From lab to greenhouse: molecular mechanisms of physiological control of plant growth

In horticulture, especially in the production of ornamental crops, it is important to grow compact-shaped plants. Achieving this year-round can be challenging, especially during cloudy and/or warm days because elongation of stems is stimulated under such conditions, resulting in spindly-shaped plants. Chemical plant growth retardants (PGRs) are used in horticulture to control plant elongation. However, the use of chemical PGRs needs to be replaced by more sustainable methods. A commonly used alternative method for controlling plant growth without the use of PGRs is realizing a negative day-night temperature difference (-DIF: cold days/warm nights), instead of a ‘normal’ +DIF regime (warm days/cold nights). Although -DIF is effective for controlling growth in many plant species, economic realization of -DIF is not always possible. Therefore, more alternative methods for controlling plant growth in greenhouses are needed. Aim of this thesis project was to increase our understanding of the molecular regulation of the growth response to -DIF. Additionally, we aimed to find ways to manipulate the components behind this regulation (e.g. by added light treatments) in order to enhance the effect of -DIF and/or identify alternative treatments for growing compact plants in greenhouses.

To study the transcriptional responses of growth-regulating components to light and temperature cues, we used firefly-luciferase (ffLUC) reporters for promoter activity of genes involved in plant growth. To this aim LUMINATOR was developed. This custom-built system was used to semi-continuously monitor ffLUC activity in four-week-old Arabidopsis reporter plants for promoter activity of PHYTOCHROME INTERACTING FACTOR 4 (PIF4), ELONGATED HYPOCOTYL 5 (HY5) and GIGANTEA (GI) under 12h light/12h dark cycles. LUMINATOR was subsequently used to study transcriptional responses of PIF4, HY5 and GI to one hour added light at the start and end of the photoperiod. Such short light pulse treatments triggered immediate and longer-lasting transcriptional responses, which were predictive for long-term plant growth responses of tomato to similar added light treatments in climate cabinets. Thus, LUMINATOR may be used to help predict long-term plant growth responses to additional light treatments. LUMINATOR was also used to monitor changes in ffLUC reporter activity in response to sudden temperature changes. However, comparing ffLUC profiles with mRNA levels of the endogenous genes showed that the effect of temperature on activity of the ffLUC enzyme itself makes results difficult to interpret.

To obtain an overview of the processes that are affected by -DIF RNA sequencing (RNAseq) analysis was performed on total mRNA and miRNA of adult Arabidopsis rosettes grown under +DIF (12h L 22°C/12h D 12°C) or -DIF (12h L 12°C/12h D 22°C) at two timepoints: end of day (ED) and end of night (EN). RNA was isolated from sink material (young sink leaves and petioles of older leaves) of Col-0 WT and the phytochrome B (PHYB) mutant phyB-9, which shows reduced sensitivity to -DIF. Analysis of differentially expressed mRNAs shows that -DIF leads to differential expression of in total 2706 genes in WT. Gene Ontology (GO)-term enrichment analysis indicates that -DIF stimulates cold acclimation processes at ED and leads to a low energy status at EN. Cell-growth processes such as cell-wall modification and water transport are downregulated at either ED or EN. Overlap between genes that are differentially expressed under -DIF and PIF-target genes indicates reduced transcriptional activity of PIF proteins under -DIF, especially at EN. Differentially expressed genes under -DIF that are related to promotion of cell growth were often not differentially expressed at EN in the phyB-9 mutant. This suggests that the transcriptional responses of these genes are causal for growth suppression under -DIF and that PHYB is involved in mediating the transcriptional response to temperature at night. Analysis of differentially expressed miRNAs and their putative target mRNAs suggests that part of the transcriptional response to -DIF is through regulation of mRNA transcript stability by miRNAs.

Previously it was shown that -DIF results in altered clock-controlled leaf movement in Arabidopsis, suggesting the timing of the circadian clock is altered under -DIF. We show that -DIF leads to changes in phase and amplitude of rhythmic expression of clock genes. Altered clock gene expression under -DIF is associated with altered regulation of starch metabolism and results in reduced starch levels in source leaves and reduced sucrose levels in sink leaves at EN, leading to induction of marker genes for carbohydrate (CH) starvation. Since it is known that plant growth is suppressed in response to CH starvation, these results suggest that the induction of CH starvation at EN may be causal for growth suppression under -DIF. In agreement with this, alternative methods that potentially induce CH starvation all lead to growth suppression in Arabidopsis.

The growth response to -DIF is associated with reduced PIF protein activity. Activity of PIFs may be (further) reduced by changing light quality by providing additional red (R) or blue (B) light. We used LUMINATOR and ffLUC reporter plants to study the transcriptional response of PIF3/4/5, the PIF-antagonist HY5 and the PIF-induced target gene INDOLE-3-ACETIC ACID INDUCIBLE 29 (IAA29) to one-hour additional B or R light at dawn and dusk. Results show that in response to additional R light at dusk IAA29 transcription is reduced throughout the night, suggesting reduced PIF protein activity. Additional B light suppressed PIF and IAA29 transcription but stimulated HY5 transcription in the morning. These results indicate that one-hour additional R or B light at dawn and dusk may be used to suppress plant growth. Indeed, stem growth in tomato plants could be suppressed by providing additional R/B light at dawn and dusk in climate cabinets and/or greenhouses. Interestingly, timing of the additional R/B light determined whether the added light had an effect on plant growth. In agreement with the transcriptional responses, growth responses of tomato to additional R light were restricted to the end of the day, while growth responses to additional B light were restricted to the start of the day.

Based on the results presented in this thesis several suggestions for novel sustainable protocols for growing compact plants in greenhouses are provided in the final chapter of this thesis.