Regulates Auxin Signaling Research Articles

Suppression of SlHDT1 expression increases fruit yield and decreases drought and salt tolerance in tomato.

Histone deacetylation, one of most important types of post-translational modification, plays multiple indispensable roles in plant growth and development and abiotic stress responses. However, little information about the roles of histone deacetylase in regulating inflorescence architecture, fruit yield, and stress responses is available in tomato. Functional characterization revealed that SlHDT1 participated in the control of inflorescence architecture and fruit yield by regulating auxin signalling, and influenced tolerance to drought andsalt stresses by governing abscisic acid (ABA) signalling. More inflorescence branches and higher fruit yield, which were influenced by auxin signalling, were observed in SlHDT1-RNAi transgenic plants. Moreover, tolerance to drought andsalt stresses was decreased in SlHDT1-RNAi transgenic lines compared with the wild type (WT). Changes in parameters related to the stress response, including decreases in survival rate, chlorophyll content, relative water content (RWC), proline content, catalase (CAT) activity and ABA content and an increase in malonaldehyde (MDA) content, were observed in SlHDT1-RNAi transgenic lines. In addition, the RNA-seq analysis revealed varying degrees of downregulation for genes such as the stress-related genes SlABCC10 and SlGAME6 and the pathogenesis-related protein P450 gene SlCYP71A1, and upregulation of the pathogenesis-related protein P450 genes SlCYP94B1, SlCYP734A7 and SlCYP94A2 in SlHDT1-RNAi transgenic plants, indicating that SlHDT1 plays an important role in the response to biotic and abiotic stresses by mediating stress-related gene expression. In summary, the data suggest that SlHDT1 plays essential roles in the regulation of inflorescence architecture and fruit yield and in the response to drought andsalt stresses.

ZmSPL10, ZmSPL14 and ZmSPL26 act together to promote stigmatic papilla formation in maize through regulating auxin signaling and ZmWOX3A expression.

Maize silk is a specialized type of stigma, covered with numerous papillae for pollen grain capture. However, the developmental process of stigmatic papillae and the underlying regulatory mechanisms have remained largely unknown. Here, we combined the cytological, genetic and molecular studies to demonstrate that three homologous genes ZmSPL10, ZmSPL14 and ZmSPL26 play a central role in promoting stigmatic papilla formation in maize. We show that their triple knockout mutants are nearly complete lack of stigmatic papilla, resulting in a severe reduction in kernel setting. Cellular examination reveals that stigmatic papilla is developed from a precursor cell, which is the smaller daughter cell resulting from asymmetric cell division of a silk epidermal cell. In situ hybridization shows that ZmSPL10, ZmSPL14 and their target genes SPI1, ZmPIN1b, ZmARF28 and ZmWOX3A are preferentially expressed in the precursor cells of stigmatic papillae. Moreover, ZmSPL10, ZmSPL14 and ZmSPL26 directly bind to the promoters of SPI1, ZmPIN1b, ZmARF28 and ZmWOX3A and promote their expression. Further, Zmwox3a knockout mutants display severe defects in stigmatic papilla formation and reduced seed setting. Collectively, our results demonstrate that ZmSPL10, ZmSPL14 and ZmSPL26 act together to promote stigmatic papilla development through regulating auxin signaling and ZmWOX3A expression.

Trichoderma-secreted anthranilic acid promotes lateral root development via auxin signaling and RBOHF-induced endodermal cell wall remodeling

Trichoderma spp. have evolved the capacity to communicate with plants by producing various secondary metabolites (SMs). Nonhormonal SMs play important roles in plant root development, while specific SMs from rhizosphere microbes and their underlying mechanisms to control plant root branching are still largely unknown. In this study, a compound, anthranilic acid (2-AA), is identified from T.guizhouense NJAU4742 to promote lateral root development. Further studies demonstrate that 2-AA positively regulates auxin signaling and transport in the canonical auxin pathway. 2-AA also partly rescues the lateral root numbers of CASP1pro:shy2-2, which regulates endodermal cell wall remodeling via an RBOHF-induced reactive oxygen species burst. In addition, our work reports another role for microbial 2-AA in the regulation of lateral root development, which is different from its better-known role in plant indole-3-acetic acid biosynthesis. In summary, this study identifies 2-AA from T.guizhouense NJAU4742, which plays versatile roles in regulating plant root development.

Role of Small RNAs in Plant Immunity

Small RNAs play a crucial role in the regulation of gene expression, operating at both the transcriptional and post-transcriptional stages. These molecules have the ability to initiate target destruction or block translation. The activation of plant immunity is frequently associated with the increased expression of growth-regulatory microRNAs (miRNAs) following the detection of pathogens. Additionally, conserved miRNAs play a crucial role in regulating auxin signaling. Plants are capable of generating two distinct categories of short RNA molecules, namely microRNAs and small interfering RNAs. Three highly prevalent miRNA families induce the generation of secondary siRNAs from 74 out of the 79 NLR transcripts that produce siRNAs. This phenomenon highlights the appeal of employing secondary siRNA-mediated control as a viable technique. Two microRNAs (miRNAs) derived from plants have been found to target virulence genes in the fungal disease Verticillium dahliae. Additionally, it has been observed that secondary small interfering RNAs (siRNAs) can increase plant protection by facilitating host-induced gene silence. TasiRNAs have been observed to exhibit enhanced mobility and resilience in the context of non-cell-autonomous silencing. The production and function of short RNAs in fungal and oomycete infections have been minimally investigated. Verticillium dahliae, has been seen to make small RNAs (sRNAs) that range in size from 18 to 25 nucleotides (nt) without a preference for one size over the other. Additionally, a specific microRNA (miRNA) known as VdmilR1, measuring 21 nt in length, has been thoroughly studied and its functional characteristics have been elucidated. Phytophthora species, which are filamentous eukaryotic microbes, have been found to inflict substantial economic losses in the fields of agriculture and forestry. RNA interference (RNAi) has the potential to regulate the expression of neighboring effector genes by inducing the creation of heterochromatin. In plants, the movement of small RNAs has been observed both locally and across extended distances. These small RNAs have the potential to enhance plant defense mechanisms against nonviral diseases by acting as systemic signaling molecules. The domain of sRNAs in plant-pathogen/parasite interactions has experienced significant advancements; nonetheless, numerous obstacles remain in the realm of trans-species gene silencing that require more investigation.

Clade-D auxin response factors regulate auxin signaling and development in the moss Physcomitrium patens.

Auxin response factors (ARFs) are a family of transcription factors that are responsible for regulating gene expression in response to changes in auxin level. The analysis of ARF sequence and activity indicates that there are 2 major groups: activators and repressors. One clade of ARFs, clade-D, is sister to clade-A activating ARFs, but are unique in that they lack a DNA-binding domain. Clade-D ARFs are present in lycophytes and bryophytes but absent in other plant lineages. The transcriptional activity of clade-D ARFs, as well as how they regulate gene expression, is not well understood. Here, we report that clade-D ARFs are transcriptional activators in the model bryophyte Physcomitrium patens and have a major role in the development of this species. Δarfddub protonemata exhibit a delay in filament branching, as well as a delay in the chloronema to caulonema transition. Additionally, leafy gametophore development in Δarfddub lines lags behind wild type. We present evidence that ARFd1 interacts with activating ARFs via their PB1 domains, but not with repressing ARFs. Based on these results, we propose a model in which clade-D ARFs enhance gene expression by interacting with DNA bound clade-A ARFs. Further, we show that ARFd1 must form oligomers for full activity.

ERF1 inhibits lateral root emergence by promoting local auxin accumulation and repressing ARF7 expression

Lateral roots (LRs) are crucial for plants to sense environmental signals in addition to water and nutrient absorption. Auxin is key for LR formation, but the underlying mechanisms are not fully understood. Here, we report that Arabidopsis ERF1 inhibits LR emergence by promoting local auxin accumulation with altered distribution and regulating auxin signaling. Loss of ERF1 increases LR density compared with the wild type, whereas ERF1 overexpression causes the opposite phenotype. ERF1 enhances auxin transport by upregulating PIN1 and AUX1, resulting in excessive auxin accumulation in the endodermal, cortical, and epidermal cells surrounding LR primordia. Furthermore, ERF1 represses ARF7 transcription, thereby downregulating the expression of cell-wall remodeling genes that facilitate LR emergence. Together, our study reveals that ERF1 integrates environmental signals to promote local auxin accumulation with altered distribution and repress ARF7, consequently inhibiting LR emergence in adaptation to fluctuating environments.

Nitric oxide-mediated S-nitrosylation of IAA17 protein in intrinsically disordered region represses auxin signaling

The phytohormone auxin plays crucial roles in nearly every aspect of plant growth and development. Auxin signaling is activated through the phytohormone-induced proteasomal degradation of the Auxin/INDOLE-3-ACETIC ACID (Aux/IAA) family of transcriptional repressors. Notably, many auxin-modulated physiological processes are also regulated by nitric oxide (NO) that executes its biological effects predominantly through protein S-nitrosylation at specific cysteine residues. However, little is known about the molecular mechanisms in regulating the interactive NO and auxin networks. Here, we show that NO represses auxin signaling by inhibiting IAA17 protein degradation. NO induces the S-nitrosylation of Cys-70 located in the intrinsically disordered region of IAA17, which inhibits the TIR1–IAA17 interaction and consequently the proteasomal degradation of IAA17. The accumulation of a higher level of IAA17 attenuates auxin response. Moreover, an IAA17C70W nitrosomimetic mutation renders the accumulation of a higher level of the mutated protein, thereby causing partial resistance to auxin and defective lateral root development. Taken together, these results suggest that S-nitrosylation of IAA17 at Cys-70 inhibits its interaction with TIR1, thereby negatively regulating auxin signaling. This study provides unique molecular insights into the redox-based auxin signaling in regulating plant growth and development.

Hydrogen sulfide alleviates salt stress through auxin signaling in Arabidopsis

Hydrogen sulfide (H2S) is a third gas transmitter following nitric oxide and carbon monoxide, that regulates plant growth, photosynthesis, and responses to abiotic stress. Auxin, a crucial plant hormone, is widely involved in stress response and plant growth processes. H2S and auxin interact with each other to regulate root growth. Whether H2S functions in the auxin signaling pathway under salt stress remains unclear. In this study, we used the double mutant of the H2S-producing enzyme-encoding gene L-cysteine desulfhydrase (lcd des1), which found that H2S was involved in alleviating salt stress through auxin signaling in Arabidopsis using physiological and transcriptomic data. RNA-seq analysis identified 267 differentially expressed genes (DEGs) in response to salt stress and revealed that H2S activated salt-related genes and limited cellular activities associated with growth through the regulation of auxin signaling. Furthermore, auxin transport-related mutants (pin347, aux1–7) and auxin signal transduction related mutants (tir1–1, arf10 16) had a significantly inhibited root length under salt stress, and root growth inhibition were alleviated after exogenous H2S treatment. Exogenous H2S treatment, however, did not alleviate inhibition in pin2 with salt treatment. Taken together, these findings suggest that H2S regulates Arabidopsis salt tolerance and inhibits root growth by regulating PIN2 expression.

Comparative phylogenomic analysis of 5’cis-regulatory elements (CREs) of miR160 gene family in diploid and allopolyploid cotton (Gossypium)

Cotton fiber development is a complex process regulated by numerous microRNAs (miRNAs), including miR160 that regulates auxin signaling in a grwoing cell. However, the evolution of cis-regulatory elements (CREs) in the 5’upstream regulatory regions of miR160 genes during cotton allopolyploidy is least known. Therefore, 1.5 kb upstream DNA sequences (−1 to −1500 bp) of miR160 genes from progenitor diploid A2 (G. arboreum), D5 (G. raimondii) and descendant allopolyploid AD1 (G. hirsutum) and AD2 (G. barbadense) cotton species were analyzed to visualize the evolutionary patterns preserved within the promoter regions of these homologous sequences. Several light-responsive, developmental-responsive, organ-specific, stress-responsive (biotic and abiotic), and hormone-responsive CREs were identified with diverse frequencies and wide distribution patterns. The structural alterations in the promoter regions were more prominent than the evolutionary changes acquired by miR160 genes, suggesting that miR160 gene sequences have been conserved throughout evolution, and the observed expression variations across cotton species are primarily due to the structural differences of their regulatory regions. The abiotic stress-responsive and hormone-responsive CREs exhibited prominent clustering at both proximal and distal ends, displaying varying frequencies. Thus, the origin of these CREs can be attributed to the genomic hybridization of diploid species that had led to the allopolyploid formation, or to the natural speciation/diversification of an allopolyploid event. Also, there is a high correlation between CREs-binding transcription factors and miR160 gene expression, suggesting a regulatory mechanism for spatiotemporal expression profiles of miR160 genes during cotton fiber development.

The Mutation of Rice MEDIATOR25, OsMED25, Induces Rice Bacterial Blight Resistance through Altering Jasmonate- and Auxin-Signaling.

Rice bacterial blight disease caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most severe diseases of rice. However, the regulatory mechanisms of rice defense against Xoo remain poorly understood. The rice MEDIATOR25, OsMED25—a subunit of the mediator multiprotein complex that acts as a universal adaptor between transcription factors (TFs) and RNA polymerase II—plays an important role in jasmonic acid (JA)-mediated lateral root development in rice. In this study, we found that OsMED25 also plays an important role in JA- and auxin-mediated resistance responses against rice bacterial blight. The osmed25 loss-of-function mutant exhibited high resistance to Xoo. The expression of JA-responsive defense-related genes regulated by OsMYC2, which is a positive TF in JA signaling, was downregulated in osmed25 mutants. Conversely, expression of some OsMYC2-independent JA-responsive defense-related genes was upregulated in osmed25 mutants. Furthermore, OsMED25 interacted with some AUXIN RESPONSE FACTORS (OsARFs) that regulate auxin signaling, whereas the mutated osmed25 protein did not interact with the OsARFs. The expression of auxin-responsive genes was downregulated in osmed25 mutants, and auxin-induced susceptibility to Xoo was not observed in osmed25 mutants. These results indicate that OsMED25 plays an important role in the stable regulation of JA- and auxin-mediated signaling in rice defense response.

Genomic identification of ARF transcription factors and expression analysis in Cannabis sativa L

Auxin response factors (ARFs) participate in the regulation of auxin signaling by binding directly to the auxin response elements (AuxREs) in the promoter regions of the downstream auxin response genes during plant development. To date, ARF genes in Cannabis sativa L. (cannabis) have not been characterized. The present study identified 21 ARF gene family members based on the genome-wide database of cannabis. Systematic analysis of these CsARFs included analysis of the gene structures, conserved domains, possible isoforms, phylogeny, cis-elements, and expression levels in various tissues and subspecies of cannabis. Currently, the function of the ARF genes in the growth and development of cannabis is largely unknown. To uncover this function, the expression patterns of the CsARF genes were analyzed in various tissues and subspecies of cannabis in detail. Combination of the results of the evolutionary tree and expression patterns of CsARFs, the functions of five CsARFs (CsARF7, CsARF8, CsARF11, CsARF19, and CsARF20) were predicted, and these genes may be involved in the development of cannabis flower. In addition, six CsARFs (CsARF3, CsARF5, CsARF7, CsARF8, CsARF10, and CsARF15) may be involved in the development of cannabis root and stem. Ultimately, CsARF13 may be involved in the development of cannabis leaf. The present study provides basic information on the CsARF genes and paves the way for further exploration of the function and mechanisms involved in the regulation of the growth and development of cannabis.

The co-chaperone HOP participates in TIR1 stabilisation and in auxin response in plants.

HOP (HSP70‐HSP90 organising protein) is a conserved family of co‐chaperones well known in mammals for its role in the folding of signalling proteins associated with development. In plants, HOP proteins have been involved in the response to multiple stresses, but their role in plant development remains elusive. Herein, we describe that the members of the HOP family participate in different aspects of plant development as well as in the response to warm temperatures through the regulation of auxin signalling. Arabidopsis hop1 hop2 hop3 triple mutant shows different auxin‐related phenotypes and a reduced auxin sensitivity. HOP interacts with TIR1 auxin coreceptor in vivo. Furthermore, TIR1 accumulation and auxin transcriptional response are reduced in the hop1 hop2 hop3 triple mutant, suggesting that HOP's function in auxin signalling is related, at least, to TIR1 interaction and stabilisation. Interestingly, HOP proteins form part of the same complexes as SGT1b (a different HSP90 co‐chaperone) and these co‐chaperones synergistically cooperate in auxin signalling. This study provides relevant data about the role of HOP in auxin regulation in plants and uncovers that both co‐chaperones, SGT1b and HOP, cooperate in the stabilisation of common targets involved in plant development.

The Auxin-Response Repressor IAA30 Is Down-Regulated in Reproductive Tissues of Apomictic Paspalum notatum.

The capacity for apomixis in Paspalum notatum is controlled by a single-dominant genomic region, which shows strong synteny to a portion of rice chromosome 12 long arm. The locus LOC_Os12g40890, encoding the Auxin/Indole-3-Acetic Acid (Aux/IAA) family member OsIAA30, is located in this rice genomic segment. The objectives of this work were to identify transcripts coding for Aux/IAA proteins expressed in reproductive tissues of P. notatum, detect the OsIAA30 putative ortholog and analyze its temporal and spatial expression pattern in reproductive organs of sexual and apomictic plants. Thirty-three transcripts coding for AUX/IAA proteins were identified. Predicted protein alignment and phylogenetic analysis detected a highly similar sequence to OsIAA30 (named as PnIAA30) present in both sexual and apomictic samples. The expression assays of PnIAA30 showed a significant down-regulation in apomictic spikelets compared to sexual ones at the stages of anthesis and post-anthesis, representation levels negatively correlated with apospory expressivity and different localizations in sexual and apomictic ovules. Several PnIAA30 predicted interactors also appeared differentially regulated in the sexual and apomictic floral transcriptomes. Our results showed that an auxin-response repressor similar to OsIAA30 is down-regulated in apomictic spikelets of P. notatum and suggests a contrasting regulation of auxin signaling during sexual and asexual seed formation.

Unravelling new roles of a tomato SIN3 homolog in leaf polarity mediated by auxin signaling and leaf growth-related transcription factors

SIN3 is a large protein providing a platform for the assembly of multiple proteins, and SIN3 acting as a repressor negatively regulates genes involved in diverse cellular functions. SIN3 has been widely studied in yeasts and animals, but its function remains largely uncharacterized in plants. In this study, we identified and characterized three SIN3 homologous genes, SlSIN3a, SlSIN3b and SlSIN3c in tomato. The transcripts of SlSIN3b and SlSIN3c were almost undetectable, while SlSIN3a was highly expressed in all examined tissues (root, hypocotyl, cotyledon, leaf and fruit). We generated knockout mutagenesis of SlSIN3a using CRISPR/Cas9 technique. The slsin3a mutant plants exhibited abaxialized leaves compared with the wild-type. RNA-seq analysis indicated that the expressions of key leaf polarity regulators (SlTCP3, and SlHZ13), auxin signaling gene (SlIAA1), as well as cell expansin gene (SlEXPB2) and cyclin genes, were significantly changed in slsin3a mutant plants as compared to wild-type. We propose that SlSIN3a may be involved in leaf polarity through regulating auxin signaling, TCP, HD-ZIP and LBD genes, therefore affecting a serious of expansin and cyclin genes. Taken together, the results reveal a new role of SIN3 in leaf polarity and provide a possible action mechanism of SlSIN3a in leaf development.

A network of transcriptional repressors modulates auxin responses.

The regulation of signalling capacity, combined with the spatiotemporal distribution of developmental signals themselves, is pivotal in setting developmental responses in both plants and animals1. The hormone auxin is a key signal for plant growth and development that acts through the AUXIN RESPONSE FACTOR (ARF) transcription factors2-4. A subset of these, the conserved class A ARFs5, are transcriptional activators of auxin-responsive target genes that are essential for regulating auxin signalling throughout the plant lifecycle2,3. Although class A ARFs have tissue-specific expression patterns, how their expression is regulated is unknown. Here we show, by investigating chromatin modifications and accessibility, that loci encoding these proteins are constitutively open for transcription. Through yeast one-hybrid screening, we identify the transcriptional regulators of the genes encoding class A ARFs from Arabidopsis thaliana and demonstrate that each gene is controlled by specific sets of transcriptional regulators. Transient transformation assays and expression analyses in mutants reveal that, in planta, the majority of these regulators repress the transcription of genes encoding class A ARFs. These observations support a scenario in which the default configuration of open chromatin enables a network of transcriptional repressors to regulate expression levels of class A ARF proteins and modulate auxin signalling output throughout development.

Salicylic acid regulates PIN2 auxin transporter hyperclustering and root gravitropic growth via Remorin-dependent lipid nanodomain organisation in Arabidopsis thaliana.

Summary To adapt to the diverse array of biotic and abiotic cues, plants have evolved sophisticated mechanisms to sense changes in environmental conditions and modulate their growth. Growth‐promoting hormones and defence signalling fine tune plant development antagonistically. During host–pathogen interactions, this defence–growth trade‐off is mediated by the counteractive effects of the defence hormone salicylic acid (SA) and the growth hormone auxin.Here we revealed an underlying mechanism of SA regulating auxin signalling by constraining the plasma membrane dynamics of PIN2 auxin efflux transporter in Arabidopsis thaliana roots.The lateral diffusion of PIN2 proteins is constrained by SA signalling, during which PIN2 proteins are condensed into hyperclusters depending on REM1.2‐mediated nanodomain compartmentalisation. Furthermore, membrane nanodomain compartmentalisation by SA or Remorin (REM) assembly significantly suppressed clathrin‐mediated endocytosis. Consequently, SA‐induced heterogeneous surface condensation disrupted asymmetric auxin distribution and the resultant gravitropic response.Our results demonstrated a defence–growth trade‐off mechanism by which SA signalling crosstalked with auxin transport by concentrating membrane‐resident PIN2 into heterogeneous compartments.

A basic/helix–loop–helix transcription factor controls leaf shape by regulating auxin signaling in apple

Climate-driven phenological change across local spatial gradients leads to leaf shape variation. At higher elevations, leaves of broadleaf species tend to become narrower, but the underlying molecular mechanism is largely unknown. In this study, a series of morphometric analyses and biochemical assays, combined with functional identification in apple, were performed. We show that the decrease in apple leaf width with increasing altitude is controlled by a basic/helix-loop-helix transcription factor (bHLH TF), MdbHLH3. The MdbHLH3-overexpressing lines have a lower transcript abundance of MdPIN1 encoding an auxin efflux carrier but a higher transcript abundance of MdGH3-2 encoding a putative auxin amido conjugate synthase, resulting in a lower free auxin concentration; feeding the transgenic leaves with exogenous auxin partially restores leaf width. MdbHLH3 transcriptionally suppresses and activates MdPIN1 and MdGH3-2, respectively, by specifically binding to their promoters. This alters auxin homeostasis and transport, consequently leading to changes in leaf shape. These findings suggest that the bHLH TF MdbHLH3 directly modulates auxin signaling in controlling leaf shape in response to local spatial gradients in apple.

Osmotic stress inhibits leaf growth of Arabidopsis thaliana by enhancing ARF-mediated auxin responses.

We investigated the interaction between osmotic stress and auxin signaling in leaf growth regulation. Therefore, we grew Arabidopsis thaliana seedlings on agar media supplemented with mannitol to impose osmotic stress and 1-naphthaleneacetic acid (NAA), a synthetic auxin. We performed kinematic analysis and flow-cytometry to quantify the effects on cell division and expansion in the first leaf pair, determined the effects on auxin homeostasis and response (DR5::β-glucuronidase), performed a next-generation sequencing transcriptome analysis and investigated the response of auxin-related mutants. Mannitol inhibited cell division and expansion. NAA increased the effect of mannitol on cell division, but ameliorated its effect on expansion. In proliferating cells, NAA and mannitol increased free IAA concentrations at the cost of conjugated IAA and stimulated DR5 promotor activity. Transcriptome analysis shows a large overlap between NAA and osmotic stress-induced changes, including upregulation of auxin synthesis, conjugation, transport and TRANSPORT INHIBITOR RESPONSE1 (TIR1) and AUXIN RESPONSE FACTOR (ARF) response genes, but downregulation of Aux/IAA response inhibitors. Consistently, arf7/19 double mutant lack the growth response to auxin and show a significantly reduced sensitivity to osmotic stress. Our results show that osmotic stress inhibits cell division during leaf growth of A.thaliana at least partly by inducing the auxin transcriptional response.

Target-mimicry based miRNA167-diminution ameliorates cotton somatic embryogenesis via transcriptional biases of auxin signaling associated miRNAs and genes

In vitro regeneration of cotton seems to be highly genotype dependent and typically ensues through the extraordinarily complex mechanism of somatic embryogenesis (SE). During the acquisition of embryogenic potential by the somatic cells, 21–24 nucleotides long regulatory microRNAs, especially miRNA167 plays a crucial role in the regulation of auxin signaling by degrading or inhibiting target mRNA molecules. To reveal the mechanism of regulation of target genes and the influence of key SE-associated miRNAs on the acquisition of embryogenic potential by the somatic cells, we employed target-mimicry approach of miR167 diminution for loss-of-function analysis encompassing callogenesis and in vitro embryogenesis stages of a fully-regenerating line of Coker 310 cultivar. Results demonstrated that miR167-diminution led to enhanced callogenesis and increased production of somatic embryos in the mimic-transformed events. In the MIM167-transformed calli, ARF6/ARF8, auxin-responsive GH3, auxin transporter Aux1, and auxin influx/efflux carrier LAX3, PIN1 and PIN2 genes showed a greater shift than control/non-transformed events, and suggested that miR167 diminution led to enhanced cellular auxin-signaling. Moreover, auxin-signaling associated microRNAs-miR160, miR393, miR166, miR156 and miR157 were differentially expressed in MIM167 mimic events indicating that diminution of miR167 also influenced the expression of these miRNAs. Evidently, miR166-LEC2 pair supplemented auxin signaling associated miR167-ARF6/ARF8 module that consequently may have resulted into substantial phenotypic transformations during embryonic transitions of cotton cultures. Coordinated and fine-scale expression-network of auxin signaling-associated miRNAs and genes in miR167-target mimic transgenic calli of cotton, directed the incessant production of somatic embryos.

Molecular Rewiring of the Jasmonate Signaling Pathway to Control Auxin-Responsive Gene Expression.

The plant hormone jasmonic acid (JA) has an important role in many aspects of plant defense response and developmental process. JA triggers interaction between the F-box protein COI1 and the transcriptional repressors of the JAZ family that leads the later to proteasomal degradation. The Jas-motif of JAZs is critical for mediating the COI1 and JAZs interaction in the presence of JA. Here, by using the protoplast transient gene expression system we reported that the Jas-motif of JAZ1 was necessary and sufficient to target a foreign reporter protein for COI1-facilitated degradation. We fused the Jas-motif to the SHY2 transcriptional repressor of auxin signaling pathway to create a chimeric protein JaSHY. Interestingly, JaSHY retained the transcriptional repressor function while become degradable by the JA coreceptor COI1 in a JA-dependent fashion. Moreover, the JA-induced and COI1-facilitated degradation of JaSHY led to activation of a synthetic auxin-responsive promoter activity. These results showed that the modular components of JA signal transduction pathway can be artificially redirected to regulate auxin signaling pathway and control auxin-responsive gene expression. Our work provides a general strategy for using synthetic biology approaches to explore and design cell signaling networks to generate new cellular functions in plant systems.