Within their natural habitat, plants are subjected to a combination of abiotic conditions that include stresses such as drought and heat. Drought and heat stress have been extensively studied; however, little is known about how their combination impacts plants. The response of Arabidopsis plants to a combination of drought and heat stress was found to be distinct from that of plants subjected to drought or heat stress. Transcriptome analysis of Arabidopsis plants subjected to a combination of drought and heat stress revealed a new pattern of defense response in plants that includes a partial combination of two multigene defense pathways (i.e. drought and heat stress), as well as 454 transcripts that are specifically expressed in plants during a combination of drought and heat stress. Metabolic profiling of plants subjected to drought, heat stress, or a combination of drought and heat stress revealed that plants subject to a combination of drought and heat stress accumulated sucrose and other sugars such as maltose and gulose. In contrast, Pro that accumulated in plants subjected to drought did not accumulate in plants during a combination of drought and heat stress. Heat stress was found to ameliorate the toxicity of Pro to cells, suggesting that during a combination of drought and heat stress sucrose replaces Pro in plants as the major osmoprotectant. Our results highlight the plasticity of the plant genome and demonstrate its ability to respond to complex environmental conditions that occur in the field.The study of abiotic stress in plants has advanced considerably in recent years. However, the majority of experiments testing the response of plants to changes in environmental conditions have focused on a single stress treatment applied to plants under controlled conditions. In contrast, in the field, a number of different stresses can occur simultaneously. These may include conditions such as high irradiance, low water availability, extreme temperature, or high salinity and may alter plant metabolism in a novel manner that may be different from that caused by each of the different stresses applied individually. The response of plants to abiotic stresses in the field may therefore be very different from that tested in the laboratory (Cushman and Bohnert, 2000;Mittler et al., 2001;Zhu, 2002). Drought and heat stress represent an excellent example of two different abiotic stresses that occur in the field simultaneously, especially in semi-arid or drought-stricken areas (Mittler et al., 2001;Moffat, 2002;Rizhsky et al., 2002). Although drought and heat stress have been extensively studied (Vierling, 1991;Ingram and Bartels, 1996; Shinozaki and YamaguchiShinozaki, 1996;Miernyk, 1999;Queitsch et al., 2000), relatively little is known about how their combination impacts plants. A number of studies examined the effect of a combination of drought and heat stress on the growth and productivity of maize, barley, sorghum, and different grasses. It was found that a combination of drought and heat stress had a significantl...

Reactive oxygen species (ROS), such as O 2 ÿ and H 2 O 2 , play a key role in plant metabolism, cellular signaling, and defense. In leaf cells, the chloroplast is considered to be a focal point of ROS metabolism. It is a major producer of O 2 ÿ and H 2 O 2 during photosynthesis, and it contains a large array of ROS-scavenging mechanisms that have been extensively studied. By contrast, the function of the cytosolic ROS-scavenging mechanisms of leaf cells is largely unknown. In this study, we demonstrate that in the absence of the cytosolic H 2 O 2 -scavenging enzyme ascorbate peroxidase 1 (APX1), the entire chloroplastic H 2 O 2 -scavenging system of Arabidopsis thaliana collapses, H 2 O 2 levels increase, and protein oxidation occurs. We further identify specific proteins oxidized in APX1-deficient plants and characterize the signaling events that ensue in knockout-Apx1 plants in response to a moderate level of light stress. Using a dominant-negative approach, we demonstrate that heat shock transcription factors play a central role in the early sensing of H 2 O 2 stress in plants. Using knockout plants for the NADPH oxidase D protein (knockout-RbohD), we demonstrate that RbohD might be required for ROS signal amplification during light stress. Our study points to a key role for the cytosol in protecting the chloroplast during light stress and provides evidence for cross-compartment protection of thylakoid and stromal/mitochondrial APXs by cytosolic APX1.

Iowa 50011 (H.L., R.M.)In nature, plants encounter a combination of environmental conditions that may include stresses such as drought or heat shock. Although drought and heat shock have been extensively studied, little is known about how their combination affect plants. We used cDNA arrays, coupled with physiological measurements, to study the effect of drought and heat shock on tobacco (Nicotiana tabacum) plants. A combination of drought and heat shock resulted in the closure of stomata, suppression of photosynthesis, enhancement of respiration, and increased leaf temperature. Some transcripts induced during drought, e.g. those encoding dehydrin, catalase, and glycolate oxidase, and some transcripts induced during heat shock, e.g. thioredoxin peroxidase, and ascorbate peroxidase, were suppressed during a combination of drought and heat shock. In contrast, the expression of other transcripts, including alternative oxidase, glutathione peroxidase, phenylalanine ammonia lyase, pathogenesis-related proteins, a WRKY transcription factor, and an ethylene response transcriptional co-activator, was specifically induced during a combination of drought and heat shock. Photosynthetic genes were suppressed, whereas transcripts encoding some glycolysis and pentose phosphate pathway enzymes were induced, suggesting the utilization of sugars through these pathways during stress. Our results demonstrate that the response of plants to a combination of drought and heat shock, similar to the conditions in many natural environments, is different from the response of plants to each of these stresses applied individually, as typically tested in the laboratory. This response was also different from the response of plants to other stresses such as cold, salt, or pathogen attack. Therefore, improving stress tolerance of plants and crops may require a reevaluation, taking into account the effect of multiple stresses on plant metabolism and defense.Under optimal conditions, cellular homeostasis is achieved by the coordinated action of many biochemical pathways. However, different pathways may have different molecular and biophysical properties, making them different in their dependence upon external conditions. Thus, during events of suboptimal conditions (stress), different pathways can be affected differently, and their coupling, which makes cellular homeostasis possible, is disrupted. This process is usually accompanied by the formation of reactive oxygen intermediates (ROIs) because of an increased flow of electrons from the disrupted pathways to the reduction of oxygen (Halliwell and Gutteridge, 1989;Noctor and Foyer, 1998; Asada, 1999; Dat et al., 2000;Mittler, 2002). One example for this process is the effect of heat shock on mitochondrial electron transfer. It was shown that during heat shock, membrane-bound complexes at the inner mitochondrial membrane are uncoupled or disrupted. Electrons from NADH produced by the soluble, and less temperature-sensitive, Krebs cycle enzymes are then channeled to the reduction of O 2 to ROI by...

Plants are sessile organisms that evolved a complex and specialized network of regulatory genes to control their response to changes in environmental conditions. It is likely that many of these regulatory genes were initially created by gene duplication and that they later acquired roles specifically related to individual pathways or stresses as well as their combination (1, 2). Different members of gene families, such as The different regulatory networks of plants are also involved in modulating the production and scavenging of reactive oxygen species (ROS) 1 in cells. These toxic intermediates of oxygen reduction not only control different plant responses to environmental and developmental cues but also potently inhibit essential metabolic pathways and may lead to cell death (6 -9). Although a number of different enzymes and proteins produce or scavenge ROS in cells, little is known about how the different regulatory networks of plants control these enzymes and proteins and modulate the steady-state level of ROS (8 -10). The steady-state level of a number of different transcripts encoding transcription factors such as MYB, WRKY, heat shock transcription factors, and different zinc finger proteins is elevated in plants in response to different forms of ROS-induced stress (11-14). However, genetic evidence supporting a direct regulatory role for these transcripts was only presented for two zinc finger proteins, Lsd1 and Lol1, which were recently found to mediate ROS signals and control programmed cell death in Arabidopsis (6), and for heat shock transcription factor 3, which was shown to enhance cytosolic ascorbate peroxidase (Apx) expression in the absence of stress (15) We are studying the response of plants to overaccumulation of ROS in cells (i.e. oxidative stress; Ref. 9). Our goal is to identify and characterize the transcription factor network that controls the response of plants to oxidative stress. To dissect and study the ROS signal transduction network of plants, we are using knock-out plants deficient in key ROS-scavenging enzymes (13,14). These plants provide an ideal experimental system to study plant responses to ROS accumulation, because they accumulate ROS and activate multiple defense mechanisms in the absence of externally applied stimuli such as stress, ROS, or ROS generators. Moreover, the ROS that accumulate in these mutants are ROS naturally produced in cells at the different cellular ROS-producing sites and not externally applied ROS that may activate additional signaling pathways, including pathogen or abiotic stress response pathways (13,14). Knock-out plants deficient in cytosolic Apx1 are of particular interest. They maintain a high steady-state level of H 2 O 2 in cells and activate ROS defense mechanisms when grown under controlled conditions (13). These plants are also altered

SummaryThe accumulation of hydrogen peroxide (H 2 O 2 ) in plants is typically associated with biotic or abiotic stresses. However, H 2 O 2 is continuously produced in cells during normal metabolism. Yet, little is known about how H 2 O 2 accumulation will affect plant metabolism in the absence of pathogens or abiotic stress. Here, we report that a de®ciency in the H 2 O 2 -scavenging enzyme, cytosolic ascorbate peroxidase (APX1), results in the accumulation of H 2 O 2 in Arabidopsis plants grown under optimal conditions. Knockout-Apx1 plants were characterized by suppressed growth and development, altered stomatal responses, and augmented induction of heat shock proteins during light stress. The inactivation of Apx1 resulted in the induction of several transcripts encoding signal transduction proteins. These were not previously linked to H 2 O 2 signaling during stress and may belong to a signal transduction pathway speci®cally involved in H 2 O 2 sensing during normal metabolism. Surprisingly, the expression of transcripts encoding H 2 O 2 scavenging enzymes, such as catalase or glutathione peroxidase, was not elevated in knockout-Apx1 plants. The expression of catalase, two typical plant peroxidases, and several different heat shock proteins was however elevated in knockout-Apx1 plants during light stress. Our results demonstrate that in planta accumulation of H 2 O 2 can suppress plant growth and development, interfere with different physiological processes, and enhance the response of plants to abiotic stress conditions. Our ®ndings also suggest that at least part of the induction of heat shock proteins during light stress in Arabidopsis is mediated by H 2 O 2 that is scavenged by APX1.

(J.S., V.S.)Abiotic stresses cause extensive losses to agricultural production worldwide. Acclimation of plants to abiotic conditions such as drought, salinity, or heat is mediated by a complex network of transcription factors and other regulatory genes that control multiple defense enzymes, proteins, and pathways. Associated with the activity of different transcription factors are transcriptional coactivators that enhance their binding to the basal transcription machinery. Although the importance of stressresponse transcription factors was demonstrated in transgenic plants, little is known about the function of transcriptional coactivators associated with abiotic stresses. Here, we report that constitutive expression of the stress-response transcriptional coactivator multiprotein bridging factor 1c (MBF1c) in Arabidopsis (Arabidopsis thaliana) enhances the tolerance of transgenic plants to bacterial infection, heat, and osmotic stress. Moreover, the enhanced tolerance of transgenic plants to osmotic and heat stress was maintained even when these two stresses were combined. The expression of MBF1c in transgenic plants augmented the accumulation of a number of defense transcripts in response to heat stress. Transcriptome profiling and inhibitor studies suggest that MBF1c expression enhances the tolerance of transgenic plants to heat and osmotic stress by partially activating, or perturbing, the ethylene-response signal transduction pathway. Present findings suggest that MBF1 proteins could be used to enhance the tolerance of plants to different abiotic stresses.

Maintaining electron flow through the photosynthetic apparatus, even in the absence of a sufficient amount of NADP ؉ as an electron acceptor, is essential for chloroplast protection from photooxidative stress. At least two different pathways are thought to participate in this process, i.e. cyclic electron flow and the water-water cycle. Although the function of the water-water cycle was inferred from a number of biochemical and physiological studies, genetic evidence for the function of this cycle is very limited. Here we show that knockdown Arabidopsis plants with suppressed expression of the key water-water cycle enzyme, thylakoid-attached copper/zinc superoxide dismutase (KD-SOD), are suppressed in their growth and development. Chloroplast size, chlorophyll content, and photosynthetic activity were also reduced in KD-SOD plants. Microarray analysis of KD-SOD plants, grown under controlled conditions, revealed changes in transcript expression consistent with an acclimation response to light stress. Although a number of transcripts involved in the defense of plants from oxidative stress were induced in KD-SOD plants, and seedlings of KD-SOD plants were more tolerant to oxidative stress, these mechanisms were unable to compensate for the suppression of the water-water cycle in mature leaves. Thus, the localization of copper/zinc superoxide dismutase at the vicinity of photosystem I may be essential for its function. Our studies provide genetic evidence for the importance of the water-water cycle in protecting the photosynthetic apparatus of higher plants from photooxidative damage.Dissipation of excess energy absorbed by the photosynthetic apparatus is a fundamental process essential for the survival of almost all photosynthetic organisms. It prevents photooxidative damage that occurs when excited chlorophyll molecules improperly transfer their higher energy state to oxygen or neighboring molecules and convert them into reactive molecules or toxic radicals (1-3). This process is especially crucial when CO 2 fixation is limited because of environmental conditions such as cold or drought. Under these conditions, the energy absorbed by the photosynthetic apparatus cannot be channeled into the reduction of CO 2 , and photooxidative damage may occur (4). Maintaining electron flow through the photosynthetic membrane, even under stressful conditions, is therefore vital for preventing damage to plant cells (2). A number of different pathways are thought to cooperate in protecting the photosynthetic apparatus from photooxidative stress. These include the zeaxanthin cycle that directly protects the antenna molecules and the cyclic electron flow and the waterwater cycle that shunt electrons through the photosynthetic apparatus and maintain the pH gradient in the chloroplast, which is essential for the function of the zeaxanthin cycle (1, 2).The water-water cycle channels electrons obtained from the splitting of water molecules at photosystem II (PSII) 1 through the photosynthetic apparatus. These electrons are transferred ...

We have previously found that expression of MARVELD1 was remarkably downregulated in multiple tumor tissues, but unclear in hepatocellular carcinoma (HCC) and its function has not been explored yet. In the present study, to uncover the underlying mechanism of MARVELD1 in the pathogenesis and development of HCC, we investigated the expression pattern of MARVELD1 and its effect on tumor proliferation in HCC. The results indicated the frequent downregulation of MARVELD1 in clinic samples and cell lines of HCC resulted from promoter methylation, as well as genetic deletion. Furthermore, treatment of MARVELD1 unexpressing Hep3B2.1-7 and PLC/PRF/5 cells with the demethylating agent 5-aza-2′ deoxycytidine restored its expression. Overexpression of MARVELD1 suppressed the proliferation of HCC cells in vitro and in vivo, whereas downregulation of endogenous MARVELD1 by shRNAs significantly enhanced these characters. MARVELD1 overexpression could enhance chemosensitivity of HCC cells to epirubicin and 10-hydroxycamptothecin. Corresponding to these results, the expression of p-ERK1/2 and cyclin D1 were decreased, whereas p16 and p53 were increased in MAR-VELD1-transfected cells. We also demonstrated that knockdown of MARVELD1 resulted in upregulation of p-ERK1/2 and cyclin D1, and downregulation of p16 and p53. Moreover, the effect of the decreased cell growth rate was significantly reversed when MAR-VELD1-overexpressing cells were trasfected with p53 or p16 siR-NA. Our findings suggest that MARVELD1 is a tumor suppressor by negatively regulating proliferation, tumor growth and chemosensitivity of HCC cells via increasing p53 and p16 in vitro and in vivo. MARVELD1 may be a potential target for HCC therapy. (Cancer Sci 2012; 103: 716-722) H epatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide, and its incidence is still rising.(1) Currently there are about 600 000 cases of HCC each year, and nearly 78% of them are from Asian countries.(2) Substantial evidence from epidemiological studies indicates that HCC is strongly associated with alcohol abuse, chronic infection with hepatitis B virus (HBV) and/or hepatitis C virus (HCV), and liver cirrhosis.(3-5) More than one million people die of liver cancer worldwide every year.(6) Few patients are diagnosed in the early stage, and <20% of HCCs can be resected completely. (7,8) Resistance to many of chemotherapy agents is a major obstacle to successful HCC treatment.(9-11) Therefore, a better understanding of the molecular mechanisms underlying HCC progression is urgently needed for leading to a more effective treatment.Genetic and epigenetic aberrations, leading to the activation of oncogenes and inactivation of tumor suppressor genes, are thought to play major roles in the pathogenesis of HCC. Recently, several MARVEL (proteins of the myelin and lymphocytes [MAL] and related proteins for vesicle trafficking and membrane link) domain-containing proteins have attracted increasing interest because they exhibit tumor suppressor activities a...