MADS-box transcription factors (TFs) are involved in multiple plant development processes and are most known during the reproductive transition and floral organ development. Very few genes have been characterized in the genome of Humulus lupulus L. (Cannabaceae), an important crop for the pharmaceutical and beverage industries. The MADS-box family has not been studied in this species yet. We identified 65 MADS-box genes in the hop genome, of which 29 encode type-II TFs (27 of subgroup MIKCC and 2 MIKC*) and 36 type-I proteins (26 α, 9 β, and 1 γ). Type-II MADS-box genes evolved more complex architectures than type-I genes. Interestingly, we did not find FLOWERING LOCUS C (FLC) homologs, a transcription factor that acts as a floral repressor and is negatively regulated by cold. This result provides a molecular explanation for a previous work showing that vernalization is not a requirement for hop flowering, which has implications for its cultivation in the tropics. Analysis of gene ontology and expression profiling revealed genes potentially involved in the development of male and female floral structures based on the differential expression of ABC homeotic genes in each whorl of the flower. We identified a gene exclusively expressed in lupulin glands, suggesting a role in specialized metabolism in these structures. In toto, this work contributes to understanding the evolutionary history of MADS-box genes in hop, and provides perspectives on functional genetic studies, biotechnology, and crop breeding.

UNLABELLED: Ethylene regulates different aspects of the plants life cycle, such as flowering, and acts as a defense signal in response to environmental stresses. Changes induced by water deficit (WD) in gene expression of the main enzymes involved in ethylene biosynthesis, 1-aminocyclopropane-1-carboxylic acid synthase (ACS) and oxidase (ACO), are frequently reported in plants. In this study, coffee (Coffea arabica) ACS and ACO family genes were characterized and their expression profiles were analyzed in leaves, roots, flower buds, and open flowers from plants under well-watered (WW) and water deficit (WD) conditions. Three new ACS genes were identified. Water deficit did not affect ACS expression in roots, however soil drying strongly downregulated ACO expression, indicating a transcriptional constraint in the biosynthesis pathway during the drought that can suppress ethylene production in roots. In floral buds, ACO expression is water-independent, suggesting a higher mechanism of control in reproductive organs during the final flowering stages. Leaves may be the main sites for ethylene precursor (1-aminocyclopropane-1-carboxylic acid, ACC) production in the shoot under well-watered conditions, contributing to an increase in the ethylene levels required for anthesis. Given these results, we suggest a possible regulatory mechanism for the ethylene biosynthesis pathway associated with coffee flowering with gene regulation in leaves being a key point in ethylene production and ACO genes play a major regulatory role in roots and the shoots. This mechanism may constitute a regulatory model for flowering in other woody species. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-022-01235-y.

The projected impact of global warming on coffee production may require the heat-adapted genotypes in the next decades. To identify cellular strategies in response to warmer temperatures, we compared the effect of elevated temperature on two commercial Coffea arabica L. genotypes exploring leaf physiology, transcriptome, and carbohydrate/protein composition. Growth temperatures were 23/19°C (day/night), as optimal condition (OpT), and 30/26°C (day/night) as a possible warmer scenario (WaT). The cv. Acauã showed lower levels of leaf temperature (Tleaf) under both conditions compared to cv. Catuaí, whereas slightly or no differences for other leaf physiological parameters. Therefore, to explore temperature responsive pathways the leaf transcriptome was examined using RNAseq. Genotypes showed a marked number of differentially-expressed genes (DEGs) under OpT, however DEGs strongly decrease in both at WaT condition indicating a transcriptional constraint. DEGs responsive to WaT revealed shared and genotype-specific genes mostly related to carbohydrate metabolism. Under OpT, leaf starch content was greater in cv. Acauã and, as WaT temperature was imposed, the leaf soluble sugar did not change in contrast to cv. Catuaí, although the levels of leaf starch, sucrose, and leaf protein decreased in both genotypes. These findings revealed intraspecific differences in the underlying transcriptional and metabolic interconnected pathways responsive to warmer temperatures, which is potentially linked to thermotolerance, and thus may be useful as biomarkers in breeding for a changing climate.

The use of transcriptomic data to make inferences about plant metabolomes is a useful tool to help the discovery of important compounds in the available biodiversity. To unveil previously undiscovered metabolites of Coffea, of phytotherapeutic and economic value, we employed 24 RNAseq libraries. These libraries were sequenced from leaves exposed to a diverse range of environmental conditions. Subsequently, the data were meticulously processed to create models of putative metabolic networks, which shed light on the production of potential natural compounds of significant interest. Then, we selected one of the predicted compounds, the L-3,4-dihydroxyphenylalanine (L-DOPA), to be analyzed by LC-MS/MS using three biological replicates of flowers, leaves, and fruits from Coffea arabica and Coffea canephora. We were able to identify metabolic pathways responsible for producing several compounds of economic importance. One of the identified pathways involved in isoquinoline alkaloid biosynthesis was found to be active and producing L-DOPA, which is a common product of POLYPHENOL OXIDASES (PPOs, EC 1.14.18.1 and EC 1.10.3.1). We show that coffee plants are a natural source of L-DOPA, a widely used medicine for treatment of the human neurodegenerative condition called Parkinsons disease. In addition, dozens of other compounds with medicinal significance were predicted as potential natural coffee products. By further refining analytical chemistry techniques, it will be possible to enhance the characterization of coffee metabolites, enabling a deeper understanding of their properties and potential applications in medicine.

microRNAs (miRNAs) are derived from self-complementary hairpin structures, while small-interfering RNAs (siRNAs) are derived from double-stranded RNA (dsRNA) or hairpin precursors. The core mechanism of sRNA production involves DICER-like (DCL) in processing the smallRNAs (sRNAs) and ARGONAUTE (AGO) as effectors of silencing, and siRNA biogenesis also involves action of RNA-Dependent RNA Polymerase (RDR), Pol IV and Pol V in biogenesis. Several other proteins interact with the core proteins to guide sRNA biogenesis, action, and turnover. We aimed to unravel the components and functions of the RNA-guided silencing pathway in a non-model plant species of worldwide economic relevance. The sRNA-guided silencing complex members have been identified in the Coffea canephora genome, and they have been characterized at the structural, functional, and evolutionary levels by computational analyses. Eleven AGO proteins, nine DCL proteins (which include a DCL1-like protein that was not previously annotated), and eight RDR proteins were identified. Another 48 proteins implicated in smallRNA (sRNA) pathways were also identified. Furthermore, we identified 235 miRNA precursors and 317 mature miRNAs from 113 MIR families, and we characterized ccp-MIR156, ccp-MIR172, and ccp-MIR390. Target prediction and gene ontology analyses of 2239 putative targets showed that significant pathways in coffee are targeted by miRNAs. We provide evidence of the expansion of the loci related to sRNA pathways, insights into the activities of these proteins by domain and catalytic site analyses, and gene expression analysis. The number of MIR loci and their targeted pathways highlight the importance of miRNAs in coffee. We identified several roles of sRNAs in C. canephora, which offers substantial insight into better understanding the transcriptional and post-transcriptional regulation of this major crop.

The effects of selenium (Se) on plant metabolism have been reported in several studies triggering plant tolerance to abiotic stresses, yet, the effects of Se on coffee plants under chilling stress are unclear. This study aimed to evaluate the effects of foliar Se application on coffee seedlings submitted to chilling stress and subsequent plant recovery. Two Coffea species, Coffea arabica cv. Arara, and Coffea canephora clone 31, were submitted to foliar application of sodium selenate solution (0.4 mg plant^-1) or a control foliar solution, then on day 2 plants were submitted to low temperature (10°C day/4°C night) for 2 days. After that, the temperature was restored to optimal (25°C day/20°C night) for 2 days. Leaf samples were collected three times (before, during, and after the chilling stress) to perform analyses. After the chilling stress, visual leaf injury was observed in both species; however, the damage was twofold higher in C. canephora. The lower effect of cold on C. arabica was correlated to the increase in ascorbate peroxidase and higher content of starch, sucrose, and total soluble sugars compared with C. canephora, as well as a reduction in reducing sugars and proline content during the stress and rewarming. Se increased the nitrogen and sulfur content before stress but reduced their content during low temperature. The reduced content of nitrogen and sulfur during stress indicates that they were remobilized to stem and roots. Se supply reduced the damage in C. canephora leaves by 24% compared with the control. However, there was no evidence of the Se effects on antioxidant enzymatic pathways or ROS activity during stress as previously reported in the literature. Se increased the content of catalase during the rewarming. Se foliar supply also increased starch, amino acids, and proline, which may have reduced symptom expression in C. canephora in response to low temperature. In conclusion, Se foliar application can be used as a strategy to improve coffee tolerance under low-temperature changing nutrient remobilization, carbohydrate metabolism, and catalase activity in response to rewarming stress, but C. arabica and C. canephora respond differently to chilling stress and Se supply.

Cultivated tomato, Solanum lycopersicum, is one of the most common fruits in the global food industry. Together with the wild tomato Solanum pennellii, it is widely used for developing better cultivars. MicroRNAs affect mRNA regulation, inhibiting its translation and/or promoting its degradation. Important proteins involved in these processes are ARGONAUTE and DICER. This study aimed to identify and characterize the genes involved in the miRNA processing pathway, miRNA molecules and target genes in both species. We validated the presence of pathway genes and miRNA in different NGS libraries and 6 miRNA families using quantitative RT-PCR. We identified 71 putative proteins in S. lycopersicum and 108 in S. pennellii likely involved in small RNAs processing. Of these, 29 and 32 participate in miRNA processing pathways, respectively. We identified 343 mature miRNAs, 226 pre-miRNAs in 87 families, including 192 miRNAs, which were not previously identified, belonging to 38 new families in S. lycopersicum. In S. pennellii, we found 388 mature miRNAs and 234 pre-miRNAs contained in 85 families. All miRNAs found in S. pennellii were unpublished, being identified for the first time in our study. Furthermore, we identified 2471 and 3462 different miRNA target in S. lycopersicum and S. pennellii, respectively.

Bud necrosis (BN) is a common disorder that affects Vitis vinifera L. and reduces its potential yield. To minimize the losses caused by BN, the double pruning management was applied in Brazilian Southeast vineyards. In this management strategy plants are pruned at the winter to promote a vegetative cycle and then, at summer, to promote the reproductive cycle at optimal environmental conditions. To investigate the relationship of BN and the double pruning management RNA-seq libraries were sequenced from healthy and necrotic tissues at four different stages of the year. The comparison of differentially expressed genes in necrotic and non-necrotic tissues showed an enhanced expression of genes related to cell death possibly induced by endophytic microorganisms in the necrotic tissues. The de novo assembly, characterization and quantification of transcripts within the RNA-seq libraries showed that genes from the endophytic fungus Alternaria alternata, responsible for the production of toxic compounds were highly expressed under BN. Here we propose a model in which unfavorable conditions and reduced carbohydrate levels in buds can promote the switch from a biotrophic lifestyle to a necrotrophic lifestyle in the endophytic fungi, which seems to be involved in the development of BN.

Recently, the siRNAs pathways, and especially reproductive phasiRNAs, have attracted attention in eudicots since their biological roles are still unknown and their biogenesis took different evolutionary pathways compared to monocots. In this work, we used Coffea arabica L., a recent allotetraploid formed from the hybridization of Coffea canephora and C. eugenioides unreduced gametes, to explore microsporogenesis and small RNAs-related pathways in a eudicot crop. First, we identified the microsporogenesis stages during anther development revealing that pre-meiosis occurs in anthers of 1.5 mm inside floral buds (FBs), whereas meiosis between 1.5 and 4.2 mm FBs, and post-meiosis in FBs larger than 4.2 mm. These stages coincide with the Brazilian winter, a period of FBs reduced growth which suggests temperature sensitivity. Next, we identified and quantified the expression of reproductive 21- and 24-nt phasiRNAs during coffee anther development together with their canonical and novel miRNA triggers, and characterized the DCL and AGO families. Our results showed that the pattern of reproductive phasiRNA abundance in C. arabica is unique among described eudicots and the canonical trigger car-miR2275 is involved in the processing of both 21- and 24-nt phasiRNAs. Fourteen DCL genes were identified, but DCL5, related to phasiRNA biosynthesis in monocots, was not, according to its specificity for monocots. Thus, our work explored the knowledge gap about microsporogenesis and related siRNAs pathways in coffee, contributing to the control of reproductive development and the improvement of fertility in eudicots.