A case of serendipity * An account is given of how a sensitive bioassay system for measurement of the neurotransmitter acetylcholine serendipitously led to the identification of adenosine triphosphate -LRB- ATP -RRB- released in vitro from active skeletal muscle . Subsequent application of the identification procedures to exercising human muscle in vivo , cardiac muscle cells in vitro , and human erythrocytes exposed to hypoxia gave rise to the general concept of ATP as a molecule that could influence cell function from the extracellular direction . Mechanisms of ATP release from cells in terms of `` trigger '' events such as mechanical distortion of the membrane , depolarization of the membrane , and exposure to hypoxia are discussed . Potential therapeutic uses of extracellular ATP in cancer therapy , radiation therapy , and a possible influence upon aging are discussed . Possible roles -LRB- distant and local -RRB- of extracellular ATP released from muscle during whole body exercise are discussed . Introduction In 1961 Boyd and Eadie -LSB- 5 -RSB- developed a highly sensitive bioassay for acetylcholine using an in situ perfusion technique of frog heart . Based on the great sensitivity of the frog heart to acetylcholine -LRB- Fig. 1 -RRB- a project was suggested by B. Katz to I. A. Boyd ; they thought it possible that the amounts of acetylcholine released from stimulated motor nerve endings in frog skeletal muscle could be directly measured . The skeletal muscle to be used was the frog sartorius , about which a lot was known regarding the distribution and population of the motor nerve terminals . At that time the association of miniature end-plate potentials -LRB- MEPPs -RRB- with the release of acetylcholine in packets -LRB- `` quanta '' -RRB- together with the anatomical existence of synaptic vesicles at the motor nerve terminals was highly plausible . Any accurate measurement of acetylcholine release from motor nerve stimulation would provide valuable evidence for -LRB- or against -RRB- the association and the establishment of the `` quantal '' theory of neuromuscular transmission . In essence the experimental approach mimicked the classic experiment of Otto Loewi , where fluid perfusing a first frog heart was used to perfuse a second heart . When the vagus nerve supplying the first heart was stimulated cardiac arrest occurred , and shortly after this the second heart also stopped beating . Loewi termed the vagal inhibitory transmitter Vagusstoff . An isolated frog sartorius was to be stimulated via the motor nerve in a bathing solution and the solution then perfused through a frog heart . When a stimulated muscle solution was perfused a surprising stimulatory response was elicited , quite unlike the anticipated inhibitory effect of acetylcholine -LRB- Fig. 1 -RRB- . Clearly this response would preclude any accurate assay for acetylcholine -LRB- as well as any successful conclusion to a thesis project aimed at substantiating the quantal theory of neuromuscular transmission ! -RRB- . Fig. 1The response of a very sensitive frog heart to graded concentrations of acetylcholine -LRB- g.ml − 1 -RRB- . A concentration of 2.5 × 10 − 13 g.ml − 1 -LRB- 1.54 × 10 − 12 mmol.ml − 1 -RRB- produced a threshold response -LSB- 19 -RSB- It was evident that for successful measurement of acetylcholine , the identification of the interfering substance , and its removal from the solution , had to be achieved . At this stage the basic question became : what substance -LRB- s -RRB- released from active skeletal muscle could possibly stimulate the frog heart ? A `` blunderbuss '' approach was adopted , by perfusing through the heart as many available substances , known to exist in muscle , to see if any of them produced the stimulatory effect . The purine monophosphates and adenosine were eliminated since they did not stimulate the heart . Electrolyte changes , including an elevated potassium level , were also ruled out . Changes in calcium levels were hardly detectable in the bathing solution . One obvious candidate as a stimulatory substance was catecholamine material . However , when the frog heart response to adrenaline was blocked by an ergot/pronethalol perfusion , the stimulatory effect remained -LRB- Fig. 3 -RRB- . Fig. 2Effect of graded concentrations of ATP -LRB- g.ml − 1 -RRB- perfused through a frog heart -LSB- 6 -RSB- . Note that low concentrations have a pure inotropic effect , while a triphasic response was produced by higher concentrations . Further analysis of this triphasic response has since been made -LSB- 17 -RSB- Indirect versus direct muscle stimulation The distribution of motor end plates in the sartorius muscle -LRB- there are no end plate regions at the pelvic end of the frog sartorius -RRB- allowed muscle fibers to be stimulated directly , without involvement of the neuromuscular junction in the activation process . Solutions bathing muscles stimulated directly produced the same stimulatory effect as the solutions from muscles stimulated via the motor nerve -LRB- indirect stimulation -RRB- . This indicated that it was highly unlikely that the stimulatory substance had as its major source the activated motor nerve terminal . Identification procedures Identification of the stimulatory substance was established using three procedures : gel filtration , enzyme action of apyrase , and firefly tail luminescence . Gel filtration chromatography The use of a gel filtration chromatography technique -LRB- Sephadex G-25 , fine grade -RRB- enabled removal of protein from the stimulated muscle solution , allowing spectral absorbance analysis . An absorbance peak obtained at 265λ was the first hint that a purine compound was present in the solution . The gel filtration technique also established a molecular weight of the stimulatory substance close to that of ATP.In the face of prevailing dogma that ATP could not cross cell membranes , perfusion with ATP was reluctantly performed . A stimulatory effect was obtained -- the author had become a citizen of the Republic of Serendip ! A dose response of the frog heart to ATP is shown in Fig. 2 . Fig. 3Comparison of the stimulatory action of a stimulated muscle solution -LRB- ` X ' -RRB- on a frog heart before and after adrenergic blockade . Vertical interrupted line , 2-h period during which heart was perfused with pronethalol hydrochloride -LRB- 10 − 6 g.ml − 1 -RRB- and ergotamine tartrate -LRB- 10 − 6 g.ml − 1 -RRB- . The stimulatory action of the stimulated muscle solution was unaffected by adrenergic blockade . Reproduced from Boyd and Forrester -LSB- 6 -RSB- , with permission of the Physiological Society Incubation with apyrase In 1962 Traverso-Cori and Cori -LSB- 61 -RSB- demonstrated that an enzyme purified from potatoes -LRB- `` apyrase '' -RRB- could selectively cleave the terminal phosphate of ATP . The stimulated muscle solution was incubated with apyrase , as were the stimulatory fractions from the gel filtration technique , and it was shown that the stimulatory effect was abolished , indicating that a phosphorylated compound was responsible for the stimulatory effect . The luciferin/luciferase test for ATP As long ago as 1947 McElroy -LSB- 38 -RSB- showed that when a crude extract of lampyrid beetles -LRB- Photinus pyralis -RRB- was exposed to ATP a light signal was generated . When the stimulated muscle solution was applied to luciferin extract a light signal was produced -LRB- see -LSB- 18 -RSB- -RRB- . Procedures applied to perfused frog musculature As this work developed the question `` does ATP appear extracellularly as a result of tissue damage ? '' was frequently asked . Dissection damage to the sartorius muscle was avoided by the use of the perfused hindlimb preparation of the frog , where instrument trauma was limited to the insertion of cannulae . The identification procedures for ATP were applied to the perfusate from stimulated -LRB- indirect -RRB- hindlimb musculature . Table 1 shows the relationship of ATP output to the frequency of motor nerve impulses -LSB- 23 -RSB- . When these amounts -LRB- 0.24 -- 1.8 nmol 100 g − 1 min − 1 -RRB- are compared to the amounts of ATP released from isolated sartorius -LRB- 50 nmol 100 g − 1 min − 1 , -LSB- 6 -RSB- -RRB- , it is seen that there is a great reduction detected in the perfusate sample , presumably as a result of exposure to greater ecto-ATPase activity that was not encountered in solutions bathing isolated muscles . Table 1Relationship of ATP output to frequency of stimulation of perfused frog limbaHzpmol volley − 1 100 g − 1pmol 100 g − 1 min − 114.024021.518051.4420103.01,800 aData from Forrester and Hassan -LSB- 23 -RSB- Procedures applied to human muscle in vivo The appearance of ATP in the perfusate from active frog hindlimb musculature raised the possibility that ATP might be released from active muscle into the circulating blood in vivo . The identification procedures were applied to human plasma obtained from resting and exercising human forearm muscle -LSB- 20 , 21 -RSB- . Figure 4 gives an example of the combined use of gel filtration chromatography , firefly luciferase , and apyrase for the identification and quantification of ATP in human plasma from exercising human forearm muscle -LSB- 22 -RSB- . Estimates of plasma ATP levels can only be approximate , since the accumulation in the blood is continuously offset by ATPase activity of both plasma and the surfaces of the erythrocyte and endothelial cells . Fig. 4Elution of ATP and plasma samples from exercising human forearm through a Sephadex column . Elution fractions were estimated with firefly extract . a ATP , 10 − 6 g.ml − 1 . b Post-occlusion human sample from a subject during forearm exercise . c Samples from another subject exercising forearm muscle , fraction at 9 ml was incubated with apyrase and retested on firefly extract . Fraction at 10 ml , incubation control . Reproduced from Forrester -LSB- 22 -RSB- , with permission of the Physiological Society Sequelae The present-day knowledge and extensive classification of purine receptors -LRB- see review by -LSB- 48 -RSB- -RRB- has vindicated the concept of an extracellular system of ATP action influencing many tissues of the body . However , the mode of ATP release from cells remains a formidable challenge -LRB- see review by -LSB- 56 -RSB- -RRB- . In the cases of skeletal muscle , cardiac muscle , and erythrocyte some specific circumstances leading to ATP release , e.g. membrane depolarization , hypoxia , and mechanical distortion -LSB- 25 -RSB- , should be addressed further . Association of ATP release from depolarizing membrane In 1962 Abood et al. -LSB- 1 -RSB- assessed outflux of various phosphates during membrane depolarization of excitable tissues . Prior to that Holton -LSB- 32 -RSB- detected ATP release from sensory nerves stimulated antidromically . An elegant experiment by Israel et al. -LSB- 33 -RSB- showed discrete release of ATP closely related to membrane depolarization in the electric organ of Torpedo . In 1973 Ohta et al. -LSB- 40 -RSB- studied and compared the mode of action of several veratrum alkaloid compounds on axonal membranes -LRB- squid giant axon -RRB- . Some of them exerted a highly specific action on the resting sodium permeability , with veratridine having the most potent effect . Two results led them to conclude that the depolarization evoked by veratridine was caused by specifically increasing the membrane permeability to sodium ions : -LRB- 1 -RRB- application of tetrodotoxin -LRB- TTX -RRB- , which specifically blocks sodium channels , restored the resting membrane potential after depolarization by veratridine and -LRB- 2 -RRB- removal of sodium ions from both external and internal phases of the axon , thus eliminating the sodium term from the Goldman-Hodgkin-Katz constant field equation describing the membrane potential -LSB- 31 -RSB- , had the result that veratridine had no depolarizing action after sodium removal . In 1977 White -LSB- 63 -RSB- demonstrated ATP release from depolarizing isolated synaptosomes prepared from rat brain . The synaptosomes were suspended in a solution containing firefly extract . Depolarization was achieved by -LRB- 1 -RRB- elevation of extracellular potassium and -LRB- 2 -RRB- with application of veratridine . A light signal was evoked in both circumstances . Prior exposure of the synaptosomes to TTX , thus blocking the sodium channels , also blocked the light signal evoked by veratridine . This suggested that the release of ATP in response to veratridine in the absence of TTX was due to membrane depolarization following the opening of sodium channels . A record of one such experiment by Tom White is shown in a previous publication -LRB- -LSB- 25 -RSB- Fig. 16b -RRB- . Hypoxia The potency of hypoxia in evoking ATP release from heart cells and erythrocytes has been demonstrated -LSB- 4 , 24 -RSB- . In the case of human erythrocyte , ATP release in response to hypoxia was very effectively blocked by the nucleoside transport blocker nitrobenzylthioinosine -LRB- NBTI -RRB- . The nucleoside transporter has now been cloned -LSB- 26 -RSB- and is thought to be a member of a group of unclassified integral membrane proteins -LRB- see review by -LSB- 8 -RSB- -RRB- . The connection between blockade of a nucleoside transporter and inhibition of nucleotide release -LRB- ATP -RRB- in response to hypoxia is unclear.Some headway has been made toward unraveling oxygen-sensing mechanisms in certain oxygen-sensitive cells . In 1988 Lopez-Barneo et al. -LSB- 36 -RSB- , studying chemoreceptive carotid body cells , demonstrated that lowered oxygen tension inhibited potassium channels , resulting in cell depolarization . The presence of oxygen-sensing potassium channels has been reported in many different cell types , for example , in pulmonary vascular myocytes , which constrict due to membrane depolarization following hypoxia -LSB- 44 -RSB- and in rat pheochromocytoma cells -LSB- 12 -RSB- . Does ATP release associated with membrane depolarization , as reported above -LSB- 63 -RSB- , occur in these cells ? Perhaps release of ATP from heart cells and erythrocytes in response to hypoxia involves the presence of oxygen-sensitive potassium channels , the sequence of events being hypoxia , leading to inhibition of oxygen-sensitive channels , followed by membrane depolarization and ATP release . To date , these potassium channels have not been identified in heart cells or erythrocytes . Mechanical distortion A broad spectrum of mechanical distortion exists , ranging from gross distortion of whole cells to the impact of suction electrodes in the patch-clamp techniques for recording of currents passing through single ion channels in the membrane . There is little doubt that mechanical distortion of the whole cell membrane can lead to the release of ATP into the immediate environment . Sprague et al. -LSB- 59 -RSB- showed a close relationship between the extent of erythrocyte deformation and ATP release . Increases in shear stress applied to endothelial cells have been shown to liberate `` vasoactive '' substances from vascular endothelial cells -LSB- 10 , 45 , 47 , 54 -RSB- . ATP release into the pulmonary vascular bed occurs within seconds after an increase in flow rate -LSB- 30 -RSB- . Grygoczyk and Hanrahan -LSB- 27 -RSB- clearly showed that minimum mechanical disturbance , such as changing the solution bathing the preparation , enhanced ATP release within seconds . Schwiebert -LSB- 55 -RSB- emphasized that the degree of suction pressure in patch-clamp techniques may determine the rate of ATP appearance . A further factor complicating physical distortion of the cell membrane is the possible involvement of the underlying cytoskeletal system . Frequency of membrane channel opening is influenced by exposure to cytochalasin B , which disrupts the cytoskeleton -LSB- 62 -RSB- . A comprehensive review by Morris -LSB- 39 -RSB- outlining the widespread occurrence of mechanosensitive ion channels in a variety of tissues is highly recommended . Perspectives Towards the search for an ATP channel in the plasma membrane Braunstein et al. -LSB- 7 -RSB- , investigating the mechanism of cell volume regulation , showed that the cystic fibrosis transmembrane conductance regulator -LRB- CFTR -RRB- restored cell volume -LRB- following hypotonic challenge -RRB- by a mechanism involving ATP release . This release could be blocked by DIDS and gadolinium , suggesting that ATP might be released through a channel separate from the CFTR . Their working hypothesis was that cell volume is sensed and transduced by an ABC transporter -LRB- ATP binding cassette -RRB- enhancing ATP release through activation of a separate but associated ATP channel . The earlier observation -LSB- 4 -RSB- that hypoxic-induced ATP release from erythrocyte was associated with `` band 3 '' membrane prompted the speculation that band 3 could be equated with the CFTR protein moiety . However , these proteins have been cloned and sequenced and are quite dissimilar in character -LSB- 34 , 52 -RSB- . ATP passage across the inner mitochondrial membrane has long been known . Thinnes et al. -LSB- 60 -RSB- have identified a voltage -- dependent anion channel -LRB- VDAC , `` porin '' -RRB- which acts as a conduit for newly synthesized ATP in and out of the mitochondrion . VDAC is present in a multitude of tissue types , including human skeletal muscle -LSB- 51 -RSB- -LRB- Table 1 -RRB- . Exercise and circulating purines An early example of the appearance of purines in the circulating blood in response to graduated whole body exercise was obtained by Peter Parkinson -LSB- 43 -RSB- . It is known that a large proportion of circulating ATP is degraded in passage through the lung -LSB- 15 -RSB- , perhaps , along with widespread ecto-ATPase activity , minimizing the effect of widespread vasodilatation and offsetting unwanted hypotension and collapse during exercise ! The products of purine degradation are probably distributed generally , especially to tissues that can not synthesize the purine ring moiety -LRB- see `` Discussion , '' -LSB- 11 -RSB- -RRB- . This phenomenon could appropriately be included in the concept of `` ATP expansion '' proposed by Abraham et al. -LSB- 2 -RSB- . This begs the question : is one of the beneficial effects of an exercise regime due to the intermittent `` internal transfusion '' of purines , including ATP , from exercising muscle to sustain `` ATP expansion '' in the tissues ? Use of ATP in cancer therapy Encouraging results have been obtained in the field of cancer therapy using extracellular ATP . A direct effect of ATP on tumor cells was demonstrated by Rapaport and coworkers -LSB- 49 , 50 -RSB- . It was shown in various human cell lines that an increase in cellular ATP pools resulted in inhibition of DNA replication in the synthesis phase of the cell cycle , with subsequent arrest of cell growth . Inhibition of cancer growth in Ehrlich tumor cells by extracellular ATP has also been found -LSB- 14 , 35 -RSB- . The dramatic weight loss seen in some forms of advanced cancer -LRB- cachexia -RRB- can be slowed with a regime of intravenous ATP -LSB- 3 -RSB- . As shown in Fig. 5 maintenance of muscle strength was also a benefit . Fig. 5Changes in body weight and muscle strength in advanced non-small-cell lung cancer after ATP administration . a Weight change . b Elbow flexor performance -LRB- top -RRB- ; knee extensor performance -LRB- bottom -RRB- . Reproduced from Agteresch et al. -LSB- 3 -RSB- , with permission Protective effect of ATP against ischemia Improved recovery of ischemic liver , kidney , and myocardial tissues after perfusion of ATP-MgCl has been shown -LSB- 9 , 37 , 41 , 58 -RSB- . Protective effect of ATP in radiation therapy Impairment of wound healing following radiation therapy has been shown to be ameliorated with use of ATP-MgCl perfusion regimes -LSB- 57 -RSB- . Although little understood , these protective effects may be associated with general elevation of tissue ATP levels , restoring the total `` energy pool '' of the body -LRB- see `` ATP expansion '' in review by -LSB- 2 -RSB- -RRB- . Aging and extracellular ATP levels The release of ATP from vascular endothelial cells in rat caudal artery is reduced with advancing age . Both spontaneous ATP release and release induced by methoxamine , an α-adrenoceptor agonist , were reduced in aged vascular endothelium -LSB- 28 -RSB- . It is interesting that enhanced release of adenyl purines from these cells was invoked by hypotension induced by exercise -LSB- 29 -RSB- . A clear example of the effect of age on the ATP content of human erythrocytes is shown in Table 1 -LSB- 46 -RSB- . Aging has also been shown to diminish phosphorus metabolites in left ventricular hypertrophy as measured by 31P magnetic resonance spectroscopy -LSB- 42 -RSB- . If aging is associated with lower tissue levels of ATP , and applied exogenous ATP can replenish these levels -LRB- `` ATP expansion '' see above -RRB- , could aging reversal be brought about by increasing the exogenous ATP levels ? At this juncture it should be noted that extracellular ATP can act as a powerful trigger of programmed cell death -LRB- apoptosis -RRB- in endothelial cells -LSB- 13 , 53 -RSB- . Perhaps the phenomenon of apoptosis is designed to prevent the Malthusian nightmare of universal immortality ! Epilogos And so what began as a project for the assessment of quantal release of acetylcholine from motor nerve terminals1 resulted in the finding that ATP was released from active skeletal muscle , leading to the establishment of a significant role for ATP in the hyperemia of exercising skeletal and heart muscle , as well as an appreciation of the widespread effects of extracellular ATP . Intrinsic to the concept of serendipity in science is the element of surprise , which , although conferring excitement to investigators , only reveals our frailty of prediction in scientific matters . Inspirata accident magis saepe quam quae speres -- What you do not expect happens more frequently than what you do expect -LRB- Plautus -RRB- .

O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B I I I O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

atp release from cells
extracellular atp
hypoxia
serendipity


Arabidopsis R2R3-MYB transcription factor AtMYB60 functions as a transcriptional repressor of anthocyanin biosynthesis in lettuce -LRB- Lactuca sativa -RRB- The MYB transcription factors play important roles in the regulation of many secondary metabolites at the transcriptional level . We evaluated the possible roles of the Arabidopsis R2R3-MYB transcription factors in flavonoid biosynthesis because they are induced by UV-B irradiation but their associated phenotypes are largely unexplored . We isolated their genes by RACE-PCR , and performed transgenic approach and metabolite analyses in lettuce -LRB- Lactuca sativa -RRB- . We found that one member of this protein family , AtMYB60 , inhibits anthocyanin biosynthesis in the lettuce plant . Wild-type lettuce normally accumulates anthocyanin , predominantly cyanidin and traces of delphinidin , and develops a red pigmentation . However , the production and accumulation of anthocyanin pigments in AtMYB60-overexpressing lettuce was inhibited . Using RT-PCR analysis , we also identified the complete absence or reduction of dihydroflavonol 4-reductase -LRB- DFR -RRB- transcripts in AtMYB60 - overexpressing lettuce -LRB- AtMYB60-117 and AtMYB60-112 lines -RRB- . The correlation between the overexpression of AtMYB60 and the inhibition of anthocyanin accumulation suggests that the transcription factorAtMYB60 controls anthocyanin biosynthesis in the lettuce leaf . Clarification of the roles of the AtMYB60 transcription factor will facilitate further studies and provide genetic tools to better understand the regulation in plants of the genes controlled by the MYB-type transcription factors . Furthermore , the characterization of AtMYB60 has implications for the development of new varieties of lettuce and other commercially important plants with metabolic engineering approaches . Introduction Flavonoid metabolism is unique to plants and produces a great number and variety of compounds including flavones , flavonols , anthocyanins , and proanthocyanins -LRB- Winkel-Shirley 2001 ; Park et al. 2004 -RRB- . These molecules play important roles in many fundamental processes in plants , such as the pigmentation of flowers and fruits , UV resistance , pollen fertility , responses to wounding and fungal attacks , and environmental stress responses -LRB- Shirley et al. 1995 ; Weisshaar and Jenkins 1998 ; Harborne and Williams 2000 ; Dixon and Piava 1995 ; Moyano et al. 1996 ; Pietta 2000 ; Cominelli et al. 2005 -RRB- . The regulation of the anthocyanin pigments in plants is particularly important in metabolic engineering applications because these compounds act as visual signals that attract the insects and animals required for pollination and seed dispersal -LRB- Holton and Cornish 1995 -RRB- . The metabolic pathways involved in anthocyanin biosynthesis are well established , and the central pathways in this process are highly conserved in plants -LRB- Shih et al. 2006 -RRB- . Moreover , studies in model plants , such as Arabidopsis and maize , have facilitated a sound understanding of the genes involved and the assembly of the relevant enzyme complexes -LRB- Winkel-Shirley 2001 -RRB- . Two groups of genes are required for anthocyanin biosynthesis : structural genes that encode the enzymes that directly participate in the biosynthetic reactions , and genes for transcription factors that regulate the expression of these structural genes and the accumulation of flavonoid metabolites . Transcription factors can act as activators or repressors of gene expression , and mediate either increases or decreases in transcription through sequence-specific DNA binding and protein-protein interactions -LRB- Broun 2004 -RRB- . MYB proteins have been identified in a large number of eukaryotes , including fungi , plants and vertebrates -LRB- Ohi et al. 1994 ; Martin and Paz-Ares 1997 ; Slamon et al. 1986 -RRB- , and have roles in a wide array of cellular processes . These include the regulation of secondary metabolism , signal transduction , cell division , and responses to plant diseases and various forms of stress -LRB- UV-B light , cold and drought -RRB- -LRB- Larkin et al. 1994 ; Borevitz et al. 2000 ; Stracke et al. 2001 ; Vailleau et al. 2002 ; Cominelli et al. 2005 -RRB- . The R2R3-MYB-related proteins activate the transcription of structural genes that function in different branches of phenylpropanoid metabolism -LRB- Martin and Paz-Ares 1997 -RRB- . The promoters of these structural genes each contain potential MYB protein recognition motifs and also bind with the basic helix-loop-helix -LRB- bHLH -RRB- domain and conserved WD40 repeat proteins -LRB- Park et al. 2007a , b ; Martin and Paz-Ares 1997 ; Winkel-Shirley 2001 -RRB- . The classification of MYB transcription factors is based on the strong conservation of imperfect repeats in the MYB DNA-binding domain of the member proteins -LRB- Martin and Paz-Ares 1997 -RRB- . In contrast to animals -LRB- R1R2R3 -RRB- , the MYB domain transcription factors found in plants are characterized by the R2R3-type MYB domain , comprising a DNA-binding domain and an activation/repression domain . Moreover , the balance between activators and repressors in this transcription factor family in plants may provide extra flexibility in terms of transcriptional control -LRB- Jin et al. 2000 -RRB- . There have been 126 R2R3 MYB genes identified in Arabidopsis thaliana , all of which exhibit strong homology within their N-terminal MYB DNA-binding domains and can be divided into 24 subgroups on the basis of their sequences -LRB- Stracke et al. 2001 ; Riechmann and Ratcliffe 2000 ; Kranz et al. 1998 -RRB- . To determine the biological roles of the MYB-related transcription factors in the production of phenylpropanoids , we isolated six R2R3-MYB proteins from Arabidopsis based on available information indicating that these genes are induced by environmental stress -LRB- UV-B irradiation -RRB- -LRB- Kranz et al. 1998 -RRB- . However , they do not have an informative phenotype , because of the structural and functional redundancy among these factors -LRB- Bouche and Bouchez 2006 -RRB- . Furthermore , although there have been other studies of anthocyanin accumulation in lettuce , one of the most popular and commercially important vegetables in the world , this process has not been characterized before at the level of transcriptional regulation . The regulation of anthocyanin accumulation in plants is also a key issue in our understanding of the regulation of leaf color . The identification of the factors that exert this control will provide valuable tools to moderate the extent and distribution of anthocyanin-derived pigmentation in plant tissues . In this study , we overexpressed the AtMYB60 gene in lettuce plants -LRB- `` Jinjachuckmyun '' cultivar -RRB- that are highly pigmented with a red color throughout their leaf tissues . Because the loss of these pigments is easily detected , the activity and repression of the MYB-type transcription factors that affect these pathways can be investigated . In the leaves of lettuce , coordinated transcriptional regulation controls virtually each step of the anthocyanin biosynthetic pathway -LRB- Park et al. 2007a -RRB- . We compared the expression of the major anthocyanin biosynthetic genes and the AtMYB60 gene by RT-PCR . We show that this transcription factor is functionally active in repressing anthocyanin accumulation and can thus generate green lettuce leaves . We also show , by functionally repressing the accumulation of this compound , that AtMYB60 plays a significant role in controlling anthocyanin biosynthesis via the inhibition of a key target gene encoding dihydroflavonol reductase -LRB- DFR -RRB- . Thus , we report here for the first time that anthocyanin biosynthesis and the transcription of the DFR gene are repressed through the overexpression of Arabidopsis R2R3-MYB transcription factors in transgenic lettuce plants . We also discuss the function of transcriptional regulators in the control of the expression of the structural protein that are required for anthocyanin biosynthesis in lettuce . Materials and methods Plant materials and growth condition To investigate the effects of exogenous AtMYB proteins on anthocyanin biosynthesis in lettuce leaves at the molecular level , Jinjachuckmyun plants , supplied by Syngenta -LRB- Seoul , Korea -RRB- , were used . The plants were grown at the National Institute of Agricultural Biotechnology in Suwon , South Korea . Fresh lettuce leaves were plucked from the plants , frozen immediately in liquid nitrogen and stored at − 80 °C until required . Isolation of AtMYB cDNAs To isolate the AtMYB genes , cDNAs were isolated from UV-B-irradiated Arabidopsis plants using the BD SMART RACE cDNA Amplification Kit -LRB- Clontech , USA -RRB- and then used as templates for PCR cloning . The following gene-specific primers were used for RACE-PCR : AtMYB4 -LRB- forward : 5 ′ - GTATGGGAAGGTCACCGTGCTGTGAGAAA-3 ′ ; reverse : 5 ′ - TATTATTTCATCTCCAAGCTTCGAAAGCC-3 ′ -RRB- , AtMYB29 -LRB- forward : 5 ′ - AAATGTCAAGAAAGCCATGTTGTGTGGGA-3 ′ ; reverse : 5 ′ - GATCATATGAAGTTCTTGTCGTCATAATC-3 ′ -RRB- , AtMYB30 -LRB- forward : 5 ′ - TAATGGTGAGGCCTCCTTGTTGTGACAAA-3 ′ ; reverse : 5 ′ - CTTCAGAAGAAATTAGTGTTTTCATCCAA-3 ′ -RRB- , AtMYB34 -LRB- forward : 5 ′ - AGATGGTGAGGACACCATGTTGCAAAGAA-3 ′ ; reverse : 5 ′ - CGTCAGACAAAGACTCCAACCATATTGTC-3 ′ -RRB- , AtMYB51 -LRB- forward : 5 ′ - GAATGGTGCGGACACCGTGTTGCAAAGCC-3 ′ ; reverse : 5 ′ - ACTCATCCAAAATAGTTATCAATTTCGTC-3 ′ -RRB- and AtMYB60 -LRB- forward : 5 ′ - AGATGGGTAGGCCTCCATGCTGTGACAAG-3 ′ ; reverse : 5 ′ - AATTAAAGCATATTAGAGAGCTCCATCAA-3 ′ -RRB- . The resulting PCR products were then cloned into the pGEM-T Easy vector -LRB- Promega , USA -RRB- and sequenced on both strands . Vector construction AtMYB coding regions were cloned into an expression vector containing the CaMV 35S promoter and the nopaline synthase 3 ′ terminator . The resulting constructs were then introduced into the binary vector 22103 -LRB- a derivative of pCAMBIA3301 ; Cambia , Australia -RRB- . The complete coding sequence of each AtMYB cDNA was amplified with a specific forward primer designed to introduce an MluI restriction site and a reverse primer designed to introduce an XbaI restriction site to allow subcloning , as follows : AtMYB4 -LRB- forward : 5 ′ - ATACGCGTATGGGAAGGTCACCGTGCTGTG-3 ′ ; reverse : 5 ′ - TATCT AGATTATTTCATCTCCAAGCTTCGA-3 ′ -RRB- , AtMYB29 -LRB- forward : 5 ′ - ATACGCGTATGTCAAGAAAGCCATGTTGTG-3 ′ ; reverse : 5 ′ - TATCTAGATCATATGAAGTTCTTGTCGTCA-3 ′ -RRB- , AtMYB30 -LRB- forward : 5 ′ - ATACGCGTATGGTGAGGCCTCCTTGTTGTG-3 ′ ; reverse : 5 ′ - TATCTAGATCAGAAGAAATTAGTGTTTTCA-3 ′ -RRB- , AtMYB34 -LRB- forward : 5 ′ - ATACGCGTATGGTGAGGACACCATGTTGCA-3 ′ ; reverse : 5 ′ - TATCTAGATCAGACAAAGACTCCAACCATA-3 ′ -RRB- , AtMYB51 -LRB- forward : 5 ′ - ATACGCGTATGGTGCGGACACCGTGTTGCA-3 ′ ; reverse : 5 ′ - TATCTAGATCATCCAAAATAGTTATCAATT-3 ′ -RRB- , AtMYB60 -LRB- forward : 5 ′ - ATACGCGTATGGGTAGGCCTCCATGCTGTG-3 ′ ; reverse : 5 ′ - TATCTAGATTAAAGCATATTAGAGAGCTCC-3 ′ -RRB- . After PCR and digestion , the MluI/XbaI fragments were cloned into the binary vector 22103 between CaMV 35S promoter and the nopaline synthase poly -LRB- A -RRB- addition site , creating the binary vector 22103-AtMYB plasmids -LRB- Fig. 2 -RRB- . These constructs were then introduced into the Agrobacteriumtumefaciens EHA105 strain using the freeze -- thaw method . Generation of AtMYB transgenic lettuce Transgenic lettuce plants were generated using the Agrobacterium-mediated transformation of cotyledons . Lettuce seeds of the Jinjachuckmyun cultivar were supplied by Syngenta -LRB- Korea -RRB- and were surface sterilized in 25 % -LRB- v/v -RRB- bleach for 7 min , washed -LRB- three changes -RRB- in sterile water , and placed on phytogel-solidified -LRB- 0.2 % , w/v -RRB- MS medium at pH 5.8 . The seeds were germinated and maintained at 25 °C -LRB- 16 h photoperiod , 200 μmol m − 2 s − 1 , daylight fluorescent tubes -RRB- . The cotyledons were excised from the 4-day-old lettuce seedling and inoculated with freshly grown A. tumefaciens . The inoculated cotyledons were then co-cultivated on MS salt medium solidified with 0.2 % purified phytogel -LRB- Sigma , USA -RRB- , for 2 days at 24 °C in the dark . The cotyledon explants were then transferred to shoot initiation medium supplemented with 400 mg l − 1 carbenicillin -LRB- Duchefa , the Netherlands -RRB- . After a washing step , these explants were cultured -LRB- ten per plate -RRB- on selection medium -LRB- MS salt 4.43 g l − 1 , sucrose 30 g l − 1 , phytogel 2 g l − 1 , kinetin 0.5 mg l − 1 , carbenicillin 400 mg l − 1 , phosphinothricin 1.0 mg l − 1 and cysteine 10 mg l − 1 -RRB- and subcultured every 2 weeks on the same medium . Shoots that regenerated from explants on medium containing phosphinothricin were rooted in rooting medium -LRB- MS salt 4.43 g l − 1 , sucrose 30 g l − 1 , phytogel 2 g l − 1 and phosphinothricin 1.0 mg l − 1 -RRB- , before transfer to the greenhouse , where they were allowed to self-pollinate and to set seed . The seeds were harvested and stored at 4 °C . Anthocyanin extraction and HPLC analysis Anthocyanin in the lettuce plants was quantified by HPLC at a detection wavelength of 510 nm , comparing the sample retention times and peaks with those of known standards . The relative quantification of each phenolic compound samples was expressed as the equivalent quantity of purified standards for each treatment . About 0.2 g of ground and freeze-dried leaves was accurately weighed and extracted with 1 ml of a solution of methanol : water -LRB- 80:20 , v/v -RRB- at 80 °C for 1 h , then sonicated for 20 min . The extracted solution was filtered into a 50-ml volumetric flask , and the flask and filter were rinsed with a solution of methanol : water -LRB- 80:20 , v/v -RRB- . The filtrate was then made up to the required volume with the same solvent . Approximately 1 mL of the sample solution was passed through a 0.4 μm filter before analysis by HPLC . A Shimadzu HPLC system with 10AD dual pumps was used , with an RP18 -LRB- LiChrospher , 250 mm × 4 mm × 5 μm -RRB- column . The HPLC parameters were as follows : column temperature 30 °C , solvent A = 0.1 % trifluoroacetic acid in water , solvent B = 98 % acetonitrile with 0.1 % trifluoroacetic acid , solvent gradient , 0 min = 0 % B , 3 min = 6 % B 12 min = 18 % B , 25 min = 25 % B , 35 min = 100 % B , 40 min = 100 % B . The flow rate was 0.35 mL min − 1 . The peaks were classified as either cyanidin or delphinidin derivatives by UV spectral analysis . RT-PCR analysis Total RNA from the transgenic and wild-type lettuce leaves was extracted using RNeasy Plant Mini Kit -LRB- Qiagen , USA -RRB- , according to the manufacturer 's instructions . To estimate the transcript levels of the exogenous AtMYB4 and AtMYB60 genes in lettuce , we used the primers 5 ′ - ATGGGAAGATCGCCTTGTTGTGAA-3 ′ -LRB- F -RRB- and 5 ′ - TCATTTCATCTCTAAGCTTCTGTAGTCCAAAA-3 ′ -LRB- R -RRB- for AtMYB4 and 5 ′ - ATGGGGAGGCCTCCTTGTTGTG-3 ′ -LRB- F -RRB- and 5 ′ - TCAATTATCGAAAAAATTAGGGTTTTCATCA-3 ′ -LRB- R -RRB- for AtMYB60 . The mRNA expression patterns of the CHS , F3H , DFR and UFGT genes were analyzed by RT-PCR with the All-in-one RT/PCR Premix -LRB- SuperBio P7003 , Suwon , Korea -RRB- . The amplification of rRNA was used as the internal control . Total RNA -LRB- 100 ng -RRB- in a volume of 20 μL containing 20 mM Tris -- HCl -LRB- pH 7.9 -RRB- , 100 mM KCl , 0.1 mM EDTA , 1 mM DTT , M-MLV reverse transcriptase -LRB- RNaseH Minus -RRB- , Super Taq Plus DNA polymerase and 10 pmol of each gene-specific amplification primer was used for RT-PCR , according to the manufacturer 's instructions . Gene-specific primers for CHS -LRB- forward : 5 ′ - GGTTTGCTCTGAGATTACAGCGGTTACC-3 ′ , reverse : 5 ′ - TCCTTGAGACCAAGCTTGAGCTCCACCT-3 ′ -RRB- , F3H -LRB- forward : 5 ′ - GAGATCTTATCAGAGGCAATGGGCCTTG-3 ′ , reverse : 5 ′ - ACAACGGCCCGATGGTCTGCGTTCTTAA-3 ′ -RRB- , DFR -LRB- forward : 5 ′ - GGTCTATGACGAGTCTCATTGGAGCGAT-3 ′ , reverse : 5 ′ - CTATCAATTGCTCCTTTGAACATCTCCT-3 ′ -RRB- , UFGT -LRB- forward : 5 ′ - TGGAGAAGCGGGTTAGACAGTTGATGGA-3 ′ , reverse : 5 ′ - TATAGCTACCATGATTCAACCAACTTCG-3 ′ -RRB- , rRNA -LRB- forward : 5 ′ - TACGGCACTGAAGGTGCCAAGCTCGTG-3 ′ , reverse : 5 ′ - CATCCTCTTGGCAGTCTTGGCGTAGGG-3 ′ -RRB- and bar -LRB- forward 5 ′ - GCCGCAGGAACCGCAGGAGT-3 ′ , reverse : 5 ′ - AGCCCGATGACAGCGACCAC-3 ′ -RRB- were used to confirm the expression levels of these genes in the lettuce plants . Reverse transcription of the total RNA was carried out at 50 °C for 30 min , and the reaction was inactivated at 96 °C for 3 min . The amplification conditions comprised 35 cycles of denaturation at 94 °C for 30 s , annealing at 60 °C for 30 s , extension at 72 °C for 2 min , and a final extension at 72 °C for 10 min . PCR was performed using a GeneAmp PCR system 9700 Cycler -LRB- Perkin-Elmer , MA , USA -RRB- . The resultant RT-PCR products were resolved on a 1 % agarose gel , stained with ethidium bromide , and photographed . The amplified products of the CHS , F3H , DFR , UFGT , rRNA and bar transcripts were 398 , 319 , 534 , 153 , 386 and 267 bp , respectively . Results Cloning of MYB-type transcription factor genes from Arabidopsis To isolate and characterize the MYB-type transcription factors in Arabidopsis -LRB- AtMYB -RRB- that play a role in phenylpropanoid metabolism , primers were designed to amplify the entire coding regions of the AtMYB genes that are induced by environmental stress -LRB- UV-B irradiation -RRB- -LRB- Meissner et al. 1999 -RRB- . Previous studies of Arabidopsis have suggested that the R2R3-MYB genes of this plant have important functions in the regulation of secondary metabolism , disease resistance , and hormonal responses -LRB- Kranz et al. 1998 -RRB- . We isolated six Arabidopsis R2R3-MYB transcription factor genes based on available information concerning the AtMYB genes induced by UV-B irradiation -LRB- Kranz et al. 1998 -RRB- . These were AtMYB4 -LRB- AF062860 , AT4G38620 -RRB- , AtMYB29 -LRB- AF062872 , At5G07690 -RRB- , AtMYB30 -LRB- AF062873 , AT3G28910 -RRB- , AtMYB34 -LRB- U66462 , AT5G60890 -RRB- , AtMYB51 -LRB- AF062887 , AT1G18570 -RRB- and AtMYB60 -LRB- AF062895 , AT1G08810 -RRB- , with apparent full-length cDNAs of 849 , 1011 , 972 , 885 , 1059 and 843 bp , respectively , encoding proteins of 283 , 337 , 324 , 295 , 353 and 281 amino acids , respectively -LRB- Fig. 1 -RRB- . The amino termini of these proteins contain the R2R3 repeats responsible for their binding to target DNA sequences and which are highly conserved among the R2R3-MYB proteins . Fig. 1Multiple alignment of AtMYB proteins . Amino acids identical in all six proteins are marked in black , amino acids found in four proteins are marked in gray . The line above indicates the R2 and R3 MYB repeats . GenBank accession numbers for the sequences : AtMYB4 -LRB- AF062860 -RRB- , AtMYB29 -LRB- AF062872 -RRB- , AtMYB30 -LRB- AF062873 -RRB- , AtMYB34 -LRB- U66462 -RRB- , AtMYB51 -LRB- AF062887 -RRB- and AtMYB60 -LRB- AF062895 -RRB- Expression of AtMYB genes in lettuce plants To examine the effects of the six isolated AtMYB genes on leaf color changes and on the anthocyanin composition of lettuce , cotyledons of the cultivar `` Jinjachuckmyun '' were transformed using the Agrobacterium tumefaciens strain EHA105 with the binary vector 22103-AtMYB -LRB- a derivative of pCAMBIA3301 ; Fig. 2 -RRB- , carrying these genes and a barstar selection marker . Shoots were regenerated from the callus of phosphinothricin-resistant transformatants . To confirm the introduction and expression of the exogenous AtMYB genes in the Jinjachuckmyun cultivars , total RNA was extracted from the leaves of six-week-old wild-type and transformed lettuce plants -LRB- T1 generation -RRB- and analyzed by RT-PCR . Fig. 2Schematic diagram of the T-DNA region of the 22103-AtMYB binary vector used in the transformation of lettuce cultivars . LB left border , RB right border , P35S Cauliflower mosaic virus 35S RNA promoter , PolyA Cauliflower mosaic virus 3 ′ UTR terminator , Tnos nopaline synthase terminator Transgenic plants were confirmed by their resistance to a barstar spray and by subsequent RT-PCR with specific primers for six AtMYB genes . Because the T1 plants of the AtMYB4 , AtMYB29 , AtMYB30 , AtMYB34 and AtMYB51 transformants were similar in leaf color and in the anthocyanin accumulation in their leaves , the AtMYB4 transformant -LRB- AtMYB4-101 -RRB- and the AtMYB60 transformant -LRB- AtMYB60-117 and AtMYB60-112 -RRB- were selected for further RT-PCR analysis to detect the expression of the structural genes that are involved in anthocyanin biosynthesis -LRB- Fig. 3 -RRB- . RT-PCR analysis was performed using six-week-old lettuce leaves of wild-type and transgenic lettuce plants . The wild-type lettuce plants had no integrated bar gene whereas each of the transgenic plants expressed bar mRNA . Analysis of the exogenously expressed AtMYB4 and AtMYB60 genes showed that AtMYB4 was specifically expressed in AtMYB4-101 and AtMYB60 in AtMYB60-117 and AtMYB60-112 . Ribosomal RNA levels were used as a normalization control . Fig. 3RT-PCR analysis confirming the transcription of the AtMYB4 , AtMYB60 and Bar genes in the indicated transgenic lettuce lines and not in the wild-type -LRB- WT -RRB- plants . rRNA was used as the control Plant morphology in AtMYB-transformed lettuce In our previous study , we demonstrated that the wild-type lettuce leaf accumulates anthocyanin , with cyanidin and delphinidin representing the major and minor components , respectively -LRB- Park et al. 2007a -RRB- . To further characterize the functions of the AtMYB genes in anthocyanin biosynthesis , we compared the morphologies of transgenic lettuce plants overexpressing these genes with that of wild-type lettuce . More than 20 independent transgenic plants , selected with barstar spray and subsequently confirmed by PCR analysis -LRB- data not shown -RRB- , were obtained with each A. tumefaciens strain . There was a large variation in the leaf color phenotypes of these transgenic lines . The AtMYB4-101 line showed normal anthocyanin accumulation and a red coloration phenotype , similar to that of the wild-type , but the AtMYB60-117 line showed different levels and patterns of anthocyanin accumulation . Under standard growth conditions , transgenic lettuce plants of the T1 generation that overexpressed AtMYB60 under the control of the cauliflower mosaic virus -LRB- CaMV -RRB- 35S promoter showed an inhibition -LRB- AtMYB60-117 -RRB- of anthocyanin accumulation -LRB- green leaves -RRB- , compared with that of wild-type plants -LRB- red leaves -RRB- -LRB- Fig. 4a , b -RRB- . Apart from this inhibition of anthocyanin biosynthesis , no other morphological or developmental abnormalities were detected in this line under standard growth conditions . These results confirm that the exogenous AtMYB60 protein was responsible for the repressed anthocyanin phenotype . Fig. 4Morphological analysis showing the inhibition of anthocyanin biosynthesis in lettuce leaves , indicated by the lack of red pigmentation , following the introduction of the Arabidopsis MYB gene , AtMYB60 : a young -LRB- 20 days after sowing -RRB- wild-type -LRB- WT -RRB- lettuce and the AtMYB60 transgenic lettuce line , AtMYB60-117 ; b morphologies of both the WT and AtMYB-overexpressing lettuce plants at a later stage of growth . Both the AtMYB4-101 and AtMYB60-117 transgenic plants are shown and reveal that AtMYB4 does not inhibit anthocyanin production High-performance liquid chromatography -LRB- HPLC -RRB- analysis of AtMYB60 transgenic lettuce shows that accumulation of anthocyanin compounds is inhibited To confirm the identity of the anthocyanins that are synthesized in lettuce after transformation with AtMYB genes , lettuce leaf tissues were harvested and analyzed by HPLC for the presence of soluble anthocyanins . The results confirmed the predominance of cyanidin in the wild-type lettuce leaves , with traces of delphinidin also evident , as we previously described -LRB- Park et al. 2007a -RRB- . The cyanidin and delphinidin peaks were detected in the AtMYB4-overexpressing lettuce -LRB- AtMYB4-101 -RRB- and wild-type lettuce . The AtMYB29 - , AtMYB30 - , AtMYB34 - and AtMYB51-overexpressing lettuce plants that showed a similar red-colored phenotype also produced corresponding cyanidin and delphinidin peaks on HPLC analysis -LRB- data not shown -RRB- . However , clear differences were evident in the patterns of anthocyanin accumulation in the AtMYB60-overexpressing lettuce plants -LRB- Fig. 5 -RRB- . With HPLC , we found that the cyanidin -LRB- red pigment -RRB- peak was absent or sharply reduced in AtMYB60-117 and AtMYB60-112 . The delphinidin -LRB- blue pigment -RRB- peak was absent in every AtMYB60-overpxpressing lines . Hence , the anthocyanin derivatives detected in lettuce leaves by HPLC can be correlated with the morphologies of the plants and with the inhibition of anthocyanin biosynthesis by the AtMYB60 protein . Fig. 5HPLC analysis of methanolic extracts from both wild-type control -LRB- WT -RRB- and AtMYB transgenic lettuce leaves -LRB- AtMYB4-101 , AtMYB60-117 and AtMYB60-112 -RRB- . HPLC chromatograms were recorded at 510 nm . Peak C corresponds to cyanidin and D represents delphinidin . Each chromatogram was generated using the same quantity of injected sample RNA expression profiles of anthocyanin biosynthetic enzymes suggest their coordinated regulation in AtMYB60 transgenic lettuce plants Based on the correlation between the plant color morphology and our HPLC analysis of the anthocyanins , we undertook to verify the effects of the AtMYB proteins on the expression of the structural genes encoding the enzymes responsible for anthocyanin biosynthesis . As shown in Figs. 4 and 6 , a dramatic leaf color change was evident in the AtMYB60-117 -LRB- green leaf -RRB- and -112 -LRB- mosaic phenotype -RRB- lines , in which anthocyanin accumulation is inhibited , and these plants showed a strong green leaf color phenotype . The enzymes that act in the anthocyanin biosynthetic pathway have been well characterized in lettuce in our previous study -LRB- Park et al. 2007a -RRB- , in which we also analyzed the expression of the structural genes encoding the relevant enzymes . Following environmental stimuli such as UV-B irradiation , the expression of chalcone synthase -LRB- CHS -RRB- , flavanone 3-hydroxylase -LRB- F3H -RRB- and dihydroflavonol 4-reductase -LRB- DFR -RRB- is positively induced , and the accumulation of anthocyanin is increased in lettuce . We also performed RT-PCR analysis using gene-specific primers in that study to analyze the expression of these structural genes . They were identified from young UV-B-irradiated lettuce leaves and designated as CHS -LRB- CV700441 -RRB- , F3H -LRB- CV700152 -RRB- , DFR -LRB- CV700105 -RRB- and UFGT -LRB- CV700246 -RRB- -LRB- Park et al. 2007a -RRB- . The transcript levels of these four anthocyanin biosynthetic genes were compared between the wild-type , AtMYB4 - and AtMYB60-overexpressing lettuce plants in the present study . Fig. 6RT-PCR analysis of the genes involved in anthocyanin biosynthesis in the leaves of wild-type and AtMYB4 - and AtMYB60-overexpressing lettuce plants . The transcripts of four biosynthetic genes involved in general anthocyanin metabolism -LRB- CHS , F3H , DFR and UFGT -RRB- were analyzed by RT-PCR in the AtMYB4-101 and AtMYB60-117 transgenic lettuce lines and were compared with the wild-type expression patterns . rRNA was used as the quantitative control Total RNA extracts were prepared from the leaves of six-week-old lettuce plants of wild-type , AtMYB4-101 and AtMYB60-117 lines and were analyzed by RT-PCR for the expression of the previously determined structural genes that are involved in the major biochemical pathways of anthocyanin biosynthesis -LRB- Fig. 6b -RRB- . No reduction in the expression of any of these genes was observed in wild-type or AtMYB4-101 lines , both of which exhibited red coloration in their leaves . Moreover , the mRNA levels of the CHS , F3H and UFGT genes , which catalyze the synthesis of chalcones , flavanones and anthocyanin , respectively , were unchanged in any of the plants . In contrast , the mRNA levels of DFR , the production of which catalyzes the conversion of dihydroquercetin and dihydrokaempferol to leucocyanidin and leucopelargonidin -LRB- of the leucoanthocyanidins -RRB- , respectively , was inhibited in the AtMYB60-117 line , which exhibits green-colored leaves with no red pigmentation . Discussion To isolate the MYB-type transcription factor genes involved in phenylpropanoid metabolism , the transgenic expression of six AtMYB genes was analyzed in lettuce . To date , a large number of transcription factors containing DNA-binding domains that are similar to those of the MYB proteins have been identified across the eukaryotic kingdom -LRB- Kranz et al. 1998 -RRB- . Several MYB transcription factors are known to regulate phenylpropanoid metabolism , and the quantities of the various end-products of the associated pathways that accumulate in specific cells , tissues and organs are thought to respresent the balanced expression of several interacting MYB proteins -LRB- Tamagnone et al. 1998 ; Espley et al. 2007 -RRB- . Recently , biochemical studies have also suggested that the MYB-type transcription factors are involved in regulating the branches of the phenylpropanoid metabolic networks in higher plants . Multiple MYBs are also thought to regulate the expression of the structural proteins that drive several downstream pathways of flavonoid metabolism -LRB- Moyano et al. 1996 -RRB- . A previous study has also shown that MYB proteins can regulate flavonoid biosynthesis via the transcriptional regulation of the gene encoding CHS , an enzyme that catalyzes the first committed step in flavonoid biosynthesis -LRB- Feldbrugge et al. 1997 -RRB- . The lettuce cultivar `` Jinjachuckmyun '' , which we used in our current transformation experiments , shows anthocyanin pigment accumulation in its leaves during development . We have shown in a recent study that environmental stresses upregulate the expression of the genes that encode anthocyanin biosynthetic enzymes in this same lettuce variety , as demonstrated by their responsiveness to UV-B irradiation -LRB- Park et al. 2007a -RRB- . The lettuce has merit as a model plant system to further characterize the functions of those gene products in anthocyanin metabolism , because of its leaf color is red under field conditions and green under chamber condition when exposed to UV-B irradiation . The findings described in this study further validate the use of lettuce as a model plant because it offers experimental versatility in terms of its genetic regulation of the inhibition of leaf pigmentation . The function of the MYB proteins as activators -LRB- green to red in a growth chamber -RRB- or repressors -LRB- red to green in the field -RRB- of anthocyanin biosynthesis can be characterized directly from a phenotypic analysis of the lettuce leaf color . This system has potential applications as a research tool in a number of areas of plant molecular biology . We show in our present experiments that the anthocyanin accumulation in leaves is strongly inhibited in AtMYB60 transgenic plants compared with that in wild-type plants -LRB- Fig. 4a , b -RRB- . These results suggest that AtMYB60 functions as a repressor of anthocyanin biosynthesis . Based upon our current findings of the inhibition of anthocyanin biosynthesis and our metabolite analysis of AtMYB60-overexpressing lettuce plants compared with the wild-type , we speculate that the synthesis of anthocyanin is principally controlled by the AtMYB60 transcription factor . The production of anthocyanin is inhibited in the AtMYB60-117 and AtMYB60-112 lines , and this is likely to underlie the green leaf phenotype of these plants . Hence , this characterization of the function of AtMYB60 represents a crucial step in furthering our understanding of the molecular regulation of anthocyanin biosynthesis and the transcription of related genes in lettuce and other plants . It is noteworthy in this context that MYB-related proteins generally act as transcriptional activators -LRB- Foos et al. 1994 -RRB- . The R2R3-MYB-related proteins have so far been shown to activate the transcription of structural proteins that act in different branches of the phenylpropanoid metabolism machinery -LRB- Martin and Paz-Ares 1997 -RRB- or that play roles in the plant response to stress signals -LRB- Cominelli et al. 2005 -RRB- . Anthocyanin expression in maize is dependent on the presence of both the MYB and MYC types of regulatory factors . It is of some interest , therefore , that in this study , the expression of AtMYB60 alone , under the control of the CaMV 35S promoter , was sufficient to repress anthocyanin accumulation in a red lettuce variety . We also investigated the effects of AtMYB60 on the expression of the different structural proteins involved in anthocyanin production , which we have identified previously in lettuce . Our results reveal that this MYB protein represses the expression of the DFR gene in the AtMYB-117 and -112 lines . Significantly , DFR is a crucial structural protein that facilitates anthocyanin pigmentation in lettuce and that no such reduction in the expression of this gene was found in the wild-type or AtMYB4-101 lines that also develop red-colored leaves . The DFR protein represents an important branching point within the anthocyanin biosynthetic pathway and catalyzes the commitment step in this process , which is the formation of leucoanthocyanidins from the substrate dihydroflavonol . This indicates that AtMYB60 specifically inhibits the flux of flavonoid intermediates toward the production of leucoanthocyanins by repressing the transcription of DFR , with a resulting green leaf phenotype -LRB- Fig. 7 -RRB- . Fig. 7Schematic representation of the relationship between AtMYB60 expression and the structural proteins that play roles in anthocyanin biosynthesis It is interesting to note that the transcriptional response to UV-B irradiation that operates through AtMYB60 focuses on the regulation of DFR expression and anthocyanin accumulation . Recently there has been some debate about the identity of the phenylpropanoids involved in the response to UV-B , which may provide the most effective components of sunscreen formulations . MYB transcription factors that are known to regulate the transcription of genes in the phenylpropanoid biosynthetic pathway have been studied previously -LRB- Jin and Martin 1999 -RRB- . The overexpression of PAP1 in Arabidopsis results in the up-regulation of the genes encoding phenylalanine ammonia-lyase -LRB- PAL -RRB- , CHS and DFR -LRB- Borevitz et al. 2000 ; Tohge et al. 2005 -RRB- . Moreover , mutations in the CHS , DFR and LDOX genes reduce the pigmentation of plants -LRB- Shirley et al. 1995 ; Abrahams et al. 2003 -RRB- , suggesting that these proteins are required for the synthesis of both anthocyanidin and proanthocyanidin . Because MYB-binding sites are present in the promoter regions of these flavonoid biosynthetic genes -LRB- Nesi et al. 2001 ; Debeaujon et al. 2003 -RRB- , it is possible that the AtMYB60 transcription factor might also specifically and directly target them . It is also possible that additional target genes that are related to phenylpropanoid metabolism can be negatively regulated by AtMYB60 when it is overexpressed in lettuce . The effects of AtMYB60 suggest that this MYB protein acts as a direct transcriptional regulator by binding to the MYB motifs common to the promoters of many structural genes in the same metabolic pathway , thus resulting in the downregulation of this pathway . The precise determination of this regulatory system at the molecular level remains to be achieved . Recently , Cominelli et al. -LRB- 2005 -RRB- reported that AtMYB60 is involved in the plant response to stress and its expression is negatively modulated during conditions of drought -LRB- Cominelli et al. 2005 -RRB- . A null mutation in AtMYB60 results in a constitutive reduction in stomatal openings and in decreased wilting under water-stress conditions . Therefore , we hypothesize that AtMYB60 has multiple functions in the regulation of anthocyanin biosynthesis and in the plant responses to different environmental conditions , including drought and exposure to UV-B light . AtMYB60 is the first MYB protein identified that functions as a transcriptional repressor of the lettuce DFR gene in anthocyanin biosynthesis , and thus extends the known roles of the R2R3-MYB proteins in secondary metabolism . Evidence from our transgenic lettuce experiments suggests that the direct repression exerted by AtMYB60 protein produces phenotypic effects in transgenic plants . The engineering of the anthocyanin biosynthetic pathway is potentially of great commercial significance . Because the AtMYB60 transcription factor should act effectively in most plant species , it can potentially be used to control anthocyanin biosynthesis in agriculturally and industrially important plants . This method of modifying the anthocyanin content in plant tissues also opens up new avenues to engineering improvements in commercial crops by altering metabolic pathways via the regulation of different structural genes in these pathways .

B O O O O O O O O B O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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