An integrated analysis of mRNA and sRNA transcriptional profiles in Coffea arabica L. roots: insights on nitrogen starvation responses.

Coffea arabica L. is an important agricultural commodity, accounting for 60% of traded coffee worldwide. Nitrogen (N) is a macronutrient that is usually limiting to plant yield; however, molecular mechanisms of plant acclimation to N limitation remain largely unknown in tropical woody crops. In this study, we investigated the transcriptome of coffee roots under N starvation, analyzing poly-A+ libraries and small RNAs. We also evaluated the concentration of selected amino acids and N-source preferences in roots. Ammonium was preferentially taken up over nitrate, and asparagine and glutamate were the most abundant amino acids observed in coffee roots. We obtained 34,654 assembled contigs by mRNA sequencing, and validated the transcriptional profile of 12 genes by RT-qPCR. Illumina small RNA sequencing yielded 8,524,332 non-redundant reads, resulting in the identification of 86 microRNA families targeting 253 genes. The transcriptional pattern of eight miRNA families was also validated. To our knowledge, this is the first catalog of differentially regulated amino acids, N sources, mRNAs, and sRNAs in Arabica coffee roots.

Homeologous genes involved in mannitol synthesis reveal unequal contributions in response to abiotic stress in Coffea arabica.

Polyploid plants can exhibit transcriptional modulation in homeologous genes in response to abiotic stresses. Coffea arabica, an allotetraploid, accounts for 75% of the world's coffee production. Extreme temperatures, salinity and drought limit crop productivity, which includes coffee plants. Mannitol is known to be involved in abiotic stress tolerance in higher plants. This study aimed to investigate the transcriptional responses of genes involved in mannitol biosynthesis and catabolism in C. arabica leaves under water deficit, salt stress and high temperature. Mannitol concentration was significantly increased in leaves of plants under drought and salinity, but reduced by heat stress. Fructose content followed the level of mannitol only in heat-stressed plants, suggesting the partitioning of the former into other metabolites during drought and salt stress conditions. Transcripts of the key enzymes involved in mannitol biosynthesis, CaM6PR, CaPMI and CaMTD, were modulated in distinct ways depending on the abiotic stress. Our data suggest that changes in mannitol accumulation during drought and salt stress in leaves of C. arabica are due, at least in part, to the increased expression of the key genes involved in mannitol biosynthesis. In addition, the homeologs of the Coffea canephora subgenome did not present the same pattern of overall transcriptional response, indicating differential regulation of these genes by the same stimulus. In this way, this study adds new information on the differential expression of C. arabica homeologous genes under adverse environmental conditions showing that abiotic stresses can influence the homeologous gene regulation pattern, in this case, mainly on those involved in mannitol pathway.

Genome of Herbaspirillum seropedicae strain SmR1, a specialized diazotrophic endophyte of tropical grasses.

The molecular mechanisms of plant recognition, colonization, and nutrient exchange between diazotrophic endophytes and plants are scarcely known. Herbaspirillum seropedicae is an endophytic bacterium capable of colonizing intercellular spaces of grasses such as rice and sugar cane. The genome of H. seropedicae strain SmR1 was sequenced and annotated by The Paraná State Genome Programme--GENOPAR. The genome is composed of a circular chromosome of 5,513,887 bp and contains a total of 4,804 genes. The genome sequence revealed that H. seropedicae is a highly versatile microorganism with capacity to metabolize a wide range of carbon and nitrogen sources and with possession of four distinct terminal oxidases. The genome contains a multitude of protein secretion systems, including type I, type II, type III, type V, and type VI secretion systems, and type IV pili, suggesting a high potential to interact with host plants. H. seropedicae is able to synthesize indole acetic acid as reflected by the four IAA biosynthetic pathways present. A gene coding for ACC deaminase, which may be involved in modulating the associated plant ethylene-signaling pathway, is also present. Genes for hemagglutinins/hemolysins/adhesins were found and may play a role in plant cell surface adhesion. These features may endow H. seropedicae with the ability to establish an endophytic life-style in a large number of plant species.

A new set of differentially expressed signaling genes is early expressed in coffee leaf rust race II incompatible interaction.

New races of coffee rust are overcoming resistance genes available in germplasm and cultivated cultivars and bringing recently some coffee-producing countries in severe economic challenge. The objective of this study was to identify the genes that are linked to host resistance to the major coffee rust race II. In our study, we have identified and studied a segregating population that has a single monogenic resistant gene to coffee rust. Coffee leaves of parents, resistant, and susceptible genotypes of the F2 generation plants were inoculated with pathogen spores. A differential analysis was performed by combined cDNA-AFLP and bulk segregant analysis (BSA) in pooled samples collected 48 and 72 h postinoculation, increasing the selectiveness for differential gene expression. Of 108 differential expressed genes, between 33,000 gene fragments analyzed, 108 differential expressed genes were identified in resistant plants. About 20 and 22 % of these resistant-correlated genes are related to signaling and defense genes, respectively. Between signaling genes, the major subclass corresponds to receptor and resistant homolog genes, like nucleotide-binding site leucine-rich repeat (NBS-LRR), Pto-like, RLKs, Bger, and RGH1A, all not previously described in coffee rust responses. The second major subclass included kinases, where two mitogen-activated kinases (MAPK) are identified. Further gene expression analysis was performed for 21 selected genes by real-time PCR gene expression analysis at 0, 12, 24, 48, and 72 h postinoculation. The expression of genes involved in signaling and defense was higher at 24 and 72 h after inoculation, respectively. The NBS-LRR was the more differentially expressed gene between the signaling genes (four times more expressed in the resistant genotype), and thraumatin (PR5) was the more expressed between all genes (six times more expressed). Multivariate analysis reinforces the significance of the temporal separation of identified signaling and defense genes: early expression of signaling genes support the hypothesis that higher expression of the signaling components up regulates the defense genes identified. Additionally the increased gene expression of these two gene sets is associated with a single monogenic resistance trait to to leaf coffee rust in the interaction characterized here.

The type III secretion system is necessary for the development of a pathogenic and endophytic interaction between Herbaspirillum rubrisubalbicans and Poaceae.

BACKGROUND: Herbaspirillum rubrisubalbicans was first identified as a bacterial plant pathogen, causing the mottled stripe disease in sugarcane. H. rubrisubalbicans can also associate with various plants of economic interest in a non pathogenic manner. RESULTS: A 21 kb DNA region of the H. rubrisubalbicans genome contains a cluster of 26 hrp/hrc genes encoding for the type three secretion system (T3SS) proteins. To investigate the contribution of T3SS to the plant-bacterial interaction process we generated mutant strains of H. rubrisubalbicans M1 carrying a Tn5 insertion in both the hrcN and hrpE genes. H. rubrisulbalbicans hrpE and hrcN mutant strains of the T3SS system failed to cause the mottled stripe disease in the sugarcane susceptible variety B-4362. These mutant strains also did not produce lesions on Vigna unguiculata leaves. Oryza sativa and Zea mays colonization experiments showed that mutations in hrpE and hrcN genes reduced the capacity of H. rubrisulbalbicans to colonize these plants, suggesting that hrpE and hrcN genes are involved in the endophytic colonization. CONCLUSIONS: Our results indicate that the T3SS of H. rubrisubalbicans is necessary for the development of the mottled stripe disease and endophytic colonization of rice.

Capsidiol-related genes are highly expressed in response to Colletotrichum scovillei during Capsicum annuum fruit development stages.

Capsicum annuum is one of the most important horticultural crops worldwide. Anthracnose disease (Colletotrichum spp.) is a major constraint for chili production, causing substantial losses. Capsidiol is a sesquiterpene phytoalexin present in pepper fruits that can enhance plant resistance. The genetic mechanisms involved in capisidiol biosynthesis are still poorly understood. In this study, a 3' RNA sequencing approach was used to develop the transcriptional profile dataset of C. annuum genes in unripe (UF) and ripe fruits (RF) in response to C. scovillei infection. Results showed 4,845 upregulated and 4,720 downregulated genes in UF, and 2,560 upregulated and 1,762 downregulated genes in RF under fungus inoculation. Four capsidiol-related genes were selected for RT-qPCR analysis, two 5-epi-aristolochene synthase (CA12g05030, CA02g09520) and two 5-epi-aristolochene-1,3-dihydroxylase genes (CA12g05070, CA01g05990). CA12g05030 and CA01g05990 genes showed an early response to fungus infection in RF (24 h post-inoculation-HPI), being 68-fold and 53-fold more expressed at 96 HPI, respectively. In UF, all genes showed a late response, especially CA12g05030, which was 700-fold more expressed at 96 HPI compared to control plants. We are proving here the first high-throughput expression dataset of pepper fruits in response to anthracnose disease in order to contribute for future pepper breeding programs.

A simple and efficient agroinfiltration method in coffee leaves (Coffea arabica L.): assessment of factors affecting transgene expression.

The establishment of a simple, rapid and efficient transient expression system is a necessary tool for the functional validation of candidate genes in coffee biotechnology. The effects of Agrobacterium strain, age of the donor plant, infiltration method, and infiltration medium on transgene expression in detached coffee leaves were evaluated. Regarding the effect of Agrobacterium strain, the expression of uidA was higher in GV3101-treated coffee disks than in LBA4404 and ATHV-treated samples. On the other hand, transient expression of uidA was significantly higher in leaf disks from young plants (6-weeks-old) (13.1 ± 1.4%) than in mature tissue (12-weeks-old) (1.6 ± 1.2%). Transient uidA expression was higher in detached coffee leaf disks from young plants infiltrated with one injection of 15 µL of Agrobacterium strain GV3101::1303 suspended in MS salts supplemented with 30 g/L sucrose, 1.9 g/L MES and 200 uM AS with subsequent sanding of the abaxial epidermis. Using the optimized protocol, expression of the uidA gene was observed 6, 24 and 48 h and 5 weeks after bacterial injection. DNA was extracted from coffee disks with positive GUS expression and specific mgfp5 and uidA fragments were amplified 5 weeks post-agroinfiltration. On the other hand, using the optimized protocol, a specific cry10Aa (500 bp) fragment was amplified in the agro-infiltrated coffee leaf disks 5 weeks post-agroinfiltration with the plasmid pB427-35S-cry10Aa. Moreover, the expression of the gene cry10Aa in two infiltrated coffee leaf disks was verified by RT-PCR and an expected 500 bp fragment was amplified.

Genome-wide association study reveals candidate genes influencing lipids and diterpenes contents in Coffea arabica L.

Lipids, including the diterpenes cafestol and kahweol, are key compounds that contribute to the quality of coffee beverages. We determined total lipid content and cafestol and kahweol concentrations in green beans and genotyped 107 Coffea arabica accessions, including wild genotypes from the historical FAO collection from Ethiopia. A genome-wide association study was performed to identify genomic regions associated with lipid, cafestol and kahweol contents and cafestol/kahweol ratio. Using the diploid Coffea canephora genome as a reference, we identified 6,696 SNPs. Population structure analyses suggested the presence of two to three groups (K = 2 and K = 3) corresponding to the east and west sides of the Great Rift Valley and an additional group formed by wild accessions collected in western forests. We identified 5 SNPs associated with lipid content, 4 with cafestol, 3 with kahweol and 9 with cafestol/kahweol ratio. Most of these SNPs are located inside or near candidate genes related to metabolic pathways of these chemical compounds in coffee beans. In addition, three trait-associated SNPs showed evidence of directional selection among cultivated and wild coffee accessions. Our results also confirm a great allelic richness in wild accessions from Ethiopia, especially in accessions originating from forests in the west side of the Great Rift Valley.

Diterpenes biochemical profile and transcriptional analysis of cytochrome P450s genes in leaves, roots, flowers, and during Coffea arabica L. fruit development.

Lipids are among the major chemical compounds present in coffee beans, and they affect the flavor and aroma of the coffee beverage. Coffee oil is rich in kaurene diterpene compounds, mainly cafestol (CAF) and kahweol (KAH), which are related to plant defense mechanisms and to nutraceutical and sensorial beverage characteristics. Despite their importance, the final steps of coffee diterpenes biosynthesis remain unknown. To understand the molecular basis of coffee diterpenes biosynthesis, we report the content dynamics of CAF and KAH in several Coffea arabica tissues and the transcriptional analysis of cytochrome P450 genes (P450). We measured CAF and KAH concentrations in leaves, roots, flower buds, flowers and fruit tissues at seven developmental stages (30-240 days after flowering - DAF) using HPLC. Higher CAF levels were detected in flower buds and flowers when compared to fruits. In contrast, KAH concentration increased along fruit development, peaking at 120 DAF. We did not detect CAF or KAH in leaves, and higher amounts of KAH than CAF were detected in roots. Using P450 candidate genes from a coffee EST database, we performed RT-qPCR transcriptional analysis of leaves, flowers and fruits at three developmental stages (90, 120 and 150 DAF). Three P450 genes (CaCYP76C4, CaCYP82C2 and CaCYP74A1) had transcriptional patterns similar to CAF concentration and two P450 genes (CaCYP71A25 and CaCYP701A3) have transcript accumulation similar to KAH concentration. These data warrant further investigation of these P450s as potential candidate genes involved in the final stages of the CAF and KAH biosynthetic pathways.

Expression of three galactinol synthase isoforms in Coffea arabica L. and accumulation of raffinose and stachyose in response to abiotic stresses.

Galactinol synthase (EC 2.4.1.123; GolS) catalyzes the first step in the synthesis of raffinose family oligosaccharides (RFOs). Their accumulation in response to abiotic stresses implies a role for RFOs in stress adaptation. In this study, the expression patterns of three isoforms of galactinol synthase (CaGolS1-2-3) from Coffea arabica were evaluated in response to water deficit, salinity and heat stress. All CaGolS isoforms were highly expressed in leaves while little to no expression were detected in flower buds, flowers, plagiotropic shoots, roots, endosperm and pericarp of mature fruits. Transcriptional analysis indicated that the genes were differentially regulated under water deficit, high salt and heat stress. CaGolS1 isoform is constitutively expressed in plants under normal growth conditions and was the most responsive during all stress treatments. CaGolS2 is unique among the three isoforms in that it was detected only under severe water deficit and salt stresses. CaGolS3 was primarily expressed under moderate and severe drought. This isoform was induced only at the third day of heat and under high salt stress. The increase in GolS transcription was not reflected into the amount of galactinol in coffee leaves, as specific glycosyltransferases most likely used galactinol to transfer galactose units to higher homologous oligosaccharides, as suggested by the increase of raffinose and stachyose during the stresses.

Transient gene expression of beta-glucuronidase in citrus thin epicotyl transversal sections using particle bombardment

Studies were carried out to optimize the conditions for transient gene expression through particle bombardment on Carrizo citrange (Citrus sinensis x Poncirus trifoliata) thin epicotyl sections. The best conditions for transient GUS expression were: M-25 tungsten particles, 1550 psi helium pressure, 9 cm distance between specimen and DNA/particle holder and culture of explants in a high osmolarity medium (0.2 M mannitol + 0.2 M sorbitol) 4 h prior and 20 h after bombardment. Under these conditions, an average of 102 blue spots per bombardment (20 explants/plate) were achieved. This protocol is currently being used for transformation of Carrizo citrange and sweet orange (Citrus sinensis)