Transcript gene (Pun1? - Plant Sciences

The genetics of heat –or pungencyin Capsicum
Marco Hernandez-Bello
PLS221
Introduction
• 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
C. annum
C. chinense ‘Habanero’
C. frutescens ‘Tabasco’
www.thechileman.org/guide_species.php
www.wikipedia.org
The Pun1 gene for pungency in pepper
encodes a putative acyltransferase
Stewart, C. et al. 2005. Plant J. 42:675-688
Background
• 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
Results
Identification of SB2-66 as candidate gene for Pun1
-
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
Results
Identification of SB2-66 as candidate gene for Pun1
-
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
Results
Characterization of cDNA and genomic sequence of Pun1
-
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
Results
Sequence analysis
-
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
Results
Regulation of AT3 expression
-
Habanero Pun1 and Bell pun1 peppers
Results
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)
Results
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?)
?
?
Results
Phylogenetic analysis
-
35 AT3 plant homologs are
functionally characterized, and
members of BAHD
-
AT3 falls in O-acetyltransferases
(ester-forming enzymes)
-
AT2 likely fruit ripening/woundinginduced, closely related to N-acyltransferases
Conclusions
• 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
Background
• 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
Results
Purification of CS
-
Correlation between CS activity
and pungency
-
CS from high pungency genotype
was purified and characterized
Results
Purification of CS
-
Crude placental protein was extracted
-
110 fractions obtained, CS assayed and bulked
-
Purification was enhanced by Sepharose column with bound vanillylamine
Results
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
High pungent C. frutescens
7d
15d
14
22
21
28
35
30
42
L/M C. annuum
50
28
45
28
Results
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
Results
Expression of csy1
-
csy1 expressed only in placenta
-
Transcript level correlated with
pungency genotypes
-
Sequences from C. frutescens &
C. annuum were similar
Results
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
35 kDa
Conclusions
• 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
Background
• 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
Results
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
Results
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
Results
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)
Results
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
Results
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%)
Results
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)
Results
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
Conclusions
• 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