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.

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.

Plant cytokinin signalling.

Cytokinin is an essential plant hormone that is involved in a wide range of plant growth and developmental processes which are controlled through its signalling pathway. Cytokinins are a class of molecules that are N(6)-substituted adenine derivatives, such as isopentenyl adenine, and trans- and cis-zeatin, which are common in most plants. The ability to perceive and respond to cytokinin occurs through a modified bacterial two-component pathway that functions via a multi-step phosphorelay. This cytokinin signalling process is a crucial part of almost all stages of plant life, from embryo patterning to apical meristem regulation, organ development and eventually senescence. The cytokinin signalling pathway involves the co-ordination of three types of proteins: histidine kinase receptors to perceive the signal, histidine phosphotransfer proteins to relay the signal, and response regulators to provide signal output. This pathway contains both positive and negative elements that function in a complex co-ordinated manner to control cytokinin-regulated plant responses. Although much is known about how this cytokinin signal is perceived and initially regulated, there are still many avenues that need to be explored before the role of cytokinin in the control of plant processes is fully understood.