Determining the ROS and the Antioxidant Status of Leaves During Cold Acclimation.

Cold slows down Calvin cycle activity stronger than photosynthetic electron transport, which supports production of reactive oxygen species (ROS). Even under extreme temperature conditions, most ROS are detoxified by the combined action of low-molecular weight antioxidants and antioxidant enzymes. Subsequent regeneration of the low-molecular weight antioxidants by NAD(P)H and thioredoxin/thiol-dependent pathways relaxes the electron pressure in the photosynthetic electron transport chain. In general, the chloroplast antioxidant system protects plants from severe damage of enzymes, metabolites, and cellular structures by both ROS detoxification and antioxidant recycling. Various methods have been developed to quantify ROS and antioxidant levels in photosynthetic tissues. Here, we summarize a series of exceptionally fast and easily applicable methods that show local ROS accumulation and provide information on the overall availability of reducing sugars, mainly ascorbate, and of thiols.

Cold priming uncouples light- and cold-regulation of gene expression in Arabidopsis thaliana.

BACKGROUND: The majority of stress-sensitive genes responds to cold and high light in the same direction, if plants face the stresses for the first time. As shown recently for a small selection of genes of the core environmental stress response cluster, pre-treatment of Arabidopsis thaliana with a 24 h long 4 °C cold stimulus modifies cold regulation of gene expression for up to a week at 20 °C, although the primary cold effects are reverted within the first 24 h. Such memory-based regulation is called priming. Here, we analyse the effect of 24 h cold priming on cold regulation of gene expression on a transcriptome-wide scale and investigate if and how cold priming affects light regulation of gene expression. RESULTS: Cold-priming affected cold and excess light regulation of a small subset of genes. In contrast to the strong gene co-regulation observed upon cold and light stress in non-primed plants, most priming-sensitive genes were regulated in a stressor-specific manner in cold-primed plant. Furthermore, almost as much genes were inversely regulated as co-regulated by a 24 h long 4 °C cold treatment and exposure to heat-filtered high light (800 µmol quanta m- 2 s- 1). Gene ontology enrichment analysis revealed that cold priming preferentially supports expression of genes involved in the defence against plant pathogens upon cold triggering. The regulation took place on the cost of the expression of genes involved in growth regulation and transport. On the contrary, cold priming resulted in stronger expression of genes regulating metabolism and development and weaker expression of defence genes in response to high light triggering. qPCR with independently cultivated and treated replicates confirmed the trends observed in the RNASeq guide experiment. CONCLUSION: A 24 h long priming cold stimulus activates a several days lasting stress memory that controls cold and light regulation of gene expression and adjusts growth and defence regulation in a stressor-specific manner.

Cold-priming of chloroplast ROS signalling is developmentally regulated and is locally controlled at the thylakoid membrane.

24 h exposure to 4 °C primes Arabidopsis thaliana in the pre-bolting rosette stage for several days against full cold activation of the ROS responsive genes ZAT10 and BAP1 and causes stronger cold-induction of pleiotropically stress-regulated genes. Transient over-expression of thylakoid ascorbate peroxidase (tAPX) at 20 °C mimicked and tAPX transcript silencing antagonized cold-priming of ZAT10 expression. The tAPX effect could not be replaced by over-expression of stromal ascorbate peroxidase (sAPX) demonstrating that priming is specific to regulation of tAPX availability and, consequently, regulated locally at the thylakoid membrane. Arabidopsis acquired cold primability in the early rosette stage between 2 and 4 weeks. During further rosette development, primability was widely maintained in the oldest leaves. Later formed and later maturing leaves were not primable demonstrating that priming is stronger regulated with plant age than with leaf age. In 4-week-old plants, which were strongest primable, the memory was fully erasable and lost seven days after priming. In summary, we conclude that cold-priming of chloroplast-to-nucleus ROS signalling by transient post-stress induction of tAPX transcription is a strategy to modify cell signalling for some time without affecting the alertness for activation of cold acclimation responses.

Preparing plants for improved cold tolerance by priming.

Cold is a major stressor, which limits plant growth and development in many parts of the world, especially in the temperate climate zones. A large number of experimental studies has demonstrated that not only acclimation and entrainment but also the experience of single short stress events of various abiotic or biotic kinds (priming stress) can improve the tolerance of plants to chilling temperatures. This process, called priming, depends on a stress "memory". It does not change cold sensitivity per se but beneficially modifies the response to cold and can last for days, months, or even longer. Elicitor factors and antagonists accumulate due to increased biosynthesis or decreased degradation either during or after the priming stimulus. Comparison of priming studies investigating improved tolerance to chilling temperatures highlighted key regulatory functions of ROS/RNS and antioxidant enzymes, plant hormones, especially jasmonates, salicylates, and abscisic acid, and signalling metabolites, such as ß- and γ-aminobutyric acid (BABA and GABA) and melatonin. We conclude that these elicitors and antagonists modify local and systemic cold tolerance by integration into cold-induced signalling cascades.

The Plant Immunity Regulating F-Box Protein CPR1 Supports Plastid Function in Absence of Pathogens.

The redox imbalanced 6 mutant (rimb6) of Arabidopsis thaliana was isolated in a genetic screening approach for mutants with defects in chloroplast-to-nucleus redox signaling. It has an atypically low activation status of the 2-Cys peroxiredoxin-A promoter in the seedling stage. rimb6 shows wildtype-like germination, seedling development and greening, but slower growth and reduced biomass in the rosette stage. Mapping of the casual mutation revealed that rimb6 carries a single nucleotide polymorphism in the gene encoding CONSTITUTIVE EXPRESSER OF PATHOGENESIS RELATED (PR) GENES 1, CPR1 (At4g12560), leading to a premature stop codon. CPR1 is known as a repressor of pathogen signaling and regulator of microtubule organization. Allelism of rimb6 and cpr1 revealed a function of CPR1 in chloroplast stress protection. Expression studies in pathogen signaling mutants demonstrated that CPR1-mediated activation of genes for photosynthesis and chloroplast antioxidant protection is, in contrast to activation of pathogen responses, regulated independently from PAD4-controlled salicylic acid (SA) accumulation. We conclude that the support of plastid function is a basic, SA-independent function of CPR1.

Cold regulation of plastid ascorbate peroxidases serves as a priming hub controlling ROS signaling in Arabidopsis thaliana.

BACKGROUND: Short cold periods comprise a challenge to plant growth and development. Series of cold stresses improve plant performance upon a future cold stress. This effect could be provoked by priming, training or acclimation dependent hardening. Here, we compared the effect of 24 h (short priming stimulus) and of 2 week long cold-pretreatment (long priming stimulus) on the response of Arabidopsis thaliana to a single 24 h cold stimulus (triggering) after a 5 day long lag-phase, to test Arabidopsis for cold primability. RESULTS: Three types of pretreatment dependent responses were observed: (1) The CBF-regulon controlled gene COR15A was stronger activated only after long-term cold pretreatment. (2) The non-chloroplast specific stress markers PAL1 and CHS were more induced by cold after long-term and slightly stronger expressed after short-term cold priming. (3) The chloroplast ROS signaling marker genes ZAT10 and BAP1 were less activated by the triggering stimulus in primed plants. The effects on ZAT10 and BAP1 were more pronounced in 24 h cold-primed plants than in 14 day long cold-primed ones demonstrating independence of priming from induction and persistence of primary cold acclimation responses. Transcript and protein abundance analysis and studies in specific knock-out lines linked the priming-specific regulation of ZAT10 and BAP1 induction to the priming-induced long-term regulation of stromal and thylakoid-bound ascorbate peroxidase (sAPX and tAPX) expression. CONCLUSION: The plastid antioxidant system, especially, plastid ascorbate peroxidase regulation, transmits information on a previous cold stress over time without the requirement of establishing cold-acclimation. We hypothesize that the plastid antioxidant system serves as a priming hub and that priming-dependent regulation of chloroplast-to-nucleus ROS signaling is a strategy to prepare plants under unstable environmental conditions against unpredictable stresses by supporting extra-plastidic stress protection.