Combined sensory, volatilome and transcriptome analyses identify a limonene terpene synthase as a major contributor to the characteristic aroma of a Coffea arabica L. specialty coffee.

BACKGROUND: The fruity aromatic bouquet of coffee has attracted recent interest to differentiate high value market produce as specialty coffee. Although the volatile compounds present in green and roasted coffee beans have been extensively described, no study has yet linked varietal molecular differences to the greater abundance of specific substances and support the aroma specificity of specialty coffees. RESULTS: This study compared four Arabica genotypes including one, Geisha Especial, suggested to generate specialty coffee. Formal sensory evaluations of coffee beverages stressed the importance of coffee genotype in aroma perception and that Geisha Especial-made coffee stood out by having fine fruity, and floral, aromas and a more balanced acidity. Comparative SPME-GC-MS analyses of green and roasted bean volatile compounds indicated that those of Geisha Especial differed by having greater amounts of limonene and 3-methylbutanoic acid in agreement with the coffee cup aroma perception. A search for gene ontology differences of ripening beans transcriptomes of the four varieties revealed that they differed by metabolic processes linked to terpene biosynthesis due to the greater gene expression of prenyl-pyrophosphate biosynthetic genes and terpene synthases. Only one terpene synthase (CaTPS10-like) had an expression pattern that paralleled limonene loss during the final stage of berry ripening and limonene content in the studied four varieties beans. Its functional expression in tobacco leaves confirmed its functioning as a limonene synthase. CONCLUSIONS: Taken together, these data indicate that coffee variety genotypic specificities may influence ripe berry chemotype and final coffee aroma unicity. For the specialty coffee variety Geisha Especial, greater expression of terpene biosynthetic genes including CaTPS10-like, a limonene synthase, resulted in the greater abundance of limonene in green beans, roasted beans and a unique citrus note of the coffee drink.

Global transcriptome profiling reveals differential regulatory, metabolic and hormonal networks during somatic embryogenesis in Coffea arabica.

BACKGROUND: Somatic embryogenesis (SE) is one of the most promising processes for large-scale dissemination of elite varieties. However, for many plant species, optimizing SE protocols still relies on a trial and error approach. We report the first global scale transcriptome profiling performed at all developmental stages of SE in coffee to unravel the mechanisms that regulate cell fate and totipotency. RESULTS: RNA-seq of 48 samples (12 developmental stages × 4 biological replicates) generated 90 million high quality reads per sample, approximately 74% of which were uniquely mapped to the Arabica genome. First, the statistical analysis of transcript data clearly grouped SE developmental stages into seven important phases (Leaf, Dedifferentiation, Primary callus, Embryogenic callus, Embryogenic cell clusters, Redifferentiation and Embryo) enabling the identification of six key developmental phase switches, which are strategic for the overall biological efficiency of embryo regeneration. Differential gene expression and functional analysis showed that genes encoding transcription factors, stress-related genes, metabolism-related genes and hormone signaling-related genes were significantly enriched. Second, the standard environmental drivers used to control SE, i.e. light, growth regulators and cell density, were clearly perceived at the molecular level at different developmental stages. Third, expression profiles of auxin-related genes, transcription factor-related genes and secondary metabolism-related genes were analyzed during SE. Gene co-expression networks were also inferred. Auxin-related genes were upregulated during dedifferentiation and redifferentiation while transcription factor-related genes were switched on from the embryogenic callus and onward. Secondary metabolism-related genes were switched off during dedifferentiation and switched back on at the onset of redifferentiation. Secondary metabolites and endogenous IAA content were tightly linked with their respective gene expression. Lastly, comparing Arabica embryogenic and non-embryogenic cell transcriptomes enabled the identification of biological processes involved in the acquisition of embryogenic capacity. CONCLUSIONS: The present analysis showed that transcript fingerprints are discriminating signatures of cell fate and are under the direct influence of environmental drivers. A total of 23 molecular candidates were successfully identified overall the 12 developmental stages and can be tested in many plant species to optimize SE protocols in a rational way.

Multiplex CRISPR/Cas9-mediated knockout of the phytoene desaturase gene in Coffea canephora.

Coffea canephora (2n = 2x = 22 chromosomes) is a species with extensive genetic diversity and desirable agronomic traits for coffee breeding programs. However, obtaining a new coffee cultivar through conventional breeding techniques may require more than 30 years of crossing cycles and selection, which hampers the effort of keeping up with market demands and rapidly proposing more resilient to climate change varieties. Although, the application of modern biotechnology tools such as precision genetic engineering technologies may enable a faster cultivar development process. Therefore, we aimed to validate the CRISPR/Cas9 system to generate mutations on a selected genotype of C. canephora, the clone 14. Embryogenic calli and a multiplex binary vector containing two sgRNAs targeting different exons of the CcPDS gene were used. The sgRNAs were under the C. canephora U6 promoter regulation. The target gene encodes phytoene desaturase, an enzyme essential for photosynthesis involved in ß-carotene biosynthesis. Somatic seedlings and embryos with albino, variegated and green phenotypes regenerated after Agrobacterium tumefaciens-mediated genetic transformation were analyzed by verifying the insertion of the Cas9 gene and later by sequencing the sgRNAs target regions in the genome of Robusta modified seedlings. Among them, 77% had the expected mutations, and of which, 50% of them had at least one target with a homozygous mutation. The genotype, temperature of co-cultivation with the bacteria, and light intensity used for subsequent embryo regeneration appeared to strongly influence the successful regeneration of plants with a mutated CcPDS gene in the Coffea genus.

Description of an Arabica Coffee Ideotype for Agroforestry Cropping Systems: A Guideline for Breeding More Resilient New Varieties.

Climate change (CC) is already impacting Arabica coffee cultivation in the intertropical zone. To deal with this situation, it is no longer possible to manage this crop using industrial agriculture techniques, which has been the main strategy implemented since the Green Revolution. Developing a more sustainable agriculture system that respects people and the environment is essential to guarantee future generations' access to natural resources. In the case of Arabica coffee, the solution has been found. Agroforestry is proposed as an ecosystem-based strategy to mitigate and adapt to CC. At least 60% of Arabica coffee is produced in agroforestry systems (AFSs), which are the most sustainable way to produce coffee. Nevertheless, AFS coffee cultivation is currently uncompetitive partly because all modern varieties, selected for full-sun intensive cropping systems, have low yields in shaded environments. Here we review the reasons why agroforestry is part of the solution to CC, and why no breeding work has been undertaken for this cropping system. Based on the literature data, for breeding purposes we also define for the first time one possible coffee ideotype required for AFS coffee cultivation. The four main traits are: (1) productivity based on F1 hybrid vigor, tree volume and flowering intensity under shade; (2) beverage quality by using wild Ethiopian accessions as female progenitors and selecting for this criterion using specific biochemical and molecular predictors; (3) plant health to ensure good tolerance to stress, especially biotic; and (4) low fertilization to promote sustainable production. For each of these traits, numerous criteria with threshold values to be achieved per trait were identified. Through this research, an ecosystem-based breeding strategy was defined to help create new F1 hybrid varieties within the next 10 years.

A comparative analysis of genomic and phenomic predictions of growth-related traits in 3-way coffee hybrids.

Genomic prediction has revolutionized crop breeding despite remaining issues of transferability of models to unseen environmental conditions and environments. Usage of endophenotypes rather than genomic markers leads to the possibility of building phenomic prediction models that can account, in part, for this challenge. Here, we compare and contrast genomic prediction and phenomic prediction models for 3 growth-related traits, namely, leaf count, tree height, and trunk diameter, from 2 coffee 3-way hybrid populations exposed to a series of treatment-inducing environmental conditions. The models are based on 7 different statistical methods built with genomic markers and ChlF data used as predictors. This comparative analysis demonstrates that the best-performing phenomic prediction models show higher predictability than the best genomic prediction models for the considered traits and environments in the vast majority of comparisons within 3-way hybrid populations. In addition, we show that phenomic prediction models are transferrable between conditions but to a lower extent between populations and we conclude that chlorophyll a fluorescence data can serve as alternative predictors in statistical models of coffee hybrid performance. Future directions will explore their combination with other endophenotypes to further improve the prediction of growth-related traits for crops.

Molecular and Environmental Triggering Factors of Pathogenicity of Fusarium oxysporum and F. solani Isolates Involved in the Coffee Corky-Root Disease.

Coffee corky-root disease causes serious damages to coffee crop and is linked to combined infection of Fusarium spp. and root-knot nematodes Meloidogyne spp. In this study, 70 Fusarium isolates were collected from both roots of healthy coffee plants and with corky-root disease symptoms. A phylogenetic analysis, and the detection of pathogenicity SIX genes and toxigenicity Fum genes was performed for 59 F. oxysporum and 11 F. solani isolates. Based on the molecular characterization, seven F. oxysporum and three F. solani isolates were assessed for their pathogenicity on coffee seedlings under optimal watering and water stress miming root-knot nematode effect on plants. Our results revealed that a drastic increment of plant colonization capacity and pathogenicity on coffee plants of some Fusarium isolates was caused by water stress. The pathogenicity on coffee of F. solani linked to coffee corky-root disease and the presence of SIX genes in this species were demonstrated for the first time. Our study provides evidence for understanding the pathogenic basis of F. oxysporum and F. solani isolates on coffee and revealed the presence of SIX and Fum genes as one of their pathogenicity-related mechanisms. We also highlight the relevance of chlorophyll, a fluorescence as an early and high-throughput phenotyping tool in Fusarium pathogenicity studies on coffee.

Photoperiod-dependent transcriptional modifications in key metabolic pathways in Coffea arabica.

Photoperiod length induces in temperate plants major changes in growth rates, morphology and metabolism with, for example, modifications in the partitioning of photosynthates to avoid starvation at the end of long nights. However, this has never been studied for a tropical perennial species adapted to grow in a natural photoperiod close to 12 h/12 h all year long. We grew Coffea arabica L., an understorey perennial evergreen tropical species in its natural 12 h/12 h and in a short 8 h/16 h photoperiod, and we investigated its responses at the physiological, metabolic and transcriptomic levels. The expression pattern of rhythmic genes, including core clock genes, was affected by changes in photoperiod. Overall, we identified 2859 rhythmic genes, of which 89% were also rhythmic in Arabidopsis thaliana L. Under short-days, plant growth was reduced, and leaves were thinner with lower chlorophyll content. In addition, secondary metabolism was also affected with chlorogenic acid and epicatechin levels decreasing, and in agreement, the genes involved in lignin synthesis were overexpressed and those involved in the flavanol pathway were underexpressed. Our results show that the 8 h/16 h photoperiod induces drastic changes in morphology, metabolites and gene expression, and the responses for gene expression are similar to those observed in the temperate annual A. thaliana species. Short photoperiod induces drastic changes in gene expression, metabolites and leaf structure, some of these responses being similar to those observed in A. thaliana.

5-CQA and Mangiferin, Two Leaf Biomarkers of Adaptation to Full Sun or Shade Conditions in Coffea arabica L.

Phenolic compounds are involved in plant response to environmental conditions and are highly present in leaves of Coffea arabica L., originally an understory shrub. To increase knowledge of C. arabica leaf phenolic compounds and their patterns in adaptation to light intensity, mature leaves of Ethiopian wild accessions, American pure lines and their relative F1 hybrids were sampled in full sun or under 50% shade field plots in Mexico and at two contrasting elevations in Nicaragua and Colombia. Twenty-one phenolic compounds were identified by LC-DAD-MS2 and sixteen were quantified by HPLC-DAD. Four of them appeared to be involved in C. arabica response to light intensity. They were consistently more accumulated in full sun, presenting a stable ratio of leaf content in the sun vs. shade for all the studied genotypes: 1.6 for 5-CQA, F-dihex and mangiferin and 2.8 for rutin. Moreover, 5-CQA and mangiferin contents, in full sun and shade, allowed for differentiating the two genetic groups of Ethiopian wild accessions (higher contents) vs. cultivated American pure lines. They appear, therefore, to be potential biomarkers of adaptation of C. arabica to light intensity for breeding programs. We hypothesize that low 5-CQA and mangiferin leaf contents should be searched for adaptation to full-sun cropping systems and high contents used for agroforestry systems.

Starmaya: The First Arabica F1 Coffee Hybrid Produced Using Genetic Male Sterility.

In the present paper, we evaluated the implementation of a seed production system based on the exploitation of male sterility on coffee. We studied specifically the combination between CIR-SM01 and Marsellesa® (a Sarchimor line), which provides a hybrid population called Starmaya. We demonstrated that the establishment of seed garden under natural pollination is possible and produces a sufficient amount of hybrid seeds to be multiplied efficiently and economically. As expected for F1 hybrid, the performances of Starmaya are highly superior to conventional cultivars. However, we observed some heterogeneity on Starmaya cultivar in the field. We confirmed by genetic marker analysis that the off-types were partly related to the heterozygosity of the CIR-SM01 clone and could not be modified. Regarding the level of rust resistance of Starmaya cv., we saw that it could be improved if Marsellesa was more fully fixed genetically. If so, we should be able to decrease significantly the percentage of rust incidence of Starmaya from 15 to 5%, which would be quite acceptable at a commercial level. Starmaya represents the proof of concept for the mass propagation of Arabica F1 hybrid seeds using male sterility. Finally, we discuss the possibility to increase the number of hybrid varieties produced by seed, exploring some initiatives to identify male sterility markers to induce male sterility on any conventional cultivar. This would definitively open up the universe of known Arabica cultivars to be used in breeding new F1 hybrids.

An Altered Circadian Clock Coupled with a Higher Photosynthesis Efficiency Could Explain the Better Agronomic Performance of a New Coffee Clone When Compared with a Standard Variety.

In a context where climate change is threatening coffee productivity, the management of coffee leaf rust is a challenging issue. Major resistant genes, which have been used for many years, are systematically being overcome by pathogens. Developing healthy plants, able to defend themselves and be productive even when attacked by the pathogen, should be part of a more sustainable alternative approach. We compared one hybrid (GPFA124), selected for its good health in various environments including a reduced rust incidence, and the cv. 'Caturra', considered as a standard in terms of productivity and quality but highly susceptible to rust, for phenotypic variables and for the expression of genes involved in the circadian clock and in primary photosynthetic metabolism. The GPFA124 hybrid showed increased photosynthetic electron transport efficiency, better carbon partitioning, and higher chlorophyll content. A strong relationship exists between chlorophyll a fluorescence and the expression of genes related to the photosynthetic electron transport chain. We also showed an alteration of the amplitude of circadian clock genes in the clone. Our work also indicated that increased photosynthetic electron transport efficiency is related to the clone's better performance. Chlorophyll a fluorescence measurement is a good indicator of the coffee tree's physiological status for the breeder. We suggest a connection between the circadian clock and carbon metabolism in coffee tree.

Coffee Somatic Embryogenesis: How Did Research, Experience Gained and Innovations Promote the Commercial Propagation of Elite Clones From the Two Cultivated Species?

Since the 1990s, somatic embryogenesis (SE) has enabled the propagation of selected varieties, Arabica F1 hybrid and Robusta clones, originating from the two cultivated coffee species, Coffea arabica and Coffea canephora, respectively. This paper shows how mostly empirical research has led to successful industrial transfers launched in the 2000s in Latin America, Africa, and Asia. Coffee SE can be considered as a model for other woody perennial crops for the following reasons: (i) a high biological efficiency has been demonstrated for propagated varieties at all developmental stages, and (ii) somaclonal variation is understood and mastered thanks to intensive research combining molecular markers and field observations. Coffee SE is also a useful model given the strong economic constraints that are specific to this species. In brief, SE faced four difficulties: (i) the high cost of SE derived plants compared to the cost of seedlings of conventional varieties, (ii) the logistic problems involved in reaching small-scale coffee growers, (iii) the need for certification, and (iv) the lack of solvency among small-scale producers. Nursery activities were professionalized by introducing varietal certification, quality control with regard to horticultural problems and somaclonal variation, and sanitary control for Xylella fastidiosa. In addition, different technology transfers were made to ensure worldwide dissemination of improved F1 Arabica hybrids and Robusta clones. Innovations have been decisive for successful scaling-up and reduction of production costs, such as the development of temporary immersion bioreactors for the mass production of pre-germinated embryos, their direct sowing on horticultural soil, and the propagation of rejuvenated SE plants by rooted mini-cuttings. Today, SE is a powerful tool that is widely used in coffee for biotechnological applications including propagation and genetic transformation. Basic research has recently started taking advantage of optimized SE protocols. Based on -omics methodologies, research aims to decipher the molecular events involved in the key developmental switches of coffee SE. In parallel, a high-throughput screening of active molecules on SE appears to be a promising tool to speed-up the optimization of SE protocols.

Influence of Environmental Conditions and Genetic Background of Arabica Coffee (C. arabica L) on Leaf Rust (Hemileia vastatrix) Pathogenesis.

Global warming is a major threat to agriculture worldwide. Between 2008 and 2013, some coffee producing countries in South and Central America suffered from severe epidemics of coffee leaf rust (CLR), resulting in high economic losses with social implications for coffee growers. The climatic events not only favored the development of the pathogen but also affected the physiological status of the coffee plant. The main objectives of the study were to evaluate how the physiological status of the coffee plant modified by different environmental conditions impact on the pathogenesis of CLR and to identify indicators of the physiological status able to predict rust incidence. Three rust susceptible genotypes (one inbred line and two hybrids) were grown in controlled conditions with a combination of thermal regime (TR), nitrogen and light intensity close to the field situation before being inoculated with the rust fungus Hemileia vastatrix. It has been demonstrated that a TR of 27-22°C resulted in 2000 times higher sporulation than with a TR of 23-18°C. It has been also shown that high light intensity combined with low nitrogen fertilization modified the CLR pathogenesis resulting in huge sporulation. CLR sporulation was significantly lower in the F1 hybrids than in the inbred line. The hybrid vigor may have reduced disease incidence. Among the many parameters studied, parameters related to photosystem II and photosynthetic electron transport chain components appeared as indicators of the physiological status of the coffee plant able to predict rust sporulation intensity. Taken together, these results show that CLR sporulation not only depends on the TR but also on the physiological status of the coffee plant, which itself depends on agronomic conditions. Our work suggests that vigorous varieties combined with a shaded system and appropriate nitrogen fertilization should be part of an agro-ecological approach to disease control.

Juvenile Coffee Leaves Acclimated to Low Light Are Unable to Cope with a Moderate Light Increase.

The understorey origin of coffee trees and the strong plasticity of Coffea arabica leaves in relation to contrasting light environments have been largely shown. The adaptability of coffee leaves to changes in light was tested under controlled conditions by increasing the illumination rate on C. arabica var. Naryelis seedlings acclimated to low light conditions and observing leaf responses at three different developmental stages (juvenile, growing and mature). Only mature leaves proved capable of adapting to new light conditions. In these leaves, different major mechanisms were found to contribute to maintaining a good photosynthetic level. With increased illumination, a high photosynthetic response was conserved thanks to fast nitrogen remobilization, as indicated by SPAD values and the photorespiration rate. Efficient photoprotection was accompanied by a great ability to export sucrose, which prevented excessive inhibition of the Calvin cycle by hexose accumulation. In contrast, in younger leaves, increased illumination caused photodamage, observable even after 9 days of treatment. One major finding was that young coffee leaves rely on the accumulation of chlorogenic acids, powerful antioxidant phenolic compounds, to deal with the accumulation of reactive oxygen species rather than on antioxidant enzymes. Due to a lack of efficient photoprotection, a poor ability to export sucrose and inadequate antioxidant protection, younger leaves seemed to be unable to cope with increased illumination. In these leaves, an absence of induced antioxidant enzyme activity was accompanied, in growing leaves, by an absence of antioxidant synthesis or, in juvenile leaves, inefficient synthesis of flavonoids because located in some epidermis cells. These observations showed that coffee leaves, at the beginning of their development, are not equipped to withstand quick switches to higher light levels. Our results confirm that coffee trees, even selected for full sunlight conditions, remain shade plants possessing leaves able to adapt to higher light levels only when mature.

Differential fine-tuning of gene expression regulation in coffee leaves by CcDREB1D promoter haplotypes under water deficit.

Despite the importance of the DREB1D gene (also known as CBF4) in plant responses to water deficit and cold stress, studies analysing its regulation by transgenic approaches are lacking. In the current work, a functional study of three CcDREB1D promoter haplotypes (named HP15, HP16 and HP17) isolated from drought-tolerant and drought-sensitive clones of Coffea canephora was carried out in plants of C. arabica stably transformed by Agrobacterium tumefaciens by analysing their ability to regulate the expression of the uidA reporter gene in response to water deficit mimicked by polyethylene glycol (-2.0 MPa) and low relative humidity treatments. A deletion analysis of their corresponding 5'-upstream regions revealed increased specificity of ß-glucuronidase activity in the polyethylene glycol and low relative humidity treatments, with high expression in leaf mesophyll and guard cells in full-length constructs. RT-qPCR assays also revealed that the HP16 haplotype (specific to clone tolerant to water deficit) had stronger and earlier activity compared with the HP15 and HP17 haplotypes. As most of the cis-regulatory elements involved in ABA-dependent and -independent networks, tissue specificity and light regulation are common to these haplotypes, we propose that their organization, as well as the nucleic acid polymorphisms present outside these boxes, may play a role in modulating activities of DREB1D promoters in guard cells.

Coffee (Coffea arabica L.).

Coffee (Coffea sp.) is a perennial plant widely cultivated in many tropical countries. It is a cash crop for millions of small farmers in these areas. As for other tree species, coffee has long breeding cycles, which makes conventional breeding programs time-consuming. For that matter, genetic transformation can be an effective way to introduce a desired trait in elite varieties or for functional genomics. In this chapter, we describe two highly efficient and reliable Agrobacterium-mediated transformation techniques developed for the C. arabica cultivated species: (1) A. tumefaciens to study and introduce genes conferring resistance/tolerance to biotic (coffee leaf rust, insects) and abiotic stress (drought, heat, seed desiccation) in fully transformed plants and (2) A. rhizogenes to study candidate gene expression for nematode resistance in transformed roots.

Indica rice cultivar IRGA 424, transformed with cry genes of B. thuringiensis, provided high resistance against Spodoptera frugiperda (Lepidoptera: Noctuidae).

Plant expression of the entomopathogenic bacteria Bacillus thuringiensis cry gene has reduced the damage created by insect pests in several economically important cultures. For this study, we have conducted genetic transformation of the indica rice "IRGA 424", via Agrobacterium tumefaciens, using the B. thuringiensis cry1Aa and cry1B genes, with the objective of obtaining rice plants resistant to the insect pests from this culture. The gene constructions harbor the promoters maize proteinase inhibitor and ubiquitin. The results showed that high concentration of the hormone 2,4-dichlorophenoxyacetic acid and agarose as the gelling agent helped the production of embryogenic calli for the analyzed cultivar. More than 80% of the obtained transformed plants revealed the integration, using polymerase chain reaction, of the cry1Aa and cry1B genes. Analysis of the expression of the heterologous protein by Western blotting revealed the expression of the Cry1B delta-endotoxin in IRGA 424 plants transformed with the ubiquitin promoter. Data showed the production and dissemination of a high number of embryogenic calli in addition to obtaining plants transformed with the cry1Aa and cry1B genes until the reproductive phase. The feed bioassays with the transformed plants and Spodoptera frugiperda (JE Smith) larvae indicated high rates of mortality to the insect target. The highest corrected mortality rate achieved under laboratory conditions with Bt-rice plants transformed with the cry1B and cry1Aa genes was 94 and 84%, respectively. Thus, our results demonstrated the great potential of transformed Bt-rice plants in controlling the damage caused by these insect pests in rice paddy fields.

An in planta, Agrobacterium-mediated transient gene expression method for inducing gene silencing in rice (Oryza sativa L.) leaves.

BACKGROUND: Localized introduction and transient expression of T-DNA constructs mediated by agro-infiltration of leaf tissues has been largely used in dicot plants for analyzing the transitivity and the cell-to cell movement of the RNAi signal. In cereals, however, the morphology of the leaf and particularly the structure of the leaf epidermis, prevent infiltration of a bacterial suspension in cells by simple pressure, a method otherwise successful in dicots leaves. This study aimed at establishing a rapid method for the functional analysis of rice genes based on the triggering of RNA interference (RNAi) following Agrobacterium-mediated transient transformation of leaves. RESULTS: Using an agro-infection protocol combining a wound treatment and a surfactant, we were able to obtain in a reliable manner transient expression of a T-DNA-borne uidA gene in leaf cells of japonica and indica rice cultivars. Using this protocol to transiently inhibit gene expression in leaf cells, we introduced hairpin RNA (hpRNA) T-DNA constructs containing gene specific tags of the phytoene desaturase (OsPDS) and of the SLENDER 1 (OsSLR1) genes previously proven to trigger RNAi of target genes in stable transformants. SiRNA accumulation was observed in the agro-infected leaf area for both constructs indicating successful triggering of the silencing signal. Accumulation of secondary siRNA was observed in both stably and transiently transformed leaf tissues expressing the HpRNA OsSLR1 construct. Gene silencing signalling was investigated in monitoring the parallel time course of OsPDS-derived mRNA and siRNA accumulation in the agro-infiltrated leaf area and adjacent systemic sectors. The sensitive RT-Q-PCR method evidenced a consistent, parallel decrease of OsPDS transcripts in both the agroinfiltred and adjacent tissues, with a time lag for the latter. CONCLUSIONS: These results indicate that the method is efficient at inducing gene silencing in the agro-infected leaf area. The transfer of low amounts of siRNA, probably occurring passively through the symplastic pathway from the agro-infected area, seemed sufficient to trigger degradation of target transcripts in the adjacent tissues. This method is therefore well suited to study the cell-to-cell movement of the silencing signal in a monocot plant and further test the functionality of natural and artificial miRNA expression constructs.

Complex regulation of two target genes encoding SPX-MFS proteins by rice miR827 in response to phosphate starvation.

Here we report on the characterization of rice osa-miR827 and its two target genes, OsSPX-MFS1 and OsSPX-MFS2, which encode SPX-MFS proteins predicted to be implicated in phosphate (Pi) sensing or transport. We first show by Northern blot analysis that osa-miR827 is strongly induced by Pi starvation in both shoots and roots. Hybridization of osa-miR827 in situ confirms its strong induction by Pi starvation, with signals concentrated in mesophyll, epidermis and ground tissues of roots. In parallel, we analyzed the responses of the two OsSPX-MFS1 and OsSPX-MFS2 gene targets to Pi starvation. OsSPX-MFS1 mRNA is mainly expressed in shoots under sufficient Pi supply while its expression is reduced on Pi starvation, revealing a direct relationship between induction of osa-miR827 and down-regulation of OsSPX-MFS1. In contrast, OsSPX-MFS2 responds in a diametrically opposed manner to Pi starvation. The accumulation of OsSPX-MFS2 mRNA is dramatically enhanced under Pi starvation, suggesting the involvement of complex regulation of osa-miR827 and its two target genes. We further produced transgenic rice lines overexpressing osa-miR827 and T-DNA knockout mutant lines in which the expression of osa-miR827 is abolished. Compared with wild-type controls, both target mRNAs exhibit similar changes, their expression being reduced and increased in overexpressing and knockout lines, respectively. This suggests that OsSPX-MFS1 and OsSPX-MFS2 are both negatively regulated by osa-miR827 abundance although they respond differently to external Pi conditions. We propose that this is a complex mechanism comprising fine tuning of spatial or temporal regulation of both targets by osa-miR827.

The mariner Mos1 transposase produced in tobacco is active in vitro.

The mariner-like transposon Mos1 is used for insertional mutagenesis and transgenesis in different animals (insects, nematodes), but has never been used in plants. In this paper, the transposition activity of Mos1 was tested in Nicotiana tabacum, but no transposition event was detected. In an attempt to understand the absence of in planta transposition, Mos1 transposase (MOS1) was produced and purified from transgenic tobacco (HMNtMOS1). HMNtMOS1 was able to perform all transposition reaction steps in vitro: binding to ITR, excision and integration of the same pseudo-transposon used in in planta transposition assays. The in vitro transposition reaction was not inhibited by tobacco nuclear proteins, and did not depend on the temperature used for plant growth. Several hypotheses are proposed that could explain the inhibition of HMNtMOS1 activity in planta.

Identification of precursor transcripts for 6 novel miRNAs expands the diversity on the genomic organisation and expression of miRNA genes in rice.

BACKGROUND: The plant miRNAs represent an important class of endogenous small RNAs that guide cleavage of an mRNA target or repress its translation to control development and adaptation to stresses. MiRNAs are nuclear-encoded genes transcribed by RNA polymerase II, producing a primary precursor that is subsequently processed by DCL1 an RNase III Dicer-like protein. In rice hundreds of miRNAs have been described or predicted, but little is known on their genes and precursors which are important criteria to distinguish them from siRNAs. Here we develop a combination of experimental approaches to detect novel miRNAs in rice, identify their precursor transcripts and genes and predict or validate their mRNA targets. RESULTS: We produced four cDNA libraries from small RNA fractions extracted from distinct rice tissues. By in silico analysis we selected 6 potential novel miRNAs, and confirmed that their expression requires OsDCL1. We predicted their targets and used 5'RACE to validate cleavage for three of them, targeting a PPR, an SPX domain protein and a GT-like transcription factor respectively. In addition, we identified precursor transcripts for the 6 miRNAs expressed in rice, showing that these precursors can be efficiently processed using a transient expression assay in transfected Nicotiana benthamiana leaves. Most interestingly, we describe two precursors producing tandem miRNAs, but in distinct arrays. We focus on one of them encoding osa-miR159a.2, a novel miRNA produced from the same stem-loop structure encoding the conserved osa-miR159a.1. We show that this dual osa-miR159a.2-osa-miR159a.1 structure is conserved in distant rice species and maize. Finally we show that the predicted mRNA target of osa-miR159a.2 encoding a GT-like transcription factor is cleaved in vivo at the expected site. CONCLUSION: The combination of approaches developed here identified six novel miRNAs expressed in rice which can be clearly distinguished from siRNAs. Importantly, we show that two miRNAs can be produced from a single precursor, either from tandem stem-loops or tandemly arrayed in a single stem-loop. This suggests that processing of these precursors could be an important regulatory step to produce one or more functional miRNAs in plants and perhaps coordinate cleavage of distinct targets in the same plant tissue.