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1.  Tomato R2R3-MYB Proteins SlANT1 and SlAN2: Same Protein Activity, Different Roles 
PLoS ONE  2015;10(8):e0136365.
Anthocyanins are water-soluble polyphenolic compounds with a high nutraceutical value. Despite the fact that cultivated tomato varieties do not accumulate anthocyanins in the fruit, the biosynthetic pathway can be activated in the vegetative organs by several environmental stimuli. Little is known about the molecular mechanisms regulating anthocyanin synthesis in tomato. Here, we carried out a molecular and functional characterization of two genes, SlAN2 and SlANT1, encoding two R2R3-MYB transcription factors. We show that both can induce ectopic anthocyanin synthesis in transgenic tomato lines, including the fruit. However, only SlAN2 acts as a positive regulator of anthocyanin synthesis in vegetative tissues under high light or low temperature conditions.
PMCID: PMC4556288  PMID: 26308527
2.  A Trihelix DNA Binding Protein Counterbalances Hypoxia-Responsive Transcriptional Activation in Arabidopsis 
PLoS Biology  2014;12(9):e1001950.
DNA binding protein controls plant transcription when oxygen is at a premium - During hypoxia, the plant transcription factor HRA1 counterbalances the upregulation of anaerobic gene expression triggered by a stabilized plant ethylene responsive factor.
Transcriptional activation in response to hypoxia in plants is orchestrated by ethylene-responsive factor group VII (ERF-VII) transcription factors, which are stable during hypoxia but destabilized during normoxia through their targeting to the N-end rule pathway of selective proteolysis. Whereas the conditionally expressed ERF-VII genes enable effective flooding survival strategies in rice, the constitutive accumulation of N-end-rule–insensitive versions of the Arabidopsis thaliana ERF-VII factor RAP2.12 is maladaptive. This suggests that transcriptional activation under hypoxia that leads to anaerobic metabolism may need to be fine-tuned. However, it is presently unknown whether a counterbalance of RAP2.12 exists. Genome-wide transcriptome analyses identified an uncharacterized trihelix transcription factor gene, which we named HYPOXIA RESPONSE ATTENUATOR1 (HRA1), as highly up-regulated by hypoxia. HRA1 counteracts the induction of core low oxygen-responsive genes and transcriptional activation of hypoxia-responsive promoters by RAP2.12. By yeast-two-hybrid assays and chromatin immunoprecipitation we demonstrated that HRA1 interacts with the RAP2.12 protein but with only a few genomic DNA regions from hypoxia-regulated genes, indicating that HRA1 modulates RAP2.12 through protein–protein interaction. Comparison of the low oxygen response of tissues characterized by different levels of metabolic hypoxia (i.e., the shoot apical zone versus mature rosette leaves) revealed that the antagonistic interplay between RAP2.12 and HRA1 enables a flexible response to fluctuating hypoxia and is of importance to stress survival. In Arabidopsis, an effective low oxygen-sensing response requires RAP2.12 stabilization followed by HRA1 induction to modulate the extent of the anaerobic response by negative feedback regulation of RAP2.12. This mechanism is crucial for plant survival under suboptimal oxygenation conditions. The discovery of the feedback loop regulating the oxygen-sensing mechanism in plants opens new perspectives for breeding flood-resistant crops.
Author Summary
Respiratory metabolism in land plants requires oxygen availability to be able to generate ATP, which is essential for biosynthetic processes. Cellular hypoxia can be triggered as a consequence of environmental events (mainly floods), anatomical constraints (low tissue permeability to gases), or elevated cellular respiration, and it is unfavorable to growth due to the resultant decline in ATP. The adaptation of plants to fluctuating oxygen levels inside tissues requires the dynamic regulation of mechanisms that ensure cell viability and ultimately organism survival, but only a few molecular components of this homeostatic network are known. Direct hypoxia-sensing entails the posttranslational stabilization of a subgroup of plant ethylene-responsive factor (ERF) transcription factors, which coordinate the expression of hypoxia-inducible genes. Turnover of these ERFs is determined by an oxygen-dependent pathway of proteasomal degradation. Here, we demonstrate that the hypoxia-inducible transcription factor gene HRA1 is transcriptionally activated upon ERF-VII RAP2.12 stabilization and encodes a trihelix DNA binding protein that functionally interacts with RAP2.12 to curtail its activity. In addition to its negative regulation of RAP2.12, HRA1 negatively regulates activation of its own promoter. This RAP2.12-HRA1 control unit allows plants to modulate the extent of the response to hypoxia, including anaerobic enzyme production, to levels that improve endurance of the stress. Our results emphasize the importance of a strategy that can counterbalance energy-inefficient survival responses.
PMCID: PMC4165759  PMID: 25226037
3.  Plant responses to flooding 
PMCID: PMC4033237  PMID: 24904621
anoxia; flooding; hypoxia; low oxygen; submergence; waterlogging
4.  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.
PMCID: PMC3634410  PMID: 23446868
anoxia; Arabidopsis thaliana; Chlamydomonas reinhardtii; hypoxia; low oxygen; N-end rule; Oryza sativa
5.  How plants sense low oxygen 
Plant Signaling & Behavior  2012;7(7):813-816.
The recent identification of the oxygen-sensing mechanism in plants is a breakthrough in plant physiology. The presence of a conserved N-terminal motif on some ethylene responsive factors (ERFs), targets the protein for post-translational modifications finally leading to degradation under normoxia and thus providing a mechanism for sensing the presence of oxygen. The stabilization of the N-terminus under low oxygen activates these ERFs, which regulate low oxygen core genes that enable plants to tolerate abiotic stress such as flooding. Additional mechanisms that signal low-oxygen probably also exist, and the production of reactive oxygen species (ROS) has been observed under low oxygen, suggesting that ROS might be part of the network involved in plant acclimation. Here, we review the most recent findings related to oxygen sensing.
PMCID: PMC3583971  PMID: 22751330
ROS; anoxia; hypoxia; low oxygen
6.  Tomato fruits: a good target for iodine biofortification 
Iodine is a trace element that is fundamental for human health: its deficiency affects about two billion people worldwide. Fruits and vegetables are usually poor sources of iodine; however, plants can accumulate iodine if it is either present or exogenously administered to the soil. The biofortification of crops with iodine has therefore been proposed as a strategy for improving human nutrition. A greenhouse pot experiment was carried out to evaluate the possibility of biofortifying tomato fruits with iodine. Increasing concentrations of iodine supplied as KI or KIO3 were administered to plants as root treatments and the iodine accumulation in fruits was measured. The influences of the soil organic matter content or the nitrate level in the nutritive solution were analyzed. Finally, yield and qualitative properties of the biofortified tomatoes were considered, as well as the possible influence of fruit storage and processing on the iodine content. Results showed that the use of both the iodized salts induced a significant increase in the fruit’s iodine content in doses that did not affect plant growth and development. The final levels ranged from a few mg up to 10 mg iodine kg - 1 fruit fresh weight and are more than adequate for a biofortification program, since 150 μg iodine per day is the recommended dietary allowance for adults. In general, the iodine treatments scarcely affected fruit appearance and quality, even with the highest concentrations applied. In contrast, the use of KI in plants fertilized with low doses of nitrate induced moderate phytotoxicity symptoms. Organic matter-rich soils improved the plant’s health and production, with only mild reductions in iodine stored in the fruits. Finally, a short period of storage at room temperature or a 30-min boiling treatment did not reduce the iodine content in the fruits, if the peel was maintained. All these results suggest that tomato is a particularly suitable crop for iodine biofortification programs.
PMCID: PMC3694224  PMID: 23818889
biofortification; iodine; iodine deficiency; potassium iodate; potassium iodide; Solanum lycopersicum L.; tomato
7.  Distinct Mechanisms Regulating Gene Expression Coexist within the Fermentative Pathways in Chlamydomonas reinhardtii 
The Scientific World Journal  2012;2012:565047.
Under dark anoxia, the unicellular green algae Chlamydomonas reinhardtii may produce hydrogen by means of its hydrogenase enzymes, in particular HYD1, using reductants derived from the degradation of intercellular carbon stores. Other enzymes belonging to the fermentative pathways compete for the same reductants. A complete understanding of the mechanisms determining the activation of one pathway rather than another will help us engineer Chlamydomonas for fermentative metabolite production, including hydrogen. We examined the expression pattern of the fermentative genes PDC3, LDH1, ADH2, PFL1, and PFR1 in response to day-night cycles, continuous light, continuous darkness, and low or high oxygen availability, which are all conditions that vary on a regular basis in Chlamydomonas' natural environment. We found that all genes except PFL1 show daily fluctuations in expression, and that PFR1 differentiated itself from the others in that it is clearly responsive to low oxygen, where as PDC3, LDH1, and ADH2 are primarily under diurnal regulation. Our results provide evidence that there exist at least three different regulatory mechanisms within the fermentative pathways and suggest that the fermentative pathways are not redundant but rather that availability of a variety of pathways allows for a differential metabolic response to different environmental conditions.
PMCID: PMC3385630  PMID: 22792045
8.  Metabolic engineering of the iodine content in Arabidopsis 
Scientific Reports  2012;2:338.
Plants are a poor source of iodine, an essential micronutrient for human health. Several attempts of iodine biofortification of crops have been carried out, but the scarce knowledge on the physiology of iodine in plants makes results often contradictory and not generalizable. In this work, we used a molecular approach to investigate how the ability of a plant to accumulate iodine can be influenced by different mechanisms. In particular, we demonstrated that the iodine content in Arabidopsis thaliana can be increased either by facilitating its uptake with the overexpression of the human sodium-iodide symporter (NIS) or through the reduction of its volatilization by knocking-out HOL-1, a halide methyltransferase. Our experiments show that the iodine content in plants results from a balance between intake and retention. A correct manipulation of this mechanism could improve iodine biofortification of crops and prevent the release of the ozone layer-threatening methyl iodide into the atmosphere.
PMCID: PMC3313481  PMID: 22468225
9.  Genomic and transcriptomic analysis of the AP2/ERF superfamily in Vitis vinifera 
BMC Genomics  2010;11:719.
The AP2/ERF protein family contains transcription factors that play a crucial role in plant growth and development and in response to biotic and abiotic stress conditions in plants. Grapevine (Vitis vinifera) is the only woody crop whose genome has been fully sequenced. So far, no detailed expression profile of AP2/ERF-like genes is available for grapevine.
An exhaustive search for AP2/ERF genes was carried out on the Vitis vinifera genome and their expression profile was analyzed by Real-Time quantitative PCR (qRT-PCR) in different vegetative and reproductive tissues and under two different ripening stages.
One hundred and forty nine sequences, containing at least one ERF domain, were identified. Specific clusters within the AP2 and ERF families showed conserved expression patterns reminiscent of other species and grapevine specific trends related to berry ripening. Moreover, putative targets of group IX ERFs were identified by co-expression and protein similarity comparisons.
The grapevine genome contains an amount of AP2/ERF genes comparable to that of other dicot species analyzed so far. We observed an increase in the size of specific groups within the ERF family, probably due to recent duplication events. Expression analyses in different aerial tissues display common features previously described in other plant systems and introduce possible new roles for members of some ERF groups during fruit ripening. The presented analysis of AP2/ERF genes in grapevine provides the bases for studying the molecular regulation of berry development and the ripening process.
PMCID: PMC3022922  PMID: 21171999
10.  Rice germination and seedling growth in the absence of oxygen 
Annals of Botany  2008;103(2):181-196.
Higher plants are aerobic organisms which suffer from the oxygen deficiency imposed by partial or total submergence. However, some plant species have developed strategies to avoid or withstand severe oxygen shortage and, in some cases, the complete absence of oxygen (tissue anoxia) for considerable periods of time.
Rice (Oryza sativa) is one of the few plant species that can tolerate prolonged soil flooding or complete submergence thanks to an array of adaptive mechanisms. These include an ability to elongate submerged shoot organs at faster than normal rates and to develop aerenchyma, allowing the efficient internal transport of oxygen from the re-emerged elongated shoot to submerged parts. However, rice seeds are able to germinate anaerobically by means of coleoptile elongation. This cannot be explained in terms of oxygen transport through an emerged shoot. This review provides an overview of anoxic rice germination that is mediated through coleoptile rather than root emergence.
Although there is still much to learn about the biochemical and molecular basis of anaerobic rice germination, the ability of rice to maintain an active fermentative metabolism (i.e. by fuelling the glycolytic pathway with readily fermentable carbohydrates) is certainly crucial. The results obtained through microarray-based transcript profiling confirm most of the previous evidence based on single-gene studies and biochemical analysis, and highlight new aspects of the molecular response of the rice coleoptile to anoxia.
PMCID: PMC2707302  PMID: 18660495
Anoxia; coleoptile; fermentative metabolism; germination; hypoxia; Oryza sativa; rice
11.  Heterologous microarray experiments allow the identification of the early events associated with potato tuber cold sweetening 
BMC Genomics  2008;9:176.
Since its discovery more than 100 years ago, potato (Solanum tuberosum) tuber cold-induced sweetening (CIS) has been extensively investigated. Several carbohydrate-associated genes would seem to be involved in the process. However, many uncertainties still exist, as the relative contribution of each gene to the process is often unclear, possibly as the consequence of the heterogeneity of experimental systems. Some enzymes associated with CIS, such as β-amylases and invertases, have still to be identified at a sequence level. In addition, little is known about the early events that trigger CIS and on the involvement/association with CIS of genes different from carbohydrate-associated genes. Many of these uncertainties could be resolved by profiling experiments, but no GeneChip is available for the potato, and the production of the potato cDNA spotted array (TIGR) has recently been discontinued. In order to obtain an overall picture of early transcriptional events associated with CIS, we investigated whether the commercially-available tomato Affymetrix GeneChip could be used to identify which potato cold-responsive gene family members should be further studied in detail by Real-Time (RT)-PCR (qPCR).
A tomato-potato Global Match File was generated for the interpretation of various aspects of the heterologous dataset, including the retrieval of best matching potato counterparts and annotation, and the establishment of a core set of highly homologous genes. Several cold-responsive genes were identified, and their expression pattern was studied in detail by qPCR over 26 days. We detected biphasic behaviour of mRNA accumulation for carbohydrate-associated genes and our combined GeneChip-qPCR data identified, at a sequence level, enzymatic activities such as β-amylases and invertases previously reported as being involved in CIS. The GeneChip data also unveiled important processes accompanying CIS, such as the induction of redox- and ethylene-associated genes.
Our Global Match File strategy proved critical for accurately interpretating heterologous datasets, and suggests that similar approaches may be fruitful for other species. Transcript profiling of early events associated with CIS revealed a complex network of events involving sugars, redox and hormone signalling which may be either linked serially or act in parallel. The identification, at a sequence level, of various enzymes long known as having a role in CIS provides molecular tools for further understanding the phenomenon.
PMCID: PMC2358903  PMID: 18416834
12.  The Use of Microarrays to Study the Anaerobic Response in Arabidopsis 
Annals of Botany  2005;96(4):661-668.
• Background and Aims The use of microarrays to characterize the transcript profile of Arabidopsis under various experimental conditions is rapidly expanding. This technique provides a huge amount of expression data, requiring bioinformatics tools to allow the proposal of working hypotheses. The aim of this study was to test the usefulness of this approach to examine the anaerobic response of Arabidopsis by evaluating the reliability of microarray data sets and by interrogation of microarray databases for the expression data of a set of anoxia-inducible genes.
•Methods User-driven software tools that display large gene expression datasets onto diagrams of metabolic pathways were used. The Genevestigator software was used to explore the expression of anoxia-inducible genes throughout the life cycle of Arabidopsis as well as relative to plant organs. T-DNA tagged mutants for selected genes identified from our microarray analysis were searched in the Arabidopsis thaliana Insertion Database, looking for insertional mutants from the Salk collection.
•Key Results The results indicate that microarray data can provide the basis for new hypotheses in the field of plant responses to anaerobiosis and also provide knowledge for a targeted screening of Arabidopsis mutants.
•Conclusions Research on plant responses to anaerobiosis can enormously benefit from the microarray technology.
PMCID: PMC4247033  PMID: 16033780
Anaerobiosis; anoxia; Arabidopsis thaliana; microarray; mutants
13.  Sugar Modulation of α‐Amylase Genes under Anoxia 
Annals of Botany  2003;91(2):143-148.
Tolerance to low oxygen availability is likely to be due to the interaction of several factors. Sugar availability is one of the elements required to support anaerobic metabolism. In cereal grains the availability of soluble sugars is limited, while starch is stored in large amounts. Degradation of starch under anoxia is therefore needed to avoid sugar starvation leading to rapid cell death. The striking difference in the ability to produce α‐amylase when comparing the anoxia‐tolerant rice (Oryza sativa L.) grains with grains of other cereals is not easily explained. Rice is able to respond to gibberellins under anoxia, but the response is too slow to explain the rapid production of α‐amylase enzyme. In the present work we demonstrated that α‐amylase production during the first 2 d after imbibition is mostly due to the activity of the Ramy3D gene, encoding for the G and H isoforms of α‐amylase. The induction of Ramy3D transcription is likely to result from a low sugar content in the grains incubated under anoxia. The ability of rice embryos to sense sugars under anoxia is reported.
PMCID: PMC4244987  PMID: 12509335
α‐amylase; anaerobiosis; anoxia; cereal; Oryza sativa; rice; sugar sensing
14.  Plant cysteine oxidases control the oxygen-dependent branch of the N-end-rule pathway 
Nature Communications  2014;5:3425.
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.
Oxygen sensing in plants is mediated by the N-end rule pathway, in which the N-terminal cysteine residue of ERF-VII transcription factors is selectively oxidised. Weits et al. identify cysteine oxidases responsible for this modification, and show that their expression is itself regulated by ERF-VII.
PMCID: PMC3959200  PMID: 24599061

Results 1-14 (14)