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.

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.

Reactive oxygen species (ROS), such as O₂⁻ and H₂O₂, 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 O₂⁻ and H₂O₂ 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 H₂O₂-scavenging enzyme ascorbate peroxidase 1 (APX1), the entire chloroplastic H₂O₂-scavenging system of Arabidopsis thaliana collapses, H₂O₂ 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 H₂O₂ 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.

Cytosolic ascorbate peroxidase 1 (Apx1) is a key H₂O₂ removal enzyme in plants. Microarray analysis of Apx1-deficient Arabidopsis plants revealed that the expression of two zinc finger proteins (Zat12 and Zat7) and a WRKY transcription factor (WRKY25) is elevated in knock-out Apx1 plants grown under controlled conditions. Because mutants lacking Apx1 accumulate H₂O₂, we examined the correlation between H₂O₂ and the expression of Zat12, Zat7, WRKY25, and Apx1. The expression of Zat12, Zat7, and WRKY25 was simultaneously elevated in cells in response to oxidative stress (i.e. H₂O₂ or paraquat application), heat shock, or wounding. In contrast, light or osmotic stress did not enhance the expression of these putative transcription factors. All stresses tested enhanced the expression of Apx1. Transgenic plants expressing Zat12 or Zat7 could tolerate oxidative stress. In contrast, transgenic plants expressing WRKY25 could not. Although the expression of Zat12, Zat7, or WRKY25 in transgenic plants did not enhance the expression of Apx1 under controlled conditions, Zat12-deficient plants were unable to enhance the expression of Apx1, Zat7, or WRKY25 in response to H₂O₂ or paraquat application. Zat12-deficient plants were also more sensitive than wild type plants to H₂O₂ application as revealed by a higher level of H₂O₂-induced protein oxidation detected in these plants by protein blots. Our results suggest that Zat12 is an important component of the oxidative stress response signal transduction network of Arabidopsis required for Zat7, WRKY25, and Apx1 expression during oxidative stress.

Reactive oxygen species (ROS) play a key signaling role in plants and are controlled in cells by a complex network of ROS metabolizing enzymes found in several different cellular compartments. To study how different ROS signals, generated in different cellular compartments, are integrated in cells, we generated a double mutant lacking thylakoid ascorbate peroxidase (tylapx) and cytosolic ascorbate peroxidase1 (apx1). Our analysis suggests that two different signals are generated in plants lacking cytosolic APX1 or tylAPX. The lack of a chloroplastic hydrogen peroxide removal enzyme triggers a specific signal in cells that results in enhanced tolerance to heat stress, whereas the lack of a cytosolic hydrogen peroxide removal enzyme triggers a different signal, which results in stunted growth and enhanced sensitivity to oxidative stress. When the two signals are coactivated in cells (i.e. tylapx/apx1), a new response is detected, suggesting that the integration of the two different signals results in a new signal that manifests in late flowering, low protein oxidation during light stress, and enhanced accumulation of anthocyanins. Our results demonstrate a high degree of plasticity in ROS signaling in Arabidopsis (Arabidopsis thaliana) and suggest the existence of redundant pathways for ROS protection that compensate for the lack of classical ROS removal enzymes such as cytosolic and chloroplastic APXs. Further investigation of the enhanced heat tolerance in plants lacking tylAPX, using mutants deficient in chloroplast-to-nuclei retrograde signaling, suggests the existence of a chloroplast-generated stress signal that enhances basal thermotolerance in plants.

Summary: The plant genome is a highly redundant and dynamic genome. Here, we show that double antisense plants lacking the two major hydrogen peroxide‐detoxifying enzymes, ascorbate peroxidase (APX) and catalase (CAT), activate an alternative/redundant defense mechanism that compensates for the lack of APX and CAT. A similar mechanism was not activated in single antisense plants that lacked APX or CAT, paradoxically rendering these plants more sensitive to oxidative stress compared to double antisense plants. The reduced susceptibility of double antisense plants to oxidative stress correlated with suppressed photosynthetic activity, the induction of metabolic genes belonging to the pentose phosphate pathway, the induction of monodehydroascorbate reductase, and the induction of IMMUTANS, a chloroplastic homologue of mitochondrial alternative oxidase. Our results suggest that a co‐ordinated induction of metabolic and defense genes, coupled with the suppression of photosynthetic activity, can compensate for the lack of APX and CAT. In addition, our findings demonstrate that the plant genome has a high degree of plasticity and will respond differently to different stressful conditions, namely, lack of APX, lack of CAT, or lack of both APX and CAT.