Compactness in Arabidopsis thaliana

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1 Compactness in Arabidopsis thaliana Effect of temperature, light and sugars on PIF expression Monique van Vegchel Supervisors Mark van Hoogdalem Sander van der Krol May 2017 December 2017 Department of Plant Physiology Wageningen University and Research Project code:

2 Abstract There is an increasing demand in the horticulture industry for compact plants as they take up less space and are aesthetically desirable. Due to increased global awareness, environmentally-friendly growing protocols are preferred to achieve compact plants. An already successful protocol is the use of DIF, a negative day-night temperature differential. By introducing a cold photoperiod and a warm dark period, plants have reduced cell elongation and thus remain compact. We aim to uncover and enhance the effect of DIF on plant elongation. Phytochrome Interacting Factors (PIFs) have been indicated as possible actors in this process. With the use of qpcr and luciferase-reporter plants, we studied the activity pattern of PIF4, PIF5 and HY5 promotors in +DIF and DIF circumstances. We show that DIF lowers PIF and HY5 promotor activity and expression, though it remains difficult to quantify this difference. In addition, adding a red or blue pulse in the morning was expected to reduce PIF promotor activity. Though a reduction in PIF activity was not observed, we did observe an increase in HY5 promotor activity. This may result in lower PIF expression, though this remains to be established. Moreover, lowering trehalose-6-phosphate (T6P) levels increases PIF4 expression in +DIF. Increasing T6P levels had no effect. The KIN10RNAi mutant, a non-functioning SnRK1 complex, showed increased PIF expression in DIF. The increase in PIF expression after lowering T6P was unexpected and remains unexplainable. Creating circumstances in which energy status is manipulated naturally PIF4 promotor activity was measured and resulted in inconclusive results. The red and blue pulses show the most promising results with regard to increasing the effects of DIF, though these are still preliminary. Further research should give conclusive results regarding their added effect on plant elongation, both in +DIF and DIF. 2

3 Table of Contents Abstract... 2 Introduction... 4 Materials and Methods... 7 Mutants... 7 Luminator... 7 Quantitative Polymerase Chain Reaction (qpcr)... 9 Results Luminator DIF lowers promotor activity and transcription. The Luminator does not accurately reflect actual PIF4, PIF5 and HY5 transcription values Red and blue pulses in morning increase PIF4, PIF5 and HY5 slightly expression in +DIF Red pulses in morning have no effect, blue pulse increase expression in DIF Sugar signalling T6P does not affect PIF3 expression. Impaired SnRK1 complex increases PIF3 expression in DIF Decreased T6P levels increase PIF4 expression in +DIF. Impaired SnRK1 complex increases PIF4 expression in DIF Decreased T6P levels increase PIF5 expression in +DIF. Impaired SnRK1 complex increases PIF5 expression Decreased T6P levels increase PIF7 expression in +DIF. Impaired SnRK1 complex increases PIF7 expression in DIF Spraying sugar results Leaf disc results Extended night lowers PIF4 and HY5 expression Discussion Conclusion Acknowledgements References Appendices A. PIF4-1 vs PIF4-2 and PIF5-6 vs PIF B. Luminator output C. Full graphs for pulse +DIF D. Full graphs for pulse -DIF E. Luciferase construct sequences F. qpcr on sugar mutants: source vs sink tissue G. Luminator settings

4 Introduction Compact plants are aesthetically desirable and take up less space in the greenhouses. To grow compact plants, the horticulture industry currently uses mostly plant growth retardants (PGR). Increasing global awareness for environmental care, however, leads to a rising demand in organic, environmental friendly methods of plant growth. New protocols have to be developed, using natural factors like light and temperature, to control plant growth processes. An example of such a protocol is the use of a negative day-night temperature differential (- DIF), where it is cold during the day and warm during the night. This treatment has already shown to reduce plant elongation (Myster and Moe 1995; Thingnaes and others 2003). Research has concluded that cell elongation, and thus plant elongation, can be influenced by light and temperature (Bours and others 2013; Carvalho and others 2002; Maas and van Hattum 1998). The family of Phytochrome Interacting Factors (PIFs) are known to play a central role in the control of cell elongation. These bhlh transcription factors were first discovered during a study on seedling deetiolation (Bae and Choi 2008; Leivar and Quail 2011). The main PIFs are PIF3, PIF4, PIF5 and PIF7. Each PIF has their own specific function, as well as a certain level of overall redundancy. PIF3 interacts with phytochromes to start light-induced phosphorylation while at the same time degrading PIF3 itself (Castillon and others 2007). PIF4 and PIF5 play a role in the auxin pathway. They control the gene expression for auxin biosynthesis and auxin signalling components (Hornitschek and others 2012). Figure 1 shows the different environmental and endogenous signals that have been shown to regulate PIF activity on transcriptional and/or translational level. These signals include light (quality and quantity), temperature, sugars, the circadian clock, and hormones. The direction of regulation is not always clear from literature (Leivar and Monte 2014). Figure 1: Interaction between light, phytochrome and PIF (Leivar and Monte 2014). Red circles indicate factors that will be studied in our experiments. Regulation of PIF activity by light quality One of the best studied pathways by which PIF activity is regulated involves the photoreceptor phytochrome B (phyb). Red light activates phyb by converting the inactive Pr form of phyb to its active Pfr form. PIF3 and PIF4, as well as PIF5 and PIF7 interact with phyb. The active phyb is able to bind to PIF proteins through a conserved motif that is specific for the Pfr form of phyb (Khanna and others 2004). This interaction leads to instable and non-functioning PIF protein, ultimately reducing PIF abundance in the cell. 4

5 PhyB influences expression of another regulator of PIFs: ELONGATED HYPOCOTYL 5 (HY5). Increased HY5 expression reduces the effect of PIF4 as HY5 competes for the same binding site on the promotor (Gangappa and Kumar 2017). As shown in figure 2, phyb and HY5 are connected through CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1). In darkness, when COP1 is active, it suppresses HY5 activity (Saijo and others 2003). When plants are exposed to light, inactive phyb converts to active phyb. Active phyb is able to bind to COP1, inactivating it in the process (Hofmann 2015). Thus in darkness HY5 is suppressed, but in light COP1 is rendered inactive. This allows HY5 instead of PIF4 to bind to the promotor. This action supresses cell elongation. phyb is not the only light-regulated receptor that regulates PIF activity. Cryptochrome 1 (Cry1), a blue light receptor in A. thaliana, represses PIF4 expression by interacting with the PIF4 protein. Due to the negative feedback PIF4 has on its own transcription, this reduces PIF4 expression in the plant. Interaction between active Cry1 and PIF4 protein results in suppression of hypocotyl elongation (Ma and others 2016). Cry1, like phyb, interacts with the COP1 complex. Upon activation due to blue light, Cry1 destabilizes the COP1 complex, thus inducing HY5 transcription (Lau and Deng 2012). Figure 2: Proposed pathway of cell elongation through the negative feedback of phyb, Cry1, and HY5 on PIF4/5 and ultimately cell elongation. Regulation of PIF activity by temperature The effect of temperature on PIF4 transcription and expression has been the subject of studies. They show that high temperatures increase PIF4 transcription (Sun and others 2012). This could be the result of the effect temperature has on phyb. High temperatures destabilize phyb, rendering it inactive (Legris and others 2016). The absence of active phyb subsequently results in increased PIF4 transcription. Upon transfer to high temperatures, PIF4-deficient mutants no longer show the elongation responses that are normally perceived in high temperatures (Koini and others 2009). There have been few studies regarding the effect of low temperatures on PIF4 transcription and expression. Lower temperatures inhibit PIF4 activity, even though PIF4 transcript is present (Kumar and others 2012), but this was shown on a post-transcriptional level. There is little known on the effect on a transcriptional level. 5

6 Regulation of PIF activity by energy status Sugar signalling pathways control plant growth and development (Lastdrager and others, 2014). A link between sugars, auxin and PIF expression has been scientifically established, though that study focussed mainly on the hexose Glc sugar (Sairanen and others 2012). The trehalose pathway has been shown to control carbohydrate utilization. This is mainly done by the trehalose-6-phosphate (T6P) intermediate. T6P is formed by the synthesis of glucose-6-phosphate and uridinediphosphate glucose (Schluepmann and others 2003). High T6P levels result in a high energy status, while low T6P levels result in a low energy status. There have been signs that Trehalose-6-Phosphate (T6P) influences PIF4 expression (Zhang and others 2009 dataset). Seedlings overexpressing T6P have suppressed PIF4 expression. T6P can be induced by glucose, but also sucrose (Lastdrager and others 2014). T6P regulates another player in the carbohydrate pathway: the protein kinase sucrose non-fermenting related kinase-1 (SnRK1). It has been shown that SnRK1 inhibits plant growth (Nunes and others 2013a). T6P inhibits the catalytic activity of SnRK1 (O Hara and others 2013). Based on literature on T6P (Padilla and others 2004) and SnRK1 signalling (Nunes and others 2013a), we propose a model in which suppression of SnRK1 by T6P results in increased PIF4/5 expression. (figure 4). Figure 3: Proposed T6P pathway. The model shows a possible connection between the T6P/SnRK1 pathway and PIF signalling. T6P would induce PIF transcription while SnRK1 suppresses it. Aim We aim to reduce plant elongation by investigating the regulation of PIFs. Our hypothesis is that lowering PIF transcription and expression, while at the same time increasing HY5 expression, will result in the desired compact plants. In short, for compact plants we want low PIF activity and high HY5 activity. This proposition is based on the functions of PIFs and HY5: stimulating and supressing elongation, respectively. As mentioned earlier, PIF (and HY5) activity is regulated by light quality, temperature and energy status. We studied how light quality, temperature, and energy status affects promoter activity of PIF4, 5 and HY5. Our ultimate goal is to identify plant growth treatments in which light quality, temperature, and/or sugars can be used to lower PIF4 and PIF5. At the same time, HY5 activity should be increased which, in theory, results in suppressing of elongation. To analyse promoter activity we used luciferase (LUC)-reporter lines. The method of LUC-reporter plants has been used for studies regarding gene expression. It uses the promotor of the gene of interest and fuses it to a 6

7 luciferase gene, which can be found in for instance click beetles or fireflies (Feeney and others 2016). The plants are placed in the Luminator, a climate cabinet that is fitted with a sensitive camera that captures the luminescence every half hour. The Luminator also enables us to change the light quality, humidity and temperature, making it possible to test +/-DIF and light quality in one machine. Materials and Methods Mutants Several different A. thaliana mutants were used during the experiments. For the Luminator experiments we had PIF4, PIF5 and HY5 mutants, where the respective promotor was attached to the luciferase gene from the firefly (appendix E) (Feeney and others 2016). For testing PIF expression in T6P mutants, A. thaliana otsa, otsb and KIN10RNAi knock-out mutants were used, as well as wildtypes of the Col-0 and Ler variety (Schluepmann and others 2003). The otsa and otsb mutants have a Col-0 background, the KIN10RNAi mutants have a Ler background. They were grown under either +DIF or DIF conditions (table 1). Luminator The PIF::LUC mutants, six replicates each, were four weeks old when they were placed for five days in the Luminator (Abeele 2017). During the treatments plants were sprayed with a 1 mm luciferin solution once in the morning and once in the afternoon. Full protocols and settings for each Luminator treatment can be found in Appendix G. +DIF vs DIF The Luminator was set to the following temperature regimes. +DIF and DIF were tested separately, each time on new plants. During the first and last hour of light during the day cycle, lights were set to 34 µmol per m 2 per second. We call this period ramping. The blue, red and far-red values were 8, 18 and 8 µmol per m 2 per second, respectively. During the rest of the day, plants received 100 µmol per m 2 per second. The blue, red and far-red values were 36, 56 and 8 µmol per m 2 per second, respectively. Table 1: Luminator treatments. +DIF: warm days and cold night, -DIF: cold days and warm nights. Each cell represents a photoperiod of 12 hours (lights on at 08:00 and off at 20:00). The dark cells represent dark periods. +DIF 22 C 12 C 22 C 12 C 22 C 12 C 22 C 12 C 22 C -DIF 12 C 22 C 12 C 22 C 12 C 22 C 12 C 22 C 12 C Red and blue pulse To test the effect of a brief red or blue pulse in the morning, PIF4:LUC and HY5:LUC plants were exposed to an extra one hour red or blue pulse for the duration of ramping. Studies have shown that application of a red 7

8 pulse during the first hour of the morning results in the same elongation suppression as a red pulse throughout the day (Gebraegziabher 2017). We therefore chose for a one-hour application of the pulses. The red and blue light values were, in turn, increased to daytime levels. This means that during a red pulse, the red value was increased from 18 to 56 µmol per m 2 per second. For a blue pulse, the value was increased from 8 to 36 µmol per m 2 per second. Other light settings remained the same. During each experiment the plants were photographed every 30 minutes (exposure time 7 minutes). The raw pictures (appendix B) obtained from the Luminator were processed using ImageJ. Each plant and one small background square were individually selected using the ROI tool, after which a MultiMeasure was performed. This resulted in a dataset of each plant showing the black/white ratio for each time point. The value of the background was subtracted in Excel to obtain the actual expression luminescence without background interference. The results were subsequently normalized, taking the luminescence value at 8:00 AM of the first morning and dividing all other values by the 8:00 AM value. This was done for each plant individually. Application of sugar To naturally induce a high energy status, we applied additional sugars to the plants. This would mimic the energy status of otsa mutants, but in a natural way and thus usable in the greenhouse. A trial experiment was performed to determine which sugar should be used and in which concentration. Glucose [3%], sucrose [5%] and trehalose [10mM] were selected for the trial due to their role in PIF expression and growth. Sorbitol was used as a control for each concentration. The sugars were tested on five-week old PIF4:LUC reporter plants. Figure 4: Rosette of A. thaliana. The leaf taken for leaf-disc is indicated in the red circle. (Online 2017) The plants were in the Luminator under +DIF conditions for three days before applying the sugar. 1 ml of sugar solution was pipetted on the plants, after which the experiment was continued for one more day. The results were normalized by dividing values by the 8:00AM values of the first morning. Next a leaf-disc experiment was set up, where a leaf was put in a luciferin-sugar solution. Leaf samples were taken of 4 week old plants and placed in a leaf disc tray. 48 Leaves were used: 24 from PIF4:LUC plants, the other 24 from 35S:LUC plants. The seventh leaf of each plant was taken consistently (figure 4). All discs were placed in the Luminator for one night in a constant temperature of 22 C in 250 µl luciferin solution [1mM]. The following morning, 50 µl of a solution of 3% glucose or 10 mm trehalose and 1 mm luciferin was added to four plants per treatment. Sorbitol was used as a control treatment. 50 µl of sorbitol, in respective concentrations, was added to two plants per treatment. The 24 remaining plants were only given a solution with luciferin, to function as a control for the sugars. The experiment was continued for the remainder of the day, after which the raw pictures were gathered for analysis. Extended night 8

9 We also wanted to mimic the lower energy status of otsb mutants in a natural way. This was attempted by exposing the plants to an extended night. PIF4:LUC and PIF5:LUC plants were placed in the Luminator for three days under constant 22 degrees C temperature to acclimate. They were exposed to normal 12h night/12h day period. After three days under normal temperatures and light regime, the night was extended for three hours, resulting in one cycle of 15h night/9h day regime. Quantitative Polymerase Chain Reaction (qpcr) Fifteen otsa, otsb and KIN10 RNAi mutants per condition were harvested between 13:00 and 15:00 PM (halfway the photoperiod), as previous experiments have shown that PIF expression was optimum around this time (figure 5B, 6B, 7B). The rosette of each plants (carbohydrate sink material) was cut out and stored in containers, separate from the bigger leaves (carbohydrate source material). The source and sink material were each pooled in groups of five individuals. This resulted in three replicates of source and sink material for each genotype in each condition. The plant material was ground to a powder while continuously kept in liquid nitrogen. RNA was extracted from the powder using the InviTrap Spin Plant RNA Mini Kit. Around 70 mg of powder per sample was used. Using the iscript TM cdna Synthesis Kit the RNA was converted into cdna, after which the cdna was diluted ten times for qpcr. We quantified PIF3, PIF4 and PIF7 mrna levels using quantitative polymerase chain reaction. ELONGATION FACTOR 1 α (Kozera and Rapacz 2013), CLATHRIN ADAPTOR PROTEIN-2 (AP2M) (Koloušková and others 2017) and AT5G08290 (mitosis protein YLS8) (Remans and others 2008) were used as reference genes. The source and sink material were first separately analysed using the -2 ( ct) method (Livak and Schmittgen 2001). This resulted in mean threshold values that could be compared between lines. There was no apparent benefit from keeping the source and sink values separated so they were combined into whole plants. This was done by calculating the ratio between the source and sink weight and adding the values in the same proportion. Separate source and sink results can be found in Appendix F. An F-test, followed by a t-test was done on the results to check for significance in +DIF vs DIF treatment within each mutant and for significance in +DIF and DIF treatments between each mutant. 9

10 Results Luminator -DIF lowers promotor activity and transcription. The Luminator does not accurately reflect actual PIF4, PIF5 and HY5 transcription values. To test the effects of DIF on promotor activity of PIF4, PIF5 and HY5, we placed four-week old PIF4::LUC, PIF5::LUC and HY5::LUC reporter lines in the Luminator. For +DIF the day temperature was set to 22 C, while the night temperature was set to 12 C. For DIF environments, these temperatures were reversed. To verify the pattern of the Luminator, the expression pattern of the genes was also tested through qpcr (figure 5B) (van Hoogdalem 2016). Two independent PIF4::LUC and PIF5::LUC lines were tested, but they showed a similar luc pattern (Appendix A). For the other experiments we only used the lines PIF4-1 and PIF5-7 as representative expression lines. A Figure 5: Comparison of PIF4 expression between Luminator (A) and qpcr (B). The qpcr shows expression in source leaves: the large outer leaves of the rosette. Luminator experiments were done on whole plants. Box in graph A represents dark period. Error bars represent SD. Data was normalized by dividing luminescence value by the value at 8:00 AM. Luminator: +DIF n = 12, -DIF n = 5. qpcr: n = 3 The results from the Luminator show a clear difference between +DIF and DIF (figure 5A). During the day, +DIF shows a steady increase in luminescence, followed by a drop in luminescence around 19:00 PM. This pattern is not observed in DIF, where luminescence remains low throughout the day, only to start increasing around 20:00 PM. This suggests that promotor activity increases during the day under normal, +DIF circumstances, while DIF suppresses promotor activity and keeps it low throughout the day. The greatest similarity between Luminator and qpcr is the observation that in both methods, values for DIF are significantly lower than +DIF. This shows that DIF does lower promotor activity and transcription, compared to +DIF. The overall pattern in qpcr is, however, still quite different than the luminescence pattern. In +DIF the transcription level steadily increases until about 13:00PM. The transcription decreases, with a sharp drop at 19:00PM. DIF also shows a different pattern in qpcr, with an increase in the middle of the day followed by a decrease towards the evening. The increase in transcription in qpcr is not observed in the Luminator. In fact, 10

11 the only increase observed in the Luminator is seen at 20:00PM, the exact moment the temperature starts to rise. This therefore suggests that the increasing luminescence is most likely caused by temperature, not actual promotor activity. A Figure 6: Comparison between Luminator (A) and qpcr (B) results for PIF5 expression. The qpcr shows expression in source leaves: the large outer leaves of the rosette. Luminator experiments were performed on whole plants. Black: +DIF, red: -DIF. Box in graph A represents dark period. Error bars represent SD. Data was normalized by dividing luminescence value by the value at 8:00 AM. Luminator: +DIF n = 6, -DIF n = 6. qpcr: n = 3 The Luminator pattern for PIF5 much resembles the pattern for PIF4 (figure 6A). As with PIF4, -DIF greatly lowers luminescence throughout the day with again an increase at 20:00PM. Luminescence in +DIF remains largely stable throughout the day, with a drop around 20:00PM. To verify the Luminator, another qpcr was performed but this time on PIF5. The qpcr pattern is very different (figure 6B). While DIF still results in lower transcription throughout the day, the pattern is largely the same as the +DIF pattern. +DIF transcription peaks at 11:00AM and decreases during the day. Around 20:00PM, transcription starts to increase again. DIF starts with a peak at the beginning of the day, but soon drops. Around 22:00PM transcription increases greatly. 11

12 A Figure 7: Comparison between Luminator (A) and qpcr (B) results for HY5 expression. The qpcr shows expression in source leaves: the large outer leaves of the rosette. Luminator experiments were performed on whole plants. Black: +DIF, red: -DIF. Box in graph A represents dark period. Error bars represent SD. Data was normalized by dividing luminescence value by the value at 8:00 AM. Luminator: +DIF n = 11, -DIF n = 5. qpcr: n = 3. Similar to the previous results, we again observe that for HY5 DIF also lowers luminescence. On the other hand, +DIF results in a gradual decrease of luminescence during the day (figure 7A). Comparing that to the qpcr pattern, we see great differences. qpcr shows that HY5 transcription is higher in DIF than +DIF during the day, even though only marginally (figure 7B). This is not observed in the Luminator. It is likely that here too, the effect of temperature on the LUC enzymatic reaction is substantial in the Luminator. This is derived from the observation that the qpcr pattern is very different from the luminescence pattern, which shows remarkable resemblance to PIF4 and PIF5. 12

13 A Red and blue pulses in morning increase PIF4, PIF5 and HY5 slightly expression in +DIF. Next, the effect of a red or blue pulse in the morning in +DIF on promotor activity was tested. The plants in the Luminator were exposed to increased red light between 8:00 AM and 9:00 AM for two days. After a cool-down day with normal morning lighting, the plants were exposed to increased blue light during the same period. This was done in regular +DIF settings. The graphs only show the morning periods, for full graphs see Appendix C. B C Figure 8: Effect of morning light pulse on PIF4 (A), PIF5 (B) and HY5 (C) in +DIF. The purple box represents the light pulse. The error bars represent the standard error. Data was normalized by dividing luminescence value by the value at 8:00 AM. Red pulse represents first red pulse administered. n = 6 Promotor activity after the red pulse remains comparable to that under normal circumstances. However, around 10:30 AM, activity after the red pulse increases for PIF4 and PIF5 as well as HY5 (figure 8A, 8B and 8C). This increase is greatest in PIF4 and PIF5, reaching up to an increase of about 0.5. The blue pulse has a similar effect on PIF4 and PIF5, though the increase in activity only reaches up to 0.3 and does not show a clear time of increase (figure 8A and 8B). In HY5, however, a blue pulse increases expression greatly throughout the morning period (figure 8C). Promotor activity shows an increase of about 1, comparing activity before the pulse and at the end of the morning. Red pulses in morning have no effect, blue pulse increase expression in DIF. The effect of a red or blue pulse in the morning in -DIF on promotor activity was also tested. The plants in the Luminator were exposed to increased red light between 8:00 AM and 9:00 AM for two days. The blue pulse was tested in a separate experiment, where the pulse was applied one morning. This was done in regular -DIF settings. The graphs only show the morning periods. For full graphs see Appendix D. 13

14 A B C Figure 9: Effect of red and blue pulse in morning on PIF4 (A), PIF5 (B) and HY5 (C) in DIF. The purple box represents the light pulse. Error bars represent standard error. Data was normalized by dividing luminescence value by the value at 8:00 AM. Red pulse represents first red pulse administered. n = 6. Figure 9 depicts the effect of the red and blue pulse in DIF. Other than +DIF, the red pulse appears to have little effect on promotor activity in DIF. Though these graphs only depict the morning period, there is also little effect for the rest of the day/night cycle (appendix D). At the end of the day it appears that PIF4, PIF5 and HY5 promotor activity is slightly increased, though there is quite an overlap in standard error. The blue pulse does show some effect on activity. This becomes most apparent in PIF5 and HY5 where the blue pulse leads to higher activity towards the end of the morning. When looking at the whole day/night cycle (appendix D), we see that this increase in PIF5 and HY5 in maintained until the experiment was ended. 14

15 Relative expression Relative expression Relative expression Relative expression Sugar signalling To test the effect of energy status on PIF expression, we harvested Col-0, otsa and otsb mutants in a Col-0 background and KIN10 RNAi mutants in a Ler background at 13:00 PM for qpcr, as well as their respective wildtypes. We used three biological replications. With the use of three reference genes, we were able to calculate the relative expression for PIF3, PIF4, PIF5 and PIF7. It was not possible to take PIF3 and PIF7 into account with the Luminator experiments, as we have not yet been able to create stable PIF3::LUC and PIF7::LUC reporter plants. The relative expression was first calculated for the sink and source material separately (appendix F) but, as there were no great differences, we decided to calculate the relative transcript levels in the whole plant. Since we only used three replicates per treatment, we mostly observe trends that have not (yet) been proven to be significant. A PIF3 B PIF * * Col-0 otsa otsb Ler Kin * * * Col-0 otsa otsb Ler Kin10 C PIF5 +DIF -DIF * Col-0 otsa otsb Ler Kin10 D PIF7 * * * Col-0 otsa otsb Ler Kin10 Figure 10: Effect of T6P and SnRK1 on PIF3 (A), PIF4 (B), PIF5 (C) and PIF7 (D) expression under +DIF (blue) and DIF (red). Results were normalized to reference genes, and according to -2( ct) method. Significance was calculated using a two-tailed t-test with a threshold of α = Black brackets show significance within a mutant line. Blue brackets show significance between mutant lines within a treatment. Error bars represent standard error. n = 3. Comparing data for each PIF should be done with caution, as expression levels are on very different relative scales. Relative expression of PIF5 can reach up to a fourfold increase, while we only measured a relative increase of for PIF3. This should be taken into account when comparing PIFs to each other. 15

16 T6P does not affect PIF3 expression. Impaired SnRK1 complex increases PIF3 expression in DIF. PIF3 expression is not affected by either higher or lower T6P levels. There is no observable difference between WT Col-0, otsa and otsb, in +DIF and -DIF. We do observe a decrease in expression comparing +DIF to DIF within each line, though this is only significant in the otsa mutants (figure 11A). An increase in PIF3 expression can be found comparing WT Ler to KIN10RNAi in DIF. This difference is not present in +DIF conditions (figure 11B). Overall this shows that the SnRK1 complex suppresses PIF3 expression, but only in DIF. Decreased T6P levels increase PIF4 expression in +DIF. Impaired SnRK1 complex increases PIF4 expression in DIF. PIF4 expression is not significantly affected by a higher or lower T6P level but there is a trend of increased PIF4 expression in +DIF under decreased T6P levels. When looking at DIF conditions, we see no apparent difference in expression between WT Col-0, otsa and otsb (figure 11B). We do see a significant change in PIF4 expression in otsa plants comparing +DIF to DIF. The other lines also show a difference between +DIF and DIF but this is not significant. There appears to be an increase in expression between Ler and KIN10RNAi in DIF, though this is not significant (figure 11B). In +DIF expression is the same with Ler as in KIN10RNAi. Decreased T6P levels increase PIF5 expression in +DIF. Impaired SnRK1 complex increases PIF5 expression. The most striking observation is the increase in PIF5 expression in the otsb mutant in +DIF, though statistically this is not significant (figure 11C). The other lines showed comparable expression levels both in +DIF and DIF. There also is no significant difference between WT Ler and KIN10 RNAi, either within the line or between lines. There may be a trend that KIN10RNAi leads to higher expression in +DIF and -DIF, but this remains uncertain (figure 11C). Decreased T6P levels increase PIF7 expression in +DIF. Impaired SnRK1 complex increases PIF7 expression in DIF. The results for PIF7 are quite different from the other PIF expression results. The most striking observation is the trend that DIF increases expression in all mutant lines except otsb. This increase in expression, however, is only significant in KIN10 RNAi (figure 11D). The otsb mutants shows an opposite expression pattern. +DIF results in a significantly higher expression of PIF7. Compared to the +DIF levels, -DIF results in significantly lower expression (figure 11D). 16

17 Spraying sugar results As a first trial, we sprayed glucose, sucrose and trehalose on plants to naturally simulate the osta mutation, which results in a high energy status. Sorbitol was used as a control substance. The sugars were tested on PIF4:LUC reporter plants that were five weeks old. The plants were in the Luminator under +DIF conditions for A three days before directly pipetting the sugars on the plants. B C Figure 11: effect of spraying glucose (A), sucrose (B) and trehalose (C) on PIF4 plants. Red: respective treatment, black: control. Arrow indicates moment of spraying. Error bars represent standard error. n = 4 Figure 11 shows that spraying sugars directly on plants had no significant effect. Not only was there almost no difference in luminescence between control plants and treated plants, there was also little difference between pre-treatment and posttreatment luminescence (figure 11A, 11B and 11C). Only trehalose showed higher luminescence than their control counterparts, but this difference is also observed before plants were treated at all (Figure 11C). Due to the lack of response, we determined that a second trial was needed, since there were many other factors that could also account for the lack of response. 17

18 Leaf disc results Because the plants were rather old and the application method of the sugar was not ideal, a leaf-disc experiment was set up. Parts of leaves of four-week old plants were cut and placed in a luciferin-sugar solution for a whole day-night cycle. A B Figure 12: Results of leaf-disc experiment on PIF4 leaves for trehalose (A) and glucose (B). Red: treatment. Black: control with sorbitol. Yellow: only luciferin. Purple triangle: time of application of sugar solution. Error bars indicate standard error. n = 4 A B Figure 13: Results of leaf-disc experiment on 35S leaves for trehalose (A) and glucose (B). Red: treatment. Black: control with sorbitol. Yellow: only luciferin. Purple triangle: time of application of sugar solution. Error bars indicate standard error. n = 4 One of the most noticeable observations from the leaf-disc experiment is the large standard error, which is especially apparent in the trehalose treatments (figure 12A and 13A). This hampers the possibility to derive any conclusions from the results. Another observation is that luminescence decreases throughout the experiment. This decrease is also observed before application of the sugar solution. It is therefore unclear how much of the decrease in luminescence and thus promotor activity is due to the sugar and how much is caused by confounding factors. During the experiment we observed that the leaf-discs were quite dry. This could account for the rapid drop in luminescence. After adding the sugar solutions, the leaves would, temporarily, be covered by liquid again. This explains why the decrease in luminescence is less steep after adding the sugars (figure 12A and 12B, figure 13A and 13B). 18

19 A Extended night lowers PIF4 and HY5 expression The extended night treatment was devised to naturally simulate the otsb mutation, which resulted in a low energy status. PIF4 and HY5 plants were placed in the Luminator, set at a constant temperature of 22 C. During the first cycle plants experienced a photoperiod of 12 hours, followed by a dark period of also 12 hours. For the second cycle, the photoperiod was set to 12 hours, while the dark period was extended to 15 hours. Figure 124: Effect of an extended dark period on PIF4 (A) and HY5 (B). Blue box: dark period. Grey box: extension of dark period. The error bar represent standard error. n = 6. PIF4 promotor activity is lowered after the extended night. During the day, promotor B activity follows a normal pattern but due to the initial decrease, daytime levels remain lower (figure 14A). HY5 promotor activity is also decreased after the extended period. An additional drop in activity is observed during the extended dark period, leading to a lower activity at the beginning of the morning. Activity increases during the morning, comparable to normal circumstances, but the altitude of this peak is greatly reduced. Overall, HY5 promotor activity remains low for the duration of the day (figure 14B). 19

20 Discussion Luminator We aimed at increasing the insight in regulation of Phytochrome Interacting Factors. We tested the influence of -DIF on PIF promotor activity through luminescent PIF mutants. With the Luminator we attempted to quantify PIF promotor activity in an efficient way, while controlling light quality and temperature. It is hard to derive solid conclusions from the Luminator results. This is mainly due to the discrepancy between the Luminator and qpcr results. We hypothesize that the qpcr represents the most reliable pattern. qpcr is based on mrna presence and is quite reliable, while the Luminator output can be influenced by many factors. The factor that best explains the difference between the Luminator and qpcr is the effect of temperature on the enzymatic luciferase reaction. This hypothesis is substantiated by the promotor activity patterns in the Luminator. PIF4, PIF5 and HY5 all show similar patterns in the Luminator in DIF environments. Each line shows a decrease in expression which lasts for the whole cold period. The normal expression fluctuations that are found in +DIF are not repeated in DIF. This suggests that the effect of temperature on luciferase overrules any promotor activity changes that may arise. In mammalian studies, LUC has been used to determine gene expression patterns. They showed that acute changes in temperature induce changes in bioluminescence, irrespective of the promotor to which the LUC-gene was expressed (Feeney and others 2016). It seems that the influence of temperature on the LUCprotein and thus luminescence is stronger than the promotor activity. In addition, qpcr is based on the presence of mrna. The Luminator quantifies promotor activity. Promotor activity does not necessarily translate linearly into protein activity. This could further explain the difference between the Luminator results and qpcr results. It is therefore important to refrain from quantitative conclusions based on the +DIF vs DIF results of the Luminator experiments. The only clear conclusion is that the Luminator and the LUC-fusion method may not accurately report changes in expression due to temperature. The reasons mentioned above do not mean that we saw no effect of DIF on PIF expression. Both the Luminator and qpcr showed a decrease in promotor activity and expression, respectively. Though the decrease cannot be quantified, it is still highly probable that promotor activity and expression do indeed decrease. This is promising for the use of DIF in plant growth protocols. Novel methods in measuring gene expression could be interesting for this line of research. Red and blue pulse Other experiments in the Luminator have dealt with light quality rather than temperature. We compared different mutant lines within the same temperature regime, while controlling light quality. They are therefore exposed to the same influence of temperature on luciferase and can therefore be used for quantitative research. The admission of a red pulse in +DIF between 8:00 AM and 9:00 AM did not greatly affect PIF4, PIF5 or HY5 promotor activity. We did observe higher PIF4 and PIF5 promotor activity during the night than the control 20

21 treatment. The second red pulse had similar expression patterns as the control treatment during the day, but decreased during the night (appendix C). This suggests that application of the red pulse does not have the desired effect of lowering PIF promotor activity at first, but continuing the treatment does result in a decrease. These observations in part confirm our hypothesis that a red pulse decreases PIF promotor activity through extra activity of phyb. It is still possible that the first red pulse results in less elongation as PIF4 and PIF5 protein are destabilized by phyb during the day. PhyB is activated further by the extra red light. This means that though PIF promotor activity itself is not affected by the red pulse, PIF expression is lowered by the extra activated phyb. Since HY5 is not affected by the red pulse, it is still able to further suppress PIF4 expression on a protein level. Where the red pulse had no effect on PIF4, PIF5 and HY5 promotor activity in DIF, the blue pulse showed different results. HY5 promotor activity was increased after a blue pulse, even in DIF. This suggests that Cry1 has an effect in colder temperatures, where phyb not. Though a blue pulse in DIF increases PIF4 promotor activity as well, it is possible that there is still a nett reduction in PIF4 expression due to the increase in HY5. This all remains speculation as the effect of low temperatures on Cry1 have not yet been shown. So far, science has qpcr on T6P/SnRK1 mutants Our results have shown that increasing the T6P levels does not affect PIF expression. This contradicts earlier research that shows that increasing T6P levels suppresses PIF4 expression (Zhang and others 2009). An explanation of this discrepancy may lay in the fact that seedlings were used, while our results are based on adult plants. Our results suggest that T6P levels are saturated in adult plants under normal conditions. Artificially increasing this will have no effect. Decreasing T6P, however, seems to have great effect on especially PIF4 expression, though only in +DIF. This result was opposite to our expectation. Since T6P increases PIF expression according to literature, we expected that decreasing this would have the opposite effect. So far we have not been able to find an explanation for this observation that coincides with literature. PIF3 on the other hand, appears to be unaffected by either higher or lower T6P. We hypothesize that PIF3 is regulated independently of T6P. Interestingly, we observed that disrupting the SnRK1 complex increases PIF expression in DIF. This means that the SnRK1 complex suppresses PIF expression, but only in DIF. This is in contrast with the effects of T6P, which are only observed in +DIF. Lower temperatures have been shown to enable the T6P/SnRK1 pathway to recover growth after stress (Nunes and others 2013b). They only speak of the effects of cold on the whole pathway but do not mention where specifically in the pathway colder temperatures have an effect. It is possible that the effect of colder temperatures is mainly felt by SnRK1, as our results suggest. The effect of changing T6P levels is the same in +DIF and DIF so temperature does not seem to be a factor. This is not the case in the KIN10RNAi mutant line, where we see an increased PIF expression in DIF. This therefore indicates that temperatures mainly affects the SnRK1 complex, not the whole T6P/SnRK1 pathway. Though qpcr resulted in interesting trends, the main draw-back of these results lays in the lack of statistical strength. We only performed measurements on three biological replicates. It is therefore hard to obtain 21

22 significant results, based solely on the number of biological replicates. We therefore recommend repeating these experiments with more replicates to determine actual, significant interactions. Spraying sugars The effect of the energy status was also tested through direct application of sugars like sucrose, glucose and trehalose, thus mimicking otsa mutant energy levels. The first trial in which we pipetted the sugars on the plants showed no change after application. This was unexpected but could be attributed to the method. It is possible that direct application of the sugars on the leafs hampered uptake and could therefore distort our measurements. A second trial was performed where we used leaf-discs placed in a solution of luciferin and glucose or trehalose, under a constant temperature of 22 C. These results proved unreliable as there was a great variation between individuals, resulting in a high standard error. This can be explained by the evaporation of solution during the experiment as we put no lid on the leaf-disc slide. No lid was put on to ensure clear pictures, but this had dramatic effect on the solution. So far, we have therefore not been able to come up with an experimental set-up to test the effects of direct application of sugars. From our results we cannot clearly indicate the effects of sugar on PIF promotor activity. This does not mean that this effect is non-existent. More research has to be done. Extended night The extended night treatment was devised to naturally induce an otsb-like environment. If this method proved to have the desired effect, lower PIF4 expression, this could be used by horticulturists as a protocol for compact plants. The qpcr results on otsb mutants were inconclusive. PIF4 expression seems to increase, though this was not statistically significant. The extended night treatment resulted in a slight decrease of PIF4, though this is based on the PIF4 promotor activity. There are several explanations that could explain this difference. Firstly, there is the difference in approach. qpcr on otsb mutants report mrna levels, while extended night in the Luminator reports PIF4 promotor activity. Promotor activity and mrna presence are not linearly linked. Secondly, there is the statistical insignificance of the qpcr results. We cannot say with certainty what the effect of a lower energy status is on PIF4, based on these results. According to theory and previous studies, an effect should be measurable. We therefore advice further research into this subject, perhaps with more replicates for the qpcr. This could give conclusive evidence into the effects of lower energy status on PIF4, which can then be linked to the Luminator observations. Future research There are still many factors that remain to be studied in order to fully understand the mechanisms behind DIF, and how to increase the effect of DIF. We have successfully mutated the LUC complex combined with the PIF4-, PIF5- and HY5 promotor but they are not the only players in the PIF regulatory pathway. Looking at our proposed model for PIF regulation PIF3 is also involved in the process. 22

23 PIF3 has been shown to function in both the phya and phyb signalling pathways (Ni and others 1998). Models have also placed PIF3 at a downstream position of PIF4 and PIF5 (Bours and others 2015). PIF3 could therefore be used as an indicator of actual PIF4 activity. This could tackle the problem we encountered earlier, where promotor activity possibly not accurately portrays protein activity. We also attempted to test indole-3-acetic acid inducible 29 (IAA29) expression, another downstream target of PIF4 (Sun and others 2013). This interaction has been proposed to be crucial for diurnal and photoperiodic control of plant growth (Kunihiro and others 2011). This could give additional information regarding actual PIF4 protein activity. There have been PIF4:PIF4:LUC transformed plants to determine actual protein activity. This construct has not successfully shown expression patterns yet. It could also be desirable to look beyond the LUC-complex due to the temperature influence on luciferase. There may be other constructs better suited for this type of experiment that are temperature-independent. This can further explored. Another course of action, though labour-intensive and perhaps not possible, is to quantify the temperature effect on the LUC protein. If this effect can be quantified, it is possible to normalize results accordingly. It could be another form of background adjustment. This course of action is hampered by the fact that we would need a reference promotor that is regulated in the same way as PIF. So far, such a reference promotor has not been identified. Conclusion We have demonstrated that DIF lowers promotor activity as well as protein expression. Our luminescence method does not accurately reflect gene expression levels. Due to the temperature dependence of the LUCgene, it is not possible to derive quantitative conclusions from the results from the +DIF vs DIF experiments. The LUC-fusion method can therefore not be used for quantitative tests based on temperature changes. The Luminator can be used for experiments based on other factors like light intensity and quality. Applying a red or blue pulse in the morning increased PIF4, PIF5 and HY5 promotor activity. It is unclear, however, if this effect is specific for PIF and HY5 promotor activity. Light-controlled genes in general could show higher promotor activity due to higher light intensity. Conclusions regarding the effect of T6P and SnRK1 should be drawn with caution. Due to the low number of repetitions, significant correlations were hard to determine. We have shown the trend that decreasing T6P levels increases PIF4, PIF5 and PIF7 expression. Increasing T6P on the other hand did not have an effect. This was surprising as we expected T6P to have a positive effect on PIF expression. Decreasing T6P levels were therefore expected to also decrease PIF expression. As this observation was not significant, it remains uncertain if these results portray an actual biological event or are just an anomaly. Since increasing T6P levels had no effect and decreasing the levels had an unexpected and uncertain effect, it is not clear where the T6P pathway should be placed with regards to PIF expression. Notably PIF3 expression is not affected by T6P at all. It is possible that PIF3 is the subject of independent regulation, regardless of PIF4 and PIF5. 23

24 An impaired SnRK1 complex increases PIF expression in DIF. SnRK1 therefore negatively regulates PIFs, but only appears to do so in DIF circumstances. So far an explanation for this difference between +DIF and DIF has not been found. qpcr of Col-0 plants also showed that DIF lowers PIF expression, compared to +DIF. Though this was not the main aim of the experiment, it does show in a reliable way that DIF lowers PIF expression. Prolonging the dark period under constant temperature resulted in a decrease in HY5 promotor activity but it had no effect on the PIF4 promotor. Prolonging the dark period to induce a state of starvation would not contribute to lowering PIF promotor activity. Furthermore, it lowers HY5 promotor activity, an undesirable result with regards to reducing plant elongation as HY5 competes with PIF4. Returning to the aim of breeding compact plants, we have shown that DIF lowers PIF promotor activity and expression with the use of qpcr and luminescence mutants, though the quantitative difference remains unclear. This suggests that PIFs are indeed one of the factors behind the effectiveness of a DIF regime. To further the efforts for compact plants, we conclude that applying a red or blue pulse will probably add to compactness. The blue pulse will possibly be more effective than the red pulse, especially in DIF environments. We expect that the effect of increased PIF4 promotor activity will be negated by the increase in HY5 promotor activity. HY5 promotor activity is only increased by a blue pulse in DIF, a red pulse showed no effect. From this we conclude that in DIF a blue pulse is more promising with regard to achieving compact plants. More research is needed on the further effects of light treatments and DIF, but our results have given a first indication of possible effects. 24

25 Acknowledgements There are several people who helped me immensely during my thesis. First of all, I would like to thank Mark for giving me this opportunity and providing me with great advice, patience and entertaining football discussions. I also want to thank Sander for keeping me alert and critical on my work. I am also grateful to Jacqueline and Mariëlle for helping me with the practical work in the laboratory. I want to thank STW and the user group for the project have to be thanked for enabling me to write this thesis. Finally I would like to thank my friends Peter Seghers and Michelle Everard. Peter was a just and critical reviewer, as well as a great sounding board during breaks. Michelle was a great help in formulating my report in such a way that the whole thing made sense. Thank you! References Abeele Cvd Measuring diurnal gene activity using the luciferase reporter system. Wageningen Universy and Research. Bae G, Choi G Decoding of light signals by plant phytochromes and their interacting proteins. Annu Rev Plant Biol 59: Bours R, Kohlen W, Bouwmeester HJ, van der Krol A Thermoperiodic Control of Hypocotyl Elongation Depends on Auxin-Induced Ethylene Signaling That Controls Downstream <em>phytochrome INTERACTING FACTOR3</em> Activity. Plant Physiology 167(2): Bours R, van Zanten M, Pierik R, Bouwmeester H, van der Krol A Antiphase light and temperature cycles affect PHYTOCHROME B-controlled ethylene sensitivity and biosynthesis, limiting leaf movement and growth of Arabidopsis. Plant Physiol 163(2): Carvalho SM, Heuvelink E, Cascais R, van Kooten O Effect of day and night temperature on internode and stem length in chrysanthemum: is everything explained by DIF? Ann Bot 90(1): Castillon A, Shen H, Huq E Phytochrome Interacting Factors: central players in phytochrome-mediated light signaling networks. Trends in Plant Science 12(11): Feeney KA, Putker M, Brancaccio M, O Neill JS In-depth Characterization of Firefly Luciferase as a Reporter of Circadian Gene Expression in Mammalian Cells. Journal of Biological Rhythms 31(6): Gangappa SN, Kumar SV DET1 and HY5 Control PIF4-Mediated Thermosensory Elongation Growth through Distinct Mechanisms. Cell Reports 18(2): Gebraegziabher H Hofmann NR A Mechanism for Inhibition of COP1 in Photomorphogenesis: Direct Interactions of Phytochromes with SPA Proteins. The Plant Cell 27(1):8-8. Hornitschek P, Kohnen MV, Lorrain S, Rougemont J, Ljung K, López-Vidriero I, Franco-Zorrilla JM, Solano R, Trevisan M, Pradervand S et al Phytochrome interacting factors 4 and 5 control seedling growth in changing light conditions by directly controlling auxin signaling. The Plant Journal 71(5): Khanna R, Huq E, Kikis EA, Al-Sady B, Lanzatella C, Quail PH A Novel Molecular Recognition Motif Necessary for Targeting Photoactivated Phytochrome Signaling to Specific Basic Helix-Loop-Helix Transcription Factors. The Plant Cell 16(11): Koini MA, Alvey L, Allen T, Tilley CA, Harberd NP, Whitelam GC, Franklin KA High temperature-mediated adaptations in plant architecture require the bhlh transcription factor PIF4. Curr Biol 19(5): Koloušková P, Stone JD, Štorchová H Evaluation of reference genes for reverse transcription quantitative real-time PCR (RT-qPCR) studies in Silene vulgaris considering the method of cdna preparation. PLOS ONE 12(8):e Kozera B, Rapacz M Reference genes in real-time PCR. Journal of Applied Genetics 54(4): Kumar SV, Lucyshyn D, Jaeger KE, Alos E, Alvey E, Harberd NP, Wigge PA Transcription factor PIF4 controls the thermosensory activation of flowering. Nature 484(7393): Kunihiro A, Yamashino T, Nakamichi N, Niwa Y, Nakanishi H, Mizuno T PHYTOCHROME-INTERACTING FACTOR 4 and 5 (PIF4 and PIF5) Activate the Homeobox ATHB2 and Auxin-Inducible IAA29 Genes in the Coincidence Mechanism Underlying Photoperiodic Control of Plant Growth of Arabidopsis thaliana. Plant and Cell Physiology 52(8): Lastdrager J, Hanson J, Smeekens S Sugar signals and the control of plant growth and development. J Exp Bot 65(3):

26 Lau OS, Deng XW The photomorphogenic repressors COP1 and DET1: 20 years later. Trends in Plant Science 17(10): Legris M, Klose C, Burgie ES, Rojas CC, Neme M, Hiltbrunner A, Wigge PA, Schafer E, Vierstra RD, Casal JJ Phytochrome B integrates light and temperature signals in Arabidopsis. Science 354(6314): Leivar P, Monte E PIFs: Systems Integrators in Plant Development. The Plant Cell 26(1): Leivar P, Quail PH PIFs: pivotal components in a cellular signaling hub. Trends Plant Sci 16(1): Livak KJ, Schmittgen TD Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25(4): Ma D, Li X, Guo Y, Chu J, Fang S, Yan C, Noel JP, Liu H Cryptochrome 1 interacts with PIF4 to regulate high temperature-mediated hypocotyl elongation in response to blue light. Proceedings of the National Academy of Sciences 113(1): Maas FM, van Hattum J Thermomorphogenic and Photomorphogenic Control of Stem Elongation in Fuchsia Is Not Mediated by Changes in Responsiveness to Gibberellins. Journal of Plant Growth Regulation 17(1): Myster J, Moe R Effect of diurnal temperature alternations on plant morphology in some greenhouse crops a mini review. Scientia Horticulturae 62(4): Ni M, Tepperman JM, Quail PH PIF3, a phytochrome-interacting factor necessary for normal photoinduced signal transduction, is a novel basic helix-loop-helix protein. Cell 95(5): Nunes C, O Hara LE, Primavesi LF, Delatte TL, Schluepmann H, Somsen GW, Silva AB, Fevereiro PS, Wingler A, Paul MJ. 2013a. The Trehalose 6-Phosphate/SnRK1 Signaling Pathway Primes Growth Recovery following Relief of Sink Limitation. Plant Physiology 162(3):1720. Nunes C, Schluepmann H, Delatte TL, Wingler A, Silva AB, Fevereiro PS, Jansen M, Fiorani F, Wiese-Klinkenberg A, Paul MJ. 2013b. Regulation of growth by the trehalose pathway: Relationship to temperature and sucrose. Plant Signaling & Behavior 8(12):e O Hara LE, Paul MJ, Wingler A How Do Sugars Regulate Plant Growth and Development? New Insight into the Role of Trehalose-6-Phosphate. Molecular Plant 6(2): Online CP. Arabidopsis Thaliana [Internet]. Padilla L, Morbach S, Krämer R, Agosin E Impact of Heterologous Expression of Escherichia coli UDP- Glucose Pyrophosphorylase on Trehalose and Glycogen Synthesis in Corynebacterium glutamicum. Remans T, Smeets K, Opdenakker K, Mathijsen D, Vangronsveld J, Cuypers A Normalisation of real-time RT-PCR gene expression measurements in Arabidopsis thaliana exposed to increased metal concentrations. Planta 227(6): Saijo Y, Sullivan JA, Wang H, Yang J, Shen Y, Rubio V, Ma L, Hoecker U, Deng XW The COP1 SPA1 interaction defines a critical step in phytochrome A-mediated regulation of HY5 activity. Genes & Development 17(21): Sairanen I, Novák O, Pěnčík A, Ikeda Y, Jones B, Sandberg G, Ljung K Soluble Carbohydrates Regulate Auxin Biosynthesis via PIF Proteins in Arabidopsis. The Plant Cell 24(12): Schluepmann H, Pellny T, van Dijken A, Smeekens S, Paul M Trehalose 6-phosphate is indispensable for carbohydrate utilization and growth in Arabidopsis thaliana. Proceedings of the National Academy of Sciences of the United States of America 100(11): Sun J, Qi L, Li Y, Chu J, Li C PIF4 Mediated Activation of YUCCA8 Expression Integrates Temperature into the Auxin Pathway in Regulating Arabidopsis Hypocotyl Growth. PLOS Genetics 8(3):e Sun J, Qi L, Li Y, Zhai Q, Li C PIF4 and PIF5 transcription factors link blue light and auxin to regulate the phototropic response in Arabidopsis. Plant Cell 25(6): Thingnaes E, Torre S, Ernstsen A, Moe R Day and Night Temperature Responses in Arabidopsis: Effects on Gibberellin and Auxin Content, Cell Size, Morphology and Flowering Time. Annals of Botany 92(4): van Hoogdalem M qpcr profiles of PIF4 and HY5 expression in +DIF and -DIF. Zhang Y, Primavesi LF, Jhurreea D, Andralojc PJ, Mitchell RAC, Powers SJ, Schluepmann H, Delatte T, Wingler A, Paul MJ Inhibition of SNF1-Related Protein Kinase1 Activity and Regulation of Metabolic Pathways by Trehalose-6-Phosphate. Plant Physiology 149(4):

27 A B Appendices A. PIF4-1 vs PIF4-2 and PIF5-6 vs PIF5-7 Figure 15: Comparison of PIF4-1 (A) and PIF4-2 (B). Both promotor activity patterns are comparable. For further experiments we chose to continue with PIF4-1. B Figure 13: Comparison of PIF5-6 (A) and PIF5-7 (B). Both promotor activity patterns are comparable. For further experiments we chose to continue with PIF

28 B. Luminator output Figure 147: A visual representation of the output of the Luminator camera. Each plant was individually selected in ImageJ with the Region of Interest manager. The level of white pixels was determined and used to calculate relative luminescence. 28