Genome-wide analysis of ZAT gene family revealed GhZAT6 regulates salt stress tolerance in G. hirsutum.

High salt environments can induce stress in different plants. The genes containing the ZAT domain constitute a family that belongs to a branch of the C2H2 family, which plays a vital role in responding to abiotic stresses. In this study, we identified 169 ZAT genes from seven plant species, including 44 ZAT genes from G. hirsutum. Phylogenetic tree analysis divided ZAT genes in six groups with conserved gene structure, protein motifs. Two C2H2 domains and an EAR domain and even chromosomal distribution on At and Dt sub-genome chromosomes of G. hirsutum was observed. GhZAT6 was primarily expressed in the root tissue and responded to NaCl and ABA treatments. Subcellular localization found that GhZAT6 was located in the nucleus and demonstrated transactivation activity during a transactivation activity assay. Arabidopsis transgenic lines overexpressing the GhZAT6 gene showed salt tolerance and grew more vigorously than WT on MS medium supplemented with 100 mmol NaCl. Additionally, the silencing of the GhZAT6 gene in cotton plants showed more obvious leaf wilting than the control plants, which were subjected to 400 mmol NaCl treatment. Next, the expressions of GhAPX1, GhFSD1, GhFSD2, and GhSOS3 were significantly lower in the GhZAT6-silenced plants treated with NaCl than the control. Based on these findings, GhZAT6 may be involved in the ABA pathway and mediate salt stress tolerance by regulating ROS-related gene expression.

Convergent Biochemical Pathways for Xanthine Alkaloid Production in Plants Evolved from Ancestral Enzymes with Different Catalytic Properties.

Convergent evolution is widespread but the extent to which common ancestral conditions are necessary to facilitate the independent acquisition of similar traits remains unclear. In order to better understand how ancestral biosynthetic catalytic capabilities might lead to convergent evolution of similar modern-day biochemical pathways, we resurrected ancient enzymes of the caffeine synthase (CS) methyltransferases that are responsible for theobromine and caffeine production in flowering plants. Ancestral CS enzymes of Theobroma, Paullinia, and Camellia exhibited similar substrate preferences but these resulted in the formation of different sets of products. From these ancestral enzymes, descendants with similar substrate preference and product formation independently evolved after gene duplication events in Theobroma and Paullinia. Thus, it appears that the convergent modern-day pathways likely originated from ancestral pathways with different inferred flux. Subsequently, the modern-day enzymes originated independently via gene duplication and their convergent catalytic characteristics evolved to partition the multiple ancestral activities by different mutations that occurred in homologous regions of the ancestral proteins. These results show that even when modern-day pathways and recruited genes are similar, the antecedent conditions may be distinctive such that different evolutionary steps are required to generate convergence.

Diversity in Composition of Bioactive Compounds Among 26 Cocoa Genotypes.

Composition of bioactive compounds in cocoa beans is critical to the sensory and nutritional quality of cocoa based products. Twenty-six cocoa bean genotypes were freshly collected from the same plantation location in Indonesia. The bioactive compounds in these raw cocoa genotypes were identified and quantified. The results showed a great diversity in the composition of bioactive compounds among the 26 cocoa samples. The concentrations of methylxanthines, epicatechin, proanthocyanidin (PA) B-type oligomers, clovamide, and anthocyanins were important variables that differentiated these genotypes. MCC 01, SUL 3, ICCRI 03, and ICS 60 genotypes had the highest contents of flavan-3-ols including PAs and have the potential to be developed for "healthy" product formulations. Some genotypes such as DR 1, DR 2, DR 38, ICS 13, KPC 1, KW 617, RCC 71, and TSH 858 could be favored by industries due to the potential to be made into end-products with brighter appearance.

Determination of optimum harvest maturity and non-destructive evaluation of pod development and maturity in cacao (Theobroma cacao L.) using a multiparametric fluorescence sensor.

BACKGROUND: A series of fluorescence indices (anthocyanin, flavonol, chlorophyll and nitrogen balance) were deployed to detect the pigments and colourless flavonoids in cacao pods of three commercial cacao (Theobroma cacao L.) genotypes (QH1003, KKM22 and MCBC1) using a fast and non-destructive multiparametric fluorescence sensor. The aim was to determine optimum harvest periods (either 4 or 5 months after pod emergence) of commercial cacao based on fluorescence indices of cacao development and bean quality. RESULTS: As pod developed, cacao exhibited a rise with the peak of flavonol occurring at months 4 and 5 after pod maturity was initiated while nitrogen balance showed a decreasing trend during maturity. Cacao pods contained high chlorophyll as they developed but chlorophyll content declined significantly on pods that ripened at month 5. CONCLUSION: Cacao pods harvested at months 4 and 5 can be considered as commercially-ready as the beans have developed good quality and comply with the Malaysian standard on cacao bean specification. Thus, cacao pods can be harvested earlier when they reach maturity at month 4 after pod emergence to avoid germinated beans and over fermentation in ripe pods harvested at month 5. © 2018 Society of Chemical Industry.

HPTLC fingerprint profile analysis of cocoa proanthocyanidins depending on origin and genotype.

Cocoa beans are rich in bioactive phytochemicals such as alkaloids, anthocyanins, as well as monomeric and oligomeric flavan-3-ols. A high performance thin layer chromatography (HPTLC) method was developed for a fast analysis of a fingerprint of bioactive compounds present in cocoa beans depending on genotype and origin. Evaluation was performed using HPTLC followed by densitometry. The best separation for a fingerprint consisting of eight phenolic compounds as markers was achieved on silica gel 60 F254 HPTLC plates with a solvent mixture of ethyl formate, formic acid, water, toluene 30/4/3/1.5 (v/v/v/v). Staining with Fast Blue Salt B enabled visualization and quantitative evaluation. Compounds of interest were confirmed by eluting zones using a TLC-MS interface. LODs were ≤ 100ng/zone for all compounds. The fingerprints obtained are useful for quality control of cocoa beans allowing their differentiation according to cocoa or chocolate sorts, varieties, and origins.

Tc-cAPX, a cytosolic ascorbate peroxidase of Theobroma cacao L. engaged in the interaction with Moniliophthora perniciosa, the causing agent of witches' broom disease.

The level of hydrogen peroxide (H2O2) in plants signalizes the induction of several genes, including that of ascorbate peroxidase (APX-EC 1.11.1.11). APX isoenzymes play a central role in the elimination of intracellular H2O2 and contribute to plant responses to diverse stresses. During the infection process in Theobroma cacao by Moniliophthora perniciosa oxidative stress is generated and the APX action recruited from the plant. The present work aimed to characterize the T. cacao APX involved in the molecular interaction of T. cacao-M. perniciosa. The peroxidase activity was analyzed in protein extracts from cocoa plants infected by M. perniciosa and showed the induction of peroxidases like APX in resistant cocoa plants. The cytosolic protein of T. cacao (GenBank: ABR68691.2) was phylogenetically analyzed in relation to other peroxidases from the cocoa genome and eight genes encoding APX proteins with conserved domains were also analyzed. The cDNA from cytosolic APX was cloned in pET28a and the recombinant protein expressed and purified (rTc-cAPX). The secondary structure of the protein was analyzed by Circular Dichroism (CD) displaying high proportion of α-helices when folded. The enzymatic assay shows stable activity using ascorbate and guaiacol as an electron donor for H2O2 reduction. The pH 7.5 is the optimum for enzyme activity. Chromatographic analysis suggests that rTc-cAPX is a homodimer in solution. Results indicate that the rTc-cAPX is correctly folded, stable and biochemically active. The purified rTc-cAPX presented biotechnological potential and is adequate for future structural and functional studies.

Understanding the genetic diversity, spatial genetic structure and mating system at the hierarchical levels of fruits and individuals of a continuous Theobroma cacao population from the Brazilian Amazon.

Understanding the mating patterns of populations of tree species is a key component of ex situ genetic conservation. In this study, we analysed the genetic diversity, spatial genetic structure (SGS) and mating system at the hierarchical levels of fruits and individuals as well as pollen dispersal patterns in a continuous population of Theobroma cacao in Pará State, Brazil. A total of 156 individuals in a 0.56 ha plot were mapped and genotyped for nine microsatellite loci. For the mating system analyses, 50 seeds were collected from nine seed trees by sampling five fruits per tree (10 seeds per fruit). Among the 156 individuals, 127 had unique multilocus genotypes, and the remaining were clones. The population was spatially aggregated; it demonstrated a significant SGS up to 15 m that could be attributed primarily to the presence of clones. However, the short seed dispersal distance also contributed to this pattern. Population matings occurred mainly via outcrossing, but selfing was observed in some seed trees, which indicated the presence of individual variation for self-incompatibility. The matings were also correlated, especially within (Ρ(p(m))=0.607) rather than among the fruits (Ρ(p(m))=0.099), which suggested that a small number of pollen donors fertilised each fruit. The paternity analysis suggested a high proportion of pollen migration (61.3%), although within the plot, most of the pollen dispersal encompassed short distances (28 m). The determination of these novel parameters provides the fundamental information required to establish long-term ex situ conservation strategies for this important tropical species.

Isolation and characterization of 2S cocoa seed albumin storage polypeptide and the corresponding cDNA.

The amine pool of cocoa is known to be an essential component for the development of the typical cocoa flavor. To better understand and to produce an intense in vitro cocoa flavor, identification of the polypeptides that are the source of the amine flavor precursor pool is essential. Chromatographic analysis of the polypeptide profile of unfermented cocoa resulted in identification of a novel storage polypeptide of M(r) 8515. The N-terminal sequence of the first 34 residues of the purified polypeptide shows similarity to 2S storage albumins of cotton and Brazil nut and sweet protein, Mabinlin. To identify the corresponding cDNA of the putative cocoa 2S albumin, 18 randomly chosen clones from the cDNA library of immature Theobroma cacao seed mRNA were sequenced, and a full-length cDNA clone encoding a protein harboring the N-terminal sequence of the novel polypeptide was selected. The open reading frame of the clone encodes a polypeptide of M(r) 17125. Comparison of the translated amino acid sequence of the precursor protein or the mature polypeptide against the Swiss-Prot and TrEMBL databases shows high sequence similarity (>52%) and identity (>38%) to many plant 2S albumins. Tryptic peptide mass fingerprinting of the purified polypeptide by high-performance liquid chromatography-electrospray ionization mass spectrometry shows 10 masses that match the expected tryptic peptides of the deduced sequence. Together with the published work on plant 2S albumin processing, the results presented here suggest that post-translational processing yields a 73-residue polypeptide (residue positions 78-150) corresponding to the 9 kDa subunit of the mature cocoa 2S albumin protein.