P H Y T O C H R O M E D I S C U S S I O N A . P . H U G H E S
Horticultural Research Laboratories, Shinfield, University of Reading, U.K.
The discussion was opened by W . L . B U T L E R ( U . S . D . A . ) who asked the question: ' W h y does the measurement of the amount of phyto- chrome in plants by spectrophotometric methods not correlate with the physiological response ? ' In dark grown seedlings phytochrome is present wholly in the PR form. It was supposed that when red light was given the phytochrome was entirely converted to the PF R form, and that in the following dark period some 20-30 per cent reverted to PR, while the remainder was lost (BUTLER, LANE and SIEGELMAN, 1963).
However, recent experiments have shown that the photostationary state in red light is about 80 per cent PF R and 20 per cent PR, consistent with the relative absorbancies of the two forms at 660 nm. Thus the PR found after the dark period is equal to that not converted initially, and the decay of PF R is due entirely to the loss of reversible phytochrome and not to reversion to PR. He pointed out that many interpretations of phytochrome effects have been based on the thermal reversion of PF R to PR, with a half-life of approximately 2 h, whereas it has now been found that there is no thermal reversion in dark-grown maize seedlings.
In cauliflower, however (bought on the market and grown in the light) the total phytochrome does not decrease when the heads are placed in the dark, but the concentration of PF R falls rapidly at first and more slowly later, due to the dark reversion to PR.
It was suggested that there are two forms of phytochrome. When dark-grown seedlings are placed in the light the phytochrome content decreases to a very low level, below that at which it can be measured reliably by spectrophotometric methods. The phytochrome then remaining may be the active form. It is noteworthy that plants typically exhibiting red-far-red displays have little phytochrome, at least at the time of the display.
W . R . B R I G G S (Stanford) also concluded that only a fraction of the phytochrome originally present in dark-grown corn seedlings is physiologically active. He described an effect of brief red irradiation
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220 P H O T O E N V I R O N M E N T
on the subsequent phototropic sensitivity of Barbecue hybrid corn seedlings that can be attributed to phytochrome and depends on the presence of Pf r. He pointed out that only a fraction of the phytochrome present in the dark-grown seedling has been converted to Pf r when the response to red light is saturated. Studies have been made of the duration of persistence of the red-light effect in an ensuing dark period following a red-light dose of about 5 χ ί ο- 1 1 Einsteins c m- 2 (which saturates the physiological response but results in no measurable Pf r formation) and a dose of i o ~7 Einsteins c m- 2 (which transforms all of the phytochrome present to the Pf r form). Pf r would be expected to persist longer in the latter case so that the phototropic sensitivity change would also be expected to persist longer; however, the time courses were identical in both cases and the sensitivity shift decayed away at about the same rate whether fractional or saturating doses had been given. He suggested that there may be two phytochrome trans- formation reactions.
(«) P,
thermal reversion
and
(b) PJ r ed > PJr > thermal destruction
Pf r is the physiologically important form, whereas Pfr is * nonsense ' phytochrome. In corn almost all the phytochrome detected initially is P|r but a small amount of Pf r is present and affecting the response.
After 24 h irradiation only about 5 - 6 per cent of the original phyto- chrome remains.
W . H . K L E I N (Smithsonian Institute) described another system controlled by phytochrome, the opening and closing of the bean plumular hook. Here a dose of 100 m J cm~2 red light will saturate the conversion of Pr to Pf r but below this level the subsequent rate of opening in the dark and the total opening after 20 h is proportional to the logarithm of the incident light. The Pf r level after the light dose and the physiological effect 20 h afterwards are correlated, but it is important to note that it is the rate of the reactions rather than the duration which is affected by the different light doses. Previously greater phytochrome responses had been attributed to longer persis- tence of reactions due to phytochrome Pf r being above a certain
D I S C U S S I O N R E P O R T 221 threshold for a longer time. When the decay of PF R was followed spectrophotometrically none was found after 4-5 h, although the opening of the hook could be totally inhibited by far-red light 8 h after the initial red treatment.
P . DE L I N T (Wageningen) put forward an hypothesis which may explain some of these results, viz. that PF R is not itself active but must be converted to another form for activity to occur. This form may be destroyed by far-red light as follows :
d a r k
Precursor > Pr ^ Pf r > Pf r (active and destroyed by far-red light) A non-saturating dose blocks the effect for up to 24 h, possibly by converting much of the available PR to PF R which becomes P £ in the dark but does not reach a level high enough for the response. This P £ must also decay in the dark. The same reactions would occur with prolonged intensity far-red light. For example, in the seeds reported by EVENARI a low percentage of PR would be converted to PF R and would be immediately destroyed. Far-red and blue light have two functions therefore, (i) the setting of the PR/ PF R ratio, and (ii) the destruction of P£. This might be an explanation of most high energy reactions.
In Avena the mesocotyl is 100 to 1000 times more sensitive to red- light treatment for phototropism than the hypocotyl on a phytochrome basis.
In an exchange of questions B.CUMMING (Canada Dept. Ag.) suggested that a daily rhythm acting through the amount of PF R in the active form might be involved. W . L . B U T L E R (U.S.D.A.) added that a change in the decay time of PF R would have a similar effect. P . DE L I N T (Wageningen) stressed that two independent forms were still not required if de novo formation was taken into account. The apparent stability of total phytochrome in certain experiments could result from synthesis balancing breakdown. BUTLER rejoined with some evidence that in cauliflower the destruction of PF R can occur both aerobically and anaerobically whereas in corn it occurred only in aerobic conditions, indicating a difference in chemical properties of the two types.
M . W I L K I N S (Norwich) described a system that is extremely sensitive to red light (660 nm). 0-5 ergs c m- 2 can double or halve the geotropic sensitivity of some completely dark-grown seedlings. In Zea seedlings red light causes a decrease in geotropic sensitivity beginning 6 h after the red-light stimulus; after 1-5 h this is no longer far-red reversible and this correlates closely with the decay of phyto- chrome observed by BUTLER et al in Zea seedlings. In aetiolated
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Avena coleoptües the responsiveness is at first increased but returns to a value similar to that of unirradiated coleoptiles after about 1-5 h.
Later, the responsiveness is decreased; this appears to persist in- definitely. The changes are elicited by a dosage of 3-3 χ i o ~1 3 Ein- steins c m- 2 and are saturated at 3-3 χ i o ~1 2 Einsteins c m- 2. The effects of red light are independent of the duration of irradiation and are reversed by far-red (740 nm) radiation. Chilling seedlings im- mediately after red irradiation causes a retention of the enhanced geotropic responsiveness. A second exposure to red light does not elicit such changes in the geotropic responsiveness of the coleoptiles.
KLEIN pointed out that measureable phytochrome would still be present in such seedlings.
A.KADMAN-ZAHAVI (Rehovot) enquired why there was so much concern over the lack of dark reversal as only one experiment had ever been explained by dark reversal. Many phytochrome reactions appeared to be limited by the lack of phytochrome precursor rather than by Pr- Pf r conversion. W . S . H I L L M A N (Yale) noted that most experiments were designed at either threshold or saturating levels and advocated measures giving half saturation. L.JAFFE (Pennsylvania) suggested that 'nonsense' phytochrome might be a dispersed form, with the active form on a membrance in a highly orientated condition.
In this case polarized light might be expected to differ from normal light in its effects. One report of such an effect existed.
H.W.SIEGELMAN ( U . S . D . A . ) concluded the discussion by outlining the variability in phytochrome behaviour that has been observed. The lifetime of Pf r is variable, the dark reversion of Pf r appears also to be highly variable and so is the amount necessary to elicit a physiological response. Consequently, he stressed that experiments on phytochrome should be simple ones if these complexities are to be unravelled.
R E F E R E N C E
BUTLER W . L . , LANE H . C . and SIEGELMAN H . W . (1963) Plant Physiol. 38, 514-19.
