The genetics of heat –or pungency- The genetics of heat –or pungency- in in CapsicumCapsicum

Marco Hernandez-BelloMarco Hernandez-Bello

PLS221PLS221

IntroductionIntroduction

• Pungency or “heat” is due to accumulation of alkaloid

capsaicin and its analogs in the placental tissue

• Capsaicin biosynthesis is restricted to Capsicum– Driven domestication of several species

• Has also an ecological role

• Pepper species and cultivars differ with respect to their

level of pungency (quantitative & qualitative)

• Capsaicin has wide applications

• Little is known about its biosynthesis (molecular, genetics,

localization, accumulation)

• Absence of pungency controlled by single recessive gene,

pun1

www.thechileman.org/guide_species.php

C. annum

C. chinense ‘Habanero’

C. frutescens ‘Tabasco’www.wikipedia.org

The Pun1 gene for pungency in pepper encodes a putative acyltransferase

Stewart, C. et al. 2005. Plant J. 42:675-688

BackgroundBackground

• Little is known about the pungency accumulation

• Absence of pungency controlled by pun1 – Single genetic source for non-pungency

– single recessive gene

– epistatic to all other pungency-related genes

– Qualitative effect on presence/absence of capsaicinoids

– Is it a master regulator of the pathway?

– Mapped to chromosome 2

– cDNA from SSH library co-segregated with pungency in C.chinense

– CAP marker has been used in breeding programs

• Objective: Cloning and characterization of gene (Pun1?)

responsible for pungency– Candidate gene approach

ResultsResults

- C. frutescens BG2816 (Pun1/Pun1) X C. annuum

cv. Maor (pun1/pun1) Bell pepper

- F2 mapping pop. (n=256)

- cDNA SB-66 mapped to same region as Pun1

Identification of SB2-66 as candidate gene for Pun1

ResultsResults

- Pungent (C. chinense, C.

frutescens & C. annuum) and

non-pungent (C. annuum)

genotypes were surveyed to

detect polymorphisms

- Gene family?

- Presence/absence band identified

SB2-66 as candidate for Pun1

Identification of SB2-66 as candidate gene for Pun1

ResultsResults

- Full cDNA and gDNA sequence from C. chinense ‘Habanero’

- C. annum ‘Thai Hot’ gene has 98% nt identity, and same structure

- Conservation of deletion is widespread

Characterization of cDNA and genomic sequence of Pun1

ResultsResults

- SMART predicted that AT3 has a acyltransferase domain (>40% similarity from

other genes in plants), belongs to BAHD superfamily

- AT1 & AT2 from Habanero fruit also showed high similarity with

acyltransferases, but not mapped to Pun1

Sequence analysis

ResultsResults

Regulation of AT3 expression

- Habanero Pun1 and Bell pun1 peppers

ResultsResults

Regulation of AT3 expression- Expression during fruit development in C. annuum Pun1 and pun1 genotypes

- Thai Hot is amenable to VIGS

- Correspondence between transcript and protein accumulation

- AT3 no detectable in Bell pepper

- AT3 is tissue specific (only in placenta but pericarp & seeds)

ResultsResults

Function of AT3 in vivo by VIGS- Construct consisted of 400bp spanning active site

- Agrobacterium-mediated transformation with tobacco rattle virus

- Environmental stress may result with an increase in pungency?

- Detection limits issues in inmmunoblot and HPLC

- All the above provide evidence that Pun1 encodes an acyltransferase involved in capsaicinoid biosynthesis (is it the capsaicin synthase?)

?

?

ResultsResults

- 35 AT3 plant homologs are

functionally characterized, and

members of BAHD

- AT3 falls in O-acetyltransferases

(ester-forming enzymes)

- AT2 likely fruit ripening/wounding-

induced, closely related to N-acyl-

transferases

Phylogenetic analysis

ConclusionsConclusions

• Pun1 is an acyltransferase (AT3) involved in capsaicinoid

biosynthesis– Capsaicin synthase?

– CS is detected in non-pungent peppers

– May be coA-dependent acyltransferase

• pun1 originated trough a deletion and has been used for

more than 300 years– Arose early in domestication?

– Its use has narrowed genetic diversity?

• AT3 activity remains to be elucidated– Mutants may identify accumulation of intermediates

– Biochem assays remain challenging

Characterization of capsaicin synthase and identification of its gene (csy1) for pungency factor capsaicin in pepper

(Capsicum sp.)

Prassad, B.C.N., et al. 2006. PNAS 103:1335-20

BackgroundBackground

• Capsaicin is biosynthesized by CS– Condensation of vanillylamine and fatty acids moieties in placenta

• Role of intermediates (8-methyl nonenoic acid) in capsaicin

biosynthesis

• Biotransformation of phenyl propanoid intermediates to

capsaicin has been demonstrated

• No reports of purification and cloning of CS gene

• Objective: Identify the gene responsible for capsaicin

biosynthesis– Enzyme-to-gene approach

ResultsResults

Purification of CS

- Correlation between CS activity

and pungency

- CS from high pungency genotype

was purified and characterized

ResultsResults

Purification of CS

- Crude placental protein was extracted

- 110 fractions obtained, CS assayed and bulked

- Purification was enhanced by Sepharose column with bound vanillylamine

ResultsResults

Expression of CS during fruit development

- Polyclonal antibodies were highly specific to CS

- CS and capsaicin levels are correlated

- CS is localized in peripherial cells of placental tissues

7d 14 21 28 35 42 50 28 28

High pungent C. frutescens L/M C. annuum

15d 22 30 45

ResultsResults

Identification of CS gene

- N-terminal amino acid sequence was determined

- Primers were design to amplify a N-terminal motif –rev

primer from SB2-66

- Gene is 981 bp, has no introns, 308 aa and predicted

38 kDa molecular mass

- NO significant homology with any reported amino acid

sequences (including acyltransferases)

cDNA gDNA

ResultsResults

Expression of csy1

- csy1 expressed only in placenta

- Transcript level correlated with

pungency genotypes

- Sequences from C. frutescens & C.

annuum were similar

ResultsResults

Heterologous expression of csy1

- csy1 was expressed in E. coli DH5a using

pRESTA vector

- CS showed higher specific activity than native

CS

- CS highly specific to substrates of CS

- csy1 function is specific to capsaicin

biosynthesis

Control

CS fruits

CS E. c

oli

35 kDa

ConclusionsConclusions

• High pungency level correlated with high levels of

capsaicin and CS activity

• CS confined to peripherial cells of placental tissue

• Levels of capsaicin and CS activity depends on genotype

• csy1 is unique to Capsicum?

QTL analysis for capsaicinoid content in Capsicum

Ben-Chaim, A., et al. 2006. TAG 113:1481-90

BackgroundBackground

• Presence/absence of capsaicinoid due to pun1

• Amount of capsaicinoid is a quantitative trait– Varieties with different levels of pungency

– Pungency level is also affected by environment

• Limited information on quantitative variation

– cap, major QTL on chromosome 7 (C. frutescens x C. annuum) F2

– No co-localization between predicted structural genes and variation

in capsaicinoid content

– cap is a regulator of the pathway or unknown structural gene?

• Objective: Identify genomic regions that may independently

control presence of single capsaincinoid analogs

– Using F2 & F3 pop’s C. frutescens BG2814-6 x C. annuum RNaky

ResultsResults

Linkage map construction

- 728 markers (SSR, AFLP, specific PCR-markers, RFLP), and candidate genes

involved in capsaicinoid biosynthesis (pAMT, COMT, Bcat)

- 12 major and 4 small linkage groups, total length 1358.7 cM

ResultsResults

Phenotypic variation and correlation among traits

- Capsaicinoids in BG2814-6 (small fruit) was 10-30X-fold than RNaky (large fruit)

- Content in F1 was higher than BG2814-6 parent (overdominance/heterosis)

- F3 families showed transgressive segregation (except fruit weight)

- Capsaicin was the most abundant (38-64%), nordihydro- the least

- Capsaicin highly correlated w/ dihydro-, and moderately w/ nordihydro-

- Capsaicin affected by environment, nordihydro- not

ResultsResults

QTL ID: capsaicin content

- Alleles from pungent parent contributed to increased capsaicin content

- cap7.2 has large QTL effect (>20%)

- Both additive (cap3.1, cap4.1) and dominant (cap7.1) gene action

- Phenotypic variation by all QTL was 24, 19, and 37% in 2001, 02 & 03

- Digenic interaction detected between cap7.1 and marker in chr 2 (NP0326)

ResultsResults

QTL ID: dihydrocapsaicin content

- Four of the 5 QTL for capsaicin, were also identified

- Alleles from pungent parent contributed to increased dihydrocapsaicin content

- Same digenic interaction as befor

ResultsResults

QTL ID: digenic interaction

- Presence of BG2814-6 alleles at both positions was correlated with the largest

increase of capsaicin (37-42%) and dihydrocapsaicin contents (24-28%)

ResultsResults

QTL ID: nordihydrocapsaicin/total content,

fruitweight- Ndhc7a.1 didn’t co-localize with QTL controlling other capsaicinoids and was

recessive

- 5 QTL for total capsaicinoid content, total7.2 has the largest effect

- 2 QTL for fruit weight, gene action dominant (fw2.1) and additive (fw3.1)

ResultsResults

Co-segregation of candidate genes w/QTL

- Genes 3A2 and BCAT (valine catabolism) co-localize with QTL

- 3A2 has motifs of hydroxyisobutyrate dehidrogenase

- BCAT involved in catabolism of branched-chain amino acids

ConclusionsConclusions

• Identified QTL may represent elements in pathway

• 2 independent QTL found– cap3.1 influenced capsaicin and total capsaicinoid content

– ndhc7a.1 affected only nordihydrocapsaicin

• Overlapping QTL suggests common genetic mechanisms

• Fruit weight QTL didn’t co-localize w/ capsaicinoid QTL

• cap7.2 likely orthologous to major QTL previously identified

• Digenic interaction may facilitate further genetic analysis