Epidermis

It occurs on the surface of plants and it is a complex tissue

consisting of epidermal cells, stomata and trichomes (hairs).

Function of epidermis:

Protect the plant from water loss in transpiration.

It acts as boundary tissue surrounding the plant.

Exchange of gases through the stomata.

Storage of water and metabolic products.

Distribution of Stomata

Plant Average number of stomata/cm2

Upper Epidermis Lower Epidermis

Apple 0 29,400

Black oak 0 58,000

Cabbage 14,100 22,600

Corn 5,200 6,800

Geranium 1,900 5,900

Mulberry 0 48,000

Pea 10,100 21,600

Scarlet oak 0 103,800

Sunflower 8,500 15,600

Wheat 3,300 1,400

Functions of MADS-box genes throughout the life cycle of Arabidopsis thaliana.

Smaczniak et al., (2012). Development. 139:3081-3098

Arabidopsis progresses through several major phase changes during its life cycle and MADS-box genes play distinct roles in the various developmental phases and transitions. Reproductive development starts with the generation of male and female haploid gametes (gametogenesis) and, after double fertilization, this results in a developmentally arrested embryo that possesses a root apical meristem (RAM) and a shoot apical meristem (SAM), enclosed within a seed. Under favorable conditions, seeds germinate and young plants go through the vegetative phase of development in which leaves are formed and plants gain size and mass. Finally, the plant is ready to flower and the floral transition stage results in the conversion of vegetative meristems into inflorescence meristems (IMs) and floral meristems (FMs) that produce floral organs. Subsequently, gametes are formed within the inner flower organs, thus completing the cycle. The MADS-box genes that are involved in each of the various stages of development are indicated.

Gas Exchange

Phases of plant development.

Huijser and Schmid (2011). Development 138:4117-4129

epidermal cells are covered by an impermeable layer :Cuticleknown as cuticle which varies in thickness. The cuticle may be smooth as in Stramonium or striated as in Belladonna.

The cuticle is formed mainly of cutin which is an aggregate of modified fatty acids, partly combined with alcohols.

Stomatal Development

• Mesogenous – guard cells and subsidiary

cells have a common origin

• Perigenous – guard cells and subsidiary

cells DO NOT have a common origin

• Mesoperigenous – some subsidiary cells

have a common origin with guard cells

Some of the common

developmental patterns

• Anomocytic – no subsidiary cells

• Diacytic – two subsidiary cells at right angles to the guard cells

• Paracytic – two or more subsidiary cells parallel the guard cells

• Actinocytic – subsidiary cells seem to radiate from guard cells

• Anisocytic – three unequal-sized subsidiary cells surround the guard cells

Plant receptor-like kinases (RLKs) and their functions

Osakabe Y et al. J. Exp. Bot. 2013;64:445-458

Receptor-like kinases (RLKs) play important roles in

perceiving the extracellular ligands and activating the

downstream pathway via phosphorylation of intracellular

serine/threonine kinase domains.

Cell fate transitions and divisions during Arabidopsis stomatal

development

Mitogen-activated protein kinase (MAPK) cascades,

which include a MAPKKK YODA, MAPK kinases MKK4,

MKK5, MKK7, and MKK9, and MAPKs MPK3 and

MPK6, act as a potential downstream pathway for ER

signalling (Wang et al., 2007).

Basic helix–loop–helix (bHLH) transcription factors

control stomatal development by serving as targets of

MAPKs.

Three paralogous bHLHs, SPEECHLESS (SPCH),

MUTE, and FAMA, are key factors regulating stomatal

development (Peterson et al., 2010).

These bHLHs control the progression of the stomatal

lineage to generate a pair of guard cells that are

sequentially differentiated from a protodermal cell,

meristemoid mother cell, meristemoid cell, and guard

mother cell. Other bHLHs involved in modulation of

stomatal development are ICE1/SCRM1 and SCRM2,

which physically interact with SPCH, MUTE, and FAMA

(Peterson et al., 2010).

Lau and Bergmann (2012). Development 139:3683-3692.

In this model, stem cells derive from the protoderm and can divide

symmetrically or asymmetrically.

Each asymmetric division produces a stomatal meristemoid and a larger sister

cell.

The larger sister cell can divide symmetrically or asymmetrically.

This balance regulates stomatal and epidermal cell number.

Pavement cells differentiate from stem cells and their derivatives.

Nadeau and Sack (2003). Trends in Plant Science 8: 294 - 299

Initiation involves the selection of a meristemoid mother cell (MMC, yellow) that then divides asymmetrically

producing a meristemoid (M, red) and a larger sister cell.

A neighbor cell (NC) is considered to be any cell next to a stoma or a precursor. When meristemoids divide

asymmetrically they regenerate a meristemoid and produce an additional NC.

When the NC divides asymmetrically it functions as an MMC and intercellular signaling orients its division. T

he resulting smaller cell is termed a satellite meristemoid (SM) to highlight the importance of this class of asymmetric

divisions in creating the minimal one-cell spacing pattern.

Each meristemoid converts into a guard mother cell (GMC) that divides symmetrically producing a stoma (S).

Pavement cells (PCs) make up a second type of terminally differentiated epidermal cell. Many PCs originate from

asymmetric divisions in the stomatal pathway.

Nadeau and Sack (2003). Trends in Plant Science 8: 294 - 299

Arabidopsis stomatal development.

(a) WT

(b) too many mouths (tmm)

(c) stomatal density and distribution1 (sdd1)

sdd1 has fewer stomata in direct contact than tmm does

(d) Confocal micrograph (inverted image) of tmm showing misoriented

asymmetric divisions that incorrectly place satellite meristemoids (∗). The satellite

meristemoids shown resulted from the asymmetric division of cells located

adjacent to two stomata and/or precursor cells, cells normally prohibited from

dividing. All scale bars=15 μm. (a) and (b) are the same magnification.

Wild-type and mutant stomatal distribution

WT tmm sdd1

Nadeau and Sack (2003). Trends in Plant Science 8: 294 - 299

(a) SDD1 is expressed (blue) in meristemoids and guard mother cells (GMCs).

(b) TMM is expressed (green) in GMCs, meristemoids and their recent sister cells.

(c) Both gene products

(1) promote meristemoid division thus delaying the transition to the GMC,

(2) act as negative regulators of neighbor cell (NC) division (bottom ‘T’),

(3) prohibit asymmetric divisions in cells next to two stomata or precursor cells (asterisk at upper right),

(4) are required for the correct placement of patterning divisions in those neighbor cells (NCs) that are

allowed to divide (top arrow).

(d) Speculative model of signaling.

Stomatal precursors broadcast an SDD1 signal (blue) that might modify or interact with an unknown

ligand (black triangles). This ligand could bind to receptor complexes (green ‘Y's) that contain TMM. The

resulting signaling through the receptor complex might set up a polarity that orients the plane of NC

division (top). Alternatively, signaling might limit the number of NC divisions (bottom).

TOO MANY MOUTHS (TMM) and STOMATAL DENSITY AND DISTRIBUTION1

(SDD1) expression patterns and functions

Nadeau and Sack (2003). Trends in Plant Science 8: 294 - 299

Receptor kinase that, together with ERL1 and ERL2, regulates aerial architecture,

including inflorescence (e.g. shoot apical meristem-originating organ shape,

elongation of the internode and pedicels), and stomatal patterning (e.g. density

and clustering)

Modulates plant transpiration efficiency by controlling stomatal density, leaf

photosynthetic capacity, epidermal cell expansion, mesophyll cell proliferation

and cell-cell contact.

May maintain development integrity in heat stress conditions. Regulates cell wall

composition and structure.

Confers resistance to the pathogenic bacteria

ERECTA

Function

The ERECTA family

Stomatal differentiation in the epidermis of plants is initiated by a series of asymmetric

cell divisions and involves cell–cell communication to establish their number and

arrangement (Peterson et al., 2010).

The ERECTA family of LRR-RLKs, consisting of ERECTA (ER), ERECTA-LIKE1 (ERL1)

and ERL2, mediate various plant developmental processes, such as cell fate specification

including stomatal development(Pillitteri and Torii, 2012).

Disruption of the ER family produced a phenotype characterized by a high density of

mispatterned stomata (Shpak et al., 2005).

The ER family together with the LRR receptor-like protein TOO MANY MOUTHS (TMM)

control stomatal patterning in a synergistic manner (Guseman et al., 2010).

Recent findings suggest that the different types of receptor–ligand pairs between

ER/TMM and EPFs may specify the different steps of stomatal development (Hara et

al., 2007, 2009; Ohki et al., 2011; Lee et al., 2012).

Mitogen-activated protein kinase (MAPK) cascades, which include a MAPKKK YODA, MAPK

kinases MKK4, MKK5, MKK7, and MKK9, and MAPKs MPK3 and MPK6, act as a potential

downstream pathway for ER signalling (Wang et al., 2007).

Basic helix–loop–helix (bHLH) transcription factors control stomatal development by serving

as targets of MAPKs.

Three paralogous bHLHs, SPEECHLESS (SPCH), MUTE, and FAMA, are key factors regulating

stomatal development (Peterson et al., 2010).

These bHLHs control the progression of the stomatal lineage to generate a pair of guard cells

that are sequentially differentiated from a protodermal cell, meristemoid mother cell, meristemoid

cell, and guard mother cell. Other bHLHs involved in modulation of stomatal development are

ICE1/SCRM1 and SCRM2, which physically interact with SPCH, MUTE, and FAMA (Peterson et

al., 2010).

The epidermal phenotype of er-family

mutants. Line drawings of mature epidermis of

wild-type (wt) (A), erl1 erl2 (B), er (C), er erl1

(D), er erl2 (E), and er erl1 erl2 (F) pedicels stage

17 are shown.

Guard cells and SLGCs are

false colored in green and pink, respectively.

Scale bar, 50 mm.

Stomatal Patterning and Differentiation by Synergistic

Interactions of Receptor Kinases

Shpak et al., (2005) 309: 290-293

(A to D) Stoichiometric nature of epistasis between ER-

family genes and TMM in stem epidermis.

tmm (A) and tmm er (B) do not differentiate stomata.

In contrast, tmm er erl1 (C) confers a recovery of stomatal

differentiation (asterisks). A tmm er erl1

erl2 quadruple mutant (D) produces high-density stomatal

clusters (dashed brackets).

(E to H) A combination-specific neomorphism revealed by

interactions of TMM with ER-family genes in silique

epidermis. tmm (E) produces guard cells (asterisks)

with a mild clustering.

whereas er (F) confers occasional

failure of guard mother cell differentiation

(bracket).

In tmm er double mutants (G), all stomatallineage

cells adopted SLGC cell fate (brackets). Again, guard cells

(asterisks) differentiate in tmm er erl1 (H). Scale bar, 50 mm.

Genetic interactions of ER-family RLKs and TMM

Shpak et al., (2005) 309: 290-293

Lau and Bergmann (2012). Development 139:3683-3692.

Ligand-receptor interactions regulate stomatal production and patterning. (A) In leaves, the secreted peptide EPF2 (pink) is produced by MMCs and early meristemoids. EPF2 is detected by the receptor-like kinase ERECTA (blue), present in protodermal cells (gray). In partnership with the receptor-like protein TMM (green), the EPF2-ERECTA pair is hypothesized to activate an intracellular signaling cascade that represses production of meristemoids (red).

(B) EPF1 (orange, top), which is secreted by late meristemoids, GMCs or GCs, interacts with ERL1 (purple). The EPF1-ERL1 pair, together with TMM, induces signaling that affects the division plane such that the secondary meristemoid (red) forms away from pre-existing stoma or stomatal precursors. Illustrated here is a GMC (orange, bottom), and the results of correct and incorrect spacing of a newly formed secondary meristemoid. The EPF1-ERL1 pair also represses meristemoid differentiation (not shown).

(C) In stems, ERf receptors (blue/purple) are subject to inadvertent activation by the EPF-related CHALf peptides (brown), which are normally produced in inner tissues for growth regulation. Stomatal lineage expression of TMM functions as a signaling insulator, repressing CHALf-mediated and promoting EPF1/2-mediated signaling. In A and B, ERECTA and ERL1 are shown as homodimers, but they may also form heterodimers with other members of the ERf.

ERECTA, which is

expressed strongly in the protodermal cells

restricts asymmetric entry division in MMCs.

ERL1 and ERL2

highly expressed later in meristemoids, GMCs and young GCs

inhibit the differentiation of meristemoids into GMCs

ERL1 might also orient asymmetric spacing division, because in mutants expressing a

kinase-deleted version of ERL1, stomata are often paired.

ERECTA and ERL1 have been shown to homo- and heterodimerize in vivo.

ERECTA, ERECTA-LIKE 1 (ERL1) and ERECTA-LIKE 2 (ERL2), together

comprising the three-membered LRR-RLK ERECTA family (ERf)

regulators of plant organ growth

control cell proliferation (Shpak et al., 2004)

proper patterning and differentiation of stomata (Shpak et al., 2005).

A mitogen-activated protein kinase (MAPK) pathway transduces and integrates intrinsic and environmental signals during stomatal production.

A signal transaction cascade involving the MAPKKK YDA, MKK4/5 and MPK3/6 is employed to repress stomatal production.

The EPFL-ERf-TMM module (including the antagonistic STOMAGEN) functioning within the stomatal lineage lies genetically upstream of YDA

The activated stomatal MAPK module can regulate stomatal development at multiple stages.

its repression of meristemoid production by MPK3/6 phosphorylation and down regulation of SPCH that is initiated upstream by the EPF2-ERECTA pair.

The downstream targets of the MAPK pathway activated by the EPF1-ERL1 pair are not known.

An intermediate signaling component in the brassinosteroid (BR) pathway, the GSK3-like kinase BIN2, interfaces with the stomatal development pathway at two levels: by phosphorylating and inhibiting YDA (thus increasing stomata) and by phosphorylating and repressing SPCH (thus decreasing stomata).

Light also regulates the number of stomata through the ubiquitin E3 ligase COP1, a central repressor in light signal transduction.

BAK1, BRI1-ASSOCIATED RECEPTOR KINASE 1; BRI1, BR INSENSITIVE 1; BSU1f, family of BRI1-SUPPRESSOR 1.

Lau and Bergmann (2012) Development 139:3683-3692

The model of ERECTA, BRI1, BAK1, and FLS signalling pathways.

Osakabe Y et al. J. Exp. Bot. 2013;64:445-458

Arabidopsis as a model organism

• Many Benefits

– Small genome (125mb)

– Rapid life cycle (6 weeks)

– Easy cultivation

– Prolific seed production

– Efficient transformation

• Using Agrobacterium tumefaciens

– Large numer of mutant lines

– A very large research community

3- Trichomes (Hairs)

Epidermal cells are sometimes extended

outwards forming projections of variable

shape and size. If it is short and conical it is

called papillae and the epidermis is

described as papillosed e.g. Coca, but if the

projections are long and well protruding

they form trichomes or hairs.

Control of GL2 expression in Arabidopsis leaves

and trichomes

Szymanski et al., (1998). Development 125: 1161-1171.

Trichomes

• Definition: A hairlike or

bristlelike outgrowth, as from

the epidermis of a plant

• Many shapes and sizes that

depend on the plant species

– Unicellular trichomes

– Multicellular trichomes

– Secretory trichomes

• Root hairs are specialized

trichomes found on roots

The development of Arabidopsis unicellular

trichomes

1. radial expansion of the trichome precursor in the plane of the leaf

2. stalk emergence and expansion

3. formation of branch structures

4. expansion of the stalk and branches

5. continued expansion of the stalk and branches, which develop pointed tips

6. mature trichome with papillate surface

Szymanski et al., (1998). Development 125: 1161-1171.

What Genes control the development of

trichomes?

• More than 20 genes are required

– The initial selection (stage1-2) of trichomal precursors depends on 2 genes.

• GLABROUS1 (GL1) a myb-class transcription factor

• TRANSPARENT TESTA GLABROUS (TTG)

WT gl1 ttg

Szymanski et al., (1998). Development 125: 1161-1171.

What Genes control the development of trichomes?

• GLABROUS2 (GL2) is

required for

subsequent

development of

trichomes

WT, early stages

WT later stages gl2 Later

stages

Szymanski et al., (1998). Development 125: 1161-1171.

The expression pattern of the GL2 gene

• In order to look at where the GL2 gene was being translated, the researchers made a special T-DNA construct

• It was composed of a 5’UTR 2,1kb fragment of the GL2 gene and the b-glucuronidase gene (GUS)

– GL2::GUS, (essentially the GUS gene under the control of the GL2 promoter region)

• This construct was then used to transform Wild type Arabidopsis plants

– The plants were then treated for GUS staining at different developmental stages and GUS activity was analyzed

A. Developing Leaves

B. Transverse Section through a

developing leaf primorida

C. Apaxial surface of a

developing leaf

D. Cross section through the

apical third of a developing

leaf

E. Emerging trichomes at the

bse of a mature leaf

F. Transverse section through

leaf base with developing

trichomes

G. Developing leaf with

trichomes at several

developmental stages

H. Mature leaf

Szymanski et al., (1998). Development 125: 1161-1171.

Relationship between GL1 and GL2

• Both the GL1 and TTG loci are required for trichome initiation, while GL2 is

required for the earliest morphogenetic events of trichome growth

– The Promoter region of GL2 contains a myb-class binding site

• GL1 is a myb-class transcription factor...

• A comparison between GL1::GUS and GL2::GUS plants was made

A. GL1::GUS

B. GL2::GUS

C. TS of apical section of a GL1::GUS plant leaf

D. TS of apical section of a GL2::GUS plant leaf

Trichome phenotype of SlCycB2-RNAi transgenic positive (Right) and negative plants (Left)

Yang et al., (2011). PNAS 108:11836-11841.

Trichome of total plants (A), young stems (B), old stems (C), magnified view of leaflet boxed in a (D), and SEM observation of leaf trichomes (E). (Scale bars: 0.5 cm.)

Expression Patterns of G. arboreum RDL1, MYB2/FIF1, and HOX3 Genes.(A) Transcript profile of

GaRDL1, GaMYB2, and GaHOX3 in plants of G. arboreum

Wang et al., (2004). Plant Cell 16:2323-2334

(A) Transcript profile of GaRDL1, GaMYB2, and GaHOX3 in plants of

G. arboreum. Relative transcript amounts of each gene were

normalized with respect to cotton Histone3 transcript levels (100%).

Mean values were obtained from three independent PCR

amplifications, and the error bars indicate the standard error of the

mean. A break in the scale (=) has been incorporated to show the

higher amount of GaRDL1 in fibers. R, roots; S, stems; L, leaves; O-0,

0-DPA ovules; F-3, 3-DPA fibers; F-6, 6-DPA fibers; F-9, 9-DPA fibers;

F-12, 12-DPA fibers; NO-6, 6-DPA naked ovules (fibers stripped off).

(B) GUS staining of Arabidopsis plants expressing RDL1-P3::GUS.

Young leaf (top), mature leaf (middle), and stem (bottom) are shown.

GaMYB2 Regulates Arabidopsis Trichome Development

(A) The Arabidopsis wild-type and a gl1 (SALK_039478) mutant seedling. (B) and (C) Trichome phenotypes of wild-type or gl1 mutant plants transformed with various chimerical genes as indicated

For intronless cDNA, a “c” was added as a suffix. Wang et al., (2004). Plant Cell 16:2323-2334

Wild-type, mutant, and transgenic Arabidopsis phenotypes

Payne et al., (2000). Genetics 156:1349-1362

(A–C) 10-day-old seedlings. (A) Wild-type Ler. (B) gl3-1 mutant in Ler background. (C) gl3-1 mutant complemented with MYC6 genomic fragment. (D–F) SEMs of fourth true leaves. (D) Wild-type WS. (E) Antisensed GL3 in WS background. (F) GL3 overexpressed in WS. (J–L) Fourth true leaves of transgenic plants produced in the ttg1-1 background. (J) GL1 overexpressed in the ttg1-1 mutant. (K) GL3 overexpressed in ttg1-1 mutant. (L) Cross in which both GL1 and GL3 are overexpressed in the ttg-1 mutant background.

Functions of MADS-box genes throughout the life cycle of Arabidopsis thaliana.

Smaczniak et al., (2012). Development. 139:3081-3098

Arabidopsis progresses through several major phase changes during its life cycle and MADS-box genes play distinct roles in the various developmental phases and transitions. Reproductive development starts with the generation of male and female haploid gametes (gametogenesis) and, after double fertilization, this results in a developmentally arrested embryo that possesses a root apical meristem (RAM) and a shoot apical meristem (SAM), enclosed within a seed. Under favorable conditions, seeds germinate and young plants go through the vegetative phase of development in which leaves are formed and plants gain size and mass. Finally, the plant is ready to flower and the floral transition stage results in the conversion of vegetative meristems into inflorescence meristems (IMs) and floral meristems (FMs) that produce floral organs. Subsequently, gametes are formed within the inner flower organs, thus completing the cycle. The MADS-box genes that are involved in each of the various stages of development are indicated.