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. 2013 Mar 5;14(4):382–388. doi: 10.1038/embor.2013.24

ORE1 balances leaf senescence against maintenance by antagonizing G2-like-mediated transcription

Mamoona Rauf 1,2,*, Muhammad Arif 1,2,*, Hakan Dortay 1, Lilian P Matallana-Ramírez 1, Mark T Waters 3, Hong Gil Nam 4, Pyung-Ok Lim 4, Bernd Mueller-Roeber 1,2,a, Salma Balazadeh 1,2,b
PMCID: PMC3615665  PMID: 23459204

Abstract

Leaf senescence is a key physiological process in all plants. Its onset is tightly controlled by transcription factors, of which NAC factor ORE1 (ANAC092) is crucial in Arabidopsis thaliana. Enhanced expression of ORE1 triggers early senescence by controlling a downstream gene network that includes various senescence-associated genes. Here, we report that unexpectedly ORE1 interacts with the G2-like transcription factors GLK1 and GLK2, which are important for chloroplast development and maintenance, and thereby for leaf maintenance. ORE1 antagonizes GLK transcriptional activity, shifting the balance from chloroplast maintenance towards deterioration. Our finding identifies a new mechanism important for the control of senescence by ORE1.

Keywords: transcription factor, senescence, chloroplast, protein–protein interaction

Introduction

Leaf senescence is a developmentally controlled process that involves extensive reprogramming and modulation of gene expression to maximize plant fitness by remobilizing nutrients from deteriorating leaves to newly growing vegetative and reproductive organs. Early and noticeable features of leaf senescence are Rubisco and chlorophyll degradation, and a decline of photosynthetic activity owing to chloroplast dismantling [1, 2]. Transcription factors (TFs) have important roles in coordinating the gene regulatory networks that underlie the senescence process [3, 4]. One of the key senescence-control TF in A. thaliana is the NAC protein ORE1 (ANAC092; At5g39610). Overexpression of ORE1 in transgenic plants triggers early senescence, while its inhibition retards senescence [5, 6]. ORE1 exerts its regulatory function by controlling the expression of various known senescence-associated genes (SAGs) [5]. Expression of ORE1 itself is controlled by at least two molecular mechanisms, one that involves currently unknown upstream TFs that determine leaf age- and abiotic stress-dependent ORE1 transcriptional activity [5], and a second one that leads to ORE1 messenger RNA degradation by transacting miR164 [6]. Both processes contribute to establishing a coordinated expression of ORE1, which is low in young, but high in aging leaves.

In an aging leaf, the onset of senescence is counterbalanced by still vaguely defined chloroplast maintenance mechanisms. Key elements in this process are the Golden2-like TFs that act as nuclear regulators of chloroplast development and maintenance by coordinating the expression of genes-encoding proteins of the photosynthetic apparatus in various plant species, including A. thaliana, Zea mays and the moss Physcomitrella patens [7, 8]. In Arabidopsis, GLK genes exist as a pair of homologous genes, GLK1 and GLK2, and they have been shown to be functionally redundant such that only glk1/glk2 double mutants show a clear phenotype [7, 8].

Herein, we report the unexpected finding that ORE1 interacts with GLK TFs at the protein level. Elevated expression of ORE1 in the presence of GLK expression strongly reduces the capacity of GLK to activate its target genes. Our data suggest a model by which ORE1 counteracts the chloroplast maintenance function of GLKs, thereby adding a new mechanism to the finely tuned control network underlying senescence.

Results and Discussion

ORE1 interacts with GLK1 and GLK2 in yeast

To identify proteins interacting with ORE1, we performed a yeast two-hybrid screen using bait construct pLexPD–ORE1 and a prey complementary DNA library derived from Arabidopsis seedlings [9]. To increase screening specificity, we only included the region downstream of the highly conserved DNA-binding domain of ORE1. We identified GLK2 as an ORE1-interacting protein (Fig 1A).

Figure 1.

Figure 1

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ORE1 interacts with GLK1 and GLK2 in vitro and in vivo. (A) Yeast two-hybrid. First row, negative control with EVs, pLex-PD and pACT2-Gal4. Rows two, three and four: results from auto-activation tests of ORE1, GLK2 and GLK1 individually fused either with LexA-DBD or Gal4-AD, co-expressed with the respective EV. Auto-activation of ORE1 (not shown) is repressed on medium supplemented with 20 mM 3AT (SDIV+3AT). The last three rows show interaction tests of ORE1 with GLK2 or GLK1, where the growth test on selection media (SDIV+3AT) and X-gal assay (SDII+X-gal) indicate an interaction of ORE1 with GLK2 or GLK1. Note, that no growth test was conducted for EV combinations on SDII+X-gal plates. (B) Western blot of in vitro pulled-down HIS::GLK2 recombinant fusion protein detected by anti-HIS monoclonal antibody (representative of three repeats). (C) Bimolecular fluorescence complementation of YFP confirming interaction between ORE1 and GLK2 in Arabidopsis mesophyll cell protoplasts prepared from rosette leaves of 4-week-old plants. Rows 1–3, negative controls. Row 1: pSPYNE-nYFP and pSPYCE-cYFP empty vectors. Row 2: pSPYNE-ORE1::nYFP and pSPYCE-cYFP empty vector. Row 3: pSPYCE-GLK2::cYFP and pSPYNE-nYFP empty vector. Row 4: mesophyll cell protoplast co-transformed with the pSPYNE-ORE1::nYFP and pSPYCE-GLK2::cYFP vectors, showing YFP signal in the nucleus (arrows). Data are representatives of four repeats. aa, amino acids; AD, activation domain; DBD, DNA-binding domain; EV, empty vector; SD, selective dropout medium; X-gal, 5-bromo-4-chloro-indolyl-β-D-galactopyranoside; 3AT, 3-amino-1,2,4-triazole.

GLK1 and GLK2 share ∼50% amino-acid sequence identity. The similarity is higher, 90% and 79%, respectively, when the two conserved regions, GARP and GCT domains, are compared [10]. This high sequence similarity prompted us to test whether GLK1 would similarly interact with ORE1, which was indeed the case when a GLK1–GAL4 activation domain fusion was employed as prey (Fig 1A).

The NAC TF ORS1 (ORE1 SISTER1; ANAC059; At3g29035) is a paralog of ORE1 [11, 12]. Within their NAM domains, both TFs share an overall amino-acid identity of 94%. We tested by two-hybrid assay for interactions of ORS1 with GLK1 or GLK2, using pLexPD-ORS1 as bait, and GLK1–GAL4 or GLK2–GAL4 as prey clones. Neither GLK1 nor GLK2 interacted with ORS1 (supplementary Fig S1 online), indicating specificity of the interaction of ORE1 with the two GLKs. Furthermore, an in vitro pull-down assay revealed that immobilized GST–ORE1 fusion protein precipitated histidine-tagged GLK2 (HIS-GLK2) protein (Fig 1B; supplementary Fig S2 online) confirming the interaction of ORE1 and GLK2.

ORE1 and GLK2 interact in planta

We next used bimolecular fluorescence complementation to test for interaction of ORE1 and GLK2 in plant cells. Yellow fluorescence was exclusively detected in nuclei of mesophyll cell protoplasts co-expressing ORE1::nYFP and GLK2::cYFP (Fig 1C).

To test the effect of ORE1 on the subcellular localization of GLK2, we introduced 35S:GLK2::GFP plasmid into mesophyll cell protoplasts prepared from VGE:ORE1::HA plants, which express haemagglutinin (HA)-tagged ORE1 protein from a methoxyfenozide (MOF)-inducible promoter (see supplementary Fig S3 online for induction of ORE1 protein after MOF induction). Confocal imaging revealed green fluorescent protein (GFP) signal in the nucleus of both MOF- and mock-treated cells (Fig 2A–D). This result clearly indicates that GLK2 is a nuclear-localized TF and that ORE1 interacts with GLK2 in the nucleus.

Figure 2.

Figure 2

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Interaction of ORE1 and GLK2 in planta. Mesophyll cell protoplasts were prepared from rosette leaves of 4-week-old plants. (A) Non-induced, non-transformed VGE:ORE1::HA protoplast (first negative control). (B) VGE:ORE1::HA protoplast induced with MOF (80 μM) and transformed with 35S:GFP plasmid (second negative control). (C) Non-induced VGE:ORE1::HA protoplast transformed with 35S:GLK2::GFP plasmid; note GFP signal in the nucleus (arrows). (D) MOF-induced VGE:ORE1::HA protoplast transformed with 35S:GLK2::GFP plasmid; nuclear GFP signal is visible (arrows). The mat green signals in chloroplasts likely arise from fluorescence of undefined metabolites. (E) Co-immunoprecipitation of ORE1::HA/GLK2::GFP protein complex from VGE:ORE1::HA protoplasts transformed either with 35S:GLK2::GFP or 35S:GFP construct; ORE1::HA expression was induced by MOF treatment for 24 h. Anti-GFP and anti-HA monoclonal antibodies were used for immunoprecipitation and immunoblot analysis of interacting protein, respectively. Protein A/G agarose beads alone were used as negative control in a Co-IP assay. Data are representatives of three repeats. GFP, green fluorescent protein; HA, haemagglutinin; IB, immunoblotting; IP, immunoprecipitation; MOF, methoxyfenozide.

We further confirmed ORE1–GLK2 interaction by co-immunoprecipitation assay. To this end, VGE:ORE1::HA mesophyll cell protoplasts transformed either with 35S:GLK2::GFP or 35S:GFP constructs were induced with MOF for 24 h. As shown in Fig 2E, immunoblot analysis detected an interaction between ORE1-HA and GLK2-GFP proteins. Thus, our data provide strong evidence for an interaction of ORE1 and GLK2 in Arabidopsis cells.

Functional consequence of ORE1 and GLK interaction

ORE1 is a key regulator of leaf senescence; its overexpression accelerates senescence while loss-of-function ore1 mutants show delayed senescence and flowering [5, 6]. In contrast, GLK1 and GLK2 regulate chloroplast development. Under long-day condition, glk1 and glk2 mutants produced fewer vegetative leaves than wild type but flowered within a similar time frame [7]. However, whether GLKs affect the timing or progression of leaf senescence has not been investigated previously. To elucidate the potential biological relevance of the ORE1–GLK interaction for senescence, we first analysed GLK transgenic lines and observed a delay of senescence, compared with wild type, in 35S:GLK1 and 35S:GLK2 overexpressors (Fig 3A), while glk1 and glk2 single mutants did not show a striking alteration in their senescence behaviour (supplementary Fig S4 online). The pale-green phenotype of the glk1/2 double mutant did not seem to alter age-dependent senescence. Delayed leaf yellowing observed in GLK overexpression plants was further confirmed by measuring senescence-related physiological and molecular parameters. Total chlorophyll content was significantly higher in GLK1 and GLK2 overexpressors than in wild-type and glk1/2 double-mutant plants 8 weeks after sowing (Fig 3B). In accordance with this, ion leakage was less prominent in GLK overexpressors than wild-type and mutant plants (Fig 3C). Expression of early- and late-senescence marker genes, SAG13 and SAG12, was downregulated, whereas expression of photosynthesis-related genes (CAB2, LHCB2.1, CAB4 and LHCB4.1) was enhanced in GLK overexpression plants compared with wild type, in accordance with their known molecular functions and the delayed senescence phenotype (Fig 3D,E).

Figure 3.

Figure 3

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Characterization of transgenic plants. (A) Phenotype of WT, 35S:ORE1/35S:GLK2 double overexpressor, 35S:ORE1, 35S:GLK1, 35S:GLK2, glk1/2 double-mutant and ore1-1 mutant plants at 29 and 54 DAS. (B) Chlorophyll content and (C) ion leakage measured 4, 6 and 8 weeks after sowing. Bars represent the mean±s.d., n=3. (D) Transcript abundance of SAG13 and SAG12 genes measured by qRT–PCR 6 and 7 weeks after sowing. (E) Transcript abundance of CAB2, LHCB2.1, CAB4 and LHCB4.1 measured 7 weeks after sowing. Heat maps indicate log2 fold-change expression ratio of selected genes in all lines compared with WT. DAS, days after sowing; qRT–PCR, quantitative reverse-transcriptase PCR; WT, wild type.

We next generated ORE1/GLK2 double overexpressors by crossing 35S:ORE1 and 35S:GLK2 lines (supplementary Fig S4D online). Under long-day condition, 35S:ORE1/35S:GLK2 plants bolted considerably earlier (5 days) and showed earlier senescence than wild-type controls (Fig 3A; supplementary Fig S4E online). Total chlorophyll was significantly lower, whereas ion leakage was higher in leaves of 35S:ORE1/35S:GLK2 lines compared with wild type (Fig 3B,C; supplementary Fig S4F,G online). Expression of SAG12 and SAG13 was higher in double overexpressors, while expression of photosynthesis-related genes was significantly reduced (Fig 3D,E). Taken together, these data demonstrate accelerated bolting and leaf senescence in transgenic plants overexpressing both ORE1 and GLK2, resembling the typical phenotype of ORE1 overexpression plants [5, 6]. This observation indicates that the interaction of ORE1 with GLKs antagonizes GLK function suggesting a negative impact of ORE1 on photosynthetic apparatus maintenance and chloroplast integrity.

Effect of ORE1/GLK2 interaction on target genes

The fact that 35S:ORE1/35S:GLK2 double overexpressors highly resemble 35S:ORE1 plants with respect to bolting and senescence suggests a possible negative influence of ORE1/GLK2 heteromerisation on the expression of GLK target genes. GLK target genes were previously reported [8]. We tested the expression of ORE1 and GLK target genes by quantitative reverse-transcriptase PCR in 35S:GLK1, 35S:GLK2, glk1/2, 35S:ORE1/35S:GLK2, 35S:ORE1, ore1-1 and wild-type plants. Expression of the ORE1 targets RNS3, SINA1, BFN1, SAG29 and VNI2 was higher in 35S:ORE1/35S:GLK2 than wild-type plants, similar to 35S:ORE1 plants. These genes did not show any significant difference in the expression in glk1/2, 35S:GLK1 and 35S:GLK2 plants compared with wild-type plants, except VNI2 that showed a slight downregulation in both overexpression lines (Fig 4A). The lack of repression of ORE1 target genes in the GLK overexpressors is fully consistent with the fact that ORE1 expression itself is not largely affected at the developmental stage analysed (Fig 4A, middle).

Figure 4.

Figure 4

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Effect of ORE1 and GLK2 on transcriptional activation of downstream target genes. (A) Expression of ORE1 and its target genes RNS3, SINA1, BFN1, SAG29 and VNI2, and expression of GLKs and GLK2 target genes LHCB3, LHCB2.2, LHCB4.2, LHCB2.4, MRU1 and Hydrolase in 7-week-old wild-type (WT), 35S:ORE1/35S:GLK2, 35S:ORE1, 35S:GLK1, 35S:GLK2, glk1/2 and ore1-1 plants (leaf number 11). Heat map indicates expression level (log2 scale) relative to WT. (B) Effect of GLK2 on ORE1 transcriptional activity towards ORE1 target genes in Col-0 protoplasts. Note that GLK2 did not affect the transcriptional activation activity of ORE1 towards its target genes. (C) Effect of ORE1 on GLK2 transcriptional activity towards GLK target genes in Col-0 protoplasts. Note that ORE1 strongly reduces the transcriptional activation capacity of GLK2 towards its photosynthesis-related target genes, while it did not affect expression of its non-photosynthetic targets (MRU1 and Hydrolase). Protoplasts were prepared from rosette leaves of 4-week-old plants. 35S:GLK2 and 35S:ORE1 effector constructs were omitted in control experiments. Luciferase signal was determined 24 h after transfection. Error bars±s.d. are given (n=3). WT, wild type.

On the other hand, expression of all six tested GLK1 and GLK2 target genes was highly upregulated in 35S:GLK1 and 35S:GLK2 plants, and strongly downregulated in the glk1/2 double mutant compared with wild type, except for MRU1 that showed no change in expression in 35S:GLK2 and glk1/2 plants. Notably, none of the GLK targets was induced in the 35S:ORE1/35S:GLK2 double overexpressor even in the presence of high transcript abundance of GLK2. This expression pattern was similar to the expression in 35S:ORE1 plants, which showed either no change in expression for some of the genes, such as LHCB4.2, MRU1 and LHCB3, or downregulation of others, such as Hydrolase, LHCB2.2 and LHCB2.4. Interestingly, expression of these genes was upregulated in the ore1-1 mutant, except for MRU1, LHCB3 and Hydrolase, which showed unaltered expression (Fig 4A). Collectively, our data demonstrate that ORE1 attenuates the transcriptional activity of GLK2.

Transactivation assays

To further analyse the effect of ORE1/GLK2 heteromers on the expression of downstream target genes, we performed luciferase-based transactivation assays using Arabidopsis mesophyll cell protoplasts. To this end, we fused the promoters of ORE1 target genes to the firefly luciferase reporter. ORE1 significantly transactivated all target gene promoters in protoplasts co-transfected with a 35S:ORE1 effector plasmid and the reporter constructs (proORE1-fLUC, proRNS3-fLUC, proSINA1-fLUC, proBFN1-fLUC, proSAG29-fLUC and proVNI2-fLUC) (Fig 4B). ORE1-mediated transcriptional activation was not altered when 35S:ORE1 was co-transfected with 35S:GLK2 effector plasmid (Fig 4B). Thus, the results obtained from luciferase-based transactivation assays further support the conclusion that ORE1/GLK2 heteromerisation does not affect the expressional regulation of ORE1 target genes.

Next, we performed transactivation assays with reporter constructs harbouring GLK target gene promoters. As shown in Fig 4C, GLK2 significantly transactivated all its target promoters (proLHCB3-fLUC, proLHCB2.2-fLUC, proLHCB4.2-fLUC, proLHCB2.4-fLUC, proMRU1-fLUC and proHydrolase-fLUC). This transcriptional activation by GLK2 significantly decreased for all photosynthetic target genes when the 35S:ORE1 effector plasmid was transfected in addition to 35S:GLK2, while the expression of non-photosynthetic GLK targets, that is, MRU1 and Hydrolase, was not affected by ORE1 (Fig 4C). This observation clearly indicates that ORE1 has an inhibitory effect on the transcriptional activity of GLK2.

Model of GLK-ORE1 action

On the basis of the experimental data obtained herein, we propose a model that can explain how ORE1 and GLKs coordinate the age-dependent alteration of the two antagonistic activities: chloroplast maintenance and initiation/progression of leaf senescence (Fig 5). During early leaf development, ORE1 transcript level is low owing to transcriptional regulation [3, 5] and post-transcriptional control by miR164 [6], whereas GLK1 and GLK2 expression is high, which transcriptionally activates genes of photosynthetic proteins involved in chloroplast development and maintenance [7, 8]. At later stages of leaf development, accumulation of ORE1 and its interaction with GLKs render inactive heterodimers unable to activate photosynthetic GLK target genes, while activation of ORE1 targets is favoured. Thus, the senescence-triggering role of ORE1 is dominant over the photosynthetic apparatus maintenance role of GLKs. Thus, A. thaliana, and possibly other plants, seem to use an intriguing and multilevel control mechanism to balance chloroplast maintenance against dismantling.

Figure 5.

Figure 5

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Model of GLK–ORE1 action. Expression of GLK target genes (green) is strongly hindered in the presence of ORE1 protein, which leads to an age-dependent decline in the expression of photosynthesis-related genes (GLK targets), while expression of ORE1 target genes (yellow) is stimulated leading to the onset and subsequent progression of senescence. ORE1 transcript abundance is regulated by developmental and stress-related parameters through currently unknown upstream TFs, as well as by miR164, which is under negative control of ethylene signalling involving EIN2 protein. TFs upstream of GLKs are currently unknown as well. TF, transcription factor.

Methods

Detailed experimental procedures are described in the supplementary information online.

Plant growth conditions. A. thaliana ecotype Col-0 was used. Seedlings were grown in soil (Einheitserde GS90; Gebrüder Patzer) in a climate-controlled chamber with 16-h day length provided by fluorescent light at 100 μmol m−2 s−1 and a day/night temperature of 22/16 °C and a relative humidity of 60/75%.

Molecular and biochemical methods. Total RNA extraction, synthesis of cDNA and quantitative reverse-transcriptase PCR were performed as described [4, 13]. cDNA quality was tested using GAPDH primers; genomic DNA contamination was checked using primers annealing to an intron of gene AGL68. ACTIN2 served as an internal control for normalization of expression data. Yeast two-hybrid screens were performed using yeast strain L40ccαU as reported [14]. The growth tests were performed on selective dropout nutrient medium SDIV lacking Leu, Trp, His and Ura; SDII medium lacking Trp and Leu was used as control. Bimolecular fluorescence complementation was performed as described [15] using Arabidopsis mesophyll cell protoplasts. DNA cloning was done using standard procedures [16]. Details on the construction of the plasmids used here are given in the supplementary information online. Primer sequences are given in supplementary Table S1 online. Expression of GST1–ORE1 and HIS–GLK2 fusion proteins in Escherichia coli strain Rosetta (DE3) pLysS (Merck), protein purification and in vitro pull-down assays for immunological detection of purified GST–ORE1 and HIS–GLK2 fusion proteins were performed as described [17, 18]. For co-immunoprecipitation of interacting protein complexes, protein was extracted from mesophyll cell protoplasts obtained from the VGE:ORE1::HA line transfected with the 35S:GFP or the 35S:GLK2::GFP construct. Anti-GFP antibody was used for immunoprecipitation. A mouse monoclonal anti-HA antibody (Santa Cruz Biotechnology) and IRDye800CW-conjugated goat anti-mouse secondary antibody (LI-COR, Bad Homburg, Germany) were used for immune blotting.

Dual-luciferase transactivation assay. Arabidopsis mesophyll cell protoplasts were prepared from rosette leaves of 4-week-old plants as described [19]. 35S:ORE1 and/or 35S:GLK2 were used as effector constructs. Reporter constructs of ORE1 target genes were individually transformed in the absence of 35S:ORE1 effector, or together with 35S:ORE1 and/or 35S:GLK2. Likewise, reporter constructs of GLK2 target genes were individually transformed in the absence of 35S:GLK2 effector, or together with 35S:GLK2 and/or 35S:ORE1. The 35S:RLuc vector [20] was used for normalization against transformation efficiency. Firefly and Renilla luciferase (RLuc) activity were assayed using the Dual Luciferase Reporter Assay System (Promega) as described [21, 22].

AGI codes. ORE1 (At5g39610); ORS1 (At3g29035); RNS3 (At1g26820); SINA1 (At3g13672); BFN1 (At1g11190); SAG29 (At5g13170); VNI2 (At5g13180); GLK1 (At2g20570); GLK2 (At5g44190); Hydrolase (At1g80280); LHCB2.2 (At2g05070); LHCB4.2 (At3g08940); LHCB2.4 (At3g27690); MRU1 (At5g35490); LHCB3 (At5g54270); SAG13 (At2g29350); SAG12 (At5g45890); CAB2 (At1g29920); CAB4 (At3g47470); LHCB2.1 (At2g05100); LHCB4.1 (At5g01530); AGL68 (At5g65080); ACTIN2 (At3g18780); and GAPDH (At3g26650).

Supplementary information is available at EMBO reports online (http://www.emboreports.org).

Supplementary Material

Supplementary Information
embor201324s1.pdf (429.4KB, pdf)
Review Process File
embor201324s2.pdf (223.2KB, pdf)

Acknowledgments

We thank the Deutsche Forschungsgemeinschaft (FOR 948; MU 1199/14-1) for funding. We thank Prof Dr Jane Langdale (University of Oxford) for providing seeds of GLK-modified plants, and Dr Eugenia Maximova (MPI-MP, Potsdam) for help with microscopy.

Author contributions: M.R. and M.A. performed the experiments, analysed the data and compiled the figures. H.D., H.G.N., L.P.M.-R., M.T.W. and P.-O.L. contributed critical analytical tools. B.M.-R. and S.B. designed the research. M.R., B.M.-R. and S.B. wrote the paper.

Footnotes

The authors declare that they have no conflict of interest.

References

  1. Nooden LD, Guiamet JJ, John I (1997) Senescence mechanisms. Physiol Plant 101: 746–753 [Google Scholar]
  2. Hörtensteiner S, Feller U (2002) Nitrogen metabolism and remobilization during senescence. J Exp Bot 53: 927–937 [DOI] [PubMed] [Google Scholar]
  3. Buchanan-Wollaston V et al. (2005) Comparative transcriptome analysis reveals significant differences in gene expression and signalling pathways between developmental and dark/starvation-induced senescence in Arabidopsis. Plant J 42: 567–585 [DOI] [PubMed] [Google Scholar]
  4. Balazadeh S, Riaño-Pachón DM, Mueller-Roeber B (2008) Transcription factors regulating leaf senescence in Arabidopsis thaliana. Plant Biol 1: 63–75 [DOI] [PubMed] [Google Scholar]
  5. Balazadeh S, Siddiqui H, Allu AD, Matallana-Ramirez LP, Caldana C, Mehrnia M, Zanor MI, Köhler B, Mueller-Roeber B (2010) A gene regulatory network controlled by the NAC transcription factor ANAC092/AtNAC2/ORE1 during salt-promoted senescence. Plant J 62: 250–264 [DOI] [PubMed] [Google Scholar]
  6. Kim JH, Woo HR, Kim J, Lim PO, Lee IC, Choi SH, Hwang D, Nam HG (2009) Trifurcate feed-forward regulation of age-dependent cell death involving miR164 in Arabidopsis. Science 323: 1053–1057 [DOI] [PubMed] [Google Scholar]
  7. Waters MT, Moylan EC, Langdale JA (2008) GLK transcription factors regulate chloroplast development in a cell-autonomous manner. Plant J 56: 432–444 [DOI] [PubMed] [Google Scholar]
  8. Waters MT, Wang P, Korkaric M, Capper RG, Saunders NJ, Langdale JA (2009) GLK transcription factors coordinate expression of the photosynthetic apparatus in Arabidopsis. Plant Cell 21: 1109–1128 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bürkle L, Meyer S, Dortay H, Lehrach H, Heyl A (2005) In vitro recombination cloning of entire cDNA libraries in Arabidopsis thaliana and its application to the yeast two-hybrid system. Funct Integr Genom 5: 175–183 [DOI] [PubMed] [Google Scholar]
  10. Bravo-Garcia A, Yasumura Y, Langdale JA (2009) Specialization of the Golden2-like regulatory pathway during land plant evolution. New Phytologist 183: 133–141 [DOI] [PubMed] [Google Scholar]
  11. Ooka H et al. (2003) Comprehensive analysis of NAC family in Oryza sativa and Arabidopsis thaliana. DNA Res 10: 239–247 [DOI] [PubMed] [Google Scholar]
  12. Balazadeh S, Kwasniewski M, Caldana C, Mehrnia M, Zanor MI, Xue GP, Mueller-Roeber B (2011) ORS1, an H2O2-responsive NAC transcription factor, controls senescence in Arabidopsis thaliana. Mol Plant 4: 346–360 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Caldana C, Scheible WR, Mueller-Roeber B, Ruzicic S (2007) A quantitative RT-PCR platform for high-throughput expression profiling of 2500 rice transcription factors. Plant Methods 3: 7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dortay H, Mehnert N, Burkle L, Schmulling T, Heyl A (2006) Analysis of protein interactions within the cytokinin-signaling pathway of Arabidopsis thaliana. FEBS J 273: 4631–4644 [DOI] [PubMed] [Google Scholar]
  15. Walter M et al. (2004) Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J 40: 428–438 [DOI] [PubMed] [Google Scholar]
  16. Skirycz A et al. (2006) DOF transcription factor AtDof1.1 (OBP2) is part of a regulatory network controlling glucosinolate biosynthesis in Arabidopsis. Plant J 47: 10–24 [DOI] [PubMed] [Google Scholar]
  17. Dortay H, Mehnert N, Burkle L, Schmulling T, Heyl A (2006) Analysis of protein interactions within the cytokinin-signaling pathway of Arabidopsis thaliana. FEBS J 273: 4631–4644 [DOI] [PubMed] [Google Scholar]
  18. Dortay H, Mueller-Roeber B (2010) A highly efficient pipeline for protein expression in Leishmania tarentolae using infrared fluorescence protein as marker. Microb Cell Fact 9: 29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sheen J (2002) A transient expression assay using Arabidopsis mesophyll protoplasts. http://genetics.mgh.harvard.edu/sheenweb/
  20. Licausi F, Weits DA, Pant BD, Scheible WR, Geigenberger P, van Dongen JT (2011) Hypoxia responsive gene expression is mediated by various subsets of transcription factors and miRNAs that are determined by the actual oxygen availability. New Phytol 190: 442–456 [DOI] [PubMed] [Google Scholar]
  21. Yoo SD, Cho YH, Sheen J (2007) Arabidopsis mesophyll protoplasts: A versatile cell system for transient gene expression analysis. Nat Protoc 2: 1565–1572 [DOI] [PubMed] [Google Scholar]
  22. Wu A et al. (2012) JUNGBRUNNEN1, a reactive oxygen species-responsive NAC transcription factor, regulates longevity in Arabidopsis. Plant Cell 24: 482–506 [DOI] [PMC free article] [PubMed] [Google Scholar]

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Supplementary Materials

Supplementary Information
embor201324s1.pdf (429.4KB, pdf)
Review Process File
embor201324s2.pdf (223.2KB, pdf)

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