The urea transporter DUR3 is differentially regulated by abiotic and biotic stresses in coffee plants.

The high costs of N fertilizers in the coffee production emphasizes the need to optimize fertilization practices and improve nitrogen use efficiency. Urea is widespread in nature, characterizing itself as a significant source of nitrogen for the growth and development of several organisms. Thus, the characterization of genes involved in urea transport in coffee plants is an important research topic for the sustainable production of this valuable cash crop. In the current study, we evaluated the expression of the DUR3 gene under abiotic and biotic stresses in coffee plants. Here, we show that the expression of a high-affinity urea transporter gene (CaDUR3) was up-regulated by N starvation in leaves and roots of two out of three C. arabica cultivars examined. Moreover, the CaDUR3 gene was differentially expressed in coffee plants under different abiotic and biotic stresses. In plants of cv. IAPAR59, CaDUR3 showed an increased expression in leaves after exposure to water deficit and heat stress, while it was downregulated in plants under salinity. Upon infection with H. vastatrix (coffee rust), the CaDUR3 was markedly up-regulated at the beginning of the infection process in the disease susceptible Catuaí Vermelho 99 in comparison with the resistant cultivar. These results indicate that besides urea acquisition and N-remobilization, CaDUR3 gene may be closely involved in the response to various stresses.

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.

Regulation of α-expansins genes in Arabidopsis thaliana seeds during post-osmopriming germination.

Seed osmopriming is a pre-sowing treatment that involves limitation of the seed water imbibition, so that pre-germinative metabolic activities proceed without radicular protrusion. This technique is used for improving germination rate, uniformity of seedling growth and hastening the time to start germination. In Arabidopsis thaliana, seed germination has been associated with the induction of enzymes involved in cell wall modifications, such as expansins. The α-expansins (EXPAs) are involved in cell wall relaxation and extension during seed germination. We used online tools to identify AtEXPA genes with preferential expression during seed germination and RT-qPCR to study the expression of five EXPA genes at different germination stages of non-primed and osmoprimed seeds. In silico promoter analysis of these genes showed that motifs similar to cis-acting elements related to abiotic stress, light and phytohormone responses are the most overrepresented in promoters of these AtEXPA genes, showing that their expression is likely be regulated by intrinsic developmental and environmental signals during Arabidopsis seed germination. The osmopriming conditioning had a decreased time and mean to 50% germination without affecting the percentage of final seed germination. The dried PEG-treated seeds showed noticeable high mRNA levels earlier at the beginning of water imbibition (18 h), showing that transcripts of all five EXPA isoforms were significantly produced during the osmopriming process. The strong up-regulation of these AtEXPA genes, mainly AtEXPA2, were associated with the earlier germination of the osmoprimed seeds, which qualifies them to monitor osmopriming procedures and the advancement of germination.

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.

Transcriptome Analysis of Leaves, Flowers and Fruits Perisperm of Coffea arabica L. Reveals the Differential Expression of Genes Involved in Raffinose Biosynthesis.

Coffea arabica L. is an important crop in several developing countries. Despite its economic importance, minimal transcriptome data are available for fruit tissues, especially during fruit development where several compounds related to coffee quality are produced. To understand the molecular aspects related to coffee fruit and grain development, we report a large-scale transcriptome analysis of leaf, flower and perisperm fruit tissue development. Illumina sequencing yielded 41,881,572 high-quality filtered reads. De novo assembly generated 65,364 unigenes with an average length of 1,264 bp. A total of 24,548 unigenes were annotated as protein coding genes, including 12,560 full-length sequences. In the annotation process, we identified nine candidate genes related to the biosynthesis of raffinose family oligossacarides (RFOs). These sugars confer osmoprotection and are accumulated during initial fruit development. Four genes from this pathway had their transcriptional pattern validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, we identified ~24,000 putative target sites for microRNAs (miRNAs) and 134 putative transcriptionally active transposable elements (TE) sequences in our dataset. This C. arabica transcriptomic atlas provides an important step for identifying candidate genes related to several coffee metabolic pathways, especially those related to fruit chemical composition and therefore beverage quality. Our results are the starting point for enhancing our knowledge about the coffee genes that are transcribed during the flowering and initial fruit development stages.

Salt stress alters the cell wall polysaccharides and anatomy of coffee (Coffea arabica L.) leaf cells.

Coffea arabica is the most important agricultural commodity in the world, and salinity is a major threat to its sustainable irrigation. Coffee leaf polysaccharides from plants subjected to salt stress were extracted and the leaves visualized through optical and electron microscopy. Alterations were detected in the monosaccharide composition of the pectin and hemicelluloses, with increases in uronic acid in all fractions. Changes in the polysaccharides were confirmed by HPSEC and FTIR. Moreover, the monolignol content was increased in the final residue, which suggests increased lignin content. The cytoplasm was altered, and the chloroplasts appeared irregular in shape. The arrangement of the stroma lamellae was disordered, and no starch granules were present. It was concluded that leaves of C. arabica under salt stress showed alterations in cell wall polysaccharides, increased monolignol content and structural damage to the cells of the mesophyll.

Heat stress causes alterations in the cell-wall polymers and anatomy of coffee leaves (Coffea arabica L.).

Coffee plants were subjected to heat stress (37 °C) and compared with control plants (24 °C). Cell wall polysaccharides were extracted using water (W), EDTA (E) and 4M NaOH (H30 and H70). In addition, monolignols were analyzed, and the leaves were observed by microscopy. Plants under heat stress accumulated higher contents of arabinose and galactose in fraction W. Xylose contents were observed to decrease in H30 fractions after the heat stress, whereas galactose and uronic acid increased. H70 fractions from plants exposed to heat stress showed increased xylose contents, whereas the contents of arabinose and glucose decreased. Differences in the molar-mass profiles of polysaccharides were also observed. The primary monolignol contents increased after the heat stress. Structural alterations in palisade cells and ultrastructural damage in chloroplasts were also observed. Our results demonstrate that the chemical profile of coffee cell-wall polymers and structural cell anatomy change under heat stress.

Nitrogen starvation, salt and heat stress in coffee (Coffea arabica L.): identification and validation of new genes for qPCR normalization.

Abiotic stresses are among the most important factors that affect food production. One important step to face these environmental challenges is the transcriptional modulation. Quantitative real-time PCR is a rapid, sensitive, and reliable method for the detection of mRNAs and it has become a powerful tool to mitigate plant stress tolerance; however, suitable reference genes are required for data normalization. Reference genes for coffee plants during nitrogen starvation, salinity and heat stress have not yet been reported. We evaluated the expression stability of ten candidate reference genes using geNorm PLUS, NormFinder, and BestKeeper softwares, in plants submitted to nitrogen starvation, salt and heat stress. EF1, EF1α, GAPDH, MDH, and UBQ10 were ranked as the most stable genes in all stresses and software analyses, while RPL39 and RPII were classified as the less reliable references. For reference gene validation, the transcriptional pattern of a Coffea non-symbiotic hemoglobin (CaHb1) was analyzed using the two new recommended and the most unstable gene references for normalization. The most unstable gene may lead to incorrect interpretation of CaHb1 transcriptional analysis. Here, we recommend two new reference genes in Coffea for use in data normalization in abiotic stresses: MDH and EF1.

FISH using a gag-like fragment probe reveals a common Ty3-gypsy-like retrotransposon in genome of Coffea species.

The genus Coffea possesses about 100 species, and the most economically important are Coffea canephora and Coffea arabica. The latter is predominantly self-compatible with 2n = 4x = 44, while the others of the genus are diploid with 2n = 2x = 22 and mostly self-incompatible. Studies using molecular markers have been useful to detect differences between genomes in Coffea; however, molecular and cytogenetic studies have produced only limited information on the karyotypes organization. We used DOP-PCR to isolate repetitive elements from genome of Coffea arabica var. typica. The pCa06 clone, containing a fragment of 775 bp length, was characterized by sequencing and used as a probe in chromosomes of C. arabica and six other species: C. canephora, Coffea eugenioides, Coffea kapakata, Coffea liberica var. dewevrei, Coffea racemosa, and Coffea stenophylla. This insert shows similarities with a gag protein of the Ty3-gypsy-like super-family. Dot blot and FISH analyses demonstrated that pCa06 is differentially accumulated between species and chromosomes. Signals appeared scattered and clustered on the chromosomes and were also associated with heterochromatic regions. While the literature shows that there is a high karyotype similarity between Coffea species, our results point out differences in the accumulation and dispersion of this Ty3-gypsy-like retrotransposon during karyotype differentiation of Coffea.