Protoplast Transactivation Research Articles

SPOTTED-LEAF7 targets the gene encoding β-Galactosidase9, which functions in rice growth and stress responses.

β-Galactosidases (Bgals) remove terminal β-D-galactosyl residues from the non-reducing ends of β-D-galactosides and oligosaccharides. Bgals are present in bacteria, fungi, animals, and plants and have various functions. Despite many studies on the evolution of BGALs in plants, their functions remain obscure. Here, we identified rice (Oryza sativa) β-galactosidase9 (OsBGAL9) as a direct target of the heat stress-induced transcription factor SPOTTED-LEAF7 (OsSPL7), as demonstrated by protoplast transactivation analysis and yeast one-hybrid and electrophoretic mobility shift assays. Knockout plants for OsBGAL9 (Osbgal9) showed short stature and growth retardation. Histochemical ß-glucuronidase (GUS) analysis of transgenic lines harboring an OsBGAL9pro:GUS reporter construct revealed that OsBGAL9 is mainly expressed in internodes at the mature stage. OsBGAL9 expression was barely detectable in seedlings under normal conditions but increased in response to biotic and abiotic stresses. Ectopic expression of OsBGAL9 enhanced resistance to the rice pathogens Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae, as well as tolerance to cold and heat stress, while Osbgal9 mutant plants showed the opposite phenotypes. OsBGAL9 localized to the cell wall, suggesting that OsBGAL9 and its plant putative orthologs likely evolved functions distinct from those of its closely related animal enzymes. Enzyme activity assays and analysis of the cell wall composition of OsBGAL9 overexpression and mutant plants indicated that OsBGAL9 has activity toward galactose residues of arabinogalactan proteins (AGPs). Our study clearly demonstrates a role for a member of the BGAL family in AGP processing during plant development and stress responses.

FtBPM3 modulates the orchestration of FtMYB11-mediated flavonoids biosynthesis in Tartary buckwheat.

Tartary buckwheat (Fagopyrum tataricum, TB) is rich in bioactive flavonoids, which have a variety of biological activities (Ghorbani, 2017). Jasmonates (JAs) are essential phytohormones, which play key roles in regulating the formation of numerous secondary metabolites, including flavonoids rutin (Zhou and Memelink, 2016; Li et al., 2019). It has been reported that JAs could induce flavonoids accumulation and identified a class of JAs-responsive R2R3-MYB TFs, such as FtMYB11, a repressor of rutin biosynthesis in TB (Zhang et al., 2018; Zhou et al., 2017). JAs leads to FtMYBs degradation by 26S proteasome pathway (Zhang et al., 2018). However, the posttranslational regulation of these FtMYBs has not been reported.

The sugarcane ShMYB78 transcription factor activates suberin biosynthesis in Nicotiana benthamiana.

A sugarcane MYB present in the culm induces suberin biosynthesis and is involved both with fatty acid and phenolics metabolism. Few transcription factors have been described as regulators of cell wall polymers deposition in C4 grasses. Particularly, regulation of suberin biosynthesis in this group of plants remains poorly understood. Here, we showed that the sugarcane MYB transcription factor ShMYB78 is an activator of suberin biosynthesis and deposition. ShMYB78 was identified upon screening genes whose expression was upregulated in sugarcane internodes undergoing suberization during culm development or triggered by wounding. Agrobacterium-mediated transient expression of ShMYB78 in Nicotiana benthamiana leaves induced the ectopic deposition of suberin and its aliphatic and aromatic monomers. Further, the expression of suberin-related genes was induced by ShMYB78 heterologous expression in Nicotiana benthamiana leaves. ShMYB78 was shown to be a nuclear protein based on its presence in sugarcane internode nuclear protein extracts, and protoplast transactivation assays demonstrated that ShMYB78 activates the promoters of the sugarcane suberin biosynthetic genes β-ketoacyl-CoA synthase (ShKCS20) and caffeic acid-O-methyltransferase (ShCOMT). Our results suggest that ShMYB78 may be involved in the transcriptional regulation of suberin deposition, from fatty acid metabolism to phenylpropanoid biosynthesis, in sugarcane internodes.

A Cytokinin Signaling Type-B Response Regulator Transcription Factor Acting in Early Nodulation.

Nitrogen-fixing root nodulation in legumes challenged with nitrogen-limiting conditions requires infection of the root hairs by soil symbiotic bacteria, collectively referred to as rhizobia, and the initiation of cell divisions in the root cortex. Cytokinin hormones are critical for early nodulation to coordinate root nodule organogenesis and the progression of bacterial infections. Cytokinin signaling involves regulation of the expression of cytokinin primary response genes by type-B response regulator (RRB) transcription factors. RNA interference or mutation of MtRRB3, the RRB-encoding gene most strongly expressed in Medicago truncatula roots and nodules, significantly decreased the number of nodules formed, indicating a function of this RRB in nodulation initiation. Fewer infection events were also observed in rrb3 mutant roots associated with a reduced Nod factor induction of the Early Nodulin 11 (MtENOD11) infection marker, and of the cytokinin-regulated Nodulation Signaling Pathway 2 (Mt NSP2) gene. Rhizobial infections correlate with an expansion of the nuclear area, suggesting the activation of endoreduplication cycles linked to the cytokinin-regulated Cell Cycle Switch 52A (Mt CCS52A) gene. Although no significant difference in nucleus size and endoreduplication were detected in rhizobia-infected rrb3 mutant roots, expression of the MtCCS52A endoreduplication marker was reduced. As the MtRRB3 expression pattern overlaps with those of MtNSP2 and MtCCS52A in roots and nodule primordia, chromatin immunoprecipitation-quantitative PCR and protoplast trans-activation assays were used to show that MtRRB3 can interact with and trans-activate MtNSP2 and MtCCS52A promoters. Overall, we highlight that the MtRRB3 cytokinin signaling transcription factor coordinates the expression of key early nodulation genes.

AtERF#111/ABR1 is a transcriptional activator involved in the wounding response.

AtERF#111/ABR1 belongs to the group X of the ERF/AP2 transcription factor family (GXERFs) and is shoot specifically induced under submergence and hypoxia. It was described to be an ABA-response repressor, but our data reveal a completely different function. Surprisingly, AtERF#111 expression is strongly responsive to wounding stress. Expression profiling of ERF#111-overexpressing (OE) plants, which show morphological phenotypes like increased root hair length and number, strengthens the hypothesis of AtERF#111 being involved in the wounding response, thereby acting as a transcriptional activator of gene expression. Consistent with a potential function outside of oxygen signalling, we could not assign AtERF#111 as a target of the PRT6N-degron pathway, even though it starts with a highly conserved N-terminal Met-Cys (MC) motif. However, the protein is unstable as it is degraded in an ubiquitin-dependent manner. Finally, direct target genes of AtERF#111 were identified by microarray analyses and subsequently confirmed by protoplast transactivation assays. The special roles of diverse members of the plant-specific GXERFs in coordinating stress signalling and wound repair mechanisms have been recently hypothesized, and our data suggest that AtERF#111 is indeed involved in these processes.

Regulation of ABI5 expression by ABF3 during salt stress responses in Arabidopsis thaliana

BackgroundBasic region/leucine zippers (bZIPs) are transcription factors (TFs) encoded by a large gene family in plants. ABF3 and ABI5 are Group A bZIP TFs that are known to be important in abscisic acid (ABA) signaling. However, questions of whether ABF3 regulates ABI5 are still present.ResultsIn vitro kinase assay results showed that Thr-128, Ser-134, and Thr-451 of ABF3 are calcium-dependent protein kinase phosphorylation sites. Bimolecular fluorescence complementation (BiFC) analysis results showed a physical interaction between ABF3 and 14-3-3ω. A Thr-451 to Ala point mutation abolished the interaction but did not change the subcellular localization. In addition, the Arabidopsis protoplast transactivation assay using a luciferase reporter exhibited ABI5 activation by either ABF3 alone or by co-expression of ABF3 and 14-3-3ω. Moreover, chromatin immunoprecipitation-qPCR results showed that in Arabidopsis, ABI5 ABA-responsive element binding proteins (ABREs) of the promoter region (between − 1376 and − 455) were enriched by ABF3 binding under normal and 150 mM NaCl salt stress conditions.ConclusionTaken together, our results demonstrated that ABI5 expression is regulated by ABF3, which could contribute to salt stress tolerance in Arabidopsis thaliana.

FtSAD2 and FtJAZ1 regulate activity of the FtMYB11 transcription repressor of the phenylpropanoid pathway in Fagopyrum tataricum.

Little is known about the molecular mechanism of the R2R3-MYB transcriptional repressors involved in plant phenylpropanoid metabolism. Here, we describe one R2R3-type MYB repressor, FtMYB11 from Fagopyrum tataricum. It contains the SID-like motif GGDFNFDL and it is regulated by both the importin protein 'Sensitive to ABA and Drought 2' (SAD2) and the jasmonates signalling cascade repressor JAZ protein. Yeast two hybrid and bimolecular fluorescence complementation assays demonstrated that FtMYB11 interacts with SAD2 and FtJAZ1. Protoplast transactivation assays demonstrated that FtMYB11 acts synergistically with FtSAD2 or FtJAZ1 and directly represses its target genes via the MYB-core element AATAGTT. Changing the Asp122 residue to Asn in the SID-like motif results in cytoplasmic localization of FtMYB11 because of loss of interaction with SAD2, while changing the Asp126 residue to Asn results in the loss of interaction with FtJAZ1. Overexpression of FtMYB11or FtMYB11D126N in F.tataricum hairy roots resulted in reduced accumulation of rutin, while overexpression of FtMYB11D122N in hairy roots did not lead to such a change. The results indicate that FtMYB11 acts as a regulator via interacting with FtSAD2 or FtJAZ1 to repress phenylpropanoid biosynthesis, and this repression depends on two conserved Asp residues of its SID-like motif.

Characterization of two TT2-type MYB transcription factors regulating proanthocyanidin biosynthesis in tetraploid cotton, Gossypium hirsutum.

Two TT2-type MYB transcription factors identified from tetraploid cotton are involved in regulating proanthocyanidin biosynthesis, providing new strategies for engineering condensed tannins in crops. Proanthocyanidins (PAs), also known as condensed tannins, are important secondary metabolites involved in stress resistance in plants, and are health supplements that help to reduce cholesterol levels. As one of the most widely grown crops in the world, cotton provides the majority of natural fabrics and is a supplemental food for ruminant animals. The previous studies have suggested that PAs present in cotton are a major contributor to fiber color. However, the biosynthesis of PAs in cotton still remains to be elucidated. AtTT2 (transparent testa 2) is a MYB family transcription factor from Arabidopsis that initiates the biosynthesis of PAs by inducing the expression of multiple genes in the pathway. In this study, we isolated two R2R3-type MYB transcription factors from Gossypium hirsutum that are homologous to AtTT2. Expression analysis showed that both genes were expressed at different levels in various cotton tissues, including leaf, seed coat, and fiber. Protoplast transactivation assays revealed that these two GhMYBs were able to activate promoters of genes encoding enzymes in the PA biosynthesis pathway, namely anthocyanidin reductase and leucoanthocyanidin reductase. Complementation experiments showed that both of the GhMYBs were able to recover the transparent testa seed coat phenotype of the Arabidopsis tt2 mutant by restoring PA biosynthesis. Ectopic expression of either of the two GhMYBs in Medicago truncatula hairy roots increased the contents of anthocyanins and PAs compared to control lines expressing the GUS gene, and expression levels of MtDFR, MtLAR, and MtANR were also elevated in lines expressing GhMYBs. Together, these data provide new insights into engineering condensed tannins in cotton.

High-Throughput Protoplast Trans-Activation (PTA) Screening to Define Transcription Factors in Auxin-Mediated Gene Regulation.

Genome sequencing and annotation studies clearly highlight the impact of transcriptional regulation in plants. However, functional characterization of the majority of transcriptional regulators remains elusive. Hence, high-throughput techniques are required to facilitate their molecular analysis. Here, we provide a detailed protocol to conduct a high-throughput protoplast trans-activation (PTA) screening, which enables simultaneous analysis of up to 95 individual transcription factor activities on a customizable promoter:LUCIFERASE reporter. This system is well suited to decipher complex transcriptional networks such as that triggered by the phytohormone auxin.

Identification of JAZ1-MYC2 Complex in Lotus corniculatus

Lotus corniculatus is exposed to many forms of biotic stresses, including pathogen and herbivore attack. Jasmonates (JAs) are lipid-based signaling molecules that play important roles in mediating secondary metabolite biosynthesis and plant defense responses. Here, we first characterized LcJAZ1 and LcMYC2, two key components of the JAs signaling pathway. Both LcJAZ1 and LcMYC2 mRNA accumulated quickly in response to JAs. Subcellular localization analysis showed that both LcJAZ1 and LcMYC2 are nuclear proteins. In addition, LcJAZ1 protein was degraded after JAs treatment, but LcMYC2 is not. Yeast two-hybrid, pull-down, and bimolecular fluorescence complementation assays demonstrated that LcJAZ1 interacts with LcMYC2 and AtMYC2. In addition, LcMYC2 interacts with AtJAZ1. The Arabidopsis protoplast transactivation assay revealed that LcJAZ1 acts as repressors of LcMYC2- or AtMYC2-mediated transcription. Our data indicate that the conserved JAZ-MYC complex contributes to the modulation of the JAs signaling pathway.

ASR5 is involved in the regulation of miRNA expression in rice.

The work describes an ASR knockdown transcriptomic analysis by deep sequencing of rice root seedlings and the transactivation of ASR cis-acting elements in the upstream region of a MIR gene. MicroRNAs are key regulators of gene expression that guide post-transcriptional control of plant development and responses to environmental stresses. ASR (ABA, Stress and Ripening) proteins are plant-specific transcription factors with key roles in different biological processes. In rice, ASR proteins have been suggested to participate in the regulation of stress response genes. This work describes the transcriptomic analysis by deep sequencing two libraries, comparing miRNA abundance from the roots of transgenic ASR5 knockdown rice seedlings with that of the roots of wild-type non-transformed rice seedlings. Members of 59 miRNA families were detected, and 276 mature miRNAs were identified. Our analysis detected 112 miRNAs that were differentially expressed between the two libraries. A predicted inverse correlation between miR167abc and its target gene (LOC_Os07g29820) was confirmed using RT-qPCR. Protoplast transactivation assays showed that ASR5 is able to recognize binding sites upstream of the MIR167a gene and drive its expression in vivo. Together, our data establish a comparative study of miRNAome profiles and is the first study to suggest the involvement of ASR proteins in miRNA gene regulation.

ETHYLENE RESPONSE FACTOR 96 positively regulates Arabidopsis resistance to necrotrophic pathogens by direct binding to GCC elements of jasmonate - and ethylene-responsive defence genes.

The ERF (ethylene responsive factor) family is composed of transcription factors (TFs) that are critical for appropriate Arabidopsis thaliana responses to biotic and abiotic stresses. Here we identified and characterized a member of the ERF TF group IX, namely ERF96, that when overexpressed enhances Arabidopsis resistance to necrotrophic pathogens such as the fungus Botrytis cinerea and the bacterium Pectobacterium carotovorum. ERF96 is jasmonate (JA) and ethylene (ET) responsive and ERF96 transcripts accumulation was abolished in JA-insensitive coi1-16 and in ET-insensitive ein2-1 mutants. Protoplast transactivation and electrophoresis mobility shift analyses revealed that ERF96 is an activator of transcription that binds to GCC elements. In addition, ERF96 mainly localized to the nucleus. Microarray analysis coupled to chromatin immunoprecipitation-PCR of Arabidopsis overexpressing ERF96 revealed that ERF96 enhances the expression of the JA/ET defence genes PDF1.2a, PR-3 and PR-4 as well as the TF ORA59 by direct binding to GCC elements present in their promoters. While ERF96-RNAi plants demonstrated wild-type resistance to necrotrophic pathogens, basal PDF1.2 expression levels were reduced in ERF96-silenced plants. This work revealed ERF96 as a key player of the ERF network that positively regulates the Arabidopsis resistance response to necrotrophic pathogens.

Functional characterization of the switchgrass (Panicum virgatum) R2R3‐MYB transcription factor PvMYB4 for improvement of lignocellulosic feedstocks

• The major obstacle for bioenergy production from switchgrass biomass is the low saccharification efficiency caused by cell wall recalcitrance. Saccharification efficiency is negatively correlated with both lignin content and cell wall ester-linked p-coumarate: ferulate (p-CA : FA) ratio. In this study, we cloned and functionally characterized an R2R3-MYB transcription factor from switchgrass and evaluated its potential for developing lignocellulosic feedstocks. • The switchgrass PvMYB4 cDNAs were cloned and expressed in Escherichia coli, yeast, tobacco and switchgrass for functional characterization. Analyses included determination of phylogenetic relations, in situ hybridization, electrophoretic mobility shift assays to determine binding sites in target promoters, and protoplast transactivation assays to demonstrate domains active on target promoters. • PvMYB4 binds to the AC-I, AC-II and AC-III elements of monolignol pathway genes and down-regulates these genes in vivo. Ectopic overexpression of PvMYB4 in transgenic switchgrass resulted in reduced lignin content and ester-linked p-CA : FA ratio, reduced plant stature, increased tillering and an approx. threefold increase in sugar release efficiency from cell wall residues. • We describe an alternative strategy for reducing recalcitrance in switchgrass by manipulating the expression of a key transcription factor instead of a lignin biosynthetic gene. PvMYB4-OX transgenic switchgrass lines can be used as potential germplasm for improvement of lignocellulosic feedstocks and provide a platform for further understanding gene regulatory networks underlying switchgrass cell wall recalcitrance.

High‐throughput protoplast transactivation (PTA) system for the analysis of Arabidopsis transcription factor function

Genomic approaches have generated large Arabidopsis open reading frame (ORF) collections. However, molecular tools are required to characterize this ORFeome functionally. A high-throughput microtiter plate-based protoplast transactivation (PTA) system has been established that can be used in a screening approach to define which transcription factor (TF) regulates a given promoter in planta. Using to this procedure, the transactivation properties of 96 TFs can be analyzed rapidly, making use of promoter:Luciferase (LUC)-reporters and luciferase imaging. Applying GATEWAY® technology, we have established a platform to assay more than 700 Arabidopsis TFs. As a proof-of-principle, the ethylene response factor (ERF) family has been studied to evaluate this system. Importantly, distinct subsets of related ERF factors were found to activate specifically the well described target promoters RD29A and PDF1.2 that are under control of DRE or GCC box cis-elements, respectively. Furthermore, several applications of the PTA system have been demonstrated, such as analysis of transcriptional repressors, salt-inducible gene expression or functional interaction of signaling molecules like kinases and TFs. This novel molecular tool will improve functional studies on transcriptional regulation in plants significantly.

WRKY Transcription Factors Involved in Activation of SA Biosynthesis Genes

BackgroundIncreased defense against a variety of pathogens in plants is achieved through activation of a mechanism known as systemic acquired resistance (SAR). The broad-spectrum resistance brought about by SAR is mediated through salicylic acid (SA). An important step in SA biosynthesis in Arabidopsis is the conversion of chorismate to isochorismate through the action of isochorismate synthase, encoded by the ICS1 gene. Also AVRPPHB SUSCEPTIBLE 3 (PBS3) plays an important role in SA metabolism, as pbs3 mutants accumulate drastically reduced levels of SA-glucoside, a putative storage form of SA. Bioinformatics analysis previously performed by us identified WRKY28 and WRKY46 as possible regulators of ICS1 and PBS3.ResultsExpression studies with ICS1 promoter::β-glucuronidase (GUS) genes in Arabidopsis thaliana protoplasts cotransfected with 35S::WRKY28 showed that over expression of WRKY28 resulted in a strong increase in GUS expression. Moreover, qRT-PCR analyses indicated that the endogenous ICS1 and PBS3 genes were highly expressed in protoplasts overexpressing WRKY28 or WRKY46, respectively. Electrophoretic mobility shift assays indentified potential WRKY28 binding sites in the ICS1 promoter, positioned -445 and -460 base pairs upstream of the transcription start site. Mutation of these sites in protoplast transactivation assays showed that these binding sites are functionally important for activation of the ICS1 promoter. Chromatin immunoprecipitation assays with haemagglutinin-epitope-tagged WRKY28 showed that the region of the ICS1 promoter containing the binding sites at -445 and -460 was highly enriched in the immunoprecipitated DNA.ConclusionsThe results obtained here confirm results from our multiple microarray co-expression analyses indicating that WRKY28 and WRKY46 are transcriptional activators of ICS1 and PBS3, respectively, and support this in silico screening as a powerful tool for identifying new components of stress signaling pathways.

β-Amylase–Like Proteins Function as Transcription Factors in Arabidopsis, Controlling Shoot Growth and Development

Plants contain β-amylase-like proteins (BAMs; enzymes usually associated with starch breakdown) present in the nucleus rather than targeted to the chloroplast. They possess BRASSINAZOLE RESISTANT1 (BZR1)-type DNA binding domains--also found in transcription factors mediating brassinosteroid (BR) responses. The two Arabidopsis thaliana BZR1-BAM proteins (BAM7 and BAM8) bind a cis-regulatory element that both contains a G box and resembles a BR-responsive element. In protoplast transactivation assays, these BZR1-BAMs activate gene expression. Structural modeling suggests that the BAM domain's glucan binding cleft is intact, but the recombinant proteins are at least 1000 times less active than chloroplastic β-amylases. Deregulation of BZR1-BAMs (the bam7bam8 double mutant and BAM8-overexpressing plants) causes altered leaf growth and development. Of the genes upregulated in plants overexpressing BAM8 and downregulated in bam7bam8 plants, many carry the cis-regulatory element in their promoters. Many genes that respond to BRs are inversely regulated by BZR1-BAMs. We propose a role for BZR1-BAMs in controlling plant growth and development through crosstalk with BR signaling. Furthermore, we speculate that BZR1-BAMs may transmit metabolic signals by binding a ligand in their BAM domain, although diurnal changes in the concentration of maltose, a candidate ligand produced by chloroplastic β-amylases, do not influence their transcription factor function.

Molecular screening tools to study Arabidopsis transcription factors

In the model plant Arabidopsis thaliana, more than 2000 genes are estimated to encode transcription factors (TFs), which clearly emphasizes the importance of transcriptional control. Although genomic approaches have generated large TF open reading frame (ORF) collections, only a limited number of these genes is functionally characterized, yet. This review evaluates strategies and methods to identify TF functions. In particular, we focus on two recently developed TF screening platforms, which make use of publically available GATEWAY®-compatible ORF collections. (1) The Arabidopsis thaliana TF ORF over-Expression (AtTORF-Ex) library provides pooled collections of transgenic lines over-expressing HA-tagged TF genes, which are suited for screening approaches to define TF functions in stress defense and development. (2) A high-throughput microtiter plate based protoplast trans activation (PTA) system has been established to screen for TFs which are regulating a given promoter:Luciferase construct in planta.