Defining the role of PIF3-like bHLH transcription factors in the integration of light and cold signalling in Arabidopsis

Lead Research Organisation: University of Edinburgh
Department Name: Sch of Biological Sciences


Details provided by lead applicant: Professor Ian Graham, Biology Dept., University of York (Reference No. E242703)

Technical Summary

We have recently discovered that cold and light control seed germination through the bHLH transcription factor SPATULA (Penfield et al., 2005 Curr Biol 15:1998-2006). SPT acts as a light stable repressor of seed germination and is required in dormant seeds for the repression of GA3 oxidase (GA3ox) gene expression. We have also shown that the related protein PIL5 represses seed germination and GA3ox expression in the dark, and have proposed a model whereby SPT and PIL5 form part of a regulatory network coupling seed germination and GA signalling to light and temperature responses. Our more recent unpublished data suggest that SPT and PIL5 act upstream of the DELLA proteins in the control of seed germination. SPT is also required post-germination in the phytochrome controlled cotyledon expansion response, which notably is also acutely temperature sensitive and is subject to DELLA control. SPT has high homology to the PIF-like bHLH transcription factors that play a key role in phytochrome- mediated light signalling. Over-expression of SPT leads to strong phyB-like phenotypes such as elongated hypocotyls. This begs the question as to whether the GA signalling based mode of action of SPT in seed dormancy control also operates at other stages in plant development. Furthermore, very little is known about the mode of action of the PIF-like bHLH TFs, and it is tempting to speculate that they also operate upstream of the growth restraining DELLA proteins. In this proposal we will build on the existing tools and complimentary skills in seed germination and PIF/phytochrome mediated signalling in the Graham and Halliday labs respectively to define the role of SPT and related bHLH transcription factors in the integration of light and cold signalling in Arabidopsis. This work should lead to further high impact publications and intellectual property that could be used to design strategies for the improvement of crop plant performance under sub-optimal environmental conditions.


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Description This project sought to interrogate the role of SPATULA (SPT), a PHYTOCHROME INTERACTING FACTOR (PIF)-like bHLH transcription factor, that operates at the confluence of light and temperature signalling. We discovered SPT is a potent antagonist of the plant hormone GA, where it acts to restrict growth in a phytochrome B-dependent manner. The other PIFs promote GA-dependent growth. We showed that SPT acts as a molecular counterbalance to the unrelated DELLA proteins, targeting a common gene subset. This molecular circuit maintains more robust growth control than either regulator alone (Josse et al. Plant Cell 2011).

We demonstrated that SPT and PIFs operate over discrete temperature ranges. SPT is an effective growth regulator at cooler temperatures (4-20?C), while other bHLHs control growth and biomass under warmer conditions (20-28?C). Transcription rate, transcript stability, protein abundance and protein modification are amongst the molecular temperature-regulated effects identified in this study (Sidaway-Lee et al. Curr Biol. 2010).

In a third strand of analysis we demonstrated that like SPT, PIF6 is expressed strongly during seed development, and that two splice variants exist, one full length (the alpha form), and a second, the beta form, in which a cryptic intron containing the potential DNA binding domain is spliced out, predicted to lead to the generation of a premature stop codon. Loss of PIF6 increases primary seed dormancy, whereas overexpression of the beta form, but not the alpha form, reduce dormancy. Our data show the potential for natural splice variants of PIF transcription factors to be important in the evolution of the control of environmental signalling in plants (Penfield et al Plant Mol Biol. 2010).

In summary- Our study illustrated that SPT and related bHLH transcription factors are important integrators of light and temperature signals. We showed that SPT was an important regulator of biomass production as well as germination. We delineated the molecular mechanisms through which these transcription factors respond to environmental signals.

Material generated
Mutant and transgenic lines generated:
spt-11 phyB-9
spt-12 phyB-9
spt-3 phyB-1
spt-11 phyE-1
spt-11 pif4-1
spt-12 pif4-1
spt-11 pif3-3
spt-11 pif7-1
spt-11 hy5-215
spt-12 hy5-215
spt-11 pif3-3 pif7-1
spt-11 pif4-2 pif7-1
spt-11 phyB-9 pif4-2
spt-11 phyB-9 pif7-1
spt-2 ga1 gai-t6 rga-t2 rgl1-1 rgl2-1 rgl3-1
spt-2 gai-t6 rga-t2 rgl1-1 rgl2-1 rgl3-1

spt-11 pRGA::GFP-RGA
spt-12 pRGA::GFP-RGA
phyB-9 pRGA::GFP-RGA
pif4-1 pRGA::GFP-RGA

Polyclonal antibodies:
Raised to SPT peptides

Constructs (ORF) generated for yeast-two-hybrid analysis (bait and prey):
Exploitation Route Our work highlighted the role of bHLH transcription factors as key regulators of germination and plant biomass. The work has application value (our collaborator Penfield filed a patent on SPT) as manipulation of the SPT pathway can lead to improvements in germination and/or biomass: important yield traits. This work provides a mechanistic insight into how environmental signal integration is achieved. It will therefore inform future studies in this and related areas.
Sectors Agriculture, Food and Drink

Description Our findings advanced understanding and helped to change the way the field views environmental signaling: toward a model where individual components integrate multiple external cues. They uncovered the molecular basis cold- regulated germination and biomass production. They also contributed directly to the inception of the BBSRC/EPSRC ROBuST project (BB/F005237/1) that took a systems approach to understand how molecular signaling is controlled by temperature.
Sector Agriculture, Food and Drink
Description Halliday-Penfield 
Organisation University of York
Country United Kingdom 
Sector Academic/University 
PI Contribution My lab contributed molecular-genetic and biochemical analysis to two initial joint publications: Penfield et al., A role for an alternative splice variant of PIF6 in the control of Arabidopsis primary seed dormancy, Plant Mol Biol. 2010 May;73(1-2):89-95; Sidaway-Lee et al., SPATULA links daytime temperature and plant growth rate. Curr Biol. 2010 Aug 24;20(16):1493-7. In the former publication we demonstrated a role for natural splice variants of PIF transcription factors in the regulation of seed dormancy. The second publication analyses temperature regulation of plant growth. Our paper shows that that only daytime temperatures affect vegetative growth, and that the transcription factor SPATULA couples morning temperature to growth rate.
Collaborator Contribution The Penfield lab contributed molecular-genetic and germination analysis to the two joint publications detailed above.
Impact Penfield et al., A role for an alternative splice variant of PIF6 in the control of Arabidopsis primary seed dormancy, Plant Mol Biol. 2010 May;73(1-2):89-95; Sidaway-Lee et al., SPATULA links daytime temperature and plant growth rate. Curr Biol. 2010 Aug 24;20(16):1493-7. The collaboration led to follow-on funding (ROBuST project) with the resulting publications: MacGregor DR, Gould P, Foreman J, Griffiths J, Bird S, Page R, Stewart K, Steel G, Young J, Paszkiewicz K, Millar AJ, Halliday KJ, Hall AJ, Penfield S. HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES1 is required for circadian periodicity through the promotion of nucleo-cytoplasmic mRNA export in Arabidopsis. Plant Cell. 2013 25(11):4391-404. Keily J, MacGregor DR, Smith RW, Millar AJ, Halliday KJ, Penfield S. Model selection reveals control of cold signalling by evening-phased components of the plant circadian clock. Plant J. 2013 76(2):247-57. Vaistij FE, Gan Y, Penfield S, Gilday AD, Dave A, He Z, Josse EM, Choi G, Halliday KJ, Graham IA. Differential control of seed primary dormancy in Arabidopsis ecotypes by the transcription factor SPATULA. Proc Natl Acad Sci U S A. 2013 110(26):10866-71. Gould PD, Ugarte N, Domijan M, Costa M, Foreman J, Macgregor D, Rose K, Griffiths J, Millar AJ, Finkenstädt B, Penfield S, Rand DA, Halliday KJ, Hall AJ. Network balance via CRY signalling controls the Arabidopsis circadian clock over ambient temperatures. Mol Syst Biol. 2013 9:650. Seaton DD, Smith RW, Song YH, MacGregor DR, Stewart K, Steel G, Foreman J, Penfield S, Imaizumi T, Millar AJ, Halliday KJ. Linked circadian outputs control elongation growth and flowering in response to photoperiod and temperature. Mol Syst Biol. 2015 11:776.
Start Year 2007
Description Press Coverage Coverage of the Sidaway-Lee et al., Curr. Biol. 2010 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact Press coverage for our Sidaway-Lee et al., Curr. Biol. 2010 publication: interviews were broadcast on BBC1 TV Scotland, BBC radio Scotland and the research was reported on Central FM and Westsound.
Year(s) Of Engagement Activity 2010
Description Women In Science Lecture, Nara, Japan 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Undergraduate students
Results and Impact Provide a forum to discuss gender inequality in science across the EU and in Japan
Year(s) Of Engagement Activity 2007