Botanical Studies (2010) 51: 325-336.
PHYSIOLOGY
Stomatal clustering, a new marker for environmental
perception and adaptation in terrestrial plants
Yi GAN, Lei ZHOU, Zhong-Ji SHEN, Zhu-Xia SHEN, Yi-Qiong ZHANG, and Gen-Xuan WANG*
Institute ofArgo-Ecology and Eco-Engineering, College of Life Science, Zhejiang University, Hangzhou, 310058, P. R.
China
(Received February 12, 2008; Accepted February 26, 2010)
ABSTRACT. "Stomatal clustering," an abnormal stomatal patterning that is formed by two or more stomata in the leaf epidermis, has been reported in more than 60 species of terrestrial plants. According to the charac­ters and distributional pattern, two different types of stomatal clusters were identified, However, calculation of R-values in 16 different plant species showed that the classical method in spatial ecology study could not distinguish between these two types of clusters. Therefore, to classify them, the term "contiguous cluster" and "non-contiguous cluster" were introduced. Their formation and ecological significance were also discussed. In order to study whether stomatal clustering occurs in response to the environment, Vicia faba L. were culti­vated under different water/salinity levels. Epidermis bioassay was conducted 2 weeks after the treatment. The results showed that drought and salt stresses significantly increased the stomatal density and stomatal index. More importantly, the occurrences of contiguous stomatal clustering also raised along the drought/salt gradi­ents. The result suggests that the stomatal clustering is correlated with environmental signals. It could serve as a new marker for environmental adaptation in terrestrial plants.
Keywords: Environmental stress; One cell spacing rule; Stomatal clustering; Stomatal development; Vicia faba L.
INTRODUCTION
Stomata are small pores located on the abaxial and adaxial sides of plant leaves; they are composed of a pair of guard cells and several subsidiary cells. This highly specialized cellular apparatus regulates the plant's car­bon dioxide assimilation and water vapor loss through changes in guard cell turgor and stomatal aperture. This is of great importance to the global carbon-water cycle and to the plant's ability to respond to environmental change (Willmer and Fricker, 1996; Hetherington and Woodward, 2003). For the past 50 years, it has been well known that the two-dimensional stomatal distribution during leaf ex­pansion is closely correlated with living environments: plants growing in arid areas tend to have fewer stomata (Croxdale, 2000; Zhang et al., 2003). Elevation of atmo­spheric carbon dioxide concentration often results in lower stomatal density (Woodward, 1987). At any rate, in most terrestrial plants, planar stomatal distribution is not random (Larkin et al., 1997). There exists a stomata-free region around each stoma, which means at least one intervening epidermal cell is always placed between two neighboring stomata. This is also known as the "one cell spacing rule" which minimizes the overlaps of stomatal gaseous diffu­sion shells (GDS) and thus ensures the optimal balance
*Corresponding author: E-mail: wanggx@zju.edu.cn; Tel: 86-0571-88206590; Fax: 86-0571-88206590.
between water loss and carbon assimilation (Korn, 1993;
Larkin et al., 1997).
However, abnormal stomatal patterning, or "stomatal cluster" as it is known, has been reported successively in certain species of Begonia, Crassulaceae, Sonnerati-aceae, and Moraceae (Payne, 1970; Metcalfe and Chalk, 1979; Hoover, 1986; Chen, 1996; Tang et al., 2002). This distribution is quite different from the normal stomatal patterns (Payne, 1970). Such stomatal clusters were re­ported in more than 60 species in Gymnosperm, Dicots, and Monocots (Metcalfe and Chalk, 1979) (see Appen­dix). The classification and morphological characters of those clusters have received less attention in recent de­cades. Interestingly, after a review of the literature and a microscopic observation, we actually found two types of stomatal clusters among those plants: type A clusters have 2 (or more) stomata placed in direct contact (without inter­vening epidermal cells between neighboring guard cells), such as: Alysicarpus procumbens Schindl. (Papilionaceae) (Kothari and Shah, 1975; Nilamoni and Parukutty, 1979),
Sonneratiaceae. alba J. Smith (Chen, 1996), Annonaceae. hongkongensis (Sun et al., 1999). While type B clusters are formed by groups of stomata that do not contact with each other (they are separated by the subsidiary cells). Those plants are Crassulaceae. lineare (Zheng and Gong, 1999), Himantandra parvifolia Bak (Baranova, 1972) and so on (see Appendix 1).
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These two types of clusters are still not well classified: the term "stomatal cluster" has been used for both types in many studies (Sachs, 1994; Tang et al., 2002; Zhao et al., 2006). Therefore, a clear definition and classification must be redrawn. In the present study, we collected the leaves of 16 plant species which contain normal stomatal patterning and the two types of clusters. Then we used the classical methods which were developed to assess stomatal distribution in ecological studies (Clark and Evans, 1954; Korn, 1993) to evaluate the difference between thesetwo stomatal cluster types.
Interestingly, many plants found to have stomatal clusters were living in arid, salty, or otherwise adverse environments: laterally contiguous stomata were found on the epidermis of Melilotus suaveolens Ledeb., which was sampled on the beach at Hangzhou Bay (Figure 1D). Similar clusters were also reported in a typical halophyte Sonneratia alba J. Smith (Sonneratiaceae) (Chen, 1996). Sedum dfredii Hance, which exhibits stomatal clusters, is a typical CAM plant with strong stress tolerance (Payne, 1970). However, fewer studies have been conducted to address questions like why so many plants bear stomatal
Figure 1. Stomatal clustering in various plant species: (A) Nail polisher imprint show laterally contiguous stomata in Cinnamo-mum camphora (Lauraceae); (B) Non-contiguous stomatal clusters in Rieger begonia (Begoniaceae); (C) Non-contiguous stomatal clusters in Sedum dfredii Hance (Crassulaceae); (D) Polarly con­tiguous stomata occasionally seen in Melilotus sua^veolens Ledeb (Leguminosae); (E) Contiguous stomatal clustering in the leaf epidermis of Vicia faba Linn. (Le-guminosae) under water stress; (F) Laterally contiguous stomata occasionally seen in Arabidopsis thaliana L. (Brassicaceae) Col-0 ecotype; (G) Large contiguous stomatal clusters in the leaf epi­dermis of Arabidopsis mutant line too many mouths. Stomatal clusters were marked with white cases. Bars in the pictures repre­sent for 25 |im.
GAN et al. ― Stomatal clustering in terrestrial plants
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clusters? Whether the occurance of stomatal clusters is a response to the environment or just a coincidence during leaf development in so many terrestrial plants? Moreover, what is the ecological significance of stomatal clustering in the natural environment? In our experiments, we hy­pothesized that the stomatal clusters could be induced by certain degrees of drought or salt stress and bear a differ­ent ecological significance according to the type of cluster­ing. To testify the hypothesis, we chose Vicia faba L. and cultivated it in three different soil moisture and salt stress levels. Stomatal density (SD), stomatal index (SI), and the occurrence of stomatal clustering for each treatment were recorded and analyzed.
MATERIALS AND METHODS
Plant material and growth condition
For test of R-value, leaves of 16 different plant spe­cies (Table 1) were collected in the Hangzhou Botanical Garden (HZBG) and Zijingang campus (ZJG) at Zhejiang University, during May to July, 2006. Sesbania grondi-flora (No. 13) was sampled at QuanTang located on the north shore of Hangzhou Bay, where the seawater salinity is 19%. Wild type (Col-0) and too many mouths (tmm) mutant lines of Arabidopsis thaliana were obtained from Arabidopsis Biological Research Center (ABRC, USA).
In the drought/salt stresses experiment, Vicia faba L. seeds were surface sterilized by soaking in 3% hydrogen peroxide for 15 min and then rinsed 5 times with sterile water. Then, they were transferred into 12-cm diameter plastic pots with vermiculite, perlite medium, and fertile soil (BeiLei Ltd., China) mixed at a 1:1:1 proportion. The
cultivars were cultivated from seeds in a green house with an 10-h light photoperiod, 18-20°C average daily tem­perature, and 70% average humidity for 3 weeks before drought and salt treatment.
Test of R-value in the leaves
According to the previous literature, a statistical method is often used to measure and classify stomatal distribution
(Clark and Evans, 1954; Korn, 1993). The R-value that
refers to the degree of observed distribution departs from random expectation is the key parameter in this method. As described by Clark and Evans, the R-value was calcu­lated using the following formula:
where p is the stomatal density in the measured area r is the distance between the two nearest neighboring sto-mata, N stands for the number of randomly measured distances between stomata (here is 15-20), rA is the mean distance between 2 nearest stomata in an given area. e is the expected distance in an random patterning. When the stomatal distribution is random, the R-value is 1. If the stomatal distribution is clustered, the R-value is near 0, and when distribution is ordered, the 7^-value should be significantly greater than 1 (Croxdale, 2000). In the pres­ent study, six replicas of the abaxial surface of leaves were prepared by applying clear nail polish to the surface and
Table 1. 16 different species for leaf R-value test.
No.
Family
Plant
Site
1
Thelypteridaceae
Macrothelypteris toressiana
HZBG
2
Pteridaceae
Pteris multifida
HZBG
3
Cryptomeria
Keteleeria oblonga Cheng
HZBG
4
Cryptomeria
Metasequoia glyptostostroboides
ZJG
5
Cycadaceae
C^ycas revoluta Thunb.
ZJG
6
Aquifoliaceae
Ilex cornuta
ZJG
7
Apocynaceae
Nerium indicum Mill.
HZBG
8
Salicaceae
Salix babylonica Linn.
ZJG
9
Oxalidaceae
Trifolium pratense Linn.
ZJG
10
Leguminosae
Sesbania grondiflora
ZJG
11
Cruciferae
Arabidopsis thaliana (Col-0)
ABRC
12
Nymphaea
Nymphaea tetragona
HZBG
13
Lauraceae
Cinnamonum camphora
ZJG
14
Leguminosae
Melilotus suaveolens Ledeb.
QuanTang
15
Ginkgoaceae
Ginkgo biloba L.
ZJG
16
Cruciferae
Arabidopsis thaliana (too many mouths)
ABRC
17
Crassulaceae
Sedum dfredii Hance
ZJG
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peeling the film off when dry. The casts were mounted for examination by light microscopy (Nikon E600, 400-fold magnification). 30 pictures were randomly taken. Then, the distance (r) among 15-20 nearest neighboring stomata were measured in each picture with ARCVIEW GIS 3.0 (Environmental Systems Research Institute, Inc. USA).
Drought and salt treatment
To control the soil moisture level, irrigation was ceased 3 weeks after seeding of V. faba. A soil moisture meter sensor HH2 (Equipped with WET1 sensor, Delta-T De­vices Ltd. UK) was used to monitor the water status. Soil moisture were controlled in three levels, that is 75%-85% field water content (FWC) as control (C), 70-60% FWC as
mild drought (M) and 50%-45% FWC as severe drought
(S). Water shortage caused by soil evaporation and plant transpiration in each level was precisely compensated each day at a fixed time. The drought treatment lasted for 10-14 days before stomatal patterning analysis. In order to simu­late the salty environment and control the soil salinity, 3 weeks after seeding, 0, 0.5%, 1% (W/V) concentration of NaCl solution were added to the cultivar. 10-15 days after salt treatment, the 7th leaves from basal part of the plant were excised, and the epidermis was peeled off and ana­lyzed with the microscope.
Assessment of stomatal patterning
After the drought/salt treatment, ten fully expanded leaves (the 7th leaves from basal part) were randomly cho-
sen, and the abaxial epidermis was peeled off. Then, the epidermis was mounted under the microscope (NIKON
E600, 400 fold magnification) and 100 fields of 0.04
square millimeters were randomly selected, and the num­ber of guard cells (Ng) and epidermal cells (Ne) in each field were counted. Stomatal density (SD) per mm2 and stomatal index (SI) were calculated as follows:
SD= 2Ng /0.04 ..........................................................(4)
SI = 2Ng/(2Ng+Ne) ....................................................(5)
In addition, the occurrence of stomatal clusters in 100 random selected fields was also counted. The incidence of stomatal clusters under each stressful environment was checked with Pearson Chi-Square Test (x2, df=2, p=0.01) in SPSS 16.0 (SPSS Inc. USA).
RESULTS
R-value could not distinguish two types of sto-matal clusters
With this method, we tested R-values in 16 species of plants (Table 2) and found most plants with normal sto-matal distribution had higher R-values (R>1). In some plants which got contiguous stomatal clusters, R-values were still larger than 1 (No.13-15 in the Table 2). This is because the occurrence of such stomatal clusters was very low in those plants. In contrast, R-values were significant­ly lower than 1 in other plants exhibiting clusters (No.16, 17in Table 2). From Table 2, it was apparent that the
Table 2. R-values in 16 observed plant species.
No.
Species
R Value
Distribution
1
Macrothelypteris toressiana
1.43 ± 0.30
Random
2
Pteris multifida
1.37 ± 0.26
Random
3
Keteleeria oblonga Cheng
1.72 0.31
Random
4
Metasequoia glyptostostroboides
1.16 ± 0.24
Random
5
Cycas revoluta Thunb.
1.25 ± 0.25
Random
6
Ilex cornuta
1.70 ± 0.30
Random
7
Nerium indicum Mill.
1.21 ± 0.27
Random
8
Salix babylonica Linn.
1.35 ± 0.23
Random
9
Trifolium pratense Linn.
1.39 ± 0.31
Random
10
Sesbania grondiflora
1.40 ± 0.23
Random
11
Arabidopsis thaliana (Col-0)
1.06 0.18
Random
12
Nymphaea tetragona
1.51 ± 0.25
Random
13
Cinnamonum camphora
1.42 ± 0.23
Contiguous Cluster
14
Melilotus suaveolens Ledeb.
1.35 ± 0.27
Contiguous Cluster
15
Ginkgo biloba Linn.
1.36 ± 0.32
Contiguous Cluster
16
Arabidopsis thaliana (tmm mutant)
0.57 0.08
Contiguous Cluster
17
Sedum dfredii Hance
0.56 ± 0.16
Non-contiguous Cluster
The majority of plants with a random stomatal distribution often have an R-value larger than one. In plants that have stomatal clusters, the R-value varies according to the type of clusters. In plants exhibiting contiguous stomatal clusters, the R-value is larger than one (except for tmm mutant), but in plants with non-contiguous stomatal clusters, the R value is smaller than 1.
GAN et al. ― Stomatal clustering in terrestrial plants
329
R-values could not distinguish between the two types of stomatal clusters. Thus, we introduced two terms: "contig­uous cluster" and "non-contiguous cluster" to distinguish between them. In most cases, the incidence of contiguous clusters is often very low across the leaf blade. Therefore, the R-values are often larger than 1 in those plants (13, 14, 15 in Table 2). Non-contiguous clusters have most of stomata arranged in groups at the whole leaf level (Figure 2). Thus, the R-values are often smaller than 1, but none of those clustered stomata "touch" each other.
Drought and salt stress induces stomatal clustering in the leaf epidermis of Vicia faba L.
Drought and salt stress significantly influenced the stomatal patterning (Table 3). Compared with control, the stomatal density (SD) increased 30% and 21% in mild and severe drought stresses, respectively. Meanwhile, the sto-matal index (SI) also increased 10.7% and 14.3% (p<0.05, n=100), which suggests more stomata were produced in a given area under drought stress as opposed to shrinkage of epidermal cells. In the salinity experiment, similar results were also observed. SD increased 15% and 36% following
application of 0.5% and 1% concentration of NaCl solu­tion (p<0.01, n=100), Meanwhile, SI increased 9.4% and 15.2% along the salinity gradient, respectively.
More importantly, the incidence of contiguous stomatal clustering in the leaf epidermis also increased along the water (x2 = 18.667, df=2, p<0.01) and salinity gradient (X2=10.988, df=2, p<0.01) (Table 3). In the drought experi­ment, only two clusters were found in the observed 469 stomata of well-watered plants while 8 (in 611 stomata observed) and 22 (in 568 stomata observed) stomatal clus­ters were found under mild and severe drought stresses, respectively. The incidence of stomatal clustering also increased along the salt gradient. 2, 6 and 14 pairs of sto-matal clusters were found in 100 randomly selected fields of the control, 0.5% and 1% salt treatments, respectively. Interestingly, most stomatal clusters observed were com­posed of two stomata placed side by side or end to end. In all fields of view analyzed, only three bigger clusters (more than three stomata) were found. Such stomatal clusters are very similar to those of the Arabidopsis thaliana mutant line four lips of which have impaired symmetric divisions
of the guard mother cell (GMC) (Yang and Sack, 1995).
Such an abnormality has also occasionally been seen in Arabidopsis thaliana Col-0 ecotype (Figure 1F).
DISCUSSION
The origin of stomatal clustering is environ­mentally responsive
In model species Arabidopsis thaliana L., initiation of guard cells involves formation of the meristemoid mother cell (MMC), which will experience three rounds of asym­metric divisions and form a meristemoid (M) with several larger sister cells. Then, the meristemoid turns into guard mother cells (GMC) and finally become a pair of guard cells (GC) through a symmetric division (Sachs, 1994; Yang and Sack, 1995; Geisler et al., 2000). Interestingly, each time asymmetric divisions are launched, a newly formed meristemoid is usually away from the existing sto-mata or precursor cells (Figure 3). Such a unique pattern­ing law backs up the "one cell spacing rule."
contiguous non-contiguous stomatal cluster stomatal cluster
Figure 2. Two different types of stomatal clusters: stomata in contiguous clusters are in direct contact with their neighbors. In non-contiguous clusters, stomata are placed in groups but do not touch each other. Contiguous clusters are occasionally seen in leaves while most stomata are grouped in non-contiguous clus­ters.
Table 3. Stomatal patterning in the leaves of Vicia faba L. under different levels of drought and salt stresses.
Treatment Drought
Well watered
SD
117.25 ± 7.08c
SI Inc 0.28 ± 0.012b
idence of clustering 2%
Pearson chi-square test X2=18.667
Mild drought
152.75 ± 8.84a
0.31 ± 0.014a
8%
df = 2
Severe drought
142 ± 5.93b
0.32 ± 0.012a
20%
p<0.01
Salt
0% Salinity
100 ± 6.41c
0.255 ± 0.015c
2%
X2=10.988
0.5% Salinity
123.75 ± 7.05b
0.306 ± 0.014b
6%
df = 2
1% Salinity
151.25 ± 8.44a
0.319 ± 0.014a
14%
p<0.01
SD and SI in each treatment were compared with one-way ANOVA (p=0.05). The incidence of clustering along the drought and salinity gradient were checked with Pearson Chi-Square test in SPSS 16.0. (df =2, p=0.01). Data labeled with different letters were statistical different.
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Figure 3. Several genes and transcriptional factors regulate the stomatal lineage in Arabidopsis thaliana. Many of them, such as YODA, MKK4/5, MAK3/6 and ICE/SCRM2, are correlated with multiple biotic/abiotic stress responses. Mutation of those genes and transcriptional factors causes excessive formation of meristemoid adjacent to the existing stoma and lead to contiguous stomatal clus­tering. On the other hand, the formation of non-contiguous stomatal clusters may initiate from the generation of new satellite meriste-moid around the existing stoma. This pattern of cell differentiation may be modulated by environmental signals, also.
Over the last 15 years, it has been known that a series of genes and transcription factors regulate the fate of pro-todermal cells during stomatal differentiation (Figure 3)
(Yang and Sack, 1995; Bergmann, 2006; Nadeau, 2009). The TMM, SDD-1, and ERECTA family are required to in­hibit the excessive cell divisions between the meristemoid
(Geisler et al., 2000; Groll et al., 2002; Shpak et al., 2005).
YODA, a Mitogen-activated protein kinase kinase kinase gene, acts as a molecular switch regulating cell identities in the epidermises (Bergmann et al., 2004). MKK4/5 and MPK3/6 act downstream of YODA and negatively regu­late the initiation of the meristemoid during asymmetrical division (Wang et al., 2007). Moreover, EPF1 encodes a small secretory peptide that secretes into the protodermal cells and inhibits the formation of a satellite meristemoid (SM) near the precursor cells (Hara et al., 2007). At last,
FOUR LIPS (FLP) and its homolog MYB88 control the
symmetrical division at the formation of the guard cells (Lai et al., 2005). These genes and proteins negatively control the cell differentiation and eliminate excessive divisions near meristemoids and persecutor cells (Nadeau, 2009). Mutation of them would bring contiguous stomatal clusters of different size and frequency. Recently, three transcriptional factors―SPCH, MUTE and FAMA―were also identified to positively regulate the initiation of asymmetrical and symmetrical divisions in the stomatal lineages
(Ohashi-Ito and Bergmann, 2006; MacAlister et al., 2007;
Pillitteri et al., 2007). At the same time, ICE1 and SCRM2 are required for the sequential actions of SPCH, MUTE,
and FAMA (Kanaoka et al., 2008).
However, we have little information on whether these genes or transcriptional factors are regulated by multiple
biotic/abiotic factors or whether the origin of stomatal clusters is affected by long-term climate change (Gray and Hetherington, 2004). It was reported that the density of epidermal cells and stomata in Scrophularia yoshimurae increased significantly in the less ventilated growth con­tainer (Chen et al., 2006). Serna & Fenoll also reported contiguous stomatal clusters were induced in the c24 eco-type of Arabidopsis thaliana growing in a sealed chamber with little gas exchange (Serna and Fenoll, 1997). These results suggest that a gaseous signal (like ethylene or a decrease in atmospheric CO2 concentration) may affect stomatal patterning. Moreover, the ERECTA gene, which co-regulates stomatal patterning with ERL1 and ERL2 (Shpak et al., 2005), has been found to modify transpira­tion efficiency under stressful environments (Masle et al., 2005). The environmentally responsive mitogen-activated protein kinases: MPK3/6 and MKK4/5, downstream components of YODA, were shown to regulate stomatal pat­terning in Arabidopsis. Intriguingly, MAPK and MAPKK also play central roles in plant abiotic and biotic stress signaling networks (Asai et al., 2002; Droillard, 2002; Pedley and Martin, 2005; Wang et al., 2007). This finding established a complete MAPK signaling cascade as a key regulator of stomatal development and patterning (Wang et al., 2007). Recently, SCRM2 were found to encode a bHLH leucine zipper protein ICE1 and contribute to freez­ing tolerance (Kanaoka et al., 2008). Those genes and proteins may serve as crucial candidates for integration of environmental signals into stomata lineages (Figure 3). In the present study, our results also show that osmotic stress caused by drought and salinity impaired normal stomatal patterning and produced more contiguous stomatal clus­ters in the leaf epidermis. Notably, the presence of such
GAN et al. ― Stomatal clustering in terrestrial plants
331
contiguous clusters in V. faba is quite similar to the Ara-bidopsis thaliana mutant line four lips, which suggest the symmetric divisions of guard mother cells during stomatal differentiation are probably impaired by osmotic stresses
(Figure 3). Components like EPF-1, FLP, and the MAP
kinase family may be involved in this process. However, despite the contiguous clusters discussed above, little is known about the origin of non-contiguous stomatal clus­tering in plants in their natural habitats (Appendix 1). We assume that the appearance of non-contiguous clustering is formed by the generation of a new satellite meristermoid around the primary stoma (Figure 3). They are all arising from one original MMC. Some position-based signals like EPF-1 may contribute to the spatial patterning of the satel­lite meristemoid and the "one cell spacing rule."
Stomatal clustering is a new marker of environmental perception and adaptation in terrestrial plants
Since adherence to the "one cell spacing rule" could reduce the overlap of stomatal gaseous diffusion shells (GDS) and ensure the optimal balance between carbon assimilation and water loss in plants (Larkin et al., 1997), why do so many plants have stomatal clusters? Did sto-matal clusters bear any ecological or evolutional advan­tage for the adaptation of terrestrial plants under natural selection? A few scientists have proposed that stomatal clusters have positive effects on plants. Hoover studied the ecological response of non-contiguous stomatal clusters in two Mexican Begonia species (B. beracleifolia & B. nelumbiifolia) and found stomatal clusters may assist in water conservation: populations growing on the rocks near waterfalls have larger clusters than those growing on well-watered soil substrates (Hoover, 1986). Tang and his col­leges reported a positive relationship between cluster size and multiple epidermis, which is a typical drought adapta­tion trait in Begonia peltatifolia (Tang et al., 2002). More­over, in Arabidopsis, exposed to high light intensities, dark adapted sdd-1 mutants which have contiguous clusters had 30% higher CO2 assimilation rates than that of the wild type (Schluter et al., 2003). Additionally, a negative correlation between the number of contiguous stomatal clusters and soil moisture level has been established in Cinnamomum camphora (Lauraceae) (Zhao et al., 2006). Our unpublished experiments on the too many mouths mu­tant of Arabidopsis thaliana showed that normal stomatal regulation was impaired by guard cell extrusion in higher number of contiguous stomatal clusters.
In light of the evidence discussed above, it is tempting to speculate that: since most normal stomata are kept in groups, in non-contiguous stomatal clusters, overlaps of GDS could reduce the total area of evaporation shells, and water loss would be kept to a minimum (Figure 4). It may be a long-term adaptation to the ever-challenging environ­ment by keeping to the "one cell spacing rule." On the oth­er hand, based on the cellular hydraulic interaction theory (Mott et al., 1997; Mott et al., 1999), the normal regulation
Figure 4. (A) The "one cell spacing rule" reduces the overlaps of Gaseous Diffusion Shells (GDS) in each stoma and balances the carbon assimilation and leaf transpiration; (B) Overlaps of GDS in non-contiguous stomatal clusters may reduce leaf tran­spiration and keep water loss at a lower level; (C) Direct contact of guard cells in contiguous stomatal clusters may cause cellular extrusion and impair normal stomatal function.
of stomata would probably be impaired due to guard cell extrusion in large contiguous stomatal clusters. Therefore, plants that have larger or a larger number of stomatal clus­ters (like too many mouths mutant of Arabidopsis) would be selected in a fluctuating environment. That may explain why contiguous clusters are so rare in most terrestrial plants. In addition, some abnormal environmental respon­sive gene expression (Like ERECTA, MKK4/5-MPK3/6)
may be involved in this process. Contiguous stomatal clus­ters are more like "scars" on the "faces" of plant leaves instead of an evolutional advance.
In conclusion, stomatal clusters are abnormal patterning in the leaves of many terrestrial plants. Evidence in mor­phology, gene regulation, and cellular signaling have dem­onstrated the delicate links between stomatal clusters and environmental factors. Nevertheless, the detailed mecha­nism still needs further exploration. Stomatal clusters, tiny marks on the leaves, leave us new clues to uncover the truth about the adaptation of terrestrial plants to the ever-challenging environment, ecologically and evolutionally.
Acknowledgement. We sincerely thank the anonymous reviewer for the insightful opinions and improvement of the language. This study is supported by Natural Science Fund of China (project 30871470, 30730020) and Min­istry of Science and Technology of China (863 program,
2006AA100202).
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Appendix 1. Stomatal clustering in terrestrial plants.
Family
Genera
Species
Stomatal cluster type
Incidence of
References*
clustering
Gymnosperm
Ginkgoaceae
Ginkgo biloba
G. biloba L.
2 or 3 contiguous stomata
Low
Chen and Li, 2004
Zamiaceae
Zamia
Z.furfuracea
2 or 3 contiguous stomata
Low
Su et al., 2003
Bennettitales
Tyrmia
T. susongensis sp. nov.
Contiguous stomata
Occasionally seen
Cao, 1998
Prynada
Pterophyllum sp.
Laterally contiguous stomata
Casuarinaceae
Casuarina
C. equisetifolia
2 contiguous stomata
Low
Torrey and Berg, 1988
Ephedraceae
Ephedra
E. foliata Boiss
2 contiguous stomata placed verti-
Occasionally seen
Pant and Mehra, 1964
cally (end to side)
Podocarpaceae
Dacrydium
D. balansae
Polarly contiguous stomata
Low
Stockey et al., 1998;
Podocarpus
D. lycopodioides
Stockey and Dacrydium,
P. madagascariensis
1992
P. woltzii
Angiosperm (Dicotyledon)
Apocynaceae
Aganosma
A. dichotoma K. Schum
Several stomata placed together
High
Patel et al., 1972
forming an non-contiguous group
Annonaceae
Annona
A. muricata L.
Contiguous stomatal placed end to Low
Sun et al., 2001
Artabotrys
A. hongkongensis
end
Desmos chinensis
Uvaria macrophylla
Asteraceae
Emilia
E. coccinea
Contiguous stomata
Low
Ndukwu and Agbagwa,
E. sonchifolia
2006
Amaryllidaceae
Agapanthus
A. umbellatus (L)
Laterally or polarly contiguous
Awasthi et al., 1984
Allium
A. cepa (L)
stomata
A. tuberosum (L)
Amaryllis
A. belladonna (U, L)
A. vittata (U, L)
Nerine
N. curvi]'olia (L)
Crinum
C. zeylanicum (U, L)
C. asiaticum (L)
Cyrtanthus
C. mackenii (L)
Cooperia
C. pedunculata (U, L)
Pancratium
P. verecundum (L)
Narcissus
N. tazetta (U)
334
Botanical Studies, Vol. 51, 2010
Appendix 1. (Continued)
Family
Genera
Species
Stomatal cluster type
Incidence of clustering
References*
Begoniaceae
Begonia
B. cavaleriei L. B. heracleifolia B. nelumbiifolia B. erythrophylla B. metallica B. semperflorens B. aridicaulis B. peltatifolia
4-6 stomata forming a non-contiguous group
High
Dehnel, 1961; Payne, 1970 Hoover, 1986; Tang et
al., 2002
Bombacaceae
Kostermansia
K. malayana
Stomata arranged in groups Juxtaposed contiguous stomata occasionally seen
High
Metcalfe and Chalk, 1979
Celatraceae
Elaeodendron
E. glaucum
Laterally contiguous stomata
High
Pant and Kidwai, 1966
Crassulaceae
Sedum
S. lineare
S. aizoon
S. polytrichoiides
S. kiangnanense
S. emargi nat um
S. alboroseum
S. dfredii Hance
2-6 Stomata forming a non-contiguous group
High
Zheng and Gong, 1999;
Payne, 1970
Cruciferae
Brassica Lepidium Raphanus
B. oleracea L. virginicum R. salivus
Stomata arranged in non-contiguous groups
High
Payne, 1970
Gesneriaceae
Gesneria
sintenisii Urb.
Stomata arranged in non-contiguous groups
High
Metcalfe and Chalk, 1979
Himantandraceae Himantandra
H. parvifolia Bak.
4-6 stomata clustered around the bases of peltate scales
High
Baranova, 1972
Ixonanthaceae
Irvingia Klainedoxa
Some species
Stomata arranged in non-contiguous groups
High
Metcalfe and Chalk, 1979
Lauraceae
Cinnamomum
C. camphora
Low
Zhao et al., 2006
Leguminosae
Melilotus
M. albus Desr.
Polarly or laterally contiguous stomata
Low
First report
Menispermaceae
Eleutharrhena
E. macrocarpa Forman
30-68 stomata arranged an island, some of them are contiguous
High
Hong et al., 2001
Melastomataceae
Calycogonium
Leandra
Ossaea
Some species
Stomata arranged in non-contiguous groups
High
Metcalfe and Chalk, 1979
Moraceae
Ficus
Some species
Stomata arranged in non-contiguous groups
High
Payne, 1970
Nyssaceae
Camptotheca
C. acuminata Dence
2 Contiguous stomata near small veins (laterally, polarly or diagonally)
Low
Xi et al., 1997
Nymphaeaceae
Nelumbo Adans. N. nucifera Gaertn
2 contiguous stomata
Low
Gupta et al., 1968
Ochnaceae
Godaya Sauvagesia
Some species
Stomata arranged in non-contiguous groups
High
Metcalfe and Chalk, 1979
Primulaceae
Primulal
P. merrilliana
   P. ciculariifolia
Laterally contiguous stomata
Occasionally seen
Chen et al., 2004
Papilionaceae
Aeschynomene
Alysicarpus
Arachis
Euchresta
Aeschynomene indica Alysicarpus -procumbens Alysicarpus -vaginalis Arachis hypogaea Desmodium Desv.
2-4 laterally or polarly contiguous Low stomata
Kothari and Shah, 1975; Nilamoni and Parukutty,
1979
GAN et al. ― Stomatal clustering in terrestrial plants
335
Appendix 1. (Continued)
Family
Genera
Species
Stomatal cluster type
Incidence of clustering
References*
Proteaceae
Banksia
Dryandra
Lambertia
Some species
Stomata arranged in non-contigu­ous groups
High
Metcalfe and Chalk, 1979
Rubiaceae
Pagamea
Some species
Stomata arranged in non-contigu­ous groups
High
Metcalfe and Chalk, 1979
Solanaceae
Capsicum
Solanum
C. annuum L.
S. melongena L.
Juxtaposed, superposed, and obliquely placed contiguous stomata
Low
Patel and Shah, 1971
Sonneratiaceae
Sonneratia
S. alba J. Smith
Contiguous stomata
Occasionally seen
Chen, 1996
Saxifragaceae
Saxifrage, Chrysosplenium
Some species
Stomata arranged in non-contigu­ous groups
High
Metcalfe and Chalk, 1979
Simaroubaceae
Castela Soulamea
Some species
Stomata arranged in non-contigu­ous groups
High
Metcalfe and Chalk, 1979
Theaceae
Cleyera Thunb.
Some species
Stomata arranged in non-contigu­ous groups
High
Metcalfe and Chalk, 1979
Verbenaceae
Stachytarpheta
Some species
Stomata arranged in non-contigu­ous groups
High
Payne, 1970
Angiosperm Monocotyledon
Araceae
Alocasia
Arisaema
Zamioculcas
Some species
Polarly contiguous stomata
Low
Pant and Kidwai, 1966
Liliaceae
Asparagus
A. gonoclados Baker Polarly or laterally contiguous A. plumosus Baker stomata A. racemosus var. javanicus
Baker A. sprengeri Regel.
Low
Gopal and Shah, 1970
Orchidaceae
Neottianthe
N. angustifolia N. calcicola N. camptoceras N. compacta N. cucullata N. gymnadenioides N. luteola N. monophylla N. oblanga N. ovata
N. pseudodiphylax N. securrdiflora
Polarly contiguous stomata
High
Sun et al., 1999
★References for Appendix 1.
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Cao, Z.Y. 1998. A study on the cuticles of some bennettital eans from the lower part of Xiangshan group in Jiangsu and Anhui Prov­inces. Acta Palaeon Tologica Sin. 37: 283-294.
Chen, L.Q. and C.S. Li. 2004. The epidermal characters and stomatal development of Ginkgo biloba. Bull. Bot. Res. 24: 417-422.
Chen, M.L., X.P. Zhang, and X.H. Li. 2004. Study on micromorphological features of Primula merrilliana and P. cicutariifolia. J. Plant Res. Environ. 13: 18-24
Chen, Z1996. The morphology and anatomy of sonneratia linn in China. J. Trop. Subtropical Bot. 4: 18-24.
Dehnel, G.S. 1961. Abnormal stomatal development in foliage leaves of Begonia aridicaulis.— Amer. J. Bot. 48: 129-133
Gopal, B.V. and G.L. Shah. 1970. Observations on Normal and Abnormal Stomatal Features in Four Species of Asparagus L. Amer. J. Bot. 57: 665-669.
Gupta, S.C., G.S. Paliwal, and R. Ahuja. 1968. The stomata of Nelumbo nucifera: formation, distribution and degeneration. Amer. J.
Bot. 55: 295-301.
Hong, Y.P., K.Y. Pan, and Z.D. Chen. 2001. Characters of leaf epidermis and their systematic significance in Menispermaceae. Acta
Bot. Sin. 43: 615-623.
336
Botanical Studies, Vol. 51, 2010
Hoover, W.S. 1986. Stomata and stomatal clusters in begonia: ecological response in two mexican species. Biotropica 18: 16-21. Kothari, M.J. and G.L. Shah. 1975. Epidermal structure and ontogeny of stomata in the Papilionaceae (Tribe Hedysareae). Bot. Ga­zette 136: 372-379.
Metcalfe, C.R. and L. Chalk. 1979. Anatomy of the dicotyledons. Second ed. Oxford: Clarendon Press.
Ndukwu, B.C. and I.O. Agbagwa. 2006. The value of leaf micromorphological in the taxonomic delimitation of Emilia cass. (Aster-
aceae) species Global. J. Pure Appl. Sci. 1: 183-187. Nilamoni, B. and B. Parukutty. 1979. Contiguous stomata in Desmodium Desv. (Papilionaceae). Current Sci. 48: 27-28. Pant, D.D. and P.F. Kidwai. 1966. Epidermal structure and ontogeny in some Celatraceae. New Phytol. 65: 288-295. Pant, D.D., and B. Mehra. 1964. Epidermal structure and development of stomata in Ephedra foliata Boiss. New Phytol. 63: 91-95. Patel, J.D., I.L. Kothari, and M.A. Pathan. 1972. Stomatal features in the leaf of Aganosma dichotoma (Roth) K. Schum. Ann. Bot. 36:
849-856.
Patel, J. D. and J. J. Shah. 1971. Studies in stomata of chilli and brinjal. Ann. Bot. 35: 1197-1203.
Payne, W.W. 1970. Helicocytic and allelocytic stomata: unrecognized patterns in the dicotyledonae. Amer. J. Bot. 57: 140-147. Stockey, R.A. and Dacrydium. 1992. Cutical micromorphology of Dacrydium (Podocarpaceae) from New Caledonia. Bot. Gazette
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Stockey, R.A., B.J. Frevel, and P. Woltz. 1998. Cutical micromorphology of Podocarpus, subgenus Podocarpus, section Scytopodium
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氣孔成簇現象,陸生植物感受和適應環境的新表徵
甘 毅  周 磊  沈仲佶  沈竹夏  張一瓊  王根軒
中國浙江大學生命科學學院農業生態與工程研究所
"氣孔簇"是一種由二到多個氣孔組成的非正常氣孔排布格局,目前在60多種陸生植物上已有
報導。根據它們的形態特徵和分佈格局,又可劃分為兩種不同的氣孔簇類型。然而,通過對16種植物
葉表皮的氣孔分佈格局進行分析發現,經典生態學上的空間分佈R係數不能很好的區分上述兩種氣孔
簇。因此,為了更好的區分它們,本文引入了"接觸型氣孔簇"和"非接觸型氣孔簇"的概念,並且分
別討論了它們的形成機理和潛在的生態意義。為了探究氣孔簇的形成是否與環境脅迫有關,蠶豆幼苗被
種植在不同的水分和鹽分梯度下。兩周後,撕去葉表皮條進行氣孔格局分析。結果表明:乾旱和鹽脅迫
明顯增加了蠶豆葉片的氣孔密度和氣孔指數。而且接觸型氣孔簇的出現頻率也明顯增加。這個結果暗示
著氣孔簇的出現與外界環境信號有關,它們可以作為陸生植物感受和適應環境的新標誌。
關鍵詞:氣孔簇;一細胞間隔法則;環境脅迫;氣孔發育;蠶豆。