Nina Sipari

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.

Chloroplast NADPH-Thioredoxin Reductase Interacts with Photoperiodic Development in Arabidopsis

Chloroplast NADPH-thioredoxin reductase (NTRC) belongs to the thioredoxin systems that control crucial metabolic and regulatory pathways in plants. Here, by characterization of T-DNA insertion lines of NTRC gene, we uncover a novel connection between chloroplast thiol redox regulation and the control of photoperiodic growth in Arabidopsis (Arabidopsis thaliana). Transcript and metabolite profiling revealed severe developmental and metabolic defects in ntrc plants grown under a short 8-h light period. Besides reduced chlorophyll and anthocyanin contents, ntrc plants showed alterations in the levels of amino acids and auxin. Furthermore, a low carbon assimilation rate of ntrc leaves was associated with enhanced transpiration and photorespiration. All of these characteristics of ntrc were less severe when plants were grown under a long 16-h photoperiod. Transcript profiling revealed that the mutant phenotypes of ntrc were accompanied by differential expression of genes involved in stomatal development, chlorophyll biosynthesis, chloroplast biogenesis, and circadian clock-linked light perception systems in ntrc plants. We propose that NTRC regulates several key processes, including chlorophyll biosynthesis and the shikimate pathway, in chloroplasts. In the absence of NTRC, imbalanced metabolic activities presumably modulate the chloroplast retrograde signals, leading to altered expression of nuclear genes and, ultimately, to the formation of the pleiotrophic phenotypes in ntrc mutant plants.

Defense-related transcription factors WRKY70 and WRKY54 modulate osmotic stress tolerance by regulating stomatal aperture in Arabidopsis

WRKY transcription factors (TFs) have been mainly associated with plant defense, but recent studies have suggested additional roles in the regulation of other physiological processes. Here, we explored the possible contribution of two related group III WRKY TFs, WRKY70 and WRKY54, to osmotic stress tolerance. These TFs are positive regulators of plant defense, and co-operate as negative regulators of salicylic acid (SA) biosynthesis and senescence. We employed single and double mutants of wrky54 and wrky70, as well as a WRKY70 overexpressor line, to explore the role of these TFs in osmotic stress (polyethylene glycol) responses. Their effect on gene expression was characterized by microarrays and verified by quantitative PCR. Stomatal phenotypes were assessed by water retention and stomatal conductance measurements. The wrky54wrky70 double mutants exhibited clearly enhanced tolerance to osmotic stress. However, gene expression analysis showed reduced induction of osmotic stress-responsive genes in addition to reduced accumulation of the osmoprotectant proline. By contrast, the enhanced tolerance was correlated with improved water retention and enhanced stomatal closure. These findings demonstrate that WRKY70 and WRKY54 co-operate as negative regulators of stomatal closure and, consequently, osmotic stress tolerance in Arabidopsis, suggesting that they have an important role, not only in plant defense, but also in abiotic stress signaling.

Multiple Roles for UV RESISTANCE LOCUS8 in Regulating Gene Expression and Metabolite Accumulation in Arabidopsis under Solar Ultraviolet Radiation

Summary: Under natural sunlight, this study demonstrates multiple and complex roles for the UV-B photoreceptor UV RESISTANCE LOCUS 8 in the acclimation of Arabidopsis plants to UV radiation. Photomorphogenic responses triggered by low fluence rates of ultraviolet B radiation (UV-B; 280–315 nm) are mediated by the UV-B photoreceptor UV RESISTANCE LOCUS8 (UVR8). Beyond our understanding of the molecular mechanisms of UV-B perception by UVR8, there is still limited information on how the UVR8 pathway functions under natural sunlight. Here, wild-type Arabidopsis (Arabidopsis thaliana) and the uvr8-2 mutant were used in an experiment outdoors where UV-A (315–400 nm) and UV-B irradiances were attenuated using plastic films. Gene expression, PYRIDOXINE BIOSYNTHESIS1 (PDX1) accumulation, and leaf metabolite signatures were analyzed. The results show that UVR8 is required for transcript accumulation of genes involved in UV protection, oxidative stress, hormone signal transduction, and defense against herbivores under solar UV. Under natural UV-A irradiance, UVR8 is likely to interact with UV-A/blue light signaling pathways to moderate UV-B-driven transcript and PDX1 accumulation. UVR8 both positively and negatively affects UV-A-regulated gene expression and metabolite accumulation but is required for the UV-B induction of phenolics. Moreover, UVR8-dependent UV-B acclimation during the early stages of plant development may enhance normal growth under long-term exposure to solar UV.

Regulatory Subunit B′γ of Protein Phosphatase 2A Prevents Unnecessary Defense Reactions under Low Light in Arabidopsis

Light is an important environmental factor that modulates acclimation strategies and defense responses in plants. We explored the functional role of the regulatory subunit B′γ (B′γ) of protein phosphatase 2A (PP2A) in light-dependent stress responses of Arabidopsis (Arabidopsis thaliana). The predominant form of PP2A consists of catalytic subunit C, scaffold subunit A, and highly variable regulatory subunit B, which determines the substrate specificity of PP2A holoenzymes. Mutant leaves of knockdown pp2a-b′γ plants show disintegration of chloroplasts and premature yellowing conditionally under moderate light intensity. The cell-death phenotype is accompanied by the accumulation of hydrogen peroxide through a pathway that requires CONSTITUTIVE EXPRESSION OF PR GENES5 (CPR5). Moreover, the pp2a-b′γ cpr5 double mutant additionally displays growth suppression and malformed trichomes. Similar to cpr5, the pp2a-b′γ mutant shows constitutive activation of both salicylic acid- and jasmonic acid-dependent defense pathways. In contrast to cpr5, however, pp2a-b′γ leaves do not contain increased levels of salicylic acid or jasmonic acid. Rather, the constitutive defense response associates with hypomethylation of DNA and increased levels of methionine-salvage pathway components in pp2a-b′γ leaves. We suggest that the specific B′γ subunit of PP2A is functionally connected to CPR5 and operates in the basal repression of defense responses under low irradiance.

Transcriptomics and Functional Genomics of ROS-Induced Cell Death Regulation by RADICAL-INDUCED CELL DEATH1

Plant responses to changes in environmental conditions are mediated by a network of signaling events leading to downstream responses, including changes in gene expression and activation of cell death programs. Arabidopsis thaliana RADICAL-INDUCED CELL DEATH1 (RCD1) has been proposed to regulate plant stress responses by protein-protein interactions with transcription factors. Furthermore, the rcd1 mutant has defective control of cell death in response to apoplastic reactive oxygen species (ROS). Combining transcriptomic and functional genomics approaches we first used microarray analysis in a time series to study changes in gene expression after apoplastic ROS treatment in rcd1. To identify a core set of cell death regulated genes, RCD1-regulated genes were clustered together with other array experiments from plants undergoing cell death or treated with various pathogens, plant hormones or other chemicals. Subsequently, selected rcd1 double mutants were constructed to further define the genetic requirements for the execution of apoplastic ROS induced cell death. Through the genetic analysis we identified WRKY70 and SGT1b as cell death regulators functioning downstream of RCD1 and show that quantitative rather than qualitative differences in gene expression related to cell death appeared to better explain the outcome. Allocation of plant energy to defenses diverts resources from growth. Recently, a plant response termed stress-induced morphogenic response (SIMR) was proposed to regulate the balance between defense and growth. Using a rcd1 double mutant collection we show that SIMR is mostly independent of the classical plant defense signaling pathways and that the redox balance is involved in development of SIMR.

The F-box protein MAX2 contributes to resistance to bacterial phytopathogens in Arabidopsis thaliana

BackgroundThe Arabidopsis thaliana F-box protein MORE AXILLARY GROWTH2 (MAX2) has previously been characterized for its role in plant development. MAX2 appears essential for the perception of the newly characterized phytohormone strigolactone, a negative regulator of polar auxin transport in Arabidopsis.ResultsA reverse genetic screen for F-box protein mutants altered in their stress responses identified MAX2 as a component of plant defense. Here we show that MAX2 contributes to plant resistance against pathogenic bacteria. Interestingly, max2 mutant plants showed increased susceptibility to the bacterial necrotroph Pectobacterium carotovorum as well as to the hemi-biotroph Pseudomonas syringae but not to the fungal necrotroph Botrytis cinerea. max2 mutant phenotype was associated with constitutively increased stomatal conductance and decreased tolerance to apoplastic ROS but also with alterations in hormonal balance.ConclusionsOur results suggest that MAX2 previously characterized for its role in regulation of polar auxin transport in Arabidopsis, and thus plant development also significantly influences plant disease resistance. We conclude that the increased susceptibility to P. syringae and P. carotovorum is due to increased stomatal conductance in max2 mutants promoting pathogen entry into the plant apoplast. Additional factors contributing to pathogen susceptibility in max2 plants include decreased tolerance to pathogen-triggered apoplastic ROS and alterations in hormonal signaling.

Epidermal UV-A absorbance and whole-leaf flavonoid composition in pea respond more to solar blue light than to solar UV radiation

Plants synthesize phenolic compounds in response to certain environmental signals or stresses. One large group of phenolics, flavonoids, is considered particularly responsive to ultraviolet (UV) radiation. However, here we demonstrate that solar blue light stimulates flavonoid biosynthesis in the absence of UV-A and UV-B radiation. We grew pea plants (Pisum sativum cv. Meteor) outdoors, in Finland during the summer, under five types of filters differing in their spectral transmittance. These filters were used to (1) attenuate UV-B; (2) attenuate UV-B and UV-A < 370 nm; (3) attenuate UV-B and UV-A; (4) attenuate UV-B, UV-A and blue light; and (5) as a control not attenuating these wavebands. Attenuation of blue light significantly reduced the flavonoid content in leaf adaxial epidermis and reduced the whole-leaf concentrations of quercetin derivatives relative to kaempferol derivatives. In contrast, UV-B responses were not significant. These results show that pea plants regulate epidermal UV-A absorbance and accumulation of individual flavonoids by perceiving complex radiation signals that extend into the visible region of the solar spectrum. Furthermore, solar blue light instead of solar UV-B radiation can be the main regulator of phenolic compound accumulation in plants that germinate and develop outdoors.

Changes in the abscisic acid levels and related gene expression during fruit development and ripening in bilberry (Vaccinium myrtillus L.)

Abscisic acid (ABA) is a natural plant hormone playing an important role in many physiological processes including fruit ripening and is also recently found to be potential for biomedical applications. This study was aimed to measure ABA levels and its biosynthesis in bilberry (Vaccinium myrtillus L.), which is one of the best sources of anthocyanins. Five ABA biosynthetic genes were isolated from bilberry and their expression profiles were studied in bilberry tissues, particularly during berry development. The level of ABA highly increased at the onset of bilberry fruit ripening, at the stage when expression of anthocyanin biosynthetic genes, chalcone synthase (VmCHS) and anthocyanidin synthase (VmANS), also increased. In fully ripe berries and leaves, ABA levels were lower but none was detected in bilberry stem or rhizome. The expression of 9-cis-epoxycarotenoid dioxygenase (VmNCED1) and putative neoxanthin synthase (VmNSY) was high in berry tissues and their expression increased markedly at the onset of berry ripening along with the accumulation of ABA. In contrast, the expression of zeaxanthin epoxidase (VmZEP), short-chain dehydrogenase/reductase (VmSDR/ABA2) and aldehyde oxidase (VmAO) were most highly associated with leaf tissues with no obvious relation to ABA content during berry development. The obtained results indicate that the ABA biosynthesis may play an important role in the regulation of ripening of non-climacteric bilberry fruits through transcriptional regulation of key ABA biosynthetic genes.

Regulation of ABA dependent wound induced spreading cell death by MYB108

Wounding results in the controlled cell death of a few rows of cells adjacent to disrupted cells resulting in physical wound closure, which combined with phenolic compound deposition, prevents water loss and pathogen entry. The control of these processes remains uncharacterized. Cell death in a mutant of Arabidopsis thaliana lacking BOTRYTIS SENSITIVE1/MYB108 (BOS1/MYB108) function was characterized utilizing physiological, cell biological and genetic methods. The bos1 mutant has a wound induced runaway cell death that includes enhanced reactive oxygen species (ROS) production that followed the extent of enhanced cell death. Exogenous abscisic acid (ABA) enhanced wound induced cell death in Col-0 plants and was sufficient to trigger cell death in bos1. Uncontrolled cell death was dependent of the production and perception of ABA. Furthermore, bos1 had altered sensitivity to and accumulation of ABA. Arabidopsis possesses a genetic program controlling the extent of wound inducible cell death. BOS1 acts as a negative regulator of ABA induced cell death, which functions in the control of this wound sealing program. This program is distinct from other known cell death programs in that it is ABA dependent, but independent of salicylate biosynthesis, ethylene, jasmonate, metacaspases and ROS derived from RBOHD and RBOHF.