Environmental control of anthocyanin production

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CHAPTER FOUR – RELATIVE CONTRIBUTIONS OF TEMPERATURE AND LIGHT TO GENE EXPRESSION RHYTHMS

Overview

The primary aim of this chapter was to elucidate the individual role that temperature and light are performing in the expression profiles obtained from the Nelson orchard experiment. The affects of these environmental stimuli are likely to be combining to give rise to the observed candidate profiles. The contribution of light and temperature were assessed by conducting a second time-series experiment where harvested apples were sampled experiencing controlled environmental conditions. Light and temperature were varied in such a way that results would reveal their relative contributions to the observed expression profiles of candidate genes.

Introduction

The assessment of light and temperature is important because identifying the hierarchy of environmental stimuli may aid apple growers in tailoring apple growth conditions to those optimised for pigment production, aiding the marketability and health properties of the fruit. From the data generated during the orchard experiment the contribution of these two factors to the results cannot be determined, so an additional experiment was required. In the orchard high light tends to coincide with high temperature and low light with low temperature. To find which of these signals is the more dominant stimuli to anthocyanin production measurements were taken in an environment where light and temperature could be controlled and the natural coincidence of these stimuli staggered or reversed, for example, high light and low temperature. By subjecting apples to different combinations of light and temperature it should be possible to distinguish their affects from one another. In order to untangle the affects of light at temperature on the results obtained in chapter three a second experiment was performed. This experiment used harvested apples placed in growth rooms where light and temperature were controlled. It was hoped that this would allow subsequent investigation to concentrate on genes which respond to the more dominant environmental signal, therefore increasing the chance of identifying the most influential factors responsible for the upstream regulation of MYB10 and the APEs.

Previous work

As well as the importance of temperature to anthocyanin production (section 3.2), the effects of light also influence apple pigmentation. Apples kept in the dark during development do not redden (Jakopic, et al., 2009). Light is a major environmental stimuli available to plants which allows them to adopt optimal strategies involving growth and developmental decisions, such as shade avoidance, circadian growth, and flowering time (Chang et al., 2011). The most effective wavelength of visible light for anthocyanin production in apple has been reported to be around 650 nm, with wavelengths between 430 and 480 nm also contributing (Arakawa, 1988). UV-B radiation also stimulates anthocyanin production, which acts as a phytoprotectant (Steyn, et al., 2009). Light responsive gene expression has been well characterised (Gilmartin et al., 1990). The influence of light on anthocyanin production in harvested apple fruit flesh has previously been measured by Bakhshi & Arakawa (2006). When apple flesh was subjected to 96 hours of constant UV-B radiation at four temperature treatments (10, 17, 24, and 30 °C) anthocyanin production was found to be light dependent and that 24 °C results in the highest production (Bakhshi & Arakawa, 2006). It has been shown in Vitis vinifera that grapes grown in the absence of light can still accumulate anthocyanin (Downey et al., 2004). However, the absence of light did change the composition of anthocyanin content, with the F3’5’H enzyme activity reduced and compensated for by F3’H. Findings found during another light experiment on apple pigmentation has found that position in the tree canopy affects individual fruit due to levels of light exposure (Jakopic, et al., 2009). Extremes of temperature and light can give rise to sunburn of apples. If fruit surface temperatures reach over 50 °C, heat induced cell death occurs (Felicetti & Schrader, 2008). At temperatures between 46 and 49 °C sunburn browning of apple tissue is seen to occur. Photo-oxidative sunburn of apples can also occur on shaded peel that is suddenly exposed to sunlight (Felicetti & Schrader, 2008). The apple sunburn studies also found it difficult to distinguish between the contribution of light and temperature to the observed results causing confusion between photo-oxidative and heat damage to the apple tissue. Finding out which factor is more important would allow growers to focus on either blocking or reflect harmful UV radiation or employing orchard management practises that keep the fruit surface temperature below sunburn threshold levels (Felicetti & Schrader, 2008). As it stands at the moment, excessive light, and or temperatures greater than 46 °C appear to result in apple tissue damage, with burning thought to occur from the light and browning and softening from high temperatures. Bagging apples during development to remove light signals significantly inhibits anthocyanin production (Figure 65) (Ju, 1998). Apples bagged until harvest, once picked could still synthesise anthocyanins when exposed to light (Ju, 1998). The capability of fruit to synthesis anthocyanin has been found to remain for 5 months after harvest, during cold storage (Ju, 1998). It appears that the general consensus regarding environmental conditions optimal for apple pigmentation is that high light and low temperatures are best for anthocyanin biosynthesis in apple peel. The high light days and low temperature nights of New Zealand’s climate perhaps explaining the highly pigmented apples that this climate produces.

Experimental design

Due to being later in the apple season, ‘Royal Gala’ had already been harvested, so a different cultivar, Scilate/Envy^TM, was used. It had to be assumed that the Scilate/Envy^TM cultivar, of which ‘Royal Gala’ is a parent, would give results similar to ‘Royal Gala’. Two hundred apples were harvested from a Hawkes Bay apple orchard and transported to Auckland overnight, where they were immediately placed in experimental conditions. The fruit were split into four equal groups and placed in one of four treatments, which varied temperature and light with 12 hour cycles. The conditions each of the four treatments received were; 1) constant 25 °C with 12 hours light and 12 hours dark (25/25:L/D), 2) constant 25 °C and constant light (25/25:L/L), 3) 12 hours at 25 °C, 12 hours at 10 °C with constant light (25/10:L/L), and 4) 25 °C light for 12 hours with 10 °C dark for 12 hours (25/10:L/D). In order to elicit the control over light and temperature regimes required for this experiment growth rooms at Plant & Food Research Mt Albert were used. Two apples from each treatment were sampled every four hours from midday April 29 to 4pm May 2 2011, giving 20 samples from each treatment over 76 hours. Sampling was performed as outlined in section 2.18. Whole apple peels were frozen in liquid nitrogen and stored at -80 °C until all samples had been taken and then the RNA was extracted (section 2.19). Growth room one (Gr1) was set to 10 °C and growth room 2 (Gr2) set to 25 °C. The light conditions were set using timers and apples moved between the rooms during the experiment as needed to suit each of the four treatments (Figure 66). Dark boxes were set up in each room to hold apples in treatments with dark periods. Temperature probes monitored representative apples from each of the growth rooms.

Temperature data

A data logger in each of the growth rooms monitored the temperature of individual apples. Data from Gr1 shows that the apples remained around 12 °C (Figure 67). The high temperatures reached by the ‘apple 3’ probe show apples coming in from the 25 °C room and the time taken for them to equilibrate. The small-scale zigzag motion of the line shows the oscillating temperature of the air conditioning unit. Data from Gr2 shows apples were held at close to 25 °C for the duration of the sampling, slightly warmer temperatures were reached depending on proximity to the light (Figure 68). The drops in temperature of the ‘apple 2’ temperature probe shows the time take for fruit moved from the 10 °C room to equilibrate. Treatment 4 (25/10:L/D) was assessed first because it subjected the fruit to the most varying environment (both light and temperature) and was therefore the most likely treatment for any rhythms to still be occurring. Any genes found to still display a rhythm could then be assessed under another temperature regime varying either light or temperature to elucidate which signal is dominant in giving rise to the observed on-tree rhythm. Graphs presented in this section show the results of this experiment compared to the on-tree expression levels found from the Nelson orchard experiment. The data is presented on the same axis so changes to the expression profile can be easily compared.

Results

To represent the APEs, two genes, DFR and UFGT, were chosen from early and late in the anthocyanin pathway. Results show that the post-harvest expression of DFR is seen to decrease and after the a few initial peaks during the day expression appeared to remain fairly constant (Figure 69). UFGT, (Figure 70) also displayed reduced expression post-harvest, remaining at fairly constant levels. The COLs were chosen to be analysed here due their strong on-tree rhythms and association with light (Putterill, et al., 1995; Tiwari, et al., 2010). COL1 expression levels remain comparable to pre-harvest levels; however the cycling present on-tree is eliminated after harvest (Figure 72). The same observation is true for COL7 (Figure 73) and COL12 (Figure 74) with both genes on-tree expression rhythms also ceasing after harvest. Temperature responsive genes were also analysed for their post-harvest activity. Expression of the CBF genes was measured due to the strong influence of heat treatment on CBF2. Post-harvest, CBF1 appears to be high during the night and low during the day (Figure 75). The opposite appears to be occurring for CBF2 where the post-harvest results show peaks during the day and low expression during the night (Figure 76). The expression of ICE1 by on-tree apples was low and once harvested the expression of ICE1 displayed a large increase in activity, becoming much more active than when on-tree (Figure 77). The expression levels of CCA1 displayed reduced activity and the loss of the slight on-tree rhythm, which tended to increase during the night and decrease during the day (Figure 78). The post-harvest expression of GI was analysed because it was being used as a positive control for rhythmic gene expression (Fowler, et al., 1999). However, it was found that even the highly rhythmic GI lost its cyclic expression after harvest (Figure 79). The expression of TOC1 (Figure 80) did not appear to exhibit much of a change between on-tree and post-harvest expression levels.

CHAPTER ONE – INTRODUCTION 
1.0 – Preface
1.1 – History of apples
1.2 – The apple industry
1.3 – Plant biosynthetic pathways
1.4 – Secondary metabolites
1.5 – Plant pigments
1.6 – The phenylproponoid pathway
1.7 – Flavonoids
1.8 – Anthocyanin
1.9 – Regulation of anthocyanin biosynthesis
1.10 – Species specific regulation of anthocyanin
1.11 – The investigated species: Apple (Malus x domestica)
1.12 – Environmental control of anthocyanin production
1.13 – Potential candidates for upstream regulation of MdMYB10
1.14 – Research aims
1.15 – Project objectives
CHAPTER TWO – MATERIALS AND METHODS
2.0 – Materials
2.1 – Plant material
2.2 – Solutions
2.3 – Buffers
2.4 – Antibiotics
2.5 – Growth media
2.6 – Restriction enzymes
2.7 – Bacterial strains
2.8 – Plasmids
2.9 – Oligonucleotide primers
2.10 – Primer design
2.11 – Automated pipetting
2.12 – Data logger
2.13 – Photometer
2.14 – Computer software
2.15 – Heating fruit
2.16 – Methods
2.17 – Research procedure
2.18 – Biological sampling
2.19 – RNA extraction
2.20 – Agarose gel electrophoresis
2.21 – DNA/RNA quantification
2.22 – DNase I digestion
2.23 – First strand cDNA synthesis
2.24 – Standard (end-point) PCR
2.25 – Real time expression analysis
2.26 – Cloning
2.27 – Restriction enzyme digest
2.28 – Ligation reactions
2.29 – Transformation
2.30 – Agrobacterium miniprep
2.31 – DNA sequencing
2.32 – Transient expression
CHAPTER THREE – NELSON TIME-SERIES
3.0 – Overview
3.1 – Introduction
3.2 – Previous work
3.3 – Experimental design
3.4 – Results
3.5 – Temperature data
3.6 – Gene candidate selection
3.7 – Expression profiles
3.8 – Expression profiles of MYB10 and anthocyanin pathway enzymes
3.9 – Chapter summary
CHAPTER FOUR – RELATIVE CONTRIBUTIONS OF TEMPERATURE AND LIGHT TO GENE EXPRESSION RHYTHMS
4.0 – Overview
4.1 – Introduction
4.2 – Previous work
4.3 – Experimental design
4.4 – Temperature data
4.5 – Results
4.6 – Chapter summary
CHAPTER FIVE – FUNCTIONAL ANALYSIS OF COL PROMOTERS 
5.0 – Overview
5.1 – Introduction
5.2 – Previous work
5.3 – Experimental design
5.4 – Promoter isolation
5.5 – Predicted promoter binding motifs
5.6 – Functional analysis
5.7 – Chapter Summary
CHAPTER SIX – DISCUSSION AND CONCLUSIONS
6.0 – Overview
6.1 – Candidate genes identified from on-tree analysis
6.2 – All measured rhythms are lost post-harvest
6.3 – COL promoters maintain cyclic activity in N. benthamiana
6.4 – Future work
6.5 – Significance of this research
CHAPTER SEVEN – SUPPLEMENTARY MATERIAL

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ENVIRONMENTALLY REGULATED CONTROL OF ANTHOCYANIN BIOSYNTHESIS IN APPLE