Cytokinin Signaling Research Articles

Phytochromes and Their Role in Diurnal Variations of ROS Metabolism and Plant Proteome.

Plants are sessile organisms forced to adapt to environmental variations recurring in a day-night cycle. Extensive research has uncovered the transcriptional control of plants' inner clock and has revealed at least some part of the intricate and elaborate regulatory mechanisms that govern plant diel responses and provide adaptation to the ever-changing environment. Here, we analyzed the proteome of the Arabidopsis thaliana mutant genotypes collected in the middle of the day and the middle of the night, including four mutants in the phytochrome (phyA, phyB, phyC, and phyD) and the circadian clock protein LHY. Our approach provided a novel insight into the diel regulations, identifying 640 significant changes in the night-day protein abundance. The comparison with previous studies confirmed that a large portion of identified proteins was a known target of diurnal regulation. However, more than 300 were novel oscillations hidden under standard growth chamber conditions or not manifested in the wild type. Our results indicated a prominent role for ROS metabolism and phytohormone cytokinin in the observed regulations, and the consecutive analyses confirmed that. The cytokinin signaling significantly increased at night, and in the mutants, the hydrogen peroxide content was lower, and the night-day variation seemed to be lost in the phyD genotype. Furthermore, regulations in the lhy and phyB mutants were partially similar to those found in the catalase mutant cat2, indicating shared ROS-mediated signaling pathways. Our data also shed light on the role of the relatively poorly characterized Phytochrome D, pointing to its connection to glutathione metabolism and the regulation of glutathione S-transferases.

Comparing adventitious root-formation and graft-unification abilities in clones of Argania spinosa.

Argania spinosa trees have attracted attention in recent years due to their high resistance to extreme climate conditions. Initial domestication activities practiced in Morocco. Here we report on selection and vegetative propagation of A. spinosa trees grown in Israel. Trees yielding relatively high amounts of fruit were propagated by rooting of stem cuttings. High variability in rooting ability was found among the 30 clones selected. In-depth comparison of a difficult-to-root (ARS7) and easy-to-root (ARS1) clone revealed that the rooted cuttings of ARS7 have a lower survival rate than those of ARS1. In addition, histological analysis of the adventitious root primordia showed many abnormal fused primordia in ARS7. Hormone profiling revealed that while ARS1 accumulates more cytokinin, ARS7 accumulates more auxin, suggesting different auxin-to-cytokinin ratios underlying the different rooting capabilities. The hypothesized relationship between rooting and grafting abilities was addressed. Reciprocal grafting was performed with ARS1/ARS7 but no significant differences in the success of graft unification between the trees was detected. Accordingly, comparative RNA sequencing of the rooting and grafting zones showed more differentially expressed genes related to rooting than to grafting between the two trees. Clustering, KEGG and Venn analyses confirmed enrichment of genes related to auxin metabolism, transport and signaling, cytokinin metabolism and signaling, cell wall modification and cell division in both regions. In addition, the differential expression of some key genes in ARS1 vs. ARS7 rooting zones was revealed. Taken together, while both adventitious root-formation and graft-unification processes share response to wounding, cell reprogramming, cell division, cell differentiation and reconnection of the vasculature, there are similar, but also many different genes regulating the two processes. Therefore an individual genotype can have low rooting capacity but good graft-unification ability.

The Plant Growth Regulator 14-OH BR Can Minimize the Application Content of CPPU in Kiwifruit (Actinidia chinensis) ‘Donghong’ and Increase Postharvest Time without Sacrificing the Yield

The application of the plant growth regulator 1-(2-chloro-4-pyridyl)-3-phenylurea (CPPU) is extensively used for red-fleshed kiwifruits or ‘Donghong’, but it has toxicological properties. Extra plant growth regulators (PGRs) were screened for partial substitution of CPPU (10 mg L−1) to the crops to minimize the CPPU content. The results showed that CPPU at a concentration of 5 mg L−1 plus 14-hydroxylated brassinosteroid (14-OH BR) at a concentration of 0.15 mg L−1 has a nearly equal effect to CPPU at a concentration of 10 mg L−1; it maintains the kiwifruit yields and quality as well as increases the postharvest time. Transcriptome sequencing data revealed that the regulation of 14-OH BR on kiwifruit growth acts mainly by activating Brassinosteroid (BR) signaling to synergistically and antagonistically stimulate the signaling of other endogenous growth regulators, including auxin (IAA), abscisic acid (ABA), cytokinin (CK), gibberellin (GA), jasmonic acid (JA) and ethylene (ET).

Transcriptome Analysis Reveals a Comprehensive Virus Resistance Response Mechanism in Pecan Infected by a Novel Badnavirus Pecan Virus.

Pecan leaf-variegated plant, which was infected with a novel badnavirus named pecan mosaic virus (PMV) detected by small RNA deep sequencing, is a vital model plant for studying the molecular mechanism of retaining green or chlorosis of virus-infected leaves. In this report, PMV infection in pecan leaves induced PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI). PMV infection suppressed the expressions of key genes of fatty acid, oleic acid (C18:1), and very-long-chain fatty acids (VLCFA) biosynthesis, indicating that fatty acids-derived signaling was one of the important defense pathways in response to PMV infection in pecan. PMV infection in pecans enhanced the expressions of pathogenesis-related protein 1 (PR1). However, the transcripts of phenylalanine ammonia-lyase (PAL) and isochorismate synthase (ICS) were downregulated, indicating that salicylic acid (SA) biosynthesis was blocked in pecan infected with PMV. Meanwhile, disruption of auxin signaling affected the activation of the jasmonic acid (JA) pathway. Thus, C18:1 and JA signals are involved in response to PMV infection in pecan. In PMV-infected yellow leaves, damaged chloroplast structure and activation of mitogen-activated protein kinase 3 (MPK3) inhibited photosynthesis. Cytokinin and SA biosynthesis was blocked, leading to plants losing immune responses and systemic acquired resistance (SAR). The repression of photosynthesis and the induction of sink metabolism in the infected tissue led to dramatic changes in carbohydrate partitioning. On the contrary, the green leaves of PMV infection in pecan plants had whole cell tissue structure and chloroplast clustering, establishing a strong antiviral immunity system. Cytokinin biosynthesis and signaling transductions were remarkably strengthened, activating plant immune responses. Meanwhile, cytokinin accumulation in green leaves induced partial SA biosynthesis and gained comparatively higher SAR compared to that of yellow leaves. Disturbance of the ribosome biogenesis might enhance the resistance to PMV infection in pecan and lead to leaves staying green.

Cytokinin signaling regulates two-stage inflorescence arrest in Arabidopsis.

To maximize reproductive success, flowering plants must correctly time entry and exit from the reproductive phase. While much is known about mechanisms that regulate initiation of flowering, end-of-flowering remains largely uncharacterized. End-of-flowering in Arabidopsis (Arabidopsis thaliana) consists of quasi-synchronous arrest of inflorescences, but it is unclear how arrest is correctly timed with respect to environmental stimuli and reproductive success. Here, we showed that Arabidopsis inflorescence arrest is a complex developmental phenomenon, which includes the arrest of the inflorescence meristem (IM), coupled with a separable "floral arrest" of all unopened floral primordia; these events occur well before visible inflorescence arrest. We showed that global inflorescence removal delays both IM and floral arrest, but that local fruit removal only delays floral arrest, emphasizing their separability. We tested whether cytokinin regulates inflorescence arrest, and found that cytokinin signaling dynamics mirror IM activity, while cytokinin treatment can delay both IM and floral arrest. We further showed that gain-of-function cytokinin receptor mutants can delay IM and floral arrest; conversely, loss-of-function mutants prevented the extension of flowering in response to inflorescence removal. Collectively, our data suggest that the dilution of cytokinin among an increasing number of sink organs leads to end-of-flowering in Arabidopsis by triggering IM and floral arrest.

Identification of MicroRNAs Related to Phytohormone Signal Transduction and Self-incompatibility of Rabbiteye Blueberry Pollen

MicroRNAs (miRNAs) related to phytohormone signal transduction and self-incompatibility may play an important role in the xenia effect. However, associated research in this area is still lacking in rabbiteye blueberry (Vaccinium ashei). In this study, we identified miRNAs, predicted their target genes, performed functional enrichment analysis of the target genes, and screened for miRNAs related to phytohormone signaling and self-incompatibility. A total of 491 miRNAs were identified, of which 27 and 67 known miRNAs as well as 274 and 416 new miRNAs were found in the rabbiteye blueberry cultivars Brightwell and Premier, respectively. Compared with ‘Premier’, 31 miRNAs were upregulated and 62 miRNAs were downregulated in ‘Brightwell’. Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis indicated that the 4985 target genes predicted were involved in biosynthesis of amino acids, plant–pathogen interaction, and spliceosome pathways. A total of 10, one, one, five, two, five, and two candidate miRNAs related to auxin, cytokinin, gibberellin, abscisic acid, ethylene, brassinosteroid, and salicylic acid signaling, respectively, in rabbiteye blueberry pollen were identified. Further analysis indicated that novel_miR_49 was a candidate miRNA related to self-incompatibility, and their target gene was maker-VaccDscaff21-snap-gene-21.37. In addition, the KEGG enrichment analysis of the target genes of novel_miR_49 showed that they were involved in the ribosome, aminoacyl-tRNA biosynthesis, and glycosylphosphatidylinositol-anchor biosynthesis pathways. The results revealed that the microRNAs of rabbiteye blueberry pollen regulated to phytohormone signal transduction and self-incompatibility signal transduction based on related to auxin, cytokinin, gibberellin, abscisic acid, ethylene, brassinosteroid, and salicylic acid signaling. Results suggest that more research of the effects of miRNAs on regulation of hormone signal transduction and self-incompatibility is necessary for elucidating the molecular mechanism of the xenia effect.

Tissue-specific transcriptome responses to Fusarium head blight and Fusarium root rot.

Fusarium head blight (FHB) and Fusarium root rot (FRR) are important diseases of small-grain cereals caused by Fusarium species. While host response to FHB has been subject to extensive study, very little is known about response to FRR and the transcriptome responses of FHB and FRR have not been thoroughly compared. Brachypodium distachyon (Bd) is an effective model for investigating host responses to both FHB and FRR. In this study the transcriptome response of Bd to F. graminearum (Fg) infection of heads and roots was investigated. An RNA-seq analysis was performed on both Bd FHB and FRR during the early infection. Additionally, an RNA-seq analysis was performed on in vitro samples of Fg for comparison with Fg gene expression in planta. Differential gene expression and gene-list enrichment analyses were used to compare FHB and FRR transcriptome responses in both Bd and Fg. Differential expression of selected genes was confirmed using RT-qPCR. Most genes associated with receptor signalling, cell-wall modification, oxidative stress metabolism, and cytokinin and auxin biosynthesis and signalling genes were generally upregulated in FHB or were downregulated in FRR. In contrast, Bd genes involved in jasmonic acid and ethylene biosynthesis and signalling, and antimicrobial production were similarly differentially expressed in both tissues in response to infection. A transcriptome analysis of predicted Fg effectors with the same infected material revealed elevated expression of core tissue-independent genes including cell-wall degradation enzymes and the gene cluster for DON production but also several tissue-dependent genes including those for aurofusarin production and cutin degradation. This evidence suggests that Fg modulates its transcriptome to different tissues of the same host.

From synthesis to the biological effect of isoprenoid 2′-deoxyriboside and 2′,3′-dideoxyriboside cytokinin analogues

Isoprenoid cytokinins are a class of naturally occurring plant signaling molecules. A series of prepared compounds derived from isoprenoid cytokinins (isopentenyladenine, trans-zeatin and cis-zeatin) with attached 2′-deoxy-d-ribose or 2′,3′-dideoxy-d-ribose at the N9 position of the purine were prepared and their biological activities were examined. Different synthetic approaches were employed. The final compounds were characterized with variety of physicochemical methods (TLC, HPLC-MS, and NMR) and their cytokinin activity was determined in classical bioassays such as Amaranthus, tobacco callus, detached wheat leaf senescence and Arabidopsis thaliana root elongation inhibition assay. In addition, compounds were screened for activation of the cytokinin signaling pathway (bacterial receptor, competitive ligand binding and ARR5::GUS assay) to provide a detailed assessment of CK structure-activity relationship. The prepared compounds were found to be non-toxic to human cells and the majority of assays exhibited the highest activity of free bases while 2′,3′-dideoxyribosides had very weak or no activity. In contrast to the free bases, all 2′-deoxyriboside derivatives were not toxic to tobacco callus even at the highest tested concentration (10−4 moL/l) and compound 1 (iPdR) induced betacyanin synthesis at higher concentration even stronger than iP free base in the Amaranthus bioassay. The general cytokinin activity pattern base > riboside >2′-deoxyriboside > 2′,3′-dideoxyriboside was distinguished.

Cytokinin modulates the metabolic network of sulfur and glutathione.

The phytohormone cytokinin is implicated in a range of growth, developmental, and defense processes. A growing body of evidence supports a crosstalk between cytokinin and nutrient signaling pathways, such as nitrate availability. Cytokinin signaling regulates sulfur-responsive gene expression, but the underlying molecular mechanisms and their impact on sulfur-containing metabolites have not been systematically explored. Using a combination of genetic and pharmacological tools, we investigated the interplay between cytokinin signaling and sulfur homeostasis. Exogenous cytokinin triggered sulfur starvation-like gene expression accompanied by a decrease in sulfate and glutathione content. This process was uncoupled from the activity of the major transcriptional regulator of sulfate starvation signaling SULFUR LIMITATION 1 and an important glutathione-degrading enzyme, γ-glutamyl cyclotransferase 2;1, expression of which was robustly up-regulated by cytokinin. Conversely, glutathione accumulation was observed in mutants lacking the cytokinin receptor ARABIDOPSIS HISTIDINE KINASE 3 and in cytokinin-deficient plants. Cytokinin-deficient plants displayed improved root growth upon exposure to glutathione-depleting chemicals which was attributed to a higher capacity to maintain glutathione levels. These results shed new light on the interplay between cytokinin signaling and sulfur homeostasis. They position cytokinin as an important modulator of sulfur uptake, assimilation, and remobilization in plant defense against xenobiotics and root growth.

Ectopic expression of WOX5 promotes cytokinin signaling and de novo shoot regeneration.

WOX5 has a potential in activating cytokinin signaling and shoot regeneration, in addition to its role in pluripotency acquisition. Thus, overexpression of WOX5 maximizes plant regeneration capacity during tissue culture. In vitro plant regeneration involves two steps: callus formation and de novo shoot organogenesis. The WUSCHEL-RELATED HOMEOBOX 5 (WOX5) homeodomain transcription factor is known to be mainly expressed during incubation on callus-inducing medium (CIM) and involved in pluripotency acquisition in callus, but whether WOX5 also affects de novo shoot regeneration on cytokinin-rich shoot-inducing medium (SIM) remains unknown. Based on the recent finding that WOX5 promotes cytokinin signaling, we hypothesized that ectopic expression of WOX5 beyond CIM would further enhance overall plant regeneration capacity, because intense cytokinin signaling is particularly required for shoot regeneration on SIM. Here, we found that overexpression of the WOX5 gene on SIM drastically promoted de novo shoot regeneration from callus with the repression of type-A ARABIDOPSIS RESPONSE REGULATOR (ARR) genes, negative regulators of cytokinin signaling. The enhanced shoot regeneration phenotypes were indeed dependent on cytokinin signaling, which were partially suppressed in the progeny derived from crossing WOX5-overexpressing plants with cytokinin-insensitive 35S:ARR7 plants. The function of WOX5 in enhancing cytokinin-dependent shoot regeneration is evolutionarily conserved, as conditional overexpression of OsWOX5 on SIM profoundly enhanced shoot regeneration in rice callus. Overall, our results provide the technical advance that maximizes in vitro plant regeneration by constitutively expressing WOX5, which unequivocally promotes both callus pluripotency and de novo shoot regeneration.

The RAC/ROP GTPase activator OsRopGEF10 functions in crown root development by regulating cytokinin signaling in rice.

RAC/Rho of plant (ROP) GTPases are major molecular switches that control diverse signaling cascades for plant growth, development, and defense. Here, we discovered a signaling node that connects RAC/ROPs to cytokinins. Rice (Oryza sativa) plants develop a fibrous root system mainly composed of crown roots. Cytokinin signaling via a phosphorelay system is critical for crown root development. We show that OsRopGEF10, which activates RAC/ROPs, acts upstream of the cytoplasmic-nuclear shuttling phosphotransfer proteins AHPs of the cytokinin signaling pathway to promote crown root development. Mutations of OsRopGEF10 induced hypersensitivity to cytokinin, whereas overexpressing this gene reduced the cytokinin response. Loss of OsRopGEF10 function reduced the expression of the response regulator gene OsRR6, a repressor of cytokinin signaling, and impaired crown root development. Mutations in OsAHP1/2 led to increased crown root production and rescued the crown root defect of Osropgef10. Furthermore, auxin activates the ROP GTPase OsRAC3, which attenuates cytokinin signaling for crown root initiation. Molecular interactions between OsRopGEF10, OsRAC3, and OsAHP1/2 implicate a mechanism whereby OsRopGEF10-activated OsRAC3 recruits OsAHP1/2 to the cortical cytoplasm, sequestering them from their phosphorelay function in the nucleus. Together, our findings uncover the OsRopGEF10-OsRAC3-OsAHP1/2 signaling module, establish a link between RAC/ROPs and cytokinin, and reveal molecular crosstalk between auxin and cytokinin during crown root development.

Anthocyanin Biosynthesis Induced by MYB Transcription Factors in Plants.

Anthocyanins act as polyphenolic pigment that is ubiquitously found in plants. Anthocyanins play a role not only in health-promoting as an antioxidant, but also in protection against all kinds of abiotic and biotic stresses. Most recent studies have found that MYB transcription factors (MYB TFs) could positively or negatively regulate anthocyanin biosynthesis. Understanding the roles of MYB TFs is essential in elucidating how MYB TFs regulate the accumulation of anthocyanin. In the review, we summarized the signaling pathways medicated by MYB TFs during anthocyanin biosynthesis including jasmonic acid (JA) signaling pathway, cytokinins (CKs) signaling pathway, temperature-induced, light signal, 26S proteasome pathway, NAC TFs, and bHLH TFs. Moreover, structural and regulator genes induced by MYB TFs, target genes bound and activated or suppressed by MYB TFs, and crosstalk between MYB TFs and other proteins, were found to be vitally important in the regulation of anthocyanin biosynthesis. In this study, we focus on the recent knowledge concerning the regulator signaling and mechanism of MYB TFs on anthocyanin biosynthesis, covering the signaling pathway, genes expression, and target genes and protein expression.

Nodule INception-independent epidermal events lead to bacterial entry during nodule development in peanut (Arachis hypogaea).

Legumes can host nitrogen-fixing rhizobia inside root nodules. In model legumes, rhizobia enter via infection threads (ITs) and develop nodules in which the infection zone contains a mixture of infected and uninfected cells. Peanut (Arachis hypogaea) diversified from model legumes c. 50-55 million years ago. Rhizobia enter through 'cracks' to form nodules in peanut roots where cells of the infection zone are uniformly infected. Phylogenomic studies have indicated symbiosis as a labile trait in peanut. These atypical features prompted us to investigate the molecular mechanism of peanut nodule development. Combining cell biology, genetics and genomic tools, we visualized the status of hormonal signaling in peanut nodule primordia. Moreover, we dissected the signaling modules of Nodule INception (NIN), a master regulator of both epidermal infection and cortical organogenesis. Cytokinin signaling operates in a broad zone, from the epidermis to the pericycle inside nodule primordia, while auxin signaling is narrower and focused. Nodule INception is involved in nodule organogenesis, but not in crack entry. Nodulation Pectate Lyase, which remodels cell walls during IT formation, is not required. By contrast, Nodule enhanced Glycosyl Hydrolases (AhNGHs) are recruited for cell wall modification during crack entry. While hormonal regulation is conserved, the function of the NIN signaling modules is diversified in peanut.

Genome-Wide Identification and Expression Analysis of Cytokinin Response Regulator (RR) Genes in the Woody Plant Jatropha curcas and Functional Analysis of JcRR12 in Arabidopsis.

The cytokinin (CK) response regulator (RR) gene family plays a pivotal role in regulating the developmental and environmental responses of plants. Axillary bud outgrowth in the perennial woody plant Jatropha curcas is regulated by the crosstalk between CK and gibberellins (GA). In this study, we first analyzed the effects of gibberellin A3 (GA3), lovastatin (a CK synthesis inhibitor), decapitation, and their interaction, on the outgrowth of axillary buds. The results indicate that lovastatin completely inhibited GA-promoted axillary bud outgrowth and partially weakened the decapitation-promoted axillary bud outgrowth. To further characterize and understand the role of CK signaling in promoting the development of female flowers and branches, we performed bioinformatics and expression analyses to characterize the CK RR gene (JcRR) family in J. curcas. A total of 14 members of the JcRR family were identified; these genes were distributed on 10 chromosomes. Phylogenetic analysis indicated that the corresponding RR proteins are evolutionarily conserved across different plant species, and the Myb-like DNA-binding domain divides the 14 members of the JcRR family into type-A and type-B proteins. Further analysis of cis-acting elements in the promoter regions of JcRRs suggests that JcRRs are expressed in response to phytohormones, light, and abiotic stress factors; thus, JcRRs may be involved in some plant development processes. Genomic sequence comparison revealed that segmental duplication may have played crucial roles in the expansion of the JcRR gene family, and five pairs of duplicated genes were all subjected to purifying selection. By analyzing RNA sequencing (RNA-seq) and quantitative reverse transcription-polymerase chain reaction (qRT–PCR) data, we characterized that the temporospatial expression patterns of JcRRs during the development of various tissues and the response of these genes to phytohormones and abiotic stress. The JcRRs were mainly expressed in the roots, while they also exhibited differential expression patterns in other tissues. The expression levels of all six type-A and one type-B JcRRs increased in response to 6-benzylaminopurine (6-BA), while the four type-B JcRRs levels decreased. The expression levels of two type-B JcRRs increased in response to exogenous GA3 treatment, while those of three type-A and three type-B JcRRs decreased. We found that type-A JcRRs may play a positive role in the continuous growth of axillary buds, while the role of type-B JcRRs might be the opposite. In response to abiotic stress, the expression levels of two type-A and three type-B JcRRs strongly increased. The overexpression of JcRR12 in Arabidopsis thaliana slightly increased the numbers of rosette branches after decapitation, but not under normal conditions. In conclusion, our results provide detailed knowledge of JcRRs for further analysis of CK signaling and JcRR functions in J. curcas.

Type-A response regulators negatively mediate heat stress response by altering redox homeostasis in Arabidopsis.

Besides the long-standing role of cytokinins (CKs) as growth regulators, their current positioning at the interface of development and stress responses is coming into recognition. The current evidence suggests the notion that CKs are involved in heat stress response (HSR), however, the role of CK signaling components is still elusive. In this study, we have identified a role of the CK signaling components type-A Arabidopsis response regulators (ARRs) in HSR in Arabidopsis. The mutants of multiple type-A ARR genes exhibit improved basal and acquired thermotolerance and, altered response to oxidative stress in our physiological analyses. Through proteomics profiling, we show that the type-A arr mutants experience a ‘stress-primed’ state enabling them to respond more efficiently upon exposure to real stress stimuli. A substantial number of proteins that are involved in the heat-acclimatization process such as the proteins related to cellular redox status and heat shock, are already altered in the type-A arr mutants without a prior exposure to stress conditions. The metabolomics analyses further reveal that the mutants accumulate higher amounts of α-and γ-tocopherols, which are important antioxidants for protection against oxidative damage. Collectively, our results suggest that the type-A ARRs play an important role in heat stress response by affecting the redox homeostasis in Arabidopsis.

Bioenergy sorghum stem growth regulation: intercalary meristem localization, development, and gene regulatory network analysis.

Bioenergy sorghum is a highly productive drought tolerant C4 grass that accumulates 80% of its harvestable biomass in approximately 4 m length stems. Stem internode growth is regulated by development, shading, and hormones that modulate cell proliferation in intercalary meristems (IMs). In this study, sorghum stem IMs were localized above the pulvinus at the base of elongating internodes using magnetic resonance imaging, microscopy, and transcriptome analysis. A change in cell morphology/organization occurred at the junction between the pulvinus and internode where LATERAL ORGAN BOUNDARIES (SbLOB), a boundary layer gene, was expressed. Inactivation of an AGCVIII kinase in DDYM (dw2) resulted in decreased SbLOB expression, disrupted IM localization, and reduced internode cell proliferation. Transcriptome analysis identified approximately 1000 genes involved in cell proliferation, hormone signaling, and other functions selectively upregulated in the IM compared with a non-meristematic stem tissue. This cohort of genes is expressed in apical dome stem tissues before localization of the IM at the base of elongating internodes. Gene regulatory network analysis identified connections between genes involved in hormone signaling and cell proliferation. The results indicate that gibberellic acid induces accumulation of growth regulatory factors (GRFs) known to interact with ANGUSTIFOLIA (SbAN3), a master regulator of cell proliferation. GRF:AN3 was predicted to induce SbARF3/ETT expression and regulate SbAN3 expression in an auxin-dependent manner. GRFs and ARFs regulate genes involved in cytokinin and brassinosteroid signaling and cell proliferation. The results provide a molecular framework for understanding how hormone signaling regulates the expression of genes involved in cell proliferation in the stem IM.

Identification and Characterization of AUXIN Response Factor Gene Family Reveals Their Regulatory Network to Respond the Multi-Hormones Crosstalk during GA-Induced Grape Parthenocarpic Berry.

Exogenous gibberellin (GA) was widely used to improve berry quality through inducing parthenocarpic seedless berries in grapes. We revealed that auxin response factors (ARFs), the key transcription factors in response to auxin, might respond to GA involving modulation of grape parthenocarpy. However, the underlying molecular mechanism in this process remains yet unclear. Here, a total of 19 VvARF members were identified in the ovaries during GA-induced grapes’ parthenocarpy. Interestingly, almost all members were GA-responsive factors, of which 9 could be classified in plant hormone signal transduction (KO04075) and involved in the tryptophan metabolic pathway (K14486). Moreover, VvARFs were predicted to have 310 interacted proteins involved in 19 KEGG pathways. Of them, 32 interacted proteins participated in the KO04075 pathway, including auxin (IAA), salicylic acid (SA), abscisic acid (ABA), cytokinin (CTK), and ethylene signaling pathways by responding to GA-mediated multi-hormone crosstalk. Further analysis demonstrated that VvARF4-2 might be the major factor in the modulation of GA-induced parthenocarpy via the crosstalk of IAA, CTK, SA, and ethylene signaling, followed by VvARF6-1 and VvARF9 involved in SA and ABA signaling pathways, respectively. Finally, we developed a VvARFs-mediated regulatory network by responding to GA-mediated multi-hormone crosstalk during grape parthenocarpy. Collectively, our findings provided novel insights into the regulatory network of VvARFs in GA-guided multi-hormone signaling to modulate grape parthenocarpy, which has great implications for the molecular breeding of high quality seedless grape berries.

The type-B response regulators ARR10, ARR12, and ARR18 specify the central cell in Arabidopsis.

In Arabidopsis thaliana, the female gametophyte consists of two synergid cells, an egg cell, a diploid central cell, and three antipodal cells. CYTOKININ INDEPENDENT 1 (CKI1), a histidine kinase constitutively activating the cytokinin signaling pathway, specifies the central cell and restricts the egg cell. However, the mechanism regulating CKI1-dependent central cell specification is largely unknown. Here, we showed that the type-B ARABIDOPSIS RESPONSE REGULATORS10, 12, and 18 (ARR10/12/18) localize at the chalazal pole of the female gametophyte. Phenotypic analysis showed that the arr10 12 18 triple mutant is female sterile. We examined the expression patterns of embryo sac marker genes and found that the embryo sac of arr10 12 18 plants had lost central cell identity, a phenotype similar to that of the Arabidopsis cki1 mutant. Genetic analyses demonstrated that ARR10/12/18, CKI1, and ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN2, 3, and 5 (AHP2/3/5) function in a common pathway to regulate female gametophyte development. In addition, constitutively activated ARR10/12/18 in the cki1 embryo sac partially restored the fertility of cki1. Results of transcriptomic analysis supported the conclusion that ARR10/12/18 and CKI1 function together to regulate the identity of the central cell. Our results demonstrated that ARR10/12/18 function downstream of CKI1-AHP2/3/5 as core factors to determine cell fate of the female gametophyte.

A conserved type III effector RipB is recognized in tobacco and contributes to Ralstonia solanacearum virulence in susceptible host plants

Ralstonia solanacearum, the causal agent of bacterial wilt, causes devastating diseases in a wide range of plants including potato, tomato, pepper and tobacco. The pathogen delivers approximately 70 type III effectors (T3Es) into plant cells during infection. In this study, we confirmed that a T3E RipB is recognized in tobacco. We further demonstrated that RipB is conserved among R. solanacearum isolates and five different ripB alleles are all recognized in tobacco. The ripB from GMI1000 was transformed into susceptible host Arabidopsis, and a defect in root development was observed in ripB-transgenic plants. Pathogen inoculation assays showed that ripB expression promoted plant susceptibility to R. solanacearum infection, indicating that RipB contributes to pathogen virulence in Arabidopsis. Expression of ripB in roq1 mutant partially suppressed reactive oxygen species production, confirming that RipB interferes with plant basal defense. Interestingly, ripB expression promoted cytokinin-related gene expression in Arabidopsis, suggesting a role of cytokinin signaling pathway in plant-R. solanacearum interactions. Finally, RipB harbors potential 14-3-3 binding motifs, but the associations between RipB and 14-3-3 proteins were undetectable in yeast two-hybrid assay. Together, our results demonstrate that multiple ripB alleles are recognized in Nicotiana, and RipB suppresses basal defense in susceptible host to promote R. solanacearum infection.

Cytokinins Induce Prehaustoria Coordinately with Quinone Signals in the Parasitic Plant Striga hermonthica.

Orobanchaceae parasitic plants are major threats to global food security, causing severe agricultural damage worldwide. Parasitic plants derive water and nutrients from their host plants through multicellular organs called haustoria. The formation of a prehaustorium, a primitive haustorial structure, is provoked by host-derived haustorium-inducing factors (HIFs). Quinones, including 2,6-dimethoxy-p-benzoquinone (DMBQ), are of the most potent HIFs for various species in Orobanchaceae, but except non-photosynthetic holoparasites, Phelipanche and Orobanche spp. Instead, cytokinin (CK) phytohormones were reported to induce prehaustoria in Phelipanche ramosa. However, little is known about whether CKs act as HIFs in the other parasitic species to date. Moreover, the signaling pathways for quinones and CKs in prehaustorium induction are not well understood. This study shows that CKs act as HIFs in the obligate parasite Striga hermonthica but not in the facultative parasite Phtheirospermum japonicum. Using chemical inhibitors and marker gene expression analysis, we demonstrate that CKs activate prehaustorium formation through a CK-specific signaling pathway that overlaps with the quinone HIF pathway at downstream in S. hermonthica. Moreover, host root exudates activated S. hermonthica CK biosynthesis and signaling genes, and DMBQ and CK inhibitors perturbed the prehaustorium-inducing activity of exudates, indicating that host root exudates include CKs. Our study reveals the importance of CKs for prehaustorium formation in obligate parasitic plants.