Wild-type Arabidopsis Plants Research Articles

PIN2 is required for the adaptation of Arabidopsis roots to alkaline stress by modulating proton secretion

Soil alkalinity is a widespread environmental problem that limits agricultural productivity. The hypothesis that an auxin-regulated proton secretion by plasma membrane H+-ATPase plays an important role in root adaption to alkaline stress was studied. It was found that alkaline stress increased auxin transport and PIN2 (an auxin efflux transporter) abundance in the root tip of wild-type Arabidopsis plants (WT). Compared with WT roots, the pin2 mutant roots exhibited much reduced plasma membrane H+-ATPase activity, root elongation, auxin transport, and proton secretion under alkaline stress. More importantly, roots of the pks5 mutant (PKS5, a protein kinase) lacking PIN2 (a pks5/pin2 double mutant) lost the previous higher proton-secretion capacity and higher elongation rate of primary roots under alkaline stress. By using Arabidopsis natural accessions with a high proton-secretion capacity, it was found that their PIN2 transcription abundance is positively related to the elongation rate of the primary root and proton-secretion capacity under alkaline stress. Taken together, our results confirm that PIN2 is involved in the PKS5-mediated signalling cascade under alkaline-stress and suggest that PIN2 is required for the adaptation of roots to alkaline stress by modulating proton secretion in the root tip to maintain primary root elongation.

The reorganization of actin filaments is required for vacuolar fusion of guard cells during stomatal opening in Arabidopsis

The reorganization of actin filaments (AFs) and vacuoles in guard cells is involved in the regulation of stomatal movement. However, it remains unclear whether there is any interaction between the reorganization of AFs and vacuolar changes during stomatal movement. Here, we report the relationship between the reorganization of AFs and vacuolar fusion revealed in pharmacological experiments, and characterizing stomatal opening in actin-related protein 2 (arp2) and arp3 mutants. Our results show that cytochalasin-D-induced depolymerization or phalloidin-induced stabilization of AFs leads to an increase in small unfused vacuoles during stomatal opening in wild-type (WT) Arabidopsis plants. Light-induced stomatal opening is retarded and vacuolar fusion in guard cells is impaired in the mutants, in which the reorganization and the dynamic parameters of AFs are aberrant compared with those of the WT. In WT, AFs tightly surround the small separated vacuoles, forming a ring that encircles the boundary membranes of vacuoles partly fused during stomatal opening. In contrast, in the mutants, most AFs and actin patches accumulate abnormally around the nuclei of the guard cells, which probably further impair vacuolar fusion and retard stomatal opening. Our results suggest that the reorganization of AFs regulates vacuolar fusion in guard cells during stomatal opening.

Functional genomics tools to decipher the pathogenicity mechanisms of the necrotrophic fungus Plectosphaerella cucumerina in Arabidopsis thaliana

The analysis of the interaction between Arabidopsis thaliana and adapted (PcBMM) and nonadapted (Pc2127) isolates of the necrotrophic fungus Plectosphaerella cucumerina has contributed to the identification of molecular mechanisms controlling plant resistance to necrotrophs. To characterize the pathogenicity bases of the virulence of necrotrophic fungi in Arabidopsis, we developed P. cucumerina functional genomics tools using Agrobacterium tumefaciens-mediated transformation. We generated PcBMM-GFP and Pc2127-GFP transformants constitutively expressing the green fluorescence protein (GFP), and a collection of random T-DNA insertional PcBMM transformants. Confocal microscopy analyses of the initial stages of PcBMM-GFP infection revealed that this pathogen, like other necrotrophic fungi, does not form an appressorium or penetrate into plant cells, but causes successive degradation of leaf cell layers. By comparing the colonization of Arabidopsis wild-type plants and hypersusceptible (agb1-1 and cyp79B2cyp79B3) and resistant (irx1-6) mutants by PcBMM-GFP or Pc2127-GFP, we found that the plant immune response was already mounted at 12-18 h post-inoculation, and that Arabidopsis resistance to these fungi correlated with the time course of spore germination and hyphal growth on the leaf surface. The virulence of a subset of the PcBMM T-DNA insertional transformants was determined in Arabidopsis wild-type plants and agb1-1 mutant, and several transformants were identified that showed altered virulence in these genotypes in comparison with that of untransformed PcBMM. The T-DNA flanking regions in these fungal mutants were successfully sequenced, further supporting the utility of these functional genomics tools in the molecular characterization of the pathogenicity of necrotrophic fungi.

The Long-Sought-After Salicylic Acid Receptors

The Long-Sought-After Salicylic Acid Receptors

Cytosolic ascorbate peroxidase 1 protects organelles against oxidative stress by wounding- and jasmonate-induced H2O2 in Arabidopsis plants

BackgroundReactive oxygen species (ROS) are not only cytotoxic compounds leading to oxidative damage, but also signaling molecules for regulating plant responses to stress and hormones. Arabidopsis cytosolic ascorbate peroxidase 1 (APX1) is thought to be a central regulator for cellular ROS levels. However, it remains unclear whether APX1 is involved in plant tolerance to wounding and methyl jasmonate (MeJA) treatment, which are known to enhance ROS production. MethodsWe studied the effect of wounding and MeJA treatment on the levels of H2O2 and oxidative damage in the Arabidopsis wild-type plants and knockout mutants lacking APX1 (KO-APX1). ResultsThe KO-APX1 plants showed high sensitivity to wounding and MeJA treatment. In the leaves of wild-type plants, H2O2 accumulated only in the vicinity of the wound, while in the leaves of the KO-APX1 plants it accumulated extensively from damaged to undamaged regions. During MeJA treatment, the levels of H2O2 were much higher in the leaves of KO-APX1 plants. Oxidative damage in the chloroplasts and nucleus was also enhanced in the leaves of KO-APX1 plants. These findings suggest that APX1 protects organelles against oxidative stress by wounding and MeJA treatment. General significanceThis is the first report demonstrating that H2O2-scavenging in the cytosol is essential for plant tolerance to wounding and MeJA treatment.

Isolation and characterization of two sorbitol transporter gene promoters in micropropagated apple plants (Malus × domestica) regulated by drought stress

The role of polyol transporters in stress tolerance in plants have been elucidated by many studies. Sorbitol transporter genes MdSOT3, MdSOT4 and MdSOT5 in apple plants, which are important for sorbitol loading and unloading, are regulated by drought stress. To further confirm the role of sorbitol transporters in stress tolerance, the constructs harboring MdSOT3 and MdSOT5 genes were introduced into wild type Arabidopsis plants (Col-0) and the Arabidopsis transformed with MdSOT3 or MdSOT5 performed higher drought stress tolerance compared to WT. In order to further understand how sorbitol transporters are involved in drought tolerance in apple plants, upstream regions of sorbitol transporter genes were isolated from apple plant source leaves by Anchored PCR from genomic DNA obtained, and then were used to drive expression of the GUS reporter in tobacco plants. The results showed that the longest fragments of MdSOT3 and MdSOT5 promoters induced the highest GUS activity under drought stress conditions. Additionally, fragments of these promoters that contain cis-acting elements known to be involved in stress response also induced GUS activity under drought stress. Taken together, our data suggest that increased MdSOT3 and MdSOT5 activity, through cis-acting elements in the promoters of these genes, play important roles in imparting tolerance to drought in micropropagated apple plants.

Monoclonal Antibodies Against Recombinant AtHOS15

Histone modifications are important components of transcriptional regulation and chromatin-based regulatory processes. In addition, WD40-repeat protein and several other components are involved in these functions. Here we present the development of monoclonal antibodies (MAbs) against Arabidopsis HOS15, a WD40-repeat protein. Mice were immunized with recombinant HOS15 (rHOS15) protein for generating MAbs via the classic hybridoma production technique. We confirmed the specific activity of anti-HOS15 MAbs by tobacco transient expression assays. Based on immunoprecipitation assays, the anti-HOS15 MAb was able to detect endogenous HOS15 in Arabidopsis wild-type plants, but not in hos15 mutant plants. Finally, the anti-HOS15 MAbs are highly sensitive for detecting endogenous HOS15 protein. They can be used for immunological and immunoprecipitation assays to support other experimental strategies. In particular, the anti-HOS15 MAbs will be essential tools to investigate the role of HOS15 in the regulation of tolerance to environmental stresses in plants.

Identification and Characterization of LHCB1 Co-Suppressed Line in Arabidopsis

To explore the function of light-harvesting complex protein (LHCP) in Arabidopsis growth and development, the Leclere and Bartel seed collection was screened. In this collection randomly cloned cDNAs are expressed under the CaMV35S promoter. A pale green line has been identified and characterized in more details. Analysis of the inserted cDNA in the pale green line showed it encodes LHCB1 proteins. The transcript level of LHCB1 in the selected line was less than that of the wild type (WT) Arabidopsis plant. Characterization of the LHCB1 co-suppressed line was achieved through its comparison with the WT plants when both were grown under normal irradiance. Then several measurements were performed such as: fresh and dry weight, chla, chlb, chla/b ratios and total chlorophyll content, soluble and insoluble sugar contents, total protein level, Hill reaction level and chlorophyll fluorescence. The LHCB1co-suppressed plants showed smaller leaf area with pale green coloration. Suppression of the LHCB1 gene significantly reduced fresh weight, chla, chlb content, total chlorophyll, and the rate of Hill reaction. Soluble and insoluble sugars, total protein, and chlorophyll fluorescence did not show significant differences between this line and WT plants. Meanwhile there was not any significant difference in fluorescence parameters between the WT and LHCB1 co-suppressed line.

A novel Medicago truncatula HD-Zip gene, MtHB2, is involved in abiotic stress responses

The HD-Zip proteins are important transcription factors participating in numerous physiological processes in plants. In this study, we identified a homeobox-leucine zipper gene from Medicago truncatula, designated MtHB2 by monitoring the expression profile of M. truncatula exposed to low temperature. A fusion protein of MtHB2 with green fluorescent protein showed localization of MtHB2 in the nucleus. To evaluate the role of MtHB2 in response to abiotic stresses, transgenic Arabidopsis plants were generated by expressing MtHB2, and the effects of abiotic stress on the transgenic and wild-type Arabidopsis plants were studied. Transgenic plants generated that constitutively expressed MtHB2 were more sensitive to drought, salt and freezing stresses than wild-type plants. The physiological mechanisms underlying the reduced tolerance of the transgenic plants to drought, salt and freezing stresses were investigated. Expression of MtHB2 in Arabidopsis resulted in the transgenic plants accumulating less amounts of Pro and soluble sugars and greater amounts of malondialdehyde (MDA) and H2O2 than their wild-type counterparts treated with and without abiotic stresses. The reduced accumulation of Pro and soluble sugars may account for the lower osmolality in the transgenic plants, thus rendering the osmo-regulation of the transgenic plants less effective, while the higher levels of MDA and H2O2 in the transgenic plants made the transgenic plants more susceptible to oxidative damage under the conditions of abiotic stress. These findings demonstrate that MtHB2 encodes a novel stress-responsive HD transcription factor that may play a negative role in regulation of abiotic stress response mechanisms.

Cloning and expression analysis of Arabidopsis TRR14 gene under salt and drought stress

TRR14 is a novel protein important in trehalose (α-D-glucosyl-[1,1]-α-D-glucopyranoside) signaling in Arabidopsis. In this research, we provided evidences to demonstrate that TRR14 plays role in Arabidopsis responses to salt and drought stress. The transgenic Arabidopsis plants over-expressing TRR14 under the control of CaMV 35S promoter were generated. Transformed lines showed higher transcript levels of TRR14 than that of Wild Type (WT) Arabidopsis plants. The RT-PCR results showed that TRR14 transcript level increased markedly by salt and drought stress both in WT and transformed lines. Further experiments indicated that the TRR14 transformed lines have unchanged seed germination, root length and chlorophyll content under stress conditions. In addition activity of oxidative enzymes like peroxidase and catalase were significantly induced in tranformed lines under salt and drought treatments. Thus, the present data indicate that a novel protein, TRR14, is involved in plant salt and drought tolerance.

Genes involved in the evolution of herbivory by a leaf-mining, Drosophilid fly.

Herbivorous insects are among the most successful radiations of life. However, we know little about the processes underpinning the evolution of herbivory. We examined the evolution of herbivory in the fly, Scaptomyza flava, whose larvae are leaf miners on species of Brassicaceae, including the widely studied reference plant, Arabidopsis thaliana (Arabidopsis). Scaptomyza flava is phylogenetically nested within the paraphyletic genus Drosophila, and the whole genome sequences available for 12 species of Drosophila facilitated phylogenetic analysis and assembly of a transcriptome for S. flava. A time-calibrated phylogeny indicated that leaf mining in Scaptomyza evolved between 6 and 16 million years ago. Feeding assays showed that biosynthesis of glucosinolates, the major class of antiherbivore chemical defense compounds in mustard leaves, was upregulated by S. flava larval feeding. The presence of glucosinolates in wild-type (WT) Arabidopsis plants reduced S. flava larval weight gain and increased egg–adult development time relative to flies reared in glucosinolate knockout (GKO) plants. An analysis of gene expression differences in 5-day-old larvae reared on WT versus GKO plants showed a total of 341 transcripts that were differentially regulated by glucosinolate uptake in larval S. flava. Of these, approximately a third corresponded to homologs of Drosophila melanogaster genes associated with starvation, dietary toxin-, heat-, oxidation-, and aging-related stress. The upregulated transcripts exhibited elevated rates of protein evolution compared with unregulated transcripts. The remaining differentially regulated transcripts also contained a higher proportion of novel genes than the unregulated transcripts. Thus, the transition to herbivory in Scaptomyza appears to be coupled with the evolution of novel genes and the co-option of conserved stress-related genes.

Comparative proteomic analysis of the Arabidopsis cbl1 mutant in response to salt stress

Many environmental stimuli, including light, biotic and abiotic stress factors, induce changes in cellular Ca(2+) concentrations in plants. Such Ca(2+) signatures are perceived by sensor molecules such as calcineurin B-like (CBL) proteins. AtCBL1, a member of the CBL family which is highly inducible by multiple stress signals, is known to function in the salt stress signal transduction pathway and to positively regulate the plant tolerance to salt. To shed light into the molecular mechanisms of the salt stress response mediated by AtCBL1, a two-dimensional DIGE proteomic approach was applied to identify the differentially expressed proteins in Arabidopsis wild-type and cbl1 null mutant plants in response to salt stress. Seventy-three spots were found altered in expression by least 1.2-fold and 50 proteins were identified by MALDI-TOF/TOF-MS, including some well-known and novel salt-responsive proteins. These proteins function in various processes, such as signal transduction, ROS scavenging, energy production, carbon fixation, metabolism, mRNA processing, protein processing and structural stability. Receptor for activated C kinase 1C (RACK1C, spot 715), a WD40 repeat protein, was up-regulated in the cbl1 null mutant, and two rack1c mutant lines showed decreased tolerance to salt stress, suggesting that RACK1C plays a role in salt stress resistance. In conclusion, our work demonstrated the advantages of the proteomic approach in studies of plant biology and identified candidate proteins in CBL1-mediated salt stress signaling network.

Photoprotection in Plants Involves a Change in Lutein 1 Binding Domain in the Major Light-harvesting Complex of Photosystem II

Nonphotochemical quenching (NPQ) is the fundamental process by which plants exposed to high light intensities dissipate the potentially harmful excess energy as heat. Recently, it has been shown that efficient energy dissipation can be induced in the major light-harvesting complexes of photosystem II (LHCII) in the absence of protein-protein interactions. Spectroscopic measurements on these samples (LHCII gels) in the quenched state revealed specific alterations in the absorption and circular dichroism bands assigned to neoxanthin and lutein 1 molecules. In this work, we investigate the changes in conformation of the pigments involved in NPQ using resonance Raman spectroscopy. By selective excitation we show that, as well as the twisting of neoxanthin that has been reported previously, the lutein 1 pigment also undergoes a significant change in conformation when LHCII switches to the energy dissipative state. Selective two-photon excitation of carotenoid (Car) dark states (Car S(1)) performed on LHCII gels shows that the extent of electronic interactions between Car S(1) and chlorophyll states correlates linearly with chlorophyll fluorescence quenching, as observed previously for isolated LHCII (aggregated versus trimeric) and whole plants (with versus without NPQ).

Synthesis of Hydroxylated Sterols in Transgenic Arabidopsis Plants Alters Growth and Steroid Metabolism

To explore mechanisms in plant sterol homeostasis, we have here increased the turnover of sterols in Arabidopsis (Arabidopsis thaliana) and potato (Solanum tuberosum) plants by overexpressing four mouse cDNA encoding cholesterol hydroxylases (CHs), hydroxylating cholesterol at the C-7, C-24, C-25, or C-27 positions. Compared to the wild type, the four types of Arabidopsis transformant showed varying degrees of phenotypic alteration, the strongest one being in CH25 lines, which were dark-green dwarfs resembling brassinosteroid-related mutants. Gas chromatography-mass spectrometry analysis of extracts from wild-type Arabidopsis plants revealed trace levels of α and β forms of 7-hydroxycholesterol, 7-hydroxycampesterol, and 7-hydroxysitosterol. The expected hydroxycholesterol metabolites in CH7-, CH24-, and CH25 transformants were identified and quantified using gas chromatography-mass spectrometry. Additional hydroxysterol forms were also observed, particularly in CH25 plants. In CH24 and CH25 lines, but not in CH7 ones, the presence of hydroxysterols was correlated with a considerable alteration of the sterol profile and an increased sterol methyltransferase activity in microsomes. Moreover, CH25 lines contained clearly reduced levels of brassinosteroids, and displayed an enhanced drought tolerance. Equivalent transformations of potato plants with the CH25 construct increased hydroxysterol levels, but without the concomitant alteration of growth and sterol profiles observed in Arabidopsis. The results suggest that an increased hydroxylation of cholesterol and/or other sterols in Arabidopsis triggers compensatory processes, acting to maintain sterols at adequate levels.

Antioxidant response of Arabidopsis plants to gamma irradiation: Genome-wide expression profiling of the ROS scavenging and signal transduction pathways

Arabidopsis presumably has few sensors for gamma-rays and few signal transduction systems that respond to them. In an effort to assess their radiation sensitivity, wild-type ( Ler) Arabidopsis plants were irradiated with various doses of gamma-rays at the vegetative (VE) and reproductive (RE) stages. 100 Gy treatment induced the higher production of siliques during both the VE and RE stages compared with non-irradiation. Treatments at doses over 200 Gy repressed shoot growth, and the plants perished under 800 Gy treatment. The results of physiological analysis using electron spin resonance (ESR) and transmission electron microscopy (TEM) revealed that increased doses of gamma-rays induce greater ROS generation. To establish the gene expression profiles after gamma irradiation and for an analysis of the antioxidant response, we employed an oligonucleotide microarray system. Different responses of genes related with ROS scavenging and signal transduction pathways by a gamma irradiation were observed. At least 33 and 42 out of all genes with significantly altered expression were associated with ROS scavenging and signal transduction pathways having an induction or repression ratio cutoff of at least 2-fold, respectively. CAT3 (At1g20620), Ferritin1 (At5g01600), Blue copper binding protein (At5g20230), and AOX putative (At1g32350) were up-regulated regardless of dosage at the VE stage. Reactive oxygen species signaling genes encoding phospholipase, zinc finger protein, WRKY DNA-binding protein, and calcium binding protein were highly expressed, evidencing changes greater than 2-fold. Our transcriptomic profile of the responses of Arabidopsis genes to gamma irradiation showed that plants evidenced altered expressions of many signal transduction and antioxidant genes, as have been seen with other environmental stresses.

A chloroplast-targeted DnaJ protein AtJ8 is negatively regulated by light and has rapid turnover in darkness

The DnaJ proteins (also called as J proteins, J domain proteins or HSP40 proteins) function as molecular co-chaperones for the HSP70 proteins. We assessed the expression of the small chloroplast-targeted DnaJ protein, the AtJ8 protein, by subjecting the wild type Arabidopsis plants to different illumination conditions. It is shown that the expression of the transcripts and proteins of the ATJ8 gene is primarily regulated at the level of transcription. When plants were incubated under high light for 3h, both the transcripts and proteins were completely abolished. Upon transfer of plants to darkness, the transcripts started rapidly accumulating, and subsequently, the AtJ8 protein became visible after 2h in darkness. Conversely, incubation of plants in darkness or under low light intensities induced expression of the ATJ8 transcripts and proteins. Feeding plants with sugars clearly decreased the transcript and protein levels, and incubation with cycloheximide revealed a rapid turnover for AtJ8 in darkness. Moreover, the AtJ8 protein was found to be nearly missing from the var1 mutant, which lacks the FTSH5 protease. It is concluded that AtJ8 is expressed mainly in darkness, is prone to a rapid turnover but is partially stabilized by the FTSH proteases.

Proteomic analysis of defense response of wildtype Arabidopsis thaliana and plants with impaired NO‐ homeostasis

In recent years, nitric oxide (NO) has been recognized as a signalling molecule of plants, being involved in diverse processes like germination, root growth, stomatal closing, and responses to various stresses. A mechanism of how NO can regulate physiological processes is the modulation of cysteine residues of proteins (S-nitrosylation) by S-nitrosoglutathione (GSNO), a physiological NO donor. The concentration of GSNO and the level of S-nitrosylated proteins are regulated by GSNO reductase, which seems to play a major role in NO signalling. To investigate the importance of NO in plant defense response, we performed a proteomic analysis of Arabidopsis wildtype and GSNO-reductase knock-out plants infected with both the avirulent and virulent pathogen strains of Pseudomonas syringae. Using 2-D DIGE technology in combination with MS, we identified proteins, which are differentially accumulated during the infection process. We observed that both lines were more resistant to avirulent infections than to virulent infections mainly due to the accumulation of stress-, redox-, and defense-related proteins. Interestingly, after virulent infections, we also observed accumulation of defense-related proteins, but no or low accumulation of stress- and redox-related proteins, respectively. In summary, we present here the first detailed proteomic analysis of plant defense response.

CGMP‐dependent ABA‐induced stomatal closure in the ABA‐insensitive Arabidopsis mutant abi1‐1

• The drought hormone abscisic acid (ABA) is widely known to produce reductions in stomatal aperture in guard cells. The second messenger cyclic guanosine 3', 5'-monophosphate (cGMP) is thought to form part of the signalling pathway by which ABA induces stomatal closure. • We have examined the signalling events during cGMP-dependent ABA-induced stomatal closure in wild-type Arabidopsis plants and plants of the ABA-insensitive Arabidopsis mutant abi1-1. • We show that cGMP acts downstream of hydrogen peroxide (H(2) O(2) ) and nitric oxide (NO) in the signalling pathway by which ABA induces stomatal closure. H(2) O(2) - and NO-induced increases in the cytosolic free calcium concentration ([Ca(2+) ](cyt) ) were cGMP-dependent, positioning cGMP upstream of [Ca(2+) ](cyt) , and involved the action of the type 2C protein phosphatase ABI1. Increases in cGMP were mediated through the stimulation of guanylyl cyclase by H(2) O(2) and NO. We identify nucleoside diphosphate kinase as a new cGMP target protein in Arabidopsis. • This study positions cGMP downstream of ABA-induced changes in H(2) O(2) and NO, and upstream of increases in [Ca(2+) ](cyt) in the signalling pathway leading to stomatal closure.

Cytokinin promotes flowering of Arabidopsis via transcriptional activation of the FT paralogue TSF

Cytokinins are involved in many aspects of plant growth and development, and physiological evidence also indicates that they have a role in floral transition. In order to integrate these phytohormones into the current knowledge of genetically defined molecular pathways to flowering, we performed exogenous treatments of adult wild type and mutant Arabidopsis plants, and analysed the expression of candidate genes. We used a hydroponic system that enables synchronous growth and flowering of Arabidopsis, and allows the precise application of chemicals to the roots for defined periods of time. We show that the application of N⁶-benzylaminopurine (BAP) promotes flowering of plants grown in non-inductive short days. The response to cytokinin treatment does not require FLOWERING LOCUS T (FT), but activates its paralogue TWIN SISTER OF FT (TSF), as well as FD, which encodes a partner protein of TSF, and the downstream gene SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1). Treatment of selected mutants confirmed that TSF and SOC1 are necessary for the flowering response to BAP, whereas the activation cascade might partially act independently of FD. These experiments provide a mechanistic basis for the role of cytokinins in flowering, and demonstrate that the redundant genes FT and TSF are differently regulated by distinct floral-inducing signals.

Dynamic imaging of glucose flux impedance using FRET sensors in wild-type Arabidopsis plants

Quantitative and dynamic analysis of metabolites and signalling molecules is limited by technical challenges in obtaining temporally resolved information at the cellular and compartmental level. Real-time information on signalling and metabolite levels with subcellular granularity can be obtained with the help of genetically encoded FRET (Förster resonance energy transfer) nanosensors. FRET nanosensors represent powerful tools for gene discovery, and analysis of regulatory networks, for example by screening mutants. However, RNA silencing has impaired our ability to express FRET nanosensors functionally in Arabidopsis plants. This drawback was overcome here by expressing the nanosensors in RNA silencing mutants. However, the use of silencing mutants requires the generation of homozygous lines deficient in RNA silencing as well as the mutation of interest and co-expression of the nanosensor. Here it is shown that dynamic changes in cytosolic glucose levels can readily be quantified in wild-type Arabidopsis plants at early stages of development (7-15 d) before silencing had a major effect on fluorescence intensity. A detailed protocol for screening 10-20 mutant seedlings per day is provided. The detailed imaging protocol provided here is suitable for analysing sugar flux in young wild-type plants as well as mutants affected in sugar signalling, metabolism, or transport using a wide spectrum of FRET nanosensors.