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

Abstract

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, F v /F m ) as well as physiology after salt treatment. Salt stress-treated crf2 plants had both reduced chlorophyll levels and lower F v /F m values compared with the WT, suggesting that CRF2 plays a role in the modulation of salt stress responses linked to photosynthesis.

Efficient mesophyll-derived protoplast manipulation system as a versatile tool for characterization of genes responding to multiple stimuli in mulberry

“…Nevertheless, the transient expression assay did not unequivocally confirm these findings, although slight changes were observed in the protoplasts expressing MaCRF2:EGFP in response to short-term (12 h) 6-BA and ABA, as well as long-term (48 h) cold, as a predominant nuclear subcellular localization was found in the transformed protoplasts without any treatments. However, our results gave hints that MaCRF2 may be involved in abiotic stress responses in addition to cytokinin regulation (Jeon et al 2016;Keshishian et al 2022;Zwack et al 2016). The transcripts of MaCRF3 were induced by all the tested hormone and abiotic stress treatments, which is in agreement with the results of transient gene expression of MaCRF3:EGFP in response to hormones and cold stress.…”

“…Among the identified four MaCRFs (MaCRF1-MaCRF4) in mulberry, three paralogs, MaCRF1, MaCRF2, and MaCRF3, showed high similarity to the Arabidopsis CRF2 (AtCRF2), which is strongly induced by cytokinin and responsive to salt and cold stress (Keshishian et al 2022). Our investigation into the promoter regions of these MaCRFs revealed the presence of diverse cis-regulatory elements, such as light-responsive elements, hormoneresponsive elements, and stress-responsive elements.…”

“…In the tree, MaCRF1, MaCRF2, and MaCRF3 clustered in the same clade with AtCRF2 (Fig. 5-B), which is known to be strongly induced by cytokinin (Rashotte et al 2006), is responsive to salt and cold stress (Jeon et al 2016;Keshishian et al 2022). On the other hand, MaCRF4 grouped alongside AtCRF5 and AtCRF6, which have been associated…”

“…However, despite the CRF designation, they play pleiotropic roles in plant development and stress response since not all CRFs are specifically cytokinin-responsive. They also respond to multiple environmental cues and other hormones such as auxin and abscisic acid, with much of these works having been conducted in Arabidopsis and tomato (Hallmark and Rashotte 2019;Keshishian et al 2022;Zwack et al 2016). However, in woody plants, the specific function of this gene family has not been elucidated yet.…”

“…The core CRF family consists of six genes, three of which have been confirmed to be transcriptionally induced by cytokinin and their proteins show rapid relocalization in the nucleus in response to cytokinin (Rashotte et al 2006). Further studies indicated that CRFs not only regulate plant growth and development through cytokinin responses, but also interact with other phytohormones (like auxin) and play significant roles in stress-related processes (such as salt, cold and oxidative stresses) (Hallmark and Rashotte 2019;Jeon et al 2016;Keshishian et al 2022). Phylogenetic analysis has shown that CRF proteins are present in land plants but absent in green algae, suggesting their importance in terrestrial adaptation (Rashotte and Goertzen 2010).…”