Leaf Proteome Analysis Research Articles

Specific adjustments in grapevine leaf proteome discriminating resistant and susceptible grapevine genotypes to Plasmopara viticola

Grapevine downy mildew is an important disease affecting crop production leading to severe yield losses. This study aims to identify the grapevine cultivar-specific adjustments of leaf proteome that allow the discrimination between resistance and susceptibility towards P. viticola (constitutive (0h) and in after inoculation (6, 12 and 24h). Leaf proteome analysis was performed using 2D difference gel electrophoresis followed by protein identification via mass spectrometry. In addition, we analysed ROS production, antioxidant capacity, lipid peroxidation and gene expression. Proteins related to photosynthesis and metabolism allowed the discrimination of resistant and susceptible grapevine cultivars prior to P. viticola inoculation. Following inoculation increase of hydrogen peroxide levels, cellular redox regulation, establishment of ROS signalling and plant cell death seem to be key points differentiating the resistant genotype. Lipid associated signalling events, particularly related to jasmonates appear also to play a major role in the establishment of resistance. The findings from this study contribute to a better understanding of genotype-specific differences that account for a successful establishment of a defence response to the downy mildew pathogen. Biological significanceHere, we present for the first time grapevine cultivar-specific adjustments of leaf proteome that allow the discrimination between resistance and susceptibility towards P. viticola (constitutive (0h) and in after inoculation (6, 12 and 24h). We have highlighted that, following inoculation, the major factors differentiating the resistant from the susceptible grapevine cultivars are the establishment of effective ROS signalling together with lipid-associated signalling events, particularly related to jasmonates. It is believed that plants infected with biotrophic pathogens suppress JA-mediated responses, however recent evidences shown that jasmonic acid signalling pathway in grapevine resistance against Plasmopara viticola. Our results corroborate those evidences and highlight the importance of lipid- signalling for an effective resistance response against the downy mildew pathogen.

A quantitative proteomic approach to highlight Phragmites sp. adaptation mechanisms to chemical stress induced by a textile dyeing pollutant

Phragmites sp. is present worldwide in treatment wetlands though the mechanisms involved in the phytoremediation remain unclear. In this study a quantitative proteomic approach was used to study the prompt response and adaptation of Phragmites to the textile dyeing pollutant, Acid Orange 7 (AO7). Previously, it was demonstrated that AO7 could be successfully removed from wastewater and mineralized in a constructed wetland planted with Phragmites sp. This azo dye is readily taken up by roots and transported to the plant aerial part by the xylem. Phragmites leaf samples were collected from a pilot scale vertical flow constructed wetland after 0.25, 3.25 and 24.25h exposure to AO7 (400mgL−1) immediately after a watering cycle used as control. Leaf soluble protein extraction yielded an average of 1560 proteins in a broad pI range (pH3–10) by two-dimensional gel electrophoresis. A time course comparative analysis of leaf proteome revealed that 40 proteins had a differential abundance compared to control (p<0.05) within a 3.25h period. After 24.25h in contact with AO7, leaf proteome was similar to control. Adaptation to AO7 involved proteins related with cellular signalling (calreticulin, Ras-related protein Rab11D and 20S proteasome), energy production and conversion (adenosine triphosphate synthase beta subunit) carbohydrate transport and metabolism (phosphoglucose isomerase, fructose-bisphosphate aldolase, monodehydroascorbate reductase, frutockinase-1 and Hypothetical protein POPTR_0003s12000g and the Uncharacterized protein LOC100272772) and photosynthesis (sedoheptulose-1,7-bisphosphatase and ferredoxin-NADP+ reductase). Therefore, the quantitative proteomic approach used in this work indicates that mechanisms associated with stress cell signalling, energy production, carbohydrate transport and metabolism as well as proteins related with photosynthesis are key players in the initial chemical stress response in the phytoremediation process of AO7.

Plant immunity induced by COS-OGA elicitor is a cumulative process that involves salicylic acid

Plant innate immunity offers considerable opportunities for plant protection but beside flagellin and chitin, not many molecules and their receptors have been extensively characterized and very few have successfully reached the field. COS-OGA, an elicitor that combines cationic chitosan oligomers (COS) with anionic pectin oligomers (OGA), efficiently protected tomato (Solanum lycopersicum) grown in greenhouse against powdery mildew (Leveillula taurica). Leaf proteomic analysis of plants sprayed with COS-OGA showed accumulation of Pathogenesis-Related proteins (PR), especially subtilisin-like proteases. qRT-PCR confirmed upregulation of PR-proteins and salicylic acid (SA)-related genes while expression of jasmonic acid/ethylene-associated genes was not modified. SA concentration and class III peroxidase activity were increased in leaves and appeared to be a cumulative process dependent on the number of sprayings with the elicitor. These results suggest a systemic acquired resistance (SAR) mechanism of action of the COS-OGA elicitor and highlight the importance of repeated applications to ensure efficient protection against disease.

Proteome Analysis of Date Palm (Phoenix dactylifera L.) under Severe Drought and Salt Stress.

Date palm cultivars differently tolerate salinity and drought stress. This study was carried out to study the response of date palm to severe salinity and drought based on leaf proteome analysis. Eighteen-month-old date palm plants were subjected to severe salt (48 g/L NaCl) and drought (82.5 g/L PEG or no irrigation) conditions for one month. Using a protein 2D electrophoresis method, 55 protein spots were analyzed using mass spectrometry. ATP synthase CF1 alpha chains were significantly upregulated under all three stress conditions. Changes in the abundance of RubisCO activase and one of the RubisCO fragments were significant in the same spots only for salt stress and drought stress with no irrigation, and oxygen-evolving enhancer protein 2 was changed in different spots. Transketolase was significantly changed only in drought stress with PEG. The expression of salt and drought stress genes of the chosen protein spots was either overexpressed or downexpressed as revealed by the high or low protein abundance, respectively. In addition, all drought tolerance genes due to no irrigation were downregulated. In conclusion, the proteome analysis of date palm under salinity and drought conditions indicated that both salinity and drought tolerance genes were differentially expressed resulting in high or low protein abundance of the chosen protein spots as a result of exposure to drought and salinity stress condition.

Stress Signaling Responses in Plants.

Stress Signaling Responses in Plants.

Leaf Proteome Analysis Reveals Prospective Drought and Heat Stress Response Mechanisms in Soybean.

Drought and heat are among the major abiotic stresses that affect soybean crops worldwide. During the current investigation, the effect of drought, heat, and drought plus heat stresses was compared in the leaves of two soybean varieties, Surge and Davison, combining 2D-DIGE proteomic data with physiology and biochemical analyses. We demonstrated how 25 differentially expressed photosynthesis-related proteins affect RuBisCO regulation, electron transport, Calvin cycle, and carbon fixation during drought and heat stress. We also observed higher abundance of heat stress-induced EF-Tu protein in Surge. It is possible that EF-Tu might have activated heat tolerance mechanisms in the soybean. Higher level expressions of heat shock-related protein seem to be regulating the heat tolerance mechanisms. This study identifies the differential expression of various abiotic stress-responsive proteins that regulate various molecular processes and signaling cascades. One inevitable outcome from the biochemical and proteomics assays of this study is that increase of ROS levels during drought stress does not show significant changes at the phenotypic level in Davison and this seems to be due to a higher amount of carbonic anhydrase accumulation in the cell which aids the cell to become more resistant to cytotoxic concentrations of H2O2.

Leaf proteome analysis provides insights into the molecular mechanisms of bentazon detoxification in rice

Bentazon is a widely used herbicide that selectively removes broad-leaf weeds by competing with plastoquinone for the binding site in the D1 protein and interrupting the PET (photosynthetic electron transfer) chain. However, monocotyledonous plants, such as rice, show strong resistance to bentazon due to CYP81A6 induction, which results in herbicide detoxification. Here, we confirmed that rice was sensitive to bentazon treatment during the initial exposure period, in which bentazon rapidly inhibited photosynthesis efficiency and electron transfer, based on results of chlorophyll fluorescence analysis. In order to gain a comprehensive, pathway-oriented, mechanistic understanding of the effects directly induced by bentazon, we employed 2D-DIGE (two-dimensional difference gel electrophoresis) to analyze the leaf proteome after 8h of bentazon treatment coupled with individual protein identification by MALDI-TOF (Matrix assisted laser desorption/ionization-time of flight) MS/MS. Proteomic analyses revealed that bentazon induced the relative upregulation or downregulation of 30 and 71 proteins (by 1.5-fold or more, p<0.05), respectively. The pathways involved include photosynthesis processes, carbohydrate metabolism, antioxidant systems, and DNA stabilization and protein folding. Protein analysis data revealed that bentazon primarily suppressed photosynthesis processes, and showed inhibitory effects on carbohydrate metabolism and ATP synthesis, whereas several stress response proteins were induced in response to bentazon. Importantly, we identified a 519kD protein containing two histidine kinase-like ATPase domains and a C3HC4 RING type zinc finger domain which may function as a transcript factor to drive expression of detoxification genes such as CYP81A6, leading to bentazon tolerance. This study identifies, for the first time, a candidate transcription factor that could up-regulate CYP81A6 expression, and provides a foundation for further research to advance our knowledge of mechanisms of bentazon resistance in rice.

Characterization of common and distinctive adjustments of wild barley leaf proteome under drought acclimation, heat stress and their combination.

In nature, plants are often exposed to combinations of different stresses at the same time, while in many laboratory studies of molecular stress induction phenomena, single stress responses are analyzed. This study aims to identify the common (i.e. more general stress-responsive) and the stress-specific adjustments of the leaf proteome of wild barley to two often co-occurring stress phenomena, i.e. in response to (long-term) drought acclimation (DA) or to (transient) heat stress (HS). In addition, we analyzed those alterations which are specific for the combination of both stresses. Leaf proteome analysis was performed using 2D difference gel electrophoresis followed by protein identification via mass spectrometry with a 1.5 threshold value of changes in relative protein contents. DA resulted in specific upregulation of proteins with cell detoxification functions, water homeostasis maintenance, amino acids synthesis and lipid metabolism and distinct forms of heat shock proteins (HSPs) and proteins with chaperon functions while proteins related to nitrogen metabolism were downregulated. This response was distinguished from the response to transient HS, which included upregulation of a broad range of HSP products. The common response to both stressors revealed upregulation of additional forms of HSPs and the downregulation of enzymes of the photosynthetic apparatus and chlorophyll binding proteins. The simultaneous exposure to both stress conditions resulted mostly in a combination of both stress responses and to unique abundance changes of proteins with yet unclear functions.

Comparative analysis of barley leaf proteome as affected by drought stress

The adaptive response of Egyptian barley land races to drought stress was analyzed using difference gel electrophoresis (DIGE). Physiological measurements and proteome alterations of accession number 15141, drought tolerant, and accession number 15163, drought sensitive, were compared. Differentially expressed proteins were subjected to MALDI-TOF-MS analysis. Alterations in proteins related to the energy balance and chaperons were the most characteristic features to explain the differences between the drought-tolerant and the drought-sensitive accessions. Further alterations in the levels of proteins involved in metabolism, transcription and protein synthesis are also indicated.

Proteomic analysis of an unsequenced plant — Mangifera indica

Mangifera indica (Mango) is an important fruit crop in tropical countries with India being the leading producer in the world. Substantial research efforts are being devoted to produce fruit that have desirable characteristics including those that pertain to taste, hardiness and resistance to pests. Characterization of the genome and proteome of mango would help in the improvement of cultivars. As the mango genome has not yet been sequenced, we employed a mass spectrometry-based approach followed by database searches of mango-derived ESTs and proteins along with proteins from six other closely related plant species to characterize its proteome. In addition to this, de novo sequencing followed by homology-based protein identification was also carried out. The LC-MS/MS analysis of the mango leaf proteome was performed using an accurate mass quadrupole time-of-flight mass spectrometer. This integrative approach enabled the identification of 1001 peptides that matched to 538 proteins. To our knowledge, this study is the first high-throughput analysis of mango leaf proteome and could pave the way for further genomic, transcriptomic and proteomic studies.

Comparative proteome analysis of high and low cadmium accumulating soybeans under cadmium stress

A comparative proteomic study was performed to unravel the protein networks involved in cadmium stress response in soybean. Ten-day-old seedlings of contrasting cadmium accumulating soybean cultivars-Harosoy (high cadmium accumulator), Fukuyutaka (low cadmium accumulator), and their recombinant inbred line CDH-80 (high cadmium accumulator) were exposed to 100 μM CdCl(2) treatment for 3 days. Root growth was found to be affected under cadmium stress in all. Varietal differences at root protein level were evaluated. NADP-dependent alkenal double bond reductase P1 was found to be more abundant in low cadmium accumulating Fukuyutaka. Leaf proteome analysis revealed that differentially expressed proteins were primarily involved in metabolism and energy production. The results indicate that both high and low cadmium accumulating cultivars and CDH-80 share some common defense strategies to cope with the cadmium stress. High abundance of enzymes involved in glycolysis and TCA cycle might help cadmium challenged cells to produce more energy necessary to meet the high energy demand. Moreover, enhanced expressions of photosynthesis related proteins indicate quick utilization of photoassimilates in energy generation. Increased abundance of glutamine synthetase in all might be involved in phytochelatin mediated detoxification of cadmium ions. In addition, increased abundance of antioxidant enzymes, namely superoxide dismutase, ascorbate peroxidase, catalase, ensures cellular protection from reactive oxygen species mediated damages under cadmium stress. Enhanced expression of molecular chaperones in high cadmium accumulating cultivar might be another additional defense mechanism for refolding of misfolded proteins and to stabilize protein structure and function, thus maintain cellular homeostasis.

Identification and characterisation of differentially expressed leaf proteins among Vitis species

Grapes are commercially grown worldwide for fresh fruit and wine. They are mainly classified into bunch and muscadine grapes. These species differ in their sugar content and composition, photosynthetic efficiency and tolerance to abiotic and biotic stresses. Grape berry relies on carbohydrates produced during photosynthesis to support its development and composition. In view of the unique physiology and genetic make-up of muscadine grape, a proteomics study was performed to increase our knowledge of Vitis leaf proteome in order to improve enological and disease tolerance characteristics of grape species. A high throughput two-dimensional gel electrophoresis (2-DE) was conducted on muscadine, bunch and hybrid bunch leaf proteins. The differentially expressed proteins were excised from 2-DE gels, subjected to in-gel trypsin digestion, and analysed in MALDI/TOF mass spectrometer. The mass spectra were collected and protein identification was performed by searching against Viridiplantae database using Matrix Science algorithm. Proteins were mapped to universal protein resource to study gene ontology. We have discovered >255 proteins with pIs between 3.5 and 8.0 and molecular weight between 12 and 100 kDa among the Vitis species. Comparative analysis of leaf proteome showed that 54 polypeptides varied qualitatively and quantitatively among the three Vitis species studied. Of these, seven proteins were unique to muscadine, two proteins were present in both muscadine and bunch, while 28 proteins were common to all the three species. Bioinformatic analysis of these proteins showed that they are involved in signal transduction pathway, transport of metabolites, energy metabolism, protein trafficking, photosynthesis and defence. We have also identified proteins unique to muscadine grape that are involved in defence and stress tolerance. In addition, photosynthesis-related proteins were found to be more abundant in Vitis vinifera grape compared to other Vitis species.

Comparative proteomic study reveals dynamic proteome changes between superhybrid rice LYP9 and its parents at different developmental stages

Heterosis is a common phenomenon in which the hybrids exhibit superior agronomic performance than either inbred parental lines. Although hybrid rice is one of the most successful apotheoses in crops utilizing heterosis, the molecular mechanisms underlying rice heterosis remain elusive. To gain a better understanding of the molecular mechanisms of rice heterosis, comparative leaf proteomic analysis between a superhybrid rice LYP9 and its parental cultivars 9311 and PA64s at tillering, flowering and grain-filling stages were carried out. A total of 384 differentially expressed proteins (DP) were detected and 297 DP were identified, corresponding to 222 unique proteins. As DP were divided into those between the parents (DPPP) and between the hybrid and its parents (DPHP), the comparative results demonstrate that proteins in the categories of photosynthesis, glycolysis, and disease/defense were mainly enriched in DP. Moreover, the number of identified DPHP involved in photosynthesis, glycolysis, and disease/defense increased at flowering and grain-filling stages as compared to that at the tillering stage. Most of the up-regulated DPHP involved in the three categories showed greater expression in LYP9 at flowering and grain-filling stages than at the tillering stage. In addition, CO2 assimilation rate and apparent quantum yield of photosynthesis also showed a greater increase in LYP9 at flowering and grain-filling stages than at the tillering stage. These results suggest that the proteins involved in photosynthesis, glycolysis, and disease/defense as well as their dynamic regulation at different developmental stages may be responsible for heterosis in rice.

Leaf proteomic analysis in cassava (Manihot esculenta, Crantz) during plant development, from planting of stem cutting to storage root formation

Tuberization in cassava (Manihot esculenta Crantz) occurs simultaneously with plant development, suggesting competition of photoassimilate partitioning between the shoot and the root organs. In potato, which is the most widely studied tuber crop, there is ample evidence suggesting that metabolism and regulatory processes in leaf may have an impact on tuber formation. To search for leaf proteins putatively involved in regulating tuber generation and/or development in cassava, comparative proteomic approaches have been applied to monitor differentially expressed leaf proteins during root transition from fibrous to tuberous. Stringent cross comparison and statistical analysis between two groups with different plant ages using Student's t test with 95% significance level revealed a number of protein spots whose abundance were significantly altered (P<0.05) during week 4 to week 8 of growth. Of these, 39 spots were successfully identified by ion trap LC-MS/MS. The proteins span various functional categories from antioxidant and defense, carbohydrate metabolism, cyanogenesis, energy metabolism, miscellaneous and unknown proteins. Results suggested possible metabolic switches in the leaf that may trigger/regulate storage root initiation and growth. This study provides a basis for further functional characterization of differentially expressed leaf proteins, which can help understand how biochemical processes in cassava leaves may be involved in storage root development.

Evaluation of Protein Extraction Methods for Vitis vinifera Leaf and Root Proteome Analysis by Two‐Dimensional Electrophoresis

An efficient protein extraction method is crucial to ensure successful separation by two-dimensional electrophoresis (2-DE) for recalcitrant plant species, in particular for grapevine (Vitis vinifera L.). Trichloroacetic acid-acetone (TCA-acetone) and phenol extraction methods were evaluated for proteome analysis of leaves and roots from the Tunisian cultivar 'Razegui'. The phenol-based protocol proved to give a higher protein yield, a greater spot resolution, and a minimal streaking on 2-DE gels for both leaf and root tissues compared with the TCA-based protocol. Furthermore, the highest numbers of detected proteins on 2-DE gels were observed using the phenol extraction from leaves and roots as compared with TCA-acetone extraction.

Leaf proteome analysis of eight Populus ×euramericana genotypes: Genetic variation in drought response and in water‐use efficiency involves photosynthesis‐related proteins

Genetic variation of leaf proteome in drought response was investigated among eight Populus xeuramericana genotypes contrasting for their leaf carbon isotope discrimination (Delta), an estimate of intrinsic water-use efficiency. Plants were grown in open field on two similar plots. Drought was induced by an 86-day irrigation cessation on one plot, whereas a second plot remained regularly irrigated. Using 2-DE, 863 reproducible spots were detected; about 60% presented at least one significant effect i.e. treatment, genotype and/or genotype by treatment interaction effect. A significant genotype by treatment interaction was detected for 62 reliably identified proteins among which, about 65% consisted in chloroplast-associated proteins either involved in the Calvin cycle or in the electron-transport chains. The other proteins were involved in oxidative stress, amino acid or protein metabolisms. Correlations between protein abundance and Delta variations were found for 45 reliably identified proteins. The abundance of ribulose-1,5-bisphosphate carboxylase/oxygenase activase isoforms scaled negatively with Delta regardless of the treatment, suggesting that a large intrinsic water-use efficiency could be due to higher abundance of ribulose-1,5-bisphosphate carboxylase/oxygenase activase. Under control condition, abundance of enzymes involved in carbon fixation was also negatively correlated with Delta, whereas abundance of enzymes involved in photorespiration or respiration was positively correlated with Delta.

Proteomics in globe artichoke: Protein extraction and sample complexity reduction by PEG fractionation

Here, we report the first leaf proteome analysis for globe artichoke. Three protein extraction protocols were tested and a reproducible Mg/NP-40-based method was established. Ribulose-1,5-biphosphate carboxylase-oxygenase (RuBisCO) is a highly abundant leaf protein, and its presence masks co-localizing, less abundant proteins. To remove RuBisCO from the sample, and thereby improve spot resolution, a PEG fractionation approach was elaborated. 2-DE profiles of various PEG fractions showed that the fractionation procedure was successful in excluding most of the RuBisCO, allowing for the detection of many low-abundance proteins. Western blot analysis was able to confirm the reduction in RuBisCO content achieved by PEG fractionation. In all, 841 distinct protein spots were detected, and 40 of these, selected from the RuBisCO region of the 2-DE profile, were successfully identified by MS. A number of homologues of these proteins also co-localize with RuBisCO in Arabidopsis thaliana.

Evaluation of three different protocols of protein extraction for Arabidopsis thaliana leaf proteome analysis by two-dimensional electrophoresis

This work was performed to compare three precipitation protocols of protein extraction for 2-DE proteomic analysis using Arabidopsis leaf tissue: TCA-acetone, phenol, and TCA-acetone-phenol. There were no statistically significant differences in protein yield between the three methods. Samples were subjected to 2-DE in the 5 to 8 pH and 14-80 kDa ranges. The TCA-acetone-phenol protocol provided the best results in terms of spot focusing, resolved spots, spot intensity, unique spots detected, and reproducibility. In all, 93 qualitative or quantitative statistically significant differential spots were found between the three protocols. The 2-DE map of TCA-acetone-phenol extracts presented more resolved spots above 40 kDa, with no pI-dependent differences observed between the three protocols. 54 spots were selected for trypsin digestion, and the peptides were analyzed by MALDI-TOF-TOF MS. After database search using peptide mass fingerprinting, and MS/MS combined search, 30 proteins were identified, the proteins from chloroplastic photosynthetic and carbohydrate metabolism being those most highly represented. From these data, we were able to conclude that each extraction protocol had its main features. Considering this, the workflow of any standard comparative proteomic experiment should include the optimization and adaptation of the protein extraction protocol to the plant tissue and to the particular objective pursued.

Leaf proteomic analysis of three rice heritable mutants after seed space flight

To explore the proteomic changes of heritable variant rice plants induced by space environment, three mutants were selected after seed space flight by comparing the phenotypes with their on-ground controls. R955 grew more tillers and became dwarf, 971-5 acquired higher grain yield and better stress resistance, 974-5 matured earlier. Leaf proteins were extracted during the tiller development and analyzed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). More than 300 proteins were detected as reproducible Coomassie Brilliant Blue stained spots with p I values from around 4.0 to 7.0. Five proteins that changed significantly over the controls were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). The main functions of these proteins were photosynthesis, stress defense and metabolism including RuBisCO activase, glycine rich RNA binding protein, peroxidase, triosephosphate isomerase and phosphoenolpyruvate carboxylase, which might be probably associated with the altered phenotypes. Quantitative analyses were also applied: less total protein spots and more down-regulated protein spots were detected in the mutants, indicating there might be a major loss of protein in heritable variant rice plants after seed space flight. These results may provide new insights to understand the biological effects of space environment to rice.

A Proteomics Study of Brassinosteroid Response in Arabidopsis

The plant steroid hormones brassinosteroids (BRs) play an important role in a wide range of developmental and physiological processes. How BR signaling regulates diverse processes remains unclear. To understand the molecular details of BR responses, we performed a proteomics study of BR-regulated proteins in Arabidopsis using two-dimensional DIGE coupled with LC-MS/MS. We identified 42 BR-regulated proteins, which are predicted to play potential roles in BR regulation of specific cellular processes, such as signaling, cytoskeleton rearrangement, vesicle trafficking, and biosynthesis of hormones and vitamins. Analyses of the BR-insensitive mutant bri1-116 and BR-hypersensitive mutant bzr1-1D identified five proteins (PATL1, PATL2, THI1, AtMDAR3, and NADP-ME2) affected both by BR treatment and in the mutants, suggesting their importance in BR action. Selected proteins were further studied using insertion knock-out mutants or immunoblotting. Interestingly about 80% of the BR-responsive proteins were not identified in previous microarray studies, and direct comparison between protein and RNA changes in BR mutants revealed a very weak correlation. RT-PCR analysis of selected genes revealed gene-specific kinetic relationships between RNA and protein responses. Furthermore BR-regulated posttranslational modification of BiP2 protein was detected as spot shifts in two-dimensional DIGE. This study provides novel insights into the molecular networks that link BR signaling to specific cellular and physiological responses.