Present address: Division of Biological Sciences, University of California, Davis, CA 95616, USA.
The lba1 mutation of UPF1 RNA helicase involved in nonsense-mediated mRNA decay causes pleiotropic phenotypic changes and altered sugar signalling in Arabidopsis
Article first published online: 1 JUN 2006
The Plant Journal
Volume 47, Issue 1, pages 49–62, July 2006
How to Cite
Yoine, M., Ohto, M.-a., Onai, K., Mita, S. and Nakamura, K. (2006), The lba1 mutation of UPF1 RNA helicase involved in nonsense-mediated mRNA decay causes pleiotropic phenotypic changes and altered sugar signalling in Arabidopsis. The Plant Journal, 47: 49–62. doi: 10.1111/j.1365-313X.2006.02771.x
- Issue published online: 1 JUN 2006
- Article first published online: 1 JUN 2006
- Received 25 January 2006; revised 23 February 2006; accepted 7 March 2006.
- alternative splicing;
- nonsense-mediated mRNA decay;
- sugar signalling;
- Top of page
- Experimental procedures
- Supporting Information
The low-beta-amylase1 (lba1) mutant of Arabidopsis thaliana has reduced sugar-induced expression of Atβ-Amy and shows pleiotropic phenotypes such as early flowering; short day-sensitive growth; and seed germination that is hypersensitive to glucose and abscisic acid and resistant to mannose. lba1 was a missense mutation of UPF1 RNA helicase involved in nonsense-mediated mRNA decay (NMD), which eliminates mRNAs with premature termination codons (PTCs), and replaces highly conserved Gly851 of UPF1 with Glu. Expression of the wild-type UPF1 in lba1 rescued not only the reduced sugar-inducible gene expression, but also early flowering and altered seed-germination phenotypes. Sugar-inducible mRNAs were over-represented among transcripts decreased in sucrose-treated lba1 compared with Col plants, suggesting that UPF1 is involved in the expression of a subset of sugar-inducible genes. On the other hand, transcripts increased in lba1, which are likely to contain direct targets of NMD, included mRNAs for many transcription factors and metabolic enzymes that play diverse functions. Among these, the level of an alternatively spliced transcript of AtTFIIIA containing PTC was 17-fold higher in lba1 compared with Col plants, and it was reduced to the level in Col by expressing the wild-type UPF1. The lba1 mutant provides a good tool for studying NMD in plants.
- Top of page
- Experimental procedures
- Supporting Information
In plants, changes in the levels of sugars cause hormone-like effects on gene expression; cell division; defensive reactions; and various developmental processes including seed germination, flowering and tuber formation. Sugar signalling in plants occurs through multiple signal transduction pathways (reviewed by Gibson, 2005; Rolland et al., 2002; Smeekens, 2000). A variety of sugar-signalling mutants of Arabidopsis have been obtained by screening strategies based on the inhibition of seed germination by high concentrations of sugar. Many of these mutants are due to mutations in genes involved in the plant's response to ethylene and abscisic acid (ABA), indicating a close link between sugar signalling during seed germination and the production of ethylene and ABA (reviewed by Rolland et al., 2002; Rook and Bevan, 2003). Direct screening of mutants based on sugar-responsive gene expression has also yielded a number of mutants, but the mutated genes responsible for these phenotypes have been identified in only a few cases (Masaki et al., 2005a,b; Tsukagoshi et al., 2005).
The low-beta-amylase 1 (lba1) mutant was originally isolated as a mutant showing reduced sugar-induced expression of Atβ-Amy, which encodes the major extrachloroplast β-amylase (Mita et al., 1997). The lba1 mutation also caused reduced sugar-induced accumulation of anthocyanin and decreased chlorophyll content without affecting the levels of sucrose (Suc) and starch (Mita et al., 1997). In addition, lba1 plants were found to exhibit various phenotypes such as early flowering, elongated seeds, short day-sensitive growth, glucose (Glc)- and ABA-hypersensitive seed germination, and mannose-resistant seed germination. In the present study we found that lba1 is a missense mutation within a single-copy gene for UPF1 RNA helicase (AtUPF1) for nonsense-mediated mRNA decay (NMD). Results of complementation experiments indicated that not only reduced sugar-induced expression of certain groups of genes in lba1, but also pleiotropic phenotypes of lba1, are due to this missense mutation of AtUPF1.
To ensure that only fully processed and error-free mRNAs are translated in the cytoplasm, eukaryotes harbour several mRNA surveillance mechanisms. The NMD pathway specifically recognizes mRNAs containing premature translational termination codons (PTCs), which could give rise to truncated and potentially harmful proteins, and targets these mRNAs for degradation (reviewed by Baker and Parker, 2004; Conti and Izaurralde, 2005). In addition to mutation-derived PTCs, various forms of natural transcripts containing PTCs are targets of NMD, and 4–10% of total genes in yeast (He et al., 2003), Drosophila (Rehwinkel et al., 2005) and human (Mendell et al., 2004) are estimated to be subjected to post-transcriptional regulation by NMD.
Three major factors in the NMD pathway (UPF1, UPF2 and UPF3) have been highly conserved during the evolution of eukaryotes. Among these factors, UPF1 RNA helicase is a key component of NMD, and interacts with UPF2 and UPF3 to form a surveillance complex (reviewed by Culbertson and Leeds, 2003; Lejeune and Maquat, 2005). The majority of mammalian NMD occurs if a pioneer round of translation terminates more than 50 nt upstream of the 3′-most exon–exon junction that has been marked with exon-junction complex during splicing (Lejeune and Maquat, 2005; Shibuya et al., 2004). However, recognition of PTCs in yeast mRNAs occurs independently of splicing if specific cis-elements are present downstream of PTCs (reviewed by González et al., 2001). Thus, although NMD is an evolutionarily conserved system and NMD factors are highly conserved, various organisms have evolved different mechanisms to discriminate PTCs from natural termination codons and to recruit NMD factors to aberrant mRNAs (Baker and Parker, 2004; Conti and Izaurralde, 2005; Culbertson and Leeds, 2003), and the target genes of NMD are not evolutionarily conserved (Mendell et al., 2004; Rehwinkel et al., 2005).
The presence of an NMD-like pathway in plants was first suggested by studies showing reduced stability of mRNAs containing PTCs from several intronless genes (reviewed by Gutierrez et al., 1999). Studies on mutants of waxy mRNA containing PTCs in rice (Oryza sativa), however, have suggested that splicing of the first intron present upstream of PTC is important for NMD of mutant waxy mRNA (Isshiki et al., 2001). Also, nonsense but not missense mutants of xantha mRNA in barley (Hordeum vulgare) appear to be subjected to rapid degradation – even the mutant mRNA contains PTC in the last exon (Gadjieva et al., 2004). Despite these findings, the mechanism of NMD in plants and its role in plant growth and development have not been clarified. In addition, there has been only a single recent study on the function of plant UPFs, which showed that UPF3 of Arabidopsis thaliana suppresses aberrantly spliced mRNAs containing PTCs (Hori and Watanabe, 2005).
Because homozygotes for the disruption of AtUPF1 are seedling-lethal (Yoine et al., 2006), the lba1 mutant provides a good tool for studying NMD in plants. Transcriptome analysis of the lba1 mutant suggested that potential targets of NMD in Arabidopsis include genes involved in diverse cellular activities, and the lba1 mutant accumulated PTC-containing transcript generated by alternative splicing.
- Top of page
- Experimental procedures
- Supporting Information
Pleiotropic phenotypes of the lba1 mutant
The lba1 mutant was first identified after screening for mutants of Arabidopsis exhibiting a reduced level of sugar-induced amylase activity in leaves caused by reduced expression of Atβ-Amy. This mutant also showed a reduced level of sugar-induced accumulation of anthocyanin, and pale green leaves with reduced chlorophyll content that could be rescued by feeding the plants sugar (Mita et al., 1997). Even after eight back-crosses to Col, lba1 plants showed a variety of growth and developmental phenotypes.
When lba1 plants that had been grown for 3 weeks on agar plates under continuous light were transferred to soil and grown further under continuous light, they bolted and flowered much earlier than Col plants (Figure 1a). On average (27 plants) the lba1 plants showed 1 cm of bolting 5.3 days (SE = 0.3) after transfer to soil, whereas the Col plants took 7.5 days (SE = 0.3) for 1 cm bolting. The lba1 plants that had been sown and grown on soil under continuous light also flowered earlier than the Col plants. In contrast, lba1 plants could not grow well under short-day conditions (Figure 1b,c). Even under continuous light, lba1 plants exhibited the pale leaf phenotype, especially when they were grown under relatively high light intensities (Figure 1d), which could be weakened by growing them under lower light intensities or with higher Suc concentrations (data not shown).
The inhibition of germination and early seedling growth of Arabidopsis by high concentrations of sugars such as Suc and Glc (Jang et al., 1997) has been used widely to monitor the response of plants to sugars, and to screen for sugar-signalling mutants (reviewed by Rolland et al., 2002; Rook and Bevan, 2003). On agar plates containing 4% Glc, lba1 seedlings showed delayed cotyledon opening and greening compared with Col seedlings. Cotyledon opening and greening of lba1 seedlings were also hypersensitive to ABA compared with Col seedlings. Germination of Arabidopsis seeds on plates containing 1% Suc is inhibited by 5 mm mannose, a non-metabolizable analog of Glc (Pego et al., 1999). Germination of lba1 seeds showed strong resistance to inhibition by mannose (Figure 1e).
Map-based identification of the lba1 mutation
The lba1 mutation has been localized at a site approximately 17 cM from DFR and 23 cM from LFY3 on chromosome 5 (Mita et al., 1997). The lba1 mutant was crossed with ecotype Wassilewskija (Ws), and F2 individuals showing low amylase activities in Suc-treated leaves were selected. Phenotypes of selected F2 individuals were subsequently confirmed by an F3 progeny test, and DNA was isolated from each individual that passed the progeny test. Mapping with CAPS and SSLP markers indicated that lba1 was located near the MQD2210 marker (Figure 2a). Further mapping of lba1 loci between the RPS4NT and SNP239 markers indicated that lba1 was located within a 61-kb region between the MQD2210 and K14A3a markers. We determined the genomic sequence of this 61-kb region from lba1. Comparison of the sequence with the corresponding sequence from Col identified a single G to A substitution in lba1 (Figure 2b). This base substitution was present in the predicted exon of At5g47010, and it could cause an exchange of the Gly851 codon (GGA) to a Glu codon (GAA) in the predicted mRNA sequence.
We prepared a dCAPS (Neff et al., 2002) primer set that can amplify genomic DNA covering the lba1 mutation site and distinguish it from the Col sequence. The upstream primer covered a sequence immediately upstream of the lba1 mutation site, and an A residue that is 3 nt upstream of the mutation site was changed to a T (Figure 2c). Amplification of lba1 genomic DNA with this primer is expected to generate an XbaI cleavage site (TCTAGA) in the PCR product, whereas the PCR product from the Col genomic DNA should contain a sequence (TCTAGG) that cannot be digested with XbaI. PCR amplification of Col and lba1 genomic DNA with this primer both generated the expected 180-bp fragment. Although XbaI did not digest the PCR product from Col DNA, digestion of the product from lba1 DNA yielded a 158-bp fragment (Figure 2c). PCR products from the genomic DNA of individual F2 progeny generated by crossing Col and lba1 were analysed following digestion with XbaI. Results of representative samples are shown in Figure 2(c). Examination of 122 F2 individuals showed a segregation of lba1+/+:lba1+/−:lba1−/− = 28:64:30 (x2 for a ratio of 1:2:1 = 6.90; P > 0.05).
LBA1 codes for UPF1 RNA helicase
The G to A mutation in lba1 was located within the 20th exon of the gene At5g47010, which is a single-copy gene for UPF1 in Arabidopsis (AtUPF1; Figure 3a). AtUPF1 shared amino acid identities of 56%, 50% and 48% with UPF1 from human, Drosophila, and Saccharomyces cerevisiae respectively (Culbertson and Leeds, 2003). The NMD RNA helicase domain of AtUPF1 contains an N-terminal Cys/His-rich region and an ATP-helicase domain (Figure 3b). The C-terminal region of AtUPF1 also contains 14 SQ (Ser–Gln) doublets. The Cys/His-rich region in the RNA helicase domain and the C-terminal SQ motifs are unique features of UPF1-type RNA helicases (Culbertson and Leeds, 2003), and AtUPF1 is distinct from other RNA helicases of Arabidopsis (Figure 3c). The G to A mutation in lba1 leads to an exchange of Gly851 within motif V of helicase superfamily I (Hodgman, 1988) to Glu (Figure 3d). Gly851 is an invariant residue in all 16 known eukaryotic UPF1s (Culbertson and Leeds, 2003; http://www.molbio.wisc.edu/culbertson/UPFsequences/UPF1.jpg). Mutation of amino acids located near the corresponding Gly in yeast UPF1 (Figure 3d) has been shown to impair its function (Leeds et al., 1992; Weng et al., 1996). The lba1 mutation was given another allele name of atupf1-1 (Yoine et al., 2006).
The levels of AtUPF1 mRNA, as determined by quantitative real-time PCR, were not significantly different among the various organs of Arabidopsis (Figure 3e). In addition, treatment of plants with 5% Suc or 100 μm ABA (data not shown) for 2 days did not significantly affect the level of AtUPF1 mRNA (Figure 3f).
Expression of AtUPF1 cDNA in the lba1 mutant
The full-length cDNA of AtUPF1 from Col was placed downstream of the cauliflower mosaic virus 35S promoter (35S:AtUPF1) and used to transform lba1 plants. In this construct, the 3′-untranslated region (3′-UTR) of AtUPF1 cDNA was replaced with the nos terminator to distinguish it from that of endogenous mRNA (Figure 4a). Total RNA was isolated from hygromycin-resistant T2 plants and subjected to quantitative real-time RT-PCR using primer set x, which specifically amplifies the C-terminal coding region of AtUPF1 mRNA, and primer set y, which specifically amplifies the 3′-UTR of AtUPF1 mRNA (Figure 4a). The levels of AtUPF1 mRNA determined with primer sets x and y are expected to represent the total amount of AtUPF1 mRNA and the amount of endogenous AtUPF1 (lba1) mRNA, respectively. Of eight independent transformants analysed, seven showed 1.8- to 3.3-fold higher levels of total AtUPF1 mRNA compared with lba1 (Figure 4a). Line L5 showed a level of AtUPF1 mRNA similar to lba1. Except for line L5, the levels of endogenous AtUPF1 (lba1) mRNA in these transformants were similar to the level in lba1.
We determined the nucleotide sequence of RT-PCR products of AtUPF1 mRNA covering the lba1 mutation site in several independent transformant lines. Both G and A residues (with G more abundant than A) were detected at the corresponding position of the RT-PCR products from L3 and L4 (Figure 4b) as well as other transformant lines. The RT-PCR product from Col produced only a signal for G at this position, whereas the product from lba1 produced only a signal for A. These results indicate that transformants contain the AtUPF1 mRNA from the transgene at levels similar to or higher than in Col, in addition to the mutant form of AtUPF1 mRNA.
Complementation of low-level expression of Atβ-Amyinlba1by35S:AtUPF1
Total amylase activities in Suc-treated T2 plants of the 35S:AtUPF1 transformant lines were higher than those in Suc-treated lba1. The above-ground parts of 3-week-old T2 plants of the transformant lines were treated with water or 5% Suc for 2 days, and total RNAs were isolated. The levels of Atβ-Amy mRNA were determined with quantitative real-time RT-PCR using UBC mRNA (Czechowski et al., 2005) as an internal reference. Although the levels of Atβ-Amy mRNA varied among individual plants, Suc-treated plants of transformant lines, except for L2, showed average levels of Atβ-Amy mRNA higher than lba1 (Figure 4c). The levels of Atβ-Amy mRNA in these transformants did not reach the levels observed in Suc-treated Col plants. The levels of Atβ-Amy mRNA in water-treated plants were not significantly different among Col, lba1 and the transformant lines. The partial recovery of expression level in the transformant lines is probably due to their expression of both wild-type and lba1 mutant forms of AtUPF1.
We also examined the expression of Din11 (Fujiki et al., 2001) in several transformant lines, because sugar-induced expression of Din11 mRNA was severely reduced in lba1. Similarly to Atβ-Amy mRNA, the levels of Din11 mRNA in Suc-treated plants of four transformant lines were all higher than those in lba1, although they were lower than the level in Col plants (Figure 4d).
Complementation of growth phenotypes of lba1 by 35S:AtUPF1
Seeds of Col, lba1, and lba1 mutants transformed with 35S:AtUPF1 were germinated and grown on soil under continuous light. Figure 5(a) shows representative plants after 8 weeks. Although only 10% of the Col plants had flowers at this stage, 83% of the lba1 plants had long bracts with flowers. Similar to Col, only 4–5% of the L3 and L4 transformant lines had developed long bracts at this stage.
Germination of lba1 seeds on plates containing 4% Glc was delayed compared with Col seeds, whereas seeds of transformant lines L3 and L4 germinated even slightly faster than Col seeds (Figure 5b). After 8 days on plates containing 4% Glc, 63% of the lba1 seedlings showed open and green cotyledons, compared with 93% of Col seeds. At this stage, most of the transformant lines examined showed >80% of seedlings with open and green cotyledons (Figure 5c). Germination of lba1 seeds was more sensitive than Col seeds to lower concentrations of ABA (Figure 5d). After 10 days on agar plates containing 0.4 μm ABA, germinated seeds accounted for, on average, 40% and 16% of Col and lba1 seeds, respectively (Figure 5e). Seeds of the transformant lines showed an even slightly higher ratio of germination compared with Col seeds on 0.4 μm ABA.
Unlike Col seeds, germination of lba1 seeds on medium containing 1% Suc was resistant to inhibition by 5 mm mannose (Figure 1e). Although the level varied depending on the line, and did not reach the level of Col seeds, seeds of the transformant lines showed increased sensitivity to inhibition by mannose compared with lba1 seeds (Figure 5f).
Reduced expression of sugar-inducible genes other than Atβ-Amyinlba1
To search for genes, other than Atβ-Amy, the expression of which is affected in lba1, we performed microarray analysis. Three-week-old Col and lba1 plants were treated with 5% Suc for 2 days, and RNAs were isolated. Microarray analysis was carried out using Agilent Arabidopsis two Oligo Microarrays and with two independently isolated pairs of RNA samples. For each pair of RNA samples from Col and lba1, replicate hybridizations were performed after dye swapping, and two independent sets of expression data for 19 675 transcript units were obtained after filtering the signal data for statistical significance. The average fold differences between lba1 and Col in dye-swap data were calculated, and the results of two biological replicates were averaged.
Thirty-two genes showed >50% reduction in transcript level in lba1 compared with Col in both biological replicates (Table S1). The sugar response of each transcript in Col plants was determined by comparing their levels in plants treated with 6% Suc or water for 6 h using microarray. Among the 32 mRNAs, 14 mRNAs including Atβ-Amy mRNA (44%), showed more than a twofold increase in response to Suc. This proportion was significantly higher than the 6.2% of total mRNAs that are Suc-inducible according to the same criteria.
The 32 genes most strongly repressed in lba1 include seven genes that encode members of several subfamilies of glycosyl hydrolases: three chitinases, two β-1,3-glucanases, β-glucosidase and Atβ-Amy (Table 1). Genes for chitinase and β-1,3-glucanase are known to be expressed in response to pathogen attack (Thomma et al., 1998), and β-glucosidase encoded by PSR3.2 is inducible by phosphate starvation (Malboobi and Lefebvre, 1997). Din11 mRNA was the most strongly reduced in lba1, and was partially recovered by 35S:AtUPF1 (Figure 4d). Expression of Din11 occurs in association with leaf senescence and is inducible by dark and sugar (Fujiki et al., 2001). Table 1 includes two other well characterized senescence-related genes, SAG12 (Lohman et al., 1994; Weaver et al., 1998) and AtFer1 (Tarantino et al., 2003). It is suggested that AtUPF1 is required for maximum expression of several groups of genes with similar function.
|AGI code||Gene description||lba1/Col (±SD)b||Suc/H2O (±SD)c|
|AT3G49620||Dark-inducible 2-oxoacid-dependent oxidase (Din11)||0.05 (0.01)||1.5 (0.3)|
|AT1G09080||Luminal binding protein 3 (BiP-3)||0.29 (0.13)||1.0 (0.5)|
|AT4G24340||Similar to poplar bark VSP||0.32 (0.04)||1.8 (0.2)|
|AT5G61160||Anthocyanin 5-aromatic acyltransferase-like protein||0.32 (0.01)||12.6 (4.1)|
|AT2G43580||Glycosyl hydrolase family 19 (chitinase)||0.32 (0.02)||2.0 (0.4)|
|AT2G43590||Glycosyl hydrolase family 19 (chitinase)||0.35 (0.01)||3.0 (1.0)|
|AT5G45890||Senescence-specific cysteine protease (SAG12)||0.35 (0.09)||0.9 (0.1)|
|AT3G47540||Glycosyl hydrolase family 19 (chitinase)||0.35 (0.01)||2.3 (0.3)|
|AT4G16260||Glycosyl hydrolase family 17 (β-1,3-glucanase)||0.36 (0.01)||1.1 (0.6)|
|AT1G66270||Glycosyl hydrolase family 1 (β-glucosidase; PSR3.2)||0.37 (0.14)||0.8 (0.2)|
|AT4G24350||Similar to poplar bark VSP||0.40 (0.02)||5.5 (2.4)|
|AT5G01600||Ferritin 1 (AtFer1)||0.41 (0.04)||0.1 (0.1)|
|AT4G15210||β-amylase (BMY1; Atβ-Amy)||0.42 (0.04)||10.6 (4.7)|
|AT3G57240||Glycosyl hydrolase family 17 (β-1,3-glucanase; BG3)||0.48 (0.02)||1.7 (0.2)|
|AT3G18000||Phosphoethanolamine N-methyltransferase 1 (AtNMT1)||5.9 (0.6)||8.3 (2.5)|
|AT4G13420||High-affinity K+ transporter (AtHAK5)||4.4 (1.3)||0.3 (0.1)|
|AT5G47880||Eukaryotic peptide chain-release factor 1-1 (eRF1-1)||4.2 (0.4)||2.2 (0.5)|
|AT3G56980||bhlh family protein, subfamily2, bHLH039 (ORG3)||3.8 (2.0)||1.0 (0.1)|
|AT1G72050||Transcription factor IIIA (AtTFIIIA)||3.4 (0.1)||1.9 (0.9)|
|AT3G56970||bHLH family protein, subfamily2, bHLH038 (ORG2)||3.3 (0.8)||0.9 (0.1)|
|AT2G41240||bHLH family protein, subfamily2, bHLH100||3.1 (0.7)||0.9 (0.1)|
|AT1G29950||bHLH family protein, subfamily13, bHLH144||3.0 (0.1)||1.2 (0.1)|
|AT5G37260||MYB family transcription factor||2.9 (0.2)||0.2 (0.1)|
|AT1G54260||Histone H1/H5 family protein||2.8 (0.1)||1.3 (0.5)|
|AT5G49500||Signal recognition particle 54-kDa protein 2 (SRP-54B)||2.8 (0.1)||1.8 (0.4)|
|AT1G04240||Auxin-responsive protein (SHY2/IAA3)||2.8 (0.2)||1.8 (0.7)|
|AT1G21160||Eukaryotic translation initiation factor eIF-2 family protein||2.8 (0.0)||0.6 (0.1)|
|AT3G08730||Ribosomal protein S6 kinase (ATPK1)||2.8 (0.2)||1.3 (0.4)|
|AT2G02990||Ribonuclease 1 (RNS1)||2.8 (0.9)||2.6 (0.8)|
|AT5G65060||MADS-box protein (MAF3)||2.8 (0.0)||0.6 (0.2)|
|AT2G18160||bZIP family transcription factor (GBF5)||2.7 (0.2)||0.4 (0.2)|
|AT4G22950||MADS-box protein (AGL19)||2.6 (0.1)||0.6 (0.2)|
|AT5G09460||bHLH family protein, subfamily13, bHLH143||2.6 (0.3)||1.5 (0.4)|
|AT1G78960||2-3-oxidosqualene-triterpene cyclase (AtLUP2)||2.4 (0.2)||0.9 (0.2)|
|AT4G33470||Histone deacetylase family protein, HDA14||2.4 (0.0)||0.9 (0.1)|
|AT1G36730||Eukaryotic translation initiation factor eIF5, putative||2.4 (0.0)||1.2 (0.4)|
|AT1G05160||ent-Kaurene oxidase (AtKAO1/CYP88A3)||2.3 (0.1)||0.8 (0.1)|
|AT5G50010||bHLH family protein,subfamily13, bHLH145||2.3 (0.1)||1.9 (0.4)|
|AT5G19400||Similar to SMG5||2.2 (0.2)||0.9 (0.1)|
|AT5G53450||Protein kinase family protein (ORG1)||2.2 (0.2)||0.6 (0.2)|
|AT1G65220||eIF4-gamma/eIF5/eIF2-epsilon domain-containing protein||2.1 (0.0)||0.7 (0.2)|
|AT3G04450||MYB family transcription factor||2.1 (0.0)||0.8 (0.3)|
Increased transcript levels of genes for a wide range of cellular activities in lba1
Seventy-five genes showed more than twofold increases in transcript levels in lba1 compared with Col in both biological replicates (Table S2). Because NMD is a pathway that leads mRNA to degradation, genes with increased transcript levels in mutants defective in NMD are likely to contain direct targets of NMD (He et al., 2003; Mendell et al., 2004; Rehwinkel et al., 2005), although they may also contain mRNAs that are upregulated indirectly by defects in NMD. Unlike mRNAs reduced in lba1, the 75 mRNAs most strongly increased in lba1 were not enriched with sugar-regulated mRNAs. They contained many genes that are involved in a wide range of cellular activities (Table 1).
Six of the 75 mRNAs encode proteins involved in translation and its related processes, including a gene for eukaryotic peptide chain-release factor 1 (eRF1-1; Chapman and Brown, 2004), and 15 of them (20%) encode transcription factors (Table 1). In particular, six are members of two subfamilies of the large basic helix–loop–helix (bHLH) family (Toledo-Ortiz et al., 2003). Two mRNAs (ORG2 and ORG3) in subfamily 2 have been shown to be direct targets of the salicylic acid-inducible Dof family transcription factor OBP2 (Kang et al., 2003). Another target of OBP2, ORG1, is also included. Table 1 includes genes for transcription factors such as AtTFIIIA involved in 5.8S rRNA transcription (Mathieu et al., 2003); SHY2/IAA3 involved in auxin response and root development (Tian et al., 2002); MAF3 (Ratcliffe et al., 2003) and AGL19 (Gan et al., 2005; Samach et al., 2000) involved in flowering control and nitrogen response; and GBF5 involved in hypo-osmolarity response (Satoh et al., 2004).
Table 1 also includes genes for enzymes involved in various metabolic processes. AtNMT1 is involved in the synthesis of phosphatidylcholine and glycine betaine (Mou et al., 2002), while AtKAO and AtLUP2 are enzymes involved in the synthesis of gibberellic acid (Helliwell et al., 1998) and triterpenoids (Husselstein-Muller et al., 2001) respectively. At HAK5 encodes a high-affinity K+ transporter, and its expression is regulated in response to K+ (Ahn et al., 2004). Taken together, these results suggest that AtUPF1 is involved in the expression of genes that participate in various aspects of plant metabolism, growth and development.
Increased level of an alternatively spliced mRNA of AtTFIIIA containing PTC in lba1
Seventy-five genes upregulated in lba1 produced transcripts with various structural features. Similarly to other eukaryotes (He et al., 2003; Mendell et al., 2004; Rehwinkel et al., 2005), natural targets of NMD in plants are likely to contain alternatively spliced mRNAs containing PTCs. Among the genes upregulated in lba1 (Table 1), AtTFIIIA produces alternatively spliced transcripts (Figure 6a). Transcript 1 is produced by skipping the third exon codes for AtTFIIIA with nine zinc fingers, similar to TFIIIA from other eukaryotes (Mathieu et al., 2003). On the other hand, transcript 2 includes the third exon, and it is predicted to produce a truncated polypeptide with two zinc fingers due to PTC in the third exon. The levels of transcript 2 of AtTFIIIA in Col, lba1 and three lines of the 35S:AtUPF1 transformants were examined using real-time RT-PCR with specific primers in the third exon. As shown in Figure 6(b), the level of transcript 2 in Suc-treated lba1 was 17-fold higher than that in Suc-treated Col plants, and the levels in the 35S:AtUPF1 transformants were reduced to nearly the same as in Col plants. To compare the relative ratio of transcripts 1 and 2 in Col and lba1, transcripts from AtTFIIIA in Suc-treated Col and lba1 were amplified by RT-PCR using primers designed in exons 2 and 4, and the amplified fragments were subjected to direct sequencing from a sequencing primer within exon 4 towards the 5′ direction. In the sequencing electropherograms (Figure 6c), both the sequence of exon 2 in transcript 1 and the sequence of exon 3 in transcript 2 appeared after the sequence of exon 4. The ratio of transcript 2 : transcript 1 was significantly higher in lba1 compared with Col. Similar results were obtained with other RNA samples.
- Top of page
- Experimental procedures
- Supporting Information
Possible effect of the Gly851-to-Glu mutation of AtUPF1 in lba1
The results presented here indicate that a change of Gly851 to Glu in AtUPF1 is responsible not only for the reduced sugar-induced expression of Atβ-Amy and other genes, but also for the visible phenotypes of the lba1/atupf1-1 mutant examined here.
UPF1 plays a central role in NMD via its RNA binding, ATP hydrolytic, and 5′ to 3′ ATP-dependent helicase activities (Czaplinski et al., 1995). In yeast, mutations in upf1 have been identified that stabilize PTC-containing his4-38 mRNA in the upf1+his4-38 strain (Leeds et al., 1992). Four of seven such mutations were conversions of amino acids near motifs V and VI: Arg779 to Cys; Arg779 to Gly; Gly787 to Asp; and Arg794 to Cys. Mutational change of Arg793 Arg794 and Thr800 Arg801 in yeast UPF1 to AlaAla abolishes NMD, and these mutant UPF1 proteins lose not only ATPase and helicase activities, but also RNA-binding activity (Weng et al., 1996). These results suggest that yeast UPF1 with mutations between Arg779 and Arg801 are defective, and that they compete with the wild-type UPF1 for some cell component. The Gly851 of AtUPF1 mutated in lba1 is located close to this region, which is important for the function.
The sugar-induced levels of Atβ-Amy and Din11 expression in the 35S:AtUPF1 transformant lines were not as high as those in Col. This could be due to partial impairment of wild-type AtUPF1 activity by the endogenous mutant form. Phenotypes of lba1, such as early flowering under continuous light and hypersensitivity of seed germination to Glc and ABA, were almost fully rescued by 35S:AtUPF1. In these cases, recovery of functional expression of the responsible gene to a threshold level may be sufficient for the rescue.
A missense mutant of UPF1 has not been identified previously in multicellular higher eukaryotes. The lba1 plants exhibit not only anomalies in gene expression, but also a variety of clearly visible phenotypes. As the Arabidopsis knockout mutant of AtUPF1 is seedling-lethal (Yoine et al., 2006), the lba1 mutant provides a good tool to study NMD and the role of UPF1 in the growth and development of plants. Additional growth and developmental phenotypes might be identified in lba1 plants under different conditions.
Role of AtUPF1 in sugar signalling
Germination of lba1 seeds was hypersensitive to Glc and ABA, and was also resistant to mannose. The lba1 mutation of AtUPF1 also caused reduced sugar-induced expression of not only Atβ-Amy but also Din11 and many other genes. These results suggest that AtUPF1-dependent post-transcriptional regulation is involved in at least a part of the sugar signalling and is required for maximum expression of a subset of sugar-inducible genes. It is unlikely that mRNAs reduced in lba1 are direct targets of NMD. Sugar-induced expression of not only Atβ-Amy, but also an intronless GUS reporter gene under control of the Atβ-Amy promoter, are reduced in lba1, suggesting that lba1 mutation affects transcription of Atβ-Amy (Mita et al., 1997). It appears that some factor encoded by a target gene of AtUPF1 is involved at some point in the sugar-signalling pathway, and altered expression of this factor affects the expression of downstream genes.
The 32 mRNAs most substantially reduced in lba1 are significantly enriched with sugar-inducible mRNAs such as two genes encoding proteins similar to bark storage proteins of poplar (Davis et al., 1993), and five genes for chitinases and β-1,3-glucanases in addition to Atβ-Amy. These genes could be involved in nutrient storage and defensive reactions. The list also contains Din11, SAG12 and AtFer1, which are expressed in senescent leaves (Fujiki et al., 2001; Lohman et al., 1994; Tarantino et al., 2003; Weaver et al., 1998), suggesting possible involvement of a target of NMD in senescence.
Transcripts of genes involved in diverse cellular activities are increased in lba1
Unlike transcripts reduced in lba1, transcripts increased in lba1 are likely to include direct targets of UPF1-dependent post-transcriptional regulation. In yeast (He et al., 2003; human (Mendell et al., 2004); and Drosophila (Rehwinkel et al., 2005) transcripts of as many as 4–10% of all genes increased more than twofold as a result of depletion of UPF1 or other NMD factors. In our experiments, transcripts of 75 genes, 0.4% of the total genes studied, increased more than twofold in lba1 compared with Col. This relatively low percentage might be due to the fact that lba1 is a missense mutant of UPF1. Furthermore, our analysis may have missed many natural targets of NMD because transcript levels in Suc-treated lba1 and Col plants were compared and transcripts with very weak hybridization signals were eliminated. Nevertheless, the transcripts most substantially increased in lba1 were not over-represented by Suc-regulated genes; rather, they contained mRNAs for a wide variety of functions and cellular activities.
The mechanisms of how PTCs in transcripts are recognized and marked to recruit NMD factors for degradation are different among yeast, Drosophila and human (reviewed by Culbertson and Leeds, 2003; Lejeune and Maquat, 2005). These organisms do not share common orthologous genes as NMD targets, except for a few orthologous groups of genes (Rehwinkel et al., 2005). One such orthologous group of genes is NMD proteins that are also involved in telomere maintenance (Lew et al., 1998). In particular, SMG5 of Drosophila and human are targets of NMD; and yeast ESB1 related to SMG5 is also an NMD target. The predicted gene At5g19400 (Table 1) showed a 2.2-fold increase in transcript level in lba1 compared with Col codes for a protein with similarities to SMG5.
Natural targets of plant NMD
Natural transcripts with diverse structural features have been identified as targets of NMD in yeast, Drosophila and human (He et al., 2003; Mendell et al., 2004; Rehwinkel et al., 2005). In both yeast and human, targets of NMD include mRNAs with upstream open reading frames (uORFs); mRNAs with nonsense codons introduced by alternative splicing; and pseudogene- or transposon-derived RNAs. In addition, NMD targets in human include many mRNAs with introns in the 3′-UTR (Mendell et al., 2004), and those in Drosophila include mRNAs regulated by stop-codon readthrough (Rehwinkel et al., 2005). Natural targets of NMD in plants are likely to contain alternatively spliced transcripts containing PTCs. Indeed, the level of alternatively spliced transcript 2 of AtTFIIIA, which contains PTC, was 17 times higher in lba1 compared with Col. Recently, Hori and Watanabe (2005) reported that an Arabidopsis knockout mutant of UPF3 shows an increased ratio of PTC+ compared with PTC− forms of alternatively spliced transcripts of five genes. The mRNAs of four of these genes showed 1.4- to 14-fold higher levels in the mutant. The levels of mRNAs for these genes, however, were not significantly different between Col and lba1 in our microarray data.
The 75 genes most strongly upregulated in lba1 (Table S2) contained eight genes (11%) that produce alternatively spliced transcripts according to the sequence database TAIR (http://www.arabidopsis.org/servlets/sv). Similarly, 11.6% of transcription units of Arabidopsis are estimated to produce alternatively spliced transcripts (Iida et al., 2004), suggesting that a large portion of targets of NMD in Arabidopsis contain mRNAs with other structural features. By clarifying the structure of transcripts increased in lba1 and characterizing the stability of various forms of transcripts in lba1, we may be able to identify structural features of plant mRNAs that elicit NMD.
- Top of page
- Experimental procedures
- Supporting Information
Plant materials and growth conditions
Arabidopsis thaliana (L.) Heynh. ecotype Columbia (Col) was used as the wild-type plant. The lba1 mutant (Mita et al., 1997) had been back-crossed with Col that contained the gl1 mutation. These plants were back-crossed an additional five times with Col plants without the gl1 mutation. At each crossing step, homozygous lba1 mutants were selected based on reduced amylase activity in leaves treated for 2 days with 5% (w/v) Suc, and selected F2 individuals were subsequently confirmed using an F3 progeny test. The abi4-1 mutant (Finkelstein, 1994) was obtained from the Arabidopsis Biological Resource Center.
Unless otherwise indicated, seeds were sterilized in sterile water, kept at 4°C for 3 days, and sown on 0.3% (w/v) gellun gum plates containing Murashige and Skoog (MS) medium (Wako, Tokyo, Japan) pH 5.7 and 100 mg l−1 myoinositol, 10 mg l−1 thiamine–HCl, 1 mg l−1 nicotinic acid, 1 mg l−1 pyridoxine HCl, and 3% (w/v) Suc. Plates were incubated in a growth chamber at 22°C under continuous light at an intensity of 50 μmol photons m−2 sec−1 . Plants were also grown on soil under continuous light or under short-day conditions (8 h light/16 h dark) at an intensity of 100 μmol photons m−2 sec−1 .
Seed germination assay
Seeds were sterilized in sterile water and incubated at 4°C for 3 days before being plated on 0.7% (w/v) agar plates containing MS medium and 4% Glc or 0.4 μm ABA at 22°C under continuous light. To examine mannose sensitivity, seeds were sown on 0.7% (w/v) agar plates containing minimal salt medium with 1% (w/v) Suc and with or without 5 mm mannose. Germination (cotyledon opening and greening) was scored daily.
Map-based cloning of lba1
For genetic mapping, the lba1 mutant was crossed with the ecotype Ws, and F2 seeds were obtained by self-pollination of the F1 plants. F2 seeds obtained by an additional two or three back-crosses with Ws were also used. Candidate homozygous lba1 mutant plants were selected based on reduced levels of amylase activity in Suc-treated leaves, and candidates were confirmed by assessing the amylase activity in more than 40 individual F3 progeny. The genomic DNA isolated from these homozygous lba1 mutant plants was subjected to genetic mapping using a combination of CAPS and SSLP markers. Where appropriate, new SSLP and CAPS markers were designed based on DNA sequences. The primers used for CAPS and SSLP markers are listed in Table S3. The nucleotide sequence of the 61-kb region of the lba1 genome covering the lba1 locus was determined using a PRISM 3100 DNA sequencer (Applied Biosystems, La Jolla, CA, USA). To determine the genotypes of plants with respect to the lba1 locus, a pair of dCAPS primers was used (Table S3).
Construction of 35S:AtUPF1 and transformation of the lba1 mutant
The cDNA clone RZL05b08, obtained from the Kazusa DNA Research Institute (Kisarazu, Chiba, Japan), was determined to be a full-length cDNA for LBA1/AtUPF1 by sequencing. The AtUPF1 cDNA was placed downstream of the cauliflower mosaic virus 35S promoter by using Gateway cloning technology (Invitrogen, Carlsbad, CA, USA) with the pGWB2 vector developed by Dr T. Nakagawa of the Research Institute for Molecular Genetics, Shimane University (Shimane, Japan). Transformation of lba1 mutant plants was carried out by vacuum infiltration with Agrobacterium tumefaciens strain C58C1 (pMP90). Hygromycin-resistant seedlings were selected on MS agar medium containing 20 mg l−1 hygromycin.
Extraction of proteins and analysis of amylase activities
The third to fourth mature leaves of 3-week-old plants were excised with a sharp razor blade. The cut edges of petioles were immersed in a solution of water or 5% (w/v) Suc and incubated for 2 days at 22°C under continuous light. Extraction of proteins, quantification of protein, and assay of total amylase activities were performed as described by Mita et al. (1997).
Preparation of RNA and quantitative real-time PCR
Three-week-old plants were treated with water or 5% (w/v) Suc for 2 days, and total RNA was isolated using an RNeasy plant mini kit (Qiagen, Valencia, CA, USA). Reverse transcription of RNA after treatment with DNase I was performed with SuperScript III RNase H reverse transcriptase (Invitrogen) using an oligo-dT primer. The levels of various mRNAs were analysed by quantitative real-time RT-PCR using an iCycler iQ (Bio-Rad, Hercules, CA, USA) with iQ SYBR Green Supermix (Bio-Rad). Primers used for real-time RT-PCR are listed in Table S3. For each pair of primers, a melting-curve analysis of the PCR product was performed to confirm that the PCR produced only one fragment. The threshold cycles were determined at which fluorescence of the PCR product-SYBR Green complex first exceeded the background level, and the relative template concentration to control was determined based on the standard curve for each cDNA. The level of each mRNA was normalized by the level of ACT2 or UBC mRNA (Czechowski et al., 2005).
Total RNA used for microarray analyses were prepared using Trizol reagent (Gibco BRL, Cleveland, OH, USA) and subsequently purified using an RNeasy mini-kit or an RNeasy plant mini-kit (Qiagen). The isolated total RNAs were used for preparation of Cy5- and Cy3-labelled cDNA probes. Samples were analysed using the Agilent Arabidopsis 2 Oligo Microarray (Agilent Technologies, Palo Alto, CA, USA). The microarray analysis was performed with two independently isolated RNA samples and assessed in each experiment by dye swapping. Feature extraction software (Agilent Technologies) was used to locate and delineate every spot in the array and to integrate each spot's intensity, filtering and normalization. The raw data files and details of labelling and hybridization experiments have been deposited in the public microarray database (http://www.ebi.ac.uk/arrayexpress) under accession number E-MEXP-541.
- Top of page
- Experimental procedures
- Supporting Information
We are grateful to Drs T. Hattori, A. Morikami and S. Ishiguro for valuable discussion and suggestions; Drs M. Nishimura and M. Hayashi of the National Institute for Basic Biology (Japan) for the use of the microarray analysis system; Dr T. Nakagawa of the Research Institute for Molecular Genetics, Shimane University for the pGWB expression vector; and Kazusa DNA Research Institute for cDNA. This work was supported in part by the Research for the Future Program of the Japan Society for the Promotion of Science (no. 00L01603) and the 21st Century COE Program from the Ministry of Education, Science, Sports, and Culture of Japan to KN.
- Top of page
- Experimental procedures
- Supporting Information
- 2004) Expression of KT/KUP genes in Arabidopsis and the role of root hairs in K+ uptake. Plant Physiol. 134, 1135–1145. , and (
- 2004) Nonsense-mediated mRNA decay: terminating erroneous gene expression. Curr. Opin. Cell Biol. 16, 293–299. and (
- 2004) Translation termination in Arabidopsis thaliana: characterisation of three versions of release factor 1. Gene, 341, 219–225. and (
- 2005) Nonsense-mediated mRNA decay: molecular insights and mechanistic variations across species. Curr. Opin. Cell Biol. 17, 316–325. and (
- 2003) Looking at mRNA decay pathways through the window of molecular evolution. Curr. Opin. Genet. Dev. 13, 207–214. and (
- 1995) Purification and characterization of the Upf1p: a factor involved in mRNA turnover. RNA, 1, 610–623. , , and (
- 2005) Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol. 139, 5–17. , , , and (
- 1993) A family of wound-induced genes in Populus shares common features with genes encoding vegetative storage proteins. Plant Mol. Biol. 23, 135–143. , , , , , and (
- 1994) Mutations at two new Arabidopsis ABA response loci are similar to the abi3 mutations. Plant J. 5, 765–771 (
- 2001) Dark-inducible genes from Arabidopsis thaliana are associated with leaf senescence and repressed by sugars. Physiol. Plant. 111, 345–352. , , , , , and (
- 2004) Nonsense-mediated mRNA decay in barley mutants allows the cloning of mutated genes by a microarray approach. Plant Physiol. Biochem. 42, 681–685. , , , and (
- 2005) Nutritional regulation of ANR1 and other root-expressed MADS-box genes in Arabidopsis thaliana. Planta, 222, 730–742. , , , and (
- 2005) Control of plant development and gene expression by sugar signaling. Curr. Opin. Plant Biol. 8, 93–102. (
- 2001) Nonsense-mediated mRNA decay in Saccharomyces cerevisiae. Gene, 274, 15–25. , , and (
- 1999) Current perspectives on mRNA stability in plants: multiple levels and mechanisms of control. Trends Plant Sci. 4, 429–438. , and (
- 2003) Genome-wide analysis of mRNAs regulated by the nonsense-mediated and 5′ to 3′ mRNA decay pathways in yeast. Mol. Cell 12, 1439–1452. , , , , and (
- 1998) Cloning of the Arabidopsis ent-kaurene oxidase gene GA3. Proc. Natl Acad. Sci. USA 95, 9019–9024. , , , , , and (
- 1988) A new superfamily of replicative proteins. Nature, 333, 22–23. (
- 2005) UPF3 suppresses aberrant spliced mRNA in Arabidopsis. Plant J. 43, 530–540. and (
- 2001) Molecular cloning and expression in yeast of 2,3-oxidosqualene-triterpenoid cyclases from Arabidopsis thaliana. Plant Mol. Biol. 45, 75–92. , and (
- 2004) Genome-wide analysis of alternative pre-mRNA splicing in Arabidopsis thaliana based on full-length cDNA sequences. Nucleic Acids Res. 32, 5096–5103. , , , , , , and (
- 2001) Nonsense-mediated decay of mutant waxy mRNA in rice. Plant Physiol. 125, 1388–1395. , , and (
- 1997) Hexokinase as a sugar sensor in higher plants. Plant Cell, 9, 5–19. , , and (
- 2003) Target genes for OBP3, a Dof transcription factor, include novel basic helix–loop–helix domain proteins inducible by salicylic acid. Plant J. 35, 362–372. , , , and (
- 2004) mega3: Integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform. 5, 150–163. , and (
- 1992) Gene products that promote mRNA turnover in Saccharomyces cerevisiae. Mol. Cell Biol. 12, 2165–2177. , , and (
- 2005) Mechanistic links between nonsense-mediated mRNA decay and pre-mRNA splicing in mammalian cells. Curr. Opin. Cell Biol. 17, 309–315. and (
- 1998) Telomere length regulation and telomeric chromatin require the nonsense-mediated mRNA decay pathway. Mol. Cell Biol. 18, 6121–6130. , and (
- 1994) Molecular analysis of natural leaf senescence in Arabidopsis thaliana. Physiol. Plant. 92, 322–328. , , and (
- 1997) A phosphate-starvation inducible beta-glucosidase gene (psr3.2) isolated from Arabidopsis thaliana is a member of a distinct subfamily of the BGA family. Plant Mol. Biol. 34, 57–68. and (
- 2005a) ACTIVATOR OF Spomin::LUC1/WRINKLED1 of Arabidopsis thaliana transactivates sugar-inducible promoters. Plant Cell Physiol. 46, 547–556. , , , , and (
- 2005b) Activation tagging of a gene for a protein with novel class of CCT-domain activates expression of a subset of sugar-inducible genes in Arabidpsis thaliana. Plant J. 43, 142–152. , , , , , and (
- 2003) Identification and characterization of transcription factor IIIA and ribosomal protein L5 from Arabidopsis thaliana. Nucleic Acids Res. 31, 2424–2433. , , , , and (
- 2004) Nonsense surveillance regulates expression of diverse classes of mammalian transcripts and mutes genomic noise. Nat. Genet. 36, 1073–1078. , , , and (
- 1997) Mutants of Arabidopsis thaliana with pleiotropic effects on the expression of the gene for β-amylase and on the accumulation of anthocyanin that are inducible by sugars. Plant J. 11, 841–851. , , and (
- 2002) Silencing of phosphoethanolamine N-methyltransferase results in temperature-sensitive male sterility and salt hypersensitivity in Arabidopsis. Plant Cell, 14, 2031–2043. , , , , , , , and (
- 2002) Web-based primer design for single nucleotide polymorphism analysis. Trends Genet. 18, 613–615. , and (
- 1999) Mannose inhibits Arabidopsis germination via a hexokinase-mediated step. Plant Physiol. 119, 1017–1023. , and (
- 2003) Analysis of the Arabidopsis mads affecting flowering gene family: MAF2 prevents vernalization by short periods of cold. Plant Cell, 15, 1159–1169. , , and (
- 2005) Nonsense-mediated mRNA decay factors act in concert to regulate common mRNA targets. RNA, 11, 1530–1544. , , , and (
- 2002) Sugar sensing and signaling in plants. Plant Cell, 14 Suppl. , S185–205. , and (
- 2003) Genetic approaches to understanding sugar-response pathways. J. Exp. Bot. 54, 495–501. and (
- 2000) Distinct roles of CONSTANS target genes in reproductive development of Arabidopsis. Science, 288, 1613–1616. , , , , , and (
- 2004) A novel subgroup of bZIP proteins functions as transcriptional activators in hypoosmolarity-responsive expression of the ProDH gene in Arabidopsis. Plant Cell Physiol. 45, 309–317. , , , and (
- 2004) eIF4AIII binds spliced mRNA in the exon junction complex and is essential for nonsense-mediated decay. Nat. Struct. Mol. Biol. 11, 346–351. , , and (
- 2000) Sugar-induced signal transduction in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 51, 49–81. (
- 2003) Differential involvement of the IDRS cis-element in the developmental and environmental regulation of the AtFer1 ferritin gene from Arabidopsis. Planta, 217, 709–716. , , , , and (
- 1998) Separate jasmonate-dependent and salicylate-dependent defense-response pathways in Arabidopsis are essential for resistance to distinct microbial pathogens. Proc. Natl Acad. Sci. USA 95, 15107–15111. , , , , , and (
- 2002) Arabidopsis SHY2/IAA3 inhibits auxin-regulated gene expression. Plant Cell, 14, 301–319. , and (
- 2003) The Arabidopsis basic/helix–loop–helix transcription factor family. Plant Cell, 15, 1749–1770. , and (
- 2005) Analysis of a sugar response mutant of Arabidopsis thaliana identified a novel B3 domain protein that functions as an active transcriptional repressor. Plant Physiol. 138, 675–685. , , , and (
- 1998) A comparison of the expression patterns of several senescence-associated genes in response to stress and hormone treatment. Plant Mol. Biol. 37, 455–469. , , and (
- 1996) Genetic and biochemical characterization of mutations in the ATPase and helicase regions of the Upf1 protein. Mol. Cell Biol. 16, 5477–5490. , and (
- 2006) Arabidopsis UPF1 RNA helicase for nonsense-mediated mRNA decay is involved in seed size control and is essential for growth. Plant Cell Physiol., 48, doi: 10.1093/pcp/pcj035. , and (
- Top of page
- Experimental procedures
- Supporting Information
Table S1 Transcripts decreased in the lba1 mutant, as identified in microarray analysis
Table S2 Transcripts increased in the lba1 mutant, as identified in microarray analysis
Table S3 Primers used
Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.
|TPJ_2771_sm_TableS1.doc||72K||Supporting info item|
|TPJ_2771_sm_TableS2.doc||124K||Supporting info item|
|TPJ_2771_sm_TableS3.doc||29K||Supporting info item|
Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.