Lecture 7 Outline

1. Lecture 7 Outline Chimeras II History of “Sports” The Chimera Concept Sectorial Chimeras Definitions Green-white-green (GWG) periclinal chimeras

2. History and Application of Grafting Plant Collectors Darwin’s travels Winkler’s Chimera Concept Types of Grafts Modern Applications

3. Grafting occurs naturally Chloroplast genomes can be transferred from one tree to the other across the graft junction Figure 1A from Stegemann et al., 2012, PNAS109:2434

4. History Explorers, like Charles Darwin, traveled to new and exotic places and collected ‘treasures’in the form of novel species of plants and animals New varieties of plants were highly prized and breeders were therefore motivated to generate new and improved stocks by making interspecific crosses by vegetative propagation (or the generation of a “sport”)

5. History A Sport was defined as, “A spontaneous change or mutation in a part of a plant that created a feature not previously known within the species, their sexual hybrids and descendants”. Sports were notoriously difficult to maintain and would often be unstable The first reproducible and stable variant was described in 1674 and was called the ‘Bizzarria orange’

6. Pietro Nati (1625-1685), who was the director of the Pisa Botanical Gardens, published one of the first descriptions of a graft chimera known as “La Bizzarria” http://www.homecitrusgrowers.co.uk/citrusvarieties/bizzarria.html Resulted from grafting a sour orange scion to a citron stock. A bud arose at the graft junction with a unique mix of parental characteristics.

7. Historical paradox: What was the nature of graft hybrids? Were the resulting hybrid branches the result of sexual hybridization or a union of vegetative tissue (somatic segregation)? The argument against sexual hybridization was the you could not produce such plants by conventional breeding methods.

8. Winkler 1907 Did experiments to test the nature of graft hybrids and, using tomato and nightshade plants, reproduced results akin to those seen previously Got shoots that were all tomato, all nightshade or 1/2 nightshade and 1/2 tomato Winkler was responsible for coining the term “chimera” (a fire breathing monster from Greek mythology that had the foreparts of a lion, torso of a goat and the hindquarters of a dragon)

9. L1 L2 L3 Convention SAM Periclinal chimeras are described according to the relative contribution of graft partners, starting with the L1 If plant A is grafted to plant B and a periclinal chimera is produced, the layer contributions are listed beginning with the L1, then the L2 and finally the L3 ABA: L1 is A, L2 is B, L3 is A BBA: L1 is B, L2 is B, L3 is A

10. Vegetative Propagation Plants can be maintained by taking cuttings and rooting them By grafting a cutting onto a stock Maintaining them in tissue culture Grafting is very important to The horticultural industry (roses) For maintaining grape varieties for wine production (root stocks resistant to pathogens) The fruit industry (apples)

11. At the SAM Two types of chimeras Within Organs Periclinal and Sectorial

12. Sectorial Chimeras • Plants with sectorial meristems show sectors that run vertically down the shoot • Meristem is divisible into circumferential lineages and therefore there must be a small number of initial cells at the top of the meristem (subset of central zone) • Sectors are not usually stable (do not persist for the lifetime of the plant)

13. Sectorial Chimeras • Position of an initial does not restrict the fate of the initial’s descendants • The position of a marked band is independent of leaf initiation and may or may not encompass a whole leaf or even part of a leaf

14. Daughter cells arising from one marked cell Sectorial Chimeras Within organs reveals division patterns and clonal relationships

15. Variegation Spontaneous sectors can arise from the disruption of pigment biosynthetic genes caused by transposon insertion and excision.

16. Definitions Genotype: The DNA constitution the genetic constitution of a cell, an organism, or an individual (i.e. the specific allele makeup of the individual) (from Wikipedia) b) the actual alleles present in an individual Phenotype: Visible consequences a) an observable characteristic b) any observable characteristic or trait of an organism (from Wikipedia)

17. Gene expression patterns In theory, every nucleus in a multicellular organism has the same genetic information but expression patterns are different. Genes are turned on and off by multiple factors (temporal/spatial). A direct relationship: gene is on, function is on or gene is off and function is off

18. Developmental Regulation A gene may be off if it resides in a cell in the L1 but not in the L2 (where you live matters!) Factors affecting gene expression Activation due to a change in fate Through cell division, an L1 daughter cell may end up in an L2 neighbourhood and hence different genes become activated and inactivated (turned on/off) Location, location, location

19. Green-white-green chimeras L1=green, L2=albino, L3=green Conventionally written as GWG What does that mean? The L1 cells are genetically green but visibly (phenotypically) white because chloroplasts do not mature (suppressed) The L2 cells are genetically albino or white (genes do not function: they have mutations) and phenotypically white The L3 cells are genetically and phenotypically green (green genes are functional)

20. Green-white-green chimeras The L2 meristematic layer is albino From Leyser and Day, Mechanisms in Plant Development. 2003 Blackwell Publishing,Chapter 3.

21. Genotype of L1, L2 and L3 L1=green, L2=albino (white), L3=green Pigments are cell autonomous markers because they are contained within a plastid Leaf in cross-section

22. Phenotype of L1, L2 and L3 L1=white (gene expression suppressed) L2=albino (genes have mutations) L3=green (genes are expressed)

23. Layer displacements L1 cells that divide periclinally (rare) will give rise to L2 daughter cells that can make chloroplast pigment while L2 cells cannot, even if displaced into the L3. L2 cells invading the L3 occurs more commonly. L1 L2 L3 layer

24. GWG Chimeras Demonstrate that…. • L1 layer is very stable and rarely invades the subjacent L2 layer • L2 cells typically remain in the subepidermal layer in most of the leaf but near leaf margins are more likely to become displaced into the L3 • If L2 cells invade, there is a compensatory reduction in cell divisions in the L3 so that the number of cell layers in the leaf remains constant • Hence one sees this fairly common pattern of variegation in garden and house plants (white margins and green centre)

25. Sectorial Chimeras are Rare • GWG to WWG, GWW or GGG sectors can occur but they tend not to be stable • GWW sector would give rise to a white sector on the stem and would run down the vertical axis of the shoot (leaf and stem tissues would be white) • GGG sector would produce a solid green band with no variegation • Seeds produced in any of these sectors would have the genotype of the L2 only

26. L1 L2 L3 What have we learned so far? SAM Meristem layers are relatively stable (L1>L2>L3). Proteins and RNAs can move between layers and there seems to be directionality. Directionality is determined on a case-by-case basis. There are tissue-specific constraints.

27. L1 L2 L3 Some additional questions SAM Who is in charge? For example, is the coordination of meristem activity due to the response of L2/L3 tissues to signals from the L1? Is there lateral movement of signals within a layer? Are there communication rules and if so, what are the rules?