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.

CEP peptide and cytokinin pathways converge on CEPD glutaredoxins to inhibit root growth.

C-TERMINALLY ENCODED PEPTIDE (CEP) and cytokinin hormones act over short and long distances to control plant responses to environmental cues. CEP and cytokinin pathway mutants share phenotypes, however, it is not known if these pathways intersect. We show that CEP and cytokinin signalling converge on CEP DOWNSTREAM (CEPD) glutaredoxins to inhibit primary root growth. CEP inhibition of root growth was impaired in mutants defective in trans-zeatin (tZ)-type cytokinin biosynthesis, transport, perception, and output. Concordantly, mutants affected in CEP RECEPTOR 1 showed reduced root growth inhibition in response to tZ, and altered levels of tZ-type cytokinins. Grafting and organ-specific hormone treatments showed that tZ-mediated root growth inhibition involved CEPD activity in roots. By contrast, root growth inhibition by CEP depended on shoot CEPD function. The results demonstrate that CEP and cytokinin pathways intersect, and utilise signalling circuits in separate organs involving common glutaredoxin genes to coordinate root growth.

A Constitutively Active Cytokinin Receptor Variant Increases Cambial Activity and Stem Growth in Poplar.

The cambial meristem is responsible for bark and wood formation in woody plants. The activity of the cambial meristem is controlled by various factors; one of them is the plant hormone cytokinin. Here, we have explored different approaches to genetically engineering cambial activity in poplar plants by the ectopic expression of a cytokinin biosynthesis gene with enhanced activity (named ROCK4) or of a gene encoding a constitutively active cytokinin receptor variant (ROCK3). Both genes are derived from Arabidopsis thaliana and were expressed in poplar trees under the control of their own promoter or the cambium-specific pHB8 promoter. pIPT3:ROCK4- and pHB8:ROCK4-expressing plants were smaller than wild-type plants and formed more lateral branches; pHB8:ROCK4 transgenic plants additionally showed an increased stem diameter. In contrast, pAHK3:ROCK3- and pHB8:ROCK3-expressing plants grew taller than wild type without an altered branching pattern and formed more cambial cells, leading to increased radial stem growth. The effectivity of ROCK3 when expressed in either secondary phloem cells or in cambial cells is consistent with a dual, tissue-autonomous and non-autonomous activity of cytokinin in regulating cambial activity. We propose ROCK3 as a novel gene to enhance biomass formation in woody plants.

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.

Increased branching independent of strigolactone in cytokinin oxidase 2-overexpressing tomato is mediated by reduced auxin transport.

Tomato production is influenced by shoot branching, which is controlled by different hormones. Here we produced tomato plants overexpressing the cytokinin-deactivating gene CYTOKININ OXYDASE 2 (CKX2). CKX2-overexpressing (CKX2-OE) plants showed an excessive growth of axillary shoots, the opposite phenotype expected for plants with reduced cytokinin content, as evidenced by LC-MS analysis and ARR5-GUS staining. The TCP transcription factor SlBRC1b was downregulated in the axillary buds of CKX2-OE and its excessive branching was dependent on a functional version of the GRAS-family gene LATERAL SUPPRESSOR (LS). Grafting experiments indicated that increased branching in CKX2-OE plants is unlikely to be mediated by root-derived signals. Crossing CKX2-OE plants with transgenic antisense plants for the strigolactone biosynthesis gene CAROTENOID CLEAVAGE DIOXYGENASE (CCD7-AS) produced an additive phenotype, indicating independent effects of cytokinin and strigolactones on increased branching. On the other hand, CKX2-OE plants showed reduced polar auxin transport and their bud outgrowth was reduced when combined with auxin mutants. Accordingly, CKX2-OE basal buds did not respond to auxin applied in the decapitated apex. Our results suggest that tomato shoot branching depends on a fine-tuning of different hormonal balances and that perturbations in the auxin status could compensate for the reduced cytokinin levels in CKX2-OE plants.

On the biological activity of cytokinin free bases and their ribosides.

MAIN CONCLUSION: The free bases of cytokinins are the biologically active forms of the hormone while cytokinin ribosides become active only upon removal of the ribose residue. Cytokinins (CKs) belong to the classical plant hormones. They were discovered more than 65 years ago, but which molecular forms possess genuine CK activity is still matter of debate. Numerous studies support the view that only the free bases are the biologically active molecules. This standpoint has been challenged in a recent review (Nguyen et al. in Planta 254: 45, 2021) proposing that also CK ribosides may have genuine own CK activity. Here we critically discuss the pros and cons of this viewpoint considering the results of biological assays, CK binding studies, 3D structural data of CK-receptor interaction and mutant analyses. It is concluded that all types of study provide clear and convincing evidence only for biological activity of free bases and not ribosides; the latter are rather a transport form of the hormone without their own biological activity.

Cytokinin regulates vegetative phase change in Arabidopsis thaliana through the miR172/TOE1-TOE2 module.

During vegetative growth plants pass from a juvenile to an adult phase causing changes in shoot morphology. This vegetative phase change is primarily regulated by the opposite actions of two microRNAs, the inhibitory miR156 and the promoting miR172 as well as their respective target genes, constituting the age pathway. Here we show that the phytohormone cytokinin promotes the juvenile-to-adult phase transition through regulating components of the age pathway. Reduction of cytokinin signalling substantially delayed the transition to the adult stage. tZ-type cytokinin was particularly important as compared to iP- and the inactive cZ-type cytokinin, and root-derived tZ influenced the phase transition significantly. Genetic and transcriptional analyses indicated the requirement of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors and miR172 for cytokinin activity. Two miR172 targets, TARGET OF EAT1 (TOE1) and TOE2 encoding transcriptional repressors were necessary and sufficient to mediate the influence of cytokinin on vegetative phase change. This cytokinin pathway regulating plant aging adds to the complexity of the regulatory network controlling the juvenile-to-adult phase transition and links cytokinin to miRNA action.

Meeting at the DNA: Specifying Cytokinin Responses through Transcription Factor Complex Formation.

Cytokinin is a plant hormone regulating numerous biological processes. Its diverse functions are realized through the expression control of specific target genes. The transcription of the immediate early cytokinin target genes is regulated by type-B response regulator proteins (RRBs), which are transcription factors (TFs) of the Myb family. RRB activity is controlled by phosphorylation and protein degradation. Here, we focus on another step of regulation, the interaction of RRBs among each other or with other TFs to form active or repressive TF complexes. Several examples in Arabidopsis thaliana illustrate that RRBs form homodimers or complexes with other TFs to specify the cytokinin response. This increases the variability of the output response and provides opportunities of crosstalk between the cytokinin signaling pathway and other cellular signaling pathways. We propose that a targeted approach is required to uncover the full extent and impact of RRB interaction with other TFs.

Root engineering in maize by increasing cytokinin degradation causes enhanced root growth and leaf mineral enrichment.

KEY MESSAGE: Root-specific expression of a cytokinin-degrading CKX gene in maize roots causes formation of a larger root system leading to higher element content in shoot organs. The size and architecture of the root system is functionally relevant for the access to water and soil nutrients. A great number of mostly unknown genes are involved in regulating root architecture complicating targeted breeding of plants with a larger root system. Here, we have explored whether root-specific degradation of the hormone cytokinin, which is a negative regulator of root growth, can be used to genetically engineer maize (Zea mays L.) plants with a larger root system. Root-specific expression of a CYTOKININ OXIDASE/DEHYDROGENASE (CKX) gene of Arabidopsis caused the formation of up to 46% more root dry weight while shoot growth of these transgenic lines was similar as in non-transgenic control plants. The concentration of several elements, in particular of those with low soil mobility (K, P, Mo, Zn), was increased in leaves of transgenic lines. In kernels, the changes in concentration of most elements were less pronounced, but the concentrations of Cu, Mn and Zn were significantly increased in at least one of the three independent lines. Our data illustrate the potential of an increased root system as part of efforts towards achieving biofortification. Taken together, this work has shown that root-specific expression of a CKX gene can be used to engineer the root system of maize and alter shoot element composition.

The Cytokinin Status of the Epidermis Regulates Aspects of Vegetative and Reproductive Development in Arabidopsis thaliana.

The epidermal cell layer of plants has important functions in regulating plant growth and development. We have studied the impact of an altered epidermal cytokinin metabolism on Arabidopsis shoot development. Increased epidermal cytokinin synthesis or breakdown was achieved through expression of the cytokinin synthesis gene LOG4 and the cytokinin-degrading CKX1 gene, respectively, under the control of the epidermis-specific AtML1 promoter. During vegetative growth, increased epidermal cytokinin production caused an increased size of the shoot apical meristem and promoted earlier flowering. Leaves became larger and the shoots showed an earlier juvenile-to-adult transition. An increased cytokinin breakdown had the opposite effect on these phenotypic traits indicating that epidermal cytokinin metabolism can be a factor regulating these aspects of shoot development. The phenotypic consequences of abbreviated cytokinin signaling in the epidermis achieved through expression of the ARR1-SRDX repressor were generally milder or even absent indicating that the epidermal cytokinin acts, at least in part, cell non-autonomously. Enhanced epidermal cytokinin synthesis delayed cell differentiation during leaf development leading to an increased cell proliferation and leaf growth. Genetic analysis showed that this cytokinin activity was mediated mainly by the AHK3 receptor and the transcription factor ARR1. We also demonstrate that epidermal cytokinin promotes leaf growth in a largely cell-autonomous fashion. Increased cytokinin synthesis in the outer layer of reproductive tissues and in the placenta enhanced ovule formation by the placenta and caused the formation of larger siliques. This led to a higher number of seeds in larger pods resulting in an increased seed yield per plant. Collectively, the results provide evidence that the cytokinin metabolism in the epidermis is a relevant parameter determining vegetative and reproductive plant growth and development.

Opening Doors for Cytokinin Trafficking at the ER Membrane.

The recent discovery of cytokinin transporters in the endoplasmic reticulum (ER) membrane provides a missing link to understand cellular cytokinin trafficking and signaling. Along with cytokinin receptors and metabolic enzymes previously found in the ER, these transporters complement the ER-confined infrastructure required for cytokinin signal generation and modulation.

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.

Cytokinin regulates the activity of the inflorescence meristem and components of seed yield in oilseed rape.

The number of flowers and seed-bearing structures formed by the inflorescence meristem and the formation of ovules in the female reproductive part of the flowers are important yield-related traits of crop plants. It has been shown that cytokinin is a pivotal factor regulating these traits. Here, we explore the impact of mutation of CYTOKININ OXIDASE/DEHYDROGENASE (CKX) genes encoding cytokinin-degrading enzymes on these yield-related traits in oilseed rape (Brassica napus L.). We describe the identification of four BnCKX3 and two BnCKX5 genes as regulators of reproductive development in the allotetraploid B. napus. RNA-seq analysis and in situ hybridization showed expression of these genes in reproductive organs. Loss-of-function mutants for each of these CKX gene copies were identified by targeting induced local lesions in genomes (TILLING) and combined by crossing. Sextuple ckx3 ckx5 mutants showed an increased cytokinin concentration and larger and more active inflorescence meristems. They also produced up to 72% more flowers with gynoecia containing 32% more ovules and up to 54% more pods on the main stem. The weight of seeds harvested from the main stem of plants grown in the greenhouse or in the field was increased by 20-32%. Our results show that cytokinin regulates inflorescence meristem and placenta activity in oilseed rape. The work demonstrates the potential to achieve yield enhancement in a dicot crop plant by modulating the cytokinin status through mutagenesis of specific CKX genes.

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.

Characterisation of the ERF102 to ERF105 genes of Arabidopsis thaliana and their role in the response to cold stress.

KEY MESSAGE: The four phylogenetically closely related ERF102 to ERF105 transcription factors of Arabidopsis thaliana are regulated by different stresses and are involved in the response to cold stress. The ETHYLENE RESPONSE FACTOR (ERF) genes of Arabidopsis thaliana form a large family encoding plant-specific transcription factors. Here, we characterise the four phylogenetically closely related ERF102/ERF5, ERF103/ERF6, ERF104 and ERF105 genes. Expression analyses revealed that these four genes are similarly regulated by different hormones and abiotic stresses. Analyses of tissue-specific expression using promoter:GUS reporter lines revealed their predominant expression in root tissues including the root meristem (ERF103), the quiescent center (ERF104) and the root vasculature (all). All GFP-ERF fusion proteins were nuclear-localised. The analysis of insertional mutants, amiRNA lines and 35S:ERF overexpressing transgenic lines indicated that ERF102 to ERF105 have only a limited impact on regulating shoot and root growth. Previous work had shown a role for ERF105 in the cold stress response. Here, measurement of electrolyte leakage to determine leaf freezing tolerance and expression analyses of cold-responsive genes revealed that the combined activity of ERF102 and ERF103 is also required for a full cold acclimation response likely involving the CBF regulon. These results suggest a common function of these ERF genes in the response to cold stress.