Growing compact plants by altering Carbohydrate. metabolism

Transcription

1 Wageningen UR Laboratory of Plant Physiology (PPH) MSc Thesis Growing compact plants by altering Carbohydrate metabolism Name: Xiao Han Register Number: Supervisors: Mark van Hoogdalem

2 Abstract It is important for modern horticulture to reduce the unit production cost by, instead of spraying plant growth regulators (PGRs), using more sustainable methods to grow compact plants. In previous studies, DIF was found to inhibit plant growth and lead to a compactness plant structure. Since the reason for it could be the starch starvation resulting from earlier starch degradation caused by a changed circadian rhythm, other treatments such as extended night, trehalose spraying, which were also found to change the starch content, might be the less cost alternative methods of -DIF. In this research, all the treatments resulted in a smaller plant with shorter leaves, smaller leaf areas, less biomass accumulations and changed starch status. Among them, -DIF was the only treatment which repress the leaf elongation by shortening the petiole. The results of this study indicate that the treatments that were tested here might influence leaf growth through the control of starch metabolism. Key words: -DIF, extended night, trehalose, starch, leaf elongation I

3 Contents Introduction... 1 Aim... 3 Research questions... 3 Approaches... 3 Hypotheses... 5 The influence of DIF... 5 The influence of Extended night... 6 The influence of Trehalose... 6 Research Methods... 7 Plant Material... 7 Systems... 7 Treatments... 8 Measurements... 9 Results Plant morphology and biomass accumulation Starch status Discussion DIF Extended night Trehalose Conclusions and recommendations Acknowledgments References II

4 Introduction Due to lower light levels, especially during fall and early spring, it can be challenging for the horticulture industry in the Netherlands to grow well shaped, compact plants in greenhouses throughout the year. By growing compact plants the plant density in the greenhouse can increase, which contributes to a reduction in energy cost of unit yield. Therefore, enhance plant density by improving plant compactness is an important issue on commercial production optimization. Plant compactness is used to describe plant morphological architecture, especially plant size. Compact plant is perceived as having a tighter structure, lower height and shorter petioles (Figure 1). Most studies about plant compactness regulation are devoted to reducing plant height, limiting shoot growth and leaf size by controlling the growth condition (temperature, light, humidity, CO2 concentration and etc.) (Heuvelink and Meeteren, 1998; Clifford et al., 2004; Hansen and Petersen, 2004), exerting stress factors (mechanical stress, cold water) or applying plant growth regulators (plant hormones, chemical growth retardants) (Armitage, 1994; Henny, 1986; Carvalho, S. M. P. et al., 2008). Figure 1 Compact plant (right) compared with loose plant (left) In greenhouses today, chemical plant growth regulators (PGRs) are most commonly used to control plant compactness. However, the use of these chemicals needs to be replaced by more sustainable ways. One way is through growing plants under DIF conditions. DIF results in reduced internode length (cell length and cell number), plant height, leaf area/size, petiole length, and to a downwards orientation of shoots and leaves (Vogelezang, 1998; H. Kok, H. van Aanholt, 2005). DIF, alters the circadian clock, which leads to a decreased downstream hormonal signals (weaken the auxin-ethylene cascade reaction) and growth stimulation 1

5 factors such as PHYTOCHROME INTERACTING FACTORs (PIFs), -especially PIF3 (Khanna et al., 2004; Bours and Kohlen, 2015). The circadian clock in plants also controls starch metabolism. During the day, starch is synthesized and stored mainly in the older (bigger) leaves. During the night, starch is broken down to provide energy to maintain growth and metabolism. For the circadian clock, temperature and light (intensity and day length) are sympathetic input signals, which could adjust the starch metabolism rate to the anticipated dawn by controlling expression of the clock genes GIGANTEA (GI), Timing of CAB expression 1 (TOC1), to make sure the plant starch reserves are not completely exhausted at the end of night, which otherwise would result in growth arrest (Stitt and Zeeman, 2012). But how does the carbohydrate/energy status relates to growth? Some studies showed that the plant growth is controlled by the complementary activity of two kinase complexes- SnRK1 and TORC1. SnRK1 (short for SNF1-related Protein Kinase1) can inhibited plant growth by activating a set of genes, while TORC1 on the contrary (Smeekens et al., 2010; Robaglia et al., 2012; Xiong and Sheen, 2014). It was found that under carbohydrate starvation or energy depleting conditions, The SnRK1 protein kinase will be activated (Smeekens et al., 2010)leading to an inhibition in growth. Thus, the activation of SnRK1 caused by a starch starvation before dawn might be the reason why plant growth was inhibited under DIF. Although DIF results in compact plants in greenhouses, there are several downsides to the treatment: heating up the greenhouses at night requires a lot of energy, the treatment can only be applied during winter and early spring, and not all species are influenced by DIF. For these reasons it is necessary to find alternative methods to inhibited plant elongation without the use of PGRs. As mentioned above, -DIF may influence plant compactness by affecting carbohydrate metabolism, it probability could be altered by other treatments which could make sense in the same way. One method could be extending the night. During night, nutrient uptake reduced since stomata are closed. This is also accompanied by starch in source leaves degrading to sustain the plant growth. As mentioned above, the starch in source leaves will be almost depleted before dawn to make sure meeting the highest energy use efficiency for growth. However, when the night is unexpectedly extended, the starch metabolism activity will continue, starch reserves will be completely exhausted, and this will result in carbohydrate starvation and plant growth arrest. Apart from DIF and extended night, trehalose spray is also a novel treatment that can influence starch status. Trehalose is a nonreducing disaccharide, which widely exists in bacteria, fungi, insect and plant. In plants, trehalose-6-phosphate (T6P) 2

6 is the mainly reactive intermediate of trehalose biosynthesis (Griffiths et al., 2016). It was found that in Arabidopsis seeding plants, T6P level has a high correlation with sucrose content, which means T6P could be used as a mark of sucrose content (Lunn et al., 2006; Fernandez et al., 2012). Starch degradation was shown to be inhibited by high T6P level (Martins et al., 2013).Besides that, the activity of SnRK1 was also found to be related with T6P by sugar signaling pathway (Lunn et al., 2006; Zhang et al., 2009). Treating plants with trehalose inhibits the transformation of T6P into trehalose, leading to an increase of T6P level which will inhibit starch degradation in source leaves. As it is known that, the starch accumulation is a result of starch biosynthesis and degradation, the reduced starch degradation will result in a higher starch level at the end of day by spraying trehalose in the daytime. Therefore, more carbohydrates will be stored as starch and less carbohydrates will be used for soluble sugar synthesis. As a result, there will be less sugar available for growth. Furthermore, it will also activate SnRK1 and inhibit growth through sugar signal pathway. Aim The aim of this project is to explore how DIF, extended nights and trehalose treatments will influence plant morphology and biomass accumulation, and try to link these to changes in starch metabolism. Research questions To study how DIF, intermittent extended night and trehalose treatments will affect plant starch status and growth in Arabidopsis, the following subquestions will be researched: 1. What is the effect of the treatments on the morphology of Arabidopsis, especially on leaf and petiole elongation? 2. How will they influence the biomass accumulation? 3. How do the treatments affect starch content in source and sink tissues in Arabidopsis rosettes? Approaches Arabidopsis is the most commonly used model plant, which has a relatively small and highly pure genome. It has many mutants and can grow very fast. Therefore, it was chosen to be the main material in this research. 3

7 Although it is known that DIF suppresses elongation of the whole plant, the whole process and how it influences separate parts of the plant has not been studied in detail. During the whole life of Arabidopsis, the role of a single leaf is changing during its ontogenesis process. Young leaves play the role of a sink, which needs to import sucrose from source leaves to sustain its growth. Other tissues, which have excess sugar such as root and old leaves, are playing the role of source. However, after the leaf area of the younger ones reach 30%~60% of their final size, they will turn into the source. In this experiment, the inner part of Arabidopsis will be treated as the sink, while outer as the source. By measuring the starch content of them, we will get an indication on how DIF influences the starch status in source and sink leaves during day and night. It will also act on the final biomass accumulation of the whole rosette. To study how it will influence the final morphology of the plant, the effects of DIF on leaf and petiole elongation will be tested. Furthermore, were are also interested in how it will influence the plant structure. It will be a challenge to find a good indicator or a good method which can be used to show the changes of plant structure caused by the application of different treatments. In previous studies, LAI has been used to represent the canopy structure, which makes sense for gas, water, and nutrient exchange between plants and ground, since it is defined as a ground-based optical measurement which equal to one-sided leaf per unit ground surface area on a horizontal orientation (Gower and Norman, 1991; J. M. Chen and J. Cihlar, 1995; Hopkinson et al., 2013). However, it is not suitable for plants like Arabidopsis which have a compact and short rosette and grown separately on Rockwool. Therefore, a new index called overlap index comes up here. It is calculated by dividing added separate leaves area by rosette overlook area. Overlap index is defined as an indicator of the plant structure in this article. It will be used to present how the changes of plant structure caused by growth will affect photosynthesis efficiency. As we know, the leaf area of every single leaf will increase during growth until individual fully matured. Thus, the detached leaf area and the rosette area from top view will keep increasing. However, the increasing rate of rosette area cannot always catch up with the detached leaf area since the younger leaves will overlap with the old leaves during growth. Therefore, the overlap index will be 1 when plants are too young to have overlap. When a plant is big enough to have an overlap on leaves, this index will pass 1 and keep increasing while the plant grows. The larger the overlap index, the less efficiently the leaf area could be used for photosynthesis. 4

8 All indicators mentioned above such as P/L ratio, overlap index will be first tested on plants from DIF condition. After that, to study if extended night and trehalose spraying will influence plant structure in the same way, the same indicators as DIF treatment will be measured on plants under those treatments. Hypotheses The influence of DIF Since the activity of auxin-ethylene cascade reaction and growth stimulation factor PIF3 will be repressed under DIF, plant elongation will be suppressed. It is suggested that the leaf length in DIF will be shorter than that in +DIF. It is also assumed that -DIF will result in a smaller petiole/leaf ratio and larger detached leaf area. This assumption is based on a figure which was shown in the PhD thesis of Ralph Bours (Bours, 2014)(Figure2). That figure was used to show the promoter activity of ACS2 on 4 weeks old plant under +DIF/-DIF in that paper. The differences of petiole/leaf length ratio under diverse DIF conditions can be obviously observed in that figure. And the added leaf area under DIF looks significantly larger than that under +DIF. The shorter petiole and larger separate leaf area under DIF will lead to a more compact plant which has a larger overlap index. Figure 2 Morphology of detached leaves under DIF/+DIF*(cited from Bours, 2014, Page 56 figure G) Plant growth is influenced by carbohydrate metabolism and sugar transportation. Under normal condition, the starch content in the plant can always keep a certain level. If starch level becomes lower than that, the plant will experience carbohydrate starvation. 5

9 We suggest that DIF will influence the carbohydrate metabolism by altering the circadian clock. The starch metabolism rate which can always adjust to the anticipated dawn controlled by gene expression of GI and TOC1 will prematurely start at daytime when it grows under DIF. Since the starch begins to degrade already half-way the photoperiod, and will last for a longer period, it will cause a carbon starvation before dawn. Therefore, less starch will be present in source leaves at the end of night. Besides that, the photosynthesis activity will be repressed due to the low temperature at daytime. It will also reduce the biomass accumulation and slow down the growth rate of the plants. However, a lower growth rate will lead to a smaller leaf area and lower leaf number, which is in contrast with what was shown in figure 2. This is something we will further investigate during this study. The influence of Extended night If state that the carbohydrate starvation caused by DIF treatments a start earlier effect, then the carbohydrate starvation at the end of an extended night will be a stop late effect. When the night is unexpectedly extended, the starch metabolism activity will continue, resulting in carbohydrates starvation at the end of the night and repression the leaf elongation. At the same time, plants subjected to an extended night treatment will accumulate fewer biomasses because of the shorter photoperiod. The inhibited growth will reduce the leaf area, leaf number, and the overlap index. The influence of Trehalose Different from DIF and extended night, trehalose spray will not result in complete exhaustion of starch at the end of the night. We suggest that when plants are treated with trehalose in the morning, the starch breakdown rate will be inhibited. So the starch level in source leaves of plants that were treated with trehalose will be higher at the end of the day than that in source leaves of plants without the treatment. As a result, less soluble sugars will be available for plant growth. So the plant will grow slower and have a lower leaf number and smaller leaf area. However, although it has a stronger starch storage function, the final biomass accumulation still will be lower because of the reduced growth rate. In this experiment, after giving a treatment, which might inhibit plant growth, the leaf elongation will also be repressed, and it will result in a reduction of leaf area. Since a smaller plant will have a smaller overlap index than a large plant, the plants after treatment could have a smaller overlap index than those without treatment if the structure of the plant was not changed by those treatments. But if the overlap index after a treatment is the same as, or even became bigger than without treatment, it might tell that the plant structure was changed by the treatment. 6

10 Research Methods Plant Material All the experiments were done with wild type Arabidopsis Col-0. Systems Growth conditions The experiments were conducted at the Laboratory of Plant Physiology of Wageningen University in Netherlands. The environmental conditions in the growth room and cabinets, in which the experiments were conducted, were as follows: B10 (Trehalose spraying and extended night experiments): 12 hours light 22 C, 12 hours dark 17 C, relative humidity 65%, light intensity μmol quanta m-2 s-1, 1.0 times Hoagland nutrient solution Weiss climate cabinets (DIF experiments): -DIF: 12 hours light 12 C, 12 hours dark 22 C, relative humidity 65%, light intensity μmol quanta m-2 s-1, 0.5 times Hoagland nutrient solution +DIF: 12 hours light 22 C, 12 hours dark 12 C, relative humidity 65%, light intensity μmol quanta m-2 s-1, 0.5 times Hoagland nutrient solution Photographing systems Pictures from Motion control system in Weiss cabinet Two IR LED light and two modified single-lens reflexes (SLR) cameras with IR filter removed are installed in the Weiss climate cabinets. SLR cameras can slide and take bird s eye view pictures of the plants. Pictures will be available directly on a specific computer which attaches with the camera through NIKON camera Control software. It was setup by hand to take a picture every two hours. The growth rate of the plant can be observed from the sequencing pictures. A ruler will be placed at the same level of the rosette in the view of the camera as a scale marker for calculating later. One picture of all the plants under +DIF and DIF was taken before moving out the plants for measuring. Pictures from the Canon camera The pictures were taking in a dark room by using a Canon EOS 60D camera. The stabilizer was switched on. The flash was switched off. The plants were photoed by manual focus. The setup of the camera was shown in Figure 3-1. Picture analyzing system -Image J Image J will be used to statistic data by analyzing the pictures achieved from motion control or manual photograph. The length and area which are recorded as pixels in 7

11 the pictures will be calculated and convert to mm or mm 2 end. by the software in the Figure 3-1 Setup of the camera. JPEG format without RAW. Sharp and Large picture which is 5184 by 3456 pixels. Treatments DIF The seeds of Arabidopsis were first stratified in 1ml MQ in 1.5ml vials with caps for 4 days before sowed. The vials were covered with aluminum paper and placed in a closed plastic box to avoid radiation in 5 cold room. In the following 4 weeks, plants were grown on Rockwool and watered with 0.5 times Hoagland nutrient solution. Those plants which were used to measure the leaf length and biomass accumulation were moved to Weiss climate cabinets one week earlier than the ones which aimed to observe the sugar status during growth. The different treatments are shown below. Plants for leaf growth and biomass measurement: 2 weeks (B10) + 1 week (+DIF, in Weiss 4) + 10 days (+DIF in Weiss 4/-DIF in Weiss 3) Plants for starch content measurement: 3 weeks (B10) + 1week (+DIF, in Weiss 4) + 4 days (+DIF in Weiss 4/-DIF in Weiss 3) After 32 days, the plants were taken out of the cabinet and used for different measurements. Extended night The extended night was reached by using a hood to cover the plants just before dawn 8

12 until 2 hours into the regular photoperiod. Normal night: 4 weeks (+DIF, in B10) Normal night (7 days a week) Extended night: 4 weeks (+DIF, in B10) Two hours extended night (Tuesday & Friday of every week, 2 times*4 weeks=8 times in total) Normal night (except Tuesday & Friday) Trehalose spray The plants were sprayed with trehalose solution (D-(+)- Trehalose dihydrate powder, SIGMA, g/mol) before 9 o clock in the morning on Tuesday and Friday of every week. 3 concentrations of solution were used in this treatment, which were 40, 20 and 10mmol/ L. Sorbitol solutions (D-SORBITOL, DUCHEFA BIOCHEMIE, g/mol) were used as an osmotic control. All the solutions contained 1% TWEEN (TWEEN 20, SIGMA-ALDRICH, 1.095g/mol, 0.06mM). Since the plants were very small in the beginning, every tray with 40 seedlings inside was only sprayed evenly with 5 ml of solution in the first week. The volume of solution rose up to 10ml from the second week. Control: week1 to 4(+DIF, in B10) Sorbitol spray (Tuesday & Friday of every week, 2*4=8 times in total) Trehalose (40mM, 10mM): week1 to 4(+DIF, in B10) Trehalose spray (Tuesday & Friday of every week, 2*4=8 times in total) 20 mm Trehalose spraying treatment: Trehalose spray (Tuesday & Friday of every week, 2*4=8 times in total) Week 1 : 5ml/40plants 10mM solution Week 2 : 10ml/40plants 10mM solution Week 3&4 : 10ml/40plants 20mM solution Measurements Morphology 1. Leaf elongation On day 32, the rosettes areas of 15 plants were photographed from the top view. Then each plant was taken apart separately. All leaves of each sample were put in one line as the order of ontogenetic sequence on a black cloth as shown in figure 3-2. Leaf number was labeled and counted manually from right to the left after photographed. However, in most cases, the first leaf and the second one had already degraded before being measured. To respect the ontogenetic sequence of leaves and make the average leaf length under the same leaf number more comparable to each other, the leaf number was switched to the right position when leaves before it 9

13 were missing/degraded. A ruler was placed in all pictures as a measuring scale. The length of leaf and petiole were analyzed by using Image J while setting the starting point and ending by hand. The leaf length was standing for the total length from bottom of petiole to the tip of the blade. The petiole length was the length from bottom of petiole to the leaf base. The yellow label showed the group and number of every sample. Figure 3-2 All the leaves detached from one plant are following ontogenetic sequence on a black cloth. A ruler is shown as measuring scale which will be used in image J. 2. Overlap index To calculate the overlap index, two numbers were needed for each sample. One was the leaf area before detached the plant. It will be called as rosette area in this thesis (figure 3-3). Another one was the leaf area after detached. It was a leaf area summation of all separate leaves of a single plant. The overlap index was the result of divided detached leaf area by rosette area. 10

14 Figure 3-3 A picture of rosette area from motion control were measuring by Image J (ROI manager). Metabolism 1. Biomass accumulation The rosettes from 5 plants were pooled together in a 12ml PP tube as one sample. Six repetitions were used to calculate the biomass accumulation of different treatments. The fresh samples were weighted on day32 just after cut off. Then they will be dried for 2 days in the oven at 70 and reweighted on day34. The result of fresh/dry weight ratio and average dry weight will be used to compare the biomass accumulation of the plant. 2. Starch content In all treatments, the samples were taken on day 33. Young leaves were treated as sink while old leaves were treated as the source. The source (outer) and sink (inner) samples were taken by using a hole puncher. The diameter of the puncher was 20 mm for all the treatments, except plants sprayed by 40mM trehalose solution. Since the sizes of those plants were too small compared to plants in other treatments, the diameter of the puncher used in that treatment was 15mm. This rough check method was shown in figure 3-4. The young leaves from 5 plants were put into the same 20ml vial with caps and stored in -80, as well as the old leaves. That was used as one sample. For every measurement, 3 samples (= 15 plants) were used as repetitions. The samples will be stored in a freezer at -80 before measuring. 11

15 Starch was extracted from solid samples as previously described by Smith and Zeeman (Smith and Zeeman, 2006) except that extraction of soluble sugars/washing of the samples was done one time in 70% MeOH and, after that, two times with MQ. For quantification we used a commercial starch assay kit (Starch (HK) Assay Kit, Sigma) and changes in absorbance at 340nm were measured using a VERSA max microplate reader. Figure 3-4. Source (outer) and sink (inner) tissues used for sugar measurements. 12

16 Results Plant morphology and biomass accumulation DIF Figure 4-1 shows that the leaf lengths of Arabidopsis grown under +DIF and DIF are significantly different (p<0.01) from leaf number 5 to leaf number 19. The leaves of plants grown under +DIF are longer than those under -DIF. The longest leaf of the whole rosette, which in some extent could be used to represent the size of the plant, under +DIF (leaf No.9 = 35.6mm) was 11.6 mm longer than that under DIF (leaf No.7= 34.2mm). At the same time, it is shown in Figure 4-2 that the P/L ratio was changed a lot by DIF. DIF treatment reduced the P/L ratio of the plant prominently (p<0.01) on the 5 th leaf to 15 th leaf. The reduction on the 2 ed leaf was also remarkable (p<0.05), but it could be ignore caused by lots of missing data (degraded leaves). Figure 4-1 Leaf Length under +DIF and DIF conditions. X-axis shows the leaf number following the order of ontogenetic sequence. Y-axis is the length of leaf in mm. Collected data is presented as mean ± S.E. Labeled with * when t-test value < Labeled with ** when t-test value <

17 Figure 4-2 Petiole/Leaf length ratio under +DIF and DIF conditions. X-axis shows the leaf number following the order of ontogenetic sequence. Y-axis is the petiole length divided by leaf length presented in ratio. Collected data is presented as mean ± S.E. Labeled with * when t-test value < Labeled with ** when t-test value <0.01. Table 4-1 shows that -DIF caused a significant reduction (p<0.01) in average rosette area and detached leaf area. It also shows that the total leaf number decreased when plants were grown under DIF. However, there was no significant difference in overlap index and Dry/Fresh weight ratio. Table 4-1 T-test on average rosette area, detached leaf area, overlap index, total leaf number and Dry/Fresh weight ratio of plants under +DIF and DIF conditions. T-test statistics +DIF -DIF t-test Average rosette area (mm2) <0.01 Average detached leaf area (mm2) <0.01 Overlap index >0.05 Average total leaf number <0.01 Dry/Fresh weight ratio >0.05 Extended night We found that leaves were substantially (p<0.05) longer under normal night in comparison with under extended night (Figure 4-3), especially the 5 th leaf to 16 th leaf (p<0.01). The longest leaf of the whole rosette grown under control conditions (leaf No.8 = 25.26mm) was 5.06 mm longer than that of the plants that were treated with extended nights (leaf No.8=20.20mm). However, different from the result of DIF treatments, although a reduction of P/L ratio was observed on leaf 11 th, 14 th, 14

18 and 15 th, in general, the P/L ratio of plants grown under control conditions and those treated with extended nights were almost the same. Figure 4-3 Leaf Length under Extended night and Normal night. X-axis shows the leaf number following the order of ontogenetic sequence. Y-axis is the length of leaf in mm. Collected data is presented as mean ± S.E. Labeled with * when t-test value < Labeled with ** when t-test value <0.01. Figure 4-4 Petiole/Leaf length ratio under Extended night and Normal night. X-axis shows the leaf number following the order of ontogenetic sequence. Y-axis is the petiole length divided by leaf length presented in ratio. Collected data is presented as mean ± S.E. Labeled with * when t-test value < Labeled with ** when t-test value <0.01 Table 4-2 shows that a smaller rosette area, detached leaf area, and lower leaf number were observed in plants that were treated with extended nights compared with 15

19 those grown under control conditions(p<0.01). There was no significant difference in overlap index and the Dry/Fresh weight ratio (p>0.05). Table 4-2 T-test on average rosette area, detached leaf area, overlap index, total leaf number and Dry/Fresh weight ratio of plants under Extended night and Normal night. T-test statistics Control Long night t-test Average rosette area (mm2) <0.01 Average detached leaf area (mm2) <0.01 Overlap index >0.05 Average total leaf number <0.01 Dry/Fresh weight ratio >0.05 Trehalose spraying 40mM Trehalose Figure 4-5 shows that the leaf length was substantially longer when plants were sprayed with 40mM sorbitol in comparison with the leaf lengths of plants that were sprayed with the same concentration of trehalose. The differences were observed on the 1 st leaf until 19 th leaf, especially for 5th leaf to 19th leaf (p<0.01). The longest leaf of the whole rosette treated with sorbitol (leaf No.8 = 25.73mm) was 4.8 mm longer than that of rosettes that had trehalose treatment (leaf No.8=20.93mm). A significant (p<0.01) reduction of P/L ratio was observed on leaf 12 th to 16 th, however, this trend was not seen on the other leaves besides 8 th leaf (figure 4-6). On balance, the P/L ratio of plants treated with 40mM sorbitol or 40mM trehalose were almost the same. Figure 4-5 Leaf Length of Arabidopsis sprayed with 40mM Sorbitol and Trehalose solution. X-axis 16

20 shows the leaf number following the order of ontogenetic sequence. Y-axis is the length of leaf in mm. Collected data is presented as mean ± S.E. Labeled with * when t-test value < Labeled with ** when t-test value <0.01. Figure 4-6 Petiole/Leaf length ratio of Arabidopsis sprayed with 40mM Sorbitol and Trehalose solution. X-axis shows the leaf number following the order of ontogenetic sequence. Y-axis is the petiole length divided by leaf length presented in ratio. Collected data is presented as mean ± S.E. Labeled with * when t-test value < Labeled with ** when t-test value <0.01. Table 4-3 shows that plants that were treated with trehalose solution, compared to plants treated with sorbitol, had a smaller rosette area, detached leaf area, and lower leaf number (p<0.01). There was no significant difference in overlap index and the Dry/Fresh weight ratio (p>0.05). Table 4-3 T-test on average rosette area, detached leaf area, overlap index, total leaf number and Dry/Fresh weight ratio of plants sprayed with 40mM Sorbitol and Trehalose solution. T-test statistics Sorbitol Trehalose t-test Average rosette area (mm2) <0.01 Average detached leaf area (mm2) <0.01 Overlap index >0.05 Average total leaf number <0.01 Dry/Fresh weight ratio >

21 10mM Trehalose When the concentration of sorbitol and trehalose was reduced to 10 mm, the results were almost the same as the results we found when plants were treated with 40mM trehalose or sorbitol. The leaf was significantly longer in plants that were treated with 10mM sorbitol in comparison with those of plants treated with the same concentration of trehalose. The longest leaf of the whole rosette under control (leaf No.9 = 33.18mm) was 5.37 mm longer than that of plants which had given the trehalose treatment (leaf No.9=27.81mm). A significant (p<0.05) reduction of P/L ratio was observed on leaf 12th to 15 th (figure 4-8), however, in general, there was no big difference in P/L ratio between plants treated with 10mM sorbitol and 10mM trehalose. Figure 4-7 Leaf Length of Arabidopsis sprayed with 10mM Sorbitol and Trehalose solution. X-axis shows the leaf number following the order of ontogenetic sequence. Y-axis is the length of leaf in mm. Collected data is presented as mean ± S.E. Labeled with * when t-test value < Labeled with ** when t-test value <

22 Figure 4-8 Petiole/Leaf length ratio of Arabidopsis sprayed with 10mM Sorbitol and Trehalose solution. X-axis shows the leaf number following the order of ontogenetic sequence. Y-axis is the petiole length divided by leaf length presented in ratio. Collected data is presented as mean ± S.E. Labeled with * when t-test value < Labeled with ** when t-test value <0.01. Table 4-4 shows that the average rosette area, detached leaf area, leaf number, and even overlap index of plants treated with 10mM trehalose were all smaller than those of plants treated with sorbitol, while there was no significant difference in Dry/fresh weight ratio between them. Table 4-4 T-test on average rosette area, detached leaf area, overlap index, total leaf number and Dry/Fresh weight ratio of plants sprayed with 10mM Sorbitol and Trehalose solution. T-test statistics Sorbitol Trehalose t-test Average rosette area (mm2) <0.01 Average detached leaf area (mm2) <0.01 Overlap index <0.01 Average total leaf number <0.01 Dry/Fresh weight ratio > mM Trehalose (combined with 10mM Trehalose) When plants were treated with 10mM trehalose during the first two weeks and then treated with 20mM trehalose for another two weeks, the lengths of the 4 th,6 th, 12 th, 21 st, 26 th, 29 th,and 15 th to 19 th leaves were substantially (p<0.05) different from those of plants treated with the same concentrations of sorbitol. This indicates that the effect of the treatment seemed to be different on older leaves compared to younger leaves. From 1 st leaf to 10 th leaf, the trend was plant sprayed with 19

23 trehalose had longer leaf length than control. But after that, from 11 th leaf and then on, the trend was on the contrary (figure 4-9). No significant difference in P/L ratio was observed in figure 4-10 except on leaf 6, 12, 14, and 16. Figure 4-9 Leaf Length of Arabidopsis sprayed with 20mM Sorbitol and Trehalose solution. X-axis shows the leaf number following the order of ontogenetic sequence. Y-axis is the length of leaf in mm. Collected data is presented as mean ± S.E. Labeled with * when t-test value < Labeled with ** when t-test value <0.01. Figure 4-10 Petiole/Leaf length ratio of Arabidopsis sprayed with 20mM Sorbitol and Trehalose solution. X-axis shows the leaf number following the order of ontogenetic sequence. Y-axis is the petiole length divided by leaf length presented in ratio. Collected data is presented as mean ± S.E. Labeled with * when t-test value < Labeled with ** when t-test value <0.01. Table 4-5 shows that, although plants sprayed with sorbitol showed a larger average 20

24 rosette area, detached leaf area, and leaf number, than those of plants treated with trehalose, these differences were not significant (P>0.05). Besides that, overlap index and dry/fresh weight ratio didn t show a significant difference. Table 4-5 T-test on average rosette area, detached leaf area, overlap index, total leaf number and Dry/Fresh weight ratio of plants sprayed with 20mM Sorbitol and Trehalose solution. T-test statistics Sorbitol Trehalose t-test Average rosette area (mm2) >0.05 Average detached leaf area (mm2) >0.05 Overlap index >0.05 Average total leaf number >0.05 Dry/Fresh weight ratio >0.05 Biomass accumulation Figure 4-11 shows the difference of dry weight between 5 treatments and their control. Four treatments, including DIF, Extended night, 40mM Trehalose, and 10mM Trehalose, all led to a reduction in dry weight compared to their control. Among them, the dry weight of plants grown under +DIF conditions was almost twice of the dry weight of plants grown under DIF conditions. Plants treated with 20mM Trehalose had higher dry weight than their controls. Figure 4-11 The Dry Weight of 5 Arabidopsis rosettes under different treatments. Two in one group could be compared: +DIF with -DIF, Normal night with Extended night, 40mM Sorbitol with 40mM Trehalose, 10mM Sorbitol with 10mM Trehalose, 20mM Sorbitol with 20mM Trehalose. The real value in grams is shown above the bar. Collected data is presented as mean ± S.E. 21

25 Starch status DIF Source leaves always had higher starch content than sink. In source leaves, the starch content increased during the day, followed by a slow and steady decrease from 17:00 until the next morning. The starch level under +DIF was always higher than under DIF, especially at the end of the night. In sink leaves, the decreasing stopped at 19:30 and started again after 21:00 under +DIF. However, no decreasing was observed in the starch level under DIF before 21:00. Different from the what was found in source leaves, in sink leaves the starch level under +DIF became lower than under DIF after 19:30 and overtook before dawn. A B Figure 4-12 Starch content in source (A) and sink (B) part of Arabidopsis rosette under +DIF and DIF during the day. 22

26 Extended night Starch content in source leaves was higher than in sink leaves at the end of night in both treatments (figure 4-13). Extended night resulted in lower starch content in both sink and source. It should be noticed that since the normal wild-type plants did not grow well in B10, the starch content of plants under extended night was compared with samples sprayed with 40mM Sorbitol which grew in the same environment at the same time period. Figure 4-13 Starch content in source and sink part of Arabidopsis rosette at 10AM under extended night compared with those at 8AM in 40mM Trehalose spraying treatment. Trehalose spraying 40mM Trehalose Figure 4-14 shows that source leaves contained more starch than sink leaves. It also shows that the 40mM trehalose led to a reduction in starch content in both sink and source leaves. Since plants treated with 40mM Trehalose looked unhealthy, there were not enough plants that could be used for taking samples in the morning of the next day after the last treatment. 23

27 Figure 4-14 Starch content in source and sink part of Arabidopsis rosette at end of the day (8PM) in 40mM Trehalose/Sorbitol spraying treatment. 10mM Trehalose It was shown that source leaves contained more starch than sink leaves. The starch level at the end of day was much higher than at the end of night. When plants were sprayed with 10mM trehalose, the starch level in both sink and source was increased during day and night. A 24

28 B Figure 4-15 Starch content in source and sink part of Arabidopsis rosette at end of the day (8PM, figure 4-15 A)/end of the next night(8am, figure 4-15 B) in 10mM Trehalose/Sorbitol spraying treatment. 25

29 Discussion DIF In this experiment, the plants grown under -DIF had shorter leafs. This could be caused by the inhibition of DIF on leaf elongation. However, due to the different light intensity in +DIF and DIF, the difference in leaf length could also be caused by higher growth rate under higher light intensity in +DIF cabinet. Based on the shorter leaf length and a smaller P/L ratio of most leaves of the plants grown under DIF conditions, we can conclude that the repression of DIF on leaf elongation was mainly induced by shortening the petiole. The influence of DIF and +DIF on leaf and petiole elongation were also studied in a related experiment by Moe and Mortensen (R. Moe, 1990). They gave a 2-hour temperature change in the morning to Euphorbia Lilo and Annette Hegg Starlight and measured the petiole length and leaf length of those plants under 0, 6 and -6 DIF conditions. Their results showed that for both of these two species, compared to plants under 0 DIF, +DIF caused stretching of the petiole while DIF shortened the petiole. Besides that, the leaf length was mostly not changed by DIF treatments. Another study showed that -DIF reduced elongation of the shoot part much more than the leafy parts of the plants (Vogelezang, 1998). Just like our experiments, these two studies also showed that DIF inhibits elongation of the petiole more than it inhibits elongation of the leaf. Meanwhile, besides the negative influence of DIF, the positive influence of +DIF on petiole elongation also plays a role in enlarging the gap. In the same study of Vogelezang, the leaf length was proved to be reduced under low light level. And the leaf thickness and chlorophyll content under high light level were shown to be higher. Thus, it could be concluded that the significant difference in leaf length between +DIF and DIF was a combined resulted of the reduction petiole length under DIF, increased petiole length under +DIF, and the increased leaf length under higher light intensity. A related study showed that the percentage of dry matter was largely increased at the high light level while not influenced by DIF strategy (Vogelezang, 1998). It means if high light intensity plays a big role in plant growth, the dry matter percentage under +DIF should be higher than under DIF. However, the dry matter percentage under two treatments showed no difference in our research. It indicated that DIF affects plant growth more than light intensity. Since the rosette area, detached leaf area, and leaf number under DIF were all shown to be reduced, it is hard to say if DIF mainly affects the growth rate or the leaf development. Some studies showed that the leaf development of Salvia 26

30 splendens and Pelargonium zonale was not significantly influenced by DIF-strategy (Vogelezang, 1998). In addition to that, our research also showed a significant reduction in dry weight under DIF (0.126g) compared to +DIF (0.241g). From the difference in photosynthesis production, the repressing effect was mainly due to a slower growth rate under -DIF. Although the leaf area decreased, the overlap index did not become smaller as was expected. This could be explained by the size of the plants in both treatments. The plants might have been too small to show a big difference in overlap index, since the two index numbers were still around 1. Another reason could be the significant difference in P/L ratio. Since the inhabitation of leaf elongation was caused by shortening the petiole, the structure of the plant will become more compact, thus led to an increase in leaves overlap. The synthetic rate of carbohydrate is limited by the nutrient uptake speed (N, Pi) during growth (Griffiths et al., 2016). Plants have higher water uptake and transpiration rate during the day. The plant will gain more nutrients from the soil. Although the synthesis and degradation of starch is a dynamic process, it mainly dominated by synthesis activity at the daytime (Smith et al., 2005). The starch produced during the day accumulates in chloroplasts and is mainly stored in source leaves. At night, starch is degraded to glucose and maltose to support the synthesis of sucrose (Maloney and Park, 2015). Then the exported sucrose will keep a constant level of carbohydrate in sink and support the growth during night (Pilkington et al., 2015; Griffiths et al., 2016). That is why the starch content increases in the daytime and decreases at night. The temperature and light intensity under +DIF are higher than under DIF. Therefore, plants accumulated more starch when grown under +DIF conditions. Starch degradation rate was found to be increased by higher temperature during night to adapt to the faster growth and higher maintenance demanding of plant (Amthor, 2000; Loveys and Scheurwater, 2002; Pilkington et al., 2015). This coincides with our results, which showed faster starch degradation under DIF than under +DIF during the night. We hypothesized that under DIF, because of the early start of starch breakdown, source leaves will contain very low amounts of starch at the end of the night. The low amount of starch in source leaves results in carbohydrate starvation and repression of growth. The results shown in figure 4-12 confirm this hypothesis. At the end of the night, source leaves of plants grown under DIF contained approximately one-fourth of the starch that is present in source leaves of +DIF grown plants. More biomass accumulated under +DIF. This might be due to a higher photosynthesis at higher temperatures during the day, accompanied by a lower respiration rate at a lower temperature during night. Furthermore, lower dry weight under DIF than +DIF could be explained by less starch accumulated in source under DIF in figure 27

31 4-12. It was also found that higher night temperature will reduce WUE (water use efficiency) and facilitate stomata closure (Neales, 1973), which will affect the biomass accumulation during night. Extended night Just like the DIF treatment, extended night also repressed elongation of the leaves. However, the decrease in petiole length was proportional to the reduction in leaf length under extended night condition. This means, the mechanism behind the compactness effect on Arabidopsis caused by extended night might be different with DIF. While DIF represses the petiole elongation, extended night repress the blade and petiole elongation equally. Leaf growth is relying on cell expansion and proliferation. Plant cells have a cellular membrane and cell wall, composed of structural components like lipids and proteins (Gibeaut and Carpita, 1991). Carbon utilization was found to be changed when night was unexpected extent. Especially for source leaf, the carbon partitioned to structural components was decreased (Kölling et al., 2015). This reduced the leaf growth severely. In addition to that, although carbon partitioned to neutral sugar content in source was increased, neutral sugar exported to sink was decreased (Kölling et al., 2015). It was reported that the leaf showed the fastest expansion at the beginning of the day in normal condition, which is accompanied by highest sugar imported from source and high speed of cell structure component synthesis (Wiese et al., 2007). Since there was a sugar starvation caused by the extended night in our research, growth in the morning was definitely repressed. Furthermore, reduced expression of genes involved in lipid and protein synthesis occurred when giving an extended night to wild-type Arabidopsis, since the phenomenon which could indicated the protein recycling was observed (Izumi et al., 2010).Besides that, differences in gene expression and metabolite levels were observed between the Arabidopsis pgm mutant, which has severely reduced starch levels, and its wild type (Gibon and Usadel, 2006; Usadel et al., 2008). The linkage between starch level and plant growth were also found to be connected with hormone regulation. Low starch level will down regulate the growth through repress the production of GA by sugar signaling (Sairanen et al., 2012; Paparelli et al., 2013). Moreover, the sugar starvation in the former night will repress the expression of ent-kaurene synthase, causing suppression of GA synthesis. Since GA was found to cooperate with photosynthesis efficiency to ensure plant growth to the optimal size under certain environment, the plants treated with an extended night were smaller than untreated plants (Davies, 2004). 28

32 In conclusion, an unanticipated extended night resulted in sugar starvation in this research. Plant growth was inhibited due to a hormone down-regulation and a defect of carbon to supply the synthesis of structure component. In an extreme case, protein degradation to ensure plant survival was even observed (Zerihun et al., 1998). One more thing that needs to be discussed here was the difference which could be seen on the 14 th and 15 th leaf. The 14th and 15th leaf under extended night were too young to have an apparent petiole, which makes most data equaled to zero. Those data show reduction the average P/L ratio of 14th and 15th leaf under the extended night. It means that the difference was caused by immature growth instead of the influence of the treatment. This was also true for plants sprayed with trehalose. In our study, extended night resulted in low starch levels in source and sink leaves. The reduction in dry weight might be caused by a day to day repression in biomass accumulation. This probably could be explained in two ways. On the one hand, the inhibited leaf growth could lead to a reduction in leaf area and photosynthesis efficiency. On the other hand, GA synthesis might have been affected by the former extended night. This might result in plant not being able to adjust their growth rate in the optimal way under certain conditions. Trehalose We found that trehalose can inhibit leaf elongation and results in a compact structure. Like discussed above, the difference in P/L ratio of the younger leaves was caused by immaturity of the plant instead of the treatment. Trehalose did not change the P/L ratio of leaves, which indicates that the trehalose treatments might not suppress leaf elongation in the same way as DIF treatments do. As it can be seen in table 4-3 and table 4-4, the average leaf area of plants sprayed with 10mM trehalose/sorbitol solution was larger than that of 40mM, suggesting that low concentration trehalose/sorbitol solution could accelerate plant growth. When looking at table 4-2, 4-3 and 4-4, it becomes clear that sorbitol accelerated plant growth while trehalose does the opposite. The average leaf area of the plants grown under the control of extended night treatment (detached leaf area= mm 2 ) was smaller than that of plants sprayed with 10mM sorbitol (detached leaf area= mm 2 ). Furthermore, the difference of leaf area (370mm 2 ) between 10mM sorbitol treated plants and control of the extended night treated plants was much larger than the difference (101mm 2 ) between 40mM sorbitol treated plants and control of the extended night treated plants. Comparing the mm 2 (10mM trehalose) with mm 2 (40mM trehalose) detached leaf area, also plant growth seems to be repressed by trehalose, depending on the concentration. Since the low concentration of trehalose and sorbitol solution only repressed the growth of plants a little, the leaf areas of those plants were much larger than 29

33 in other treatments. Among the five treatments, it only showed a significant (p<0.01) difference in overlap index in this treatment. This could be explained by the fact that leaf area of these plants were large enough to make a difference in overlap index. That difference was more due to the accelerative influence of Sorbitol than the inhibitive influence of Trehalose, since the difference in detached leaf area(370mm 2 ) of plants treated with 10mM or 40mM sorbitol, was 6 times higher than the difference in detached leaf area of plant treated with 10mM or 40mM trehalose (64mm 2 ). It can be seen that, no matter when plants grow under extended night or sprayed with 40mM or 20mM trehalose, the overlap index was always around 1 ( between 1 and 1.12). It means that the plants were still too young. They were not large enough to have a big overlap index which could change a lot induced by different treatments. That would be the main reason why there was not a significant difference in overlap index under all these treatments beside 10mM trehalose. The inhibition effect of 40mM trehalose on leaf expansion matched with the results of the study by Hanhong Bae, which showed that 30mM exogenous trehalose affect root and leaf growth of 2-week-old Arabidopsis grown ion MS agar media (Bae et al., 2005).The restricted growth could be caused by the Low-sucrose signing pathway when increasing T6P level artificially (Figueroa and Lunn, 2016). Trehalose spraying leads to an increase in T6P level, since Tre6P is the intermediate of trehalose synthesis, which is synthesized by trehalose-phosphate synthase (TPS) and dephosphorylated to trehalose by trehalose-phosphate phosphatase (TPP). Higher T6P will inhibited starch degradation through regulating sugar signaling pathways. It was shown that an increase of T6P in ethanol-inducible TPS plants at the beginning of the photoperiod led to a higher starch content at the end of day (Martins et al., 2013). These findings match with our results from our 10mM trehalose treatment experiments (figure 4-15 A).However, the starch synthesis and carbon partition seemed to be only slightly influenced by T6P level. In addition to that, recently, Figueroa and Lunn (2016) explained in their research that an increased T6P level could drive photo assimilates into organic and amino acid, away from sucrose. As a result plants perceived a low sucrose signaling which will activate SnRK1, leading to growth inhibition. The increase in SnRK1 activity will repress the energy consumption, thus accelerated the starch accumulation (Figueroa and Lunn, 2016).That could be the reason of a higher starch level in source and sink at the end of day after sprayed with 10mM trehalose. In the same article(figueroa and Lunn, 2016), they also argued that under artificial high T6P level, there will be a conflict sugar signal caused by multiplicity Sucrose-signaling pathways. Although plants get different input signals which could match or opposite to the real sugar level, the plant growth will still depend on actual sugar availability. This means that the inhibited growth of the treated 30

34 plants we saw in our research, was probably mainly due to the lower sugar level caused by trehalose spraying. Martins et al. also reported the T6P level in ethanol induced plants was still about 2 times higher than control after 4h in darkness while it was induced at middle of the day(martins et al., 2013).This indicates that the T6P level changed by treatment could last for a quite long period. The trehalose spraying in the morning might be affect the growth until the next day. The constantly inhibition on starch degradation during the day led to a higher starch level in sink and source part in 10mM trehalose compared to control. But surprisely, the starch content of plants sprayed with 40mM trehalose was lower than control. This might be caused by a defence response of the plants, induced by the high concentration of trehalose. This could also explain the large variation in size and appearance of plants that were treated with 40mM trehalose. As what was shown in figure 5a and figure 5b, most plants sprayed with 40mM was extremely small. And about 7/40 was dead before taking samples. The plants were divided into 4 classes, which were extra small (SS), small (S), medium (M), Large (L), before measuring (figure 5c). M was the common size of the plants which were used for leaf measurement in the end. Since SS and S were too small for starch sampling by puncher check method, the L plants were used to determine starch levels. It is possible that those L plants showed normal growth because the treatment did not affect them. 5a 5b 5c 31