Glyoxalase I activity affects Arabidopsis sensitivity to ammonium nutrition.

KEY MESSAGE: Elevated methylglyoxal levels contribute to ammonium-induced growth disorders in Arabidopsis thaliana. Methylglyoxal detoxification pathway limitation, mainly the glyoxalase I activity, leads to enhanced sensitivity of plants to ammonium nutrition. Ammonium applied to plants as the exclusive source of nitrogen often triggers multiple phenotypic effects, with severe growth inhibition being the most prominent symptom. Glycolytic flux increase, leading to overproduction of its toxic by-product methylglyoxal (MG), is one of the major metabolic consequences of long-term ammonium nutrition. This study aimed to evaluate the influence of MG metabolism on ammonium-dependent growth restriction in Arabidopsis thaliana plants. As the level of MG in plant cells is maintained by the glyoxalase (GLX) system, we analyzed MG-related metabolism in plants with a dysfunctional glyoxalase pathway. We report that MG detoxification, based on glutathione-dependent glyoxalases, is crucial for plants exposed to ammonium nutrition, and its essential role in ammonium sensitivity relays on glyoxalase I (GLXI) activity. Our results indicated that the accumulation of MG-derived advanced glycation end products significantly contributes to the incidence of ammonium toxicity symptoms. Using A. thaliana frostbite1 as a model plant that overcomes growth repression on ammonium, we have shown that its resistance to enhanced MG levels is based on increased GLXI activity and tolerance to elevated MG-derived advanced glycation end-product (MAGE) levels. Furthermore, our results show that glyoxalase pathway activity strongly affects cellular antioxidative systems. Under stress conditions, the disruption of the MG detoxification pathway limits the functioning of antioxidant defense. However, under optimal growth conditions, a defect in the MG detoxification route results in the activation of antioxidative systems.

Metabolic control of arginine and ornithine levels paces the progression of leaf senescence.

Leaf senescence can be induced by stress or aging, sometimes in a synergistic manner. It is generally acknowledged that the ability to withstand senescence-inducing conditions can provide plants with stress resilience. Although the signaling and transcriptional networks responsible for a delayed senescence phenotype, often referred to as a functional stay-green trait, have been actively investigated, very little is known about the subsequent metabolic adjustments conferring this aptitude to survival. First, using the individually darkened leaf (IDL) experimental setup, we compared IDLs of wild-type (WT) Arabidopsis (Arabidopsis thaliana) to several stay-green contexts, that is IDLs of two functional stay-green mutant lines, oresara1-2 (ore1-2) and an allele of phytochrome-interacting factor 5 (pif5), as well as to leaves from a WT plant entirely darkened (DP). We provide compelling evidence that arginine and ornithine, which accumulate in all stay-green contexts-likely due to the lack of induction of amino acids (AAs) transport-can delay the progression of senescence by fueling the Krebs cycle or the production of polyamines (PAs). Secondly, we show that the conversion of putrescine to spermidine (SPD) is controlled in an age-dependent manner. Thirdly, we demonstrate that SPD represses senescence via interference with ethylene signaling by stabilizing the ETHYLENE BINDING FACTOR1 and 2 (EBF1/2) complex. Taken together, our results identify arginine and ornithine as central metabolites influencing the stress- and age-dependent progression of leaf senescence. We propose that the regulatory loop between the pace of the AA export and the progression of leaf senescence provides the plant with a mechanism to fine-tune the induction of cell death in leaves, which, if triggered unnecessarily, can impede nutrient remobilization and thus plant growth and survival.

Modulating the activities of chloroplasts and mitochondria promotes adenosine triphosphate production and plant growth.

Efficient photosynthesis requires a balance of ATP and NADPH production/consumption in chloroplasts, and the exportation of reducing equivalents from chloroplasts is important for balancing stromal ATP/NADPH ratio. Here, we showed that the overexpression of purple acid phosphatase 2 on the outer membranes of chloroplasts and mitochondria can streamline the production and consumption of reducing equivalents in these two organelles, respectively. A higher capacity of consumption of reducing equivalents in mitochondria can indirectly help chloroplasts to balance the ATP/NADPH ratio in stroma and recycle NADP+, the electron acceptors of the linear electron flow (LEF). A higher rate of ATP and NADPH production from the LEF, a higher capacity of carbon fixation by the Calvin-Benson-Bassham (CBB) cycle and a greater consumption of NADH in mitochondria enhance photosynthesis in the chloroplasts, ATP production in the mitochondria and sucrose synthesis in the cytosol and eventually boost plant growth and seed yields in the overexpression lines.

In planta study of photosynthesis and photorespiration using NADPH and NADH/NAD+ fluorescent protein sensors.

The challenge of monitoring in planta dynamic changes of NADP(H) and NAD(H) redox states at the subcellular level is considered a major obstacle in plant bioenergetics studies. Here, we introduced two circularly permuted yellow fluorescent protein sensors, iNAP and SoNar, into Arabidopsis thaliana to monitor the dynamic changes in NADPH and the NADH/NAD+ ratio. In the light, photosynthesis and photorespiration are linked to the redox states of NAD(P)H and NAD(P) pools in several subcellular compartments connected by the malate-OAA shuttles. We show that the photosynthetic increases in stromal NADPH and NADH/NAD+ ratio, but not ATP, disappear when glycine decarboxylation is inhibited. These observations highlight the complex interplay between chloroplasts and mitochondria during photosynthesis and support the suggestions that, under normal conditions, photorespiration supplies a large amount of NADH to mitochondria, exceeding its NADH-dissipating capacity, and the surplus NADH is exported from the mitochondria to the cytosol through the malate-OAA shuttle.

ATP compartmentation in plastids and cytosol of Arabidopsis thaliana revealed by fluorescent protein sensing.

Matching ATP:NADPH provision and consumption in the chloroplast is a prerequisite for efficient photosynthesis. In terms of ATP:NADPH ratio, the amount of ATP generated from the linear electron flow does not meet the demand of the Calvin-Benson-Bassham (CBB) cycle. Several different mechanisms to increase ATP availability have evolved, including cyclic electron flow in higher plants and the direct import of mitochondrial-derived ATP in diatoms. By imaging a fluorescent ATP sensor protein expressed in living Arabidopsis thaliana seedlings, we found that MgATP2- concentrations were lower in the stroma of mature chloroplasts than in the cytosol, and exogenous ATP was able to enter chloroplasts isolated from 4- and 5-day-old seedlings, but not chloroplasts isolated from 10- or 20-day-old photosynthetic tissues. This observation is in line with the previous finding that the expression of chloroplast nucleotide transporters (NTTs) in Arabidopsis mesophyll is limited to very young seedlings. Employing a combination of photosynthetic and respiratory inhibitors with compartment-specific imaging of ATP, we corroborate the dependency of stromal ATP production on mitochondrial dissipation of photosynthetic reductant. Our data suggest that, during illumination, the provision and consumption of ATP:NADPH in chloroplasts can be balanced by exporting excess reductants rather than importing ATP from the cytosol.

Nitrogen Source Dependent Changes in Central Sugar Metabolism Maintain Cell Wall Assembly in Mitochondrial Complex I-Defective frostbite1 and Secondarily Affect Programmed Cell Death.

For optimal plant growth, carbon and nitrogen availability needs to be tightly coordinated. Mitochondrial perturbations related to a defect in complex I in the Arabidopsis thalianafrostbite1 (fro1) mutant, carrying a point mutation in the 8-kD Fe-S subunit of NDUFS4 protein, alter aspects of fundamental carbon metabolism, which is manifested as stunted growth. During nitrate nutrition, fro1 plants showed a dominant sugar flux toward nitrogen assimilation and energy production, whereas cellulose integration in the cell wall was restricted. However, when cultured on NH4⁺ as the sole nitrogen source, which typically induces developmental disorders in plants (i.e., the ammonium toxicity syndrome), fro1 showed improved growth as compared to NO3- nourishing. Higher energy availability in fro1 plants was correlated with restored cell wall assembly during NH4⁺ growth. To determine the relationship between mitochondrial complex I disassembly and cell wall-related processes, aspects of cell wall integrity and sugar and reactive oxygen species signaling were analyzed in fro1 plants. The responses of fro1 plants to NH4⁺ treatment were consistent with the inhibition of a form of programmed cell death. Resistance of fro1 plants to NH4⁺ toxicity coincided with an absence of necrotic lesion in plant leaves.

Darkened Leaves Use Different Metabolic Strategies for Senescence and Survival.

In plants, an individually darkened leaf initiates senescence much more rapidly than a leaf from a whole darkened plant. Combining transcriptomic and metabolomic approaches in Arabidopsis (Arabidopsis thaliana), we present an overview of the metabolic strategies that are employed in response to different darkening treatments. Under darkened plant conditions, the perception of carbon starvation drove a profound metabolic readjustment in which branched-chain amino acids and potentially monosaccharides released from cell wall loosening became important substrates for maintaining minimal ATP production. Concomitantly, the increased accumulation of amino acids with a high nitrogen-carbon ratio may provide a safety mechanism for the storage of metabolically derived cytotoxic ammonium and a pool of nitrogen for use upon returning to typical growth conditions. Conversely, in individually darkened leaf, the metabolic profiling that followed our 13C-enrichment assays revealed a temporal and differential exchange of metabolites, including sugars and amino acids, between the darkened leaf and the rest of the plant. This active transport could be the basis for a progressive metabolic shift in the substrates fueling mitochondrial activities, which are central to the catabolic reactions facilitating the retrieval of nutrients from the senescing leaf. We propose a model illustrating the specific metabolic strategies employed by leaves in response to these two darkening treatments, which support either rapid senescence or a strong capacity for survival.

The redox control of photorespiration: from biochemical and physiological aspects to biotechnological considerations.

Photorespiration is a complex and tightly regulated process occurring in photosynthetic organisms. This process can alter the cellular redox balance, notably via the production and consumption of both reducing and oxidizing equivalents. Under certain circumstances, these equivalents, as well as reactive oxygen or nitrogen species, can become prominent in subcellular compartments involved in the photorespiratory process, eventually promoting oxidative post-translational modifications of proteins. Keeping these changes under tight control should therefore be of primary importance. In order to review the current state of knowledge about the redox control of photorespiration, we primarily performed a careful description of the known and potential redox-regulated or oxidation sensitive photorespiratory proteins, and examined in more details two interesting cases: the glycerate kinase and the glycine cleavage system. When possible, the potential impact and subsequent physiological regulations associated with these changes have been discussed. In the second part, we reviewed the extent to which photorespiration contributes to cellular redox homeostasis considering, in particular, the set of peripheral enzymes associated with the canonical photorespiratory pathway. Finally, some recent biotechnological strategies to circumvent photorespiration for future growth improvements are discussed in the light of these redox regulations.

Dissecting the Metabolic Role of Mitochondria during Developmental Leaf Senescence.

The functions of mitochondria during leaf senescence, a type of programmed cell death aimed at the massive retrieval of nutrients from the senescing organ to the rest of the plant, remain elusive. Here, combining experimental and analytical approaches, we showed that mitochondrial integrity in Arabidopsis (Arabidopsis thaliana) is conserved until the latest stages of leaf senescence, while their number drops by 30%. Adenylate phosphorylation state assays and mitochondrial respiratory measurements indicated that the leaf energy status also is maintained during this time period. Furthermore, after establishing a curated list of genes coding for products targeted to mitochondria, we analyzed in isolation their transcript profiles, focusing on several key mitochondrial functions, such as the tricarboxylic acid cycle, mitochondrial electron transfer chain, iron-sulfur cluster biosynthesis, transporters, as well as catabolic pathways. In tandem with a metabolomic approach, our data indicated that mitochondrial metabolism was reorganized to support the selective catabolism of both amino acids and fatty acids. Such adjustments would ensure the replenishment of α-ketoglutarate and glutamate, which provide the carbon backbones for nitrogen remobilization. Glutamate, being the substrate of the strongly up-regulated cytosolic glutamine synthase, is likely to become a metabolically limiting factor in the latest stages of developmental leaf senescence. Finally, an evolutionary age analysis revealed that, while branched-chain amino acid and proline catabolism are very old mitochondrial functions particularly enriched at the latest stages of leaf senescence, auxin metabolism appears to be rather newly acquired. In summation, our work shows that, during developmental leaf senescence, mitochondria orchestrate catabolic processes by becoming increasingly central energy and metabolic hubs.

The origin of cytosolic ATP in photosynthetic cells.

In photosynthetically active cells, both chloroplasts and mitochondria have the capacity to produce ATP via photophosphorylation and oxidative phosphorylation, respectively. Thus, theoretically, both organelles could provide ATP for the cytosol, but the extent, to which they actually do this, and how the process is regulated, both remain unclear. Most of the evidence discussed comes from experiments with rapid fractionation of isolated protoplasts subjected to different treatments in combination with application of specific inhibitors. The results obtained indicate that, under conditions where ATP demand for photosynthetic CO2 fixation is sufficiently high, the mitochondria supply the bulk of ATP for the cytosol. In contrast, under stress conditions where CO2 fixation is severely limited, ATP will build up in chloroplasts and it can then be exported to the cytosol, by metabolite shuttle mechanisms. Thus, depending on the conditions, either mitochondria or chloroplasts can supply the bulk of ATP for the cytosol. This supply of ATP is discussed in relation to the idea that mitochondrial functions may be tuned to provide an optimal environment for the chloroplast. By balancing cellular redox states, mitochondria can contribute to an optimal photosynthetic capacity.

Reduced mitochondrial malate dehydrogenase activity has a strong effect on photorespiratory metabolism as revealed by 13C labelling.

Mitochondrial malate dehydrogenase (mMDH) catalyses the interconversion of malate and oxaloacetate (OAA) in the tricarboxylic acid (TCA) cycle. Its activity is important for redox control of the mitochondrial matrix, through which it may participate in regulation of TCA cycle turnover. In Arabidopsis, there are two isoforms of mMDH. Here, we investigated to which extent the lack of the major isoform, mMDH1 accounting for about 60% of the activity, affected leaf metabolism. In air, rosettes of mmdh1 plants were only slightly smaller than wild type plants although the fresh weight was decreased by about 50%. In low CO2 the difference was much bigger, with mutant plants accumulating only 14% of fresh weight as compared to wild type. To investigate the metabolic background to the differences in growth, we developed a (13)CO2 labelling method, using a custom-built chamber that enabled simultaneous treatment of sets of plants under controlled conditions. The metabolic profiles were analysed by gas- and liquid- chromatography coupled to mass spectrometry to investigate the metabolic adjustments between wild type and mmdh1 The genotypes responded similarly to high CO2 treatment both with respect to metabolite pools and (13)C incorporation during a 2-h treatment. However, under low CO2 several metabolites differed between the two genotypes and, interestingly most of these were closely associated with photorespiration. We found that while the glycine/serine ratio increased, a concomitant altered glutamine/glutamate/α-ketoglutarate relation occurred. Taken together, our results indicate that adequate mMDH activity is essential to shuttle reductants out from the mitochondria to support the photorespiratory flux, and strengthen the idea that photorespiration is tightly intertwined with peripheral metabolic reactions.

Hexokinase 1 is required for glucose-induced repression of bZIP63, At5g22920, and BT2 in Arabidopsis.

Simple sugars, like glucose (Glc) and sucrose (Suc), act as signals to modulate the expression of hundreds of genes in plants. Frequently, however, it remains unclear whether this regulation is induced by the sugars themselves or by their derivatives generated in the course of carbohydrate (CH) metabolism. In the present study, we tested the relevance of different CH metabolism and allocation pathways affecting expression patterns of five selected sugar-responsive genes (bZIP63, At5g22920, BT2, MGD2, and TPS9) in Arabidopsis thaliana. In general, the expression followed diurnal changes in the overall sugar availability. However, under steady growth conditions, this response was hardly impaired in the mutants for CH metabolizing/ transporting proteins (adg1, sex1, sus1-4, sus5/6, and tpt2), including also hexokinase1 (HXK1) loss- and gain-of-function plants-gin2.1 and oe3.2, respectively. In addition, transgenic plants carrying pbZIP63::GUS showed no changes in reporter-gene-expression when grown on sugar under steady-state conditions. In contrast, short-term treatments of agar-grown seedlings with 1% Glc or Suc induced pbZIP63::GUS repression, which became even more apparent in seedlings grown in liquid media. Subsequent analyses of liquid-grown gin2.1 and oe3.2 seedlings revealed that Glc -dependent regulation of the five selected genes was not affected in gin2.1, whereas it was enhanced in oe3.2 plants for bZIP63, At5g22920, and BT2. The sugar treatments had no effect on ATP/ADP ratio, suggesting that changes in gene expression were not linked to cellular energy status. Overall, the data suggest that HXK1 does not act as Glc sensor controlling bZIP63, At5g22920, and BT2 expression, but it is nevertheless required for the production of a downstream metabolic signal regulating their expression.

In comparison with nitrate nutrition, ammonium nutrition increases growth of the frostbite1†Arabidopsis mutant.

Ammonium nutrition inhibits the growth of many plant species, including Arabidopsis thaliana. The toxicity of ammonium is associated with changes in the cellular redox state. The cellular oxidant/antioxidant balance is controlled by mitochondrial electron transport chain. In this study, we analysed the redox metabolism of frostbite1 (fro1) plants, which lack mitochondrial respiratory chain complex I. Surprisingly, the growth of fro1 plants increased under ammonium nutrition. Ammonium nutrition increased the reduction level of pyridine nucleotides in the leaves of wild-type plants, but not in the leaves of fro1 mutant plants. The observed higher activities of type II NADH dehydrogenases and cytochrome c oxidase in the mitochondrial electron transport chain may improve the energy metabolism of fro1 plants grown on ammonium. Additionally, the observed changes in reactive oxygen species (ROS) metabolism in the apoplast may be important for determining the growth of fro1 under ammonium nutrition. Moreover, bioinformatic analyses showed that the gene expression changes in fro1 plants significantly overlap with the changes previously observed in plants with a modified apoplastic pH. Overall, the results suggest a pronounced connection between the mitochondrial redox system and the apoplastic pH and ROS levels, which may modify cell wall plasticity and influence growth.

Activity of the mitochondrial pyruvate dehydrogenase complex in plants is stimulated in the presence of malate.

The effect of malate on the steady-state activity of the pea (Pisum sativum L.) and barley (Hordeum vulgare L.) leaf pyruvate dehydrogenase complex (PDC) has been studied in isolated mitochondria. The addition of malate was found to be stimulatory for the mitochondrial PDC, however there was no stimulation of chloroplast PDC. The stimulation was saturated below 1mM malate and was apparently related to а partially activated complex, which activity increased in the presence of malate by about twofold. Malate also reversed the reduction of PDC activity in the presence of glycine. Based on the obtained kinetic data, we suggest that the effect of malate is rather not a direct activation of PDC but involves the establishment of NAD-malate dehydrogenase equilibrium, decreasing concentration of NADH and relieving its inhibitory effect of PDC.

In response to partial plant shading, the lack of phytochrome A does not directly induce leaf senescence but alters the fine-tuning of chlorophyll biosynthesis.

Phytochrome is thought to control the induction of leaf senescence directly, however, the signalling and molecular mechanisms remain unclear. In the present study, an ecophysiological approach was used to establish a functional connection between phytochrome signalling and the physiological processes underlying the induction of leaf senescence in response to shade. With shade it is important to distinguish between complete and partial shading, during which either the whole or only a part of the plant is shaded, respectively. It is first shown here that, while PHYB is required to maintain chlorophyll content in a completely shaded plant, only PHYA is involved in maintaining the leaf chlorophyll content in response to partial plant shading. Second, it is shown that leaf yellowing associated with strong partial shading in phyA-mutant plants actually correlates to a decreased biosynthesis of chlorophyll rather than to an increase of its degradation. Third, it is shown that the physiological impact of this decreased biosynthesis of chlorophyll in strongly shaded phyA-mutant leaves is accompanied by a decreased capacity to adjust the Light Compensation Point. However, the increased leaf yellowing in phyA-mutant plants is not accompanied by an increase of senescence-specific molecular markers, which argues against a direct role of PHYA in inducing leaf senescence in response to partial shade. In conclusion, it is proposed that PHYA, but not PHYB, is essential for fine-tuning the chlorophyll biosynthetic pathway in response to partial shading. In turn, this mechanism allows the shaded leaf to adjust its photosynthetic machinery to very low irradiances, thus maintaining a positive carbon balance and repressing the induction of leaf senescence, which can occur under prolonged periods of shade.

The function of glycine decarboxylase complex is optimized to maintain high photorespiratory flux via buffering of its reaction products.

Oxidation of glycine in photorespiratory pathway is the major flux through mitochondria of C3 plants in the light. It sustains increased intramitochondrial concentrations of NADH and NADPH, which are required to engage the internal rotenone-insensitive NAD(P)H dehydrogenases and the alternative oxidase. We discuss here possible mechanisms of high photorespiratory flux maintenance in mitochondria and suggest that it is fulfilled under conditions where the concentrations of glycine decarboxylase reaction products NADH and CO2 achieve an equilibrium provided by malate dehydrogenase and carbonic anhydrase, respectively. This results in the removal of these products from the glycine decarboxylase multienzyme active sites and in the maintenance of their concentrations at levels sufficiently low to prevent substrate inhibition of the reaction.

Long-term ammonium nutrition of Arabidopsis increases the extrachloroplastic NAD(P)H/NAD(P)(+) ratio and mitochondrial reactive oxygen species level in leaves but does not impair photosynthetic capacity.

Ammonium nutrition has been suggested to be associated with alterations in the oxidation-reduction state of leaf cells. Herein, we show that ammonium nutrition in Arabidopsis thaliana increases leaf NAD(P)H/NAD(P)(+) ratio, reactive oxygen species content and accumulation of biomolecules oxidized by free radicals. We used the method of rapid fractionation of protoplasts to analyse which cellular compartments were over-reduced under ammonium supply and revealed that observed changes in NAD(P)H/NAD(P)(+) ratio involved only the extrachloroplastic fraction. We also showed that ammonium nutrition changes mitochondrial electron transport chain activity, increasing mitochondrial reactive oxygen species production. Our results indicate that the functional impairment associated with ammonium nutrition is mainly associated with redox reactions outside the chloroplast.

The impact of light intensity on shade-induced leaf senescence.

Plants often have to cope with altered light conditions, which in leaves induce various physiological responses ranging from photosynthetic acclimation to leaf senescence. However, our knowledge of the regulatory pathways by which shade and darkness induce leaf senescence remains incomplete. To determine to what extent reduced light intensities regulate the induction of leaf senescence, we performed a functional comparison between Arabidopsis leaves subjected to a range of shading treatments. Individually covered leaves, which remained attached to the plant, were compared with respect to chlorophyll, protein, histology, expression of senescence-associated genes, capacity for photosynthesis and respiration, and light compensation point (LCP). Mild shading induced photosynthetic acclimation and resource partitioning, which, together with a decreased respiration, lowered the LCP. Leaf senescence was induced only under strong shade, coinciding with a negative carbon balance and independent of the red/far-red ratio. Interestingly, while senescence was significantly delayed at very low light compared with darkness, phytochrome A mutant plants showed enhanced chlorophyll degradation under all shading treatments except complete darkness. Taken together, our results suggest that the induction of leaf senescence during shading depends on the efficiency of carbon fixation, which in turn appears to be modulated via light receptors such as phytochrome A.

Quantitative analysis of photosynthetic carbon metabolism in protoplasts and intact leaves of barley. Determination of carbon fluxes and pool sizes of metabolites in different cellular compartments.

Rates of carbon fluxes and pool sizes of photosynthetic metabolites in different cellular compartments of barley protoplasts were calculated from the time curves of their labeling in the medium of ¹4CO2. Using membrane filtration procedure, kinetics of ¹4C incorporation into the products of steady-state photosynthesis was determined separately in chloroplasts, mitochondria and cytosol of barley protoplasts illuminated for different periods in the air containing ¹4CO2. To extract the quantitative information, analytical labeling functions P(t) describing the dependence of ¹4C content in the primary, intermediate and end products of a linear reaction chain upon the duration of tracer feeding have been derived. The parameters of these functions represent pool sizes of metabolites and rates of carbon fluxes. The values of these parameters were determined by fitting the experimental labeling curves to the functions P(t) by means of non-linear regression procedure. To elucidate the possible effects of fractionation on the photosynthetic carbon metabolism, the parameters of protoplasts were compared with corresponding values in intact leaves of barley.

Leaf senescence is accompanied by an early disruption of the microtubule network in Arabidopsis.

The dynamic assembly and disassembly of microtubules (MTs) is essential for cell function. Although leaf senescence is a well-documented process, the role of the MT cytoskeleton during senescence in plants remains unknown. Here, we show that both natural leaf senescence and senescence of individually darkened Arabidopsis (Arabidopsis thaliana) leaves are accompanied by early degradation of the MT network in epidermis and mesophyll cells, whereas guard cells, which do not senesce, retain their MT network. Similarly, entirely darkened plants, which do not senesce, retain their MT network. While genes encoding the tubulin subunits and the bundling/stabilizing MT-associated proteins (MAPs) MAP65 and MAP70-1 were repressed in both natural senescence and dark-induced senescence, we found strong induction of the gene encoding the MT-destabilizing protein MAP18. However, induction of MAP18 gene expression was also observed in leaves from entirely darkened plants, showing that its expression is not sufficient to induce MT disassembly and is more likely to be part of a Ca(2+)-dependent signaling mechanism. Similarly, genes encoding the MT-severing protein katanin p60 and two of the four putative regulatory katanin p80s were repressed in the dark, but their expression did not correlate with degradation of the MT network during leaf senescence. Taken together, these results highlight the earliness of the degradation of the cortical MT array during leaf senescence and lead us to propose a model in which suppression of tubulin and MAP genes together with induction of MAP18 play key roles in MT disassembly during senescence.