Francesco Licausi

Oxygen sensing in plants is mediated by an N-end rule pathway for protein destabilization

The majority of eukaryotic organisms rely on molecular oxygen for respiratory energy production. When the supply of oxygen is compromised, a variety of acclimation responses are activated to reduce the detrimental effects of energy depletion. Various oxygen-sensing mechanisms have been described that are thought to trigger these responses, but they each seem to be kingdom specific and no sensing mechanism has been identified in plants until now. Here we show that one branch of the ubiquitin-dependent N-end rule pathway for protein degradation, which is active in both mammals and plants, functions as an oxygen-sensing mechanism in Arabidopsis thaliana. We identified a conserved amino-terminal amino acid sequence of the ethylene response factor (ERF)-transcription factor RAP2.12 to be dedicated to an oxygen-dependent sequence of post-translational modifications, which ultimately lead to degradation of RAP2.12 under aerobic conditions. When the oxygen concentration is low—as during flooding—RAP2.12 is released from the plasma membrane and accumulates in the nucleus to activate gene expression for hypoxia acclimation. Our discovery of an oxygen-sensing mechanism opens up new possibilities for improving flooding tolerance in crops.

Transcript Profiling of the Anoxic Rice Coleoptile

Rice (Oryza sativa) seeds can germinate in the complete absence of oxygen. Under anoxia, the rice coleoptile elongates, reaching a length greater than that of the aerobic one. In this article, we compared and investigated the transcriptome of rice coleoptiles grown under aerobic and anaerobic conditions. The results allow drawing a detailed picture of the modulation of the transcripts involved in anaerobic carbohydrate metabolism, suggesting up-regulation of the steps required to produce and metabolize pyruvate and its derivatives. Sugars appear to play a signaling role under anoxia, with several genes indirectly up-regulated by anoxia-driven sugar starvation. Analysis of the effects of anoxia on the expansin gene families revealed that EXPA7 and EXPB12 are likely to be involved in rice coleoptile elongation under anoxia. Genes coding for ethylene response factors and heat shock proteins are among the genes modulated by anoxia in both rice and Arabidopsis (Arabidopsis thaliana). Identification of anoxia-induced ethylene response factors is suggestive because genes belonging to this gene family play a crucial role in rice tolerance to submergence, a process closely related to, but independent from, the ability to germinate under anoxia. Genes coding for some enzymes requiring oxygen for their activity are dramatically down-regulated under anoxia, suggesting the existence of an energy-saving strategy in the regulation of gene expression.

Making sense of low oxygen sensing

Plant-specific group VII Ethylene Response Factor (ERF) transcription factors have emerged as pivotal regulators of flooding and low oxygen responses. In rice (Oryza sativa), these proteins regulate contrasting strategies of flooding survival. Recent studies on Arabidopsis thaliana group VII ERFs show they are stabilized under hypoxia but destabilized under oxygen-replete conditions via the N-end rule pathway of targeted proteolysis. Oxygen-dependent sequestration at the plasma membrane maintains at least one of these proteins, RAP2.12, under normoxia. Remarkably, SUB1A, the rice group VII ERF that enables prolonged submergence tolerance, appears to evade oxygen-regulated N-end rule degradation. We propose that the turnover of group VII ERFs is of ecological relevance in wetland species and might be manipulated to improve flood tolerance of crops.

HRE1 and HRE2, two hypoxia-inducible ethylene response factors, affect anaerobic responses in Arabidopsis thaliana.

Plants often experience challenging hypoxic conditions imposed by soil waterlogging or complete flooding. In rice, Sub1A, a flooding-induced ethylene responsive factor (ERF) plays a crucial role in submergence tolerance. In this study, we examined two Arabidopsis Hypoxia Responsive ERF genes (HRE1 and HRE2), belonging to the same ERF group as Sub1A. Transgenic Arabidopsis plants, which over-expressed HRE1, showed an improved tolerance of anoxia, whereas a double-knockout mutant hre1hre2 was more susceptible than the wild type. HRE1 over-expressing plants showed an increased activity in the fermentative enzymes pyruvate decarboxylase and alcohol dehydrogenase together with increased ethanol production under hypoxia, but not in normoxia. Whole-genome microarray analyses suggested that an over-expression of HRE1, but not HRE2, increased the induction of most anaerobic genes under hypoxia. Real-time quantitative (q)PCR analyses confirmed a positive effect of HRE1 over-expression on several anaerobic genes, whereas the double-knockout mutant hre1hre2 showed a decreased expression in the same genes after 4 h of hypoxia. Single-knockout mutants did not show significant differences from the wild type. We found that the regulation of HRE1 and HRE2 by low oxygen relies on different mechanisms, since HRE1 requires protein synthesis to be induced while HRE2 does not. HRE2 is likely to be regulated post-transcriptionally by mRNA stabilization. We propose that HRE1 and HRE2 play a partially redundant role in low oxygen signalling in Arabidopsis thaliana, thus improving the tolerance of the plant to the stress by enhancing anaerobic gene expression and ethanolic fermentation.

Glycolysis and the tricarboxylic acid cycle are linked by alanine aminotransferase during hypoxia induced by waterlogging of Lotus japonicus.

The role of nitrogen metabolism in the survival of prolonged periods of waterlogging was investigated in highly flood-tolerant, nodulated Lotus japonicus plants. Alanine production revealed to be a critical hypoxic pathway. Alanine is the only amino acid whose biosynthesis is not inhibited by nitrogen deficiency resulting from RNA interference silencing of nodular leghemoglobin. The metabolic changes that were induced following waterlogging can be best explained by the activation of alanine metabolism in combination with the modular operation of a split tricarboxylic acid pathway. The sum result of this metabolic scenario is the accumulation of alanine and succinate and the production of extra ATP under hypoxia. The importance of alanine metabolism is discussed with respect to its ability to regulate the level of pyruvate, and this and all other changes are discussed in the context of current models concerning the regulation of plant metabolism.

Genomic and transcriptomic analysis of the AP2/ERF superfamily in Vitis vinifera

BackgroundThe 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.ResultsAn 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.ConclusionsThe 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.

Hypoxia responsive gene expression is mediated by various subsets of transcription factors and miRNAs that are determined by the actual oxygen availability

• Reduced oxygen availability is not only associated with flooding, but occurs also during growth and development. It is largely unknown how hypoxia is perceived and what signaling cascade is involved in activating adaptive responses. • We analysed the expression of over 1900 transcription factors (TFs) and 180 microRNA primary transcripts (pri-miRNAs) in Arabidopsis roots exposed to different hypoxic conditions by means of quantitative PCR. We also analysed the promoters of genes induced by hypoxia with respect to over-represented DNA elements that can act as potential TF binding sites and their in vivo interaction was verified. • We identified various subsets of TFs that responded differentially through time and in an oxygen concentration-dependent manner. The regulatory potential of selected TFs and their predicted DNA binding elements was validated. Although the expression of pri-miRNAs was differentially regulated under hypoxia, only one corresponding mature miRNA changed accordingly. Putative target transcripts of the miRNAs were not significantly affected. • Our results show that the regulation of hypoxia-induced genes is controlled via simultaneous interaction of various combinations of TFs. Under anoxic conditions, an additional set of TFs is induced. Regulation of gene expression via miRNAs appears to play a minor role during hypoxia.

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.

Oxygen Sensing and Signaling

Oxygen is an indispensable substrate for many biochemical reactions in plants, including energy metabolism (respiration). Despite its importance, plants lack an active transport mechanism to distribute oxygen to all cells. Therefore, steep oxygen gradients occur within most plant tissues, which can be exacerbated by environmental perturbations that further reduce oxygen availability. Plants possess various responses to cope with spatial and temporal variations in oxygen availability, many of which involve metabolic adaptations to deal with energy crises induced by low oxygen. Responses are induced gradually when oxygen concentrations decrease and are rapidly reversed upon reoxygenation. A direct effect of the oxygen level can be observed in the stability, and thus activity, of various transcription factors that control the expression of hypoxia-induced genes. Additional signaling pathways are activated by the impact of oxygen deficiency on mitochondrial and chloroplast functioning. Here, we describe the molecular components of the oxygen-sensing pathway.

Comparative study of RNA-seq-and Microarray-derived coexpression networks in Arabidopsis thaliana

MOTIVATION Coexpression networks are data-derived representations of genes behaving in a similar way across tissues and experimental conditions. They have been used for hypothesis generation and guilt-by-association approaches for inferring functions of previously unknown genes. So far, the main platform for expression data has been DNA microarrays; however, the recent development of RNA-seq allows for higher accuracy and coverage of transcript populations. It is therefore important to assess the potential for biological investigation of coexpression networks derived from this novel technique in a condition-independent dataset. RESULTS We collected 65 publicly available Illumina RNA-seq high quality Arabidopsis thaliana samples and generated Pearson correlation coexpression networks. These networks were then compared with those derived from analogous microarray data. We show how Variance-Stabilizing Transformed (VST) RNA-seq data samples are the most similar to microarray ones, with respect to inter-sample variation, correlation coefficient distribution and network topological architecture. Microarray networks show a slightly higher score in biology-derived quality assessments such as overlap with the known protein-protein interaction network and edge ontological agreement. Different coexpression network centralities are investigated; in particular, we show how betweenness centrality is generally a positive marker for essential genes in A.thaliana, regardless of the platform originating the data. In the end, we focus on a specific gene network case, showing that although microarray data seem more suited for gene network reverse engineering, RNA-seq offers the great advantage of extending coexpression analyses to the entire transcriptome.