Secret Message at the Plant Surface - USP · 5. Chandra S, Kable EP, Morrison GH, Webb WW. Calcium...
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DO NOT DISTRIBUTE. e1 Plant Signaling & Behavior 2007; Vol. 2 Issue 5 Addendum Secret Message at the Plant Surface [Plant Signaling & Behavior 2:5, e1-e3, EPUB Ahead of Print: http://www.landesbioscience.com/journals/psb/abstract.php?id=4523; September/October 2007]; ©2007 Landes Bioscience Hernán Boccalandro 1 Jorge Casal 2 Laura Serna 3, * 1 Facultad de Ciencias Agrarias; Universidad Nacional de Cuyo; Mendoza, Argentina 2 21Feva Facultad de Agronomía; Universidad de Buenos Aires y Conicet; Buenos Aires, Argentina 3 Facultad de Ciencias del Medio Ambiente e ICAM; Universidad de Castilla-La Mancha; Toledo, Spain *Correspondence to: Laura Serna; Facultad de Ciencias del Medio Ambiente; Universidad de Castilla-La Mancha; Toledo E-45071 Spain; Tel.: +34.925.2688.00; Fax: +34.925.2688.40; Email: [email protected] Original manuscript submitted: 06/01/07 Manuscript accepted: 06/01/07 This manuscript has been published online, prior to printing for Plant Signaling & Behavior, Volume 2, Issue 5. Definitive page numbers have not been assigned. The current citation is: Plant Signaling & Behavior 2007; 2(5): http://www.landesbioscience.com/journals/psb/abstract.php?id=4523 Once the issue is complete and page numbers have been assigned, the citation will change accordingly. KEY WORDS Stomata, phototropins, autonomous func- tion, blue light, cellular irradiation Addendum to: Use of Confocal Laser as Light Source Reveals Stomata- Autonomous Function R.C. Cañamero, H. Boccalandro, J. Casal and L. Serna PLoS ONE 2006; 1:e36 ABSTRACT In general, stomata open during the day and close at night. This behavior has a crucial importance because it maximizes the update of CO 2 for photosynthesis and minimizes the water loss. Blue light is one of the environmental factors that regulates this process. Certainly, when either entire plants or epidermal strips adapted to the dark are exposed to blue light, the stomata open widely their pores. But, what does happen if we illuminate individual stomata instead of peels or entire plants? In the inaugural issue of PLoS ONE, we have answered this question by irradiating individual stomata with a laser attached to a confocal microscope. Our study not only demonstrates that the stomata function independently from the behavior of their neighbors, and illuminates the implica‑ tion of the blue light receptors PHOTOTROPIN1 and PHOTOTROPIN2 in such response. It also gives clues about the physiological relevancy of this behavior. ILLUMINATING INDIVIDUAL CELLS Driven by the desire to image biological events occurring in living organisms, Marvin Minsky, a pioneer in artificial intelligence and robotics, developed the basic concept of confocal microscopy in the mid-1950s. From its invention, we have experienced a tremen- dous explosion in its number of applications. Some of the most conventional ones include live cell imaging by attaching fluorochromes to subcellular components. 1-3 Confocal microscopy is also used to analyze functions, such as pH gradients and membrane potentials, and to measure intracellular changes in ion concentrations of molecules such as calcium, sodium, magnesium, zinc and potassium. 3-6 Imaging protein-protein interactions is also a target, among many others, of this versatile technique. 3,7 But confocal microscopy can be used not only to visualize the structural details and the dynamics of cellular processes, and to create images in three dimensions, it can also be used to modify these dynamics and/or structures. For example, twelve years ago, an unfiltered laser beam coupled to a confocal microscope was used to modify the cellular structure of the Arabidopsis root by ablating individual cells. 8 The response of the root to the ablation experiments allowed uncovering the cell signalling underlying its develop- ment. 8 Obviously, confocal microscopy can also be used to irradiate individual cells with a laser bean of defined wavelength and intensity. Because both light quality and quantity modulate many developmental and physiological processes at a cellular (stomatal opening) and even subcellular level (chloroplast movement), laser confocal microscopy can be used to deep into the cell and/or sub-cell signalling mechanisms underlying such as processes. For example, it is known that when either entire plants or epidermal strips adapted to dark are exposed to blue light, the stomata open their pores. 9-11 But, does anything happen if we illuminate individual stomata? Given that blue light induces stomatal opening exhib- iting an action spectrum with a maximum at 450-nm and two minor peaks at 420-nm and 470-nm (ref. 12), specific confocal laser lines can be selected as light source of individual cells to study the cell signalling underlying this response. THE AUTONOMY OF THE STOMATAL RESPONSE Plants open stomata to allow carbon dioxide uptake. However, when they do it, tissues lose part of their precious water. Opening the stomata is then a crucial physiological decision, which is tightly controlled by both endogenous and environmental signals. 13 The environmental factors that regulate stomatal movements include both blue and red light, carbon dioxide concentrations and atmospheric humidity. 13 Little is known
Transcript of Secret Message at the Plant Surface - USP · 5. Chandra S, Kable EP, Morrison GH, Webb WW. Calcium...
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ANDES BIOSCI
ENCE.
DO NOT DIST
RIBUTE.
e1 PlantSignaling&Behavior 2007;Vol.2Issue5
Addendum
Secret Message at the Plant Surface
[PlantSignaling&Behavior2:5,e1-e3,EPUBAheadofPrint:http://www.landesbioscience.com/journals/psb/abstract.php?id=4523;September/October2007];©2007LandesBioscience
Hernán Boccalandro1
Jorge Casal2 Laura Serna3,*1Facultad de Ciencias Agrarias; Universidad Nacional de Cuyo; Mendoza, Argentina
2 21Feva Facultad de Agronomía; Universidad de Buenos Aires y Conicet; Buenos Aires, Argentina
3Facultad de Ciencias del Medio Ambiente e ICAM; Universidad de Castilla-La Mancha; Toledo, Spain
*Correspondence to: Laura Serna; Facultad de Ciencias del Medio Ambiente; Universidad de Castilla-La Mancha; Toledo E-45071 Spain; Tel.: +34.925.2688.00; Fax: +34.925.2688.40; Email: [email protected]
Original manuscript submitted: 06/01/07Manuscript accepted: 06/01/07
This manuscript has been published online, prior to printing for Plant Signaling & Behavior, Volume 2, Issue 5. Definitive page numbers have not been assigned. The current citation is: Plant Signaling & Behavior 2007; 2(5):http://www.landesbioscience.com/journals/psb/abstract.php?id=4523Once the issue is complete and page numbers have been assigned, the citation will change accordingly.
Key wordS
Stomata, phototropins, autonomous func-tion,bluelight,cellularirradiation
Addendum to:
Use of Confocal Laser as Light Source Reveals Stomata-Autonomous Function
R.C. Cañamero, H. Boccalandro, J. Casal and L. Serna
PLoS ONE 2006; 1:e36
ABStrACtIn general, stomata open during the day and close at night. This behavior has a
crucial importance because it maximizes the update of CO2 for photosynthesis and minimizes the water loss. Blue light is one of the environmental factors that regulates this process. Certainly, when either entire plants or epidermal strips adapted to the dark are exposed to blue light, the stomata open widely their pores. But, what does happen if we illuminate individual stomata instead of peels or entire plants? In the inaugural issue of PLoS ONE, we have answered this question by irradiating individual stomata with a laser attached to a confocal microscope. Our study not only demonstrates that the stomata function independently from the behavior of their neighbors, and illuminates the implica‑tion of the blue light receptors PHOTOTROPIN1 and PHOTOTROPIN2 in such response. It also gives clues about the physiological relevancy of this behavior.
ILLumInAtIng IndIvIduAL CeLLSDrivenbythedesiretoimagebiologicaleventsoccurringinlivingorganisms,Marvin
Minsky,apioneer inartificial intelligenceand robotics,developed thebasicconceptofconfocalmicroscopyinthemid-1950s.Fromitsinvention,wehaveexperiencedatremen-dousexplosioninitsnumberofapplications.Someofthemostconventionalonesincludelive cell imaging by attaching fluorochromes to subcellular components.1-3 Confocalmicroscopyisalsousedtoanalyzefunctions,suchaspHgradientsandmembranepotentials,andtomeasureintracellularchangesinionconcentrationsofmoleculessuchascalcium,sodium,magnesium,zincandpotassium.3-6Imagingprotein-proteininteractionsisalsoatarget,amongmanyothers,ofthisversatiletechnique.3,7
Butconfocalmicroscopycanbeusednotonly tovisualize the structuraldetailsandthedynamicsofcellularprocesses,andtocreateimagesinthreedimensions,itcanalsobeused tomodify thesedynamicsand/or structures.Forexample, twelveyears ago, anunfiltered laserbeamcoupledtoaconfocalmicroscopewasusedtomodifythecellularstructureof theArabidopsisrootbyablating individualcells.8Theresponseof theroottotheablationexperimentsalloweduncoveringthecellsignallingunderlyingitsdevelop-ment.8Obviously,confocalmicroscopycanalsobeusedtoirradiateindividualcellswithalaserbeanofdefinedwavelengthandintensity.Becausebothlightqualityandquantitymodulatemanydevelopmentalandphysiologicalprocessesatacellular(stomatalopening)andevensubcellularlevel(chloroplastmovement),laserconfocalmicroscopycanbeusedtodeepintothecelland/orsub-cellsignallingmechanismsunderlyingsuchasprocesses.Forexample,itisknownthatwheneitherentireplantsorepidermalstripsadaptedtodarkareexposedtobluelight,thestomataopentheirpores.9-11But,doesanythinghappenifweilluminateindividualstomata?Giventhatbluelightinducesstomatalopeningexhib-itinganactionspectrumwithamaximumat450-nmandtwominorpeaksat420-nmand470-nm(ref.12),specificconfocallaserlinescanbeselectedaslightsourceofindividualcellstostudythecellsignallingunderlyingthisresponse.
tHe Autonomy of tHe StomAtAL reSponSePlants open stomata to allow carbon dioxide uptake. However, when they do it,
tissueslosepartoftheirpreciouswater.Openingthestomataisthenacrucialphysiologicaldecision, which is tightly controlled by both endogenous and environmental signals.13The environmental factors that regulate stomatal movements include both blue andred light, carbon dioxide concentrations and atmospheric humidity.13 Little is known
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about the mechanisms by which stomata sense red light, carbondioxide or atmospheric humidity. In contrast, blue-light stomatalperception is a well-characterized process.The blue-light receptorsPHOTOTROPIN1 (PHOT1) and PHOTOTROPIN2 (PHOT2)mediate stomatal opening in a redundant manner:9 when peels ofsinglemutantsare illuminatedwithblue light, theyretainthewildtyperesponsebyopeningtheirpores,butphot1phot2doublemutantlacksuchresponse.Itisknownthatinthedark,phototropinslocateto the plasma membrane.14-20 However, blue light illuminationinducesthereleaseofPHOT1tothecytoplasm21andtheassociationofPHOT2withtheGolgiapparatus.20
Equippedwithbotha458-nmlineanda476-nmlineofanargonlaserattachedtoaconfocalmicroscope,weaddressedthecellsignal-lingmechanismunderlyingthestomatalresponsetotheblue-light.Whenindividualstomatawereilluminated,theyopenedtheirpores(Fig.1).However,theirnonirradiatedneighborsremainedunaltered(Fig.1).Thisistellingusthatstomatafunctionautonomouslyinthebluelightresponse,andthatthesignalthattriggersstomatalopeningdoesnottransmitamongstomata.Thelaterconclusioncontrastswithelegantworkdonebyothers,whichhighlightsthatstomatalresponsesto several stimuli seem tobedictatedby thebehavior ofneighborstomata.22-25Thefactthattheirradiationofneighborepidermalcellsdidnotinduceopeningoftheadjacentstoma,extendstheabsenceofcellsignallingfromnonstomatalcellstostomatalones.
To unravel the hypothetical implications of both PHOT1 andPHOT2 in the autonomous stomatal opening to the blue lightresponse,wetookacombinatorialapproachbyilluminatingindividualcellsinthephot1phot2doublemutant.Wefoundthattheinductionofthestomatalopeninginthewildtypewasdisruptedinthedoublemutant(Fig.1),whichledustoproposethatthestomatalautonomydepends on PHOT1 and/or PHOT2. Certainly, PHOT1 wasreleasedfromthecellmembranetothecytoplasminirradiatedcells,illuminatingthecellularmechanismthatunderliesthisresponse.
Figure 1. Stoma autonomy in its blue light‑induced response. When indi‑vidual stomata of wild‑type seedlings were blue‑light irradiated with a laser coupled to a confocal microscope, they opened they pores. Unexpectedly, their dark‑adapted neighbors experienced no change. This finding uncovers the stomatal autonomy in its blue‑light opening, which contrasts with other works suggesting that stomatal opening is dictated by that of neighbor stomata. In addition, this induction of the stomatal opening in the wild type was disrupted in the phot1phot2 double mutant, which indicates that the stomatal autonomy depends on phototropins.
Figure 2. Photosynthetic active radiation (PAR) reaching the soil surface of understorey patches of (A) 20 x 20 cm2, (B) 4 x 4 cm2 and (C) 0.4 x 0.4 cm2. PAR reaching the patch containing the light grey bar in (A), and the dark grey in (B), are detailed in (B and C) respectively. PAR was measured with a radiometer Licor 250. The punctual sensor was placed in the centre of every patch. PAR measurements in (C) were obtained covering the sensor with an aluminium foil containing a 1mm diameter‑hole in the centre to perform milimetric measurements. PAR determinations in (C) were corrected multi‑plying the PAR values by a factor, which was obtained from the quotient between the PAR value determined under direct solar radiation with the uncovered sensor and he PAR value obtained with the cover. Sensor was mounted on a microscope to make sharply movements.
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e3 PlantSignaling&Behavior 2007;Vol.2Issue5
tHe pHySIoLogICAL reLevAnCyBut, why did nature invent this behavior? While penetrating
thought a forest, light suffers a reduction in quantity but, giventhe heterogeneous nature of the canopies, highly illuminated areasreceiving sunflecks and deeply shaded areas can be observed atdifferentscalesonthesoilsurface(Fig.2).Thispatternisdynamicthankstothemovementofthefoliagebywindandthechangesinsolar elevation throughout the photoperiod. Leaf surface is then amobile mosaic constituted by lighted and shadowed patches. Bymeasuringirradiancelevelsintransectsacrosstheboundarybetweenshaded and light leaf areas,weobserved that the transition canberelatively abrupt. This leads to large differences in light in shortdistances, which are in the range of the average distance betweenstomata.Then,theautonomousstomatabehaviorwouldcontributeto optimize the balance between water loss and CO2 acquisition,allowingtheopeningonlyoftheilluminatedpairedguardcells.
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