Final Report Summary - ARABISHADE (Molecular cross-talk of light perception and development for plant adaptation to the environment)
1. Publishable summary/final publishable summary report
Project overview: Plants sense the presence of competing vegetation as a change in light quality, i.e. a reduced red to far-red (R: FR) ratio. The responses to shade are generally referred to as the shade avoidance syndrome (SAS), and involve various developmental changes intended to overgrow or survive neighboring plants. Molecularly, after perception of plant proximity, the phytochrome photoreceptors rapidly modulate the expression of several dozens of phytochrome rapidly regulated (PAR) genes that encode partially redundant positive and negative regulators controlling SAS responses forming a transcriptional web. The major objective of this project is to understand the SAS, with an emphasis on the definition of the architecture of the SAS regulatory network (i.e. understanding the relationship between its components). In addition, the proposed approaches might help to deepen into the molecular basis of the cross-talk between shade perception and endogenous transcriptional networks, such as those controlling hormone signaling and/or other developmental responses.
Specific research objectives: In particular, the project aims at:
(1. 1.) Identification and functional analysis of new SAS components. Unpublished work from the host lab has identified a likely constituent of the nuclear pore complex, Dracula2 (DRA2), as an important regulator of the SAS. Site(s), mode(s) and steps of DRA2 action in planta have to be clarified by subcellular localisation to definitely establish the identity of DRA2 as a nucleoporin. To this end a construct to overexpress under the control of the 35S promoter the DRA2 gene fused to the green fluorescent reporter protein (GFP) had to be generated (P35S: DRA2-GFP).
(1.2.) Generation of new genetic tools to increase our knowledge of the SAS transcriptional network. PAR2 is a negative regulator, BEE1 a positive regulator of the SAS the lab is interested in. P35S: BEE1-GR transgenic seedlings have to be generated to identify primary target genes of these SAS transcription factors by means of microarray analysis combined with DEX and CHX application. P35S: PAR2-GR (in analogy to P35S: PAR1-GR) will be used to further confirm similar or different function vs PAR1 in the SAS transcriptional network.
(1.3.) Complementation of athb4hat3 double mutation by transgenic ATHB4-GR. The HD-Zip class-II subfamily PAR genes ATHB4 and HAT3 are complex regulators of the SAS. ATHB4 acts redundantly with its paralogue HAT3 as transcription factors to simulated shade. The double mutant athb4hat3 has thin and lancet- shaped cotyledons and no hypocotyl elongation response to shade. By genetic crossing we wanted to find out whether the double mutation athb4hat3 can be complemented by ATHB4 generated by P35S: ATHB4-GR and thus rescue the ATHB4 wild-type phenotype (controlled by DEX). This would yield a further confirmation of ATHB4/HAT3 functional redundancy and open the possibility to use this genetic approach to test the biological activity of truncated forms of ATHB4 generated in the lab.
(2.) Comparison of the molecular mechanisms involved in the response to plant proximity in shade-avoidance (Arabidopsis thaliana) and shade-tolerant (Cardamine hirsuta) species. This novel objective aims to obtain genetic and molecular evidence on whether there are shared or unique mechanisms that govern plant responses to vegetation proximity between model species that either avoid (A. thaliana) or tolerate (C. hirsuta) shade.
Results and prospects (1. 1.). A complicated multi-stage cloning procedure was run through and at the end the entire and correct Dracula2-cDNA (3126 bp) was obtained (P35S: DRA2-GFP). The GFP activity will be visualised by two different approaches: (i) by transient expression in onion epidermal cells (transformed with the shoot gun) and (ii) in the resulting transgenic seedlings (P35S: DRA2-GFP) transformed via Agrobacterium. Transgenic seedlings will allow for future complementation studies of the DRA2 mutation. Together, these experiments will allow to definitely establish the identity of DRA2 as a nucleoporin.
(1. 2.). PAR2- and BEE1-cDNAs were cloned into binary vectors and Arabidopsis plants were Agrobacterium- transformed. Transgenic plants were selected by genotyping (T1), T-DNA insertion analyses (T2) by hygromicine Hg resistance and + DEX phenotype, a dark green dwarf phenotype (P35S: PAR2-GR) and longer hypocotyls vs wild-type plants (P35S: BEE1-GR). The same for T3 selection for homozygosis. P35S: PAR2-GR transgenic seedlings (6 lines) and P35S:BEE1-GR transgenic seedlings (eight lines) were obtained and available in the lab.
(1. 3.). By genetic crossing the homozygous triple mutant lines (P35S: ATHB4-GR x hat3 x athb4) were achieved. The DEX-inducible reversion of the lancet-phenotype did not work, i.e. no complementation occurred probably because the transgene lost its functionality (by silencing). Slight + DEX dwarf seedlings were selected and genotyped for being homozygous for one of the mutant alleles and heterozygous for the other one, i.e. athb4/+; hat3 or athb4; hat3/+. Further selection in the next generation for the strongest P35S: ATHB4-GR transgene activity (dwarf) and crossing again for the triple mutant is expected to finally yield a high rate of complementation of the double mutation athb4; hat3 by functional P35S:ATHB4-GR and thus rescue the ATHB4 wild-type phenotype (controlled by DEX treatments).
(2.). To elucidate the molecular and physiological basis of the differences in response to shade between these model plant species, we quantified SAS physiological responses in C. hirsuta and A. thaliana and related them to the expression of relevant SAS genes of different functional groups. Hypocotyl elongation (physiological response) with shade was clearly weaker in C. hirsuta and positively correlated with the weaker hypocotyl responsiveness to picloram (PIC), a synthetic auxin. Expression of negative SAS regulator marker genes analysed (HFR1, PIL1, PAR2, ATHB2, ATHB4) showed significant induction with shade in both species without substantial differences between species. Together, these results suggested a different wiring of the phytochrome signal with the transcriptional network, which executes the responses, e.g. the network of auxines. Therefore we tested the expression of shade- relevant auxin genes like SAUR15 & SAUR68 and genes situated at different stages of the IAA biosynthesis pathway, which had shown rapid and strong induction with simulated shade, such as IAA1, IAA19 & IAA29 and YUCCA5 (YUC5), YUC8 & YUC9. IAA19 & IAA29 exhibited induction (1h of W+FR) twice as high in A. thaliana vs C. hirsuta and we will further pursue this issue. The expression of SAS positive regulators (BIM1, BIM2, BEE1) will provide information if there is an altered balance of expression of positive and negative key SAS regulators in C. hirsuta vs A. thaliana. In collaboration with the group of Prof Miltos Tsiantis we are realising RNA illumina sequencing (RNA seq) to obtain global expression profiles in C. hirsuta vs A. thaliana. To find out whether the differences in hypocotyl elongation in response to simulated shade between shade-avoidance (A. thaliana) and shade-tolerant species (C. hirsuta) is related to differential phyA action, i.e. inhibition of C. hirsuta hypocotyl elongation by phyA action, we also try to reduce phyA levels in C. hirsuta (for A. thaliana, phyA mutants are available in the lab). This is done in parallel by a genetic screening looking for phyA mutants in C. hirsuta (using EMS mutagenised seed population) and by silencing phyA in transgenic plants of C. hirsuta (using Ch-phyA_RNAi constructs). phyA protein levels in C. hirsuta and A. thaliana seedlings are analysed (by westerns) to account for different stability.
Socio-economic impact: Transfer of the information generated in this project to species of commercial value might have a positive impact in biotechnological companies. Transfer of the knowledge generated in this project to energy crops, such as low-input non-food plants, might have a positive impact in the production of biofuels. Biofuel should be produced in large quantities without reducing food supplies. If produced on agriculturally marginal lands or in shaded areas with minimal fertilizer and pesticide input, fuel supplies with greater environmental benefits than either petroleum or current food-based biofuels will result. Understanding of light response pathways might help to guide breeding programs towards the creation of energy crops that are able to produce increased biomass in the form of cellulose under high plant densities.
Project overview: Plants sense the presence of competing vegetation as a change in light quality, i.e. a reduced red to far-red (R: FR) ratio. The responses to shade are generally referred to as the shade avoidance syndrome (SAS), and involve various developmental changes intended to overgrow or survive neighboring plants. Molecularly, after perception of plant proximity, the phytochrome photoreceptors rapidly modulate the expression of several dozens of phytochrome rapidly regulated (PAR) genes that encode partially redundant positive and negative regulators controlling SAS responses forming a transcriptional web. The major objective of this project is to understand the SAS, with an emphasis on the definition of the architecture of the SAS regulatory network (i.e. understanding the relationship between its components). In addition, the proposed approaches might help to deepen into the molecular basis of the cross-talk between shade perception and endogenous transcriptional networks, such as those controlling hormone signaling and/or other developmental responses.
Specific research objectives: In particular, the project aims at:
(1. 1.) Identification and functional analysis of new SAS components. Unpublished work from the host lab has identified a likely constituent of the nuclear pore complex, Dracula2 (DRA2), as an important regulator of the SAS. Site(s), mode(s) and steps of DRA2 action in planta have to be clarified by subcellular localisation to definitely establish the identity of DRA2 as a nucleoporin. To this end a construct to overexpress under the control of the 35S promoter the DRA2 gene fused to the green fluorescent reporter protein (GFP) had to be generated (P35S: DRA2-GFP).
(1.2.) Generation of new genetic tools to increase our knowledge of the SAS transcriptional network. PAR2 is a negative regulator, BEE1 a positive regulator of the SAS the lab is interested in. P35S: BEE1-GR transgenic seedlings have to be generated to identify primary target genes of these SAS transcription factors by means of microarray analysis combined with DEX and CHX application. P35S: PAR2-GR (in analogy to P35S: PAR1-GR) will be used to further confirm similar or different function vs PAR1 in the SAS transcriptional network.
(1.3.) Complementation of athb4hat3 double mutation by transgenic ATHB4-GR. The HD-Zip class-II subfamily PAR genes ATHB4 and HAT3 are complex regulators of the SAS. ATHB4 acts redundantly with its paralogue HAT3 as transcription factors to simulated shade. The double mutant athb4hat3 has thin and lancet- shaped cotyledons and no hypocotyl elongation response to shade. By genetic crossing we wanted to find out whether the double mutation athb4hat3 can be complemented by ATHB4 generated by P35S: ATHB4-GR and thus rescue the ATHB4 wild-type phenotype (controlled by DEX). This would yield a further confirmation of ATHB4/HAT3 functional redundancy and open the possibility to use this genetic approach to test the biological activity of truncated forms of ATHB4 generated in the lab.
(2.) Comparison of the molecular mechanisms involved in the response to plant proximity in shade-avoidance (Arabidopsis thaliana) and shade-tolerant (Cardamine hirsuta) species. This novel objective aims to obtain genetic and molecular evidence on whether there are shared or unique mechanisms that govern plant responses to vegetation proximity between model species that either avoid (A. thaliana) or tolerate (C. hirsuta) shade.
Results and prospects (1. 1.). A complicated multi-stage cloning procedure was run through and at the end the entire and correct Dracula2-cDNA (3126 bp) was obtained (P35S: DRA2-GFP). The GFP activity will be visualised by two different approaches: (i) by transient expression in onion epidermal cells (transformed with the shoot gun) and (ii) in the resulting transgenic seedlings (P35S: DRA2-GFP) transformed via Agrobacterium. Transgenic seedlings will allow for future complementation studies of the DRA2 mutation. Together, these experiments will allow to definitely establish the identity of DRA2 as a nucleoporin.
(1. 2.). PAR2- and BEE1-cDNAs were cloned into binary vectors and Arabidopsis plants were Agrobacterium- transformed. Transgenic plants were selected by genotyping (T1), T-DNA insertion analyses (T2) by hygromicine Hg resistance and + DEX phenotype, a dark green dwarf phenotype (P35S: PAR2-GR) and longer hypocotyls vs wild-type plants (P35S: BEE1-GR). The same for T3 selection for homozygosis. P35S: PAR2-GR transgenic seedlings (6 lines) and P35S:BEE1-GR transgenic seedlings (eight lines) were obtained and available in the lab.
(1. 3.). By genetic crossing the homozygous triple mutant lines (P35S: ATHB4-GR x hat3 x athb4) were achieved. The DEX-inducible reversion of the lancet-phenotype did not work, i.e. no complementation occurred probably because the transgene lost its functionality (by silencing). Slight + DEX dwarf seedlings were selected and genotyped for being homozygous for one of the mutant alleles and heterozygous for the other one, i.e. athb4/+; hat3 or athb4; hat3/+. Further selection in the next generation for the strongest P35S: ATHB4-GR transgene activity (dwarf) and crossing again for the triple mutant is expected to finally yield a high rate of complementation of the double mutation athb4; hat3 by functional P35S:ATHB4-GR and thus rescue the ATHB4 wild-type phenotype (controlled by DEX treatments).
(2.). To elucidate the molecular and physiological basis of the differences in response to shade between these model plant species, we quantified SAS physiological responses in C. hirsuta and A. thaliana and related them to the expression of relevant SAS genes of different functional groups. Hypocotyl elongation (physiological response) with shade was clearly weaker in C. hirsuta and positively correlated with the weaker hypocotyl responsiveness to picloram (PIC), a synthetic auxin. Expression of negative SAS regulator marker genes analysed (HFR1, PIL1, PAR2, ATHB2, ATHB4) showed significant induction with shade in both species without substantial differences between species. Together, these results suggested a different wiring of the phytochrome signal with the transcriptional network, which executes the responses, e.g. the network of auxines. Therefore we tested the expression of shade- relevant auxin genes like SAUR15 & SAUR68 and genes situated at different stages of the IAA biosynthesis pathway, which had shown rapid and strong induction with simulated shade, such as IAA1, IAA19 & IAA29 and YUCCA5 (YUC5), YUC8 & YUC9. IAA19 & IAA29 exhibited induction (1h of W+FR) twice as high in A. thaliana vs C. hirsuta and we will further pursue this issue. The expression of SAS positive regulators (BIM1, BIM2, BEE1) will provide information if there is an altered balance of expression of positive and negative key SAS regulators in C. hirsuta vs A. thaliana. In collaboration with the group of Prof Miltos Tsiantis we are realising RNA illumina sequencing (RNA seq) to obtain global expression profiles in C. hirsuta vs A. thaliana. To find out whether the differences in hypocotyl elongation in response to simulated shade between shade-avoidance (A. thaliana) and shade-tolerant species (C. hirsuta) is related to differential phyA action, i.e. inhibition of C. hirsuta hypocotyl elongation by phyA action, we also try to reduce phyA levels in C. hirsuta (for A. thaliana, phyA mutants are available in the lab). This is done in parallel by a genetic screening looking for phyA mutants in C. hirsuta (using EMS mutagenised seed population) and by silencing phyA in transgenic plants of C. hirsuta (using Ch-phyA_RNAi constructs). phyA protein levels in C. hirsuta and A. thaliana seedlings are analysed (by westerns) to account for different stability.
Socio-economic impact: Transfer of the information generated in this project to species of commercial value might have a positive impact in biotechnological companies. Transfer of the knowledge generated in this project to energy crops, such as low-input non-food plants, might have a positive impact in the production of biofuels. Biofuel should be produced in large quantities without reducing food supplies. If produced on agriculturally marginal lands or in shaded areas with minimal fertilizer and pesticide input, fuel supplies with greater environmental benefits than either petroleum or current food-based biofuels will result. Understanding of light response pathways might help to guide breeding programs towards the creation of energy crops that are able to produce increased biomass in the form of cellulose under high plant densities.