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.

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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 7^th 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 7^th 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 (N_g) and epidermal cells (N_e) 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 士 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 士 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 士 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.

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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 asym國 metrical 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 com國 ponents 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

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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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Larkin, J.C., M.D. Marks, J. Nadeau, and F. Sack. 1997. Epidermal cell fate and patterning in leaves. Plant Cell 9:

1109-1120.

MacAlister, C.A., K. Ohashi-Ito, and D.C. Bergmann. 2007.

Transcription factor control of asymmetric cell divisions that establish the stomatal lineage. Nature 445: 537-540.

Masle, J., S.R. Gilmore, and G.D. Farquhar. 2005. The ERECTA

gene regulates plant transpiration efficiency in Arabidopsis.

Nature 436: 866-870.

Metcalfe, C.R. and L. Chalk. 1979. Anatomy of the dicotyledons. Second ed. Oxford: Clarendon Press.

Mott, K.A., F. Denne, and J. Powell. 1997. Interactions among stomata in response to perturbations humidity. Plant Cell

Environ. 20: 1098-1107. Mott, K.A., J.C. Shope, and T.N. Buckley. 1999. Effects of

humidity on light-induced stomatal opening:evidence for hydraulic coupling among stomata. J. Exp. Bot. 50:

1207-1213.

Nadeau, J.A. 2009. Stomatal development: new signals and fate determinants. Current Opinion in Plant Biol. 12: 29-35.

Nilamoni, B. and B. Parukutty. 1979. Contiguous stomata in Desmodium Desv. (Papilionaceae). Current Sci. (India) 48:

27-28.

Ohashi-Ito, K. and D.C. Bergmann. 2006. Arabidopsis FAMA controls the final proliferation/differentiation switch during

stomatal development. Plant Cell 18: 2493-2505.

Payne, W.W. 1970. Helicocytic and allelocytic stomata: unrecognized patterns in the dicotyledonae. Amer. J. Bot. 57:

140-147.

Pedley, K.F. and G.B. Martin. 2005. Role of mitogen-activated protein kinases in plant immunity. Curr. Opin. Plant Biol. 8:

541-547.

Pillitteri, L.J., D.B. Sloan, N.L. Bogenschutz, and K.U. Torii.

2007. Termination of asymmetric cell division and differentiation of stomata. Nature 445: 501-505.

Sachs, T. 1994. Both cell lineages and cell interactions contribute to stomatal patterning. Int. J. Plant Sci. 155: 245-247.

Schluter, U., M. Muschak, D. Berger, and T. Altmann. 2003.

Photosynthetic performance of an Arabidopsis mutant with elevated stomatal density (sdd1-1) under different light regimes. J. Exp. Bot. 54: 867-874.

Serna, L. and C. Fenoll. 1997. Tracing the ontogeny of stomatal clusters in Arabidopsis with molecular markers. Plant J. 12:

747-755.

GAN et al. ― Stomatal clustering in terrestrial plants

333

Shpak, E.D., J.M. McAbee, L.J. Pillitteri, and K.U. Torii. 2005. Stomatal patterning and differentiation by synergistic interactions of receptor kinases. Science 309: 290-293.

Sun, T., Y. Hu, and K. Lang. 1999. A Study on Micromorpholog-ical Characters of Leaf Epidermis of Neottianthe in China.

Acta Bot. Yunn. 21: 57-62.

Tang, M., Y.X. Hu, J.X. Lin, and X.B. Jin. 2002. Developmental mechanism and distribution pattern of stomatal clusters in Begonia peltatifolia. Acta Bot. Sin. 44: 384-390.

Wang, H., N. Ngwenyama, Y. Liu, J.C. Walker, and S. Zhang. 2007. Stomatal development and patterning are regulated by environmentally responsive mitogen-activated protein kinases in Arabidopsis. Plant Cell 19: 63-73.

Willmer, C. and M. Fricker. 1996. Stomata. Second ed. London: Chapman & Hall.

Woodward, F.I. 1987. Stomatal numbers are sensitive to increases in CO2 from preindustrial levels. Nature 327: 617-618.

Yang, M. and F.D. Sack. 1995. The too many mouths and four lips Mutations Affect Stomatal Pnoduction in Arabidopsis.

Plant Cell 7: 2227-2239.

Zhang, X.Y., H.M. Yang, and G.X. Wang. 2003. Stomatal density and distribution of spring wheat leaves under different planting densities and soil moisture levels. Acta Phytoecol.

Sin. 27: 133-136.

Zhao, X.Z., Y.S. Yang, and Z.X. Shen. 2006. Stomatal clustering in Cinnamomum camphora. South Africa J. Bot. 72:

565-569.

Zheng, Y. and J. Gong. 1999. A leaf epidermis study on tweleve species of Sedum in Anhui. Bull. Bot. Res. 19: 292-297.

Appendix 1. Stomatal clustering in terrestrial plants.


Family	Genera	Species	Stomatal cluster type	Incidence of	References*
Family	Genera	Species	Stomatal cluster type	clustering	References*
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.

Awasthi, D.K., V. Kumar, and R. Rawat. 1984. Stomatal studies in Amaryllidaceae with sepcial reference to stomatal abnormalities. Proc. Indian Acad. Sci (Plant Sci.) 93: 629-633.

Baranova, M. 1972. Systematic anatomy of the leaf epidermis in the magnoliaceae and some related families. Taxon 21: 447-469.

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, Z丄1996. 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

151: 138-149.

Stockey, R.A., B.J. Frevel, and P. Woltz. 1998. Cutical micromorphology of Podocarpus, subgenus Podocarpus, section Scytopodium

(Podocarpaceae) of Madagascar and South Africa. Int. J. Plant Sci. 159: 923-940. Su, J. X., F. Liao, and YY Huang. 2003. The leaf epidermis of three species in Zamiaceae. Acta Bot. Yunn. 25: 596-602. Sun, T.X., Y.S. Hu, and K.Y. Lang. 1999. A study on micromorphological characters of leaf epidermis of Neottianthe in China. Acta

Bot. Yunn. 21: 57-62.

Sun, T.X., S. Zhao, and X.Y. Zhuang. 2001. Leaf epidermal structure in 10 species of Annonaceae. J. Trop. Subt. Bot. 9: 194-200. Tang, M., Y.X. Hu, J.X. Lin, and X.B. Jin. 2002. Developmental mechanism and distribution pattern of stomatal clusters in Begonia

peltatifolia. Acta Bot. Sin. 44: 384-390.

Torrey, J.G. and H. Berg. 1988. Some morphological features for generic characterization among the Casuarinaceae. Amer. J. Bot. 75:

295-301.

Xi, M.L., S.K. Bao, and D.D. Lin. 1997 The epidermal structure of Camptotheca acuminata leaf. J. South west China Normal Univ.

(Natural Science) 22: 58-61.

Zhao, X.Z., Y.S. Yang, and Z.X. Shen. 2006. Stomatal clustering in Cinnamomum camphora. South Africa. J. Bot. 72: 565-569. Zheng, Y. and J. Gong. 1999. A leaf epidermis study on tweleve species of sedum in AnHui. Bull. Bot. Res. 19: 292-297.

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。



	332	Botanical Studies, Vol. 51, 2010

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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。