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ORE1 balances leaf senescence against maintenance by antagonizing G2-like-mediated transcription
(2013)
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.
The NAC transcription factor ORE1 is a key regulator of senescence in Arabidopsis thaliana. Here, we demonstrate that senescence-induced and cell death-associated BIFUNCTIONAL NUCLEASE1 (BFN1) is a direct downstream target of ORE1, revealing a previously unknown regulatory cascade.Senescence is a highly regulated process that involves the action of a large number of transcription factors. The NAC transcription factor ORE1 (ANAC092) has recently been shown to play a critical role in positively controlling senescence in Arabidopsis thaliana; however, no direct target gene through which it exerts its molecular function has been identified previously. Here, we report that BIFUNCTIONAL NUCLEASE1 (BFN1), a well-known senescence-enhanced gene, is directly regulated by ORE1. We detected elevated expression of BFN1 already 2 h after induction of ORE1 in estradiol-inducible ORE1 overexpression lines and 6 h after transfection of Arabidopsis mesophyll cell protoplasts with a 35S:ORE1 construct. ORE1 and BFN1 expression patterns largely overlap, as shown by promoterreporter gene (GUS) fusions, while BFN1 expression in senescent leaves and the abscission zones of maturing flower organs was virtually absent in ore1 mutant background. In vitro binding site assays revealed a bipartite ORE1 binding site, similar to that of ORS1, a paralog of ORE1. A bipartite ORE1 binding site was identified in the BFN1 promoter; mutating the cis-element within the context of the full-length BFN1 promoter drastically reduced ORE1-mediated transactivation capacity in transiently transfected Arabidopsis mesophyll cell protoplasts. Furthermore, chromatin immunoprecipitation (ChIP) demonstrates in vivo binding of ORE1 to the BFN1 promoter. We also demonstrate binding of ORE1 in vivo to the promoters of two other senescence-associated genes, namely SAG29/SWEET15 and SINA1, supporting the central role of ORE1 during senescence.
Water deficit (drought stress) massively restricts plant growth and the yield of crops; reducing the deleterious effects of drought is therefore of high agricultural relevance. Drought triggers diverse cellular processes including the inhibition of photosynthesis, the accumulation of cell‐damaging reactive oxygen species and gene expression reprogramming, besides others. Transcription factors (TF) are central regulators of transcriptional reprogramming and expression of many TF genes is affected by drought, including members of the NAC family. Here, we identify the NAC factor JUNGBRUNNEN1 (JUB1) as a regulator of drought tolerance in tomato (Solanum lycopersicum). Expression of tomato JUB1 (SlJUB1) is enhanced by various abiotic stresses, including drought. Inhibiting SlJUB1 by virus‐induced gene silencing drastically lowers drought tolerance concomitant with an increase in ion leakage, an elevation of hydrogen peroxide (H2O2) levels and a decrease in the expression of various drought‐responsive genes. In contrast, overexpression of AtJUB1 from Arabidopsis thaliana increases drought tolerance in tomato, alongside with a higher relative leaf water content during drought and reduced H2O2 levels. AtJUB1 was previously shown to stimulate expression of DREB2A, a TF involved in drought responses, and of the DELLA genes GAI and RGL1. We show here that SlJUB1 similarly controls the expression of the tomato orthologs SlDREB1, SlDREB2 and SlDELLA. Furthermore, AtJUB1 directly binds to the promoters of SlDREB1, SlDREB2 and SlDELLA in tomato. Our study highlights JUB1 as a transcriptional regulator of drought tolerance and suggests considerable conservation of the abiotic stress‐related gene regulatory networks controlled by this NAC factor between Arabidopsis and tomato.
Water deficit (drought stress) massively restricts plant growth and the yield of crops; reducing the deleterious effects of drought is therefore of high agricultural relevance. Drought triggers diverse cellular processes including the inhibition of photosynthesis, the accumulation of cell-damaging reactive oxygen species and gene expression reprogramming, besides others. Transcription factors (TF) are central regulators of transcriptional reprogramming and expression of many TF genes is affected by drought, including members of the NAC family. Here, we identify the NAC factor JUNGBRUNNEN1 (JUB1) as a regulator of drought tolerance in tomato (Solanum lycopersicum). Expression of tomato JUB1 (SlJUB1) is enhanced by various abiotic stresses, including drought. Inhibiting SlJUB1 by virus-induced gene silencing drastically lowers drought tolerance concomitant with an increase in ion leakage, an elevation of hydrogen peroxide (H2O2) levels and a decrease in the expression of various drought-responsive genes. In contrast, overexpression of AtJUB1 from Arabidopsis thaliana increases drought tolerance in tomato, alongside with a higher relative leaf water content during drought and reduced H2O2 levels. AtJUB1 was previously shown to stimulate expression of DREB2A, a TF involved in drought responses, and of the DELLA genes GAI and RGL1. We show here that SlJUB1 similarly controls the expression of the tomato orthologs SlDREB1, SlDREB2 and SlDELLA. Furthermore, AtJUB1 directly binds to the promoters of SlDREB1, SlDREB2 and SlDELLA in tomato. Our study highlights JUB1 as a transcriptional regulator of drought tolerance and suggests considerable conservation of the abiotic stress-related gene regulatory networks controlled by this NAC factor between Arabidopsis and tomato.
Maturation of fleshy fruits such as tomato (Solanum lycopersicum) is subject to tight genetic control. Here we describe the development of a quantitative real-time PCR platform that allows accurate quantification of the expression level of approximately 1000 tomato transcription factors. In addition to utilizing this novel approach, we performed cDNA microarray analysis and metabolite profiling of primary and secondary metabolites using GC-MS and LC-MS, respectively. We applied these platforms to pericarp material harvested throughout fruit development, studying both wild-type Solanum lycopersicum cv. Ailsa Craig and the hp1 mutant. This mutant is functionally deficient in the tomato homologue of the negative regulator of the light signal transduction gene DDB1 from Arabidopsis, and is furthermore characterized by dramatically increased pigment and phenolic contents. We choose this particular mutant as it had previously been shown to have dramatic alterations in the content of several important fruit metabolites but relatively little impact on other ripening phenotypes. The combined dataset was mined in order to identify metabolites that were under the control of these transcription factors, and, where possible, the respective transcriptional regulation underlying this control. The results are discussed in terms of both programmed fruit ripening and development and the transcriptional and metabolic shifts that occur in parallel during these processes.
The control of gene expression by transcriptional regulators and other types of functionally relevant DNA transactions such as chromatin remodeling and replication underlie a vast spectrum of biological processes in all organisms. DNA transactions require the controlled interaction of proteins with DNA sequence motifs which are often located in nucleosome-depleted regions (NDRs) of the chromatin. Formaldehyde-assisted isolation of regulatory elements (FAIRE) has been established as an easy-to-implement method for the isolation of NDRs from a number of eukaryotic organisms, and it has been successfully employed for the discovery of new regulatory segments in genomic DNA from, for example, yeast, Drosophila, and humans. Until today, however, FAIRE has only rarely been employed in plant research and currently no detailed FAIRE protocol for plants has been published. Here, we provide a step-by-step FAIRE protocol for NDR discovery in Arabidopsis thaliana. We demonstrate that NDRs isolated from plant chromatin are readily amenable to quantitative polymerase chain reaction and next-generation sequencing. Only minor modification of the FAIRE protocol will be needed to adapt it to other plants, thus facilitating the global inventory of regulatory regions across species.