Constitutively expressed ERF-VII transcription factors redundantly activate the core anaerobic response in Arabidopsis thaliana.

Plant adaptation to hypoxic conditions is mediated by the transcriptional activation of genes involved in the metabolic reprogramming of plant cells to cope with reduced oxygen availability. Recent studies indicated that members of the group VII of the Ethylene Responsive Transcription Factor (ERFs) family act as positive regulators of this molecular response. In the current study, the five ERF-VII transcription factors of Arabidopsis thaliana were compared to infer a hierarchy in their role with respect to the anaerobic response. When the activity of each transcription factor was tested on a set of hypoxia-responsive promoters, RAP2.2, RAP2.3 and RAP2.12 appeared to be the most powerful activators. RAP2.12 was further dissected in transactivation assays in Arabidopsis protoplasts to identify responsible regions for transcriptional activation. An ultimate C-terminal motif was identified as sufficient to drive gene transcription. Finally, using realtime RT-PCR in single and double mutants for the corresponding genes, we confirmed that RAP2.2 and RAP2.12 exert major control upon the anaerobic response.

Universal stress protein HRU1 mediates ROS homeostasis under anoxia.

Plant survival is greatly impaired when oxygen levels are limiting, such as during flooding or when anatomical constraints limit oxygen diffusion. Oxygen sensing in Arabidopsis thaliana is mediated by Ethylene Responsive Factor (ERF)-VII transcription factors, which control a core set of hypoxia- and anoxia-responsive genes responsible for metabolic acclimation to low-oxygen conditions. Anoxic conditions also induce genes related to reactive oxygen species (ROS). Whether the oxygen-sensing machinery coordinates ROS production under anoxia has remained unclear. Here we show that a low-oxygen-responsive universal stress protein (USP), Hypoxia Responsive Universal Stress Protein 1 (HRU1), is induced by RAP2.12 (Related to Apetala 2.12), an ERF-VII protein, and modulates ROS production in Arabidopsis. We found that HRU1 is strongly induced by submergence, but that this induction is abolished in plants lacking RAP2.12. Mutation of HRU1 through transfer DNA (T-DNA) insertion alters hydrogen peroxide production, and reduces tolerance to submergence and anoxia. Yeast two-hybrid and bimolecular fluorescence complementation (BiFC) analyses reveal that HRU1 interacts with proteins that induce ROS production, the GTPase ROP2 and the NADPH oxidase RbohD, pointing to the existence of a low-oxygen-specific mechanism for the modulation of ROS levels. We propose that HRU1 coordinates oxygen sensing with ROS signalling under anoxic conditions.

The stability and nuclear localization of the transcription factor RAP2.12 are dynamically regulated by oxygen concentration.

Plants often experience low oxygen conditions as the consequence of reduced oxygen availability in their environment or due to a high activity of respiratory metabolism. Recently, an oxygen sensing pathway was described in Arabidopsis thaliana which involves the migration of an ERF transcription factor (RAP2.12) from the plasma membrane to the nucleus upon hypoxia. Moreover, RAP2.12 protein level is controlled through an oxygen-dependent branch of the N-end rule pathway for proteasomal degradation. Inside the nucleus, RAP2.12 induces the expression of genes involved in the adaptation to reduced oxygen availability. In the present study, we describe the oxygen concentration and time-resolved characterization of RAP2.12 activity. A reduction of the oxygen availability to half the concentration in normal air is sufficient to trigger RAP2.12 relocalization into the nucleus, while nuclear accumulation correlates with the first induction of the molecular response to hypoxia. Nuclear presence of RAP2.12 may not only depend on relocalization of existing protein, but involves de novo synthesis of the transcription factor as well. After re-oxygenation of the tissue, degradation of RAP2.12 in the nucleus was observed within 3 h, concomitant with reduction in hypoxia responsive gene transcripts to normoxic levels.

Plant cysteine oxidases control the oxygen-dependent branch of the N-end-rule pathway.

In plant and animal cells, amino-terminal cysteine oxidation controls selective proteolysis via an oxygen-dependent branch of the N-end rule pathway. It remains unknown how the N-terminal cysteine is specifically oxidized. Here we identify plant cysteine oxidase (PCO) enzymes that oxidize the penultimate cysteine of ERF-VII transcription factors by using oxygen as a co-substrate, thereby controlling the lifetime of these proteins. Consequently, ERF-VII proteins are stabilized under hypoxia and activate the molecular response to low oxygen while the expression of anaerobic genes is repressed in air. Members of the PCO family are themselves targets of ERF-VII transcription factors, generating a feedback loop that adapts the stress response according to the extent of the hypoxic condition. Our results reveal that PCOs act as sensor proteins for oxygen in plants and provide an example of how proactive regulation of the N-end rule pathway balances stress response to optimal growth and development in plants.

Nighttime sugar starvation orchestrates gibberellin biosynthesis and plant growth in Arabidopsis.

A plant's eventual size depends on the integration of its genetic program with environmental cues, which vary on a daily basis. Both efficient carbon metabolism and the plant hormone gibberellin are required to guarantee optimal plant growth. Yet, little is known about the interplay between carbon metabolism and gibberellins that modulates plant growth. Here, we show that sugar starvation in Arabidopsis thaliana arising from inefficient starch metabolism at night strongly reduces the expression of ent-kaurene synthase, a key regulatory enzyme for gibberellin synthesis, the following day. Our results demonstrate that plants integrate the efficiency of photosynthesis over a period of days, which is transduced into a daily rate of gibberellin biosynthesis. This enables a plant to grow to a size that is compatible with its environment.

Low oxygen response mechanisms in green organisms.

Low oxygen stress often occurs during the life of green organisms, mostly due to the environmental conditions affecting oxygen availability. Both plants and algae respond to low oxygen by resetting their metabolism. The shift from mitochondrial respiration to fermentation is the hallmark of anaerobic metabolism in most organisms. This involves a modified carbohydrate metabolism coupled with glycolysis and fermentation. For a coordinated response to low oxygen, plants exploit various molecular mechanisms to sense when oxygen is either absent or in limited amounts. In Arabidopsis thaliana, a direct oxygen sensing system has recently been discovered, where a conserved N-terminal motif on some ethylene responsive factors (ERFs), targets the fate of the protein under normoxia/hypoxia. In Oryza sativa, this same group of ERFs drives physiological and anatomical modifications that vary in relation to the genotype studied. The microalga Chlamydomonas reinhardtii responses to low oxygen seem to have evolved independently of higher plants, posing questions on how the fermentative metabolism is modulated. In this review, we summarize the most recent findings related to these topics, highlighting promising developments for the future.

Misexpression of a chloroplast aspartyl protease leads to severe growth defects and alters carbohydrate metabolism in Arabidopsis.

The crucial role of carbohydrate in plant growth and morphogenesis is widely recognized. In this study, we describe the characterization of nana, a dwarf Arabidopsis (Arabidopsis thaliana) mutant impaired in carbohydrate metabolism. We show that the nana dwarf phenotype was accompanied by altered leaf morphology and a delayed flowering time. Our genetic and molecular data indicate that the mutation in nana is due to a transfer DNA insertion in the promoter region of a gene encoding a chloroplast-located aspartyl protease that alters its pattern of expression. Overexpression of the gene (oxNANA) phenocopies the mutation. Both nana and oxNANA display alterations in carbohydrate content, and the extent of these changes varies depending on growth light intensity. In particular, in low light, soluble sugar levels are lower and do not show the daily fluctuations observed in wild-type plants. Moreover, nana and oxNANA are defective in the expression of some genes implicated in sugar metabolism and photosynthetic light harvesting. Interestingly, some chloroplast-encoded genes as well as genes whose products seem to be involved in retrograde signaling appear to be down-regulated. These findings suggest that the NANA aspartic protease has an important regulatory function in chloroplasts that not only influences photosynthetic carbon metabolism but also plastid and nuclear gene expression.

Reactive oxygen species-driven transcription in Arabidopsis under oxygen deprivation.

Reactive oxygen species (ROS) play an important role as triggers of gene expression during biotic and abiotic stresses, among which is low oxygen (O(2)). Previous studies have shown that ROS regulation under low O(2) is driven by a RHO-like GTPase that allows tight control of hydrogen peroxide (H(2)O(2)) production. H(2)O(2) is thought to regulate the expression of heat shock proteins, in a mechanism that is common to both O(2) deprivation and to heat stress. In this work, we used publicly available Arabidopsis (Arabidopsis thaliana) microarray datasets related to ROS and O(2) deprivation to define transcriptome convergence pattern. Our results show that although Arabidopsis response to anoxic and hypoxic treatments share a common core of genes related to the anaerobic metabolism, they differ in terms of ROS-related gene response. We propose that H(2)O(2) production under O(2) deprivation is a trait present in a very early phase of anoxia, and that ROS are needed for the regulation of a set of genes belonging to the heat shock protein and ROS-mediated groups. This mechanism, likely not regulated via the N-end rule pathway for O(2) sensing, is probably mediated by a NADPH oxidase and it is involved in plant tolerance to the stress.

The extreme dwarf phenotype of the GA-sensitive mutant of sunflower, dwarf2, is generated by a deletion in the ent-kaurenoic acid oxidase1 (HaKAO1) gene sequence.

A dwarf mutant, dw arf 2 (dw2), was isolated from sunflower (Helianthus annuus). The most obvious alterations of dw2 plants were the lack of stem growth, reduced size of leaves, petioles and flower organs, retarded flower development. Pollen and ovules were produced but the filaments failed to extrude the anthers from the corolla. The dw2 phenotype was mainly because of reduced cell size. In dw2 leaves, the dark-green color was not so much due to higher pigment content, but was correlated with a changed leaf morphology. The mutant responded to the application of bioactive gibberellins (GAs). The levels of ent-7α-hydroxykaurenoic acid, GA(19), GA(20) and GA(1) in dw2 seedlings were severely decreased relative to those in its wild type (WT). ent-Kaurenoic acid was actively converted to ent-7α-hydroxykaurenoic acid in WT plants but quite poorly in dw2 plants. All together these data suggested that the dw2 mutation severely reduced the flux through the biosynthetic pathway leading to active GAs by hampering the conversion of ent-kaurenoic acid to GA(12). Two ent-kaurenoic acid oxidase (KAO) genes were identified. HaKAO1 was expressed everywhere in sunflower organs, while HaKAO2 was mainly expressed in roots. We demonstrated that a DNA deletion in HaKAO1 of dw2 generated aberrant mRNA-splicing, causing a premature stop codon in the amino acid sequence. In dw2 calli, Agrobacterium-mediated transfer of WT HaKAO1 cDNA restored the WT endogenous levels of GAs. In segregating BC(1) progeny, the deletion co-segregated with the dwarf phenotype. The deletion was generated near to a breakpoint of a more complex chromosome rearrangement.