Stomatal Opening in Light of Different Wavelengths: Effects of Blue Light Independent of Carbon Dioxide Concentration

Stomatal opening in Xanthium pennsylvanicum was found to besignificantly greater in blue light than in red. Experimentsin which leaves were placed in a closed system and allowed toestablish their own steady-state carbon dioxide concentrationshowed that when the CO₂ concentration was about the same asthat in red, opening was much greater in blue light. Blue lightof low intensity could cause as great an opening as red of higherintensity, even though the CO₂ concentration was much higherin blue. Stomatal opening in light is considered as involvingat least two reactions: (1) a response to the removal of CO₂by photosynthesis; (2) a response to blue light not dependenton the removal of CO₂. Blue light became increasingly effective, relative to red, asthe length of night was increased over the range 2 to 14 hours.This might, in part, explain previously observed effects ofnight length on rate of opening in light. The initial very rapid phase of closure in darkness appearedto be independent of CO₂ accumulation, for it was not preventedby flushing the intercellular spaces with air free of CO₂. Itis suggested that closure in darkness, like opening in light,should be considered as involving components both dependentupon, and independent of, CO₂ concentration.     

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: 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.     

Light Actions in the Germination of Cocklebur Seeds: I. DIFFERENCES IN THE LIGHT RESPONSES OF THE UPPER AND LOWER SEEDS

Germination responses to light were studied in the upper andlower seeds of cocklebur (Xanthium pennsylvanicum Wallr.). Thelower seed was dark-germinating and negatively photoblastic;the upper one had a red-light (R) requirement and was positivelyphotoblastic. Germination of the lower seeds was inhibited bya prolonged single irradiation with R, blue (B) or far-red (FR)light applied during imbibition. The maximal inhibitory effectof a single irradiation occurred 9 h and 13 h after the startof soaking at 33 °C and 23 °C, respectively. However,the inhibitory effect of R differed from that of B and FR, byonly delaying germination. A single exposure to B or FR lightcould be replaced by intermittent B or FR irradiation, and theireffects were repeatedly reversible by the following R irradiation.If the upper seeds were not exposed to R during imbibition,they failed to germinate even at 33 °C which was optimalfor germination, and the promotive effect of R increased withdelay of its application time. The photoperceptive locus incocklebur seeds was the axial tissue for all B, R and FR. Lightreceived by the cotyledonary tissue had little effect. Germinationdimorphism in response to light is discussed with respect tothe phytochrome content and the ageing of axial tissues. Key words: Blue light, Dimorphism, Far red light, Germination, Red light, Xanthium seed     

Alterations in light-induced stomatal opening in a starch-deficient mutant of Arabidopsis thaliana L. deficient in chloroplast phosphoglucomutase activity

Stomatal responses to light of Arabidopsis thaliana wild-type plants and mutant plants deficient in starch (phosphoglucomutase deficient) were compared in gas exchange experiments. Stomatal density, size and ultrastructure were identical for the two phenotypes, but no starch was observed in guard cells of the mutant plants whatever the time of day. The overall extent of changes in stomatal conductance during 14 h light–10 h dark cycles was similar for the two phenotypes. However, the slow endogenous stomatal opening occurring in darkness in the wild type was not observed in the mutant plants. Stomata in the mutant plants responded much more slowly to blue light (70 μmol m^?2 s^?1) though the response to red light (250 μmol m^?2 s^?1) was similar to that of wild-type plants. In paradermal sections, stomatal responses to red light (300 μmol m^?2 s^?1) were weak for wild-type plants as well as for mutant plants. Stomatal opening was greater under low blue light (75 μmol m^?2 s^?1) than under red light for the two genotypes. However, in mutant plants, a high chloride concentration (50 mol m^?3) was necessary to achieve the same stomatal aperture as observed for the wild-type plants. These results suggest that starch metabolism, via the synthesis of a counter-ion to potassium (probably malate), is required for full stomatal response to blue light but is not involved in the stomatal response to red light.     

Effect of Light Quality on Stomatal Opening in Leaves of Xanthium strumarium L

Flux response curves were determined at 16 wavelengths of light for the conductance for water vapor of the lower epidermis of detached leaves of Xanthium strumarium L. An action spectrum of stomatal opening resulted in which blue light (wavelengths between 430 and 460 nanometers) was nearly ten times more effective than red light (wavelengths between 630 and 680 nanometers) in producing a conductance of 15 centimoles per square meter per second. Stomata responded only slightly to green light. An action spectrum of stomatal responses to red light corresponded to that of CO₂ assimilation; the inhibitors of photosynthetic electron transport, cyanazine (2-chloro-4[1-cyano-1-methylethylamino]-6-ethylamino-s-triazine) and 3-(3,4-dichlorophenyl)-1,1-dimethylurea, eliminated the response to red light. This indicates that light absorption by chlorophyll is the cause of stomatal sensitivity to red light. Determination of flux response curves on leaves in the normal position (upper epidermis facing the light) or in the inverted position (lower epidermis facing the light) led to the conclusion that the photoreceptors for blue as well as for red light are located on or near the surfaces of the leaves; presumably they are in the guard cells themselves.     

Different stomatal responses of maize leaves after blue or red illumination under anoxia

Abstract In normal air, illumination with a low level of blue or red light (40 μmol m^?2 s^?1) did not induce stomatal opening in maize plantlets. In CO₂-free air, 40 μmol m^?2 s^?1 of blue or red light promoted an enhancement in stomatal opening. At the same quantum flux, blue light was more efficient than red light and stomatal closure occurred more rapidly with a significantly shorter lag phase after blue light. Anoxia inhibited light-dependent stomatal opening, even under 320 μmol m^?2 s^?1 illumination. However, after 60 min of illumination with 40 μmol m^?2 s^?1 of blue light in anoxia, transient stomatal opening was observed when the plant was returned to darkness and normal air. This transient stomatal opening was weaker after pretreatment with red light. We conclude that a blue-light-dependent process induced under anoxia leads to stomatal opening provided oxygen is present. Possible mechanisms associated with blue-light-effect and the nature of the oxygen-consuming processes are discussed.     

Abaxial and Adaxial Stomatal Responses to Light of Different Wavelengths and to Phenylacetic Acid on Isolated Epidermes of Commelina communis L.

Abaxial and adaxial stomatal responses to light of differentwavelengths and to phenylacetic acid (PAA), a molecule knownto form complexes with irradiated flavins, were examined onisolated epidermes of Commelina communis L. Blue light was superiorto red and green in promoting opening. Potassium accumulationand malate production were common to both abaxial and adaxialstomatal cells, but the photosensitivity was markedly higherin the former than in the latter. PAA suppressed opening andpotassium accumulation in guard cells, but hardly affected thelevel of epidermal malate; CO₂-free air failed to reverse thesesuppressions. The PAA-effect was more substantial in blue lightthan in red, green or darkness; thus, a flavin photoreceptoris indicated. Because of the overall effect of PAA under allconditions it is suggested that, in addition to its interactionwith blue light reception, PAA also has a more general effecton guard cells.     

Properties of the Signal Transduction Pathway in the Blue Light Response of Stomatal Guard Cells of Vicia faba and Commelina benghalensis

Blue light-dependent proton extrusion in guard cell protoplastsfrom Vicia faba and light-dependent stomatal opening in theepidermis of Commelina benghalensis are inhibited by the calmodulin(CaM) antagonist, N-(6-aminohexyl)-5-chloro-l-naphthalenesulfononamide(W-7) and the myosin light chain kinase (MLCK) inhibitor, 1-(5-iodonaphthalene-1-sulfonyl)-lH-hexahydro-1,4-diazepine (ML-7) [Shimazaki, K., Kinoshita, T.and Nishimura, M. (1992) Plant Physiol. 99: 1416]. We now suggestthat the inhibition occurs in the blue light signaling pathwaywithout affecting the proton pump. Addition of fusicoccin (FC),an activator of H⁺-ATPase, to the protoplasts and the epidermiswhose blue light-dependent proton extrusion and light-dependentstomatal opening had been inhibited by W-7 and ML-7, inducedboth proton extrusion and stomatal opening, respectively. Bluelight-dependent proton extrusion was inhibited by K-252a, awide-range inhibitor of protein kinases, and KT5926, a selectiveinhibitor of MLCK. FC induced proton extrusion in the presenceof K-252a and KT5926. In contrast, phenylmercuric acetate (PMA),carbonyl cyanide-m-chlorophenylhydrazone (CCCP) and N, N'-dicyclohexylcarbodiimide(DCCD) inhibited both the proton extrusion and stomatal opening,but FC did not induce the responses. These results suggest thatW-7, ML-7, K-252a and KT5926 inhibit the signal transductionprocess by which the perception of blue light is transducedinto activation of the proton pump in guard cells, and thatMLCK or MLCK-like protein is involved in the blue light responseof stomata. The possibility that calcium-dependent, calmodulinindependent protein kinase [Harper, J.F. et al. (1991) Science252: 951] functions rather than MLCK in the blue light responseof stomata should be noted, however. (Received July 23, 1993; Accepted September 30, 1993)     

Steady-state stomatal responses of C3 and C4 species to blue light fraction: Interactions with CO2 concentration

Blue light induced stomatal opening has been studied by applying a short pulse (~5 to 60 s) of blue light to a background of saturating photosynthetic red photons, but little is known about steady-state stomatal responses. Here we report stomatal responses to blue light at high and low CO₂ concentrations. Steady-state stomatal conductance (g_s) of C₃ plants increased asymptotically with increasing blue light to a maximum at 20% blue (120 μmol m⁻² s⁻¹). This response was consistent from 200 to 800 μmol mol⁻¹ atmospheric CO₂ (C_a). In contrast, blue light induced only a transient stomatal opening (~5 min) in C₄ species above a C_a of 400 μmol mol⁻¹. Steady-state g_s of C₄ plants generally decreased with increasing blue intensity. The net photosynthetic rate of all species decreased above 20% blue because blue photons have lower quantum yield (moles carbon fixed per mole photons absorbed) than red photons. Our findings indicate that photosynthesis, rather than a blue light signal, plays a dominant role in stomatal regulation in C₄ species. Additionally, we found that blue light affected only stomata on the illuminated side of the leaf. Contrary to widely held belief, the blue light-induced stomatal opening minimally enhanced photosynthesis and consistently decreased water use efficiency.     

Suppression of Stomatal Opening in Leaves Treated with Abscisic Acid

Small doses of abscisic acid (approximately 0.02 µg cm^-2of leaf) applied to the leaf surface as a 10^-4 M solution causedmarked stomatal closure in Xanthium pennsylvanicum, and theeffect persisted for up to 9 days after application. Similareffects were found when 10^-4 M abscisic acid was supplied todetached tobacco leaves via their petioles. CO²-free air didnot cause a reversal of the closure, and it was therefore concludedthat the effect was not due simply to an increase in the intercellularCO²concentration; a more direct effect on the stomatal apparatusis suggested. It is considered that abscisic acid could playan endogenous role in the control of stomatal aperture, andthat this, and/or related substances, might be more useful as‘anti-transpirants’ than the phytotoxic substancescurrently employed for this purpose.     

The effect of wavelength of light on stomatal opening

The effect of broad band green, blue and red light on stomatal opening of Vicia faba L. (broad bean) leaves was examined. In air, blue light caused greater stomatal opening than red light. In air with green light stomata were only slightly opened. In a nitrogen atmosphere red light caused greater opening than blue light, and green light caused only slight opening. Opening in air or nitrogen atmosphere in red or blue light was inhibited by the uncoupler CCCP, while the photosynthetic inhibitor DCMU inhibited opening in air but not in nitrogen atmosphere. We concluded that more than one light activated metabolic pathway can supply the energy needed to effect stomatal opening and that different pathways are operative under different conditions.     

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.     

Carbon dioxide requirements for phytochrome action in photoperiodism and seed germination

The effect of interrupting darkness with red light in the presence or absence of 0.03% CO₂ was studied in relation to flowering of Xanthium pennsylvanicum and germination of light-sensitive lettuce seeds. The results indicate that CO₂ is essential for red light to be effective in either process.     

Multi-level Modeling of Light-Induced Stomatal Opening Offers New Insights into Its Regulation by Drought

Plant guard cells gate CO₂ uptake and transpirational water loss through stomatal pores. As a result of decades of experimental investigation, there is an abundance of information on the involvement of specific proteins and secondary messengers in the regulation of stomatal movements and on the pairwise relationships between guard cell components. We constructed a multi-level dynamic model of guard cell signal transduction during light-induced stomatal opening and of the effect of the plant hormone abscisic acid (ABA) on this process. The model integrates into a coherent network the direct and indirect biological evidence regarding the regulation of seventy components implicated in stomatal opening. Analysis of this signal transduction network identified robust cross-talk between blue light and ABA, in which [Ca²⁺]_c plays a key role, and indicated an absence of cross-talk between red light and ABA. The dynamic model captured more than 10³¹ distinct states for the system and yielded outcomes that were in qualitative agreement with a wide variety of previous experimental results. We obtained novel model predictions by simulating single component knockout phenotypes. We found that under white light or blue light, over 60%, and under red light, over 90% of all simulated knockouts had similar opening responses as wild type, showing that the system is robust against single node loss. The model revealed an open question concerning the effect of ABA on red light-induced stomatal opening. We experimentally showed that ABA is able to inhibit red light-induced stomatal opening, and our model offers possible hypotheses for the underlying mechanism, which point to potential future experiments. Our modelling methodology combines simplicity and flexibility with dynamic richness, making it well suited for a wide class of biological regulatory systems.     

Identification and Content of 1-Malonylaminocyclopropanecarboxylic Acid in Germinating Cocklebur Seeds

A major conjugate of 1-aminocyclopropanecarboxylic acid in germinatingcocklebur (Xanthium pennsylvanicum Wallr.) seeds was isolatedand identified as 1-malonylaminocyclopropanecarboxylic acid(MACC). The change in MACC content during the germination periodof this seed also was examined. (Received November 4, 1983; Accepted March 15, 1984)     

The Role of the Oxygen Permeability of the Seed Coat in the Dormancy of Seed of Xanthium pennsylvanicum Wallr

A method for direct identification and quantitative measurementsof mixed or pure gases diffusing through seed coats was devisedto test the hypothesis that the dormancy of Xanthium pennsylvanicumseeds is caused by oxygen-impermeable seed coats. The diffusionof oxygen through seed coats of X. pennsylvanicum was shownto obey Fick's first law. Oxygen diffused through the lowerand upper seed coats at the same rate. Imbibed lower and upperseeds showed essentially equal oxygen uptake rates before radicleemergence. This uptake was lower than the rate at which oxygencan diffuse into the seed. Therefore upper seeds are not dormantbecause of seed coat restriction of oxygen diffusion. The relationshipsof oxygen with other factors involved in seed dormancy are discussed.     

Slow photosynthetic induction and low photosynthesis in Paphiopedilum armeniacum are related to its lack of guard cell chloroplast and peculiar stomatal anatomy

Paphiopedilum and Cypripedium are close relatives in the subfamily Cypripedioideae. Cypripedium leaves contain guard cell chloroplasts, whereas Paphiopedilum do not. It is unclear whether the lack of guard cell chloroplasts affects photosynthetic induction, which is important for understory plants to utilize sunflecks. To understand the role of guard cell chloroplasts in photosynthetic induction of Paphiopedilum and Cypripedium, the stomatal anatomy and photosynthetic induction of Paphiopedilum armeniacum and Cypripedium flavum were investigated at different ratios of red to blue light. The highest stomatal opening and photosynthesis of intact leaves in P. armeniacum were induced by irradiance enriched with blue light. Its stomatal opening could be induced by red light 250 µmol m^?2 s^?1, but the magnitude of stomatal opening was lower than those at the other light qualities. However, the stomatal opening and photosynthesis of C. flavum were highly induced by mixed blue and red light rather than pure blue or red light. The two orchid species did not differ in stomatal density, but P. armeniacum had smaller stomatal size than C. flavum. The stomata of P. armeniacum were slightly sunken into the leaf epidermis, while C. flavum protruded above the leaf surface. The slower photosynthetic induction and lower photosynthetic rate of P. armeniacum than C. flavum were linked to the lack of guard cell chloroplasts and specific stomatal structure, which reflected an adaptation of Paphiopedilum to periodic water deficiency in limestone habitats. These results provide evidence for the morphological and physiological evolution of stomata relation for water conservation under natural selection.     

Effects of Natural and Artificial Light in Arctic Latitudes on Long- and Short-day Plants as Revealed by Growth Analysis

The effects on growth and flowering of two short-day and twolong-day plants when grown under different conditions of illuminationare described. The plants fully investigated were Kalanchoeblossfeldiana and Xanthium pennsylvanicum and the annual varietiesof Hyoscyamus niger and Beta vulgaris. Wintex barley, Iberisumbellata, and tomato were also grown in some selected treatments.The conditions investigated comprised continuous full daylight(24 hours), full daylight for the whole of the daily photoperiodand full daylight for half the photoperiods, the other halfconsisting of either daylight reduced by shading or light fromincandescent lamps or fluorescent tubes (daylight-matching type),all of the same low intensity. Two lengths of photoperiod wereused for each species, one nearly optimal for flowering, theother closer to the critical day-length; and the order of thelow and high light treatments was varied. These factors werecombined factorially. Data were collected (or derived) for the following characteristics,though not always for all the species grown: dry weights, leafareas, heights, water contents, epidermal cell sizes, net assimilationrates, times to flowering, leaf-number increments until flowering,numbers of inflorescences, stomatal apertures, and leaf postures. Among other effects, the data revealed that in all four speciesinvestigated the adverse effects on over-all growth to be expectedfrom reduction of the daily photoperiod or of the intensityof illumination are in fact minimized. This compensation waseffected mainly by large increases in leaf areas, even thoughin all cases half the daily photoperiod consisted of full daylight.There are indications that increased efficiencies (net assimilationrates) may also have been involved. The leaf-area increasesappear to have been due to increased cell size rather than cellnumber and a close positive correlation with water content wasfound. The most striking among the effects on flowering was the failureof sugar-beet to bolt when half of its photoperiod (totals of20 and 14 hours) consisted of light from fluorescent lamps.The flowering of barley and Hyoscyamus was also delayed considerablyunder these conditions. The deficiency of red in the spectrumof the fluorescent light is believed to have been the cause.By contrast, the flowering of Iberis, a crucifer, was not affected.     

Effects of Photoperiodic Induction and Debudding in Xanthium pennsylvanicum and of Partial Defoliation in Phaseolus vulgaris on Rates of Net Photosynthesis and Stomatal Conductances

Xanthium pennsylvanicum plants received four treatments in thefactorial experiment (a) debudded v. not debudded, and (b) longdays . photoinductive short days. Rates of net photosynthesis,carbon dioxide compensation points (), and stomatal conductanceswere assessed after 8 days and before leaf growth or stomatalsize were appreciably affected. Leaf size, stomatal frequencies,and lengths of guard cells were estimated at this time and again22 flays after treatment. Debudding alone slightly Increased stomatal conductance; inductionalone had a similar but larger effect. Debudding and inductiontogether caused a more than additive increase in net photosynthesisat 8 days, with marked decreases in . At 22 days this combinationcaused more than additive increases in leaf size and guard-celllengths while stomatal frequencies had decreased. Induction alone directly increased stomatal conductance andthis may be responsible for the increase in photosynthesis;but debudding alone may directly affect photosynthesis by increasingthe supply of cytokinins to the leaves. The positive interactionof these factors in photosynthesis could not be explained interms of stomatal conductance and a synergism between cytokininsand a photoperiodically induced hormone is suggested. In Phaseolus vulgaris plants, 4 days after partial defoliation,stomatal conductances and rates of net photosynthesis increasedgreatly in the remaining leaflets.