IPD3, a master regulator of arbuscular mycorrhizal symbiosis, affects genes for immunity and metabolism of non-host Arabidopsis when restored long after its evolutionary loss.

Arbuscular mycorrhizal symbiosis (AM) is a beneficial trait originating with the first land plants, which has subsequently been lost by species scattered throughout the radiation of plant diversity to the present day, including the model Arabidopsis thaliana. To explore if elements of this apparently beneficial trait are still present and could be reactivated we generated Arabidopsis plants expressing a constitutively active form of Interacting Protein of DMI3, a key transcription factor that enables AM within the Common Symbiosis Pathway, which was lost from Arabidopsis along with the AM host trait. We characterize the transcriptomic effect of expressing IPD3 in Arabidopsis with and without exposure to the AM fungus (AMF) Rhizophagus irregularis, and compare these results to the AM model Lotus japonicus and its ipd3 knockout mutant cyclops-4. Despite its long history as a non-AM species, restoring IPD3 in the form of its constitutively active DNA-binding domain to Arabidopsis altered expression of specific gene networks. Surprisingly, the effect of expressing IPD3 in Arabidopsis and knocking it out in Lotus was strongest in plants not exposed to AMF, which is revealed to be due to changes in IPD3 genotype causing a transcriptional state, which partially mimics AMF exposure in non-inoculated plants. Our results indicate that molecular connections to symbiosis machinery remain in place in this nonAM species, with implications for both basic science and the prospect of engineering this trait for agriculture.

Long- and short-read sequencing methods discover distinct circular RNA pools in Lotus japonicus.

Circular RNAs (circRNAs) are covalently closed single-stranded RNAs, generated through a back-splicing process that links a downstream 5' site to an upstream 3' end. The only distinction in the sequence between circRNA and their linear cognate RNA is the back splice junction. Their low abundance and sequence similarity with their linear origin RNA have made the discovery and identification of circRNA challenging. We have identified almost 6000 novel circRNAs from Lotus japonicus leaf tissue using different enrichment, amplification, and sequencing methods as well as alternative bioinformatics pipelines. The different methodologies identified different pools of circRNA with little overlap. We validated circRNA identified by the different methods using reverse transcription polymerase chain reaction and characterized sequence variations using nanopore sequencing. We compared validated circRNA identified in L. japonicus to other plant species and showed conservation of high-confidence circRNA-expressing genes. This is the first identification of L. japonicus circRNA and provides a resource for further characterization of their function in gene regulation. CircRNAs identified in this study originated from genes involved in all biological functions of eukaryotic cells. The comparison of methodologies and technologies to sequence, identify, analyze, and validate circRNA from plant tissues will enable further research to characterize the function and biogenesis of circRNA in L. japonicus.

Emergent molecular traits of lettuce and tomato grown under wavelength-selective solar cells.

The integration of semi-transparent organic solar cells (ST-OSCs) in greenhouses offers new agrivoltaic opportunities to meet the growing demands for sustainable food production. The tailored absorption/transmission spectra of ST-OSCs impacts the power generated as well as crop growth, development and responses to the biotic and abiotic environments. To characterize crop responses to ST-OSCs, we grew lettuce and tomato, traditional greenhouse crops, under three ST-OSC filters that create different light spectra. Lettuce yield and early tomato development are not negatively affected by the modified light environment. Our genomic analysis reveals that lettuce production exhibits beneficial traits involving nutrient content and nitrogen utilization while select ST-OSCs impact regulation of flowering initiation in tomato. These results suggest that ST-OSCs integrated into greenhouses are not only a promising technology for energy-neutral, sustainable and climate-change protected crop production, but can deliver benefits beyond energy considerations.

Re-engineering a lost trait: IPD3, a master regulator of arbuscular mycorrhizal symbiosis, affects genes for immunity and metabolism of non-host Arabidopsis when restored long after its evolutionary loss.

Arbuscular mycorrhizal symbiosis (AM) is a beneficial trait originating with the first land plants, which has subsequently been lost by species scattered throughout the radiation of plant diversity to the present day, including the model Arabidopsis thaliana. To explore why an apparently beneficial trait would be repeatedly lost, we generated Arabidopsis plants expressing a constitutively active form of Interacting Protein of DMI3, a key transcription factor that enables AM within the Common Symbiosis Pathway, which was lost from Arabidopsis along with the AM host trait. We characterize the transcriptomic effect of expressing IPD3 in Arabidopsis with and without exposure to the AM fungus (AMF) Rhizophagus irregularis, and compare these results to the AM model Lotus japonicus and its ipd3 knockout mutant cyclops-4. Despite its long history as a non-AM species, restoring IPD3 in the form of its constitutively active DNA-binding domain to Arabidopsis altered expression of specific gene networks. Surprisingly, the effect of expressing IPD3 in Arabidopsis and knocking it out in Lotus was strongest in plants not exposed to AMF, which is revealed to be due to changes in IPD3 genotype causing a transcriptional state which partially mimics AMF exposure in non-inoculated plants. Our results indicate that despite the long interval since loss of AM and IPD3 in Arabidopsis, molecular connections to symbiosis machinery remain in place in this nonAM species, with implications for both basic science and the prospect of engineering this trait for agriculture.

The double flower variant of yellowhorn is due to a LINE1 transposon-mediated insertion.

As essential organs of reproduction in angiosperms, flowers, and the genetic mechanisms of their development have been well characterized in many plant species but not in the woody tree yellowhorn (Xanthoceras sorbifolium). Here, we focused on the double flower phenotype in yellowhorn, which has high ornamental value. We found a candidate C-class gene, AGAMOUS1 (XsAG1), through bovine serum albumin sequencing and genetics analysis with a Long Interpersed Nuclear Elements 1 (LINE1) transposable element fragment (Xsag1-LINE1-1) inserted into its second intron that caused a loss-of-C-function and therefore the double flower phenotype. In situ hybridization of XsAG1 and analysis of the expression levels of other ABC genes were used to identify differences between single- and double-flower development processes. These findings enrich our understanding of double flower formation in yellowhorn and provide evidence that transposon insertions into genes can reshape plant traits in forest trees.

High-throughput detection of T-DNA insertion sites for multiple transgenes in complex genomes.

BACKGROUND: Genetic engineering of crop plants has been successful in transferring traits into elite lines beyond what can be achieved with breeding techniques. Introduction of transgenes originating from other species has conferred resistance to biotic and abiotic stresses, increased efficiency, and modified developmental programs. The next challenge is now to combine multiple transgenes into elite varieties via gene stacking to combine traits. Generating stable homozygous lines with multiple transgenes requires selection of segregating generations which is time consuming and labor intensive, especially if the crop is polyploid. Insertion site effects and transgene copy number are important metrics for commercialization and trait efficiency. RESULTS: We have developed a simple method to identify the sites of transgene insertions using T-DNA-specific primers and high-throughput sequencing that enables identification of multiple insertion sites in the T1 generation of any crop transformed via Agrobacterium. We present an example using the allohexaploid oil-seed plant Camelina sativa to determine insertion site location of two transgenes. CONCLUSION: This new methodology enables the early selection of desirable transgene location and copy number to generate homozygous lines within two generations.

Gene sdaB Is Involved in the Nematocidal Activity of Enterobacter ludwigii AA4 Against the Pine Wood Nematode Bursaphelenchus xylophilus.

Bursaphelenchus xylophilus, a plant parasitic nematode, is the causal agent of pine wilt, a devastating forest tree disease. Essentially, no efficient methods for controlling B. xylophilus and pine wilt disease have yet been developed. Enterobacter ludwigii AA4, isolated from the root of maize, has powerful nematocidal activity against B. xylophilus in a new in vitro dye exclusion test. The corrected mortality of the B. xylophilus treated by E. ludwigii AA4 or its cell extract reached 98.3 and 98.6%, respectively. Morphological changes in B. xylophilus treated with a cell extract from strain AA4 suggested that the death of B. xylophilus might be caused by an increased number of vacuoles in non-apoptotic cell death and the damage to tissues of the nematodes. In a greenhouse test, the disease index of the seedlings of Scots pine (Pinus sylvestris) treated with the cells of strain AA4 plus B. xylophilus or those treated by AA4 cell extract plus B. xylophilus was 38.2 and 30.3, respectively, was significantly lower than 92.5 in the control plants treated with distilled water and B. xylophilus. We created a sdaB gene knockout in strain AA4 by deleting the gene that was putatively encoding the beta-subunit of L-serine dehydratase through Red homologous recombination. The nematocidal and disease-suppressing activities of the knockout strain were remarkably impaired. Finally, we revealed a robust colonization of P. sylvestris seedling needles by E. ludwigii AA4, which is supposed to contribute to the disease-controlling efficacy of strain AA4. Therefore, E. ludwigii AA4 has significant potential to serve as an agent for the biological control of pine wilt disease caused by B. xylophilus.

Microbial Interactions Within Multiple-Strain Biological Control Agents Impact Soil-Borne Plant Disease.

Major losses of crop yield and quality caused by soil-borne plant diseases have long threatened the ecology and economy of agriculture and forestry. Biological control using beneficial microorganisms has become more popular for management of soil-borne pathogens as an environmentally friendly method for protecting plants. Two major barriers limiting the disease-suppressive functions of biocontrol microbes are inadequate colonization of hosts and inefficient inhibition of soil-borne pathogen growth, due to biotic and abiotic factors acting in complex rhizosphere environments. Use of a consortium of microbial strains with disease inhibitory activity may improve the biocontrol efficacy of the disease-inhibiting microbes. The mechanisms of biological control are not fully understood. In this review, we focus on bacterial and fungal biocontrol agents to summarize the current state of the use of single strain and multi-strain biological control consortia in the management of soil-borne diseases. We discuss potential mechanisms used by microbial components to improve the disease suppressing efficacy. We emphasize the interaction-related factors to be considered when constructing multiple-strain biological control consortia and propose a workflow for assembling them by applying a reductionist synthetic community approach.

Construction and Setup of a Bench-scale Algal Photosynthetic Bioreactor with Temperature, Light, and pH Monitoring for Kinetic Growth Tests.

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic performance of microalgae-based biofuel production. Models that estimate microalgal growth under different conditions can help to optimize PBR design and operation. To be effective, the growth parameters used in these models must be accurately determined. Algal growth experiments are often constrained by the dynamic nature of the culture environment, and control systems are needed to accurately determine the kinetic parameters. The first step in setting up a controlled batch experiment is live data acquisition and monitoring. This protocol outlines a process for the assembly and operation of a bench-scale photosynthetic bioreactor that can be used to conduct microalgal growth experiments. This protocol describes how to size and assemble a flat-plate, bench-scale PBR from acrylic. It also details how to configure a PBR with continuous pH, light, and temperature monitoring using a data acquisition and control unit, analog sensors, and open-source data acquisition software.

Amino Acids Are an Ineffective Fertilizer for Dunaliella spp. Growth.

Autotrophic microalgae are a promising bioproducts platform. However, the fundamental requirements these organisms have for nitrogen fertilizer severely limit the impact and scale of their cultivation. As an alternative to inorganic fertilizers, we investigated the possibility of using amino acids from deconstructed biomass as a nitrogen source in the genus Dunaliella. We found that only four amino acids (glutamine, histidine, cysteine, and tryptophan) rescue Dunaliella spp. growth in nitrogen depleted media, and that supplementation of these amino acids altered the metabolic profile of Dunaliella cells. Our investigations revealed that histidine is transported across the cell membrane, and that glutamine and cysteine are not transported. Rather, glutamine, cysteine, and tryptophan are degraded in solution by a set of oxidative chemical reactions, releasing ammonium that in turn supports growth. Utilization of biomass-derived amino acids is therefore not a suitable option unless additional amino acid nitrogen uptake is enabled through genetic modifications of these algae.

Accumulation of medium-chain, saturated fatty acyl moieties in seed oils of transgenic Camelina sativa.

With its high seed oil content, the mustard family plant Camelina sativa has gained attention as a potential biofuel source. As a bioenergy crop, camelina has many advantages. It grows on marginal land with low demand for water and fertilizer, has a relatively short life cycle, and is stress tolerant. As most other crop seed oils, camelina seed triacylglycerols (TAGs) consist of mostly long, unsaturated fatty acyl moieties, which is not desirable for biofuel processing. In our efforts to produce shorter, saturated chain fatty acyl moieties in camelina seed oil for conversion to jet fuel, a 12:0-acyl-carrier thioesterase gene, UcFATB1, from California bay (Umbellularia californica Nutt.) was expressed in camelina seeds. Up to 40% of short chain laurate (C12:0) and myristate (C14:0) were present in TAGs of the seed oil of the transgenics. The total oil content and germination rate of the transgenic seeds were not affected. Analysis of positions of these two fatty acyl moieties in TAGs indicated that they were present at the sn-1 and sn-3 positions, but not sn-2, on the TAGs. Suppression of the camelina KASII genes by RNAi constructs led to higher accumulation of palmitate (C16:0), from 7.5% up to 28.5%, and further reduction of longer, unsaturated fatty acids in seed TAGs. Co-transformation of camelina with both constructs resulted in enhanced accumulation of all three medium-chain, saturated fatty acids in camelina seed oils. Our results show that a California bay gene can be successfully used to modify the oil composition in camelina seed and present a new biological alternative for jet fuel production.

ROSY1, a novel regulator of gravitropic response is a stigmasterol binding protein.

The gravitropic bending in plant roots is caused by asymmetric cell elongation. This requires an asymmetric increase in cell surface and therefore plasma membrane components such as lipids, sterols, and membrane proteins. We have identified an early gravity-regulated protein in Arabidopsis thaliana root apices that binds stigmasterol and phosphoethanolamines. This root-specific protein interacts with the membrane transport protein synaptotagmin-1 and was therefore named InteractoR Of SYnaptotagmin1 (ROSY1). While interactions between ML-domain proteins with membrane transport proteins and their impact have been reported from animal cell systems, this is the first report of such an interaction in a plant system. Homozygous mutants of ROSY1 exhibit decreased basipetal auxin transport, a faster root gravitropic response, and an increase in salt stress tolerance. Our results suggest that ROSY1 plays a role in root gravitropism, possibly by facilitating membrane trafficking and asymmetric cell elongation via its interaction with synaptotagmin-1.

A photorespiratory bypass increases plant growth and seed yield in biofuel crop Camelina sativa.

BACKGROUND: Camelina sativa is an oilseed crop with great potential for biofuel production on marginal land. The seed oil from camelina has been converted to jet fuel and improved fuel efficiency in commercial and military test flights. Hydrogenation-derived renewable diesel from camelina is environmentally superior to that from canola due to lower agricultural inputs, and the seed meal is FDA approved for animal consumption. However, relatively low yield makes its farming less profitable. Our study is aimed at increasing camelina seed yield by reducing carbon loss from photorespiration via a photorespiratory bypass. Genes encoding three enzymes of the Escherichia coli glycolate catabolic pathway were introduced: glycolate dehydrogenase (GDH), glyoxylate carboxyligase (GCL) and tartronic semialdehyde reductase (TSR). These enzymes compete for the photorespiratory substrate, glycolate, convert it to glycerate within the chloroplasts, and reduce photorespiration. As a by-product of the reaction, CO2 is released in the chloroplast, which increases photosynthesis. Camelina plants were transformed with either partial bypass (GDH), or full bypass (GDH, GCL and TSR) genes. Transgenic plants were evaluated for physiological and metabolic traits. RESULTS: Expressing the photorespiratory bypass genes in camelina reduced photorespiration and increased photosynthesis in both partial and full bypass expressing lines. Expression of partial bypass increased seed yield by 50-57 %, while expression of full bypass increased seed yield by 57-73 %, with no loss in seed quality. The transgenic plants also showed increased vegetative biomass and faster development; they flowered, set seed and reached seed maturity about 1 week earlier than WT. At the transcriptional level, transgenic plants showed differential expression in categories such as respiration, amino acid biosynthesis and fatty acid metabolism. The increased growth of the bypass transgenics compared to WT was only observed in ambient or low CO2 conditions, but not in elevated CO2 conditions. CONCLUSIONS: The photorespiratory bypass is an effective approach to increase photosynthetic productivity in camelina. By reducing photorespiratory losses and increasing photosynthetic CO2 fixation rates, transgenic plants show dramatic increases in seed yield. Because photorespiration causes losses in productivity of most C3 plants, the bypass approach may have significant impact on increasing agricultural productivity for C3 crops.

Algal ancestor of land plants was preadapted for symbiosis.

Colonization of land by plants was a major transition on Earth, but the developmental and genetic innovations required for this transition remain unknown. Physiological studies and the fossil record strongly suggest that the ability of the first land plants to form symbiotic associations with beneficial fungi was one of these critical innovations. In angiosperms, genes required for the perception and transduction of diffusible fungal signals for root colonization and for nutrient exchange have been characterized. However, the origin of these genes and their potential correlation with land colonization remain elusive. A comprehensive phylogenetic analysis of 259 transcriptomes and 10 green algal and basal land plant genomes, coupled with the characterization of the evolutionary path leading to the appearance of a key regulator, a calcium- and calmodulin-dependent protein kinase, showed that the symbiotic signaling pathway predated the first land plants. In contrast, downstream genes required for root colonization and their specific expression pattern probably appeared subsequent to the colonization of land. We conclude that the most recent common ancestor of extant land plants and green algae was preadapted for symbiotic associations. Subsequent improvement of this precursor stage in early land plants through rounds of gene duplication led to the acquisition of additional pathways and the ability to form a fully functional arbuscular mycorrhizal symbiosis.

A novel gateway-compatible binary vector series (PC-GW) for flexible cloning of multiple genes for genetic transformation of plants.

The rapidly advancing field of plant synthetic biology requires transforming plants with multiple genes. This has sparked a growing interest in flexible plant transformation vectors, which can be used for multi-gene transformations. We have developed a novel binary vector series, named the PC-GW series (GenBank: KP826769-KP826773), for Agrobacterium-mediated plant transformation. The PC-GW vectors use the pCAMBIA vector backbone, and contain NPTII, hpt, bar, mCherry or egfp genes as selectable markers for plant transformation. In a modified multiple cloning site (MCS) of the T-DNA region, we have placed the attR1, attR2 and ccdB sequences for rapid cloning of one to four genes by Gateway™-assisted recombination. In addition, we have introduced four meganuclease sites, and other restriction sites for multi-gene vector construction. Finally, we have placed a CaMV 35S promoter and a 35S terminator on the 5' and 3' ends of the MCS. The CaMV 35S promoter is flanked by PstI restriction sites that can be used to replace it with another promoter sequence if needed. The PC-GW vectors provide choices for selectable markers, cloning methods, and can accommodate up to eight gene constructs in a single T-DNA, thereby significantly reducing the number of transformations or crosses needed to generate multi-transgene expressing plants.

Interaction of Temperature and Photoperiod Increases Growth and Oil Content in the Marine Microalgae Dunaliella viridis.

Eukaryotic marine microalgae like Dunaliella spp. have great potential as a feedstock for liquid transportation fuels because they grow fast and can accumulate high levels of triacylgycerides with little need for fresh water or land. Their growth rates vary between species and are dependent on environmental conditions. The cell cycle, starch and triacylglycerol accumulation are controlled by the diurnal light:dark cycle. Storage compounds like starch and triacylglycerol accumulate in the light when CO2 fixation rates exceed the need of assimilated carbon and energy for cell maintenance and division during the dark phase. To delineate environmental effects, we analyzed cell division rates, metabolism and transcriptional regulation in Dunaliella viridis in response to changes in light duration and growth temperatures. Its rate of cell division was increased under continuous light conditions, while a shift in temperature from 25 °C to 35 °C did not significantly affect the cell division rate, but increased the triacylglycerol content per cell several-fold under continuous light. The amount of saturated fatty acids in triacylglycerol fraction was more responsive to an increase in temperature than to a change in the light regime. Detailed fatty acid profiles showed that Dunaliella viridis incorporated lauric acid (C12:0) into triacylglycerol after 24 hours under continuous light. Transcriptome analysis identified potential regulators involved in the light and temperature-induced lipid accumulation in Dunaliella viridis.

Methods for RNA profiling of gravi-responding plant tissues.

Plant transcriptional responses to gravity stimulation by reorientation are among the fastest measured in any tissue or species. Upon reorientation, changes in abundance of specific mRNAs can be measured within seconds or minutes, for plastid or nuclear encoded genes, respectively. Identifying fast gravity-induced transcripts has been made possible by the development of high-throughput technology for qualitative and quantitative RNA analysis. RNA profiling has undergone further rapid development due to its enormous potential in basic sciences and medical applications. We describe here the current and most widely used methods to profile the changes in an entire transcriptome by high-throughput sequencing of RNA fractions (RNAseq) and single gene transcript analysis using real-time quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR).

High-throughput RNA sequencing of pseudomonas-infected Arabidopsis reveals hidden transcriptome complexity and novel splice variants.

We report the results of a genome-wide analysis of transcription in Arabidopsis thaliana after treatment with Pseudomonas syringae pathovar tomato. Our time course RNA-Seq experiment uses over 500 million read pairs to provide a detailed characterization of the response to infection in both susceptible and resistant hosts. The set of observed differentially expressed genes is consistent with previous studies, confirming and extending existing findings about genes likely to play an important role in the defense response to Pseudomonas syringae. The high coverage of the Arabidopsis transcriptome resulted in the discovery of a surprisingly large number of alternative splicing (AS) events--more than 44% of multi-exon genes showed evidence for novel AS in at least one of the probed conditions. This demonstrates that the Arabidopsis transcriptome annotation is still highly incomplete, and that AS events are more abundant than expected. To further refine our predictions, we identified genes with statistically significant changes in the ratios of alternative isoforms between treatments. This set includes several genes previously known to be alternatively spliced or expressed during the defense response, and it may serve as a pool of candidate genes for regulated alternative splicing with possible biological relevance for the defense response against invasive pathogens.

The SOS4 pyridoxal kinase is required for maintenance of vitamin B6-mediated processes in chloroplasts.

Vitamin B(6) (pyridoxal 5'-phosphate and its vitamers) is an important cofactor in numerous enzymatic reactions. In spite of its importance, the consequences of altering vitamin B(6) content on plant growth and development are not well understood. This study compares two mutants for vitamin B(6)-metabolizing enzymes in Arabidopsis thaliana: a pdx1.3 mutant in the de novo synthesis pathway and a salvage pathway sos4 mutant that accumulates more vitamin B(6). We show that despite a difference in total B(6) content in leaf tissue, both mutants share similar phenotypes, including chlorosis, decreased size, altered chloroplast ultrastructure, and root sensitivity to sucrose. Assay of B(6) vitamer content from isolated chloroplasts showed that, despite differing B(6) vitamer content in whole leaf tissue, both mutants share a common deficiency in total and phosphorylated vitamers in chloroplasts. One of the splice variants of the SOS4 proteins was shown to be located in the chloroplast. Our data indicate that some of the phenotypic consequences shared between the pdx1.3 and sos4 mutants are due to B(6) deficiency in chloroplasts, and show that SOS4 is required for maintenance of phosphorylated B(6) vitamer concentrations in chloroplasts. Further, our data are consistent with a diffusion model for transport of vitamin B(6) into chloroplasts.

An ER-targeted calcium-binding peptide confers salt and drought tolerance mediated by CIPK6 in Arabidopsis.

Different plant organelles have high internal stores of Ca(2+) compared to the cytoplasm and could play independent roles in stress responses or signal transduction. We used a GFP fusion with the C-domain of calreticulin, which shows low-affinity, high capacity Ca(2+) binding in the ER, as a calcium-binding peptide (CBP) to specifically increase stores in the ER and nucleus. Despite the presence of a signal sequence and KDEL retention sequence, our work and previous studies (Brandizzi et al. Plant Journal 34:269-281, 2003) demonstrated both ER and nuclear localization of GFP-CBP. Under normal conditions, GFP-CBP-expressing lines had ~25% more total Ca(2+) and higher levels of chlorophyll and seed yield than wild type and GFP controls. CBP-expressing plants also had better survival under intermittent drought or high salt treatments and increased root growth. One member of the CIPK (calcineurin B-like interacting protein kinase) gene family, CIPK6, was up-regulated in CBP-expressing plants, even under non-stress conditions. A null mutation in cipk6 abolished the increased stress tolerance of CBP-transgenic plants, as well as the CBP-mediated induction of two stress-associated genes, DREB1A and RD29A, under non-stress conditions. Although this suggested that it was the induction of CIPK6, rather than localized changes in Ca(2+), that resulted in increased survival under adverse conditions, CIPK6 induction still required Ca(2+). This work demonstrates that ER (or nuclear) Ca(2+) can directly participate in signal transduction to alter gene expression. The discovery of a method for increasing Ca(2+) levels without deleterious effects on plant growth may have practical applications.