Joost T. van Dongen

On the origins of nitric oxide

Nitric oxide (NO) is widely recognized for its role as signaling compound. However, the metabolic mechanisms that determine changes in the level of NO in plants are only poorly understood, despite this knowledge being crucial to understanding the signal function of NO. To date, at least seven possible pathways of NO biosynthesis have been described for plants, although the molecular and enzymatic components are resolved for only one of these. Currently, this represents the most significant bottleneck for NO research. In this review, we provide an overview of the multiplicity of NO production and scavenging pathways in plants. Furthermore, we discuss which areas should be focused on in future studies to investigate the origin of fluctuations in the level of NO in plants.

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.

SNF1-related kinases allow plants to tolerate herbivory by allocating carbon to roots

Herbivore attack elicits costly defenses that are known to decrease plant fitness by using resources that are normally slated for growth and reproduction. Additionally, plants have evolved mechanisms for tolerating attack, which are not understood on a molecular level. Using 11C-photosynthate labeling as well as sugar and enzyme measurements, we found rapid changes in sink–source relations in the annual Nicotiana attenuata after simulated herbivore attacks, which increased the allocation of sugars to roots. This herbivore-induced response is regulated by the β-subunit of an SnRK1 (SNF1-related kinase) protein kinase, GAL83, transcripts of which are rapidly down-regulated in source leaves after herbivore attack and, when silenced, increase assimilate transport to roots. This C diversion response is activated by herbivore-specific elicitors and is independent of jasmonate signaling, which regulates most of the plants defense responses. Herbivore attack during early stages of development increases root reserves, which, in turn, delays senescence and prolongs flowering. That attacked GAL83-silenced plants use their enhanced root reserves to prolong reproduction demonstrates that SnRK1 alters resource allocation so that plants better tolerate herbivory. This tolerance mechanism complements the likely defensive value of diverting resources to a less vulnerable location within the plant.

Symbiotic leghemoglobins are crucial for nitrogen fixation in legume root nodules but not for general plant growth and development

Hemoglobins are ubiquitous in nature and among the best-characterized proteins. Genetics has revealed crucial roles for human hemoglobins, but similar data are lacking for plants. Plants contain symbiotic and nonsymbiotic hemoglobins; the former are thought to be important for symbiotic nitrogen fixation (SNF). In legumes, SNF occurs in specialized organs, called nodules, which contain millions of nitrogen-fixing rhizobia, called bacteroids. The induction of nodule-specific plant genes, including those encoding symbiotic leghemoglobins (Lb), accompanies nodule development. Leghemoglobins accumulate to millimolar concentrations in the cytoplasm of infected plant cells prior to nitrogen fixation and are thought to buffer free oxygen in the nanomolar range, avoiding inactivation of oxygen-labile nitrogenase while maintaining high oxygen flux for respiration. Although widely accepted, this hypothesis has never been tested in planta. Using RNAi, we abolished symbiotic leghemoglobin synthesis in nodules of the model legume Lotus japonicus. This caused an increase in nodule free oxygen, a decrease in the ATP/ADP ratio, loss of bacterial nitrogenase protein, and absence of SNF. However, LbRNAi plants grew normally when fertilized with mineral nitrogen. These data indicate roles for leghemoglobins in oxygen transport and buffering and prove for the first time that plant hemoglobins are crucial for symbiotic nitrogen fixation.

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.

The effect of geometry on three-dimensional tissue growth.

Tissue formation is determined by uncountable biochemical signals between cells; in addition, physical parameters have been shown to exhibit significant effects on the level of the single cell. Beyond the cell, however, there is still no quantitative understanding of how geometry affects tissue growth, which is of much significance for bone healing and tissue engineering. In this paper, it is shown that the local growth rate of tissue formed by osteoblasts is strongly influenced by the geometrical features of channels in an artificial three-dimensional matrix. Curvature-driven effects and mechanical forces within the tissue may explain the growth patterns as demonstrated by numerical simulation and confocal laser scanning microscopy. This implies that cells within the tissue surface are able to sense and react to radii of curvature much larger than the size of the cells themselves. This has important implications towards the understanding of bone remodelling and defect healing as well as towards scaffold design in bone tissue engineering.

Transcript and metabolite profiling of the adaptive response to mild decreases in oxygen concentration in the roots of Arabidopsis plants.

Background and Aims Oxygen can fall to low concentrations within plant tissues, either because of environmental factors that decrease the external oxygen concentration or because the movement of oxygen through the plant tissues cannot keep pace with the rate of oxygen consumption. Recent studies document that plants can decrease their oxygen consumption in response to relatively small changes in oxygen concentrations to avoid internal anoxia. The molecular mechanisms underlying this response have not been identified yet. The aim of this study was to use transcript and metabolite profiling to investigate the genomic response of arabidopsis roots to a mild decrease in oxygen concentrations. Methods Arabidopsis seedlings were grown on vertical agar plates at 21, 8, 4 and 1 % (v/v) external oxygen for 0·5, 2 and 48 h. Roots were analysed for changes in transcript levels using Affymetrix whole genome DNA microarrays, and for changes in metabolite levels using routine GC-MS based metabolite profiling. Root extension rates were monitored in parallel to investigate adaptive changes in growth. Key Results The results show that root growth was inhibited and transcript and metabolite profiles were significantly altered in response to a moderate decrease in oxygen concentrations. Low oxygen leads to a preferential up-regulation of genes that might be important to trigger adaptive responses in the plant. A small but highly specific set of genes is induced very early in response to a moderate decrease in oxygen concentrations. Genes that were down-regulated mainly encoded proteins involved in energy-consuming processes. In line with this, root extension growth was significantly decreased which will ultimately save ATP and decrease oxygen consumption. This was accompanied by a differential regulation of metabolite levels at short- and long-term incubation at low oxygen. Conclusions The results show that there are adaptive changes in root extension involving large-scale reprogramming of gene expression and metabolism when oxygen concentration is decreased in a very narrow range.

Regulation of Respiration and Fermentation to Control the Plant Internal Oxygen Concentration

Plant internal oxygen concentrations can drop well below ambient even when the plant grows under optimal conditions. Using pea (Pisum sativum) roots, we show how amenable respiration adapts to hypoxia to save oxygen when the oxygen availability decreases. The data cannot simply be explained by oxygen being limiting as substrate but indicate the existence of a regulatory mechanism, because the oxygen concentration at which the adaptive response is initiated is independent of the actual respiratory rate. Two phases can be discerned during the adaptive reaction: an initial linear decline of respiration is followed by a nonlinear inhibition in which the respiratory rate decreased progressively faster upon decreasing oxygen availability. In contrast to the cytochrome c pathway, the inhibition of the alternative oxidase pathway shows only the linear component of the adaptive response. Feeding pyruvate to the roots led to an increase of the oxygen consumption rate, which ultimately led to anoxia. The importance of balancing the in vivo pyruvate availability in the tissue was further investigated. Using various alcohol dehydrogenase knockout lines of Arabidopsis (Arabidopsis thaliana), it was shown that even under aerobic conditions, alcohol fermentation plays an important role in the control of the level of pyruvate in the tissue. Interestingly, alcohol fermentation appeared to be primarily induced by a drop in the energy status of the tissue rather than by a low oxygen concentration, indicating that sensing the energy status is an important component of optimizing plant metabolism to changes in the oxygen availability.

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.

Alternative oxidase: a defence against metabolic fluctuations?

An increasing number of oscillating or fluctuating cellular systems have been recently described following the adaptation of fluorescent technology. In diverse organisms, these variously involve signalling factors, heat production, central metabolism and reactive oxygen species (ROS). In response to many plant stresses and primarily via the influence of ROS, changes in mRNA and protein levels or in vivo activity of alternative oxidase are often observed. However, in several investigations, a lack of correlation between the mRNA, protein and in vivo activity has been evident. This discrepancy has made it questionable whether the induction of alternative oxidase has importance in regulating alternative pathway activity in vivo, or being diagnostic for a role of alternative oxidase in stress tolerance and ROS avoidance. Here, we suggest a role of alternative oxidase in counteracting deleterious short-term metabolic fluctuations, especially under stress conditions. This model emphasizes the importance of peak activity for establishing protein levels and allows an amalgamation of the present status of physiological, cellular and molecular knowledge.