The genetics of heat –or pungency- in Capsicum

1. The genetics of heat –or pungency- in Capsicum Marco Hernandez-Bello PLS221

2. 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

4. C. annum C. chinense ‘Habanero’ C. frutescens ‘Tabasco’ www.thechileman.org/guide_species.php www.wikipedia.org

5. The Pun1 gene for pungency in pepper encodes a putative acyltransferase Stewart, C. et al. 2005. Plant J. 42:675-688

6. 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

7. 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

8. 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

9. 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

10. 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

11. Results Regulation of AT3 expression • Habanero Pun1 and Bell pun1 peppers

12. 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)

13. 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?) ? ?

14. 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/wounding- induced, closely related to N-acyl-transferases

15. 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

16. 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

17. 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

18. Results Purification of CS • Correlation between CS activity and pungency • CS from high pungency genotype was purified and characterized

19. 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

20. 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 L/M C. annuum 7d 14 21 28 35 42 50 28 28 15d 22 30 45

21. 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

22. Results Expression of csy1 • csy1 expressed only in placenta • Transcript level correlated with pungency genotypes • Sequences from C. frutescens & C. annuum were similar

23. Results Heterologous expression of csy1 • csy1 wasexpressed 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. coli 35 kDa

24. 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?

25. QTL analysis for capsaicinoid content in Capsicum Ben-Chaim, A., et al. 2006. TAG 113:1481-90

26. 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

27. 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

28. 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) • F3families 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

29. 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)

30. 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

31. 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%)

32. 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)

33. Results Co-segregation of candidate genes w/QTL • Genes 3A2 and BCAT (valine catabolism) co-localize with QTL • 3A2has motifs of hydroxyisobutyrate dehidrogenase • BCAT involved in catabolism of branched-chain amino acids

34. 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