See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/259985690

Longitudinal Photosynthetic Gradient in Crust Lichens' Thalli

Article  in  Microbial Ecology · January 2014

DOI: 10.1007/s00248-014-0366-9 · Source: PubMed

CITATIONS

9READS

101

5 authors, including:

Some of the authors of this publication are also working on these related projects:

Investigating novel mechanisms in regulation of resistance to apoptosis in T-cell lymphoma and Acute Myeloid Leukemia View project

low dimensional photocatalyst View project

Li Wu

The Ohio State University

123 PUBLICATIONS   5,497 CITATIONS   

SEE PROFILE

Gaoke Zhang

Wuhan University of Technology

309 PUBLICATIONS   9,944 CITATIONS   

SEE PROFILE

Shubin Lan

Chinese Academy of Sciences

36 PUBLICATIONS   777 CITATIONS   

SEE PROFILE

Delu Zhang

Wuhan University of Technology

49 PUBLICATIONS   1,414 CITATIONS   

SEE PROFILE

All content following this page was uploaded by Delu Zhang on 03 December 2014.

The user has requested enhancement of the downloaded file.

https://www.researchgate.net/publication/259985690_Longitudinal_Photosynthetic_Gradient_in_Crust_Lichens%27_Thalli?enrichId=rgreq-6c89ecbc1d457fd4569315ac85d343d8-XXX&enrichSource=Y292ZXJQYWdlOzI1OTk4NTY5MDtBUzoxNzAzMDIyMjcwOTk2NDhAMTQxNzYxNDYyNDA5MQ%3D%3D&el=1_x_2&_esc=publicationCoverPdfhttps://www.researchgate.net/publication/259985690_Longitudinal_Photosynthetic_Gradient_in_Crust_Lichens%27_Thalli?enrichId=rgreq-6c89ecbc1d457fd4569315ac85d343d8-XXX&enrichSource=Y292ZXJQYWdlOzI1OTk4NTY5MDtBUzoxNzAzMDIyMjcwOTk2NDhAMTQxNzYxNDYyNDA5MQ%3D%3D&el=1_x_3&_esc=publicationCoverPdfhttps://www.researchgate.net/project/Investigating-novel-mechanisms-in-regulation-of-resistance-to-apoptosis-in-T-cell-lymphoma-and-Acute-Myeloid-Leukemia?enrichId=rgreq-6c89ecbc1d457fd4569315ac85d343d8-XXX&enrichSource=Y292ZXJQYWdlOzI1OTk4NTY5MDtBUzoxNzAzMDIyMjcwOTk2NDhAMTQxNzYxNDYyNDA5MQ%3D%3D&el=1_x_9&_esc=publicationCoverPdfhttps://www.researchgate.net/project/low-dimensional-photocatalyst?enrichId=rgreq-6c89ecbc1d457fd4569315ac85d343d8-XXX&enrichSource=Y292ZXJQYWdlOzI1OTk4NTY5MDtBUzoxNzAzMDIyMjcwOTk2NDhAMTQxNzYxNDYyNDA5MQ%3D%3D&el=1_x_9&_esc=publicationCoverPdfhttps://www.researchgate.net/?enrichId=rgreq-6c89ecbc1d457fd4569315ac85d343d8-XXX&enrichSource=Y292ZXJQYWdlOzI1OTk4NTY5MDtBUzoxNzAzMDIyMjcwOTk2NDhAMTQxNzYxNDYyNDA5MQ%3D%3D&el=1_x_1&_esc=publicationCoverPdfhttps://www.researchgate.net/profile/Li-Wu?enrichId=rgreq-6c89ecbc1d457fd4569315ac85d343d8-XXX&enrichSource=Y292ZXJQYWdlOzI1OTk4NTY5MDtBUzoxNzAzMDIyMjcwOTk2NDhAMTQxNzYxNDYyNDA5MQ%3D%3D&el=1_x_4&_esc=publicationCoverPdfhttps://www.researchgate.net/profile/Li-Wu?enrichId=rgreq-6c89ecbc1d457fd4569315ac85d343d8-XXX&enrichSource=Y292ZXJQYWdlOzI1OTk4NTY5MDtBUzoxNzAzMDIyMjcwOTk2NDhAMTQxNzYxNDYyNDA5MQ%3D%3D&el=1_x_5&_esc=publicationCoverPdfhttps://www.researchgate.net/institution/The-Ohio-State-University?enrichId=rgreq-6c89ecbc1d457fd4569315ac85d343d8-XXX&enrichSource=Y292ZXJQYWdlOzI1OTk4NTY5MDtBUzoxNzAzMDIyMjcwOTk2NDhAMTQxNzYxNDYyNDA5MQ%3D%3D&el=1_x_6&_esc=publicationCoverPdfhttps://www.researchgate.net/profile/Li-Wu?enrichId=rgreq-6c89ecbc1d457fd4569315ac85d343d8-XXX&enrichSource=Y292ZXJQYWdlOzI1OTk4NTY5MDtBUzoxNzAzMDIyMjcwOTk2NDhAMTQxNzYxNDYyNDA5MQ%3D%3D&el=1_x_7&_esc=publicationCoverPdfhttps://www.researchgate.net/profile/Gaoke-Zhang?enrichId=rgreq-6c89ecbc1d457fd4569315ac85d343d8-XXX&enrichSource=Y292ZXJQYWdlOzI1OTk4NTY5MDtBUzoxNzAzMDIyMjcwOTk2NDhAMTQxNzYxNDYyNDA5MQ%3D%3D&el=1_x_4&_esc=publicationCoverPdfhttps://www.researchgate.net/profile/Gaoke-Zhang?enrichId=rgreq-6c89ecbc1d457fd4569315ac85d343d8-XXX&enrichSource=Y292ZXJQYWdlOzI1OTk4NTY5MDtBUzoxNzAzMDIyMjcwOTk2NDhAMTQxNzYxNDYyNDA5MQ%3D%3D&el=1_x_5&_esc=publicationCoverPdfhttps://www.researchgate.net/institution/Wuhan-University-of-Technology?enrichId=rgreq-6c89ecbc1d457fd4569315ac85d343d8-XXX&enrichSource=Y292ZXJQYWdlOzI1OTk4NTY5MDtBUzoxNzAzMDIyMjcwOTk2NDhAMTQxNzYxNDYyNDA5MQ%3D%3D&el=1_x_6&_esc=publicationCoverPdfhttps://www.researchgate.net/profile/Gaoke-Zhang?enrichId=rgreq-6c89ecbc1d457fd4569315ac85d343d8-XXX&enrichSource=Y292ZXJQYWdlOzI1OTk4NTY5MDtBUzoxNzAzMDIyMjcwOTk2NDhAMTQxNzYxNDYyNDA5MQ%3D%3D&el=1_x_7&_esc=publicationCoverPdfhttps://www.researchgate.net/profile/Shubin-Lan?enrichId=rgreq-6c89ecbc1d457fd4569315ac85d343d8-XXX&enrichSource=Y292ZXJQYWdlOzI1OTk4NTY5MDtBUzoxNzAzMDIyMjcwOTk2NDhAMTQxNzYxNDYyNDA5MQ%3D%3D&el=1_x_4&_esc=publicationCoverPdfhttps://www.researchgate.net/profile/Shubin-Lan?enrichId=rgreq-6c89ecbc1d457fd4569315ac85d343d8-XXX&enrichSource=Y292ZXJQYWdlOzI1OTk4NTY5MDtBUzoxNzAzMDIyMjcwOTk2NDhAMTQxNzYxNDYyNDA5MQ%3D%3D&el=1_x_5&_esc=publicationCoverPdfhttps://www.researchgate.net/institution/Chinese_Academy_of_Sciences?enrichId=rgreq-6c89ecbc1d457fd4569315ac85d343d8-XXX&enrichSource=Y292ZXJQYWdlOzI1OTk4NTY5MDtBUzoxNzAzMDIyMjcwOTk2NDhAMTQxNzYxNDYyNDA5MQ%3D%3D&el=1_x_6&_esc=publicationCoverPdfhttps://www.researchgate.net/profile/Shubin-Lan?enrichId=rgreq-6c89ecbc1d457fd4569315ac85d343d8-XXX&enrichSource=Y292ZXJQYWdlOzI1OTk4NTY5MDtBUzoxNzAzMDIyMjcwOTk2NDhAMTQxNzYxNDYyNDA5MQ%3D%3D&el=1_x_7&_esc=publicationCoverPdfhttps://www.researchgate.net/profile/Delu-Zhang?enrichId=rgreq-6c89ecbc1d457fd4569315ac85d343d8-XXX&enrichSource=Y292ZXJQYWdlOzI1OTk4NTY5MDtBUzoxNzAzMDIyMjcwOTk2NDhAMTQxNzYxNDYyNDA5MQ%3D%3D&el=1_x_4&_esc=publicationCoverPdfhttps://www.researchgate.net/profile/Delu-Zhang?enrichId=rgreq-6c89ecbc1d457fd4569315ac85d343d8-XXX&enrichSource=Y292ZXJQYWdlOzI1OTk4NTY5MDtBUzoxNzAzMDIyMjcwOTk2NDhAMTQxNzYxNDYyNDA5MQ%3D%3D&el=1_x_5&_esc=publicationCoverPdfhttps://www.researchgate.net/institution/Wuhan-University-of-Technology?enrichId=rgreq-6c89ecbc1d457fd4569315ac85d343d8-XXX&enrichSource=Y292ZXJQYWdlOzI1OTk4NTY5MDtBUzoxNzAzMDIyMjcwOTk2NDhAMTQxNzYxNDYyNDA5MQ%3D%3D&el=1_x_6&_esc=publicationCoverPdfhttps://www.researchgate.net/profile/Delu-Zhang?enrichId=rgreq-6c89ecbc1d457fd4569315ac85d343d8-XXX&enrichSource=Y292ZXJQYWdlOzI1OTk4NTY5MDtBUzoxNzAzMDIyMjcwOTk2NDhAMTQxNzYxNDYyNDA5MQ%3D%3D&el=1_x_7&_esc=publicationCoverPdfhttps://www.researchgate.net/profile/Delu-Zhang?enrichId=rgreq-6c89ecbc1d457fd4569315ac85d343d8-XXX&enrichSource=Y292ZXJQYWdlOzI1OTk4NTY5MDtBUzoxNzAzMDIyMjcwOTk2NDhAMTQxNzYxNDYyNDA5MQ%3D%3D&el=1_x_10&_esc=publicationCoverPdf

SOIL MICROBIOLOGY

Longitudinal Photosynthetic Gradient in Crust Lichens’ Thalli

Li Wu & Gaoke Zhang & Shubin Lan & Delu Zhang &Chunxiang Hu

Received: 10 September 2013 /Accepted: 6 January 2014 /Published online: 30 January 2014# Springer Science+Business Media New York 2014

Abstract In order to evaluate the self-shading protection forinner photobionts, the photosynthetic activities of three crustlichens were detected using Microscope-Imaging-PAM. Thefalse color images showed that longitudinal photosyntheticgradient was found in both the green algal lichen Placidiumsp. and the cyanolichen Peltula sp. In longitudinal direction,all the four chlorophyll fluorescence parameters Fv/Fm, Yield,qP, and rETR gradually decreased with depth in the thalli ofboth of these two lichens. In Placidium sp., qN values de-creased with depth, whereas an opposite trend was found inPeltula sp. However, no such photosynthetic heterogeneitywas found in the thalli ofCollema sp. in longitudinal direction.Microscope observation showed that photobiont cells arecompactly arranged in Placidium sp. and Peltula sp. whileloosely distributed in Collema sp. It was considered that thelongitudinal photosynthetic heterogeneity was ascribed to theresult of gradual decrease of incidence caused by the compactarrangement of photobiont cells in the thalli. The resultsindicate a good protection from the self-shading for the innerphotobionts against high radiation in crust lichens.

Introduction

As an important component of biological soil crusts (BSCs),lichens are exposed to harsh desert stresses in global arid andsemiarid regions, where extremely strong radiation (includingboth UV and visible light) always brings damages to living

cells. Excess energy caught by light-harvesting complexes caninduce the formation of triplet excited chlorophyll molecules(3Chl*) and also the reactive oxygen species (ROS) [1], whichcan attack photosynthetic reaction centers and cause the deg-radation of D1 proteins of photosystem II (PSII) [2]. Someother studies also propose that light can directly affect oxygen-evolving complexes, and ROS inhibits the repair of damagedPSII by suppressing the de novo synthesis of proteins [2]. As amutagenic factor, UV can cause damages to DNA, lipids, andproteins [3]. Even under no stress conditions, ROS can also beproduced by normal metabolic activities such as respiration andphotosynthesis, and enhanced by environmental stresses [4].

To cope with high radiation, lichens develop many strate-gies. Unlike higher plants, lichens lack epidermic cells con-taining high content of flavonoid polyphenolics to resist highradiation [5, 6]. Compact cortex formed by symbiotic fungicompensates this lack in some extent. All kinds of UV-screening compounds, such as scytonemin [7], mycosporine-like amino acids [7], phenolics [8, 9], melanin, and parietin[10], secreted by lichens can effectively reduce the transmis-sion of radiation [8, 11], and the reducing effect is moreeffective in dehydrated thalli [11]. Buffoni Hall et al. foundthat just due to the accumulation of UV-screening compoundphenolics in Cladonia arbuscula (Wallr.) Flot. ssp. mitis(Sandst.) Ruoss, the UV transmission in the thalli was effec-tively reduced; therefore, both the UV radiation at 280 and310 nm decreased to 0 at 30–40 μm depth in the thalli [8].Gauslaa and Solhaug [11] found that more UV-screeningpigments are deposited in sun-exposed Lobaria pulmonaria,so that sun-exposed thalli had lower transmission efficiencythan the shade-adapted ones. The transmission efficiency ofUV radiation at 280–340 nm was about 2 % in the sun-exposed thalli (both the hydrated and dehydrated ones),whereas the transmission efficiency gradually increased withwavelength and reached a peak (approximately 57 %) at600 nm [11]. Other research demonstrates that the apotheciain Teloschistes lacunosus (P. Rupr.) Savicz (the current nameis Seirophora lacunosa (Rupr.) Frödén) quench as high as91.5 % of the incident photosynthetically active radiation

L. Wu :G. ZhangSchool of Resources and Environmental Engineering, WuhanUniversity of Technology, Wuhan 430072, China

L. Wu : S. Lan : C. Hu (*)Key Laboratory of Algal Biology, Institute of Hydrobiology, ChineseAcademy of Sciences, Wuhan 430072, Chinae-mail: cxhu@ihb.ac.cn

D. ZhangSchool of Sciences, Wuhan University of Technology,Wuhan 430070, China

Microb Ecol (2014) 67:888–896DOI 10.1007/s00248-014-0366-9

(PAR) [12]. Lichens can also dissipate excess energy caughtby antennae in the form of nonradiation energy [13–16].

For crust lichens in desert regions, high radiation is acommon and inescapable stress. Together with other environ-mental stresses, high radiation severely restricts the survivaland development of organisms in these regions. Althoughlichens developed many adaptation strategies to high radia-tion, Gauslaa and Solhaug [11] considered that inner symbi-otic algae received rather incomplete protection [8]. Actually,different lichen species are different in thallus thickness, cor-tex, pigments types and contents, and photobiont species andcompaction, and these differences are likely to lead to differ-ent adaptation abilities and physical characteristics. Therefore,this study detects the photosynthetic activities of differentlichen thalli in longitudinal directions, aiming at evaluatingthe self-shading protection given to the phototrophic cells bytheir position in the thalli against high radiation in desertenvironments.

Materials and Methods

Samples

All the samples used in this study were collected from anonirrigated area on the north side of the railway at theShapotou Desert Research and Experimentation Station ofthe Chinese Academy of Sciences (37°32′N and 105°02′E, Tengger Desert, Ningxia Hui Autonomous Region ofChina). The samples were collected in June 2009, and thisexperiment was conducted in November 2010. The crustsamples were kept in the desiccators after collection.According to our field investigation, this area is dominat-ed by cyanolichen soil crusts, more than 80 % of whichare Collema spp. Other cyanolichens and green algallichens only occupy a small proportion. Thereby threecrust lichens were selected in this experiment, includingtwo cyanolichens, Collema sp. and Peltula sp., respective-ly, and a green algal lichen, Placidium sp. The details areshown in Table 1.

Methods

Photosynthetic Recovery of Crust Lichens and SamplePreparation

The selected lichen soil crusts with intact lichens were fullyrehydrated with sterilized distilled water and then weretransported to a greenhouse (25±2 °C) to naturally dry again(for 2 days). This process was defined as a rehydration/desiccation cycle. A cool white light lamp was used to supplylight. Then these crusts that experienced rehydration/desiccation cycle as described were rehydrated to recover their

photosynthetic activities in the light (40 μmol photonsm−2 s−1).After a recovery period of about 42–48 h, the lichen thalli wereseparated from the crusts and sliced into thin pieces in longitu-dinal direction with a scalpel under stereomicroscope. Thenthese slices were put on the slides and detected withMicroscope-Imaging-PAM later.

Table 1 Characteristics of the three selected crust lichens

Lichen Type Color Photoboint Coverageon crusts

Placidium sp. Squamulose Brown green alga

Chlorophyll a Fluorescence Measurement

Microscope-Imaging-PAM was used to detect the photosyn-thetic activities of crust lichens in this experiment. Differentfrom other versions of pulse amplitude modulation (PAM)fluorometry, Microscope-Imaging-PAM connects with a mi-croscope, so that it allows a rather small fluorescence imagingarea (830×613 μm), using false color images to exhibit theheterogeneity of a chlorophyll fluorescence parameter overthe whole imaging area.

After a period of dark adaptation (about 10 min), aweak light lower than 1 μmol photons m−2 s−1 wasapplied to induce the minimal fluorescence Fo, and thena saturating light pulse ~3000 μmol photons m−2 s−1 wasused to determine the maximal fluorescence Fm. Thesetwo fluorescence values (Fo and Fm) were used to calcu-late several chlorophyll fluorescence parameters in the fol-lowingmeasurement.When illuminated for a period of time, asaturating light pulse (3000 μmol photons m−2 s−1) was alsosupplied to determine Fm’, and the stable fluorescence Fsunder this light intensity would also be recorded at the sametime. Different chlorophyll fluorescence parameters,reflecting the photosynthetic activities of PSII, are calculatedas follows:

Fv = Fm ¼ Fm−Foð Þ = FmYield ΦIIð Þ ¼ Fm’−Fsð Þ=Fm’rETR ¼ Yield � PARqP ¼ Fm’−Fsð Þ= Fm’−Fo’ð ÞqN ¼ Fm−Fm’ð Þ= Fm−Fo’ð Þ

Fo’ is the minimum fluorescence yield in light-adaptedstate, and it cannot be imaged with the existing imagingsystem. Therefore, Fo’ is estimated through an equation ac-cording to Oxborough and Baker [17]. All these chlorophyllfluorescence parameters will be calculated by software auto-matically and presented in the form of false color images.

Fv/Fm is the maximum quantum yield of PSII primaryphotochemistry [18], and is always stable in unstressed plants[19]. Yield (ФPSII) is the effective quantum yield of PSII in thelight, reflecting the light energy that is absorbed by PSII andused in photochemistry [17, 20]. Both Fv/Fm and Yield are thecharacteristics of photosystems and are not linked with theamount of chlorophyll [21]. Therefore, the estimation of elec-tron transport rate is a relative one, defined as rETR, giving anoverall estimation of the photosynthetic performance. The“photochemical quenching” qP is, by definition, a value closeto Yield superficially, but not exactly the same. Yield is theabsorbed light energy being used in photochemistry, while qPprovides an indication of open PSII centers under a certainlight [17, 20]. The parameter qN refers to the energy dissipa-tion in the form of heat [22], reflecting the protective abilityagainst high radiation.

Rapid light curve (RLC) is a powerful tool to assess pho-tosynthetic activities. RLC can assess not only the presentphotosynthetic capacity but also the samples’ potential activ-ity over a wide range of ambient light [21]. RLC shows therETR variations as a function of PAR. In our experiment, ninePAR levels (0, 17, 26, 53, 81, 154, 200, 255, and310 μmol photons m−2 s−1) were set with a preinstalledsoftware routine, which was used to measure the RLC. Theillumination duration for each PAR level was 10 s. One thing

.50

.55

.60

.65

.70

.1

.2

.3

.4

.5

0.00 .02 .04 .06 .08 .10 .12 .14.2

.3

.4

.5

.6

.7

.8

0.00 .02 .04 .06 .08 .10 .12 .14.30

.35

.40

.45

.50

.55

Fv/

Fm

Yie

ld

qP qN

Depth (mm) Depth (mm)

y= -59.72x3+15.27x2-1.842x+0.668

R2=0.93

y= 12.39x2-3.773x+0.446

R2=0.984

y=17.51x2-5.81x+0.795

R2=0.978

y=175.3x3-29.46x2-0.326x+0.528

R2=0.905

Fig. 2 Curve estimation betweendifferent chlorophyll fluorescenceparameters and the depth ofphotoboint layer in Placidium sp.at 108 μmol photons m−2 s−1 (theupper border of photoboint layerwas treated as 0-μm depth)

890 L. Wu et al.

that must be pointed was that the Fv/Fm values were deter-mined in the dark after 10-min dark adaptation, and wedisplayed the result together with Yield, and the result ofYield at 0 μmol m−2 s−1 was actually the result of Fv/Fm. Infact, Yield is the absorbed light energy being used in photo-chemistry; therefore, the actual Yield value was 0 in the dark.

Data Analysis

Variance of each parameter between the upper and lowerlayers of a lichen thallus from low- to high-PAR level wasanalyzed using paired samples t test. Variance of each param-eter between different layers of lichen thalli was analyzedusing one-way ANOVA. All the above data analyses werecarried out using SPSS 13.0. The curve estimations betweenthe chlorophyll fluorescence parameters and the depth oflichen thallus were carried out using Sigmaplot 11.0.

Results

In this study, more than five replicate samples were studied foreach lichen species, and similar trends were found amongdifferent replicates for each lichen species. Therefore, we justshowed the false color results of photosynthetic characteristicsof one sample for each lichen species.

Placidium sp.

The longitudinal photosynthetic gradient was evident inPlacidium sp. thallus, as shown in Fig. 1. This figure showsthe false color images of three parameters (Yield, qP, and qN) inPlacidium sp. during the RLC measurement. Different colorsrepresented different values of the chlorophyll fluorescenceparameters. The results showed the obvious heterogeneity ofeach chlorophyll fluorescence parameter in longitudinal direc-tion under a certain PAR level. Especially, once PAR exceeded53 μmol photons m−2 s−1, the heterogeneity was particularlyobvious. At each radiation intensity, the upper layer ofphotobiont had the higher Yield, qP, and qN values than therelative lower layer (P

and the lower band was red. Once the PAR reached154 μmol photons m−2 s−1, the upper half band was red andthe lower half disappeared. The results of curve estimationbetween fluorescence parameters and the depth of thephotobiont layer (we took the results at 81 μmol m−2 s−1 foran example) showed that all the three parameters, Fv/Fm,Yield, and qP, linearly decreased with the depth of photobiontlayer, whereas qN gradually increased with the depth ofphotobiont layer, and the increasing rate of early stage(depth

40- to 60-μm thickness of the thalli in Peltula sp., so that thislayer showed brown color, whereas the photobiont cells underthis thickness showed blue-green color without any depositedUV-screening pigment (Fig. 7c). Alcian blue staining resultshowed that Collema sp. lacked the upper cortex, and theupper surface contained polysaccharide materials, but notthe symbiotic hyphae (Fig. 7f, white arrow). Dark brownUV-screening pigments mainly concentrated the thallus sur-face of Collema sp. (Fig. 7e), while it was also found that theinner symbiotic cyanobacterial filaments did not show freshblue-green color as the photobiont of Peltula sp. (Fig. 7c, e).

Discussion

Longitudinal stratification is a common phenomenon inBSCs, and light is an important environmental factor affecting

the distribution of photosynthetic organisms [23–25].Different from most of the crust organisms, lichens directlydistribute on the crust surface, suffering high radiation un-avoidably. Self-shading is an important strategy for lichens toresist high radiation including both UV and visible light.Because of the gradual attenuation of incident light, thephotobiont cells of the upper layer receive more light thanthe lower layer in lichen thalli. Our results showed that Fv/Fm,Yield, and qP gradually decreased with the depth in bothPlacidium sp. and Peltula sp. thalli under a certain PAR level(from low to high PAR). The results imply that crust lichensreceive a relative complete protection from self-shading effectagainst high-light stress (including visible light and UV-radiation).

There are many studies on the self-shading effects in li-chen’s resistant ability against high radiation [7–11]. Gauslaaand Solhaug [11] considered that the inner photobiont cells ofthe sun-exposed thalli received rather incomplete protection.However, our results seemingly do not support their view-point. If the protection is not complete, the relative upper layershould have lower Fv/Fm, Yield, and qP values, because theyreceive much more solar radiation (especially of UV) than thelower layer. The fact is just the opposite. In both Placidium sp.and Peltula sp., the Yield and qP values gradually decreasedwith thallus depth in the longitudinal direction from the low tohigh PAR, so did Fv/Fm. Additionally, we also found that UV-screening pigments deposited in the upper 40- to 60-μmthickness of the thalli in Peltula sp., and no such pigmentwas found around the photobiont cells below this depth. Aswe know, it is an important strategy for cyanobacteriumChroococcidiopsis to secrete scytonemin, a brown pigment,in resisting UV stress [7, 26]. Therefore, we consider thatphotobiont in Peltula sp. receives protection against UV radi-ation well, that is the self-shading protection for crust lichens’photobionts is complete. Other researchers’ study also dem-onstrated that the UV radiation at 280 and 310 nm decreasedto 0 at 30- to 40-μm depth in lichen thalli [8], and this depthwas basically coincident with both the pigment-depositeddepth in Peltula sp. and the upper cortex thickness inPlacidium sp. in the present study.

As far as we know, it is the first study on the longitudinalphotosynthetic characteristics of lichens, and it provides somenew information that differs from previous observations.Although many experts have detected the radiation attenua-tion within the lichen thalli [8, 11, 27], the effect of attenuatedlight on photobiont has not yet been evaluated. The possibilitywas once proposed that both the position and thickness ofphotobiont layer in the lichen thallus could be determined bythe PAR attenuation [27]. The PAR attenuation at the upperborder of the photobiont layer would be low enough and at thelower border, it would be high enough (above the photosyn-thetic compensation point) for the growth of photobionts [27].Our results of the gradual decreasing photosynthetic activity

Fig. 5 Series of images (Yield, qP, and qN) measured during a rapid lightcurve (RLC) of cyanolichen Collema sp. over seven PAR levels. Differentcolors (bar at the bottom) indicate different values of each parameter, and themaximum value of each parameter has been adjusted to 1. All these picturesare a longitudinal section of the lichen thallus with its uppermost surface up

Longitudinal Photosynthetic Gradient in Crust Lichens’ Thalli 893

in the longitudinal direction imply that the PAR intensity at theupper border of the photobiont layer was not high enough tocause obvious damages to photobiont cells. Additionally, a

significantly inverse relationship between the amount of sym-biotic alga and the intracellular chlorophyll concentration wasfound in the lichen family Umbilicariaceae, and this result was

Yie

ld

0.0

.1

.2

.3

.4

.5

.6

rET

R

0

2

4

6

8

IsidiumUpper layerLower layer

0 50 100 150 200 250 300 0 50 100 150 200 250 300

qP

-.2

0.0

.2

.4

.6

.8

1.0

qN0.0

.2

.4

.6

IsidiumUpper layerLower layer

IsidiumUpper layerLower layer

IsidiumUpper layerLower layer

Isidium

Upper layer

Lower layer

PAR µ mol photons m-2 s-1 PAR µ mol photons m-2 s-1

Fig. 6 Change of chlorophyll fluorescence parameters (Yield, rETR, qPand qN) of Collema sp. thallus under different photosynthetically activeradiation (PAR). For each parameter, the data change is compared among

the isidium, upper layer and lower layer, three different regions of thethallus. In each region (the isidium, upper layer or lower layer), three tofour data areas are selected as shown in the colored picture on the right

100 µm 30 µm

30 µm

100 µm 10 µm

30 µm

a b

c d

fe

Fig. 7 The inner structures of thethree crust lichens. a, bPlacidiumsp., c, dPeltula sp., and e, fCollema sp. b, d the magnificationpictures of the circled areas in aandc, respectively. The red arrow in bindicates the polysaccharides layeroutside the upper cortex; the whiteone indicates the upper cortex withdeposited UV-screening pigments.The red arrow in c indicates thephotoboint cells that showed theblue-green color. The arrow in dindicates the photoboint cells closeto the uppermost surface of lichenthallus. e the picture of Collema sp.under microscope without anytreatment, and fwas stained withAlcian blue. The arrows in e andf indicate the filaments of thephotoboint distributing within thepolysaccharides matrix of thethallus

894 L. Wu et al.

ascribed to the mechanism of avoiding the excess loss ofvisible light caused by pigment self-shading [28]. Both ourresults in this study and other results mentioned above supportour viewpoint that inner photobiont cells receive relativecomplete protection against high radiation including both theUVand visible radiation.

The upper layer having higher photosynthetic activitiessuggests a good adaptation of crust lichens to desert environ-ments. It has been demonstrated that the light compensation ofcrust lichens will change with the water content; therefore, nomatter how limited the water resource is, it still can be effec-tively used by crust lichens to fix carbon [29]. When limitedwater occurs, the upper layer of photobiont with high photo-synthetic efficiency has an advantage to activate photosyn-thetic activity preferentially, so that more carbon will be fixed,and this characteristic maybe is an important strategy for thesurvival and development of crust lichens in desert regions.

Our results showed that not all the crust lichens had thelongitudinal photosynthetic gradient phenomenon. The chlo-rophyll fluorescence parameters Fv/Fm, Yield, and qP did notexhibit obvious change with the depth in Collema sp. thalli.We ascribe the difference in longitudinal photosynthetic gra-dient between lichen species to the different inner structures ofthe thalli, and it has no relationship with the specific species ofthe photobiont (a cyanobacterium or a green alga). The obser-vation results under microscope showed that the photobiontcells are compactly arranged in Placidium sp. and Peltula sp.thalli, and UV-screening pigments are mainly distributed inthe upper 40-μm thickness (brown), under which photobiontcells showed green (Placidium sp.) or blue-green color(Peltula sp.). However, photobiont cells are loosely distributedin Collema sp. The brown UV-screening pigments are mainlyconcentrated in the thallus’ surface, while the inner symbioticcyanobacterial filaments did not show fresh blue-green coloras the photobiont in Peltula sp., and this result was ascribed tothe UV-screening pigments secretion around thesecyanobacterial filaments. In addition, the brown UV-screening pigments were also found near the lower surfaceof Collema sp. thalli (result not shown here). In some otherspecies of the same genus Collema having much thicker thallithan the species used in this study, we also found the photo-synthetic gradient phenomenon in longitudinal direction (datanot shown). Therefore, we speculate that the lower photobiontcell density leading to relative homogeneous light circum-stance may be the cause of the disappearance of photosynthet-ic gradient in Collema sp. Additionally, the photosyntheticgradient phenomenon is also found in several other crustlichens in the same area (data not shown); therefore, it is acommon but not absolute characteristic.

The results of microscope observation of the lichen struc-tures also showed us an interesting phenomenon: all the crustcyanolichens in our study field lacked upper cortices, and theupper border of the photobiont layer was quite near the

uppermost surface of the thalli. To the contrary, green algallichens’ photobionts receive protection from the upper corti-ces, where UV-screening pigments are deposited. Thecyanolichens found in our study area were with Scytonemasp., Nostoc sp., and Chroococcidiopsis sp., as photobionts,respectively. According to our observation, besides forminglichens with fungi, the free-living forms of the above threecyanobacteria are also widely distributed in arid and semiaridregions [30, 31]. Especially, Nostoc and Scytonema are evendirectly distributed on crust surface [24, 25, 31], while greenalgae are distributed at the relative deeper depth [24]. Theseresults imply that free-living cyanobacteria have a strongability in resisting high radiation in desert regions. However,water ecosystems are considered to be the evolutionary originof green algae, and their free-living forms without the protec-tion from symbiosis rarely dominate purely terrestrial ecosys-tems [11]. From this perspective, the protection from fungi isindispensable for the green algal photobiont, whereas is non-essential for cyanobiont of crust lichens. Therefore, we spec-ulate that the symbiotic relationship is not uniform for alllichen species, and it may have a close relationship with thespecies of inner photobionts.

In general, longitudinal gradient phenomenon exists notonly in the whole BSCs system [24, 25], but also in single-crust organisms such as the lichens in this study. The photo-synthetic gradient in crust lichens is the intuitive reflection ofself-shading protection given to the phototrophic cells by theirposition in the thalli. Additionally, our study implies that innerphotobiont cells of crust lichens receive complete protectionfrom self-shading effect against high radiation in desertregions.

Acknowledgments This study was kindly supported by grants fromthe China Postdoctoral Science Foundation (2013 M542077), NationalNatural Science Foundation of China (Nos. 31300100 and 31170464),and National Forestry Public Welfare Industry Research project(201404204).

References

1. Foyer CH, Lelandais M, Kunert KJ (1994) Photooxidative stress inplants. Physiol Plant 92(4):696–717

2. Murata N, Takahashi S, Nishiyama Y, Allakhverdiev SI (2007)Photoinhibition of photosystem II under environmental stress.BBA-Bioenergetics 1767:414–421

3. He YY, Häder P (2002) Reactive oxygen species and UV-B: effect oncyanobacteria. Photochem. Photobio Sci 1:729–736

4. Weissman L, Garty J, Hochman A (2005) Rehydration of the lichenRamalina lacera results in production of reactive oxygen species andnitric oxide and a decrease in antioxidants. Appl Environ Microbiol7(4):2121–2129

5. Li J, Ou–Lee TM, Raba R, Amundson RG, Last RL (1993)Arabidopsis flavonoid mutants are hypersensitive to UV-B irradia-tion. Plant Cell 5:171–179

Longitudinal Photosynthetic Gradient in Crust Lichens’ Thalli 895

6. Hutzler P, Fischbach R, Heller W, Jungblut TP, Reuber S, Schmitz R,Veit M, Weissenbock G, Schnitzler J-P (1998) Tissue localization ofphenolic compounds in plants by confocal laser scanning microsco-py. J Exp Bot 49:953–965

7. Büdel B, Karsten U, Garcia-Pichel F (1997) Ultraviolet-absorbing scytonemin and mycosporine-like amino acid deriv-atives in exposed, rock-inhabiting cyanobacterial lichens.Oecologia 112:165–172

8. Buffoni Hall RS, Bornman JF, Björn LO (2002) UV-induced changesin pigment content and light penetration in the fruticose lichenCladonia arbuscula ssp. Mitis. J Photochem Photobiol B 66:13–20

9. Bjerke JW, Gwynn–Jones D, Callaghan TV (2005) Effects of en-hanced UV-B radiation in the field on the concentration of phenolicsand chlorophyll fluorescence in two boreal and arctic–alpine lichens.Environ Exp Bot 53:139–149

10. SolhaugKA,Gauslaa Y, Nybakken L, BilgerW (2003) UV-inductionof sun-screening pigments in lichens. New Phytol 158:91–100

11. Gauslaa Y, Solhaug KA (2001) Fungal melanins as a sun screen forsymbiotic green algae in the lichen Lobaria pulmonaria. Oecologia126:462–471

12. Del Prado R, Sancho LG, Kappen L (2001) Photosynthetic perfor-mance of photosymbiotic ascomata in the lichen Teloschisteslacunosus. Flora 196:261–268

13. Demmig–Adams B, Adams WW III, Green TGA, Czygan F-C,Lange OL (1990) Differences in the susceptibility to light stress intwo lichens forming a phycosymbiodeme, one partner possessing andone lacking the xanthophyll cycle. Oecologia 84:451–456

14. Heber U, Azarkovich M, Shuvalov V (2007) Activation of mecha-nisms of photoprotection by desiccation and by light: poikilohydricphotoautotrophs. J Exp Bot 58(11):2745–2759

15. Kopecky J, Azarkovich M, Pfündel EE, Shuvalov VA (2005) HeberU Thermal dissipation of light energy is regulated differently and bydifferent mechanisms in lichens and higher plants. Plant Biol 7:156–167

16. Sundberg B, Campbell D, Palmqvist K (1997) Predicting CO2 gainand photosynthetic light acclimation from fluorescence yield andquenching in cyano-lichens. Planta 201:138–145

17. Oxborough K, Baker NR (1997) Resolving chlorophyll a fluores-cence images of photosynthetic efficiency into photochemical andnon-photochemical components calculation of qP and Fv’/Fm’without measuring Fo’. Photosynth Res 54:135–142

18. Baker NR (2008) Chlorophyll fluorescence: a probe of photosynthe-sis in vivo. Annu Rev Plant Biol 59:89–113

19. Björkman O, Demming B (1987) Photon yield of O2 evolution andchlorophyll fluorescence characteristics at 77°K among vascularplants of diverse origins. Planta 170:489–504

20. Maxwell K, Johnson GN (2000) Chlorophyll fluorescence—a prac-tical guide. J Exp Bot 51(345):659–668

21. Ralph PJ, Gademann R (2005) Rapid light curves: A powerful tool toassess photosynthetic activity. Aquat Bot 82:222–237

22. White AJ, Critchley C (1999) Rapid light curves: A new fluorescencemethod to assess the state of the photosynthetic apparatus.Photosynth Res 59:63–72

23. Garcia–Pichel F, Johnson SL, Youngkin D, Belnap J (2003) Small-scale longitudinal distribution of bacterial biomass and diversity inbiological soil crusts from arid lands in the Colorado Plateau. MicrobEcol 46:312–321

24. Hu CX, Zhang DL, Huang ZB, Liu YD (2003) The longitudinalmicrodistribution of cyanobacteria and green algae within desertcrusts and the development of the algal crusts. Plant Soil 257:97–111

25. Wu L, Lan SB, Zhang DL, Hu CX (2011) Small-scale longitudinaldistribution of algae and structure of lichen soil crusts. Microb Ecol62:715–724

26. Dillon JG, Tatsumi CM, Tandingan PG, Castenholz RW (2002)Effect of environmental factors on the synthesis of scytonemin, aUV-screening pigment, in a cyanobacterium (Chroococcidiopsis sp.).Arch Microbiol 177:322–331

27. Büdel B, LangeOL (1994) The role of cortical and epinecral layers inthe lichen genus Peltula. Cryptogam Bot 4:262–269

28. Valladares F, Sancho LG, Ascaso C (1996) Functional analysis of theintrathalline and intracellular chlorophyll concentrations in the lichenfamily Umbilicariaceae. Ann Bot 78(4):471–477

29. Lange OL, Green TGA, Melzer B, Meyer A, Zellner H (2006) Waterrelations and CO2 exchange of the terrestrial lichen Teloschistescapensis in the Namib fog desert: Measurements during two seasonsin the field and under controlled conditions. Flora 201:268–280

30. Caiola M.G., Billi D.: Chroococcidiopsis from desert to mars. In:Seckbach J (ed) Algae and cyanobacteria in extreme environments.Springer, pp 555–568 (2007)

31. Lan SB, Wu L, Zhang DL, Hu CX (2012) Successional stages ofbiological soil crusts and their microstructure variability in Shapotouregion (China). Environ Earth Sci 65:77–88

896 L. Wu et al.

View publication statsView publication stats

https://www.researchgate.net/publication/259985690

Longitudinal Photosynthetic Gradient in Crust Lichens’ ThalliAbstractIntroductionMaterials and MethodsSamplesMethodsPhotosynthetic Recovery of Crust Lichens and Sample PreparationChlorophyll a Fluorescence MeasurementData Analysis

ResultsPlacidium sp.Peltula sp.Collema sp.Inner Structures of Crust Lichens

DiscussionReferences