Studies in Stomatal Behaviour: VI. AN INVESTIGATION OF THE LIGHT RESPONSES OF WHEAT STOMATA WITH THE ATTEMPTED ELIMINATION OF CONTROL BY THE MESOPHYLL

An apparatus is described enabling light responses of wheatstomata to be followed by periodical porometer readings, betweenwhich the substomatal cavities can be swept with carbon dioxide-freeair forced in through the stomata. This is held to eliminatethe influence of changes in the carbon dioxide content of theintercellular space atmosphere brought about by photosynthesisand respiration in the mesophyll. Using this apparatus in anexperiment of ‘Latin square’ design with 6-foldreplication of three light treatments, a statistically significantresponse to light was found in a leaf area subjected to varyinglight intensities from 90 to 800 f.c. This response was reflectedin a similar significant effect, though of smaller magnitude,occurring in a similarly swept ‘control’ area 2cm. distant and maintained at a constant light intensity of270 f.c, the intervening portion of leaf being illuminated with800 f.c. throughout. It is argued that this transmission ofa light effect from one area to another does not occur via theintercellular spaces, nor by means of carbon dioxide gradientsfrom cell to cell. It is concluded from the data that thereis an indirect effect of light and perhaps also a direct effectupon the guard cells themselves; it is probable that neitherof the two effects concerned operates by reduction of the internalcarbon dioxide content in the guard cells.     

Studies in Stomatal Behaviour: II. THE ROLE OF STARCH IN THE LIGHT RESPONSE OF STOMATA

The relationship between starch-content and aperture in thestomata of Pelargonium has been investigated by a quantitativetechnique. Heath's suggestion of an inherent diurnal rhythmin starch-content is confirmed, and the light effect which hasbeen the subject of previous contradictory reports is foundto be dependent on external humidity. When humidity is high,light (which in these experiments is confounded with reducedC0₂-content) causes a striking reduction in stomatal starch;when humidity is low, light has no effect on starch, but itseffect on aperture is unchanged. No evidence for any dependenceof aperture on carbohydrate status was obtained, and it is suggestedthat the function of carbohydrate changes in stomata is, asearlier suggested by Kisselew, the amplifying and stabilizingof changes primarily controlled by other factors.     

Studies in Stomatal Behaviour: V. THE ROLE OF CARBON DIOXIDE IN THE LIGHT RESPONSE OF STOMATA

Experiments are described in which the responses of wheat stomatato carbon dioxide concentration (0·00, 0·01, 0·02,0·03 per cent.), light intensity (275, 625, 975 f.c.),and rate of air flow (2, 5, 12·5 l./hr.) were studied. Reduction of carbon dioxide concentration from 0·03 to0·01 per cent. resulted in marked stomatal opening; furtherreduction to 0·00 per cent, was accompanied by a slightbut not significant closure. These effects were found at alllight intensities and rates of flow, except at 975 f.c. with2 l./hr. air flow, where no effect of carbon dioxide concentrationwas detected. This last is attributed to excessive depletionof the carbon dioxide supply by assimilation. The apparent lowerlimit of 0·01 per cent, carbon dioxide causing maximalstomatal opening is discussed in relation to recent assimilationexperiments. Increase of light intensity caused considerable stomatal opening,this effect being as great with air of 0·00 and 0·01per cent. as with higher concentrations of carbon dioxide. Thissuggests an effect of light on stomatal movement other thanthat exerted indirectly through photosynthesis by the mesophylcells. Increased rate of flow of dry air caused closure of the stomata;this was shown to be a drying effect and was absent when moistair was used.     

Studies in Stomatal Behaviour: V. THE ROLE OF CARBON DIOXIDE IN THE LIGHT RESPONSE OF STOMATA

It was found that stomata on illuminated leaves, both of Pelargoniumand wheat, opened much wider where the leaf surface was enclosedin a small volume of air, as in a normal porometer cup, thanelsewhere. This was shown for both species by the infiltrationmethod, and for Pelargonium by Lloyd's method and direct microscopicalobservation also. The effect was shown not to be due to pressure of the porometercup or glass plate on the leaf, or to temperature differences,nor directly to the lack of movement or high humidity of theenclosed air. A considerable body of data was collected which appeared tosupport the hypothesis that the wide opening was due to accumulationof some volatile substance produced by the leaf, but all theresults were also consistent with the view that it was causedby reduction in the carbon dioxide content of the enclosed airbelow the normal 0·03 per cent. owing to photosynthesis.Further crucial experiments with both the porometer and infiltrationmethods left virtually no doubt that the latter hypothesis wascorrect. This extreme sensitivity of stomata to carbon dioxide concentrationwithin the range 0·03 per cent. to zero is discussedin relation to their operation in nature, and a possible biologicaladvantage is suggested. The bearing of the effect upon porometer investigations is alsodiscussed and it is concluded that for all quantitative or semi-quantitativeexperimentation it is essential to use a cup detached betweenreadings, or at least swept with air such as surrounds the restof the leaf, and to have the upper leaf surface above the cuparea freely exposed or similarly swept. For qualitative investigationof the light response of stomata the traditional form of cupmay be used. The importance is stressed of allowing porometer readings toreach equilibrium under one set of conditions before changingto another, when investigating the ‘closing’ or‘opening’ effects of external factors. Several subsidiary effects, observed in the course of the investigation,are discussed; in particular an effect of humidity upon therate of response to other factors.     

Studies in Stomatal Behaviour: III. THE SENSITIVITY OF STOMATA TO MECHANICAL SHOCK

If Pelargonium stomata, wide open under a porometer-cup, aresubjected to a mechanical shock and then darkened, subsequentclosure is markedly accelerated. The effect persists for atleast 18 hours and, provided a relatively lengthy dark periodquickly follows the shock, is unaffected by subsequent lightperiods. If, however, the shock is followed by a light periodof about 2 hours, subsequent closure is decelerated, and thisdeceleration similarly persists through subsequent dark periods.The effects can largely be explained on the basis of shock-inducedrespiratory changes, if it is postulated that photosynthesisinhibits respiration in chlorophyllous tissue. The effect ofthe shock is not instantaneous, the new pattern of behaviournot being discernible in either case until the stimulated tissuehas been exposed to light or darkness for perhaps an hour. Itappears that in occasional sensitive leaves such phenomena maybe induced by the slight shock involved in fitting a porometer-cup,and the conditions of light or darkness to which the cup issubjected immediately after fixing may then influence its subsequentbehaviour.     

Studies in Stomatal Behaviour: X. AN INVESTIGATION OF RESPONSES TO LOW-INTENSITY ILLUMINATION AND TEMPERATURE IN XANTHIUM PENNSYLVANICUM

Two experiments are described in which stomatal sensitivityto low-intensity white light was studied for Xanthium pennsylvanicumWall. In the first experiment a daylength extension for 7, 9, or 15hrs. was given using 10, 40, or 160 lux to shorten a basic 16-hr.night, which was also given at its full length as a tenth treatment.Measurements were made of stomatal opening ability on the morningfollowing the different treatments. With a 15-hr. extensionthere was at all intensities a significant response, shown bya reduced rate of opening in the morning. With a 9-hr. extensionusing 40 or 160 lux, opening ability was reduced, but 9 hrs.of 10 lux was insufficinet to produce a detectable effect. The7-hr. extension was ineffective at all three intensities. In the second experiment stomatal behaviour was observed during20 hrs. of either darkness or 10 lux at four temperatures (15,22, 29, and 36°C.). During 20 hrs. of darkness there wasnight opening at all temperatures, but at lower temperaturesit began sooner and lasted longer. These responses to temperaturedid not fit a simple linear relationship, there being a significantcubic term revealed by non-linear regression analysis. Thiscould be explained if the response was considered in terms ofthe magnitude of the change in temperature (from 25°C.)at the beginning of the experiment; there appeared to be sometemperature compensation over a limited range. in 10 lux, nightopening was suppressed at 29° and 36°, but at 15°it was apparently unaffected by the light; at 22° it wasnot completely suppressed by 10 lux but the time of its occurrencewas delayed. Effects of light and temperature are discussed in relation toan endogenous rhythm in darkness which was previoulsy shownto operate in Xanthium pennsylvanicum (Part IX). It is considered that to explain effects of very low intensitylight it will be necessary to recognize a ‘low intensityresponse’ by stomata, which does not operate via changesin guard-cell carbon dioxide.     

Studies in Stomatal Behaviour: II THE ROLE OF STARCH IN THE LIGHT RESPONSE OF STOMATA PART 4. VARIATION UNDER CONSTANT CONDITIONS

A quantitative study has been made of the extent and natureof the variation in starch-content and aperture of the stomataof Pelargonium. It is shown that distribution over the surfaceof a single leaf is substantially normal in form; that underexperimental conditions in common use there is a highly significantdifference between groups of stomata on the lower epidermisof a single leaf; that the variation between different leaveson the same plant, or between similar leaves on different plants,does not differ significantly from that between strips fromthe same leaf; that not less than 20 stomata should be measuredon each strip; and that pore-width is a satisfactory measureof the area of the stomatal aperture.     

Studies in Stomatal Behaviour: IV. THE WATER-RELATIONS OF THE EPIDERMIS

1. It is shown that a dry external atmosphere exerts the followingeffects on stomatal movement:

1. A striking accelaration ofclosure in darkness.
2. A slight acceleration of opening in light.
3. If the water-supply to the leaf is impaired, an inabilitytomaintain full opening in the light.

Conversely, a saturatedexternal atmosphere induces sluggishness of movement and a tendencyto incomplete closure in darkness.

1. These results are consideredto support La Rue's contentionthat the epidermal water-supplyis drawn solely by lateral movementfrom the main veins, andnot from the underlying mesophyll.The stomatal phenomena themselvesdo not appear capable of anysimple explanation based on currentknowledge of guard-cellphysiology.
2. The biological significanceof these results is discussed, withparticular reference tothe problem of xeromorphic structures,for which a new interpretationis suggested.

     

Studies in Stomatal Behaviour: XI. FURTHER OBSERVATIONS ON RESPONSES TO NIGHT LENGTH

Stomatal opening in high-intensity light after long and shortnights was measured with the differential transpiration porometerand the Wheatstone bridge porometer on attached and detachedleaves of Xanthium pennsylvanicuin. Results from both porometersshowed that the rate of opening was markedly affected by night-lengthtreatment, but there was no effect on time of onset of openingor on the maximum aperture achieved. Attached and detached leavesshowed practically the same responses. In another experimentdifferent night-length treatments were simultaneously appliedto two leaves on the same plant. They apparently reacted independentlyto the treatments. Night-length effects on rate of opening arediscussed in relation to recent work by other authors on bananaand Vicia faba.     

Studies in Stomatal Behaviour: XII. OPENING IN HIGH TEMPERATURE IN DARKNESS

Xanthium pennsylvanicum exhibited a small stomatal opening (‘nightopening’) towards the end of a long night at 27? C. Experimentsare described in which a temperature increase from 27? to 36?,given during the period of night opening, caused the stomatato open widely for several hours. The degree of opening firstachieved was comparable with that observed in light of 1,000lux, but high temperature was less efficient than light formaintaining opening. Openmg was greater in mature than in youngleaves. The opening did not appear to be due to water strainunder the high temperature. It was found that temperature-induced opening was much greaterafter a long night (16 hours) than after a short night (fourhours). From this it is deduced that the opening is affectedby the endogenous rhythm which occurs in darkness (this wasstudied in previous work). The results obtained here contrastsharply with some obtained previously (and confirmed here) inwhich different temperatures were given throughout the night.The differences can probably be explained in terms of temperatureeffects on the endogenous rhythm. The stomata retained their normal responses to carbon dioxideand carbon dioxide-free air during temperature-induced opening.Thus the opening must occur in spite of any temperature stimulationof respiration. An experiment on the effect of temperature on stomatal closurein response to darkness is also described. Closure was significantlyslower at 36? than at 27? C.     

A STUDY OF THE BACTERICIDAL ACTION OF ULTRA VIOLET LIGHT : III. THE ABSORPTION OF ULTRA VIOLET LIGHT BY BACTERIA

The simple conclusion of former investigators that the shorter the wave length of ultra violet light the greater the bactericidal action is in error. A study with measured monochromatic energy reveals a characteristic curve of bactericidal effectiveness with a striking maximum between 260 and 270 m.µ. The reciprocal of this abiotic energy curve suggests its close relation to specific light absorption by some single essential substance in the cell. Methods are described for determining the absorption curve, or absorption coefficients, of intact bacteria. These curves for S. aureus and B. coli have important points of similarity and of difference with the reciprocals of the curves of bactericidal incident energy, and point the way in a further search for the specific substance, or substances, involved in the lethal reaction.     

PHOTOCHEMISTRY OF VISUAL PURPLE : I. THE KINETICS OF THE DECOMPOSITION OF VISUAL PURPLE BY LIGHT.

1. Visual purple solutions are prepared under such conditions that the bleaching reaction is irreversible. 2. A method is described for the colorimetric estimation of very small quantities of visual purple. By this means the kinetics of the bleaching reaction are investigated. 3. The results show that the course of the decomposition follows that of a monomolecular reaction, without any measurable period of induction or after effect.     

Studies in Stomatal Behaviour: VII. EFFECTS OF ANAEROBIC CONDITIONS UPON STOMATAL MOVEMENT--A TEST OF WILLIAMS'S HYPOTHESIS OF STOMATAL MECHANISM

An experiment was carried out to investigate stomatal responsesin wheat to four ‘closing treatments’, viz. highcarbon dioxide concentration, darkness, dry air and nil, eachgiven under both aerobic and anaerobic conditions. Thus theeffect of lack of oxygen on the closing (or opening) tendencywas estimated. Changes in calculated from resistance porometer readings were used as data and reasonsare given for thinking this is the best available measure forinvestigating stomatal dynamics in wheat. Williams's hypothesisdemands that lack of oxygen should cause stomatal opening orprevent closure; the present experiment shows that anaerobicconditions significantly increase the closing tendency when‘closing treatments’ are first applied. There isalso some suggestion that oxygen-lack itself tends to causeclosure in the absence of any other ‘closing treatment’.Williams's hypothesis in its original form is thus disproved(for wheat) but the present results would be consistent withan ‘active’ uptake of water by the guard cells contributingto stomatal opening. A nearly significant interaction betweencarbon dioxide and oxygen suggests that under anaerobic conditionsa ‘closing substance’ may perhaps be formed, forexample, by the union of some intermediate in glycolysis withcarbon dioxide.     

Studies in Stomatal Behaviour: IX. PHOTOPERIODIC EFFECTS ON RHYTHMIC PHENOMENA IN XANTHIUM PENNSYLVANICUM

An investigation of the effects of different day-length treatmentson stomatal behaviour in Xanthium pennsylvanicum Wall. has shownthat there are differences in long-and short-day treatmentssimilar to those first reported by Schwabe (1952) for Chrysanthemumand Kalanchoe, viz. stomatal opening towards the end of thenight in short days, but not in long days (short night precededby a period of low intensity illumination). In Xanthium therewere in addition very marked differences in rates of stomatalopening in the morning after different lengths of night. Theseeffects were not persistent, there being an immediate reversalupon a change from long- to shrot-day treatment, or vice versa. Further investigation showed that there was an endogenous rhythmaffecting the stomata in continuous darkness; rate of openingwas slow after very short nights, but became greater with extensionof the night to 14–16 hrs., this being the first ‘peak’of an ‘opening ability’ rhythm. The rhythm diedout rapidly and the second cycle was much reduced in amplitude.The period of the rhythm appeared to be approximately 24 hrs.The phase was set mainly by the time of onset of darkness, butthe duration of the pretreatment with low intensity illuminationwas also important—prolonging this was found to reudcethe time in darkness before the first peak. Each hour of lightof 1,500 lux given before darkness was found to be equivalentot approximately 0.3 hr. to darkness. The predominant effect of the length of the preceding nightwas on the slope of the opening curves rather than on the timeof onset of opening following illumination. The first peak of the opening ability rhythm was often manifestedeven in continuous darkness by a period of ‘night opening’of the stomata. The results are discussed and compared with those of other authorsand attempts are made to relate them to theories of the stomatalmechanism.     

Studies in Extension Growth: II. THE LIGHT-GROWTH RESPONSES OF VICIA FABA L

1. The changes of the rate of elongation of the stem of darkgrown Vicia faba seedlings, after illumination by white light,have been measured by auxanometer. 2. Illumination of the plumular hook region, including the apex,causes a reduction in the extension rate of the tissue below.This has been called the ‘primary phase’, and itis complete within three hours of illumination. In the rangeof exposure used, the new rate may be between 50 and 20 percent, of the original rate. 3. Illumination of the extending tissue alone causes a similarprimary phase, which is followed by a period of accelerationduring the third hour after illumination. This period has beencalled the ‘reaction phase’, and it reaches itspeak rate, which is of the order of 60 per cent. of the originalrate before treatment, about 4 hours after illumination. 4. Illumination of the whole plumule causes a primary phasefollowed by a reaction phase of more variable form and size. 5. The magnitude of the primary phase appears to increase withthe exposure. There is an optimum exposure for the productionof the reaction phase in the region of 200 foot-candle seconds. 6. The significance of these results in the interpretation ofetiolation phenomena is discussed.