Apoplastic reactive oxygen species transiently decrease auxin signaling and cause stress-induced morphogenic response in Arabidopsis.

Reactive oxygen species (ROS) are ubiquitous signaling molecules in plant stress and development. To gain further insight into the plant transcriptional response to apoplastic ROS, the phytotoxic atmospheric pollutant ozone was used as a model ROS inducer in Arabidopsis (Arabidopsis thaliana) and gene expression was analyzed with microarrays. In contrast to the increase in signaling via the stress hormones salicylic acid, abscisic acid, jasmonic acid (JA), and ethylene, ROS treatment caused auxin signaling to be transiently suppressed, which was confirmed with a DR5-uidA auxin reporter construct. Transcriptomic data revealed that various aspects of auxin homeostasis and signaling were modified by apoplastic ROS. Furthermore, a detailed analysis of auxin signaling showed that transcripts of several auxin receptors and Auxin/Indole-3-Acetic Acid (Aux/IAA) transcriptional repressors were reduced in response to apoplastic ROS. The ROS-derived changes in the expression of auxin signaling genes partially overlapped with abiotic stress, pathogen responses, and salicylic acid signaling. Several mechanisms known to suppress auxin signaling during biotic stress were excluded, indicating that ROS regulated auxin responses via a novel mechanism. Using mutants defective in various auxin (axr1, nit1, aux1, tir1 afb2, iaa28-1, iaa28-2) and JA (axr1, coi1-16) responses, ROS-induced cell death was found to be regulated by JA but not by auxin. Chronic ROS treatment resulted in altered leaf morphology, a stress response known as "stress-induced morphogenic response." Altered leaf shape of tir1 afb2 suggests that auxin was a negative regulator of stress-induced morphogenic response in the rosette.

Nitric oxide modulates ozone-induced cell death, hormone biosynthesis and gene expression in Arabidopsis thaliana.

Nitric oxide (NO) is involved together with reactive oxygen species (ROS) in the activation of various stress responses in plants. We have used ozone (O3) as a tool to elicit ROS-activated stress responses, and to activate cell death in plant leaves. Here, we have investigated the roles and interactions of ROS and NO in the induction and regulation of O3-induced cell death. Treatment with O3 induced a rapid accumulation of NO, which started from guard cells, spread to adjacent epidermal cells and eventually moved to mesophyll cells. During the later time points, NO production coincided with the formation of hypersensitive response (HR)-like lesions. The NO donor sodium nitroprusside (SNP) and O3 individually induced a large set of defence-related genes; however, in a combined treatment SNP attenuated the O3 induction of salicylic acid (SA) biosynthesis and other defence-related genes. Consistent with this, SNP treatment also decreased O3-induced SA accumulation. The O3-sensitive mutant rcd1 was found to be an NO overproducer; in contrast, Atnoa1/rif1 (Arabidopsis nitric oxide associated 1/resistant to inhibition by FSM1), a mutant with decreased production of NO, was also O3 sensitive. This, together with experiments combining O3 and the NO donor SNP suggested that NO can modify signalling, hormone biosynthesis and gene expression in plants during O3 exposure, and that a functional NO production is needed for a proper O3 response. In summary, NO is an important signalling molecule in the response to O3.

Unequally redundant RCD1 and SRO1 mediate stress and developmental responses and interact with transcription factors.

RADICAL-INDUCED CELL DEATH1 (RCD1) is an important regulator of stress and hormonal and developmental responses in Arabidopsis thaliana. Together with its closest homolog, SIMILAR TO RCD-ONE1 (SRO1), it is the only Arabidopsis protein containing the WWE domain, which is known to mediate protein-protein interactions in other organisms. Additionally, these two proteins contain the core catalytic region of poly-ADP-ribose transferases and a conserved C-terminal domain. Tissue and subcellular localization data indicate that RCD1 and SRO1 have partially overlapping functions in plant development. In contrast mutant data indicate that rcd1 has defects in plant development, whereas sro1 displays normal development. However, the rcd1 sro1 double mutant has severe growth defects, indicating that RCD1 and SRO1 exemplify an important genetic principle - unequal genetic redundancy. A large pair-wise interaction test against the REGIA transcription factor collection revealed that RCD1 interacts with a large number of transcription factors belonging to several protein families, such as AP2/ERF, NAC and basic helix-loop-helix (bHLH), and that SRO1 interacts with a smaller subset of these. Full genome array analysis indicated that in many cases targets of these transcription factors have altered expression in the rcd1 but not the sro1 mutant. Taken together RCD1 and SRO1 are required for proper plant development.

Ozone and nitric oxide interaction in Arabidopsis thaliana: a role for ethylene?

Nitric oxide (NO) is involved together with reactive oxygen species (ROS) in the activation of various stress responses in plants. However, the biochemical mechanisms by which ROS and NO participate, and the potential interaction between these molecules are still unclear. Ozone (O(3)) can be used as a tool to elicit ROS-activated stress responses and to activate cell death in plant leaves. We have recently shown that O(3) induced a rapid accumulation of NO in Arabidopsis leaves and at late time points NO production coincided with the formation of hypersensitive response like lesions. Experiments using O(3) and the NO-donor SNP alone or in combination indicated that both molecules are capable of activating a large set of stress related genes. In combined treatment, NO attenuated O(3)-induction of salicylic acid (SA) biosynthetic and signaling genes, and reduced SA accumulation. In addition, NO can elevate the levels of ethylene in several mutants. Thus, NO is a modifier of ROS signaling.

Ozone-induced programmed cell death in the Arabidopsis radical-induced cell death1 mutant.

Short, high-concentration peaks of the atmospheric pollutant ozone (O(3)) cause the formation of cell death lesions on the leaves of sensitive plants. Numerous similarities between the plant responses to O(3) and pathogens suggest that O(3) triggers hypersensitive response-like programmed cell death (PCD). We examined O(3) and superoxide-induced cell death in the O(3)-sensitive radical-induced cell death1 (rcd1) mutant. Dying cells in O(3)-exposed rcd1 exhibited several of the typical morphological characteristics of the hypersensitive response and PCD. Double-mutant analyses indicated a requirement for salicylic acid and the function of the cyclic nucleotide-gated ion channel AtCNGC2 in cell death. Furthermore, a requirement for ATPases, kinases, transcription, Ca(2+) flux, caspase-like proteolytic activity, and also one or more phenylmethylsulfonyl fluoride-sensitive protease activities was shown for the development of cell death lesions in rcd1. Furthermore, mitogen-activated protein kinases showed differential activation patterns in rcd1 and Columbia. Taken together, these results directly demonstrate the induction of PCD by O(3).

Stress hormone-independent activation and nuclear translocation of mitogen-activated protein kinases in Arabidopsis thaliana during ozone exposure.

Changing environmental conditions, atmospheric pollutants and resistance reactions to pathogens cause production of reactive oxygen species (ROS) in plants. ROS in turn trigger the activation of signaling cascades such as the mitogen-activated protein kinase (MAPK) cascade and accumulation of plant hormones, jasmonic acid, salicylic acid (SA), and ethylene (ET). We have used ozone (O3) to generate ROS in the apoplast of wild-type Col-0 and hormonal signaling mutants of Arabidopsis thaliana and show that this treatment caused a transient activation of 43 and 45 kDa MAPKs. These were identified as AtMPK3 and AtMPK6. We also demonstrate that initial AtMPK3 and AtMPK6 activation in response to O3 was not dependent on ET signaling, but that ET is likely to have secondary effects on AtMPK3 and AtMPK6 function, whereas functional SA signaling was needed for full-level AtMPK3 activation by O3. In addition, we show that AtMPK3, but not AtMPK6, responded to O3 transcriptionally and translationally during O3 exposure. Finally, we show in planta that activated AtMPK3 and AtMPK6 are translocated to the nucleus during the early stages of O3 treatment. The use of O3 to induce apoplastic ROS formation offers a non-invasive in planta system amenable to reverse genetics that can be used for the study of stress-responsive MAPK signaling in plants.

Arabidopsis RADICAL-INDUCED CELL DEATH1 belongs to the WWE protein-protein interaction domain protein family and modulates abscisic acid, ethylene, and methyl jasmonate responses.

Experiments with several Arabidopsis thaliana mutants have revealed a web of interactions between hormonal signaling. Here, we show that the Arabidopsis mutant radical-induced cell death1 (rcd1), although hypersensitive to apoplastic superoxide and ozone, is more resistant to chloroplastic superoxide formation, exhibits reduced sensitivity to abscisic acid, ethylene, and methyl jasmonate, and has altered expression of several hormonally regulated genes. Furthermore, rcd1 has higher stomatal conductance than the wild type. The rcd1-1 mutation was mapped to the gene At1g32230 where it disrupts an intron splice site resulting in a truncated protein. RCD1 belongs to the (ADP-ribosyl)transferase domain-containing subfamily of the WWE protein-protein interaction domain protein family. The results suggest that RCD1 could act as an integrative node in hormonal signaling and in the regulation of several stress-responsive genes.