Photosynthesis Research

80:

Kluwer Academic Publishers. Printed in the Netherlands.

421

Minireview

Pictorial demonstrations of photosynthesis

Roger P. Hangarter

∗

& Howard Gest

Department of Biology, Indiana University, Bloomington, IN 47405, USA;

∗

Author for correspondence(e-mail: rhangart@bio.indiana.edu;fax:

1-812-855-6082)

Received 15 April 2003; accepted in revised form 14 May 2003

Key words:

action spectra, chlorophyll ﬂuorescence, chloroplast movement, Theodor Engelmann, Hans Molisch,photoinhibition, photosynthesis, starch, David Walker, Julius von Sachs

Abstract

Theodor Engelmann’s experiments in 1882 provided the ﬁrst recorded visual demonstration of light wavelengthsthat are absorbed by photosynthetic pigments. Later, starch images in intact leaves were used to demonstratephotosynthesis in green plants. Similarly, light-induced chloroplast movements can form images in leaves as aresult of changes in light transmittance through leaves and photoinhibition can form images that can be visualizedby whole leaf chlorophyll ﬂuorescence. This paper provides a brief account of how photosynthesis has been usedto create an assortment of ‘living images’ that offer stunning demonstrations of various aspects of photosynthesis.

Introduction

The invention of photography began in the early1800s but the first biological subjects for photo-graphic publication happened to be photosyntheticorganisms, namely, ‘British algae,’ when in 1843,the botanist Anna Atkins started issuing her photo-graphically illustrated ‘British Algae: Cyanotype Im-pressions’ (Atkins 1843). Prior to the developmentof long-lasting photographic methods, documentationof biological specimens was limited by the ability of illustrators and artists to capture fine details. Oneof the primary inventors of photography, WilliamHenry Fox Talbot, who was also a botanist, wasdriven to develop stable photographic technologiesnot so much by the desire to understand the sci-entific principles behind the chemistry of photographybut by the desire to use light to reveal propertiesof objects that were not previously perceived or re-corded (Thomas 1997). Similarly, in their desire toreveal processes previously not perceived, photosyn-thesis researchers have on occasion, found cleverways to create ‘living images,’ somewhat analogousto photographs, that reveal fundamental properties of photosynthesis.

Early action spectra

Theodor Engelmann (1843–1909) almost certainlydescribed the first experiments that employed light-induced images of living systems as a means of ob-taining new insights into the process of photosynthesis(Engelmann 1882, 1883). For his experiments, he hada microscope specially modified to project a micro-spectrum on the plane of a specimen on a microscopeslide. The microspectrum was used to illuminate indi-vidual strands of the filamentous green alga,

Chlado- phora

in which each cell is nearly completely andevenly ﬁlled by a chloroplast. To measure photosyn-thetic oxygen production, the

Chladophora

filamentswere dispersed on a microscope slide in a suspen-sion containing aerotactic bacteria. By increasingthe light being delivered through the microspectrum,Engelmann was able to observe the bacteria move asoxygen was produced, and after a short time theyaccumulated most abundantly at regions of the algalfilamentsthat wereilluminatedwith blueandredlight.His drawings of the response of these living organ-isms provided a striking illustration of the first ac-tion spectrum of oxygenic photosynthesis (Figure 1).Engelmann also used bacterial motility to determine

422

Figure 1.

Engelmann’s drawing of his action spectrum for oxy-genic photosynthesis. A portion of a

Cladophora

ﬁlament is shownwith swarming bacteria (

B. termo

) in a microspectum of light. Thechloroplasts, which ﬁll the cells uniformly, were omitted. The ab-sorption bands of chlorophyll in the red (between B and C) and theblue/violet (F) are indicated by stippling. Reproduced from Kamen(1986).

whichcomponentsofplantcells functionedas lightre-ceptors for photosyntheticoxygenproduction.For thishe used a modiﬁedmicroscopecondenserthat allowedhim to expose small parts of photosynthetically activecells of the green alga

Spirogyra

, in which chloro-plasts only occupy parts of each cell, to an extremelythin ray of light while in a suspension of bacteria.He observed that bacteria moved and concentratedin areas wherever parts of a chloroplast were illumi-nated whereas the illumination of other parts of thecell resulted in no such aggregations.Thus, with his clever use of living organisms andlight Engelmann was able to create living ‘pictures’that formed the basis for the first action spectrumshowing chlorophyll as the pigment driving photo-synthesis and he demonstrated that chloroplasts werethe cellular site of photosynthesis. Kamen (1986) hasbeautifully described and discussed the life and worksof Engelmann.The general validity of the action spectrum ob-tained by Engelmann was confirmed by Hans Molisch(1856–1937; see Molisch 1907) and, of course, wasgreatly refined over the years to identify the differentchlorophylls and accessory pigments that constitutethe complete light harvesting and energy-convertingassemblies in a variety of photosynthetic organisms.

Visualizing photosynthetic action by starchproduction

In his pioneering work on the photosynthetic produc-tion and dark utilization of starch in green plants,

Figure 2.

Starch picture of Dr Jan Ingen-Housz, one of the dis-coverers of photosynthesis, on a geranium leaf. The image wasprepared by William Ruf and Howard Gest using a variationof Molisch’s method (see Gest 1991). Light from a slide projector was passed through a photographic negative of an engrav-ing of Ingen-Housz, and focused on a leaf (depleted of starchby prior incubation in darkness). After extracting pigments fromthe leaf with boiling 80% alcohol, starch granules were stainedwith an I

–KI solution. The Latin inscription at the bottomrefers to Dr Ingen-Housz’s fame as a ‘smallpox inoculator.’ Amovie of the procedure for making starch pictures can be seen atwww.cells.de/cellseng/medienarchiv/archiv/d1157.htm.

Julius von Sachs (1832–1897; see von Sachs 1864)used an iodine stain, which disclosed starch in leavesas blue or violet granules. By masking parts of leaveswith foil, he was able to demonstrate that light wasrequiredforstarchformation.Unlikethe bacteriospec-trogramm (see later) of Engelmann, which had to berecorded in drawings, the starch-stained leaves result-ed in fairly stable images of photosynthetic action.Molisch (1914) expanded on von Sachs’ work by de-veloping ‘starch pictures’ in intact leaves by usingactual photographic negatives as masks over the illu-minated leaves. The level of detail in terms of both thegradations in shading that could be reproduced in hisstarch pictures and the spatial resolution were aston-ishingat thetime. Insuchstarchimages, theresolutionis related to the size and number of starch grainsproduced in each chloroplast. Thus, the starch is anal-ogous to silver grains in a conventional photograph or

423pixels in digital images. Molisch’s simple techniqueprovides such a dramatic representation of photosyn-thesis in green plants that it is widely used in teachinglaboratories and books to demonstrate the process(Edwards and Walker 1983; Walker 1992, see pp. 55–58). Walker’s favorite starch picture was a reproduc-tion, on a geranium leaf, of a masterpiece ‘Innocence’by Pierre-Paul Proudhon. An example of a starch pic-ture using a modiﬁed version of Molisch’s techniqueis shown in Figure 2.

Imaging with chloroplast movements

Light-induced changes in the cellular distribution ororientation of chloroplasts have been observed inspecies of algae, mosses, ferns, and angiosperms.Under low light conditions, chloroplasts accumu-late along the cell walls that are perpendicular tothe incident light. Under high light conditions, theyaccumulate along the walls that are parallel to theincident light. These are the regions of plant leaf cells where internal fluence rates of light are thehighest and lowest, respectively, and it is believedlikely that the light-induced chloroplast movementsserve an adaptive function. In algae, moss andferns, both red and blue light can cause chloroplastmovements. In terrestrial angiosperms, chloroplastmovements have been shown to be blue-light-specific(Inoue and Shibata 1973) but the red/far-red phyto-chrome photoreceptors appear to play a role in mod-ulating the response (DeBlasio et al. 2003). In anyevent, chloroplast movements can have visible ef-fects on light transmittance and reflectance propertiesof leaves. For example, Wada and Sugai (1994) ex-posed ferngametophytesmaskedwith stenciled lettersto high fluence rates of light and produced visibleimages of the letters in the fern gametophytes. In amore recent study in which the blue light photore-ceptor phot2 was described, Kagawa et al. (2001; seetheir accompanyingcoverpicture)showedthat imagescould also be observed in

Arabidopsis

leaves. Thelight-inducedchloroplastmovementsare sensitive androbust enough that by using masks made from black and white photographs,it is possible to obtain detailedimages that can come surprisingly close to the reso-lution observable with the starch images, as shown inthe image of Norman Good in Figure 3. In the caseof chloroplast movement images, the grain size is thatof the individual chloroplasts rather than the starchgranules contained within them. Moreover, chloro-

Figure 3.

Image of Norman E. Good (1917–1992) in a leaf createdby light-induced chloroplast movements. Norman E. Good was aphotosynthesis pioneer who was instrumental in deciphering photo-phosphorylation and other segments of the photosynthetic electrontransfer chain. He also created the ‘Good buffers’ (see Hangarterand Ort 1992). To create this image, a

Coleus l

eaf was placed on awet paper towel, covered with a laser-printed transparency and illu-minated with a slide projector for 30min. The leaf was then photo-graphed using blue backlighting. The image in the living leaf resul-ted from light-dependent changes in the cellular position of chloro-plasts. The lighter areas are those that received the most light sothat the chloroplasts moved to the edges of the cells allowing morelight to be transmitted through the leaf in those areas. ‘Gray-scale’resolution is possible since in areas receiving non-saturating intens-ities, there is a gradient of chloroplast movement in different celllayers. See http://sunﬂower.bio.indiana.edu/

∼

rhangart/plantmotionfor more details about creating images with chloroplast movements.

plast movement-induced images can be observed inliving leaves without the need for chemical stains andthe same leaf can be used over and over to create newimages simply by changing the masking image andre-exposing the leaf to light.

Chlorophyll ﬂuorescence images