Functional Characterization of ent-Copalyl Diphosphate Synthase and Kaurene Synthase Genes from Coffea arabica L.

The biochemical profile of coffee beans translates directly into quality traits, nutraceutical and health promoting properties of the coffee beverage. Ent-kaurene is the ubiquitous precursor for gibberellin biosynthesis in plants, but it also serves as an intermediate in specialized (i.e., secondary) diterpenoid metabolism that leads to a diversity of more than 1,000 different metabolites. Nutraceutical effects on human health attributed to diterpenes include antioxidant, anticarcinogenic, and anti-inflammatory properties. Cafestol (CAF) and kahweol (KAH) are two diterpenes found exclusively in the Coffea genus. Our objective was to identify and functionally characterize genes involved in the central step of ent-kaurene production. We identified 17 putative terpene synthase genes in the transcriptome of Coffea arabica. Two ent-copalyl diphosphate synthase (CaCPS) and three kaurene synthase (CaKS) were selected and manually annotated. Transcript expression profiles of CaCPS1 and CaKS3 best matched the CAF and KAH metabolite profiles in different tissues. CaCPS1 and CaKS3 proteins were heterologously expressed and functionally characterized. CaCPS1 catalyzes the cyclization of geranylgeranyl diphosphate (GGPP) to ent-copalyl diphosphate (ent-CPP), which is converted to ent-kaurene by CaKS3. Knowledge about the central steps of diterpene formation in coffee provides a foundation for future characterization of the subsequent enzymes involved in CAF and KAH biosynthesis.

Genetic Progress of Seed Yield and Nitrogen Use Efficiency of Brazilian carioca Common Bean Cultivars Using Bayesian Approaches.

Common bean (Phaseolus vulgaris L.) is one of the most important crops worldwide and is considered an essential source of proteins, fibers, and minerals in the daily diet of several countries. Nitrogen (N) is considered the most important nutrient for common bean crop. On the other hand, the reduction of chemical fertilizers is a global challenge, and the development of cultivars with more N use efficiency (NUsE) is considered one of the main strategies to reduce the amount of N fertilizers. Genetic progress of NUsE has been reported in several crops; however, there was still no quantity in common bean. In this study, our goal was to analyze the genetic progress of seed yield (SY) and NUsE-related traits of 40 carioca common bean cultivars release from 1970 to 2017 in eight environments under low (zero) or high N (40 kg ha-1) in top-dressing. Genetic progress, principal component analysis, correlations among traits, and cultivar stability were analyzed using Bayesian approaches. The lowest values of the deviance information criterion (DIC) for the full model tested indicated the presence of the genotype × N × environment interaction for all evaluated traits. Nitrogen utilization efficiency (NUtE) and nitrogen uptake efficiency (NUpE) were the traits that most contributed to discriminate cultivars. The genetic progress of SY under high N (0.53% year-1, 95% HPD = 0.39; 0.65% year-1) was similar to that obtained in low N conditions (0.48% year-1, 95% HPD = 0.31; 0.64% year-1). These results indicate that modern cultivars do not demand more N fertilizers to be more productive. In addition, we observed a high genetic variability for NUsE-related traits, but there was no genetic progress for these variables. SY showed negative correlation with seed protein content (Prot) in both N conditions, and there was no reduction in Prot in modern cultivars. Both modern and old cultivars showed adaptability and stability under contrasting N conditions. Our study contributed to improve our knowledge about the genetic progress of common bean breeding program in Brazil in the last 47 years, and our data will help researchers to face the challenge of increase NUsE and Prot in the next few years.

Low-Copy Genes in Terpenoid Metabolism: The Evolution and Expression of MVK and DXR Genes in Angiosperms.

Terpenoids are a diverse class of metabolites that impact plant metabolism in response to environmental cues. They are synthesized either via a predominantly cytosolic (MVA) pathway or a plastidic pathway (MEP). In Arabidopsis, several enzymes from the MVA and MEP pathways are encoded by gene families, excluding MVK and DXR, which are single-copy genes. In this study, we assess the diversity, evolution and expression of DXR and MVK genes in selected angiosperms and Coffea arabica in particular. Evolutionary analysis revealed that DXR and MVK underwent purifying selection, but the selection effect for DXR was stronger than it was for MVK. Digital gene expression (DGE) profile analysis of six species revealed that expression levels of MVK in flowers and roots were high, whereas for DXR peak values were observed in leaves. In C. arabica, both genes were highly expressed in flowers, and CaDXR was upregulated in response to methyl jasmonate. C. arabica DGE data were validated by assessing gene expression in selected organs, and by plants treated with hexanoic acid (Hx) using RT-qPCR. MVK expression was upregulated in roots treated with Hx. CaDXR was downregulated in leaves by Hx treatment in a genotype-specific manner, indicating a differential response to priming.

Transcriptional patterns of Coffea arabica L. nitrate reductase, glutamine and asparagine synthetase genes are modulated under nitrogen suppression and coffee leaf rust.

This study evaluated the transcriptional profile of genes related to nitrogen (N) assimilation in coffee plants susceptible and resistant to rust fungi under N sufficiency and N suppression. For this purpose, we inoculated young coffee leaves with Hemileia vastatrix uredospores and collected them at 0, 12, 24 and 48 hours post-inoculation (HPI) to evaluate the relative expressions of genes encoding cytosolic glutamine synthetase (CaGS1 ), plastid glutamine synthetase (CaGS2 ), nitrate reductase (CaNR), and asparagine synthetase (CaAS). The genes exhibited distinct patterns of transcriptional modulation for the different genotypes and N nutritional regimes. The resistant genotype (I59) presented high levels of transcription in response to pathogen inoculation for CaNR and CaGS1 genes, evaluated under N sufficiency in the initial moments of infection (12 HPI). The gene CaGS1 also showed a peak at 48 HPI. The susceptible genotype (CV99) showed increased transcript rates of CaNR at 12 and 24 HPI in response to rust inoculation. The transcriptional patterns observed for CV99, under N suppression, were high levels for CaAS and CaGS2 at all post-inoculation times in response to coffee leaf rust disease. In addition, CaGS1 was up-regulated at 48 HPI for CV99. Cultivar I59 showed high transcript levels at 12 HPI for CaAS and peaks at 24 and 48 HPI for CaGS2 in inoculated samples. Consequently, total chlorophyl concentration was influenced by N suppression and by rust infection. Regarding enzyme activities in vitro for glutamine synthetase and CaNR, there was an increase in infected coffee leaves (I59) and under N sufficiency. Moreover, CV99 was modulated in both N nutritional regimes for GS activity in response to rust. Our results indicate that N transport genes trigger a differential modulation between genotypes through the action of rust disease.