Characterization of Specialized Metabolites Involved in Black Pod Rot Resistance in Theobroma Cacao
Open Access
- Author:
- Knollenberg, Benjamin
- Graduate Program:
- Plant Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 17, 2020
- Committee Members:
- Mark Guiltinan, Dissertation Advisor/Co-Advisor
Mark Guiltinan, Committee Chair/Co-Chair
Siela N. Maximova, Dissertation Advisor/Co-Advisor
Surinder Chopra, Committee Member
Andrew David Patterson, Outside Member
Teh-Hui Kao, Program Head/Chair - Keywords:
- cacao
phytophthora
clovamide
SCPL
black pod rot
hydroxycinnamic acid amide
HCAA
disease resistance
specialized metabolite
secondary metabolite
UGT - Abstract:
- Cacao (Theobroma cacao, chocolate tree) is an important tropical crop that provides the raw ingredients for chocolate production and provides an important source of income to over five million farmers worldwide. Production of cacao is continually limited by diseases, with global annual crop losses estimated between thirty and forty percent. Oomycete (water mold) pathogens in the genus Phytophthora are the most widespread and economically destructive of the cacao pathogens, causing a disease known as black pod rot (BPR). Increasing cacao resistance to BPR is the most promising strategy for reducing yield losses due to the disease but requires an understanding of cacao defense mechanisms of cacao against BPR. Plant specialized metabolites are well known to contribute to plant defense and have been underexploited as breeding targets in cacao. In this work, the metabolite composition of a BPR tolerant and a BPR susceptible genotype were compared by untargeted metabolomics using liquid chromatography – tandem mass spectrometry (LC-MS/MS). This analysis revealed that the tolerant genotype ‘Scavina 6’ (‘Sca6’) accumulates clovamide, a hydroxycinnamic acid amide (HCAA), and several related compounds in leaf tissue at much higher concentrations that the susceptible genotype ‘Imperial College Selection 1’ (‘ICS1’). Both genotypes tested produced equivalent amounts of clovamide in fruit peel, but sulfated HCAAs accumulated to high levels only in ‘Sca6’ in this tissue. These results implicated clovamide and HCAAs as important contributors to the difference in resistance between the two genotypes, so clovamide was synthesized and characterized for its role in defense. Clovamide was found to be a substrate for cacao polyphenol oxidase (PPO), which is involved in reactive quinone formation, protein cross linking, and oxidative browning. Furthermore, clovamide inhibited protease and pectinase in vitro, activities associated with pathogenicity of Phytophthora spp. Clovamide also inhibited growth of three Phytophthora species in vitro. Based on these activities of clovamide and the high relative accumulation of clovamide in ‘Sca6’ I determined that clovamide is an important resistance factor in cacao and a promising breeding target for enhancing resistance. Since clovamide appears important for BPR resistance in cacao, I sought to elucidate cacao genes involved in its biosynthesis by combining metabolomics (LC-MS/MS) and transcriptomics (RNA-Seq) analysis of ‘Sca6’ and ‘ICS1’ leaves of three developmental stages and functional characterization of candidate clovamide biosynthetic genes. By correlating clovamide abundance with that of over 1,000 metabolites identified in extracts by LC-MS/MS, I found a strong negative correlation between clovamide and several flavonoid glucosides. Based on this observation I hypothesized that clovamide biosynthesis directs flux of UDP-glucose away from flavonoid glucoside production and therefore proceeds through a pathway requiring UDP-glucose dependent glycosyltransferase (UGT) and serine carboxypeptidase-like acyltransferase (SCPL-AT) activities. Following this hypothesis, I identified three cacao UGT and three SCPL-AT candidate genes based on expression patterns consistent with HCAA accumulation. All possible UGT/SCPL-AT candidate combinations were co-expressed in N. benthamiana to identify only one combination that resulted in clovamide production: cacao gene IDs (Criollo v2 reference genome) Tc04v2_g000570 (UGT) and Tc02v2_g013480 (SCPL-AT). The UGT candidate, Tc04v2_g000570 was more highly expressed in ‘Sca6’ than ‘ICS1’ (~5.6-fold, p < 0.00001) and was also found to have a premature stop codon in the coding sequence of the ‘ICS1’ allele, resulting in a truncated predicted protein sequence lacking a conserved UGT domain identified by InterProScan. Taken together, these results demonstrate that differential expression and allelic variation in the UGT Tc04v2_g000570 are the likely cause of the difference in leaf clovamide content between ‘Sca6’ and ‘ICS1’. The proposed UGT/SCPLAT pathway for clovamide biosynthesis in cacao is in contrast to the 4-CL/BAHD-AT pathway reported in red clover. BAHD- and SCPL-acyltransferases are evolutionarily distinct protein families, meaning that the two species evolved clovamide biosynthesis completely independently. In order to identify other genes controlling leaf clovamide content that were not detected using the transcriptomics/metabolomics approach, a quantitative trait loci (QTL) analysis for clovamide content was performed in an F2 population derived from ‘Sca6’ and ‘ICS1’. Leaf clovamide content was quantified in 116 F2 trees by HPLC-DAD and clovamide was found to be segregating. Another hydroxycinnamic acid derivative, chlorogenic acid (CGA), was also segregating. Clovamide and CGA content had a slight but significant negative correlation in the F2 population, so CGA was included as a covariate in QTL analyses. Two QTL were identified for clovamide, including one on chromosome 1 (“QTL1”) and one on chromosome 4 (“QTL4”). A three-factor model including QTL1, QTL4, and CGA explained 20.91% of variation in leaf clovamide content (p = 8.34E-05), suggesting that environmental factors or other undetected loci are also contributing to the variation. RNA-Seq data from ‘Sca6’ and ‘ICS1’ leaves was analyzed to identify differentially expressed genes (DEGs) in the two QTL. QTL4 contained several DEGs including Tc04v2_g000570, the UDP-glucose dependent glycosyltransferase (UGT) previously identified as a clovamide biosynthetic gene using the metabolomics/transcriptomics approach described above. QTL4 also contained an uncharacterized chloroplastic ABC transporter with a suspected role in clovamide precursor transport that was more highly expressed in ‘Sca6’ than ‘ICS1’ (~4-fold higher, p < 0.05). QTL1 also contained several DEGs including one uncharacterized serine carboxypeptidase-like acyltransferase (SCPL-AT) that had slightly higher expression in ‘Sca6’ than ‘ICS1’ (~0.9-fold, p < 0.05). A different cacao SCPL-AT was previously identified as a clovamide biosynthetic gene, described above. Surprisingly, the two clovamide QTL overlapped perfectly with two previously identified self-incompatibility loci in cacao, suggesting a potential but currently tenuous connection between clovamide biosynthesis and self-incompatibility in cacao. Overall, this work identifies a new resistance factor in cacao, clovamide, that should be explored as a breeding target and metabolic selectable marker in cacao breeding programs. Furthermore, it contributes to our broader understanding of plant specialized metabolite biosynthesis by identifying an example of convergent evolution in clovamide biosynthesis, which proceeds by an SCPL-AT in cacao and a BAHD-AT in red clover. This work also generates new questions about the potential role of clovamide in self-incompatibility in cacao that warrant further exploration. The integration of metabolomics, transcriptomics, and QTL analysis presented in this work may provide a road map for future attempts to identify important defense metabolites and elucidate their biosynthetic pathways in other systems.