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Plants have evolved intricate signaling pathways, which operate as networks governed by feedback to deal with stressors. Nevertheless, the sophisticated molecular mechanisms underlying these routes still need to be comprehended, and experimental validation poses significant challenges and expenses. Consequently, computational hypothesis evaluation gains prominence in understanding plant signaling dynamics. Biosensors are genetically modified to emit light when exposed to a particular hormone, such as abscisic acid (ABA), enabling quantification. We developed computational models to simulate the relationship between ABA concentrations and bioluminescent sensors utilizing the Hill equation and ordinary differential equations (ODEs), aiding better hypothesis development regarding plant signaling. Based on simulation results, the luminescence intensity was recorded for a concentration of 47.646 RLUs for 1.5 µmol, given the specified parameters and model assumptions. This method enhances our understanding of plant signaling pathways at the cellular level, offering significant benefits to the scientific community in a cost-effective manner. The alignment of these computational predictions with experimental results emphasizes the robustness of our approach, providing a cost-effective means to validate mathematical models empirically. The research intended to correlate the bioluminescence of biosensors with plant signaling and its mathematical models for quantified detection of specific plant hormone ABA.

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Melia azedarach demonstrates strong salt tolerance and thrives in harsh saline soil conditions, but the underlying mechanisms are poorly understood. In this study, we analyzed gene expression under low, medium, and high salinity conditions to gain a deeper understanding of adaptation mechanisms of M. azedarach under salt stress. The GO (gene ontology) analysis unveiled a prominent trend: as salt stress intensified, a greater number of differentially expressed genes (DEGs) became enriched in categories related to metabolic processes, catalytic activities, and membrane components. Through the analysis of the category GO:0009651 (response to salt stress), we identified four key candidate genes (CBL7, SAPK10, EDL3, and AKT1) that play a pivotal role in salt stress responses. Furthermore, the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis revealed that DEGs were significantly enriched in the plant hormone signaling pathways and starch and sucrose metabolism under both medium and high salt exposure in comparison to low salt conditions. Notably, genes involved in JAZ and MYC2 in the jasmonic acid (JA) metabolic pathway were markedly upregulated in response to high salt stress. This study offers valuable insights into the molecular mechanisms underlying M. azedarach salt tolerance and identifies potential candidate genes for enhancing salt tolerance in M. azedarach.

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In this comprehensive genome-wide study, we identified and classified 83 Xylanase Inhibitor Protein (XIP) genes in wheat, grouped into five distinct categories, to enhance understanding of wheat's resistance to Fusarium head blight (FHB), a significant fungal threat to global wheat production. Our analysis reveals the unique distribution of XIP genes across wheat chromosomes, particularly at terminal regions, suggesting their role in the evolutionary expansion of the gene family. Several XIP genes lack signal peptides, indicating potential alternative secretion pathways that could be pivotal in plant defense against FHB. The study also uncovers the sequence homology between XIPs and chitinases, hinting at a functional diversification within the XIP gene family. Additionally, the research explores the association of XIP genes with plant immune mechanisms, particularly their linkage with plant hormone signaling pathways like abscisic acid and jasmonic acid. XIP-7A3, in particular, demonstrates a significant increase in expression upon FHB infection, highlighting its potential as a key candidate gene for enhancing wheat's resistance to this disease. This research not only enriches our understanding of the XIP gene family in wheat but also provides a foundation for future investigations into their role in developing FHB-resistant wheat cultivars. The findings offer significant implications for wheat genomics and breeding, contributing to the development of more resilient crops against fungal diseases.

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KEY MESSAGE: Yeast extract-induced oxidative stress in Sorbus aucuparia suspension cells leads to the biosynthesis of various hormones, which activates specific signaling pathways that augments biphenyl phytoalexin production. Pathogen incursions pose a significant threat to crop yield and can have a pronounced effect on agricultural productivity and food security. Biphenyl phytoalexins are a specialized group of secondary metabolites that are mainly biosynthesized by Pyrinae plants as a defense mechanism against various pathogens. Despite previous research demonstrating that biphenyl phytoalexin production increased dramatically in Sorbus aucuparia suspension cells (SASCs) treated with yeast extract (YE), the underlying mechanisms remain poorly understood. To address this gap, we conducted an in-depth, multi-omics analysis of transcriptome, proteome, and metabolite (including biphenyl phytoalexins and phytohormones) dynamics in SASCs exposed to YE. Our results indicated that exposure to YE-induced oxidative stress in SASCs, leading to the biosynthesis of a range of hormones, including jasmonic acid (JA), jasmonic acid isoleucine (JA-ILE), gibberellin A4 (GA4), indole-3-carboxylic acid (ICA), and indole-3-acetic acid (IAA). These hormones activated specific signaling pathways that promoted phenylpropanoid biosynthesis and augmented biphenyl phytoalexin production. Moreover, reactive oxygen species (ROS) generated during this process also acted as signaling molecules, amplifying the phenylpropanoid biosynthesis cascade through activation of the mitogen-activated protein kinase (MAPK) pathway. Key genes involved in these signaling pathways included SaBIS1, SaBIS2, SaBIS3, SaPAL, SaB4H, SaOMT, SaUGT1, SaLOX2, SaPR1, SaCHIB1, SaCHIB2 and SaCHIB3. Collectively, this study provided intensive insights into biphenyl phytoalexin accumulation in YE-treated SASCs, which would inform the development of more efficient disease-resistance strategies in economically significant cultivars.

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Kentucky bluegrass (Poa pratensis L.) is an important cool season turfgrass species with a high cold tolerance, but it is sensitive to drought. It is valuable for the applications of Kentucky bluegrass to improve its drought tolerance. However, little is known about the underlying drought mechanism. In the present study, transcriptomic profiling in the roots and leaves of the Kentucky bluegrass cultivar 'Qinghai', in response to osmotic stress in the form of treatment with 2 h and 50 h of 25% (v/v) PEG-6000, was analyzed. The results showed that a large number of genes were significantly up-regulated or down-regulated under osmotic stress. The majority of genes were up-regulated in leaves but down-regulated in roots after 2 h and 50 h of osmotic stress, among them were 350 up-regulated DEGs and 20 down-regulated DEGs shared in both leaves and roots. GO and KEGG analysis showed that carbohydrate metabolism, polyamine and amino acid metabolism and the plant hormone signaling pathway were enriched in the leaves and roots of 'Qinghai' after osmotic stress. The genes involving in carbohydrate metabolism were up-regulated, and sucrose, trehalose and raffinose levels were consistently increased. The genes involved in polyamine and amino acid metabolism were up-regulated in leaves in response to osmotic stress and several amino acids, such as Glu, Met and Val levels were increased, while the genes involved in photosynthesis, carbon fixation and citrate cycle in leaves were down-regulated. In addition, the genes involved in plant hormone biosynthesis and signal transduction were altered in leaves after osmotic stress. This study provided promising candidate genes for studying drought mechanisms in 'Qinghai' and improving the drought tolerance of Kentucky bluegrass and drought-sensitive crops.

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Mango is an important tropical fruit with the reputation of "Tropical Fruit King." It is widely cultivated in tropical and subtropical regions. Mango bacterial leaf spot, which is caused by Xanthomonas critis pv. mangiferaeindicae (Xcm), poses a great threat to the development of mango planting industry. In this study, we used RNA sequencing and data-independent acquisition techniques to compare the transcriptome and proteome of the highly resistant cultivar "Renong No.1" (RN) and the highly susceptible cultivar "Keitt" (KT) in response to Xcm infection at different stages (0, 2, and 6 days). A total of 14,397 differentially expressed genes (DEGs) were identified in the transcriptome of the two varieties, and 4,400 and 8,926 genes were differentially expressed in RN and KT, respectively. Among them, 217 DEGs were related to plant hormone signaling pathway, and 202 were involved in the maintenance of cellular redox homeostasis. A total of 3,438 differentially expressed proteins (DEPs) were identified in the proteome of the two varieties. Exactly 1,542 and 1,700 DEPs were detected in RN and KT, respectively. In addition, 39 DEPs were related to plant hormone signaling pathway, whereas 68 were involved in the maintenance of cellular redox homeostasis. Through cross-validation of the two omics, 1,470 genes were found to be expressed in both groups, and a large number of glutathione metabolism-related genes, such as HSP26-A, G6PD4, and GPX2, were up-regulated in both omics. Peroxisome-related genes, such as LACS6, LACS9, PED1, GLO4, and HACL, were up-regulated or down-regulated in both omics. ABCB11, SAPK2, MYC2, TAG7, PYL1, and other genes related to indole-3-acetic acid and abscisic acid signal transduction and plant-pathogen interaction were up-regulated or down-regulated in both omics. We also used weighted gene co-expression network analysis to combine physiological and biochemical data (superoxide dismutase and catalase activity changes) with transcriptome and proteome data and finally identified three hub genes/proteins (SAG113, SRK2A, and ABCB1) that play an important role in plant hormone signal transduction. This work was the first study of gene/protein changes in resistant and susceptible mango varieties, and its results improved our understanding of the molecular mechanism of mango resistance to Xcm.

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Introduction: P. ternata is a perennial herb of the family Araceae that grows in China and has various medicinal properties and applications. At present, the artificial cultivation of P. ternata is constrained by seedling propagation. To address the problems of low seedling breeding propagation efficiency and high cost, our group has developed a highly efficient cultivation technology for "hydroponic cuttings of P. ternata "for the first time. P. ternata is used as the source material and is grown in a hydroponic system, increasing the seedling production rate 10-fold compared with the traditional cultivation mode. However, the callus formation mechanism in cuttings from hydroponic cultivation is still remains unclear. Methods: In order to better understand the biological process of callus formation in cuttings from hydroponic P. ternata, anatomical characterization, endogenous hormone content determination and transcriptome sequencing were performed on five callus stages from early growth to early senescence. Results: Regarding the four major hormones during the callus developmental stages of P. ternata hydroponic cuttings, cytokinins showed an increasing trend during callus formation. IAA(indole-3-acetic acid) and abscisic acid contents increased at 8d and then decreased, while jasmonic acid content gradually decreased. A total of 254137 unigenes were identified by transcriptome sequencing in five callus formation stages. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the differentially expressed genes (DEGs) that differentially expressed unigenes were involved in various plant hormone signaling and hormone synthesis-related pathways. The expression patterns of 7 genes were validated using quantitative real-time PCR. Discussion: This study presented integrated transcriptomic and metabolic analysis approach to obtain insights into the underlying biosynthetic mechanisms and function of key hormones involved in the callus formation process from hydroponic P. ternata cuttings.

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Partial mycoheterotrophic i.e., mixotrophic, plants are the species which partially depend on mycorrhizal fungi for its nutrients. Although some of these plants are known to show plasticity in the degree of fungal dependence induced by the changes in light condition, the genetic background of this plasticity is largely unsolved. Here, we investigated the relationships between environmental conditions and nutrient sources based on 13C and 15N enrichment in mixotrophic orchid Cymbidium goeringii. We also shaded them for 2 months and evaluated the effect of light condition on the nutrient sources based on the abundance of 13C and 15N and the gene expressions by RNA-seq based de novo assembly. The shading had no effect on isotope enrichment, possibly because of the translocation of carbon and nitrogen from the storage organs. Gene expression analysis showed the upregulation of genes involved in jasmonic acid response in leaves of the shaded plants, which suggests that the jasmonic acid played an important role in regulation of degree of dependence against the mycorrhizal fungi. Our results suggest that mixotrophic plants might be controlling their dependency against the mycorrhizal fungi by a common mechanism with the autotrophic plants.

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The selection and breeding of deep rooting and drought-tolerant varieties has become a promising approach for improving the yield and adaptability of potato (Solanum tuberosum L.) in arid and semiarid areas. Therefore, the discovery of root-development-related genes and drought tolerance signaling pathways in potato is important. In this study, we used deep-rooting (C119) and shallow-rooting (C16) potato genotypes, with different levels of drought tolerance, to achieve this objective. Both genotypes were treated with 150 mM mannitol for 0 h (T0), 2 h (T2), 6 h (T6), 12 h (T12), and 24 h (T24), and their root tissues were subjected to comparative transcriptome analysis. A total of 531, 1571, 1247, and 3540 differentially expressed genes (DEGs) in C16 and 1531, 1108, 674, and 4850 DEGs in C119 were identified in T2 vs. T0, T6 vs. T2, T12 vs. T6, and T24 vs. T12 comparisons, respectively. Gene expression analysis indicated that a delay in the onset of drought-induced transcriptional changes in C16 compared with C119. Functional enrichment analysis revealed genotype-specific biological processes involved in drought stress tolerance. The metabolic pathways of plant hormone transduction and MAPK signaling were heavily involved in the resistance of C16 and C119 to drought, while abscisic acid (ABA), ethylene, and salicylic acid signal transduction pathways likely played more important roles in C119 stress responses. Furthermore, genes involved in root cell elongation and division showed differential expression between the two genotypes under drought stress. Overall, this study provides important information for the marker-assisted selection and breeding of drought-tolerant potato genotypes.

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Catharanthus roseus produces terpenoid indole alkaloids (TIAs) of high medicinal importance. The current research focuses on finding an efficient production system such as cell suspension cultures for high TIA concentrations. Catharanthus roseus cambial meristematic cells (CMCs) offer multiple advantages over dedifferentiated cells (DDCs) regarding growth, homogeneity, and shear resistance. Our lab has established a CMC culture system induced by C. roseus cambium. We determined the concentrations of TIAs in CMCs and DDCs. CMCs produced significantly higher concentrations of total alkaloids, vindoline, vinblastine, catharanthine, and ajmalicine as compared to DDCs. We then performed Illumina HiSeq transcriptome sequencing of CMCs and DDCs and explored the differential transcriptomic signatures. Of the 96,004 unigenes, 9,564 were differentially expressed between the 2 cell suspension types. These differentially expressed genes (DEGs) were enriched in 137 KEGG pathways. Most importantly, genes from the indole alkaloid biosynthesis and the upstream pathways i.e., tryptophan metabolism, monoterpenoid biosynthesis, tropane, piperidine, and pyridine alkaloid biosynthesis, and terpenoid backbone biosynthesis showed differential transcriptomic signatures. Remarkably, the expression of genes associated with plant hormone biosynthesis, signaling, and MAPK signaling pathways was relatable to the different TIA concentrations in CMCs and DDCs. These results put forward multiple target genes, transcription factors, and regulators to develop a large-scale TIA production system using C. roseus CMCs.

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Oxidation of membrane lipids by reactive oxygen species (ROS) or O2/lipoxygenase leads to the formation of various bioactive compounds collectively called oxylipins. Reactive carbonyl species (RCS) are a group of oxylipins that have the α,ß-unsaturated carbonyl structure, including acrolein and 4-hydroxy-(E)-2-nonenal. RCS provides a missing link between ROS stimuli and cellular responses in plants via their electrophilic modification of proteins. The physiological significance of RCS in plants has been established based on the observations that the RCS-scavenging enzymes that are overexpressed in plants or the RCS-scavenging chemicals added to plants suppress the plants' responses to ROS, i.e., photoinhibition, aluminum-induced root damage, programmed cell death (PCD), senescence, abscisic acid-induced stomata closure, and auxin-induced lateral root formation. The functions of RCS are thus a key to ROS- and redox-signaling in plants. The chemical species involved in distinct RCS signaling/damaging phenomena were recently revealed, based on comprehensive carbonyl determinations. This review presents an overview of the current status of research regarding RCS signaling functions in plants and discusses present challenges for gaining a more complete understanding of the signaling mechanisms.

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Chinese cork oak (Quercus variabilis) is a widely distributed and highly valuable deciduous broadleaf tree from both ecological and economic perspectives. Seeds of this species are recalcitrant, i.e., sensitive to desiccation, which affects their storage and long-term preservation of germplasm. However, little is known about the underlying molecular mechanism of desiccation sensitivity of Q. variabilis seeds. In this study, the seeds were desiccated with silica gel for certain days as different treatments from 0 (Control) to 15 days (T15) with a gradient of 1 day. According to the seed germination percentage, four key stages (Control, T2, T4, and T11) were found. Then the transcriptomic profiles of these four stages were compared. A total of 4,405, 4,441, and 5,907 differentially expressed genes (DEGs) were identified in T2 vs. Control, T4 vs. Control, and T11 vs. Control, respectively. Among them, 2,219 DEGs were overlapped in the three comparison groups. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that these DEGs were enriched into 124 pathways, such as "Plant hormone signal transduction" and "Glycerophospholipid metabolism". DEGs related to hormone biosynthesis and signal transduction (ZEP, YUC, PYR, ABI5, ERF1B, etc.), stress response proteins (LEA D-29, HSP70, etc.), and phospholipase D (PLD1) were detected during desiccation. These genes and their interactions may determine the desiccation sensitivity of seeds. In addition, group specific DEGs were also identified in T2 vs. Control (PP2C62, UNE12, etc.), T4 vs. Control (WRKY1-like, WAK10, etc.), and T11 vs. Control (IBH1, bZIP44, etc.), respectively. Finally, a possible work model was proposed to show the molecular regulation mechanism of desiccation sensitivity in Q. variabilis seeds. This is the first report on the molecular regulation mechanism of desiccation sensitivity of Q. variabilis seeds using RNA-Seq. The findings could make a great contribution to seed storage and long-term conservation of recalcitrant seeds in the future.

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BACKGROUND: Activated charcoal (AC) is highly adsorbent and is often used to promote seedling growth in plant tissue culture; however, the underlying molecular mechanism remains unclear. In this study, root and leaf tissues of 10-day-old seedlings grown via immature embryo culture in the presence or absence of AC in the culture medium were subjected to global transcriptome analysis by RNA sequencing to provide insights into the effects of AC on seedling growth. RESULTS: In total, we identified 18,555 differentially expressed genes (DEGs). Of these, 11,182 were detected in the roots and 7373 in the leaves. In seedlings grown in the presence of AC, 9460 DEGs were upregulated and 7483 DEGs were downregulated in the presence of AC as compared to the control. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed 254 DEG-enriched pathways, 226 of which were common between roots and leaves. Further analysis of the major metabolic pathways revealed that AC stimulated the expression of nine genes in the phenylpropanoid biosynthesis pathway, including PLA, CYP73A, COMT, CYP84A, and 4CL, the protein products of which promote cell differentiation and seedling growth. Further, AC upregulated genes involved in plant hormone signaling related to stress resistance and disease resistance, including EIN3, BZR1, JAR1, JAZ, and PR1, and downregulated genes related to plant growth inhibition, including BKI1, ARR-B, DELLA, and ABF. CONCLUSIONS: Growth medium containing AC promotes seedling growth by increasing the expression of certain genes in the phenylpropanoid biosynthesis pathway, which are related to cell differentiation and seedling growth, as well as genes involved in plant hormone signaling, which is related to resistance.

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Blue mold, caused by Penicillium expansum, is an important postharvest disease of apple, and can result in significant economic losses. The present study investigated the interaction between P. expansum and wounded apple fruit tissues during the early stages of the infection. Spores of P. expansum became activated one hour post-inoculation (hpi), exhibited swelling at 3 hpi, and the germ tubes were found entering into apple tissues at 6 hpi. RNA-seq was performed on samples of P. expansum and apple fruit tissue collected at 1, 3, and 6 hpi. The main differentially expressed genes (DEGs) that were identified in P. expansum were related to interaction, cell wall degradation enzymes, anti-oxidative stress, pH regulation, and effectors. Apple tissues responded to the presence of P. expansum by activating pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) at 1 hpi, then activated effector-triggered immunity (ETI) at 3 hpi. This research provides new information on the interaction between P. expansum and apple fruit tissue at an early stage of the infection process.

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Reactive oxygen species (ROS) and plant hormones play important roles in regulating plant growth and stress responses as signaling molecules. Abscisic acid (ABA) is known as the key regulator of both abiotic and biotic stress responses. During stress responses, ABA is known to regulate ROS production, indicating that important crosstalk occurs between ROS and ABA signaling. We recently reported that MYB30, an MYB-type transcription factor, regulates root cell elongation under ROS signaling. In this study, we analyzed the molecular interaction between ROS and ABA signal during for root development, which is mediated through MYB30 transcriptional regulation. We showed that MYB30-regulated root cell elongation was mediated by ROS production under ABA signaling. Our findings will provide one piece of evidence of the complex cross talk between ROS and hormone signaling that regulates root development.

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Fruits have been traditionally classified into two categories based on their capacity to produce and respond to ethylene during ripening. Fruits whose ripening is associated to a peak of ethylene production and a respiration burst are referred to as climacteric, while those that are not are referred to as non-climacteric. However, an increasing body of literature supports an important role for ethylene in the ripening of both climacteric and non-climacteric fruits. Genome and transcriptomic data have become available across a variety of fruits and we leverage these data to compare the structure and transcriptional regulation of the ethylene receptors and related proteins. Through the analysis of four economically important fruits, two climacteric (tomato and apple), and two non-climacteric (grape and citrus), this review compares the structure and transcriptional regulation of the ethylene receptors and related proteins in both types of fruit, establishing a basis for the annotation of ethylene-related genes. This analysis reveals two interesting differences between climacteric and non-climacteric fruit: i) a higher number of ETR genes are found in climacteric fruits, and ii) non-climacteric fruits are characterized by an earlier ETR expression peak relative to sugar accumulation.

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Phytohormones tightly regulate plant growth by integrating changing environmental and developmental cues. Although the key players have been identified in many plant hormonal pathways, the molecular mechanisms and mode of action of perception and signaling remain incompletely resolved. Characterization of protein partners of known signaling components provides insight into the formed protein complexes, but, unless quantification is involved, does not deliver much, if any, information about the dynamics of the induced or disrupted protein complexes. Therefore, in proteomics research, the discovery of what actually triggers, regulates or interrupts the composition of protein complexes is gaining importance. Here, tandem affinity purification coupled to mass spectrometry (TAP-MS) is combined with label-free quantification (LFQ) to a highly valuable tool to detect physiologically relevant, dynamic protein-protein interactions in Arabidopsis thaliana cell cultures. To demonstrate its potential, we focus on the signaling pathway of one of the most recently discovered phytohormones, strigolactones.

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C-repeat binding factors (CBF) are a subfamily of AP2 transcription factors that play critical roles in the regulation of plant cold tolerance and growth in low temperature. In the present work, we sought to perform a detailed investigation into global transcriptional regulation of plant hormone signaling associated genes in transgenic plants engineered with CBF genes. RNA samples from Arabidopsis thaliana plants overexpressing two CBF genes, CBF2 and CBF3, were subjected to Illumina HiSeq 2000 RNA sequencing (RNA-Seq). Our results showed that more than half of the hormone associated genes that were differentially expressed in CBF2 or CBF3 transgenic plants were related to auxin signal transduction and metabolism. Most of these alterations in gene expression could lead to repression of auxin signaling. Accordingly, the IAA content was significantly decreased in young tissues of plants overexpressing CBF2 and CBF3 compared with wild type. In addition, genes associated with the biosynthesis of Jasmonate (JA) and Salicylic acid (SA), as well as the signal sensing of Brassinolide (BR) and SA, were down-regulated, while genes associated with Gibberellin (GA) deactivation were up-regulated. In general, overexpression of CBF2 and CBF3 negatively affects multiple plant hormone signaling pathways in Arabidopsis. The transcriptome analysis using CBF2 and CBF3 transgenic plants provides novel and integrated insights into the interaction between CBFs and plant hormones, particularly the modulation of auxin signaling, which may contribute to the improvement of crop yields under abiotic stress via molecular engineering using CBF genes.

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Vascular phloem loading has long been recognized as an essential step in the establishment of a systemic virus infection. In this study, an interaction between the replication protein of tobacco mosaic virus (TMV) and phloem-specific auxin/indole acetic acid (Aux/IAA) transcriptional regulators was found to modulate virus phloem loading in an age-dependent manner. Promoter expression studies show that in mature tissues TMV 126/183-kDa-interacting Aux/IAAs predominantly express and accumulate within the nuclei of phloem companion cells (CCs). Furthermore, CC Aux/IAA nuclear localization is disrupted upon infection with an interacting virus. In situ analysis of virus spread shows that the inability to disrupt Aux/IAA CC nuclear localization correlates with a reduced ability to load into the vascular tissue. Subsequent systemic movement assays also demonstrate that a virus capable of disrupting Aux/IAA localization is significantly more competitive at moving out of older plant tissues than a noninteracting virus. Similarly, CC expression and overaccumulation of a degradation-resistant Aux/IAA-interacting protein was found to inhibit TMV accumulation and phloem loading selectively in flowering plants. Transcriptional expression studies demonstrate a role for Aux/IAA-interacting proteins in the regulation of salicylic and jasmonic acid host defense responses as well as virus-specific movement factors, including pectin methylesterase, that are involved in regulating plasmodesmata size-exclusion limits and promoting virus cell-to-cell movement. Combined, these findings indicate that TMV directs the reprogramming of auxin-regulated gene expression within the vascular phloem of mature tissues as a means to enhance phloem loading and systemic spread.

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Eukaryotic two-component signaling involves the His-Asp-His-Asp multistep phosphorelay (MSP). In Arabidopsis thaliana, cytokinin-mediated MSP signaling intermediates include histidine kinases (HKs), histidine phosphotransfer proteins (Hpts) and response regulators (RRs). The structure-function relationship of interaction between Hpt (e.g. AHP1) and RR (e.g. ARR4) is poorly understood. Using a homology model and yeast two-hybrid analysis, we identified key amino acids of ARR4 at the AHP1-ΔARR4((16-175)) interaction interface. Mutating them in Arabidopsis (arr3,4,5,6,8,9 hextuple mutant background) and performing root length assays provided functional relevance, and coimmunoprecipitation (coIP) assay provided biochemical evidence for the interaction. The homology model mimics crystal structures of Hpt-RR complexes. Mutating selected interface residues of ARR4 either abolished or destabilized the interaction. D45A and Y96A mutations weakened interaction with AHP1, and exhibited weaker rescue of root elongation in the hextuple mutants. CoIP analysis using cytokinin-treated transgenic Arabidopsis seedlings provided biochemical evidence for weakened AHP1-ARR4 interaction. The relevance of the selected residues for the interaction was further validated in two independent pairs of Hpt-RR proteins from Arabidopsis and rice (Oryza sativa). Our data provide evidence of a link between Hpt-RR interaction affinity and regulation of downstream functions of RRs. This establishes a structure-function relationship for the final step of a eukaryotic MSP signal cascade.

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