L20 - Stomatal development

Question 1

What are the three main cell types in the leaf epidermis?

What is one restriction on stomata formation?

Why does this lecture focus on stomata?

Answer 1

• Pavement cells, stomata, trichomes
• Stomata must be spaced apart by at least one cell
• Provides a good case study to understand patterning, environmental regulation of development within an organ

Question 2

Give the 5 steps for stomatal development. Include the name of the cells at each step and the TFs that move the cell through each stage

What type of TFs are all the TFs involved in this pathway?

Sketch the relevant diagram

Answer 2

1) Begin as undifferentiated protodermal cells

2) Differentiate to meristemoid mother cells (MMCs) that express speechless (SPCH) TF

3) MMCs divide many times
- Each time a meristemoid and stomatal lineage ground cell (SLGC) are made

4) Meristemoid then differentiates to become a guard mother cell (GMC) through MUTE TF

5) GMC divides once to make pore, regulated by FAMA TF

• All bHLH transcription factors

Question 3

What do SPEECHLESS (SPCH) mutants look like? What is YODA and what do YODA mutants look like?

Answer 3

• SPCH mutants have no stomata or meristemoids
• Over expression line has many small meristemoid cells

YODA:
- Normally, SPCH present in both daughter cells, then kept only in meristemoid
- YODA down-regulates SPCH in SLGC, even in SPCH overexpression
- YODA mutant has many stomata, plant is small

Question 4

What do MUTE mutants look like?

Answer 4

• MUTE mutant doesn’t make stomata
• Makes many meristemoids that never differentiate to stomata
• In MUTE over expression every cell becomes a stomata

Question 5

What do FAMA mutants look like?

Answer 5

• FAMA ensures one cell division occurs and then cell cycle is shut down
• FAMA mutant = multiple divisions in guard cell lineage, non-functional
• Overexpression = all cells become guard cells

Question 6

What other transcription factors work in coordination with SPCH, MUTE and FAMA?
What do mutations in these produce?

Answer 6

• The binding partners of SPCH, MUTE and FAMA are SCRM and SCRM2 (also bHLH TFs)
• Dominant SCRM-D mutation = every cell is stomata
• MUTE SCRM-D double mutants = endodermis highly enriched w/ small meristemoids
• Scrm-D also enhances stacks of GMC-like cells in seen in FAMA mutant
• Indicates that SCRM is upstream of MUTE and FAMA

Question 7

How is it ensured that the stomata are spaced out by at least one cell?

Draw the relevant diagram

Answer 7

• Meristemoid divisions help space stomata out by generating SLGCs that aren’t part of stomatal lineage

BASL used to orient this division:
- BASL forms crescent in meristemoid pre division
- BASL crescent pushes nucleus away
- Causes asymmetric division as cell division must pass through the nucleus
- Smaller cell away from BASL crescent inherits stomatal fate
- Stomata end up surrounded by SLGCs

(See diagram on pg 26)

Question 8

How do diffusible signals also act to space stomata out?

Answer 8

• The peptides EPF1/2 are produced by stomatal lineage cells, perceived by ERECTA receptors and require TOO MANY MOUTHS (TMM) to repress stomata formation

EPF2:
- Produced by MMCs and early Ms
- Diffuses to neighbouring cells, binds to ER receptor and downregulates SPCH
- Fewer cells enter stomatal lineage = fewer stomata

EPF1:
- Produced by later meristemoids, GMCs and stomata
- Bound by ERL1 receptor in surrounding cells and downregulates MUTE
- Surrounding cells can’t differentiate into stomata

Question 9

Describe how the regulator STOMAGEN acts?

Answer 9

STOMAGEN
- Produced in the mesophyll
- Diffuses to epidermis
- Promotes SPCH by inhibiting ER, which usually inhibits SPCH
- May also help coordinate air space formation with stomatal development

Question 10

What environmental factor can influence the density of stomata in a leaf?

Answer 10

• CO2, light, temperature and drought

Question 11

How does CO2 concentration affect stomatal density?

Answer 11

High CO2 conc. leads to reduced stomatal density via two mechanisms:
1) HIC gene (High CO2)
2) Carbonic-anhydrase-encoding genes CA1 and CA4, downregulates SPCH via EPF2

Question 12

How does temperature affect stomatal density?

Answer 12

High temp. reduces stomatal density via PIF4 which downregulates SPCH

Question 13

How does drought affect stomatal density?

Answer 13

To prevent water loss, drought induces stomata closure and reduces stomatal density
- Under drought, plants produce and accumulate more ABA in guard cells, inducing closure
- Increased ABA reduces expression of SPCH and MUTE, reducing stomatal density

Question 14

How many times have stomata evolved?

Describe the plants in which stomata have been lost

Answer 14

• Stomata only evolved once, so conserved pathways in many species
• Evidence of stomata in most extinct and extant species, excluding liverworts (stomata lost)

Stomata not required and key genes therefore lost in following cases:
1) Single cell layer organs
2) Underwater organs
- Some amphibious plants respond to environment
- E.g. Rorippa aquatica make stomata in leaves in air but not in submerged leaves

Question 15

Describe the diversity of stomata

Answer 15

Some diversity in guard cell shape and arrangement of cells around the stomata
- Grasses don’t have amplifying divisions
- Grass stomata have subsidiary cells (increases efficiency and ensures spacing)
- Only some grass cell files able to make stomata (ensures spacing)

Question 16

How can a knowledge of stomata be used for crop efficiency?

Answer 16

• Conserved stomatal pathways between plants means knowledge from model plants applies
• E.g. reducing stomatal density by increasing EPFL1 in rice can improve water efficiency + plant survival