Growth of silver birch (Betula pendula) plantlets with altered phenolic metabolism under enhanced UV-B

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GROWTH OF SILVER BIRCH (Betula pendula) PLANTLETS WITH ALTERED PHENOLIC METABOLISM UNDER

ENHANCED UV-B

ROSE ASGHAR

Master of Science Thesis University of Eastern Finland

Department of Environmental and Biological Science Biology

2017

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UNIVERSITY OF EASTERN FINLAND

Department of Biology

ROSE ASGHAR: Growth of silver birch (Betula pendula) plantlets with altered phenolic metabolism under enhanced UV-B

M.Sc. Thesis, 32 pp., 40 ECTs August 2017

ABSTRACT

Certain secondary metabolites (e.g. flavonoids) are assumed to absorb the UV-radiation and thus protect the plants from its harmful effects. They are formed through phenylpropanoid pathway. The final product of this phenylpropanoid pathway is proanthocyanidins (PAs).

The present study uses a greenhouse experiment to check the changes which occur in the growth of (Betula pendula Roth) modified by changing the PA pathway genes expression of young plants under enhanced UV-B.

The expression of anthocyanidin reductase (ANR), dihydroflavonol reductase (DFR) and anthocyanidin synthase (ANS) were inhibited using the RNA interference method in 12 lines of silver birch (B. pendula). The non-modified line (wild, BPM5) was used as a control line for modification.

The length and diameter of the ANRi, DFRi and ANSi birches grown under enhanced UVB and ambient (=control) UV-B were studied. Some lines of silver birch showed decreased growth because of enzyme restriction. Although the effect of elevated UV-B was minor on the birch lines with modified phenolics, there is the great variation in growth responses within ANRi, DFRi and ANSi birch lines. On the other hand, DFRi lines grew more in diameter compared to control line under enhanced UVB. We can assume that in DFRi lines, restricted gene expression leads to increased production of UVB absorbing phenolic compounds (flavonoids and phenolics acid) which is protecting its diameter growth.

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ABBREVIATIONS

ANR anthocyanidin reductase

ANRi birches birches with ANR gene inhibition with RNA interference method

ANS anthocyanidin synthase

ANSi birches birches with ANS gene inhibition with RNA interference method

DFR dihydroflavonol 4-reductase

ANR Betula pendula ANR gene

ANS Betula pendula ANS gene

BPM5 early-flowering birch (Betula pendula) clone number 5

CFCs Chlorofluorocarbons

DFR Betula pendula DFR gene

DFRi birches in which the DFR gene was inhibited using the RNA interference method

mRNA messenger RNA

ODS ozone depleting substances

PA proanthocyanidin, condensed tannin

UV ultraviolet

UV-C ultraviolet C radiation

UV-B ultraviolet B radiation

UV-A ultraviolet A radiation

WMO World Meteorological Organization

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1 INTRODUCTION

The notion that the anthropogenic emissions of chlorofluorocarbons (CFCs) are the reason for the ozone depletion emerged in the early 1970s (Previdi and Polvani 2014). The amount of ultraviolet-B radiation (UV-B) is rising on the surface of earth due to this ozone depletion (Shindell et al.

1998).

Ozone layer depletion has been notably greater at high latitudes (towards the North and South Poles) than at low latitudes (Madronich et al. 1998). It has been found in recent past, that ozone layer is recovering due to the Montreal Protocol and amendments (IPCC 2014, Previdi and Pol- vani 2017). However, the process is slow, and it will take many decades to recover (Newman et al. 2006). Generally, plants have to face the sunlight for a long duration on daily basis compared to animals or humans (Shirley 1996). The UV emissions are divided into three spectral regions UV-C, UV-B, and UV-A based on their wavelengths (e.g. Madronich et al. 1998). Short wavelength radiations are more dangerous than longer wavelength. UV-C (100-280 nm) is of shortest wavelength, so it is the most dangerous radiation. Caldwell and Flint (1994) reported that ozone is good at absorbing shorter wavelengths, i.e. UV-C is totally absorbed by atmospheric oxygen and ozone and unable to reach the earth surface. Also, UV-C is not affected by the ozone depletion. UV-A (315-400 nm) is of longest UV-wavelength and hardly absorbed by ozone so significant amount of UV-A is transmitted to the earth surface (Hockberger 2002). Although UV-A is of the large wavelength, it is also damaging to some extent. On the other hand, UV-B is of short wavelength, 280-315 nm, and ozone absorbs most of it, but the little fraction of it reaching earth’s surface is enough to cause damage of serious concerns.

1.1 UVB and general plant responses

UV radiation is a key component of the environment that has various effects on plant growth and development (e.g. Jansen and Bornman 2012). UV-B part of solar spectrum can damage the plants at the cellular level through photochemical reactions (Caldwell and Flint 1994). Therefore, UV- B is causing more serious damage than UV-A and UV-C to all living organism on earth (Caldwell et al. 2003, Hockberger 2002). In northern Europe, plants will be more affected by UV-B as that region is most affected by the ozone depletion (Shindell et al. 1998).

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Enhanced UV-B have several harmful effects on plants, i.e. it damages DNA, proteins, and mem- branes, affects transpiration and photosynthesis, and ultimately reduces biomass accumulation and growth of plants (Teramura and Sullivan 1994; Jenkins, 2009). All plants are not exposed to high levels of UV-B. The plants which are present in environments with intense UV-B and have prolonged exposure to UV-B are the most affected. For example, plants receive more UV-B in the spring time and especially during the midday, i.e. time of day of peak exposure to sunlight.

The snow cover in the northern hemisphere will reflect effectively UV-B radiation increasing the possible UV-B stress for evergreen plants (Caldwell 1968).

Plants have developed morphological and biochemical mechanisms to cope with harmful UV radiation. Morphological responses to enhanced UV-B include reduced leaf size, thickening of leaves, curling and bronzing of leaves (Kostina et al. 2001, Robson et al. 2015). Biochemical mechanisms include the formation and accumulation of secondary metabolites under UV stress which helps the plants to protect and acclimatize to UV-B stress (e.g. Wilson et al. 2001, Ko- tilainen et al. 2008, Jenkins 2014). They are mostly phenolic compounds, e.g. anthocyanins and other flavonoids, and are induced by exposure to UV (de la Rosa et al.2001, Tegelberg et al. 2001, Kostina et al. 2001, Jansen 2002, Kotilainen et al. 2009; Jenkins, 2009; Morales et al. 2010,).

1.2 Phenolics as protective mechanism against enhanced UVB

Phenolics work as defensive compounds in plants against different biotic and abiotic threats (Dixon and Paiva 1995). In higher plants, phenolic compounds are produced through phenylpropanoid pathway from phenylalanine (Fig. 1) and can be classified in phenolic acids, flavonoids, anthocyanidins and proanthocyanidins (PA). They perform various biological functions in plants, i.e. act as antioxidants, are responsible for the color of flowers and provide defense against various stresses (Bowsher et al. 2008, Julkunen-Tiitto et al. 2015). Production of these flavonoids and phenolic compounds is one of the widely occurring responses and competent mechanism in plants to defend against UV-B (Mazza et al. 2000, Tegelberg et al. 2001). The damaging UV-B radiation is filtered by flavonoids and phenolic acid in the epidermis of leaves (Cen and Bornman 1993).

Mutant lines of Arabidopsis with decreased levels of flavonoids show increased sensitivity to enhanced UV radiation (Bieza and Lois 2001, Li et al. 1993). Li et al. (1993) have reported that

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Arabidopsis tt5 mutants (in which CHI is blocked) with low levels of flavonoids were more af- fected by the UVB than wild-type plants. Whereas, the mutant with high levels of flavonoids showed tolerance to UVB which was even lethal to the wild type.

Figure 1. A schematic and simplified presentation of phenylpropanoid pathway (PAL, L-phenylalanine ammonia-lyase; C4H, cinnamate-4hydroxylase; 4CL, 4-coumarate; CHS, chalcone synthase; CHI, chalcone isomerase; FNS, flavone synthase; F3H, flavanone-3-hydroxylase;

FLS, flavonol synthase; DFR, dihydroflavonol reductase; LAR, leucoanthocyanidin reductase;

ANS, anthocyanidin synthase, ANR, anthocyanidin reductase).

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B. pendula is extensively used as a model organism among woody plants in experimental botany.

Recently, genome sequence of the B. pendula was mapped by Salojärvi et al. (2017). Silver birch is an important deciduous tree species, both ecologically and economically, and used by the pulp, paper, and plywood industries. Many studies have been done on silver birch trees to investigate the effects of UVB and the role of secondary metabolites in plants (Keinänen et al. 1999, De La Rosa et al. 2001, Tegelberg et al. 2001, Keski-Saari et al.2005). Various indoor and outdoor experiments with silver birch have shown that with increased UV-B the concentration of many UV- B absorbing phenolics in the leaves also increased (De La Rosa et al. 2001, Lavola et al. 2000).

Leaves of silver birch have displayed high UV-B absorption capacity (Tegelberg et al. 2001).

However, not all the phenolics of silver birch are responsive to increased UV-B radiation (Lavola et al. 1997).

There are many studies on conifer species regarding UV-B effects (Laakso et al. 2000, Lavola et al. 2003, Turtola et al. 2006). According to Turtola et al. (2006) long term exposure to enhanced UV-B has no effect on the growth and phenolic concentration of Scots pine and Norway spruce seedlings grown outdoors. In an indoor experiment with enhanced UV-B, Scots pine seedlings reduced their growth (Lavola et al. 2003). Similarly, reduced growth has also been reported in Populus, under high levels of UV-B (Xu et al. 2010). Randriamanana et al. (2015a) reported gender-specific responses of plants under enhanced UV-B. According to Randriamanana et al.

(2015a) the males of Salix myrsinifolia plants are not as tolerant to UV-B as females.

1.3 RNA interference method (RNAi) and its effect on phenylpropanoid pathway in RNAi modified silver birch lines

RNA interference method is used to artificially silence of the expression of the targeted genes in genetically modified plants (Dixon et al. 2013). Generally, the double-stranded RNA (dsRNA) is engineered which bears the known gene sequence, and injected it into the cell with different methods. One of the best-known method of the insertion of this dsRNA is with viral vector (Baul- combe, 1999). As this viral vector replicates, so do the dsRNA in the cell; therefore, dsRNA activation is ensured. As it enters the cell, the dsRNA is automatically cleaved by the enzyme named “dicer” into small fragments called short interfering RNAs (siRNA) (Zamore et al. 2000).

siRNAs are then assembled with the protein component (Meister and Tuschl, 2004) called RNA-

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induced silencing complex (RISC).Then the antisense RNA along with RISC complex comple- mentary bind with mRNA. That breaks the mRNA into two pieces so it cannot make the protein (Pratt and MacRae, 2009). The schematic mechanism is shown in Fig 2.

Figure 2: The mechanism of RNA interfering methods according to Meister and Tuschl (2004) (dsRNA, double-stranded RNA; siRNA, small interfering RNA; RISC, RNA interfering silenc-

ing complex; mRNA, messenger RNA).

ANR, ANS and DFR are the main enzymes in the phenylpropanoid pathway, which leads to the production of PAs (Figure 1). Restriction of any of these enzymes with RNAi can disturb the pathway. Many plant studies have used RNA interference technique to investigate the functioning of these enzymes. RNA restriction of ANR shunted the phenylpropanoid pathway route towards

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the production of flavonoids (Kosonen et al. 2015). For example, high levels of flavonol glycosides (in stem) and phenolic acids, platyphyllosides and flavones (both stem and leaves) has been observed in ANRi birch lines (Kosonen et al. 2015).

According to Lim et al. (2016) RNA restriction of two DFR genes in tobacco leads to upregulation of flavonol pathway. There was higher accumulation of dihydroflavonols, flavonols (quercetin and kaempferol) and total flavonoids in all transgenic plant lines compared to control lines. Sim- ilarly, according to Wang et al. (2013) RNA restriction of DFR in purple sweet potato plant. This RNA restriction reduced the anthocyanin and increased accumulation of flavonols (Quercetin-3- O-hexose-hexoside and quercetin-3-O-glucoside) in the metabolic pathway.

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2 OBJECTIVES AND HYPOTHESES

This thesis is investigating how growth parameters are affected by the restriction of enzyme expression. In this thesis, we use DFRi, ANSi and ANRi lines where the PA have been reduced by using RNA-interference.

The aims of the study were:

(1) To find out how the young, greenhouse-grown plantlets of B. pendula plants will survive and grow under enhanced UVB.

(2) To examine changes, which occur in the growth of early flowering B. pendula with changes in PA pathway gene expression (ANR, ANS, and DFR)

3) To investigate how RNA-interference affects the growth of young B. pendula plantlets under enhanced UV-B.

Our hypothesis was that the modified lines of silver birch with less PAs produce more flavonoids, which protect the plants from UVB and thus modified lines are more resistant to UVB measured by growth parameters.

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3 MATERIALS AND METHODS

3.1 Plant Material

Plant material used in this experiment consisted of micro-propagated plantlets of early-flowering silver birch (Betula pendula). Some of the lines were genetically modified by RNA-interference.

In total, 13 lines of silver birch were used.

One of the lines was a control line BPM5 in which all enzymes are working, and which was used as a parent line for modification. In the remaining 12 lines, the expression of either DFR (dihydroflavonol reductase), ANS (anthocyanidin synthase) or ANR (anthocyanidin reductase), had been suppressed with RNA interference (RNAi). There were 4 DFR-restricted lines (1266, 1271, 1290, and 1294; Fig. 3), 4 ANS-restricted lines (1204, 1214, 1226, and 1232), and 4 lines with ANR-restriction (1183, 1179, 1168, and 1201; Fig. 3). We choose these enzymes for RNAi because they are the key enzyme in the biosynthesis of PAs through the phenylpropanoid biosynthetic pathway.

Figure 3: Plantlets grown in the greenhouse in Joensuu DFRi

ANRi

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3.2 Cultivation of birches

The experiment was performed at the greenhouse located at Science Park in Joensuu, Eastern Finland. After five weeks of rooting, micro-propagated plantlets of the silver birch were planted in mini-greenhouse trays with 0.08-l pots (5 x 5.5 x 5 cm depth) containing (50:50) mixture of vermiculite and fertilized Sphagnum peat. The plantlets were watered twice a week with fertilized water. They were then acclimated for two weeks to greenhouse conditions, varying around 55%

relative humidity and 23°C. After acclimation, these plantlets were planted in 1.55 l pots (EC15, Pöppelmann) filled with a mixture of 67% peat (Kekkilä puutarhaturve/garden peat) and 33%

vermiculite on 31st, May 2016. These plantlets were watered when needed with fertilized water until the 11th June 2016, after which the seedlings were watered with clean tap water.

The humidity in the greenhouse, and the maximum and minimum temperature since the previous measurement, were recorded daily around noon. The optimal temperature set during the experiment was between 20-25°C. The highest measured temperature was 45.9℃, and the minimum was 13.1℃. This much fluctuation in temperature is due to uncontrolled humidity in the greenhouse. The maximum 95% and minimum 20% humidity were also recorded in the greenhouse during the experiment.

3.3 Experimental setup

The plantlets in a greenhouse, were placed under high dose of UVB radiation from 13th, June until 5th, August 2016. The experiment lasted 52 days, i.e. seven weeks and three days. The greenhouse was divided into two compartments by putting a plastic screen impermeable to UV radiation to the center. Each side contained 65 plants providing in total 130 plants. However, some of the DFRi plants died early in the experiment, so few additional individuals of DFRi were planted to 1.55 l pots in the first week of the experiment (Table 3). Two UVB lamps provided the UVB treatment. In each compartment, one lamp was hanging in the aluminum frames 60 cm above the plants. The lamps were covered with two types of UVB filters: cellulose diacetate filter which stop UVC, and polyester filter that stops UVB. The amount of UVB the different sides received was controlled by adding the polyester filter to the control side, which stops the UVB and after which control side plants had been given the ambient dose. Because the UVB lamps emitted UVA the control plants were also supplied with additional UVA using polyester filter.

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3.4 Treatment

Enhanced UVB treatment received 32 % more UVB compared to the control treatment. The daily amount of UVB the control treatment received was 3,8 kJ/m², and the enhanced UVB -treatment received 5,016 kJ/m². However, the greenhouse walls did permeate only a little UVB. We used ten replicate plantlets from each of the 13 lines, randomly assigned to the enhanced UV-B treatment (5 plantlets/line) and the control treatment (5 plantlets/line). During the experiment, the locations of plants within treatment and between the treatments were changed once a week to avoid the effects of the location in the greenhouse.

There were two phases of the treatment - 1st phase and 2nd phase. The UVB treatment was given between 11am-1pm. The timing may be critical for the biological effects of UVB. It is also reported by (Balasaraswathy et al. 2002) that between 11.30am and 1.30pm the amount of UVB is very high

.

Figure 4: Experimental setup in greenhouse, Joensuu

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Phase I

In the 1st phase, the UVB was provided to plants in both control and treatment side by switching on the UV lamps for 1 hour and 25 minutes.

Phase II

In the 2nd phase, after one and half hour control the side lamp was covered with the polyester filter in addition to cellulose diacetate filter. This polyester filter stops the UVB light and only let UVA reach plants. All the plantlets received the same dose of UVA irradiance. After adding this filter, UVB lamps were again switched on both (control and enhanced) sides for 30 minutes.

Figure 5: Picture taken outside the greenhouse, when the lamps were on

3.5 Growth measurements and data analysis

The silver birch growth under enhanced UVB and control side was checking by measuring height and stem diameter at the base of the stem once a week (every Wednesday). The measurements were taken from the main branch because of the bushy growth form of these plants. We have measured the lengths of the plants with ruler and diameter by using caliper.

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3.6 Determination of condensed tannins (PAs)

The condensed tannin concentration of plant material was analyzed with the acid-butanol assay in spring 2016 (Thitz, preliminary data). The standard used to convert absorbance values to concentrations was for purified tannins from Betula nana. Average tannin level from several leaves/stems sampled from several plantlets growing in one micropropagation jar were measured (Figure 8a, 8b, 8c). All the samples were from plants much younger than in the experiment, and there was only one replicate.

3.7 Statistical analyses

We have used IBMSPSS Statistics^® Version 21 to do all the statistical data analyses. Linear mixed models were used to study the enhanced UVB effect on length and diameter of the modified plant lines and the control line. The length and diameter of the plant individuals in the first measurement were used as a covariate. Treatment, covariates (initial diameter or length), and lines (nested within enzyme-modification) are the fixed variable whereas length and diameter are the dependent variables in linear mixed models. Pairwise comparisons (least significant difference method) were used to find the differences between the levels of statistically significant factors.

The threshold for significance is P<0.05.

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4 RESULTS

Inhibition of enzymes had a clear effect on the length and diameter of the modified plants (Table 1, p < 0.05, Figures 7). when different initial sizes (Diameter and Length on week 1 in Table 1, p

< 0.05) of plants were considered. The enhanced UVB did not affect the length of plants (Table 1, p > 0.05) compared to the plants in the control treatment, but it has a significant effect on the diameter (Table 1, p < 0.05, Figure 7), when initial size of plants was considered. Figure 6a and 6b present the length and diameter development of silver birches during the experiment.

Table 1: The treatment effect and enzyme restriction effects on length and diameter of plants.

Stars denote statistically significant factors as * for 0.01 < P < 0.05, ** for P < 0.01

Length F P

Diameter F P Line (nested within

restricted enzyme) 11.237 0.000 15.047 0.000

Treatment 0.397 0.529 5.321 0.021*

Diameter on week 1 57.437 0.000**

Length on week 1 175.797 0.000**

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Figure 6: Length (A) and diameter (B) of plantlets grown in the greenhouse for 11.5 weeks after the last micropropagation, measured weekly during the experiment starting on 13th of June and ending on 3rd August. BPM5 is unmodified control line. ANR, ANS, and DFR are the three- restricted enzyme, four lines within each group.

4.1 Growth difference between lines

Silver birch line ANR 1168 showed maximum growth in height and diameter compared to the other ANRi lines, control line, ANSi lines and DFRi lines. When we look at the rest of the three ANRi lines (1179, 1183, and 1201) they showed less growth in sizes compared to control line.

DFRi plant lines showed minimum growth in height and diameter compared to ANR, ANS, and DFR (Figure 7). However, ANSi lines have grown like the control line both in height and diameter.

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Figure 7: Mean length (A) and diameter (B) of silver birch lines with modified phenolic metabolism.

BPM5 is the unmodified control line. From other lines, enzymes ANR, ANS and DFR have been restricted with RNA-interference. The bars with similar letters are not different based on pairwise comparison (LSD, p<0.05). Error bars are standard error of means (n=10)

Note: We have 5 individuals of each line in the control and the treatment. There were no significant interactions of line*treatment, so the results from 10 plants of the same line are put together in this figure.

4.2 Difference in growth between treatments

The effect of elevated UV-B was statistically insignificant on the length of the plants. (Table 1, figure 7a). However, a slight effect of enhanced UVB can be seen in the diameter of plants. BPM5 plants, where all the enzymes were working, had lower diameter under enhanced UVB than in the control treatment (Figure 7b). On the other hand, DFRi plants line in which DFR gene expression was reduced had higher diameter under enhanced UVB compared to the DFRi plant line under control side (Figure 7b). In the end of the first week of the experiment some of the plants from DFRi started to wilt. The amount of dead DFRi plants did not differ between control and enhanced UVB treatments (Table 3).

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Table 2: Lengths and Diameter average ± SEM) of plants on last week under both treatments Plant lines on

8^th week

Length under control UVB (cm)

Length under Enhanced UVB (cm)

Diameter under control UVB (mm)

Diameter under enhanced UVB (mm)

BPM5 69.5 ± 1.90 68.5 ± 1.60 8.00 ± 0.31 7.66 ± 0.13 ANR 48.1 ± 4.54 46.6 ± 4.86 4.76 ± 0.48 4.84 ± 0.59

ANS 76.9 ± 1.22 73.5 ± 1.44 8.01 ± 0.15 6.46 ± 0.46

DFR 15.9 ± 1.15 15.0 ± 1.44 1.44 ± 0.11 1.70 ± 0.16

Table 3: Number of DFR dead plants on week 8^th

Total no. of plants (n)

DFRi plant lines

No. of dead plants in each

line

Total dead plants each

side

% of dead plants Control UVB

(n=24)

1266 4

9 37,5%

1290 3

1294 2

1271 0

Enhanced UVB (n=24)

1266 5

9 37,5%

1290 3

1294 1

1271 0

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4.3 Condensed tannins concentrations in the plant lines

Among ANRi plant lines, ANRi line 1179 has higher condensed tannins level in leaves and the same in the stem than the control lines. Both ANRi 1201 and ANRi 1183, has lower condensed tannins level compared to the control line both in stem and leaves. Whereas, in ANRi 1168 the condensed level is same in leave but less in stem than control line. (Figure 8a).

For ANS-restricted lines ANSi 1232, ANSi 1226 and ANSi 1204 have almost same condensed tannin concentration both in leaves and stem but less than the control line (Figure 8b). Within DFR restricted lines DFR1271 has the high condensed tannins levels compared to other DFR lines and DFR 1266 lines have the lowest level (Figure 8c). However, all the DFR enzyme restricted lines have lower condensed tannins than (BPM5) control line.

Figure 8a: Concentrations of condensed tannins in leaves and stem of ANR-restricted plants and BPM5.

0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00

1183 1201 1179 1168 BPM5

mg/g

condensed tannins in ANR and BPM5

leaves stems

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Figure 8b: Concentrations of condensed tannins in leaves and stem of ANS-restricted plants and BPM5

Figure 8c: Concentrations of condensed tannins in leaves and stem of DFR-restricted plants and BPM5.

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5 DISCUSSIONS

5.1 Effect of enzyme restriction

According to Mocellin and Provenzano (2004) the gene expression level cannot be entirely blocked with mRNA restriction method. Dr. Mika Lännenpää, who has done the genetic modification, measured the RNA expression for ANR, DFR and ANS lines in 2010-2011 (Kosonen et al. 2015). The ANR-restricted lines (1179, 1183, and 1201) showed approximately 33% of the expression level of ANR compared to control line (Kosonen et.al 2015). Lännenpää (unpublished results) reported that the expression of ANR 1168 line was only 0,8% of that in the control line in 2010, but when he repeated the measurement again in 2011, the expression in 1168 was 70%

of the expression in the control line (data not shown).

According to the expression level report made by Dr. Mika Lännenpää, there was also high expression level variability in DRF and ANS lines due to mRNA restriction. In some cases, there was more expression (e.g. DFR1266, ANS 1226) than in the control line. There was also a high variability in expression between individuals within the line, as was shown by DFR1294. Possible reasons for high variability in expression results could be the sampling of leaves with different age or from different individuals and the time interval between samples (up to two days; during this time the conditions may vary, and RNA is very short-lived). It is also possible that plants have several genes encoding the same function.

In our experiment the ANR-inhibited birch lines (1183, 1179 and 1201) showed reduced growth compared to control line (Figure 8). Similar reduced growth results have been seen in Kosonen et al. (2015) for the same birch lines on ANR enzyme restriction in which the ANR 1179, 1183, and 1201 plant lines grew slowly and having a dwarf growth form. The formation of catechins are catalyzed by LAR whereas epicatechins are catalyzed by ANR (Figure 1; Xie et al. 2003).

Both catechins and epicatechins are precursor of PAs. PAs can be formed via epicatechins (Abra- hams et al. 2003), or they may be formed only via catechin or via both catechins and epicatechins.

Kosonen et al. (2015) also found that the levels of catechins were high in ANRi lines and only catechins were detected in both control and ANRi lines of silver birch. As epicatechins were not present it might be possible that they are either not present in birches or they are utilized to make condensed tannins (Kosonen et al.2015)

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Connection between PAs and reduced growth can be partly explained by increased production of the upstream phenolics (i.e. flavonol glycosides) in ANR-restricted birch lines (Kosonen et al.

2015). As flavonoids inhibit the transportation of auxin (Brown et al. 2001, Peer and Murphy 2007) which promotes shoot elongation (Hopkins et al. 2002) accumulation of flavonoids may cause the dwarf phenotypes observed in our experiment. This could be the reason for the low growth of ANRi or DFRi plant lines.

Anthocyanins are responsible for the plant color, and protect plants against UV damage and other biotic and abiotic stress (Petroni and Tonelli 2011). Kovinich et al. (2012) and Fischer et al.

(2014) have studied blocked ANR expression in strawberry and soybean which resulted in the change of direction of metabolic flow into the anthocyanin and the flavonol pathway. The purpose of these soybean and strawberry studies was to check how ANR inhibition affects the phenotype of the plants. Strawberry and soybean are not closely related to silver birch but this explains that some effects of RNA inhibition of ANR might be the same in all other species as well.

As downregulation of ANRi in strawberry leads to the high accumulation of anthocyanin. Fischer et al. (2014) reported that due to ANR inhibition some strawberry transgenic lines have more prominent purple color in flower and green color in unripen strawberries than the control lines.

This redirection of the metabolic flux and change of color on blocking ANR are like Kosonen et al. (2015) results, who observed that the ANR restricted plants turned reddish and brownish and there was high anthocyanin accumulation. The same phenotype occurs also in my experiment for three ANR-restricted lines i.e. ANR 1201, showed dark red color, ANR 1183 showed average red and ANR 1179 less red. We did not measure the levels of anthocyanin for these lines, but since the effect of ANR inhibition on flavonoids in strawberry and soybean (Kovinich et al. 2012, Fischer et al. 2014) was like that observed in silver birch (Kosonen et al. 2015), we can suppose that the prominent red color in these ANR lines is caused by increased level of anthocyanins.

The growth of the fourth ANRi 1168 line was clearly different from other ANRi lines. It grew taller and thicker than the control line. According to preliminary butanol tests, the leaves of ANRi 1168 and the BPM5 (control line) had similar concentration of condensed tannins in spring 2016 (Figure 8). It seems that the ANR expression level of 1168 is not inhibited enough to reduce the PA levels. Preliminary butanol tests imply that the ANR-inhibition in 1168 was not working in spring 2016. It seems that the birch line ANR 1168 may have lost the inhibition when it was

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micropropagated repeatedly or, the inhibition of ANR works fine, but ANRi 1168 produces its condensed tannins via LAR and catechins.

DFRi silver birch lines also showed the reduced growth. which might be caused by the fact that the dihydroflavonol 4-reductase (DFR) is the first enzyme which may affect the production of PA in the phenolic pathway (Dixon & Paiva 1995, Xie et al. 2004). Dihydroflavonol is the substrate of DFR (Figure 1) and can produce flavonols, when catalyzed by FLS. On the other hand, the leucoanthocyanidin produced from DFR can be converted to proanthocyanidins by leucoanthocyanidin reductase (LAR) (Davies et al. 2003). They are the two main branches in flavonoid pathways. As a result, plant will produce more upstream small flavonols which may in return have affected decreased growth via increased carbon demand for flavonoids at the expense of growth (e.g. Bryant et al. 1983). Also, there will be no accumulation of anthocyanins which act as anti- oxidant (Wang et al.1997), and protect plants from light. This will make the plants weak to with- stand the abiotic stress for long. Therefore, all above could be the reasons for that the downregulation of DFR gene expression strongly affected the growth of DFRi plant lines. It also demonstrates the importance of DFR in the phenylpropanoid pathway. Shirley et al. (1995) reported that the silencing of the DFR gene in the tt3 mutants of Arabidopsis makes it unable to store the brown tannins of anthocyanins and proanthocyanidins in their seeds. Reduced anthocyanin accumulation is reported in transgenic purple sweet potato due to the restriction of DFR gene expression (Wang et al. 2013).

When we look at the ANS restricted lines, they showed same growth as the control line. The condensed tannins concentration for these ANSi plant lines was less than for the control line (Figure 6b) when analyzed before the experiment. The expression levels varied within the ANS lines taken in 2010-2011 by Mika Lännenpää as mentioned earlier. The tannin concentration levels show that the mRNA restriction of ANS was working well, even though the expression levels in 2010-2011 were highly variable. As we have not measured condensed tannins concentration and expression for these ANSi plant lines after the experiment, one can assume that with repeated micro-propagation these plant lines have greater ANS expression which can be the reason of their good growth. The samples collected after the experiment might provide more information on the relationship between ANS expression, condensed tannins concentration and growth in older sap- lings. The variable growth within enzyme-restricted lines could be due to variation in expression levels.

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5.2 UV-B effects on length and diameter

In my experiment the BPM5 (the control line), which has all the enzymes working, has a smaller diameter under enhanced UV-B than under the control treatment. The reduction of stem diameter under enhanced UVB in BPM5 is similar with Tegelberg et al. (2001) results. Tegelberg et al.

(2001) have used a normal silver birch which also showed reduced growth of stem diameter under supplement UV-B. Decreased growth of plants under enhanced UV-B were also observed in some other experiments. (Jansen 2002, Caldwell et al. 2003, Kosonen et al.2015). However, when the plants become mature they become less responsive to the UVB (Hunt & McSeveny, 2000) The age of the plants and the amount of UV-B are also important how vulnerable the plants are or how much they are damaged. According to Bryant & Julkunen-Tiitto (1995), young plants need more protection against harsh conditions during this developmental stage, which is why they use most of their energy to produce protective agents. Early developmental stage and high allocation to chemical protection in young, unmodified silver birches could explain the reduction in growth of BPM5 under high UV-B, even though the effects on height were not statistically significant. Generally, plants under enhanced UV-B showed reduced growth. Enhanced UV-B stim- ulates the production of flavonoids which inhibit the IAA transport (Huang et al.1997, Brown et al. 2001); therefore, the reduction in growth under enhanced UV-B could also be caused by the inhibition of indole acetic acid, both in modified and unmodified plants.

At the end of the first week of the experiment few plants of DFRi lines died. The symptoms observed in dying plants were brown pigmentation and wilting of the stem. However, same number of plants died under both treatments which means there is no difference in the death percent- age of DFR restricted birch plants between the treatments (Table 4). This shows that the increased UV-B did not caused the death of these DFR lines. One possible reason might be that the DFRi plants didn’t adapt to the new environment when transferred to the big pots (Figure 3). Therefore, the change of the place might have affected their growth as well. RNA restriction is responsible for their small growth, and it may make DFRi plants more vulnerable to light. There is also pos- sibility that the growth of DFRi plants were affected from occasional high temperature or low humidity in the greenhouse.

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5.3 Possible errors of the study

Optimally, repeated measurements would be always made by the same person. As measurements were taken by many individuals this might have caused the errors in the data.

Secondly, there were some plants which grow very tall and fast and there were some plants that were dwarf and grew slowly in height. The biologically effective UV-B dose depends on the distance from the lamps supplying the radiation. The lamps were lifted to maintain the same distance between the tallest plants and the lamps. For the first weeks, the smallest plants did not receive the same amount of the UV-B as did the tall plants. The smallest plants were lifted 15 cm on 6th July and an additional 35 cm in 28^th July to obtain more homogeneous conditions for all experimental plants.

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6 CONCLUSIONS

Based on the results, we can conclude that all these three enzymes ANR, ANS and DFR are very important for the normal growth of the plants. DFRi plants died more rapidly during experiment, which points out the significance of DFR for the growth of plants particularly in the early juvenile phases. In other words, the restriction of DFR seems more fatal for the plants compared to the restriction of ANR and ANS. As our results of reduced growth (length and diameter) due to enzyme restriction match to the findings of Kosonen et al. (2015), it is likely that the PA levels are also reduced, and as a result small molecular mass phenolics are increased in the modified lines.

This is the most probable reason why their growth is reduced. Phenylalanine pathways need proper coordinated expression of all three enzymes to produce final product (PAs). The UV-B effect on growth seen in this experiment is minor. This may be partly because of the short-term experiment where only small changes can be seen. More pronounced impacts of the enhanced UV-B on plants could be seen in long-term experiments. We could conclude that the line effect (the restriction of enzymes in the phenylpropanoid pathway) affected the growth more than the enhanced UV-B.

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7 ACKNOWLEDGMENTS

I would like to thank everyone from the bottom of my heart which, in their own way, has helped me along the way of completing my thesis, with special thanks to

My main supervisor Dr. Riitta Julkunen-Tiitto for giving me this chance to work with her and to learn about the phenolics along with UV-B. This thesis work was not possible without her support and helpful advice.

My Co-Supervisor Paula Thitz for helping me with the statistics and having the patience to guide and support me all the way, with her valuable and prudent advice, I learned a lot from her.

My Friend Dr. Jenna Lihavainen for all her encouragement, valuable discussions and questions, which opened my mind. Antti Tenkanen for his helpful guidance about article searching and stuff.

Dr. Sari Kontunen-Soppela for all her kind support and guidance which helped me to complete my degree and Dr. Jarkko Akkanen for allowing me to borrow department monitor which was very helpful in writing my thesis.

All the staff and students at the department for their support and for creating a friendly working environment.

My family and Friends, especially my twin sisters Prim Asghar, without whose support this thesis would never have possible. My godmother, my Äiti Rita Lipponen for all her love, support and for bearing my mood swings during this time.

My godmother Dr. Paulina kainulainen and Tyna Pesonen for their care which helped me to survive here in Finland, million miles away from home.

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