Lecture 11 - The role of the dorsoventral axis

Question 1

What is knotted-1?

Answer 1

• developmental gene
• member of the homeobox gene family
• not expressed in cells that are going to become leaves or in the outer layer of the SAM
• finte expression pattern

Question 2

What is the mutant phenotype of knotted?

Answer 2

• knotted leaf structures

- most mutations are dominant (couldn’t find loss of phenotype)

Question 3

Where is knotted-1 mRNA and protein found?

Answer 3

mRNA
-in situ hybridisation (antisense labelled mRNA probe binds to native RNA form dsRNA -> colour reachtion)
-localised to centre of the meristem, not the outer layer L1
Protein
-immunolocalisation (antibody raised to KN1 protein)
-protein in the meristem AND the outerlayer

Transported by plasmodesmata
none in leaves in each case

Question 4

What is the member of the knotted class of homeodomain proteins in Arabidopsis?

Answer 4

STM

Question 5

What is the WT and mutant phenotype of STM?

Answer 5

WT
-trichores in the cotelydon (single celled hair cells)

stm

• lack SAM
• initiated meristem further down the cotelydon

Question 6

Where is STM expressed and what is its purpose?

Answer 6

• expressed early, throughout the meristem but not in cells that are destined to become leaves
• specifies meristematic fate

Question 7

How do plants build organs?

Answer 7

1. Cells become identified to become leaves in the shoot apical meristem by the downregulation of the expression of STM
   - leaves differentiate into leaf primordia on the flank of the meristem
   - differentiation of SAM cells pushes cells destined to becomes leaves onto the flanks of the meristem
   - produces initially symmetrical mounds of tissue
2. Mounds of tissue begin to flattern along the dorsoventral (abaxial-adaxial axis) axis, and the dv axis develops
3. leaf primordia then begin to expand laterally
4. leaf elaborated along the proximal-distal axis

2 Key axis’ involved:
-dorsoventral
-proximo-distal
Evidence for dorso-ventral axis

Question 8

Where is the dorsoventral axis strong and what is the pattern in these structures?

Answer 8

Clear in the adult leaf
Clear distinction between the dorsal and ventral regions
-Dorsal: palisade parenchyma + upper epidermis
-Ventral: spongy parenchyma + lower epidermis
Vascular tissue also patterned in a dorsoventral manner
-Xylem vascular bundle more dorsal than the more ventral phloem (in the central portion of the leaf)
Epidermal surfaces
-Dorsal: trichores
-Ventral: stomata

Question 9

What is the evidence for proximo-distal polarity (leaf)?

Answer 9

-very small leaves were irradiated leading to mutations of not green tissue (yellow)
-sections developing in the leaves were not of the same size
Earlier irradiated: larger patches
Later irradiated: smaller patches

Conclusions

• cell division happens rapidly early in development, expansion happens after
• patches were smallest towards the tip of the leaf than the base -> cell divisions happen slower at the tip of the leaves
• poximal distal axis is required to pattern the leaf -> cell cycle arrest runs down the top of the leaf to the base corresponding to the proximal distal axis

Question 10

What evidence is there for the radial polarity of leaves?

Answer 10

• took cross section through leaf about to emerge
• youngest leaf showed radial symmetry
• evidence for dv as can recognise the top from the bottom
• radial patterning is evident before others

Question 11

What experiment confirmed the radial axis of leaves?

Answer 11

• worked out where the next leaf would initiate
• made a cut and inserted a piece of non permeable material to stop the centre of the meristem communicating with the primordial leaf
• confirms the radial axis as once detatched from the merisetem produces a radial leaf which does not broaden laterally
• also must be a signal from the centre of the meristem to pattern the leaf

Question 12

What is the summary of the axes of leaf development?

Answer 12

Meristem signal passes from the centre to the developing leaves to pattern the dorsoventral axis (through Pahn/As1)
As1 is established in the developing lead to downregulate STM

Question 13

What are the three developmentally axis required to pattern the leaf?

Answer 13

• dorsoventral axis
• promial distal axis
• radial axis

Question 14

What is the genetic evidence for the dorsoventrality of leaves?

Answer 14

Phantastica

• mutant loses its dorsoventral axis almost entirely
• leaves are entirely radial

Question 15

What is phantastica?

Answer 15

• gene required for the growth and dorsoventralilty of lateral organs in antirrhinium majus
• encodes a MYB transcription factor
• mutants show radialised or needle like leaves - are abaxialised and lact dorsoventral polarity
• problems in specifying dorsal tissue within the leaf

Question 16

What, leading to ectopic axis of growth, confirms that the dorsoventral axis drives lateral growth?

Answer 16

Novel interactions between abaxial and adaxial tissues in phan mutants (lead to ectopic axies of growth)
-when juxtapose ventral tissue against dorsal tissue, results in ectopic growth of tissue like the edge of the leaf

Question 17

What is PHAN required for?

Answer 17

Dorsalising function

-specifies dorsal cell fate within the leaf

Question 18

Where is the phantastica gene expressed?

Answer 18

-in initiating leaves
(STM has a reciprocal pattern to phan)
-expressed as soon as cells have been chosen to become leaves

Question 19

What does establishment of leaf polarity involve?

Answer 19

Coordinated expression of transcription factors

• phan has interactions with other genes that have other expression patterns
  eg. KAN (defines abaxial fate) and PHAN (Adaxial fate) have mutually supressive function
• Phan initiated from a meristem derived signal

Question 20

What is the function of asymetric leaves 1 (AS1)?

Answer 20

-mediates leaf patterning and stem cell funtion in arabidopsis

Question 21

What is the interaction between STM and AS1?

Answer 21

-have antagonistic functions
-STM important for maintaining meristematic stem cells
-AS1 determines which cells become leaves
as1 mutant - not particularly stong phenotype
stm mutant - lacks meristem
as1 and stm mutant - resurrects the stm phenotype
as1 and stm - flower

Question 22

What is knox?

Answer 22

STM family

Question 23

Where is AS1 expressed?

Answer 23

strongly in the cotelydon