Sex determination in cucumber Anandkumar Surendrarao VC221: Vegetable crop breeding May 10, 2006

Sex determination in cucumber

Anandkumar Surendrarao

VC221: Vegetable crop breeding

May 10, 2006

Perfect flowers or Hermaphroditic flowersBoth male and female reproductive parts are present on the same flower

Perfect flowers or Hermaphroditic flowersBoth male and female reproductive parts are present on the same flower

Monoecious plants – Imperfect flowersSeparate male and female flowers are present on the same plant

Dioecious plants – Imperfect flowersMale and female flowers are present on individual separate plants

Dioecious plants – Imperfect flowersMale and female flowers are present on individual separate plants

Sex determination in cucumber

♂flower

♀flower

ABC model of floral development

Paper #1

Developmental arrest of whorl 4 in male and whorl 3 in female flowers

Cucumber floral MADS box gene expression and sequenceProbing female cDNA library with petunia MADS box gene

Class C

Class B

amino-acid conservation amongst MADS box genes

CUM1 = Ath AGAMOUS (69%)

CUM1 = Antirhinum PLENA (71%)

CUM26 = Ath PISTLLATA (69%)

CUM26 = Antirhinum GLOBOSA (70%)

CUM26 = Petunia FLORAL BINDING PROTEIN 1 (71%)

In situ hybridization analyses of CUM1 and CUM26 expression in wild type male and female flowers

with antisense probe to divergent 3’ UTR sequence

CUM1 – class C, whorls 3 and 4; CUM26 – class B, whorls 2 and 3

Wild type

Expression of homeotic genes is observed even in arrested primordia

gp mutant flower phenotypes at 22°C

A-D

♂ sepal-sepal-flower-X♀ sepal-sepal-X-carpel

gp mutant flower phenotypes at ≥ 30°C

E-J

♂ sepal-sepal-carpel-X ♀ sepal-sepal-X-carpel

In situ hybridization analyses of CUM1 and CUM26 expression in gp mutant male flowers

gp mutant

CUM26 = GP = class B mutant

CUM1 – class C, whorls 3 and 4; CUM26 – class B, whorls 2 and 3

22°C35°C

22°C35°C

CUM1 hypermorph (over-

expression)

CUM1 hypomorph(co-suppression)

Unisexual to bisexualfloral conversion

ABC model of floral development

Selective repression of male or female reproductive organs Selective repression of male or female reproductive organs depends on floral whorl position rather than organ identitydepends on floral whorl position rather than organ identity

Paper #2

Genetic and environmental control of cucumber sex determination

Genotypes

Gynoecious - F-M- - ♀

Andromonoecious - ffmm - ♂ and ♀

Monoecious - ffM- - ♂ and ♀

Hermaphrodite - F-mm - ♀

Ethylene and ethephon – induction of ♀ flowers

AVG and AgNO3 – induction of ♂ flowers

Sex of different cultivars used in this study

Development of flower buds in gynoecious cucumber plants

Development of flower buds in gynoecious cucumber plants

Development of flower buds in monoecious cucumber plants

Development of flower buds in monoecious cucumber plants

AVG masculinizes between node 8 and 13, Ethephon feminizes between nodes 10 and 14

Floral stages immediately before and after differentiation of stamen primordiaare responsive to both AVG and ethephon treatments

Monoecious Gynoecious Andromonoecious Monoecious

Antisense CS ACS2

Antisense CS ERS

Antisense CS ETR1

Antisense CS ETR2

Sense CS ACS2

Sense CS ERS

Sense CS ETR1

Sense CS ETR2

In situ hybridization results

The expression patterns for The expression patterns for CS-ACS2CS-ACS2, , CS-ERSCS-ERS, , CS-ETR1CS-ETR1, , and and CS-ETR2 CS-ETR2 are all different among monoecious, are all different among monoecious, gynoecious and andromonoecious plants.gynoecious and andromonoecious plants.

CS-ACS2 CS-ACS2 and and CS-ETR2CS-ETR2 are expressed in identical are expressed in identical domains in monoecious plants and overlapping domains domains in monoecious plants and overlapping domains in gynoecious plants.in gynoecious plants.

In andromonoecious plants, none of the ethylene receptors In andromonoecious plants, none of the ethylene receptors transcripts accumulated in the stamen primordia.transcripts accumulated in the stamen primordia.

Atleast one ethylene receptor transcript is expressed in the Atleast one ethylene receptor transcript is expressed in the stamen and pistil primordia of monoecious and stamen and pistil primordia of monoecious and gynoecious flowers, and pistil primordium of gynoecious flowers, and pistil primordium of andromonoecious flowers.andromonoecious flowers.

Cells producing and sensing ethylene are identical. Eg. Cells producing and sensing ethylene are identical. Eg. Overlapping CS-ACS2 and CS-ETR2 mRNA expression Overlapping CS-ACS2 and CS-ETR2 mRNA expression in monoecious and gynoecious plants, direct in monoecious and gynoecious plants, direct determination of female flowers by inducing pistil determination of female flowers by inducing pistil development.development.

Cells producing and sensing ethylene are adjacent. Eg. Cells producing and sensing ethylene are adjacent. Eg. mRNA expression of CS-ACS2 in adaxial side of petals mRNA expression of CS-ACS2 in adaxial side of petals but all the receptors in stamen primordia in monoecious but all the receptors in stamen primordia in monoecious plants. (diffusion?)plants. (diffusion?)

Cells producing and sensing ethylene are distant. Eg. Cells producing and sensing ethylene are distant. Eg. mRNA expression of mRNA expression of CS-ACS2CS-ACS2 in pistil primordia but that in pistil primordia but that of receptors in the stamen primordia. (diffusion?)of receptors in the stamen primordia. (diffusion?)

Paper #3

What are the downstream targets of the sex determination machinery that allow the selective arrest of stamen and pistil primordia development?

Use suppression subtractive hybridization on NILs of gynoecious (FFMMaa), hermaphrodite (FFmmaa), androecious (ffMMaa) and monoecious (ffMMA-) genotypes.

AgNO3 induced male flowers in gynoecious plants, and ethephon induced female flowers in hermaphrodite plants used for SSH.

Controls for SSH were female and male flowers from gynoecious and androecious plants respectively.

Results from SSH

Selection of 21/178 clones by dot blot analyses

11/21 differentially expressed in hermaphrodite buds – Clone #38 is putative CS nt sugar epimerase

10/21 differentially expressed in gynoecious plants

Putative sugar nt epimerase expressed lower in gynoecious than in hermaphrodite plants

Floral buds Leaves

Putative sugat nt epimerase expressed higher in natural/induced male flowers compared to

natural/induced female flowers

Monoecious + no treatment ♂ plants + ethephon ♀ plants + AgNO3

No detectable polymorphisms at gDNA level between gynoecious and hermaphrodite plants

Southern blot hybridization with 19 different restriction enzymes

Mechanistic role for sugar nt epimerase in stamen primordia outgrowth and arrest

UDP glucose-4-epimerase converts UDP-glucose to UDP-galactose.

These are required for the synthesis of AGPs (Arabino-Galactan proteins) and cell wall polysaccharides that are necessary for cell wall expansion and therefore primordial outgrowth.