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. www.thechileman.org/guide_species.php C. annum C. chinense ‘Habanero’ C. frutescens ‘Tabasco’ 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 -C. frutescens BG2816 (Pun1/Pun1) X C. annuum cv. Maor (pun1/pun1) Bell pepper -F 2 mapping pop. (n=256) -cDNA SB-66 mapped to same region as Pun1 Identification of SB2-66 as candidate gene for Pun1

8. Results -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

9. Results -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

10. Results -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

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 -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

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 7d 14 21 28 35 42 50 28 28 High pungent C. frutescensL/M C. annuum 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) cDNAgDNA

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 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 ControlCS 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) F 2 –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 F 2 & F 3 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 F 1 was higher than BG2814-6 parent (overdominance/heterosis) -F 3 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

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 -3A2 has 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