The ethylene response factor (ERF) family in Arabidopsis thaliana comprises 122 members in 12 groups, yet the biological functions of the majority remain unknown. Of the group IX ERFs, the IXc subgroup has been studied the most, and includes ERF1, ERF14 and ORA59, which play roles in plant innate immunity. Here we investigate the biological functions of two members of the less studied IXb subgroup: ERF5 and ERF6. In order to identify potential targets of these transcription factors, microarray analyses were performed on plants constitutively expressing either ERF5 or ERF6. Expression of defense genes, JA/Et-responsive genes and genes containing the GCC box promoter motif were significantly upregulated in both ERF5 and ERF6 transgenic plants, suggesting that ERF5 and ERF6 may act as positive regulators of JA-mediated defense and potentially overlap in their function. Since defense against necrotrophic pathogens is generally mediated through JA/Et-signalling, resistance against the fungal necrotroph Botrytis cinerea was examined. Constitutive expression of ERF5 or ERF6 resulted in significantly increased resistance. Although no significant difference in susceptibility to B. cinerea was observed in either erf5 or erf6 mutants, the erf5 erf6 double mutant showed a significant increase in susceptibility, which was likely due to compromised JA-mediated gene expression, since JA-induced gene expression was reduced in the double mutant. Taken together these data suggest that ERF5 and ERF6 play positive but redundant roles in defense against B. cinerea. Since mutual antagonism between JA/Et and salicylic acid (SA) signalling is well known, the UV-C inducibility of an SA-inducible gene, PR-1, was examined. Reduced inducibilty in both ERF5 and ERF6 constitutive overexepressors was consistent with suppression of SA-mediated signalling, as was an increased susceptibility to avirulent Pseudomonas syringae. These data suggest that ERF5 and ERF6 may also play a role in the antagonistic crosstalk between the JA/Et and SA signalling pathways.

Summary Arabidopsis SENSITIVE TO FREEZING6 (SFR6) controls cold‐ and drought‐inducible gene expression and freezing‐ and osmotic‐stress tolerance. Its identification as a component of the MEDIATOR transcriptional co‐activator complex led us to address its involvement in other transcriptional responses. Gene expression responses to Pseudomonas syringae, ultraviolet‐C (UV‐C) irradiation, salicylic acid (SA) and jasmonic acid (JA) were investigated in three sfr6 mutant alleles by quantitative real‐time PCR and susceptibility to UV‐C irradiation and Pseudomonas infection were assessed. sfr6 mutants were more susceptible to both Pseudomonas syringae infection and UV‐C irradiation. They exhibited correspondingly weaker PR (pathogenesis‐related) gene expression than wild‐type Arabidopsis following these treatments or after direct application of SA, involved in response to both UV‐C and Pseudomonas infection. Other genes, however, were induced normally in the mutants by these treatments. sfr6 mutants were severely defective in expression of plant defensin genes in response to JA; ectopic expression of defensin genes was provoked in wild‐type but not sfr6 by overexpression of ERF5. SFR6/MED16 controls both SA‐ and JA‐mediated defence gene expression and is necessary for tolerance of Pseudomonas syringae infection and UV‐C irradiation. It is not, however, a universal regulator of stress gene transcription and is likely to mediate transcriptional activation of specific regulons only.

Summary• The Arabidopsis protein SENSITIVE TO FREEZING-6 (AtSFR6) is required for cold-and drought-inducible expression of COLD-ON REGULATED (COR) genes and, as a consequence, AtSFR6 is essential for osmotic stress and freezing tolerance in Arabidopsis. Therefore, orthologues of AtSFR6 in crop species represent important candidate targets for future manipulation of stress tolerance. We identified and cloned a homologue of AtSFR6 from rice (Oryza sativa), OsSFR6, and confirmed its orthology in Arabidopsis.• OsSFR6 was identified by homology searches, and a full-length coding region isolated using reverse transcription polymerase chain reaction (RT-PCR) from Oryza sativa cDNA. To test for orthology, OsSFR6 was expressed in an Arabidopsis sfr6 loss-of-function mutant background, and restoration of wild-type phenotypes was assessed.• Searching the rice genome revealed a single homologue of AtSFR6. Cloning and sequencing the OsSFR6 coding region showed OsSFR6 to have 61.7% identity and 71.1% similarity to AtSFR6 at the predicted protein sequence level. Expression of OsSFR6 in the atsfr6 mutant background restored the wild-type visible phenotype, as well as restoring wild-type levels of COR gene expression and tolerance of osmotic and freezing stresses.• OsSFR6 is an orthologue of AtSFR6, and thus a target for future manipulation to improve tolerance to osmotic and other abiotic stresses.

Developing stress tolerant crops to cope with the rapid environmental degradation that is occurring is an absolute requirement in order to provide enough food for growing population. One of the basic genetic engineering approaches currently being used to improve crop stress tolerance is generation of transgenic plants by introducing novel genes into the genome of agriculturally important crops or altering the expression of existing genes. Understanding stress response signaling pathways is the prime requirement to manipulate stress tolerance of crop plants by this approach. SFR6 (SENSITIVE TO FREEZING6) is one of plant mediator protein which has identified first with its involvement to tolerance against freezing in Arabidopsis. The freezing sensitivity of SFR6 mutant is lack of expression of downstream genes in CBF cold response pathway. SFR6 also mediates tolerance to osmotic stress induced by drought and salinity. However, the over expression of SFR6 in wild type Arabidopsis did not increase COR (Cold on regulated) gene expression under ambient temperature. Therefore, the effect of SFR6 on cold gene expression was further characterized in this study. Results showed that over expression of SFR6 did not alter CBF1 expression levels under ambient temperature as well as COR gene expression under cold and osmotic stress indicating SFR6 alone cannot use as a molecular tool to improve stress tolerance of crop plants. Further over expression of both SFR6 and CBF1 also did not have additive effect on COR gene expression. Therefore, further studies of the mechanism of SFR6 on COR gene regulation need to be conducted to evaluate the potential of use of SFR6 as a molecular tool to increase stress tolerance of crop plants.

SFR6 (SENSITIVE TO FREEZING6) is one of plant mediator proteins which has been identified first with its involvement to tolerance against freezing in Arabidopsis. The freezing sensitive sfr6 mutant is lack of expression of downstream genes in CBF cold response pathway. SFR6 also mediates tolerance to osmotic stress induced by drought and salinity. However, sfr6-1 mutant shows normal levels of CBF gene expression suggesting that SFR6 acts downstream of CBF transcription. The over-expression of CBF1 and CBF2 in the sfr6-1 mutant did not increase downstream cold gene expression. Further, SFR6 does not affect the level of accumulation of CBF protein suggesting that SFR6 may effect on localization of CBF proteins in Arabidopsis. Therefore, the localization of CBF1 protein in the sfr6-1 mutant was examined in this study. sfr6-1 and Col-0 plants were crossed with wild type plants over-expressing 35S::CBF1::GFP and F3 generation was used to visualize CBF1 localization. However, the localization of CBF1 protein was not altered in sfr6-1 mutant compared to the wild type Arabidopsis indicating that the involvement of SFR6 for COR gene expression needs to be further studied.