CYTOKININ RESPONSE FACTOR 2 is involved in modulating the salt stress response.

Cytokinin has strong connections to development and a growing role in the abiotic stress response. Here we show that CYTOKININ RESPONSE FACTOR 2 (CRF2) is additionally involved in the salt (NaCl) stress response. CRF2 promoter-GUS expression indicates CRF2 involvement in the response to salt stress as well as the previously known cytokinin response. Interestingly, CRF2 mutant seedlings are quite similar to the wild type (WT) under non-stressed conditions yet have many distinct changes in response to salt stress. Cytokinin levels measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) that increased in the WT after salt stress are decreased in crf2, potentially from CRF2 regulation of cytokinin biosynthesis genes. Ion content measured by inductively coupled plasma optical emission spectrometry (ICP-OES) was increased in the WT for Na, K, Mn, Ca and Mg after salt stress, whereas the corresponding Ca and Mg increases are lacking in crf2. Many genes examined by RNA-seq analysis were altered transcriptionally by salt stress in both the WT and crf2, yet interestingly approximately one-third of salt-modified crf2 transcripts (2655) showed unique regulation. Different transcript profiles for salt stress in crf2 compared with the WT background was further supported through an examination of co-expressed genes by weighted gene correlation network analysis (WGCMA) and principal component analysis (PCA). Additionally, Gene Ontology (GO) enrichment terms found from salt-treated transcripts revealed most photosynthesis-related terms as only being affected in crf2, leading to an examination of chlorophyll levels and the efficiency of photosystem II (via the ratio of variable fluorescence to maximum fluorescence, Fv /Fm ) as well as physiology after salt treatment. Salt stress-treated crf2 plants had both reduced chlorophyll levels and lower Fv /Fm values compared with the WT, suggesting that CRF2 plays a role in the modulation of salt stress responses linked to photosynthesis.

Three Strains of Tobacco etch virus Distinctly Alter the Transcriptome of Apical Stem Tissue in Capsicum annuum during Infection.

Tobacco etch virus (TEV; genus Potyvirus) is flexuous rod shaped with a single molecule of single-stranded RNA and causes serious yield losses in species in the Solanaceae. Three TEV strains (HAT, Mex21, and N) are genetically distinct and cause different disease symptoms in plants. Here, a transcriptomic RNA sequencing approach was taken for each TEV strain to evaluate gene expression of the apical stem segment of pepper plants during two stages of disease development. Distinct profiles of Differentially Expressed Genes (DEGs) were identified for each TEV strain. DEG numbers increased with degree of symptom severity: 24 from HAT, 1190 from Mex21, and 4010 from N. At 7 days post-inoculation (dpi), when systemic symptoms were similar, there were few DEGs for HAT- and Mex21-infected plants, whereas N-infected plants had 2516 DEGs. DEG patterns from 7 to 14 dpi corresponded to severity of disease symptoms: milder disease with smaller DEG changes for HAT and Mex21 and severe disease with larger DEG changes for N. Strikingly, in each of these comparisons, there are very few overlapping DEGs among the TEV strains, including no overlapping DEGs between all three strains at 7 or 14 dpi.

Clade III cytokinin response factors share common roles in response to oxidative stress responses linked to cytokinin synthesis.

Cytokinin response factors (CRFs) are transcription factors that are involved in cytokinin (CK) response, as well as being linked to abiotic stress tolerance. In particular, oxidative stress responses are activated by Clade III CRF members, such as AtCRF6. Here we explored the relationships between Clade III CRFs and oxidative stress. Transcriptomic responses to oxidative stress were determined in two Clade III transcription factors, Arabidopsis AtCRF5 and tomato SlCRF5. AtCRF5 was required for regulated expression of >240 genes that are involved in oxidative stress response. Similarly, SlCRF5 was involved in the regulated expression of nearly 420 oxidative stress response genes. Similarities in gene regulation by these Clade III members in response to oxidative stress were observed between Arabidopsis and tomato, as indicated by Gene Ontology term enrichment. CK levels were also changed in response to oxidative stress in both species. These changes were regulated by Clade III CRFs. Taken together, these findings suggest that Clade III CRFs play a role in oxidative stress response as well as having roles in CK signaling.

Cytokinin N-glucosides: Occurrence, Metabolism and Biological Activities in Plants.

Cytokinins (CKs) are a class of phytohormones affecting many aspects of plant growth and development. In the complex process of CK homeostasis in plants, N-glucosylation represents one of the essential metabolic pathways. Its products, CK N7- and N9-glucosides, have been largely overlooked in the past as irreversible and inactive CK products lacking any relevant physiological impact. In this work, we report a widespread distribution of CK N-glucosides across the plant kingdom proceeding from evolutionary older to younger plants with different proportions between N7- and N9-glucosides in the total CK pool. We show dramatic changes in their profiles as well as in expression levels of the UGT76C1 and UGT76C2 genes during Arabidopsis ontogenesis. We also demonstrate specific physiological effects of CK N-glucosides in CK bioassays including their antisenescent activities, inhibitory effects on root development, and activation of the CK signaling pathway visualized by the CK-responsive YFP reporter line, TCSv2::3XVENUS. Last but not least, we present the considerable impact of CK N7- and N9-glucosides on the expression of CK-related genes in maize and their stimulatory effects on CK oxidase/dehydrogenase activity in oats. Our findings revise the apparent irreversibility and inactivity of CK N7- and N9-glucosides and indicate their involvement in CK evolution while suggesting their unique function(s) in plants.

Cytokinin isopentenyladenine and its glucoside isopentenyladenine-9G delay leaf senescence through activation of cytokinin-associated genes.

Cytokinins (CKs) are well-known as a class of phytohormones capable of delaying senescence in detached leaves. However, CKs are often treated as a monolithic group of compounds even though dozens of CK species are present in plants with varied degrees of reported biological activity. One specific type of CK, isopentenyladenine base (iP), has been demonstrated as having roles in delaying leaf senescence, inhibition of root growth, and promoting shoot regeneration. However, its N-glucosides isopentenyladenine-7- and -9-glucoside (iP7G, iP9G) have remained understudied and thought of as inactive cytokinins for several decades, despite their relatively high concentrations in plants such as the model species Arabidopsis thaliana. Here we show that iP and one of its glucosides, iP9G, are capable of delaying senescence in leaves, though the glucosides having little to no activity in other bioassays. Additionally, we performed the first transcriptomic study of iP-delayed cotyledon senescence which shows that iP is capable of upregulating photosynthetic genes and downregulating catabolic genes in detached cotyledons. Transcriptomic analysis also shows iP9G has limited effects on gene expression, but that the few affected genes are CK-related and are similar to those seen from iP effects during senescence as seen for the type-A response regulator ARR6. These findings suggest that iP9G functions as an active CK during senescence.

trans-Zeatin-N-glucosides have biological activity in Arabidopsis thaliana.

Cytokinin is an indispensable phytohormone responsible for physiological processes ranging from root development to leaf senescence. The term "cytokinin" refers to several dozen adenine-derived compounds occurring naturally in plants. Cytokinins (CKs) can be divided into various classes and forms; base forms are generally considered to be active while highly abundant cytokinin-N-glucosides (CKNGs), composed of a CK base irreversibly conjugated to a glucose molecule, are considered inactive. However, results from early CK studies suggest CKNGs do not always lack activity despite the perpetuation over several decades in the literature that they are inactive. Here we show that exogenous application of trans-Zeatin-N-glucosides (tZNGs, a specific class of CKNGs) to Arabidopsis results in CK response comparable to the application of an active CK base. These results are most apparent in senescence assays where both a CK base (tZ) and tZNGs (tZ7G, tZ9G) delay senescence in cotyledons. Further experiments involving root growth and shoot regeneration revealed tZNGs do not always have the same effects as tZ, and have largely distinct effects on the transcriptome and proteome. These data are in contrast to previous reports of CKNGs being inactive and raise questions about the function of these compounds as well as their mechanism of action.

Review - Cytokinin Response Factors: Responding to more than cytokinin.

Cytokinin Response Factors (CRFs) are a family of transcription factors which make up a side branch of the classical cytokinin two-component signaling pathway. CRFs were originally identified and have been primarily studied in Arabidopsis thaliana, although orthologs have be found throughout all land plants. Research into the evolution of CRFs as sub-group members of the larger APETALA2/Ethylene Response Factor (AP2/ERF) family has yielded interesting and useful insights related to the functional roles of CRFs in plants. Recent studies of CRFs suggest that these transcription factors are a lot more than just a group of cytokinin related genes and play important roles in both plant development and environmental stress response. This review focuses on recent advances in understanding the roles of CRFs beyond cytokinin, in reproductive development and abiotic stress response, as well as to other environmental cues.

Salt and oxidative stresses uniquely regulate tomato cytokinin levels and transcriptomic response.

Cytokinins are well-known to be involved in processes responsible for plant growth and development. More recently, these hormones have begun to be associated with stress responses as well. However, it is unclear how changes in cytokinin biosynthesis, signaling, or transport relate to stress effects. This study examines in parallel how two different stresses, salt, and oxidative stress, affect changes in both cytokinin levels and whole plant transcriptome response. Solanum lycopersicum seedlings were given a short-term (6 hr) exposure to either salt (150 mM NaCl) or oxidative (20 mM H2O2) stress and then examined to determine both changes in cytokinin levels and transcriptome. LC-MS/MS was used to determine the levels of 22 different types of cytokinins in tomato plants including precursors, active, transported, and conjugated forms. When examining cytokinin levels we found that salt treatment caused an increase in both active and inactive cytokinin levels and oxidative stress caused a decrease in these levels. RNA-sequencing analyses of these same stress-treated tissues revealed 6,643 significantly differentially expressed genes (DEGs). Although many DEGs are similar between the two stresses, approximately one-third of the DEGs in each treatment were unique to that stress. Several cytokinin-related genes were among the DEGs. Examination of photosystem II efficiency revealed that cytokinins affect physiological response to stress in tomato, further validating the changes in cytokinin levels seen in planta.

Cytokinin Response Factor 6 Represses Cytokinin-Associated Genes during Oxidative Stress.

Cytokinin is a phytohormone that is well known for its roles in numerous plant growth and developmental processes, yet it has also been linked to abiotic stress response in a less defined manner. Arabidopsis (Arabidopsis thaliana) Cytokinin Response Factor 6 (CRF6) is a cytokinin-responsive AP2/ERF-family transcription factor that, through the cytokinin signaling pathway, plays a key role in the inhibition of dark-induced senescence. CRF6 expression is also induced by oxidative stress, and here we show a novel function for CRF6 in relation to oxidative stress and identify downstream transcriptional targets of CRF6 that are repressed in response to oxidative stress. Analysis of transcriptomic changes in wild-type and crf6 mutant plants treated with H2O2 identified CRF6-dependent differentially expressed transcripts, many of which were repressed rather than induced. Moreover, many repressed genes also show decreased expression in 35S:CRF6 overexpressing plants. Together, these findings suggest that CRF6 functions largely as a transcriptional repressor. Interestingly, among the H2O2 repressed CRF6-dependent transcripts was a set of five genes associated with cytokinin processes: (signaling) ARR6, ARR9, ARR11, (biosynthesis) LOG7, and (transport) ABCG14. We have examined mutants of these cytokinin-associated target genes to reveal novel connections to oxidative stress. Further examination of CRF6-DNA interactions indicated that CRF6 may regulate its targets both directly and indirectly. Together, this shows that CRF6 functions during oxidative stress as a negative regulator to control this cytokinin-associated module of CRF6-dependent genes and establishes a novel connection between cytokinin and oxidative stress response.