholar
- Smith CW, Chu TT, Nadal-Ginard B: Scanning and competition between AGs are involved in 3' splice site selection in mammalian introns. Mol Cell Biol. 1993, 13 (8): 4939-4952.PubMedPubMed CentralView ArticleGoogle Scholar
- Tanaka A, Mita S, Ohta S, Kyozuka J, Shimamoto K, Nakamura K: Enhancement of foreign gene expression by a dicot intron in rice but not in tobacco is correlated with an increased level of mRNA and an efficient splicing of the intron. Nucleic Acids Res. 1990, 18 (23): 6767-6770. 10.1093/nar/18.23.6767.PubMedPubMed CentralView ArticleGoogle Scholar
- Palusa SG, Reddy AS: Extensive coupling of alternative splicing of pre-mRNAs of serine/arginine (SR) genes with nonsense-mediated decay. New Phytol. 185 (1): 83-89. 10.1111/j.1469-8137.2009.03065.x.Google Scholar
- Kurepa J, Toh EA, Smalle JA: 26S proteasome regulatory particle mutants have increased oxidative stress tolerance. Plant J. 2008, 53 (1): 102-114. 10.1111/j.1365-313X.2007.03322.x.PubMedView ArticleGoogle Scholar
- Ueda M, Matsui K, Ishiguro S, Sano R, Wada T, Paponov I, Palme K, Okada K: The HALTED ROOT gene encoding the 26S proteasome subunit RPT2a is essential for the maintenance of Arabidopsis meristems. Development. 2004, 131 (9): 2101-2111. 10.1242/dev.01096.PubMedView ArticleGoogle Scholar
- Palusa SG, Ali GS, Reddy AS: Alternative splicing of pre-mRNAs of Arabidopsis serine/arginine-rich proteins: regulation by hormones and stresses. Plant J. 2007, 49 (6): 1091-1107. 10.1111/j.1365-313X.2006.03020.x.PubMedView ArticleGoogle Scholar
- Zhang PG, Huang SZ, Pin AL, Adams KL: Extensive Divergence in Alternative Splicing Patterns After Gene and Genome Duplication During the Evolutionary History of Arabidopsis. Mol Biol Evol.Google Scholar
- Simpson CG, Fuller J, Maronova M, Kalyna M, Davidson D, McNicol J, Barta A, Brown JW: Monitoring changes in alternative precursor messenger RNA splicing in multiple gene transcripts. Plant J. 2008, 53 (6): 1035-1048. 10.1111/j.1365-313X.2007.03392.x.PubMedView ArticleGoogle Scholar
- Moll C, von Lyncker L, Zimmermann S, Kagi C, Baumann N, Twell D, Grossniklaus U, Gross-Hardt R: CLO/GFA1 and ATO are novel regulators of gametic cell fate in plants. Plant J. 2008, 56 (6): 913-921. 10.1111/j.1365-313X.2008.03650.x.PubMedView ArticleGoogle Scholar
- Gross-Hardt R, Kagi C, Baumann N, Moore JM, Baskar R, Gagliano WB, Jurgens G, Grossniklaus U: LACHESIS restricts gametic cell fate in the female gametophyte of Arabidopsis. PLoS Biol. 2007, 5 (3): e47-10.1371/journal.pbio.0050047.PubMedPubMed CentralView ArticleGoogle Scholar
- Fu H, Doelling JH, Rubin DM, Vierstra RD: Structural and functional analysis of the six regulatory particle triple-A ATPase subunits from the Arabidopsis 26S proteasome. Plant J. 1999, 18 (5): 529-539. 10.1046/j.1365-313X.1999.00479.x.PubMedView ArticleGoogle Scholar
- Beyer A: Sequence analysis of the AAA protein family. Protein Sci. 1997, 6 (10): 2043-2058. 10.1002/pro.5560061001.PubMedPubMed CentralView ArticleGoogle Scholar
- Lareau LF, Green RE, Bhatnagar RS, Brenner SE: The evolving roles of alternative splicing. Curr Opin Struct Biol. 2004, 14 (3): 273-282. 10.1016/j.sbi.2004.05.002.PubMedView ArticleGoogle Scholar
- Filichkin SA, Priest HD, Givan SA, Shen R, Bryant DW, Fox SE, Wong WK, Mockler TC: Genome-wide mapping of alternative splicing in Arabidopsis thaliana. Genome Res. 2010, 20 (1): 45-58. 10.1101/gr.093302.109.PubMedPubMed CentralView ArticleGoogle Scholar
- Pickrell JK, Marioni JC, Pai AA, Degner JF, Engelhardt BE, Nkadori E, Veyrieras JB, Stephens M, Gilad Y, Pritchard JK: Understanding mechanisms underlying human gene expression variation with RNA sequencing. Nature. 2010, 464 (7289): 768-772. 10.1038/nature08872.PubMedPubMed CentralView ArticleGoogle Scholar
- Shendure J, Ji H: Next-generation DNA sequencing. Nat Biotechnol. 2008, 26 (10): 1135-1145. 10.1038/nbt1486.PubMedView ArticleGoogle Scholar
- Smalle J, Vierstra RD: The ubiquitin 26S proteasome proteolytic pathway. Annu Rev Plant Biol. 2004, 55: 555-590. 10.1146/annurev.arplant.55.031903.141801.PubMedView ArticleGoogle Scholar
- Vierstra RD: The ubiquitin-26S proteasome system at the nexus of plant biology. Nat Rev Mol Cell Biol. 2009, 10 (6): 385-397. 10.1038/nrm2688.PubMedView ArticleGoogle Scholar
- Hershko A, Ciechanover A: The ubiquitin system. Annu Rev Biochem. 1998, 67: 425-479. 10.1146/annurev.biochem.67.1.425.PubMedView ArticleGoogle Scholar
- Kurepa J, Smalle JA: Structure, function and regulation of plant proteasomes. Biochimie. 2008, 90 (2): 324-335. 10.1016/j.biochi.2007.07.019.PubMedView ArticleGoogle Scholar
- Simon M, Loudet O, Durand S, Berard A, Brunel D, Sennesal FX, Durand-Tardif M, Pelletier G, Camilleri C: Quantitative trait loci mapping in five new large recombinant inbred line populations of Arabidopsis thaliana genotyped with consensus single-nucleotide polymorphism markers. Genetics. 2008, 178 (4): 2253-2264. 10.1534/genetics.107.083899.PubMedPubMed CentralView ArticleGoogle Scholar
- Alexander MP: Differential staining of aborted and nonaborted pollen. Stain Technol. 1969, 44 (3): 117-122.PubMedGoogle Scholar
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