Fructose sensitivity is suppressed in Arabidopsis by the transcription factor ANAC089 lacking the membrane-bound domain

Abstract

In living organisms sugars not only provide energy and carbon skeletons but also act as evolutionarily conserved signaling molecules. The three major soluble sugars in plants are sucrose, glucose, and fructose. Information on plant glucose and sucrose signaling is available, but to date no fructose-specific signaling pathway has been reported. In this study, sugar repression of seedling development was used to study fructose sensitivity in the Landsberg erecta (Ler)/Cape Verde Islands (Cvi) recombinant inbred line population, and eight fructose-sensing quantitative trait loci (QTLs) (FSQ1–8) were mapped. Among them, FSQ6 was confirmed to be a fructose-specific QTL by analyzing near-isogenic lines in which Cvi genomic fragments were introgressed in the Ler background. These results indicate the existence of a fructose-specific signaling pathway in Arabidopsis. Further analysis demonstrated that the FSQ6-associated fructose-signaling pathway functions independently of the hexokinase1 (HXK1) glucose sensor. Remarkably, fructose-specific FSQ6 downstream signaling interacts with abscisic acid (ABA)- and ethylene-signaling pathways, similar to HXK1-dependent glucose signaling. The Cvi allele of FSQ6 acts as a suppressor of fructose signaling. The FSQ6 gene was identified using map-based cloning approach, and FSQ6 was shown to encode the transcription factor gene Arabidopsis NAC (petunia No apical meristem and Arabidopsis transcription activation factor 1, 2 and Cup-shaped cotyledon 2) domain containing protein 89 (ANAC089). The Cvi allele of FSQ6/ANAC089 is a gain-of-function allele caused by a premature stop in the third exon of the gene. The truncated Cvi FSQ6/ANAC089 protein lacks a membrane association domain that is present in ANAC089 proteins from other Arabidopsis accessions. As a result, Cvi FSQ6/ANAC089 is constitutively active as a transcription factor in the nucleus.

In plants, sugars provide the energy and carbon skeletons needed for growth and in addition act as crucial signaling molecules that affect growth, development, and response to the (a)biotic environment (1–3). Plant cells harbor sugar-sensing and -signaling systems that regulate the expression of thousands of genes and control the metabolic processes needed for growth (2–4). These sugar-response systems are known to interact with other signaling pathways, such as those for light, phytohormones, stress, and nutrients (1).

The neutral sugars sucrose, glucose, and fructose are central to metabolism in plants and in other organisms as well. So far, detailed information is available only on glucose sensing, and it has been shown that the hexokinase 1 (HXK1) enzyme acts as a glucose sensor (5, 6). Sucrose-specific signaling also was demonstrated because the effect of sucrose cannot be mimicked by glucose and/or fructose (7), but so far no information on sucrose-sensing systems is available. A signaling function for fructose has been proposed (8, 9), but no convincing experimental evidence on such fructose-specific signaling is available.

In Arabidopsis early seedling development is arrested by high concentrations of exogenous glucose or sucrose. This observation has been used in several screens to identify mutants defective in sugar sensing or signaling (10, 11). This rapid and convenient phenotypic screen allowed the isolation of many mutants with altered sugar responses and the subsequent identification of the genes involved. In this way the glucose-insensitive (gin) phenotype of the hxk1/gin2 mutant (6) was established, and a network of HXK1-dependent glucose signaling and abscisic acid (ABA) and ethylene biosynthesis and signaling was identified (10–17).

Fructose is a major soluble monosaccharide in plant. Fructose is produced from sucrose by invertases and sucrose synthases. Like glucose, fructose can repress the expression of photosynthesis genes (18). Most likely, fructose is phosphorylated mainly by fructokinases (FRKs), and a member of that gene family is present in Arabidopsis. The putative regulatory role of FRK in fructose signaling was investigated first in tomato (9, 19). Inhibition of the tomato fructokinase 2 (LeFRK2) interferes with development of phloem and xylem by impairing callose deposition, thus reducing transport of sugars and water (20). In this way LeFRK2 affects tomato stem and root growth and the normal development of flowers, fruits, and seeds. These experiments illustrate the important role of FRK in metabolism, but a signaling function for FRKs or for fructose could not be established.

Here, a fructose-specific signaling pathway is proposed by the identification of Arabidopsis quantitative trait loci (QTLs) in the Landsberg erecta (Ler)/Cape Verde Islands (Cvi) recombinant inbred line (RIL) population that displays altered fructose-specific sensitivity. This fructose-signaling pathway is HXK1 independent, but, remarkably, the fructose signal feeds into the same downstream ABA-signaling pathway as the glucose/HXK signal. The Cvi fructose-sensing QTL allele 6 (FSQ6) was cloned by a map-based cloning approach and shown to encode the Arabidopsis NAC (petunia No apical meristem and Arabidopsis transcription activation factor 1, 2 and Cup-shaped cotyledon 2) domain containing protein 89 (ANAC089) gene. Functional analysis indicates that the Cvi FSQ6/ANAC089 allele is a gain-of-function allele that represses the fructose-induced ABA-signaling pathway.

Results

Identification of Fructose-Sensitivity QTLs in the Ler/Cvi RIL Population.

High concentrations of glucose and sucrose repress Arabidopsis seedling development. The identified gin and sugar-insensitive (sis) mutants are insensitive to glucose and/or sucrose repression of cotyledon and shoot development (10, 11). High concentrations of fructose similarly produce pale seedlings, and it was found that the Cvi accession is more sensitive than the Ler accession to high fructose levels (Fig. 1A). Sensitivity of seedling development to fructose was quantified in the available Ler/Cvi RIL population, and the data were used for QTL mapping (21). A total of eight QTLs for fructose sensitivity were detected [logarithm of the odds (LOD) score >2.5]. These QTLs were named “fructose-sensing QTL 1–8” (FSQ1 to FSQ8) (Fig. 1B and Table S1). Interestingly, the Cvi alleles of FSQ2, FSQ4, FSQ5, FSQ7, and FSQ8 increase fructose sensitivity, whereas the Cvi alleles of FSQ1, FSQ3, and FSQ6 decrease fructose sensitivity.

Fig. 1.

Identification and confirmation of FSQs. (A) Fructose-sensitivity phenotypes of Ler and Cvi accessions. Seeds were grown on agar-solidified 1/2 MS containing 6% fructose for 9 d at 22 °C under continuous light. (B) QTL map of fructose sensitivity in the RIL population of Ler/Cvi. The LOD score threshold was 2.5. The FSQ loci are indicated by solid black bars representing the two-LOD support interval; up or down arrowhead of the bar indicates that the Cvi allele increases or decreases fructose sensitivity. The LOD score peak is indicated by an arrowhead next to the interval bars. (C) Genotypes of NILs used for confirmation of QTLs. Black bars indicate the integrated Cvi genomic segment in the Ler background. (D) Fructose-sensitivity phenotypes of the NILs presented in C. Seeds of Ler, Cvi, and the NILs were grown on 1/2 MS containing fructose concentrations as indicated for 9 d at 22 °C under continuous light. Seedling development was scored, and the percentage of normally developed seedlings with green cotyledons is presented.

Four available near-isogenic lines (NILs) were used to test the FSQ1, FSQ3, FSQ6, and FSQ7+FSQ8 target genomic regions of Cvi in the Ler background. The fructose sensitivity phenotypes of Ler/Cvi NIL (LCN)1-2 (FSQ1), LCN3-1 (FSQ3), LCN5-7 (FSQ6), and LCN5-14 (FSQ7+FSQ8) (22) (Fig. 1C), were investigated to confirm the QTL results from the RIL population. LCN1-2, LCN3-1, and LCN5-7 were less sensitive to fructose than the Ler parent, whereas LCN5-14 was more sensitive to fructose (Fig. 1D). These results fully confirmed the fructose-sensitivity phenotypes of FSQ1, FSQ3, FSQ6, and FSQ7+FSQ8 QTLs.

FSQ6 Defines a Fructose-Specific Signaling Pathway.

Comparison of the fructose-sensitivity QTLs with those published previously for glucose sensing (23) in the same population showed that FSQ3, FSQ4, FSQ6, and FSQ8 are fructose specific. Among these, the fructose sensitivity of FSQ6 had the highest LOD score and was confirmed using the NIL line LCN5-7. Fructose specificity of FSQ6 was confirmed by evaluating the sensitivities to glucose and sucrose in LCN5-7. Whereas LCN5-7 showed a fructose-insensitive phenotype (Fig. 2 A and E), its glucose sensitivity was identical to that of Ler (Fig. 2 B and F). LCN5-7 also was tested on sucrose; interestingly, LCN5-7 also showed a sucrose-insensitive phenotype (Fig. 2 C and G). Fructose released by sucrose hydrolysis probably inhibits Ler seedling development. Sugar-repressed seedling development is independent of osmotic conditions imposed by the high sugar levels, because growth on equal levels of sorbitol is not inhibited (Fig. 2 D and H). The results suggest that FSQ6 affects a fructose-specific signaling pathway.

Fig. 2.

FSQ6 is specifically sensitive to fructose. (A and E) Fructose sensitivity of Ler (A) and NIL line LCN5-7 (E). The seeds were grown on agar-solidified 1/2 MS containing 6.5% fructose, and incubated under continuous light for 7 d. The percentages of green seedlings of Ler and LCN5-7 were 2.2 ± 2.3% and 87.2 ± 7.2%, respectively. (B and F) Glucose sensitivity of Ler (B) and LCN5-7 (F). The seeds were grown as in A and E but on 6% glucose. The percentages of green seedlings of Ler and LCN5-7 were 5.3 ± 4.9% and 7.1 ± 8.7%, respectively. (C and G) Sucrose sensitivity of Ler (C) and LCN5-7 (G). The seeds were grown as in A and E but in the presence of 8% sucrose. The percentages of green seedlings of Ler and LCN5-7 were 12.6 ± 9.7% and 69.6 ± 11.4%, respectively. (D and H) Ler (D) and LCN5-7 (H) osmotic control experiments. The seeds were grown as in A and E but in the presence of 6.5% sorbitol. Ler and LCN5-7 seedlings showed 100% greening. Seedlings shown are representative of three independent experiments in each condition.

Seedling development of F₁ populations derived from reciprocal crosses between Ler and LCN5-7 grown on high fructose levels was tested. The reciprocal F₁ populations showed a fructose-insensitive phenotype similar to LCN5-7 (Fig. S1), indicating that the Cvi FSQ6 allele is dominant.

Fructose Signaling Is HXK1 Independent.

The different phenotypes of LCN5-7 on glucose and fructose imply the existence of a fructose-specific signaling pathway. The fructose-sensitivity phenotype of the glucose sensor protein HXK1 was investigated in the gin2-1/hxk1 mutant (5, 6) to investigate a possible relation between fructose signaling and HXK1-dependent glucose signaling. The gin2-1/hxk1 mutant displayed a wild-type phenotype when grown on fructose (Fig. 3 A and B), showing that fructose signaling represents a separate and HXK1- independent signaling pathway in Arabidopsis.

Fig. 3.

Analysis of the fructose-specific signaling pathway. (A–G) Fructose sensitivities of Ler (A), gin2-1 (B), Col (C), aba2-1 (D), abi4-1 (E), ctr1-1 (F), and ein2-1 (G). The seeds were grown under continuous light for 7 d on agar-solidified 1/2 MS containing 6.5% fructose. (H) CAB2 and PC gene expression in Ler, Ler expressing the Cvi FSQ6 gene (see Fig. 4A), and gin2-1 grown as in A–G in the presence of fructose, glucose, or sorbitol as indicated. Expression levels of CAB2 and PC were calculated relative to the reference gene PP2A (40). Values represent the average of three independent biological repeats. Bars indicate SE.

HXK1-mediated glucose signaling depends on and interacts with the ABA- and ethylene hormonal-signaling pathways. Therefore, the fructose-sensitivity phenotypes were investigated in mutants defective in ABA biosynthesis and signaling and in ethylene constitutive or defective signaling mutants. These mutants represent genes that are part of the sugar/ABA-signaling network that controls seedling development (10, 12, 14, 17). Remarkably, ABA-deficient mutants (Fig. 3D and Fig. S2B), ABA-signaling mutants (Fig. 3E and Fig. S2 C and D), and mutants demonstrating constitutive ethylene-signaling (ctr1) (Fig. 3F) showed a fructose-insensitive phenotype, whereas the ein2 ethylene-signaling mutants were more sensitive to fructose (Fig. 3G). These results show that fructose signaling depends on ABA and ethylene signaling, similar to reports for glucose signaling.

In seedlings, photosynthesis genes are repressed by sugars via the ABA–insensitve (ABI) signaling pathway. Sugar responsiveness of chlorophyll A/B binding 2 (CAB2) and plastocyanin (PC), was investigated in different genetic backgrounds to evaluate further the relation between glucose- and fructose-signaling pathways and the role of FSQ6 in sugar-signaling networks. High concentrations of glucose or fructose repress CAB2 and PC expression in the Ler accession relative to the sorbitol osmotic control (Fig. 3H), with glucose being more effective than fructose. In the hxk1/gin2 glucose-sensing mutant (Ler accession) CAB2 and PC are insensitive to glucose repression, confirming previous reports (6). In Ler expressing the Cvi FSQ6 gene (see below), glucose effectively represses CAB2 and PC. In the hxk1/gin2 mutant, fructose has no effect on the expression of CAB2 and PC genes as compared with the control treatment, but in the presence of fructose, the expression of both CAB2 and PC is about twofold higher in Ler expressing Cvi FSQ6 than in the Ler parent (Fig. 3H). Importantly, CAB2 and PC expression respond differentially to glucose and fructose in Ler, hxk1/gin2, and Ler containing Cvi FSQ6, supporting at the gene-expression level the finding that glucose and fructose are perceived through separate sensing systems.

FSQ6 Locus Encodes the NAC Domain Transcription Factor ANAC089.

A map-based approach was used to clone the FSQ6 gene. FSQ6 was narrowed to an 11.8-kb region on chromosome 5 in 5,023 F₂ plants with newly developed markers (Fig. S3 and Table S2). This region encodes four annotated genes, namely At5g22270 (unknown protein), At5g22280 (unknown protein), At5g22290 (Arabidopsis NAC domain containing protein 89, ANAC089), and At5g22300 (Nitrilase 4) (Fig. S3). These four genes were used in a genetic complementation assay, and only the transformation of the Cvi At5g22290 gene (ANAC089) into Ler showed fructose-insensitive transgenic seedlings (Fig. 4A). Importantly, Ler lines transformed with the Ler At5g22290 allele remained fructose sensitive (Fig. 4B).

Fig. 4.

A C-terminal truncation of the Cvi FSQ6/ANAC089 protein results in the fructose-insensitivity phenotype. (A–D) Fructose sensitivities of transgenic Ler containing the genomic Cvi FSQ6/ANAC089 sequence (A), the genomic Ler FSQ6/ANAC089 sequence (B), the CaMV35S Cvi FSQ6/ANAC089 cDNA construct (C), and the CaMV35S Ler FSQ6/ANAC089 cDNA (D). The seeds were grown on agar-solidified 1/2 MS with 6.5% fructose and incubated under continuous light for 7 d. (E) Amino acid sequences of Ler and Cvi FSQ6/ANAC089 proteins. Green underlined letters indicate the NAC domains, and red letters indicate the amino acid changes between the two alleles. (F) Point mutations introduced in the Ler FSQ6/ANAC089 cDNA sequence. These mutations were introduced in the Ler accession under the control of the Ler FSQ6/ANAC089 promoter. (G–K) Fructose sensitivities of the transgenic Ler lines were tested. (G) Control construct A, (H) mutation B, (I) mutation C, (J) mutation D, and (K) mutation E. The seeds were grown on agar-solidified 1/2 MS with 6.5% fructose and were incubated under continuous light for 7 d.

The function of Ler FSQ6/ANAC089 was investigated by studying the fsq6/anac089 mutant in the Ler background. Line GT19225 has a dissociation (Ds) insertion early in the coding region of FSQ6/ANAC089 (Fig. S4 A and B). Expression analysis indicated GT19225 is a knockout of FSQ6/ANAC089 (Fig. S4C). Interestingly, the fructose sensitivity of GT19225 was similar to that of the Ler parent (Fig. S4D). The GT19225 line was transformed with both Ler and Cvi alleles of FSQ6/ANAC089, and fructose sensitivity was tested in the transgenic lines. Transgenic GT19225 lines transformed with the Ler FSQ6/ANAC089 allele showed fructose sensitivity similar to the parent GT19225 line (Fig. S4 D and E), whereas transgenic GT19225 lines transformed with the Cvi FSQ6/ANAC089 allele showed fructose-insensitive phenotypes (Fig. S4F). These results indicate that the Cvi FSQ6/ANAC089 allele is a gain-of-function allele affecting fructose signaling.

A Premature Stop Codon in the Cvi FSQ6 Allele Is Responsible for the Gain-of-Function Phenotype.

The genomic and cDNA sequences of FSQ6/ANAC089 in Ler and Cvi were determined, and the sequences of the Columbia (Col) allele were retrieved from the Arabidopsis Information Resource (TAIR) site (www.arabidopsis.org). In the Ler, Cvi, and Col accessions FSQ6/ANAC089 encodes four exons and three introns. SNPs and insertion/deletion polymorphisms are present in both the promoter and coding regions. Transgenic Ler lines with the cauliflower mosaic virus (CaMV) 35S promoter-driven cDNA of Cvi FSQ6/ANAC089 also showed fructose-insensitive phenotypes (Fig. 4C), whereas CaMV 35S promoter-driven cDNA of Ler FSQ6/ANAC089 did not (Fig. 4D), showing that a polymorphism in the coding region conferred the fructose-insensitive phenotype.

The Ler FSQ6/ANAC089 allele encodes a 340-residue polypeptide that is identical to the Col-0 sequence (Fig. 4E) (www.arabidopsis.org). Comparison of the nucleotide sequences in the coding regions of the Ler and Cvi FSQ6/ANAC089 alleles uncovered 10 nucleotide changes, including three SNPs in exon 1, four in exon 2, one in exon 3, and one in exon 4 (Fig. S5A). SNPs in exon 1 and exon 3 produced amino acid changes: S15 (Ler) to A15 (Cvi) and S180 (Ler) to L180 (Cvi) (Fig. 4E). These amino acid changes were outside the NAC domain. In the Cvi allele a 1-bp deletion resulted in a premature stop codon in exon 3 (Fig. S5A), leading to a truncated protein of 224 amino acid residues (Fig. 4E).

The polymorphism in the Cvi allele resulting in fructose insensitivity was identified by introducing all seven combinations of the three mutations (S15A, S180L, and S225*) into the Ler cDNA and transferring the constructs into Ler under the control of the Ler FSQ6/ANAC089 promoter (Fig. 4F and Fig. S5B). Only transgenic lines with the premature stop mutation showed fructose-insensitive phenotypes (Fig. 4 G–K). The S15A and S180L mutations did not affect the fructose-sensitivity phenotype (Fig. S5 C–E). Thus, the gain-of-function Cvi FSQ6/ANAC089 allele is caused by a truncation of the protein.

Additional screening of 100 accessions from different geographical regions using a cleaved amplified polymorphic sequences (CAPS) marker that specifically detects the Cvi 1-bp deletion allelic difference (Table S2 and Fig. S5F) did not uncover further accessions with this deletion. Moreover, this 1-bp deletion haplotype was not found in the Arabidopsis Genome 1001 sequence collection (http://signal-genet.salk.edu/atg1001/3.0/gebrowser.php). These results indicate that the 1-bp deletion is rare in natural populations and might be specific for Cvi, perhaps because of the geographical isolation of Cape Verde Islands. The ecological significance of the allele therefore is unclear, and further studies including other plant species are needed to confirm its uniqueness.

Cvi FSQ6/ANAC089 Protein Is a Constitutively Active Nuclear Transcription Factor.

ANAC089 belongs to OsNAC8 subgroup of group I of NAC transcription factors (24). In the C-terminal part of the protein (amino acid residues 321–340) an alpha helix is predicted, suggesting the presence of a membrane-bound domain in ANAC089 (http://aramemnon.botanik.uni-koeln.de/) (25). The membrane-bound domain retains the transcription factor in the cytoplasm (26). Transgenic Ler plants with a CaMV35S promoter-driven GFP-fused Ler and Cvi FSQ6/ANAC089 constructs were generated. Transgenic lines expressing GFP::Ler FSQ6/ANAC089 fusion protein were as fructose sensitive as wild-type Ler, whereas expression of GFP::Cvi FSQ6/ANAC089 fusion protein made the transgenic plants insensitive to fructose, suggesting that the N-terminal GFP tag does not affect functional properties of the proteins (Fig. S6 A and B). The cellular locations of the GFP-fused Ler and Cvi FSQ6/ANAC089 proteins were studied using confocal laser scanning microscopy. The Ler FSQ6/ANAC089 was located in cytoplasm (Fig. 5 A–C), as previously reported (27). The premature stop codon in ANAC089 results in the gain-of-function mutation in the Cvi FSQ6/ANAC089 protein and the absence of the C-terminal 116 amino acid residues, including the membrane-anchoring domain. Importantly, the absence of the membrane-anchoring domain in the Cvi FSQ6/ANAC089 protein resulted in nuclear localization (Fig. 5 D and F).

Fig. 5.

The Cvi FSQ6/ANAC089 protein is a nuclear transcription factor. (A) Fluorescence image, (B) bright-field image, and (C) merged GFP fluorescence and bright-field image of root cell expressing the GFP::Ler FSQ6/ANAC089 fusion protein. (D) Fluorescence image, (E) bright-field image, and (F) merged GFP fluorescence and bright-field image of root cell expressing the GFP::Cvi FSQ6/ANAC089 fusion protein. (G and H) Fructose sensitivities of transgenic Ler overexpressing 1–204 amino acid residues of Cvi (G) and Ler (H) FSQ6/ANAC089. The seeds were grown on agar-solidified 1/2 MS with 6.5% fructose and were incubated under continuous light for 7 d.

The transcriptional activity of different FSQ6/ANAC089 proteins was studied in a yeast assay system. Expression of both Ler FSQ6/ANAC089-BD (DNA-binding domain) and Cvi FSQ6/ANAC089-BD fusion proteins in yeast showed strong activation of the reporter gene (Fig. S6C). Transactivation by the full-length Ler FSQ6/ANAC089-BD protein was in the same range as in the C-terminal truncated (residues 225–340) Ler protein, suggesting that the C-terminal 116 amino acids of the Ler FSQ6/ANAC089 protein did not affect the transcriptional potential (Fig. S6C). Interestingly, a further C-terminal deletion of 20 amino acids up to position 204 in the Cvi and Ler Δ225–340 proteins resulted in a complete loss of transactivation activity (Fig. S6C). Transgenic Ler lines that express amino acid residues 1–224 of either Cvi or Ler FSQ6/ANAC089 proteins with transcription-activating activity in yeast assay were fructose insensitive (Fig. 4 C and I), whereas transgenic Ler lines that overexpress amino acid residues 1–205 of either Cvi or Ler FSQ6/ANAC089 proteins without transcription-activating activity in yeast assay did not display fructose-insensitive phenotypes (Fig. 5 G and H). These finding suggest that amino acids 205–224 are essential for the gain-of-function phenotype of the Cvi FSQ6/ANAC089 protein and promote transcriptional activity. The transcription-activating function of FSQ6/ANAC089 probably is required for the fructose-insensitive phenotype.

Discussion

Glucose and fructose are major monosaccharides in plants and in many other organisms as well. In plants glucose and fructose are metabolized differently and have different metabolic effects. Therefore, sensing of glucose and fructose by separate systems seems important to monitor these sugars at the cellular level. Like glucose, fructose probably is an evolutionarily conserved signaling molecule in plants (9), fungi (28), and mammals (29). However, in plants convincing evidence for the signaling function of fructose is lacking. In this study, several QTLs were identified that allow seedling development on high fructose levels while retaining normal glucose sensitivity, providing convincing evidence for fructose-mediated signaling processes in Arabidopsis. Fructose sensing is independent of the well-studied HXK1-mediated glucose-sensing system. Remarkably, ABA-deficient mutants and ABA- and ethylene-signaling mutants show fructose-sensitivity phenotypes similar to those observed for HXK1-mediated glucose sensitivity (10, 12, 17, 30, 31). Therefore, both fructose and glucose sensing depend on and interact with the same downstream ABA- and ethylene-signaling pathways. In seedlings, this sugar/ABA-signaling pathway represses photosynthesis genes such as CAB2 and PC (6, 32, 33). Importantly, CAB2 and PC are differentially regulated by glucose and fructose in the hxk1/gin2 and in the Ler expressing Cvi ANAC089, providing molecular evidence for separate glucose- and fructose-sensing systems.

The fructose-specific FSQ6 QTL encodes the NAC transcription factor ANAC089. Genetic evidence shows that the Cvi FSQ6/ANAC089 is a gain-of-function allele that affects fructose signaling. Transformation of Ler with the CaMV35S promoter-driven Cvi FSQ6/ANAC089 cDNA resulted in fructose-insensitive seedling development, showing that polymorphisms in the amino acid-coding region are important for the phenotype. Detailed analysis showed that a premature stop codon in the Cvi allele is responsible for the gain-of-function phenotype.

FSQ6/ANAC089 encodes a membrane-bound NAC transcription factor (24, 25). The C-terminal region of FSQ6/ANAC089 harbors a helical membrane-association domain (25). Controlled proteolytic release of membrane-bound transcription factors is emerging as a versatile way of inducing rapid transcriptional changes in plants, e.g., in response to environmental changes (26). Recently, the functions of several membrane-bound NAC transcription factors were documented as well as their stimulus-dependent nuclear translocation (34–37). The Cvi FSQ6/ANAC089 protein lacks the membrane anchor because of the premature stop mutation. The Ler FSQ6/ANAC089 is located in the cytoplasm (27), precluding a nuclear function as a transcriptional activator. The absence of the membrane-bound domain in the Cvi FSQ6/ANAC089 protein allows its redistribution to the nucleus. In yeast, both the Ler and Cvi FSQ6/ANAC089 proteins function as potent transcriptional activators, and the transcriptional activation function depends on the presence of amino acid residues 205–224. These amino acids are essential for the fructose-insensitive phenotypes both in the Cvi and the truncated Ler FSQ6/ANAC089 proteins. Thus, the nuclear-located Cvi FSQ6/ANAC089 probably activates genes mediating the fructose-insensitive phenotype seedling.

The LCN5-7 NIL line and other Cvi FSQ6/ANAC089 genetic material generated in this study also showed sucrose insensitivity, similar to fructose insensitivity. Ler seedling development probably is inhibited by extensive fructose and glucose release from sucrose resulting from invertase and sucrose synthase activities. Alternatively, the fructosyl moiety of sucrose might be sensed directly by a fructose-specific sensor, but this possibility remains to be investigated.

In conclusion, an unidentified HXK1-independent fructose-signaling pathway has been discovered in Arabidopsis. This fructose-signaling pathway interacts with ABA- and ethylene-signaling pathways, as does the HXK1-dependent glucose-signaling pathway. The Cvi FSQ6 locus affects this fructose-specific signaling pathway and depends on the ABA-signaling pathway. FSQ6 encodes the ANAC089 NAC domain transcription factor. The Cvi FSQ6/ANAC089 allele is a gain-of-function allele in which a premature stop codon in the protein coding region of the Cvi allele is responsible for the gain-of-function phenotype. The premature stop results in a C-terminally truncated protein that lacks the membrane-attachment domain and relocates to the nucleus, where it probably functions as a transcriptional activator. Identification of target genes of Cvi FSQ6/ANAC089 may provide clues for a better understanding of this fructose-signaling pathway.

Materials and Methods

Plant Materials.

The genetically characterized RIL population from a cross between Ler and Cvi was used for QTL analysis (21). Four NILs, LCN1-2, LCN3-1, LCN5-7, and LCN5-14 (22), were used to confirm relevant fructose-sensing QTLs. The fsq6/anac089 knockout line, GT19225, was obtained from the Martienssen laboratory at Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (http://genetrap.cshl.org/). The insertion was confirmed by PCR using the primer specified in legend of Fig. S4. Other mutants were ordered from Arabidopsis Biological Resource Center (ABRC) or European Arabidopsis Stock Centre (NASC).

Analysis of Fructose Sensitivity.

Surface-sterilized seeds were stratified in 0.1% agarose for 4 d at 4 °C in the dark, followed by plating on 1/2 MS medium, pH 5.8, solidified with 0.8% agar. Different concentrations of sugars and sorbitol were added as indicated. Plates were incubated at 22 °C under continuous fluorescent light for 7 d, and the sugar sensitivities were determined by scoring the percentages of greening and uninhibited development of seedlings.

QTL Analysis and Fine Mapping of FSQ6.

QTL analysis was performed using MapQTL as described (23, 38). FSQ6 initially was mapped between T6G21 and K8E10 in 113 fully arrested F₂ individuals derived from the crossing between LCN5-7 and Ler. These two molecular markers were used to detect recombinants in 5,023 F₂ individuals. New markers were developed on the basis of the sequenced BAC clone MDW9 (Table S2) and were used to determine the genotypes of the recombinants. F₄ seeds from homozygous F₃ recombinant plants were used to test the fructose sensitivity and determine the FSQ6 genotypes. The CAPS marker detecting the 1-bp deletion difference between Ler and Cvi is shown in Table S2.

Constructs.

A 5,800-bp genomic fragment of Cvi and Ler FSQ6/ANAC089, including 2,563 bp upstream of the start codon and a 1,792-bp sequence downstream of the stop codon, was amplified by PCR using primers “Compl” (Table S3). The PCR product was digested with BamHI and PstI and cloned into pCAMBIA2301 (www.cambia.org). To generate the CaMV35S:: FSQ6/ANAC089 cDNA vector, a 1,022-bp cDNA fragment of FSQ6/ANAC089 was PCR amplified with the primers “cDNA” (Table S3). The PCR product was digested by BamH I/Pst I and cloned downstream of the CaMV35S promoter in pCAMBIA1301-OE (modified from pCAMBIA1301).

For GFP fusion constructs, the codon sequences of FSQ6/ANAC089 from cDNA of Cvi and Ler were amplified with primers “CDSgateway” (Table S3) and were recombined into pDONR-zeo vector using Gateway BP Clonase (Invitrogen). The DNA fragment containing the desirable sequence was recombined into pMDC45 (GFP tag at the N terminus) using Gateway LR Clonase (Invitrogen).

The CaMV35S promoter of pCAMBIA1301-OE was replaced with a 2,000-bp EcoRI/KpnI fragment of the Ler FSQ6/ANAC089 promoter. The Ler FSQ6/ANAC089 cDNA was amplified as before and was cloned into pMD18. Point mutations were introduced into this construct via gene splicing by overlap extension PCR. The fragments of mutated Ler FSQ6/ANAC089 cDNA were isolated by BamHI/PstI digestion and inserted into the modified expression vector to obtain the point mutant constructs.

Transactivation Activity Assay.

The transactivation activity assay was performed as described (39). To create pLer, pLerΔC225-340, pLerΔC205-340, pCvi, and pCviΔC205-224, the full-length coding sequence and the C terminus deletions of FSQ6/NAC089 based on the Ler and Cvi sequences were PCR amplified. The PCR products were digested with BamH I and Pst I and were cloned into pGBKT7 to fuse to the DNA-binding domain. All plasmids were transformed into yeast strain PJ69-4A. The overnight yeast colonies were diluted to an OD₆₀₀ of 0.5, serially diluted, and dropped on either -Trp SD medium or -Trp/-His/-Ade SD medium. The primer sequences are listed in Table S3.

Real-Time RT-PCR.

Total RNA was isolated using the Spectrum Plant Total RNA Kit from Sigma Aldrich and was DNase treated (Fermentas, http://www.fermentas.com) to remove genomic DNA. One-microgram aliquots were reverse transcribed using M-MLV (Promega, http://www.promega.com) according to the manufacturer's instructions. Five microliters of cDNA were used per real-time PCR using 10 μL of Power Cybergreen mix from Applied Biosystems (http://www.appliedbiosystems.com) and 2.5 μL of 5-μM diluted primers. Real-time PCR was performed using the Applied Biosystems 7900HT Fast Real-Time PCR system sequence detector. Expression levels of CAB2 and PC genes were calculated relative to protein phosphatae 2A (PP2A) (40) levels using the Q-gene method that takes into account the relative efficiencies of the different primer pairs (41, 42). The sequences of the primers used for the amplification of PP2A, CAB2, and PC are presented in Table S4.