Identification and functional characterization of the sugarcane (Saccharum spp.) AMT2-type ammonium transporter ScAMT3;3 revealed a presumed role in shoot ammonium remobilization.

Sugarcane (Saccharum spp.) is an important crop for sugar and bioethanol production worldwide. To maintain and increase sugarcane yields in marginal areas, the use of nitrogen (N) fertilizers is essential, but N overuse may result in the leaching of reactive N to the natural environment. Despite the importance of N in sugarcane production, little is known about the molecular mechanisms involved in N homeostasis in this crop, particularly regarding ammonium (NH4 +), the sugarcane's preferred source of N. Here, using a sugarcane bacterial artificial chromosome (BAC) library and a series of in silico analyses, we identified an AMMONIUM TRANSPORTER (AMT) from the AMT2 subfamily, sugarcane AMMONIUM TRANSPORTER 3;3 (ScAMT3;3), which is constitutively and highly expressed in young and mature leaves. To characterize its biochemical function, we ectopically expressed ScAMT3;3 in heterologous systems (Saccharomyces cerevisiae and Arabidopsis thaliana). The complementation of triple mep mutant yeast demonstrated that ScAMT3;3 is functional for NH3/H+ cotransport at high availability of NH4 + and under physiological pH conditions. The ectopic expression of ScAMT3;3 in the Arabidopsis quadruple AMT knockout mutant restored the transport capacity of 15N-NH4 + in roots and plant growth under specific N availability conditions, confirming the role of ScAMT3;3 in NH4 + transport in planta. Our results indicate that ScAMT3;3 belongs to the low-affinity transport system (Km 270.9 µM; Vmax 209.3 µmol g-1 root DW h-1). We were able to infer that ScAMT3;3 plays a presumed role in NH4 + source-sink remobilization in the shoots via phloem loading. These findings help to shed light on the functionality of a novel AMT2-type protein and provide bases for future research focusing on the improvement of sugarcane yield and N use efficiency.

Functional characterization of the sugarcane (Saccharum spp.) ammonium transporter AMT2;1 suggests a role in ammonium root-to-shoot translocation.

AMMONIUM TRANSPORTER/METHYLAMMONIUM PERMEASE/RHESUS (AMT) family members transport ammonium across membranes in all life domains. Plant AMTs can be categorized into AMT1 and AMT2 subfamilies. Functional studies of AMTs, particularly AMT1-type, have been conducted using model plants but little is known about the function of AMTs from crops. Sugarcane (Saccharum spp.) is a major bioenergy crop that requires heavy nitrogen fertilization but depends on a low carbon-footprint for competitive sustainability. Here, we identified and functionally characterized sugarcane ScAMT2;1 by complementing ammonium uptake-defective mutants of Saccharomyces cerevisiae and Arabidopsis thaliana. Reporter gene driven by the ScAMT2;1 promoter in A. thaliana revealed preferential expression in the shoot vasculature and root endodermis/pericycle according to nitrogen availability and source. Arabidopsis quadruple mutant plants expressing ScAMT2;1 driven by the CaMV35S promoter or by a sugarcane endogenous promoter produced significantly more biomass than mutant plants when grown in NH4 + and showed more 15N-ammonium uptake by roots and nitrogen translocation to shoots. In A. thaliana, ScAMT2;1 displayed a Km of 90.17 µM and Vmax of 338.99 µmoles h-1 g-1 root DW. Altogether, our results suggest that ScAMT2;1 is a functional high-affinity ammonium transporter that might contribute to ammonium uptake and presumably to root-to-shoot translocation under high NH4 + conditions.

Root nitrate uptake in sugarcane (Saccharum spp.) is modulated by transcriptional and presumably posttranscriptional regulation of the NRT2.1/NRT3.1 transport system.

KEY MESSAGE: Nitrate uptake in sugarcane roots is regulated at the transcriptional and posttranscriptional levels based on the physiological status of the plant and is likely a determinant mechanism for discrimination against nitrate. Sugarcane (Saccharum spp.) is one of the most suitable energy crops for biofuel feedstock, but the reduced recovery of nitrogen (N) fertilizer by sugarcane roots increases the crop carbon footprint. The low nitrogen use efficiency (NUE) of sugarcane has been associated with the significantly low nitrate uptake, which limits the utilization of the large amount of nitrate available in agricultural soils. To understand the regulation of nitrate uptake in sugarcane roots, we identified the major canonical nitrate transporter genes (NRTs-NITRATE TRANSPORTERS) and then determined their expression profiles in roots under contrasting N conditions. Correlation of gene expression with 15N-nitrate uptake revealed that under N deprivation or inorganic N (ammonium or nitrate) supply in N-sufficient roots, the regulation of ScNRT2.1 and ScNRT3.1 expression is the predominant mechanism for the modulation of the activity of the nitrate high-affinity transport system. Conversely, in N-deficient roots, the induction of ScNRT2.1 and ScNRT3.1 transcription is not correlated with the marked repression of nitrate uptake in response to nitrate resupply or high N provision, which suggested the existence of a posttranscriptional regulatory mechanism. Our findings suggested that high-affinity nitrate uptake is regulated at the transcriptional and presumably at the posttranscriptional levels based on the physiological N status and that the regulation of NRT2.1 and NRT3.1 activity is likely a determinant mechanism for the discrimination against nitrate uptake observed in sugarcane roots, which contributes to the low NUE in this crop species.

Auxin-driven ecophysiological diversification of leaves in domesticated tomato.

Heterobaric leaves have bundle sheath extensions (BSEs) that compartmentalize the parenchyma, whereas homobaric leaves do not. The presence of BSEs affects leaf hydraulics and photosynthetic rate. The tomato (Solanum lycopersicum) obscuravenosa (obv) mutant lacks BSEs. Here, we identify the obv gene and the causative mutation, a nonsynonymous amino acid change that disrupts a C2H2 zinc finger motif in a putative transcription factor. This mutation exists as a polymorphism in the natural range of wild tomatoes but has increased in frequency in domesticated tomatoes, suggesting that the latter diversified into heterobaric and homobaric leaf types. The obv mutant displays reduced vein density, leaf hydraulic conductance and photosynthetic assimilation rate. We show that these and other pleiotropic effects on plant development, including changes in leaf insertion angle, leaf margin serration, minor vein density, and fruit shape, are controlled by OBV via changes in auxin signaling. Loss of function of the transcriptional regulator AUXIN RESPONSE FACTOR 4 (ARF4) also results in defective BSE development, revealing an additional component of a genetic module controlling aspects of leaf development important for ecological adaptation and subject to breeding selection.

Increased branching independent of strigolactone in cytokinin oxidase 2-overexpressing tomato is mediated by reduced auxin transport.

Tomato production is influenced by shoot branching, which is controlled by different hormones. Here we produced tomato plants overexpressing the cytokinin-deactivating gene CYTOKININ OXYDASE 2 (CKX2). CKX2-overexpressing (CKX2-OE) plants showed an excessive growth of axillary shoots, the opposite phenotype expected for plants with reduced cytokinin content, as evidenced by LC-MS analysis and ARR5-GUS staining. The TCP transcription factor SlBRC1b was downregulated in the axillary buds of CKX2-OE and its excessive branching was dependent on a functional version of the GRAS-family gene LATERAL SUPPRESSOR (LS). Grafting experiments indicated that increased branching in CKX2-OE plants is unlikely to be mediated by root-derived signals. Crossing CKX2-OE plants with transgenic antisense plants for the strigolactone biosynthesis gene CAROTENOID CLEAVAGE DIOXYGENASE (CCD7-AS) produced an additive phenotype, indicating independent effects of cytokinin and strigolactones on increased branching. On the other hand, CKX2-OE plants showed reduced polar auxin transport and their bud outgrowth was reduced when combined with auxin mutants. Accordingly, CKX2-OE basal buds did not respond to auxin applied in the decapitated apex. Our results suggest that tomato shoot branching depends on a fine-tuning of different hormonal balances and that perturbations in the auxin status could compensate for the reduced cytokinin levels in CKX2-OE plants.

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.

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.

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.

RNA interference as a gene silencing tool to control Tuta absoluta in tomato (Solanum lycopersicum).

RNA interference (RNAi), a gene-silencing mechanism that involves providing double-stranded RNA molecules that match a specific target gene sequence, is now widely used in functional genetic studies. The potential application of RNAi-mediated control of agricultural insect pests has rapidly become evident. The production of transgenic plants expressing dsRNA molecules that target essential insect genes could provide a means of specific gene silencing in larvae that feed on these plants, resulting in larval phenotypes that range from loss of appetite to death. In this report, we show that the tomato leafminer ( Tuta absoluta ), a major threat to commercial tomato production, can be targeted by RNAi. We selected two target genes (Vacuolar ATPase-A and Arginine kinase) based on the RNAi response reported for these genes in other pest species. In view of the lack of an artificial diet for T. absoluta, we used two approaches to deliver dsRNA into tomato leaflets. The first approach was based on the uptake of dsRNA by leaflets and the second was based on "in planta-induced transient gene silencing" (PITGS), a well-established method for silencing plant genes, used here for the first time to deliver in planta-transcribed dsRNA to target insect genes. Tuta absoluta larvae that fed on leaves containing dsRNA of the target genes showed an ∼60% reduction in target gene transcript accumulation, an increase in larval mortality and less leaf damage. We then generated transgenic 'Micro-Tom' tomato plants that expressed hairpin sequences for both genes and observed a reduction in foliar damage by T. absoluta in these plants. Our results demonstrate the feasibility of RNAi as an alternative method for controlling this critical tomato pest.

De novo transcriptome assembly and analysis to identify potential gene targets for RNAi-mediated control of the tomato leafminer (Tuta absoluta).

BACKGROUND: Providing double-stranded RNA (dsRNA) to insects has been proven to silence target genes, and this approach has emerged as a potential method to control agricultural pests by engineering plants to express insect dsRNAs. A critical step of this technology is the screening of effective target genes essential for insect development and/or survival. The tomato leafminer (Tuta absoluta Meyrick) is a major Solanum lycopersicum (tomato) pest that causes significant yield losses and has recently invaded Europe, from where it is spreading at an alarming rate. To explore RNA interference (RNAi) against T. absoluta, sequence information on potential target genes is necessary, but only a few sequences are available in public databases. RESULTS: We sequenced six libraries from RNA samples from eggs, adults, and larvae at four stages, obtaining an overall total of around 245 million reads. The assembled T. absoluta transcriptome contained 93,477 contigs with an average size of 1,574 bp, 59.8 % of which presented positive Blast hits, with 19,995 (21.4 %) annotated by gene ontology. From the transcriptome, most of the core genes of the RNAi mechanism of Lepidoptera were identified indicating the potential suitability of T. absoluta for gene silencing. No contigs displayed significant similarity with a RNA-dependent RNA Polymerase. Genes from the juvenile hormone and ecdysteroid biosynthetic pathways were identified, representing potential target genes for systemic silencing. Comparisons of transcript profiles among stages revealed 1,577 genes differentially expressed at earlier larval stages, from which potential gene targets were identified. Five of these genes were evaluated using in vitro transcribed dsRNA absorbed by tomato leaflets, which were fed to 1(st) instar T. absoluta larvae, resulting in significant reduction of larval body weight while exhibiting significant knockdown for three of the genes. CONCLUSIONS: The transcriptome we generated represents a valuable genomic resource for screening potential gene targets that affect the development or survival of T. absoluta larvae. Five novel genes that showed greater expression at the 1(st) larval stage were demonstrated to be effective potential RNAi targets by reducing larval weight and can be considered good candidates for use in RNAi-mediated crop protection.

Regulatory components involved in altering lateral root development in response to localized iron: evidence for natural genetic variation.

On the search for sparingly available nutrients, plants may alter their root architecture to improve soil exploration. So far, the examples for root system modifications induced by a heterogeneous availability of nutrients have been reported for the macronutrients nitrogen (N) and phosphorous (P). In an attempt to extend this type of knowledge to other nutrients, we recently provided evidence that Arabidopsis roots are able to sense a local availability of the micronutrient iron (Fe) and to respond with lateral root elongation into the Fe-containing patch. This specific root response was caused by enhanced elongation of cells leaving the root meristem and was dependent on an AUX1-mediated auxin accumulation in the lateral root apices. In this report, we compare mechanisms underlying this response with those known for other nutrients and show that a substantial genotypic variation exists among accessions of A. thaliana in the responsiveness of lateral roots toward localized Fe supplies.

Localized iron supply triggers lateral root elongation in Arabidopsis by altering the AUX1-mediated auxin distribution.

Root system architecture depends on nutrient availability, which shapes primary and lateral root development in a nutrient-specific manner. To better understand how nutrient signals are integrated into root developmental programs, we investigated the morphological response of Arabidopsis thaliana roots to iron (Fe). Relative to a homogeneous supply, localized Fe supply in horizontally separated agar plates doubled lateral root length without having a differential effect on lateral root number. In the Fe uptake-defective mutant iron-regulated transporter1 (irt1), lateral root development was severely repressed, but a requirement for IRT1 could be circumvented by Fe application to shoots, indicating that symplastic Fe triggered the local elongation of lateral roots. The Fe-stimulated emergence of lateral root primordia and root cell elongation depended on the rootward auxin stream and was accompanied by a higher activity of the auxin reporter DR5-ß-glucuronidase in lateral root apices. A crucial role of the auxin transporter AUXIN RESISTANT1 (AUX1) in Fe-triggered lateral root elongation was indicated by Fe-responsive AUX1 promoter activities in lateral root apices and by the failure of the aux1-T mutant to elongate lateral roots into Fe-enriched agar patches. We conclude that a local symplastic Fe gradient in lateral roots upregulates AUX1 to accumulate auxin in lateral root apices as a prerequisite for lateral root elongation.

Convergence of developmental mutants into a single tomato model system: 'Micro-Tom' as an effective toolkit for plant development research.

BACKGROUND: The tomato (Solanum lycopersicum L.) plant is both an economically important food crop and an ideal dicot model to investigate various physiological phenomena not possible in Arabidopsis thaliana. Due to the great diversity of tomato cultivars used by the research community, it is often difficult to reliably compare phenotypes. The lack of tomato developmental mutants in a single genetic background prevents the stacking of mutations to facilitate analysis of double and multiple mutants, often required for elucidating developmental pathways. RESULTS: We took advantage of the small size and rapid life cycle of the tomato cultivar Micro-Tom (MT) to create near-isogenic lines (NILs) by introgressing a suite of hormonal and photomorphogenetic mutations (altered sensitivity or endogenous levels of auxin, ethylene, abscisic acid, gibberellin, brassinosteroid, and light response) into this genetic background. To demonstrate the usefulness of this collection, we compared developmental traits between the produced NILs. All expected mutant phenotypes were expressed in the NILs. We also created NILs harboring the wild type alleles for dwarf, self-pruning and uniform fruit, which are mutations characteristic of MT. This amplified both the applications of the mutant collection presented here and of MT as a genetic model system. CONCLUSIONS: The community resource presented here is a useful toolkit for plant research, particularly for future studies in plant development, which will require the simultaneous observation of the effect of various hormones, signaling pathways and crosstalk.

Ammonium triggers lateral root branching in Arabidopsis in an AMMONIUM TRANSPORTER1;3-dependent manner.

Root development is strongly affected by the plant's nutritional status and the external availability of nutrients. Employing split-root systems, we show here that local ammonium supply to Arabidopsis thaliana plants increases lateral root initiation and higher-order lateral root branching, whereas the elongation of lateral roots is stimulated mainly by nitrate. Ammonium-stimulated lateral root number or density decreased after ammonium or Gln supply to a separate root fraction and did not correlate with cumulative uptake of (15)N-labeled ammonium, suggesting that lateral root branching was not purely due to a nutritional effect but most likely is a response to a sensing event. Ammonium-induced lateral root branching was almost absent in a quadruple AMMONIUM TRANSPORTER (qko, the amt1;1 amt1;2 amt1;3 amt2;1 mutant) insertion line and significantly lower in the amt1;3-1 mutant than in the wild type. Reconstitution of AMT1;3 expression in the amt1;3-1 or in the qko background restored higher-order lateral root development. By contrast, AMT1;1, which shares similar transport properties with AMT1;3, did not confer significant higher-order lateral root proliferation. These results show that ammonium is complementary to nitrate in shaping lateral root development and that stimulation of lateral root branching by ammonium occurs in an AMT1;3-dependent manner.