Chemical priming of plant defense responses to pathogen attacks.

Plants can acquire an improved resistance against pathogen attacks by exogenous application of natural or artificial compounds. In a process called chemical priming, application of these compounds causes earlier, faster and/or stronger responses to pathogen attacks. The primed defense may persist over a stress-free time (lag phase) and may be expressed also in plant organs that have not been directly treated with the compound. This review summarizes the current knowledge on the signaling pathways involved in chemical priming of plant defense responses to pathogen attacks. Chemical priming in induced systemic resistance (ISR) and systemic acquired resistance (SAR) is highlighted. The roles of the transcriptional coactivator NONEXPRESSOR OF PR1 (NPR1), a key regulator of plant immunity, induced resistance (IR) and salicylic acid signaling during chemical priming are underlined. Finally, we consider the potential usage of chemical priming to enhance plant resistance to pathogens in agriculture.

Photoperiod Stress in Arabidopsis thaliana Induces a Transcriptional Response Resembling That of Pathogen Infection.

Plants are exposed to regular diurnal rhythms of light and dark. Changes in the photoperiod by the prolongation of the light period cause photoperiod stress in short day-adapted Arabidopsis thaliana. Here, we report on the transcriptional response to photoperiod stress of wild-type A. thaliana and photoperiod stress-sensitive cytokinin signaling and clock mutants and identify a core set of photoperiod stress-responsive genes. Photoperiod stress caused altered expression of numerous reactive oxygen species (ROS)-related genes. Photoperiod stress-sensitive mutants displayed similar, but stronger transcriptomic changes than wild-type plants. The alterations showed a strong overlap with those occurring in response to ozone stress, pathogen attack and flagellin peptide (flg22)-induced PAMP triggered immunity (PTI), which have in common the induction of an apoplastic oxidative burst. Interestingly, photoperiod stress triggers transcriptional changes in jasmonic acid (JA) and salicylic acid (SA) biosynthesis and signaling and results in increased JA, SA and camalexin levels. These responses are typically observed after pathogen infections. Consequently, photoperiod stress increased the resistance of Arabidopsis plants to a subsequent infection by Pseudomonas syringae pv. tomato DC3000. In summary, we show that photoperiod stress causes transcriptional reprogramming resembling plant pathogen defense responses and induces systemic acquired resistance (SAR) in the absence of a pathogen.

The Photoperiod Stress Response in Arabidopsis thaliana Depends on Auxin Acting as an Antagonist to the Protectant Cytokinin.

Fluctuating environmental conditions trigger adaptive responses in plants, which are regulated by phytohormones. During photoperiod stress caused by a prolongation of the light period, cytokinin (CK) has a protective function. Auxin often acts as an antagonist of CK in developmental processes and stress responses. Here, we investigated the regulation of the photoperiod stress response in Arabidopsis thaliana by auxin and its interaction with CK. Transcriptome analysis revealed an altered transcript abundance of numerous auxin metabolism and signaling genes after photoperiod stress treatment. The changes appeared earlier and were stronger in the photoperiod-stress-sensitive CK receptor mutant arabidopsis histidine kinase 2 (ahk2),3 compared to wild-type plants. The concentrations of indole-3-acetic acid (IAA), IAA-Glc and IAA-Asp increased in both genotypes, but the increases were more pronounced in ahk2,3. Genetic analysis revealed that the gain-of-function YUCCA 1 (YUC1) mutant, yuc1D, displayed an increased photoperiod stress sensitivity. In contrast, a loss of the auxin receptors TRANSPORT-INHIBITOR-RESISTANT 1 (TIR1), AUXIN SIGNALING F-BOX 2 (AFB2) and AFB3 in wild-type and ahk2,3 background caused a reduced photoperiod stress response. Overall, this study revealed that auxin promotes response to photoperiod stress antagonizing the protective CK.

Characterization of CHARK, an unusual cytokinin receptor of rice.

The signal transduction of the plant hormone cytokinin is mediated by a His-to-Asp phosphorelay. The canonical cytokinin receptor consists of an extra cytoplasmic hormone binding domain named cyclase/histidine kinase associated sensory extracellular (CHASE) and cytoplasmic histidine kinase and receiver domains. In addition to classical cytokinin receptors, a different type receptor-named CHASE domain receptor serine/threonine kinase (CHARK)-is also present in rice. It contains the same ligand binding domain as other cytokinin receptors but has a predicted Ser/Thr-instead of a His-kinase domain. Bioinformatic analysis indicates that CHARK is a retrogene and a product of trans-splicing. Here, we analyzed whether CHARK can function as a bona fide cytokinin receptor. A biochemical assay demonstrated its ability to bind cytokinin. Transient expression of CHARK in protoplasts increased their response to cytokinin. Expression of CHARK in an Arabidopsis receptor double mutant complemented its growth defects and restored the ability to activate cytokinin response genes, clearly demonstrating that CHARK functions as a cytokinin receptor. We propose that the CHARK gene presents an evolutionary novelty in the cytokinin signaling system.

The Photoperiod: Handling and Causing Stress in Plants.

The photoperiod, which is the length of the light period in the diurnal cycle of 24 h, is an important environmental signal. Plants have evolved sensitive mechanisms to measure the length of the photoperiod. Photoperiod sensing enables plants to synchronize developmental processes, such as the onset of flowering, with a specific time of the year, and enables them to alleviate the impact of environmental stresses occurring at the same time every year. During the last years, the importance of the photoperiod for plant responses to abiotic and biotic stresses has received increasing attention. In this review, we summarize the current knowledge on the signaling pathways involved in the photoperiod-dependent regulation of responses to abiotic (freezing, drought, osmotic stress) and biotic stresses. A central role of GIGANTEA (GI), which is a key player in the regulation of photoperiod-dependent flowering, in stress responses is highlighted. Special attention is paid to the role of the photoperiod in regulating the redox state of plants. Furthermore, an update on photoperiod stress, which is caused by sudden alterations in the photoperiod, is given. Finally, we will review and discuss the possible use of photoperiod-induced stress as a sustainable resource to enhance plant resistance to biotic stress in horticulture.

Light acts as a stressor and influences abiotic and biotic stress responses in plants.

Light is important for plants as an energy source and a developmental signal, but it can also cause stress to plants and modulates responses to stress. Excess and fluctuating light result in photoinhibition and reactive oxygen species (ROS) accumulation around photosystems II and I, respectively. Ultraviolet light causes photodamage to DNA and a prolongation of the light period initiates the photoperiod stress syndrome. Changes in light quality and quantity, as well as in light duration are also key factors impacting the outcome of diverse abiotic and biotic stresses. Short day or shady environments enhance thermotolerance and increase cold acclimation. Similarly, shade conditions improve drought stress tolerance in plants. Additionally, the light environment affects the plants' responses to biotic intruders, such as pathogens or insect herbivores, often reducing growth-defence trade-offs. Understanding how plants use light information to modulate stress responses will support breeding strategies to enhance crop stress resilience. This review summarizes the effect of light as a stressor and the impact of the light environment on abiotic and biotic stress responses. There is a special focus on the role of the different light receptors and the crosstalk between light signalling and stress response pathways.

Photoperiod stress induces an oxidative burst-like response and is associated with increased apoplastic peroxidase and decreased catalase activities.

Periodic changes of light and dark regulate numerous processes in plants. Recently, a novel type of stress caused by an extended light period has been described in Arabidopsis thaliana and was named photoperiod stress. Although photoperiod stress causes the induction of numerous stress response genes of which many are indicators of oxidative stress, the exact timing and mechanisms involved in dealing with this stress have not yet been investigated. We describe the response of the cellular redox system in wild-type Arabidopsis, the photoperiod stress sensitive cytokinin receptor mutant ahk2 ahk3 and the clock mutant cca1 lhy. Photoperiod stress caused several changes in the ROS scavenging system including a reduction of the ascorbic acid (AsA) redox status and strong peroxide formation during the night following the extended photoperiod. The changes were associated with reduced catalase (CAT) and increased apoplastic peroxidase (PRX) activities. Consistently, the expression of the apoplastic PRX genes PRX4, PRX33, PRX34 and PRX71 was strongly induced by photoperiod stress. We show that extending the light period by only few hours causes a stress response during the following night suggesting that the photoperiod stress response might occur in a natural setting.

Root-derived trans-zeatin cytokinin protects Arabidopsis plants against photoperiod stress.

Recently, a novel type of abiotic stress caused by a prolongation of the light period-coined photoperiod stress-has been described in Arabidopsis. During the night after the prolongation of the light period, stress and cell death marker genes are induced. The next day, strongly stressed plants display a reduced photosynthetic efficiency and leaf cells eventually enter programmed cell death. The phytohormone cytokinin (CK) acts as a negative regulator of this photoperiod stress syndrome. In this study, we show that Arabidopsis wild-type plants increase the CK concentration in response to photoperiod stress. Analysis of cytokinin synthesis and transport mutants revealed that root-derived trans-zeatin (tZ)-type CKs protect against photoperiod stress. The CK signalling proteins ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 2 (AHP2), AHP3 and AHP5 and transcription factors ARABIDOPSIS RESPONSE REGULATOR 2 (ARR2), ARR10 and ARR12 are required for the protective activity of CK. Analysis of higher order B-type arr mutants suggested that a complex regulatory circuit exists in which the loss of ARR10 or ARR12 can rescue the arr2 phenotype. Together the results revealed the role of root-derived CK acting in the shoot through the two-component signalling system to protect from the negative consequences of strong photoperiod stress.

Ethylene-independent promotion of photomorphogenesis in the dark by cytokinin requires COP1 and the CDD complex.

The transition of skotomorphogenesis to photomorphogenesis is induced by the perception of light, and is characterized by the inhibition of hypocotyl elongation and opening of cotyledons. Although it is known that the plant hormone cytokinin inhibits hypocotyl elongation in dark-grown Arabidopsis plants when applied in high concentrations, it is unclear to what extent this response is the result of cytokinin alone or cytokinin-induced ethylene production. Here, we show that cytokinin-induced inhibition of hypocotyl elongation is largely independent of ethylene and suggest a close connection between the cytokinin two-component system and the light-signaling networks. We show that this cytokinin signal is mainly mediated through the cytokinin receptor ARABIDOPSIS HISTIDINE KINASE3 and the ARABIDOPSIS RESPONSE REGULATOR1 in combination with ARR12. Interestingly, mutation of CONSTITUTIVELY PHOTOMORPOGENIC1 (COP1), DE-ETIOLATED1, and CYTOKININ INSENSITIVE4/COP10 renders plants insensitive to cytokinin, and these factors are indispensable for the transcriptional response during cytokinin-induced de-etiolation, indicating that a functional light-signaling pathway is essential for this cytokinin response. In addition, the effect of cytokinin on hypocotyl elongation is strongly dependent on the light conditions, with higher light intensities causing a switch in the response to cytokinin from an inhibitor to a promoter of hypocotyl elongation.

Cytokinin action in response to abiotic and biotic stresses in plants.

The phytohormone cytokinin was originally discovered as a regulator of cell division. Later, it was described to be involved in regulating numerous processes in plant growth and development including meristem activity, tissue patterning, and organ size. More recently, diverse functions for cytokinin in the response to abiotic and biotic stresses have been reported. Cytokinin is required for the defence against high light stress and to protect plants from a novel type of abiotic stress caused by an altered photoperiod. Additionally, cytokinin has a role in the response to temperature, drought, osmotic, salt, and nutrient stress. Similarly, the full response to certain plant pathogens and herbivores requires a functional cytokinin signalling pathway. Conversely, different types of stress impact cytokinin homeostasis. The diverse functions of cytokinin in responses to stress and crosstalk with other hormones are described. Its emerging roles as a priming agent and as a regulator of growth-defence trade-offs are discussed.

Novel Stress in Plants by Altering the Photoperiod.

Recent work has shown that changing the photoperiod induces stress in Arabidopsis thaliana. It has particularly dramatic consequences in cytokinin-deficient plants and clock mutants. Here, we argue that studying the impact of an altered photoperiod will provide novel insights into the circadian clock, factors regulating it, and pathways under its control.

Analysis of CFB, a cytokinin-responsive gene of Arabidopsis thaliana encoding a novel F-box protein regulating sterol biosynthesis.

Protein degradation by the ubiquitin-26S proteasome pathway is important for the regulation of cellular processes, but the function of most F-box proteins relevant to substrate recognition is unknown. We describe the analysis of the gene Cytokinin-induced F-box encoding (CFB, AT3G44326), identified in a meta-analysis of cytokinin-related transcriptome studies as one of the most robust cytokinin response genes. F-box domain-dependent interaction with the E3 ubiquitin ligase complex component ASK1 classifies CFB as a functional F-box protein. Apart from F-box and transmembrane domains, CFB contains no known functional domains. CFB is expressed in all plant tissues, predominantly in root tissue. A ProCFB:GFP-GUS fusion gene showed strongest expression in the lateral root cap and during lateral root formation. CFB-GFP fusion proteins were mainly localized in the nucleus and the cytosol but also at the plasma membrane. cfb mutants had no discernible phenotype, but CFB overexpressing plants showed several defects, such as a white upper inflorescence stem, similar to the hypomorphic cycloartenol synthase mutant cas1-1. Both CFB overexpressing plants and cas1-1 mutants accumulated the CAS1 substrate 2,3-oxidosqualene in the white stem tissue, the latter even more after cytokinin treatment, indicating impairment of CAS1 function. This suggests that CFB may link cytokinin and the sterol biosynthesis pathway.

Cytokinin Regulates the Etioplast-Chloroplast Transition through the Two-Component Signaling System and Activation of Chloroplast-Related Genes.

One of the classical functions of the plant hormone cytokinin is the regulation of plastid development, but the underlying molecular mechanisms remain elusive. In this study, we employed a genetic approach to evaluate the role of cytokinin and its signaling pathway in the light-induced development of chloroplasts from etioplasts in Arabidopsis (Arabidopsis thaliana). Cytokinin increases the rate of greening and stimulates ultrastructural changes characteristic for the etioplast-to-chloroplast transition. The steady-state levels of metabolites of the tetrapyrrole biosynthesis pathway leading to the production of chlorophyll are enhanced by cytokinin. This effect of cytokinin on metabolite levels arises due to the modulation of expression for chlorophyll biosynthesis genes such as HEMA1, GUN4, GUN5, and CHLM Increased expression of HEMA1 is reflected in an enhanced level of the encoded glutamyl-tRNA reductase, which catalyzes one of the rate-limiting steps of chlorophyll biosynthesis. Mutant analysis indicates that the cytokinin receptors ARABIDOPSIS HIS KINASE2 (AHK2) and AHK3 play a central role in this process. Furthermore, the B-type ARABIDOPSIS RESPONSE REGULATOR1 (ARR1), ARR10, and ARR12 play an important role in mediating the transcriptional output during etioplast-chloroplast transition. B-type ARRs bind to the promotors of HEMA1 and LHCB6 genes, indicating that cytokinin-dependent transcription factors directly regulate genes of chlorophyll biosynthesis and the light harvesting complex. Together, these results demonstrate an important role for the cytokinin signaling pathway in chloroplast development, with the direct transcriptional regulation of chlorophyll biosynthesis genes as a key aspect for this hormonal control.

Circadian Stress Regimes Affect the Circadian Clock and Cause Jasmonic Acid-Dependent Cell Death in Cytokinin-Deficient Arabidopsis Plants.

The circadian clock helps plants measure daylength and adapt to changes in the day-night rhythm. We found that changes in the light-dark regime triggered stress responses, eventually leading to cell death, in leaves of Arabidopsis thaliana plants with reduced cytokinin levels or defective cytokinin signaling. Prolonged light treatment followed by a dark period induced stress and cell death marker genes while reducing photosynthetic efficiency. This response, called circadian stress, is also characterized by altered expression of clock and clock output genes. In particular, this treatment strongly reduced the expression of CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY). Intriguingly, similar changes in gene expression and cell death were observed in clock mutants lacking proper CCA1 and LHY function. Circadian stress caused strong changes in reactive oxygen species- and jasmonic acid (JA)-related gene expression. The activation of the JA pathway, involving the accumulation of JA metabolites, was crucial for the induction of cell death, since the cell death phenotype was strongly reduced in the jasmonate resistant1 mutant background. We propose that adaptation to circadian stress regimes requires a normal cytokinin status which, acting primarily through the AHK3 receptor, supports circadian clock function to guard against the detrimental effects of circadian stress.

Regulation of chloroplast development and function by cytokinin.

A role of the plant hormone cytokinin in regulating the development and activity of chloroplasts was described soon after its discovery as a plant growth regulator more than 50 years ago. Its promoting action on chloroplast ultrastructure and chlorophyll synthesis has been reported repeatedly, especially during etioplast-to-chloroplast transition. Recently, a protective role of the hormone for the photosynthetic apparatus during high light stress was shown. Details about the molecular mechanisms of cytokinin action on plastids are accumulating from genetic and transcriptomic studies. The cytokinin receptors AHK2 and AHK3 are mainly responsible for the transduction of the cytokinin signal to B-type response regulators, in particular ARR1, ARR10, and ARR12, which are transcription factors of the two-component system mediating cytokinin functions. Additional transcription factors linking cytokinin and chloroplast development include CGA1, GNC, HY5, GLK2, and CRF2. In this review, we summarize early and more recent findings of the long-known relationship between the hormone and the organelle and describe crosstalk between cytokinin, light, and other hormones during chloroplast development.

A novel protective function for cytokinin in the light stress response is mediated by the Arabidopsis histidine kinase2 and Arabidopsis histidine kinase3 receptors.

Cytokinins are plant hormones that regulate diverse processes in plant development and responses to biotic and abiotic stresses. In this study, we show that Arabidopsis (Arabidopsis thaliana) plants with a reduced cytokinin status (i.e. cytokinin receptor mutants and transgenic cytokinin-deficient plants) are more susceptible to light stress compared with wild-type plants. This was reflected by a stronger photoinhibition after 24 h of high light (approximately 1,000 µmol m(-2) s(-1)), as shown by the decline in maximum quantum efficiency of photosystem II photochemistry. Photosystem II, especially the D1 protein, is highly sensitive to the detrimental impact of light. Therefore, photoinhibition is always observed when the rate of photodamage exceeds the rate of D1 repair. We demonstrate that in plants with a reduced cytokinin status, the D1 protein level was strongly decreased upon light stress. Inhibition of the D1 repair cycle by lincomycin treatment indicated that these plants experience stronger photodamage. The efficiency of photoprotective mechanisms, such as nonenzymatic and enzymatic scavenging systems, was decreased in plants with a reduced cytokinin status, which could be a cause for the increased photodamage and subsequent D1 degradation. Additionally, slow and incomplete recovery in these plants after light stress indicated insufficient D1 repair. Mutant analysis revealed that the protective function of cytokinin during light stress depends on the Arabidopsis histidine KINASE2 (AHK2) and AHK3 receptors and the type B Arabidopsis response regulator1 (ARR1) and ARR12. We conclude that proper cytokinin signaling and regulation of specific target genes are necessary to protect leaves efficiently from light stress.

Evaluation of the photosynthetic activity in transgenic tobacco plants with altered endogenous cytokinin content: lessons from cytokinin.

Cytokinin is known to be involved in many processes related to plastid development and function but the exact role of cytokinin in photosynthesis remains elusive. To investigate more profoundly the effects of cytokinin in this process, the photosynthetic activity of transgenic Pssuipt and 35S:CKX1 tobacco (Nicotiana tabacum) plants with respectively elevated and reduced endogenous cytokinin content was evaluated. Pigment analysis indicated that elevated endogenous cytokinin content resulted in increased pigment content. Functional analysis of the photosynthetic apparatus by chlorophyll a fluorescence and in vitro electron transport measurements clearly showed that changing the endogenous cytokinin content affects the activity of the photosynthetic apparatus. Surprisingly, both an increase as well as a decrease in cytokinin content results in a better photosynthetic performance. Quenching analysis revealed that the initial responses of the photosynthetic apparatus on a dark-light transition are not affected by changed cytokinin content. However, it has an effect on the further kinetic behavior. Taken together, we suggest that cytokinins can induce structural changes in the different parts of the electron transport chain as also demonstrated by the in vitro electron transport measurements.

Analysis of the photosynthetic apparatus in transgenic tobacco plants with altered endogenous cytokinin content: a proteomic study.

BACKGROUND: Cytokinin is a plant hormone that plays a crucial role in several processes of plant growth and development. In recent years, major breakthroughs have been achieved in the elucidation of the metabolism, the signal perception and transduction, as well as the biological functions of cytokinin. An important activity of cytokinin is the involvement in chloroplast development and function. Although this biological function has already been known for 50 years, the exact mechanisms remain elusive. RESULTS: To elucidate the effects of altered endogenous cytokinin content on the structure and function of the chloroplasts, chloroplast subfractions (stroma and thylakoids) from transgenic Pssu-ipt and 35S:CKX1 tobacco (Nicotiana tabacum) plants with, respectively, elevated and reduced endogenous cytokinin content were analysed using two different 2-DE approaches. Firstly, thykaloids were analysed by blue-native polyacrylamide gel electrophoresis followed by SDS-PAGE (BN/SDS-PAGE). Image analysis of the gel spot pattern thus obtained from thylakoids showed no substantial differences between wild-type and transgenic tobacco plants. Secondly, a quantitative DIGE analysis of CHAPS soluble proteins derived from chloroplast subfractions indicated significant gel spot abundance differences in the stroma fraction. Upon identification by MALDI-TOF/TOF mass spectrometry, these proteins could be assigned to the Calvin-Benson cycle and photoprotective mechanisms. CONCLUSION: Taken together, presented proteomic data reveal that the constitutively altered cytokinin status of transgenic plants does not result in any qualitative changes in either stroma proteins or protein complexes of thylakoid membranes of fully developed chloroplasts, while few but significant quantitative differences are observed in stroma proteins.

Selection of plastid- and nuclear-encoded reference genes to study the effect of altered endogenous cytokinin content on photosynthesis genes in Nicotiana tabacum.

Selection and use of appropriate reference genes as internal controls in real-time reverse transcription PCR (RT-PCR) assays is highly important for accurate quantification of gene expression levels. Since some photosynthetic genes are encoded in the nuclear genome and others in the chloroplast genome, we evaluated both nuclear- and plastid-encoded candidate reference genes. Six plastid-encoded candidate reference genes were derived from Arabidopsis microarray data and three plastid- and five nuclear-encoded reference genes were derived from literature. Cytokinins influence photosynthetic gene expression, so we evaluated the expression stability of the candidate reference genes in transgenic Nicotiana tabacum plants with elevated or diminished cytokinin content. We found that the most reliable strategy makes use of plastid-encoded genes for normalizing plastid photosynthetic genes and nuclear-encoded reference genes for normalizing nuclear photosynthetic genes. Compared to the use of nuclear reference genes only, this approach assimilates any effects on transcriptional activity of chloroplasts or number of chloroplast. The best expression stabilities in Nicotiana tabacum were observed for the plastid-encoded references genes Nt-RPS3, Nt-NDHI and Nt-IN1 and for the nuclear-encoded genes Nt-ACT9, Nt-alphaTUB and Nt-SSU. These genes may be suitable for normalization of photosynthetic genes under other experimental conditions in Nicotiana tabacum, and orthologues of these genes may be suitable candidates for normalizing photosynthetic gene expression in other species.