Stomata in Lepidium

Variations in adaxial epidermis

Anatomical Study for The Leaf Epidermis of The Genus Lepidium L. in Iraq

by Kadhem T. A., Alnomani R. M. (2017)

Thulfiqar Abbas Kadhem, Ruqayah Manoon Alnomani,


In RJPBCS 8(2): 768-773 – ISSN: 0975-8585 –

Variations in abaxial epidermis


The aim of the study was to investigate the detail epidermal characteristics of the leaf for six species of the genus Lepidium in Iraq. The species investigated include L.aucheri, L. latifoliaum, L. perfoliatum, L. persicum, L. ruderale and L. sativum

The qualitative parameters, such as straight or sinuous, thin or thick anticlinal cell walls, types of stomata apparatus and trichomes types were studied by using light microscopy(LM). Also quantitative traits such as stomatal index, length, width, epidermal length and width was described in details. The results show high covariance in the leaf attributes which can be of great taxonomic significance


Stomata and gas supply in plants

The stomatal apparatus: The path of the gas supply in plants

by Robertson R. N. (1936)

Linnean Macleay Fellow


In that remote age when the ancestors of our present day plants left the aquatic environment and became adapted to hind conditions, they had to undergo severe changes. Whereas they had been accustomed to being surrounded by water, they had, under the new conditions, to resist the drying effects of the atmosphere. If they had developed a water-proof skin the difficulty would have been solved: but a water-proof skin is also a gas-proof skin; and a plant, like an animal, needs to have gaseous oxygen to enable it to respire ; unlike an animal, it also needs gaseous carbon dioxide (which it takes in and makes into starch, cellulose, sugars, proteins and most of those substances which constitute the plant body). Thus a gas-proof skin would mean that the plant would be unable to get the gas necessary for its food or the gas necessary for its energy.

The plants were virtually faced with two kinds of death-on the one hand by drying up, on the other hand by starving to death through lack of gas. The situation was saved by a compromise, and the result was the delicate complicated structure which is visible in a leaf under the microscope. This resultant development of leaf structure was by no means sudden, and it has taken many, many years for all the diverse leaf forms to evolve.

In the light of our knowledge of the behaviour of stomates, let us look at the

possible functions for this behaviour. Early observers assumed that the stomates- since they were the passage for diffusion of water vapour; and since they opened and shut at different times-were delicate controllers of transpiration (the loss of water vapour from the leaves).

Critical experimental work soon showed this assumption was unjustified. Attacks were made by Lloyd, who is at present visiting Sydney and lecturing under the auspices of the University Extension Board, and by Knight. Knight showed that transpiration is controlled to a large extent by the water content of the leaf; a high water content tends to high transpiration rate; low water content tends to low transpiration rate. At the same time stomatal aperture is not reduced by slight water deficit in the leaf. The plant, indeed, has an internal regulating mechanism which, as Maximov says, is.far quicker to act and far surpasses in its effect the regulative action of the closing stomates. This, of course, does not mean that the closure of stomates is not important from the point of view of moisture conservation. It is important, and helps the plant considerably, but the stomates must be considered as conservers of moisture during hard times, and certainly not, under ordinary conditions, as controllers of the transpiration rate. Loftfield considers that the effects of external factors are not masked by the closure of stomates until the stomates are more than half closed.

The evolution of the stomatal apparatus

Cryo-scanning electron micrographs of freeze-fractured hornwort gametophytes (a–c) and sporophytes (d–i): Anthoceros agrestis(a,c,d–f); Folioceros fusiformis (b); Leiosporoceros dussii (g); Megaceros enigmaticus (h); Dendroceros granulatus (i). Sections through thalli showing mucilage-filled cavities (asterisk). (cNostoccolony. (d,g) Intercellular spaces are initially liquid-filled (asterisk) but become gas-filled (e, arrowed) following stomatal opening. (f) Columella with gas-filled (asterisk) intercellular spaces. (h,i) Young (h) and mature (i) sporophytes of astomate taxa, showing complete absence of intercellular spaces in the assimilatory layers which collapse and dry (i). Scale bars: (a,b) 200 µm; (d,e,g) 50 µm; (c,f,h,i) 20 µm.

The evolution of the stomatal apparatus: intercellular spaces and sporophyte water relations in bryophytes—two ignored dimensions

by Duckett J. G., Pressel S. (2017)

Jeffrey G. Duckett, Silvia Pressel,


In Phil. Transact. Roy. Soc. B. 2017 –

Cryo-scanning electron micrographs of freeze-fractured moss sporophytes: Physcomitrella patens (a,b); Physcomitrium pyriforme(c,d); Lyellia crispa (e,f). (a,c) Young sporophytes with liquid-filled (asterisk) intercellular spaces. (e) Gas (arrowed) gradually replaces their initially liquid-filled content following stomatal opening, as evidenced by the presence of intercellular spaces only partially filled with liquid (asterisk in f). Liquid is first lost from the substomatal cavities (b; S, stoma) until the entire intercellular space system becomes gas-filled (d). Scale bars: (c,d) 100 µm; (a,e) 50 µm; (b,d) 20 µm.


Cryo-scanning electron microscopy shows that nascent intercellular spaces (ICSs) in bryophytes are liquid-filled, whereas these are gas-filled from the outset in tracheophytes except in the gametophytes of Lycopodiales. ICSs are absent in moss gametophytes and remain liquid-filled in hornwort gametophytes and in both generations in liverworts. Liquid is replaced by gas following stomatal opening in hornworts and is ubiquitous in moss sporophytes even in astomate taxa.

Cryo-scanning electron micrographs of freeze-fractured moss sporophytes: Polytrichum juniperinum (a,b); Mnium hornum (c); Atrichum undulatum (d); Pogonatum aloides (e,f). (a,b) Unopened (a) and open (b) stoma subtended by a gas-filled intercellular space. (c) Sunken stoma subtended by a liquid-filled intercellular space. (d–f) In astomate taxa, intercellular spaces are also initially liquid-filled (asterisk, e) and the same process of liquid replacement by gas occurs in their fully expanded capsules (d,f). Scale bars: (f) 200 µm; (a–e) 20 µm.

New data on moss water relations and sporophyte weights indicate that the latter are homiohydric while X-ray microanalysis reveals an absence of potassium pumps in the stomatal apparatus. The distribution of ICSs in bryophytes is strongly indicative of very ancient multiple origins. Inherent in this scenario is either the dual or triple evolution of stomata. The absence, in mosses, of any relationship between increases in sporophyte biomass and stomata numbers and absences, suggests that CO2 entry through the stomata, possible only after fluid replacement by gas in the ICSs, makes but a minor contribution to sporophyte nutrition. Save for a single claim of active regulation of aperture dimensions in mosses, all other functional and structural data point to the sporophyte desiccation, leading to spore discharge, as the primeval role of the stomatal apparatus.

Stomata in Pongamia (Papilionaceae)

Cotyledonary stomatal types of Pongamia pinnata
 (45 x 10X). A, Young anomocytic stoma with fivesubsidiaries; B, young staurocytic stomata; C and D, tetracytic stomata and E, One anisotricytic, onetetracytic and a anomocytic stomata in close proximity; F, Anomocytic with seven subsidiaries. A, B and C,Stomatal pore developing. Subsidiaries shown by asterisks
Surface views of young cotyledon showing apparently no stomata on the outer surface (A) and the presenceof stomata on the inner surface (B). 10 X 10 X magnification.

Seedling characteristics of Pongamia pinnata (L.) Pierre (Papilionaceae)

by Khan D., Zaki M. J., Shaukat S. S., Sahitio Z.A. (2015)

In INT. J. BIOL.12 (3): 457-479 –

The paracytic stomata with cuticular striations seen on the dorsal surface of simple leaf (near veinlet) of 10-day old seedling – a rare feature (A and B) and Common paracytic stomata on ventral surface (C). Note onestoma with unequal subsidiaries (shown in circle). Magnification: 45 x 10 X


Seedling characteristics of Pongamia pinnata (L.) Pierre are described. Its seeds were collected from a tree growing in the Campus of University of Karachi and germinated without any dormancy breaking treatment in pots filled with garden sandy loam soil maintained at 75% MWHC. The seeds were sown in surface layer of soil not below than 1 cm. Seedlings of10- and 60 days were studied. The seedling was of Phanerocotylar –  Epigeal Reserve type. The major allocation of biomass in 10-day old seedlings was in cotyledons followed by leaves, roots and stem. Tap root had profuse laterals. Cotyledons large, more or less sessile, green fleshy – food laden, concave inside and convex outside, with no visible venation and retained with the seedling for some 50 – 60 days after emergence.

Few stomatal types seen on the ventral surface of lateral leaflet of trifoliate leaf. A and B, Differentially developing stomata of anomocytic type on young leaflet with five and seven subsidiaries. C, a stoma with cuticular striations. D, an anomocytic stoma besides paracytic stomata. E, normal paracytic stomata and large anomocytic stoma with cuticular striations and seven subsidiaries of various sizes. Lobation of epidermal cells develops with maturity. Subsidiaries shown by the asterisks. (Magnification 45 x 10 X) except E, 45 x15X).

The hypocotyl short, green, shining. Epicotyl longer, pubescent. The basal stem may sometimes be pruinose. The primary leaf simple, alternate. There is a great irregularity with respect to the first appearance of imparipinnate trifoliate leaf with ovate leaflets. It may arise after 2, 4 or 6-7 simple leaves.Leaf stipulate, apex acute (at times acuminate, basally obtuse. Aspect ratio 0.6 – 0.67. Venation brachidodromous. Angle of divergence (AOD) moderate between 1o and 2o veins and wide between 2o and 3o veins. Areole small and veinlets endings linear. There were four types of trichomes on very young stipules and leaves –  

1) Short flat, bent near base, curved, brown, pointed-at-the-apex and unicellular, non-glandular trichomes,

2) Septate multicellular non-glandular trichomes,

3) Very long and thin non-glandular trichomes and

4) Stalked capitate uniseriate filiform non-glandular trichomes. Leaves multistomatic ventrally; dorsally generally devoid of stomata except few rarely. Stomata were identified on the basis of Prabhakar (2004).

A few rare stomatal types on the ventral surface of terminal leaflet of trifoliate leaf of
 P. pinnata.
A, hemitricyclic paracytic stomata with distinct type of subsidiaries; B and C, large anomocytic stomata havinglarge more or less indistinct subsidiaries with less wavy contours; D, An arrested stomatal aperture (a of paracytic stoma); E, A paracytic hemitricyclic stomata with distinct as well as indistinct subsidiaries.Subsidiaries shown by the asterisks.

Paracytic, anomocytic, staurocytic, tetracytic and anisocytic stomata were present on the inner surface of cotyledon.Hypocotyl had paracytic stomata. Epicotylar stomata included paracytic, anisocytic, anomocytic and anisotricytic types where as paracytic, anisocytic, anomocytic, staurocytic and tetracytic characterized the ventral surface of leaf. 

Epicotylar (A) and cotyledonary (B and C) stomata (on inner surface of cotyledons) of
 P. pinnata seedlings as seen under45 x 10 X magnification. Epicotyl (A) had tetracytic, paracytic, anisocytic and anomocytic) and anisotricytic types ofstomata. Number of the subsidiaries in cotyledonary stomata (B) varied from 2 to 9. C, Three abutting stomata withoutsubsidiaries in between. Note the unusual division of one subsidiary (Fig. 17B: 25). Diagrams not drawn to scale.

Paracytic type of stoma characterized P. pinnata and was common amongst all the organs. Abnormal stomata included paracytic stomata with common subsidiary and contiguous paracytic stomata with no subsidiaries. Paracytic hemitricyclic stoma was also present but very rarely. Stomata density on ventral surface of leaves averaged to 211.59 ± anomocytic stomata were larger than the paracytic stomata.

Common and less frequent foliar stomatal types on ventral surface of leaves of
 Pongamia pinnata
 seedlings – as seen under 45 x 15 X magnification. Note wavy cell contours of subsidiaries as well as the pavement epidermal cells of leaf and varying orientation of the stomatal long axis. Diagrams not drawn toscale. Note the cell wall developing in one of the subsidiary (shown by the asterisk). Paracytic (1A, 1B, 4),anisocytic (5, 6), anisotricytic (2), tetracytic (3, 7), anomocytic (8, 9) and anomocytic with cuticular striations(10)

The precise control of stomatal terminal division

A conserved but plant-specific CDK-mediated regulation of DNA replication protein A2 in the precise control of stomatal terminal division

by Yang K., Zhu L., Wang H., Jiang M., Xiao C., Hu X., Vanneste S., Dong J., Le J. (2019)

Kezhen Yang, Lingling Zhu, Hongzhe Wang, Min Jiang, Chunwang Xiao, Xiangyang Hu, Steffen Vanneste, Juan Dong, Jie Le




The Arabidopsis R2R3-MYB transcription factor FOUR LIPS (FLP) is the first identified key transcription factor regulating stomatal development. By screening and analyzing a genetic suppressor of flp stomatal defects, we found that FSP1/RPA2a, which encodes a core subunit of Replication Protein A (RPA) complexes, acts downstream of B1-type Cyclin-Dependent Kinases (CDKB1s). This ensures that terminal division during stomatal development will produce a pair of kidney-shaped guard cells to compose a functional stomatal complex. We demonstrate that the CDK-mediated phosphorylation at the N terminus of RPA2a is essential for the RPA functions in cell cycle control and response to DNA damage. We provide direct evidence for the existence of an evolutionarily conserved, but plant-specific, RPA regulatory pathway in plants.


The R2R3-MYB transcription factor FOUR LIPS (FLP) controls the stomatal terminal division through transcriptional repression of the cell cycle genes CYCLIN-DEPENDENT KINASE (CDKB1s (CDKB1s), CDKA;1, and CYCLIN A2s (CYCA2s). We mutagenized the weak mutant allele flp-1 seeds with ethylmethane sulfonate and screened out a flp-1suppressor 1 (fsp1) that suppressed the flp-1 stomatal cluster phenotype. FSP1encodes RPA2a subunit of Replication Protein A (RPA) complexes that play important roles in DNA replication, recombination, and repair. Here, we show that FSP1/RPA2afunctions together with CDKB1s and CYCA2s in restricting stomatal precursor proliferation, ensuring the stomatal terminal division and maintaining a normal guard-cell size and DNA content. Furthermore, we provide direct evidence for the existence of an evolutionarily conserved, but plant-specific, CDK-mediated RPA regulatory pathway. Serine-11 and Serine-21 at the N terminus of RPA2a are CDK phosphorylation target residues. The expression of the phosphorylation-mimic variant RPA2aS11,21/D partially complemented the defective cell division and DNA damage hypersensitivity in cdkb1;1 1;2 mutants. Thus, our study provides a mechanistic understanding of the CDK-mediated phosphorylation of RPA in the precise control of cell cycle and DNA repair in plants.

The potential of stomatal density as a tool for breeding wheat plants that are better able to withstand water-restricted environments

Stomatal density is reduced in wheat (Triticum aestivum) lines overexpressing TaEPF1. (A) Stomatal densities of wild-type cv Fielder and three independently transformed TaEPF1-OE lines were measured in fully expanded leaf 1. Comparison of stomatal densities was performed using a one-way ANOVA and Fisher’s LSD test, Means that are not significantly different from each other (P<0.05) are indicated with the same letter (n=5–8 leaves). Bars=SE. (B) Image of wild-type wheat epidermis of the abaxial side of leaf 1, with stomatal files labelled (purple arrows). (C) as in (B) but a TaEPF1 transgenic plant (TaEPF-OE3) with stomatal files (purple arrows) and arrested precursor cells (green arrows). (D–G) Example images of arrested or altered cell division patterns in TaEPF-OE3 in positions predicted to form stomata normally. Scale bars (B, C)=20 µm, (D–G)=5 µm.

Reduced stomatal density in bread wheat leads to increased water-use efficiency

by Dunn J., Hunt L., Afsharinafar M., Al Meselmani M., Mitchell A., Howells R., Wallington E., Fleming A. J., Gray J. E. (2019)

Jessica Dunn 1, Lee Hunt 1, Mana Afsharinafar 1, Moaed Al Meselmani 1, Alice Mitchell 1, Rhian Howells 2, Emma Wallington 2, Andrew J Fleming 3, Julie E Gray 1

1 Molecular Biology & Biotechnology Department, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK
2 The John Bingham Laboratory, NIAB, Huntingdon Road, Cambridge CB3 0LE, UK
3 Department of Animal and Plant Sciences, University of Sheffield, Sheffield S10 2TN, UK

In Journal of Experimental Botany, erz248 –


Wheat is a staple crop, frequently cultivated in water-restricted environments. Improving crop water-use efficiency would be desirable if grain yield can be maintained. We investigated whether a decrease in wheat stomatal density via the manipulation of epidermal patterning factor (EPF) gene expression could improve water-use efficiency. Our results show that severe reductions in stomatal density in EPF-overexpressing wheat plants have a detrimental outcome on yields. However, wheat plants with a more moderate reduction in stomatal density (i.e. <50% reduction in stomatal density on leaves prior to tillering) had yields indistinguishable from controls, coupled with an increase in intrinsic water-use efficiency. Yields of these moderately reduced stomatal density plants were also comparable with those of control plants under conditions of drought and elevated CO2. Our data demonstrate that EPF-mediated control of wheat stomatal development follows that observed in other grasses, and we identify the potential of stomatal density as a tool for breeding wheat plants that are better able to withstand water-restricted environments without yield loss.