the quality and commercial value of american cranberry fruit 
 ( vaccinium macrocarpon ait ) are determined by their color 
 .
anthocyanins have important therapeutic values , 
 including antitumor [ 2 , 
 3 ] , antiulcer , antioxidant , 
 and anti - inflammatory activities .
six anthocyanins have 
 been reported in cranberries based on the high - performance liquid 
 chromatography ( hplc ) analysis of acid - alcohol fruit extracts on 
 reversed - phase c18 column . these are cyanidin 3-galactoside , 
 cyanidin 3-glucoside , cyanidin 3-arabinoside , peonidin 
 3-galactoside , peonidin 3-glucoside , and peonidin 3-arabinoside 
 [ 6 , 7 ] . 

the proportions of individual anthocyanins in 
 cranberry fruit may affect the color stability of cranberry 
 products such as juice and sauce [ 8 , 
 9 ] .
yan et al 
 reported that cyanidin 3-galactoside showed 
 antioxidant activity superior to six other monoglycosides of 
 quercetin and myricetin isolated from cranberry fruit as well as 
 vitamin e by evaluating compounds for 
 1,1-diphenyl-2-picrylhydrazyl radical - scavenging activity and 
 ability to inhibit low - density lipoprotein oxidation in vitro .
light has been shown to be the most important environmental 
 factor influencing anthocyanin biosynthesis in plants , 
 although in some species , such as vitis vinifera cv . 

phytochromes are among the most extensively researched 
 photoreceptors which sense light , and are known to be involved in 
 anthocyanin biosynthesis [ 13 , 
 14 , 15 ] .
phytochromes respond 
 to red ( 660  nm ) and far - red ( 730  nm ) light , and direct 
 plant gene expression by switching between the red - absorbing form 
 ( pr ) and the far - red absorbing form ( pfr ) .
previously , we have 
 examined the effect of various wavelengths of light on the 
 development of the cranberry plant and anthocyanin biosynthesis 
 in cranberries which were still attached to the plant .
we have 
 observed that red light stimulates flowering and anthocyanin 
 biosynthesis in cranberry plant and fruit , respectively . in general , leaves and stems decrease light exposure for berries 
 lower on the plant .
preharvest anthocyanin content of cranberries 
 at the bottom and the top of the plant varies 
 significantly , primarily due to the differences in light 
 accessibility .
water - harvesting has become a common practice in the cranberry 
 industry , and it is accomplished by flooding the cranberry bog 
 with water to float the buoyant fruit for easy collection . 

however , potential effects on the berries due to the water - harvest 
 technique have not been studied systematically .
one study did show 
 that prolonged fruit immersion increases fungal rot of the berries 
 . during the water - harvest ,
cranberries on the surface of 
 the water receive the same or more light than the fruit still 
 attached to the plant . in this paper
, we evaluate the effects of 
 natural light , red light , and far - red light on individual as well 
 as total anthocyanin levels in cranberry fruit under conditions 
 that mimic water - harvesting .
cranberries were exposed to 
 different light conditions and individual anthocyanin content was 
 analyzed .
different light conditions , natural light ( 48  h ) , 
 natural light ( 24  h ) , dark , red light , and far - red light , are 
 indicated in the bottom .
percentage of anthocyanin increased in submerged , 
 harvested cranberries exposed to different light conditions in 
 comparison with the control , which was kept in the dark .
* p < .02 ; * * .05 < p < .1 ; * * * .1 < p < .5
cranberries ( vaccinium macrocarpon ait , cv  early 
 black  ) used in this study were obtained from the bog of the 
 cranberry experiment station , the university of massachusetts , 
 east wareham , mass , in october 1999 .
red light , with a photon fluence rate of 12  mole 
 ms , was obtained from six 40-w 
 fluorescent tubes ( f48t12/r-660/ho , red , general electric 
 company , usa ) filtered through a red plastic sheet ( roscolux 
 color filter # 27 , rosco laboratories , port chester , ny ) . 

far - red light , with a photon fluence rate of 
 5  mole ms , 
 was obtained from 500-w 
 brilliant white light halogen double - ended quartz fcl bulbs 
 ( osram sylvania products inc , winchester , ky ) filtered through 
 3  mm far - red plastic ( type frf700 , west lakes plastics , 
 lenni , pa ) .
light sources in each case were kept at a distance of 
 0.8  meter from the berries .
all light measurements were 
 made with a model il1400a radiometer / photometer ( international 
 light , inc , newburyport , mass ) .
cranberries were taken from a flooded bog after the harvest 
 machine had knocked the fruit loose from the vines and selected 
 in approximate same size and color for experiment in order to 
 avoid variability in the anthocyanin content .
the fruit were 
 randomly divided into five groups and held in beakers containing 
 water .
two sizes of beakers ( 1000  ml and 250  ml ) were 
 used . the 1000  ml beaker ( diameter : 11.6  cm ) contained 
 800  ml of water , and approximate 34 berries forming just one 
 layer on the surface of the water were placed in the beaker .
the 
 250  ml beaker ( diameter : 7.5  cm ) contained 200  ml of 
 water , and approximate 17 berries forming just one layer on the 
 surface of the water were placed in the beaker .
two groups in the 
 1000  ml beakers were placed in a nursery area outside the 
 laboratory and received a cycle of daylight for 24 and 48 hours , 
 respectively .
the remaining three groups in the 250  ml beakers 
 were placed in a temperature - controlled darkroom at 
 20c .
one of these 250  ml beakers was kept in the 
 darkroom and was used as a control sample .
the other two , also 
 kept in the darkroom , received 30  minutes of red light or 
 far - red light per day , respectively , for two days .
the berries 
 from the two groups placed outside were collected after 24 and 48 
 hours , respectively , and the fruit from the three groups placed 
 in the temperature - monitored room were individually collected 
 after 48 hours .
eight grams of the berries from each group were 
 weighed and homogenized in 10  ml of ethanol : 1.5 
 n hcl ( 85 : 15 , v / v ) to extract the anthocyanins overnight at 
 4c .
the sample extracts were 
 filtered through 0.2  m filters before 
 injection into the hplc column .
hplc analyses of anthocyanins were carried out on a waters 515 
 dual pump hplc system , equipped with a 996-photodiode - array 
 detector and a c18 column ( 4.6  150  mm ) with 
 5  m particle size ( waters corporation , milford , mass ) . 

the software used to control the hplc system and data analysis was 
 millennium 32 ( waters corporation , milford , mass ) .
elution was 
 carried out using a mobile phase formed by a linear gradient of 
 ( a ) h2o - acetic acid ( 10 : 1 ) and ( b ) methanol - acetic 
 acid ( 10 : 1 ) , with 100% ( a ) at 0 minute to 40% ( a ) and 60% 
 ( b ) at 20 minutes . the flow rate was fixed at 0.2  ml / min . 

anthocyanin content was calculated in 
 absolute quantities using the extinction coefficient 
 ( 1  cm ) at 535  nm as 982 .
cranberries ( vaccinium macrocarpon ait , cv  early 
 black  ) used in this study were obtained from the bog of the 
 cranberry experiment station , the university of massachusetts , 
 east wareham , mass , in october 1999 .
red light , with a photon fluence rate of 12  mole 
 ms , was obtained from six 40-w 
 fluorescent tubes ( f48t12/r-660/ho , red , general electric 
 company , usa ) filtered through a red plastic sheet ( roscolux 
 color filter # 27 , rosco laboratories , port chester , ny ) . 

far - red light , with a photon fluence rate of 
 5  mole ms , 
 was obtained from 500-w 
 brilliant white light halogen double - ended quartz fcl bulbs 
 ( osram sylvania products inc , winchester , ky ) filtered through 
 3  mm far - red plastic ( type frf700 , west lakes plastics , 
 lenni , pa ) .
light sources in each case were kept at a distance of 
 0.8  meter from the berries .
all light measurements were 
 made with a model il1400a radiometer / photometer ( international 
 light , inc , newburyport , mass ) .
cranberries were taken from a flooded bog after the harvest 
 machine had knocked the fruit loose from the vines and selected 
 in approximate same size and color for experiment in order to 
 avoid variability in the anthocyanin content .
the fruit were 
 randomly divided into five groups and held in beakers containing 
 water .
two sizes of beakers ( 1000  ml and 250  ml ) were 
 used . the 1000  ml beaker ( diameter : 11.6  cm ) contained 
 800  ml of water , and approximate 34 berries forming just one 
 layer on the surface of the water were placed in the beaker . the 
 250  ml beaker ( diameter : 7.5  cm ) contained 200  ml of 
 water , and approximate 17 berries forming just one layer on the 
 surface of the water were placed in the beaker .
two groups in the 
 1000  ml beakers were placed in a nursery area outside the 
 laboratory and received a cycle of daylight for 24 and 48 hours , 
 respectively .
the remaining three groups in the 250  ml beakers 
 were placed in a temperature - controlled darkroom at 
 20c .
one of these 250  ml beakers was kept in the 
 darkroom and was used as a control sample .
the other two , also 
 kept in the darkroom , received 30  minutes of red light or 
 far - red light per day , respectively , for two days .
the berries 
 from the two groups placed outside were collected after 24 and 48 
 hours , respectively , and the fruit from the three groups placed 
 in the temperature - monitored room were individually collected 
 after 48 hours .
eight grams of the berries from each group were 
 weighed and homogenized in 10  ml of ethanol : 1.5 
 n hcl ( 85 : 15 , v / v ) to extract the anthocyanins overnight at 
 4c .
the sample extracts were 
 filtered through 0.2  m filters before 
 injection into the hplc column .
hplc analyses of anthocyanins were carried out on a waters 515 
 dual pump hplc system , equipped with a 996-photodiode - array 
 detector and a c18 column ( 4.6  150  mm ) with 
 5  m particle size ( waters corporation , milford , mass ) . 

the software used to control the hplc system and data analysis was 
 millennium 32 ( waters corporation , milford , mass ) .
elution was 
 carried out using a mobile phase formed by a linear gradient of 
 ( a ) h2o - acetic acid ( 10 : 1 ) and ( b ) methanol - acetic 
 acid ( 10 : 1 ) , with 100% ( a ) at 0 minute to 40% ( a ) and 60% 
 ( b ) at 20 minutes .
anthocyanin content was calculated in 
 absolute quantities using the extinction coefficient 
 ( 1  cm ) at 535  nm as 982 .
composition of anthocyanins plays a role in their therapeutic 
 effects . 
 although six anthocyanins have been identified 
 in cranberries [ 21 , 22 , 
 23 ] , biosynthesis of those individual 
 anthocyanins in response to environmental conditions such as 
 light is not understood . in an attempt to elucidate anthocyanin 
 biosynthesis ,
we measured total as well as individual anthocyanin 
 content in cranberry fruit under different light conditions in an 
 experimental setting designed to mimic water - harvesting conditions . statistical analysis ( student t test ) was performed to detect 
 the statistical difference between total anthocyanin 
 content under natural light ( 48-hour and 24-hour ) , red light , and 
 far - red light conditions and that under dark 
 conditions .
table 1 shows that the total anthocyanin 
 level varied significantly ( > 98% confidence level 
 ( p<.02 ) ) when the submerged , harvested cranberries 
 were exposed to various light conditions .
the total anthocyanin 
 content of berries before exposure to any experimental light 
 conditions was 18.95  0.88 , and was the same as the 
 control that was kept in the dark .
compared to the 
 control , cranberries exposed to one 24-hour day - night cycle had 
 75.3% higher anthocyanin content , and berries exposed 
 to a 48-hour day - night cycle posted only a small further increase 
 ( 87.2% ) .
red and far - red light had substantially less effect 
 on total anthocyanin biosynthesis than natural light ( 7587% vs. 
 3542% ) .
red light increased total anthocyanin content 
 ( 41.5% ) more than far - red light ( 34.7% ) .
separation of cranberry anthocyanins by reverse - phase hplc 
 revealed six anthocyanins which were assumed to be 
 cyanidin 3-galactoside , cyanidin 3-glucoside , 
 cyanidin 3-arabinoside , peonidin 3-galactoside , 
 peonidin 3-glucoside , and peonidin 3-arabinoside 
 ( table 2 ) according to previous reports 
 [ 6 , 24 ] . 

the relative amounts of the six anthocyanins in the control 
 samples which were kept in the dark ( figure 1 ) are 
 consistent with earlier reports [ 6 , 
 24 ] .
variation in 
 different individual anthocyanins under different light 
 conditions was also subjected to the statistical analysis 
 ( student - test ) , which showed significant differences except for 
 the cyanidin 3-glucoside under each light condition , and for 
 cyanidin 3-galactoside under red light and far - red light 
 conditions , as shown in table 2 .
compared with the 
 dark conditions , the natural light conditions enhanced all the 
 anthocyanins substantially , 72%100% ( in the 48-hour cycle ) , 
 except for the cyanidin 3-glucoside , which increased by 54% 
 ( table 2 ) , whereas the red and far - red light had the 
 most prominent effect on cyanidin 3-glucoside and 
 peonidin 3-arabinoside , showing 7092% increase 
 ( table 2 ) .
the biosynthesis of cyanidin 3-galactoside 
 was least affected by red and far - red light , showing only 29 and 
 17% increase , respectively ( table 2 ) .
although 
 experimental conditions in our previous study   were 
 different ( 30 minutes of light treatments per day for eight 
 days ) , results had shown that red light and sunlight increased 
 anthocyanin biosynthesis more than   the far - red light did , 
 consistently with the conclusions of the present study .
however , 
 in the above two   cases  cranberry fruit that were still 
 attached to the plant and cranberry fruit that were no longer 
 attached to the plant , the effect of far - red illumination 
 appeared to be close to the effect of red light , not similar to 
 the dark control .
exposure of etiolated normal 
 seedlings of brassica rapa to red 
 light and far - red light showed that far - red illumination enhanced 
 more anthocyanin synthesis than red light .
study of 
 different phytochromes in arabidopsis photomorphogenesis has 
 shown that phytochrome a regulates plant responses to far - red 
 light irradiation , whereas phytochrome b plays a predominant role 
 in responses to red light irradiation .
therefore , it is 
 considered that coactions between different photoreceptors 
 involved in the effects of red light and far - red light on 
 anthocyanin content in cranberry fruit are 
 as coactions between the photoreceptors involved in flavonoid 
 biosynthesis .
in addition , anthocyanins contain two parts in their structures : 
 aglycones and sugars .
the biosynthesis of anthocyanins was 
 catalyzed by several enzymes from pla ( phenylalanine 
 ammonia - lyase ) , c4h ( cinnamic acid 4-hydroxylase ) , 4cl 
 ( 4-coumarate : coa ligase ) , chs ( chalcone synthase ) , chi ( chalcone 
 isomerase ) , f3h ( flavanone 3 -hydroxylase ) , dfr 
 ( dihydroflavonol 4-reductase ) , as ( anthocyanin synthase ) through 
 3gt ( udp - glucose : flavonoid 3-o - glycosyl transferase ) .
chs is the 
 first committed enzyme of flavonoid biosynthesis . as is the first 
 committed enzyme of anthocyanin biosynthesis .  
the expressions of 
 the above enzymes are regulated differently by environments such 
 as light and temperature .
this results in the disproportional 
 increase of different anthocyanins such as peonidin 3-glucoside 
 compared to cyanidin 3-glucoside , due to the different aglycones 
 although same sugar ; or cyanidin 3-galactoside compared to 
 cyanidin-3-glucoside , due to the different sugars although same aglycon .
this study demonstrates that during water - harvesting conditions , 
 where the berries are no longer attached to the plant , exposure 
 of the berries to light still results in increased anthocyanin 
 levels .
this study also shows that levels of individual 
 anthocyanins increase differently following different light 
 exposure such as natural light , red light , and far - red light . the 
 variation in composition of anthocyanin
this 
 study also contributes to our understanding of cranberry 
 anthocyanin biosynthesis under water - harvesting conditions .
 anthocyanins are a group of plant antioxidants 
 known for their therapeutic use . 
 the effects of natural light , 
 red light , and far - red light on individual as well as total 
 anthocyanin content in cranberry fruit ( vaccinium 
 macrocarpon ait ) were examined in an experimental setting 
 designed to mimic water - harvesting conditions . 
 the reversed - phase 
 high - performance liquid chromatography ( hplc ) method was used to 
 separate and analyze the anthocyanins . 
 in contrast to the 
 case of the control sample that was kept in the dark , 
 natural light increased the total anthocyanin level by 75.3% 
 and 87.2% after 24 and 48 hours of water immersion , 
 respectively . 
 red light and far - red light increased the total 
 anthocyanin level by 41.5% and 34.7% , respectively . 
 the 
 amount of each individual anthocyanin increased differently under 
 natural light , red light , and far - red light , suggesting that 
 expressions of enzymes that catalyze the anthocyanin biosynthesis 
 are regulated differently by environments . 
INTRODUCTION
MATERIALS AND METHODS
Plants
Light sources
Experimental setting
HPLC analyses
RESULTS AND DISCUSSION