GCC Box Research Articles

Investigating the Role of OsHDT701 and Other Blast-Associated Negative Regulatory Genes in Indica Rice Cultivar Ranjit Using Combined Wet Lab and Computational Approaches.

Rice blast, caused by Magnaporthe oryzae, severely impacts global rice production. Understanding the role of the host's blast negative regulatory genes is crucial for combating this disease. We studied the expression of seven rice blast negative regulatory genes (previously characterized in ssp. japonica) in susceptible (Ranjit and Mahsuri) and tolerant/resistant (Shahsarang 1 and IR64) indica rice cultivars during M. oryzae infection. The selected genes showed differential expression patterns, with OsHDT701 (histone H4 deacetylase) downregulated in tolerant/resistant and upregulated in susceptible cultivars. Promoter analysis of OsHDT701 in cv. Ranjit revealed biotic stress-responsive and M. oryzae effector binding motifs (MoHTR2 and MoSPAB1). Protein-DNA docking showed an interaction for both the effectors and promoter sequences. Furthermore, a strong interaction between the OsERF922 transcription factor (a negative regulator of rice blast) and GCC box at the promoter suggests OsERF922-mediated regulation of OsHDT701. Next, a comparative analysis of ssp. japonica (Nipponbare) OsHDT701 CDS to 11 indica rice cultivars, including Ranjit, detected an SNP (T-A) at position 591. Moreover, the structure modeling and protein-protein interaction reveal that OsHDT701 may form a complex with other histone deacetylases to regulate defense-related gene expression. Taken together, our study unveils new possibilities for OsHDT701 regulation mechanisms during M. oryzae infection.

Comprehensive expression analysis of ERF transcription factors during chilling acclimation in Saintpaulia

AbstractSaintpaulia (Saintpaulia ionantha), a popular indoor ornamental potted plant, is native to the highlands of Kenya and Tanzania where temperatures rarely fall below 4 °C. Chilling injury during cultivation and transportation is a major commercial problem in Saintpaulia. In this study, we investigated chilling acclimation in Saintpaulia ‘Kilauea’. Plants grown at 20 °C (14 h light/10 h dark) displayed rapid and severe chilling injury after 24-h exposure to 4 °C. However, chilling injury at 4 °C could be dramatically reduced by pre-treating the plants at 10 °C but not at 6 °C. From whole genome analysis, 161 ethylene-responsive factors (ERFs) were identified and classified into 12 clades according to existing reports. Among these ERFs, 43, 8, and 4 ERFs were upregulated at 12, 24, and 48 h after 10 °C treatment, respectively. Most of these ERFs had GCC box and/or DRE/CRT core motifs-like sequences in their upstream regions. Finally, we compared the expression of ERFs between the treatments for 24 h at 10 °C, an effective temperature for chilling acclimation, and 6 °C, an ineffective temperature. The results showed that the expression of all six ERFs we investigated was increased by the 10 °C treatment, but not or only barely increased by the 6 °C treatment. This study suggests that Saintpaulia, a subtropical plant, can acclimate to low temperatures and that ERF upregulation is involved in chilling acclimation.

Nuclear factors NF-YC3 and NF-YBs positively regulate arbuscular mycorrhizal symbiosis in tomato.

The involvement of nuclear factor Y (NF-Y) in transcriptional reprogramming during arbuscular mycorrhizal symbiosis has been demonstrated in several plant species. However, a comprehensive picture is lacking. We showed that the spatial expression of NF-YC3 was observed in cortical cells containing arbuscules via the cis-regulatory element GCC boxes. Moreover, the NF-YC3 promoter was transactivated by the combination of CYCLOPS and autoactive calcium and calmodulin-dependent kinase (CCaMK) via GCC boxes. Knockdown of NF-YC3 significantly reduced the abundance of all intraradical fungal structures and affected arbuscule size. BCP1, SbtM1, and WRI5a, whose expression associated with NF-YC3 levels, might be downstream of NF-YC3. NF-YC3 interacted with NF-YB3a, NF-YB5c, or NF-YB3b, in yeast (Saccharomyces cerevisiae) and in planta, and interacted with NF-YA3a in yeast. Spatial expression of three NF-YBs was observed in all cell layers of roots under both mock and mycorrhizal conditions. Simultaneous knockdown of three NF-YBs, but not individually, reduced the fungal colonization level, suggesting that there might be functional redundancy of NF-YBs to regulate AM symbiosis. Collectively, our data suggest that NF-YC3 and NF-YBs positively regulate AM symbiosis in tomato, and arbuscule-related NF-YC3 may be an important downstream gene of the common symbiosis signaling pathway.

FaERF2 activates two β-1,3-glucanase genes to enhance strawberry resistance to Botrytis cinerea

Ethylene response factor (ERF) is a class of plant-specific transcription factors that play an important role in plant growth, development, and stress response. However, the underlying mechanism of strawberry ERFs in pathogenic responses against Botrytis cinerea (B. cinerea) remains largely unclear. In this study, we isolated FaERF2, a nucleus-localized ERF transcription factor from Fragaria x ananassa. Transiently overexpressing FaERF2 in strawberry fruits significantly enhances their resistant ability to B. cinerea, while silencing FaERF2 in strawberry fruits enhances their susceptibility to B. cinerea. In addition, we found that FaERF2 could directly bind to the cis-acting element GCC box in the promoters of two β-1,3-glucanase genes, FaBG-1 and FaBG-2, and activate their expression. Finally, both strawberry fruits transient expression followed by B. cinerea inoculation assays and recombinant protein incubation tests collectively substantiated the inhibitory effect of FaBG-1 and FaBG-2 on B. cinerea mycelium growth. These results revealed the molecular regulation mechanism of FaERF2 in response to B. cinerea and laid foundations for creating disease-resistance strawberry cultivar through genome editing approach.

Molecular regulation of oil gland development and biosynthesis of essential oils in Citrus spp.

Secretory structures in terrestrial plants serve as reservoirs for a variety of secondary metabolites. Among these, the secretory cavity of the Rutaceae family is notable for containing essential oils with a wide range of applications. However, the molecular basis underlying secretory cavity development is unknown. Here, we reveal a molecular framework for Citrus oil gland formation. Using genetic mapping and genome editing, we demonstrated that this process requires LATE MERISTEM IDENTITY1 (LMI1), a key regulator of leaf serration. A conserved GCC box element of the LMI1 promoter recruits DORNROSCHEN-like (DRNL) for transcriptional activation. This DRNL-LMI1 cascade triggers MYC5 activation, facilitating the development of oil glands and the biosynthesis of essential oils. Our findings spotlight cis-regulatory divergence within leaf shape genes, propelling novel functional tissue formation.

NtERF4 promotes the biosynthesis of chlorogenic acid and flavonoids by targeting PAL genes in Nicotiana tabacum.

Chlorogenic acid (CGA) and flavonoids are important secondary metabolites, which modulate plant growth and development, and contribute to plant resistance to various environmental stresses. ERF4 has been shown to be a repressor of anthocyanin accumulation in grape, but its full roles in regulating the biosynthesis of other phenylpropanoid compounds still needs to be further studied. In the present study, two NtERF4 genes were identified from N. tabacum genome. The expression level of NtERF4a was higher than that of NtERF4b in all the tobacco tissues examined. Over-expression of NtERF4a significantly promoted the accumulation of CGA and flavonoids in tobacco leaves, while silencing of NtERF4a significantly repressed the biosynthesis of CGA and flavonoids. RNA-seq analysis of NtERF4a-OE and WT plants revealed 8 phenylpropanoids-related differentially expressed genes (DEGs), including 4 NtPAL genes that encode key enzymes in the phenylpropanoid pathway. Activation of NtERF4a-GR fusion protein in tobacco significantly induced the transcription of NtPAL1 and NtPAL2 in the presence of protein synthesis inhibitor. Chromatin immunoprecipitation and Dual-Luc assays further indicated that NtERF4a could bind to the GCC box presented in the promoters of NtPAL1 and NtPAL2, thereby activating their transcription. Moreover, ectopic expression of NtERF4a induced the transcription of NtGSK1, NtMYC2, and NtJAZ3 genes, and enhanced the resistance of tobacco seedlings to salt and drought stresses, indicating multiple roles of NtERF4a in plants. Our findings revealed new roles of NtERF4a in modulating the accumulation of phenylpropanoid compounds in tobacco, and provided a putative target for improving phenylpropanoids synthesis and stress resistance in plants.

Knockout of transcription factor OsERF65 enhances ROS scavenging ability and confers resistance to rice sheath blight.

Rice sheath blight (ShB) is a devastating disease that severely threatens rice production worldwide. Induction of cell death represents a key step during infection by the ShB pathogen Rhizoctonia solani. Nonetheless, the underlying mechanisms remain largely unclear. In the present study, we identified a rice transcription factor, OsERF65, that negatively regulates resistance to ShB by suppressing cell death. OsERF65 was significantly upregulated by R. solani infection in susceptible cultivar Lemont and was highly expressed in the leaf sheath. Overexpression of OsERF65 (OsERF65OE) decreased rice resistance, while the knockout mutant (oserf65) exhibited significantly increased resistance against ShB. The transcriptome assay revealed that OsERF65 repressed the expression of peroxidase genes after R. solani infection. The antioxidative enzyme activity was significantly increased in oserf65 plants but reduced in OsERF65OE plants. Consistently, hydrogen peroxide content was apparently reduced in oserf65 plants but accumulated in OsERF65OE plants. OsERF65 directly bound to the GCC box in the promoter regions of four peroxidase genes and suppressed their transcription, reducing the ability to scavenge reactive oxygen species (ROS). The oserf65 mutant exhibited a slight decrease in plant height but increased grain yield. Overall, our results revealed an undocumented role of OsERF65 that acts as a crucial regulator of rice resistance to R. solani and a potential target for improving both ShB resistance and rice yield.

The MKK3–MPK7 cascade phosphorylates ERF4 and promotes its rapid degradation to release seed dormancy in Arabidopsis

Seeds establish dormancy to delay germination until the arrival of a favorable growing season. In this study, we identify a fate switch comprised of the MKK3–MPK7 kinase cascade and the ethylene response factor ERF4 that is responsible for the seed state transition from dormancy to germination. We show that dormancy-breaking factors activate the MKK3–MPK7 module, which affects the expression of some α-EXPANSIN (EXPA) genes to control seed dormancy. Furthermore, we identify a direct downstream substrate of this module, ERF4, which suppresses the expression of these EXPAs by directly binding to the GCC boxes in their exon regions. The activated MKK3–MPK7 module phosphorylates ERF4, leading to its rapid degradation and thereby releasing its inhibitory effect on the expression of these EXPAs. Collectively, our work identifies a signaling chain consisting of protein phosphorylation, degradation, and gene transcription , by which the germination promoters within the embryo sense and are activated by germination signals from ambient conditions.

The transcription factor NtERF13a enhances abiotic stress tolerance and phenylpropanoid compounds biosynthesis in tobacco

The AP2/ERF (APETALA2/ETHYLENE RESPONSE FACTOR) transcription factors play multiple roles in modulating the biosynthesis of diverse specialized metabolites in response to various environmental stresses. ERF13 has been shown to participate in plant resistance to biotic stress as well as in repressing the synthesis of fatty acid. However, its full roles in regulating plant metabolism and stress resistance still remains to be further studied. In this study, we identified two NtERF genes from N. tabacum genome that belong to Ⅸa subgroup of ERF family. Over-expression and knock-out of NtERF13a showed that NtERF13a could enhance plant resistance to salt and drought stresses, as well as promoted the biosynthesis of chlorogenic acid (CGA), flavonoids, and lignin in tobacco. Transcriptome analysis between WT and NtERF13a-OE plants revealed 6 differentially expressed genes (DEGs) that encode enzymes catalyzing the key steps of phenylpropanoid pathway. Chromatin immunoprecipitation, Y1H, and Dual-Luc assays further clarified that NtERF13a could directly bind to the fragments containing GCC box or DRE element in the promoters of NtHCT, NtF3'H, and NtANS genes to induce the transcription of these genes. Knock-out of NtHCT, NtF3'H, or NtANS in the NtERF13a-OE background significantly repressed the increase of phenylpropanoid compound contents caused by over-expression of NtERF13a, indicating that the promotion of NtERF13a on the phenylpropanoid compound contents depends on the activity of NtHCT, NtF3'H, and NtANS. Our study demonstrated new roles of NtERF13a in promoting plant resistance to abiotic stresses, and provided a promising target for modulating the biosynthesis of phenylpropanoid compounds in tobacco.

Overexpression of OsERF106MZ promotes parental root growth in rice seedlings by relieving the ABA-mediated inhibition of root growth under salinity stress conditions

BackgroundRoots are essential for plant growth and have a variety of functions, such as anchoring the plant to the ground, absorbing water and nutrients from the soil, and sensing abiotic stresses, among others. OsERF106MZ is a salinity-induced gene that is expressed in germinating seeds and rice seedling roots. However, the roles of OsERF106MZ in root growth remain poorly understood.ResultsHistochemical staining to examine β-glucuronidase (GUS) activity in transgenic rice seedlings harboring OsERF106MZp::GUS indicated that OsERF106MZ is mainly expressed in the root exodermis, sclerenchyma layer, and vascular system. OsERF106MZ overexpression in rice seedlings leads to an increase in primary root (PR) length. The phytohormone abscisic acid (ABA) is thought to act as a hidden architect of root system structure. The expression of the ABA biosynthetic gene OsAO3 is downregulated in OsERF106MZ-overexpressing roots under normal conditions, while the expression of OsNPC3, an AtNPC4 homolog involved in ABA sensitivity, is reduced in OsERF106MZ-overexpressing roots under both normal and NaCl-treated conditions. Under normal conditions, OsERF106MZ-overexpressing roots show a significantly reduced ABA level; moreover, exogenous application of 1.0 µM ABA can suppress OsERF106MZ-mediated root growth promotion. Additionally, OsERF106MZ-overexpressing roots display less sensitivity to ABA-mediated root growth inhibition when treated with 5.0 µM ABA under normal conditions or exposed to NaCl-treated conditions. Furthermore, chromatin immunoprecipitation (ChIP)-qPCR and luciferase (LUC) reporter assays showed that OsERF106MZ can bind directly to the sequence containing the GCC box in the promoter region of the OsAO3 gene and repress the expression of OsAO3.ConclusionsOsERF106MZ may play a role in maintaining root growth for resource uptake when rice seeds germinate under salinity stress by alleviating ABA-mediated root growth inhibition.

The transcription factors VaERF16 and VaMYB306 interact to enhance resistance of grapevine to Botrytis cinerea infection.

Botrytis cinerea is a fungus that infects cultivated grape (Vitis vinifera); the identification and characterization of resistance mechanisms in the host is of great importance for the grape industry. Here, we report that a transcription factor in the ethylene‐responsive factor (ERF) family (VaERF16) from Chinese wild grape (Vitis amurensis ‘Shuang You’) is expressed during B. cinerea infection and in response to treatments with the hormones ethylene and methyl jasmonate. Heterologous overexpression of VaERF16 in Arabidopsis thaliana substantially enhanced resistance to B. cinerea and the bacterium Pseudomonas syringae DC3000 via the salicylic acid and jasmonate/ethylene signalling pathways. Yeast two‐hybrid, bimolecular fluorescence complementation, and co‐immunoprecipitation assays indicated that VaERF16 interacts with the MYB family transcription factor VaMYB306. Overexpression of VaERF16 or VaMYB306 in grape leaves increased resistance to B. cinerea and caused an up‐regulation of the defence‐related gene PDF1.2, which encodes a defensin‐like protein. Conversely, silencing of either gene resulted in increased susceptibility to B. cinerea. Yeast one‐hybrid and dual‐luciferase assays indicated that VaERF16 increased the transcript levels of VaPDF1.2 by binding directly to the GCC box in its promoter. Notably, VaMYB306 alone did not bind to the VaPDF1.2 promoter, but the VaERF16–VaMYB306 transcriptional complex resulted in higher transcript levels of VaPDF1.2, suggesting that the proteins function through their mutual interaction. Elucidation of this regulatory module may be of value in enhancing resistance of grapevine to B. cinerea infection.

Function Analysis of the ERF and DREB Subfamilies in Tomato Fruit Development and Ripening.

APETALA2/ethylene responsive factors (AP2/ERF) are unique regulators in the plant kingdom and are involved in the whole life activity processes such as development, ripening, and biotic and abiotic stresses. In tomato (Solanum lycopersicum), there are 140 AP2/ERF genes; however, their functionality remains poorly understood. In this work, the 14th and 19th amino acid differences in the AP2 domain were used to distinguish DREB and ERF subfamily members. Even when the AP2 domain of 68 ERF proteins from 20 plant species and motifs in tomato DREB and ERF proteins were compared, the binding ability of DREB and ERF proteins with DRE/CRT and/or GCC boxes remained unknown. During fruit development and ripening, the expressions of 13 DREB and 19 ERF subfamily genes showed some regular changes, and the promoters of most genes had ARF, DRE/CRT, and/or GCC boxes. This suggests that these genes directly or indirectly respond to IAA and/or ethylene (ET) signals during fruit development and ripening. Moreover, some of these may feedback regulate IAA or ET biosynthesis. In addition, 16 EAR motif-containing ERF genes in tomato were expressed in many organs and their total transcripts per million (TPM) values exceeded those of other ERF genes in most organs. To determine whether the EAR motif in EAR motif-containing ERF proteins has repression function, their EAR motifs were retained or deleted in a yeast one-hybrid (YIH) assay. The results indicate that most of EAR motif-containing ERF proteins lost repression activity after deleting the EAR motif. Moreover, some of these were expressed during ripening. Thus, these EAR motif-containing ERF proteins play vital roles in balancing the regulatory functions of other ERF proteins by completing the DRE/CRT and/or GCC box sites of target genes to ensure normal growth and development in tomato.

AP2/ERF Transcription Factor NbERF-IX-33 Is Involved in the Regulation of Phytoalexin Production for the Resistance of Nicotiana benthamiana to Phytophthora infestans.

Plants recognize molecular patterns unique to a certain group of microbes to induce effective resistance mechanisms. Elicitins are secretory proteins produced by plant pathogenic oomycete genera including Phytophthora and Pythium. Treatment of INF1 (an elicitin produced by P. infestans) induces a series of defense responses in Nicotiana species, including reactive oxygen species (ROS) production, transient induction of ethylene production, hypersensitive cell death and accumulation of the sesquiterpenoid phytoalexin capsidiol. In this study, we analyzed the expression profiles of N. benthamiana genes after INF1 treatment by RNAseq analysis. Based on their expression patterns, N. benthamiana genes were categorized into 20 clusters and 4,761 (8.3%) out of 57,140 genes were assigned to the clusters for INF1-induced genes. All genes encoding enzymes dedicated to capsidiol production, 5-epi-aristolochene (EA) synthase (NbEAS, 10 copies) and EA dehydrogenase (NbEAH, 6 copies), and some genes for ethylene production, such as 1-aminocyclopropane 1-carboxylate (ACC) synthase (NbACS) and ACC oxidase (NbACO), were significantly upregulated by INF1 treatment. Analysis of NbEAS1 and NbEAS4 promoters revealed that AGACGCC (GCC box-like motif) is the essential cis-element required for INF1-induced expression of NbEAS genes. Given that the GCC box is known to be targeted by ERF (ethylene-responsive factor) transcription factors, we created a complete list of N. benthamiana genes encoding AP2/ERF family transcription factors, and identified 45 out of 337 AP2/ERF genes in the clusters for INF1-induced genes. Among INF1-induced NbERF genes, silencing of NbERF-IX-33 compromised resistance against P. infestans and INF1-induced production of capsidiol. Recombinant NbERF-IX-33 protein can bind to the promoter sequence of NbEAS4, suggesting that NbERF-IX-33 is a transcription factor directly regulating the expression of genes for phytoalexin production.

LcERF19, an AP2/ERF transcription factor from Litsea cubeba, positively regulates geranial and neral biosynthesis.

The APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factors (TFs) are involved in the regulation of specialized terpenoid biosynthesis. However, the AP2/ERF TFs in Litsea cubeba have not been characterized and their role in the biosynthesis of terpenoids is unknown. Here, 174 LcAP2/ERF TFs were identified in L. cubeba and categorized into four subfamilies: 27 AP2, 7 RAV, 1 Soloist, and 139 ERF. Transcriptomic and qRT-PCR assays both showed that the expression levels of LcERF19 were similar to that of terpene synthase LcTPS42 in the pericarp, which is related to the synthesis of geranial and neral in L. cubeba. LcERF19 was further shown to encode a nuclear-localized protein and its expression was strongly induced by jasmonate. Yeast one-hybrid and dual-luciferase assays showed that LcERF19 associated with GCC box elements of the LcTPS42 promoter and promoted its activity. Transient overexpression of LcERF19 in L. cubeba and overexpression of LcERF19 in tomato resulted in a significant increase in geranial and neral. Our findings show that LcERF19 enhances geranial and neral biosynthesis through activation of LcTPS42 expression, which provides a strategy to improve the flavor of tomato and other fruits.

The key regulator LcERF056 enhances salt tolerance by modulating reactive oxygen species-related genes in Lotus corniculatus

BackgroundThe APETALA2/ethylene response factor (AP2/ERF) family are important regulatory factors involved in plants’ response to environmental stimuli. However, their roles in salt tolerance in Lotus corniculatus remain unclear.ResultsHere, the key salt-responsive transcription factor LcERF056 was cloned and characterised. LcERF056 belonging to the B3–1 (IX) subfamily of ERFs was considerably upregulated by salt treatment. LcERF056-fused GFP was exclusively localised to nuclei. Furthermore, LcERF056- overexpression (OE) transgenic Arabidopsis and L. corniculatus lines exhibited significantly high tolerance to salt treatment compared with wild-type (WT) or RNA interference expression (RNAi) transgenic lines at the phenotypic and physiological levels. Transcriptome analysis of OE, RNAi, and WT lines showed that LcERF056 regulated the downstream genes involved in several metabolic pathways. Chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) and yeast one-hybrid (Y1H) assay demonstrated that LcERF056 could bind to cis-element GCC box or DRE of reactive oxygen species (ROS)-related genes such as lipid-transfer protein, peroxidase and ribosomal protein.ConclusionOur results suggested that the key regulator LcERF056 plays important roles in salt tolerance in L. corniculatus by modulating ROS-related genes. Therefore, it may be a useful target for engineering salt-tolerant L. corniculatus or other crops.

The transcription factor ORA59 exhibits dual DNA binding specificity that differentially regulates ethylene- and jasmonic acid-induced genes in plant immunity.

Jasmonic acid (JA) and ethylene (ET) signaling modulate plant defense against necrotrophic pathogens in a synergistic and interdependent manner, while JA and ET also have independent roles in certain processes, e.g. in responses to wounding and flooding, respectively. These hormone pathways lead to transcriptional reprogramming, which is a major part of plant immunity and requires the roles of transcription factors. ET response factors are responsible for the transcriptional regulation of JA/ET-responsive defense genes, of which ORA59 functions as a key regulator of this process and has been implicated in the JA-ET crosstalk. We previously demonstrated that Arabidopsis (Arabidopsis thaliana) GDSL LIPASE 1 (GLIP1) depends on ET for gene expression and pathogen resistance. Here, promoter analysis of GLIP1 revealed ERELEE4 as the critical cis-element for ET-responsive GLIP1 expression. In a yeast one-hybrid screening, ORA59 was isolated as a specific transcription factor that binds to the ERELEE4 element, in addition to the well-characterized GCC box. We found that ORA59 regulates JA/ET-responsive genes through direct binding to these elements in gene promoters. Notably, ORA59 exhibited a differential preference for GCC box and ERELEE4, depending on whether ORA59 activation is achieved by JA and ET, respectively. JA and ET induced ORA59 phosphorylation, which was required for both activity and specificity of ORA59. Furthermore, RNA-seq and virus-induced gene silencing analyses led to the identification of ORA59 target genes of distinct functional categories in JA and ET pathways. Our results provide insights into how ORA59 can generate specific patterns of gene expression dynamics through JA and ET hormone pathways.

A Rapid Pipeline for Pollen- and Anther-Specific Gene Discovery Based on Transcriptome Profiling Analysis of Maize Tissues.

Recently, crop breeders have widely adopted a new biotechnology-based process, termed Seed Production Technology (SPT), to produce hybrid varieties. The SPT does not produce nuclear male-sterile lines, and instead utilizes transgenic SPT maintainer lines to pollinate male-sterile plants for propagation of nuclear-recessive male-sterile lines. A late-stage pollen-specific promoter is an essential component of the pollen-inactivating cassette used by the SPT maintainers. While a number of plant pollen-specific promoters have been reported so far, their usefulness in SPT has remained limited. To increase the repertoire of pollen-specific promoters for the maize community, we conducted a comprehensive comparative analysis of transcriptome profiles of mature pollen and mature anthers against other tissue types. We found that maize pollen has much less expressed genes (>1 FPKM) than other tissue types, but the pollen grain has a large set of distinct genes, called pollen-specific genes, which are exclusively or much higher (100 folds) expressed in pollen than other tissue types. Utilizing transcript abundance and correlation coefficient analysis, 1215 mature pollen-specific (MPS) genes and 1009 mature anther-specific (MAS) genes were identified in B73 transcriptome. These two gene sets had similar GO term and KEGG pathway enrichment patterns, indicating that their members share similar functions in the maize reproductive process. Of the genes, 623 were shared between the two sets, called mature anther- and pollen-specific (MAPS) genes, which represent the late-stage pollen-specific genes of the maize genome. Functional annotation analysis of MAPS showed that 447 MAPS genes (71.7% of MAPS) belonged to genes encoding pollen allergen protein. Their 2-kb promoters were analyzed for cis-element enrichment and six well-known pollen-specific cis-elements (AGAAA, TCCACCA, TGTGGTT, [TA]AAAG, AAATGA, and TTTCT) were found highly enriched in the promoters of MAPS. Interestingly, JA-responsive cis-element GCC box (GCCGCC) and ABA-responsive cis-element-coupling element1 (ABRE-CE1, CCACC) were also found enriched in the MAPS promoters, indicating that JA and ABA signaling likely regulate pollen-specific MAPS expression. This study describes a robust and straightforward pipeline to discover pollen-specific promotes from publicly available data while providing maize breeders and the maize industry a number of late-stage (mature) pollen-specific promoters for use in SPT for hybrid breeding and seed production.

AtMPK6-induced phosphorylation of AtERF72 enhances its DNA binding activity and interaction with TGA4/OBF4 in Arabidopsis.

The ethylene-responsive element binding factor (ERF) family is a large family of transcription factors involved in plant development and environmental stress responses. We previously reported the identification of 29 putative substrates of Mitogen-activated Protein Kinase3 (AtMPK3), AtMPK4 and AtMPK6, based on a solid-phase phosphorylation screening using a lambda phage expression library in Arabidopsis thaliana. In this study, a putative MPK substrate, AtERF72 (At3g16770), was strongly phosphorylated by AtMPK6 on the serine residue at position 151 (Ser151). AtERF72 binds to the GCC box (AGCCGCC) in the promoters of several pathogenesis-related (PR) genes and activates their transcription. We also show that the DNA-binding activity of AtERF72 is enhanced upon phosphorylation by AtMPK6 in vitro. In addition, transient co-expression experiments in Arabidopsis protoplasts revealed that effector constructs expressing a mutant variant of AtERF72, AtERF72S151D (carrying a Ser to aspartic acid [Asp] substitution at amino acid position 151) showed higher expression of the β-glucuronidase (GUS) reporter gene driven by the GCC box element than effector constructs expressing the wild-type AtERF72. Furthermore, yeast two-hybrid assays revealed that the interaction between AtERF72S151D and TGA4/OBF4 was stronger than that between wild-type AtERF72 and TGA4/OBF4. Since AtERF72S151D is equivalent to AtERF72 phosphorylated by AtMPK6 at Ser151, these results suggest that the phosphorylation of AtERF72 by AtMPK6 triggers an event of transcriptional regulation from defence signalling in Arabidopsis.

Transcriptome profiling reveals the occurrence mechanism of bisexual flowers in melon (Cucumis melo L.)

Most cultivated melons are andromonoecies in which male flowers arose both in main stem and lateral branches but bisexual flowers only emerged from the leaf axils of lateral branches. However, bisexual flowers emerged in leaf axils of main stem after ethephon treatment. Therefore, the mechanism regulating the occurrence of bisexual flowers were investigated by performing transcriptome analysis in two comparison sets: shoot apex of main stem (MA) versus that of lateral branches (LA), and shoot apex of main stem after ethephon treatment (Eth) versus control (Cont). KEGG results showed that genes involved in “plant hormone signal transduction”, “MAPK signaling pathway” and “carbon metabolism” were significantly upregulated both in LA and Eth. Further, details of DEGs involved in ethylene signaling pathway were surveyed and six genes were co-upregulated in two comparison sets. Among these, CmERF1, downstream in ethylene signaling pathway, showed the most significantly difference and expressed higher in bisexual buds than that in male buds. Furthermore, fifteen DEGs were found to contain GCC box or CRT/DRE cis-element for CmERF1 in their putative promoter region, and these DEGs involved in several plant hormones signaling pathway, camalexin synthesis, carbon metabolism and plant pathogen interaction.

Two Alternative Splicing Variants of AtERF73/HRE1, HRE1α and HRE1β, Have Differential Transactivation Activities in Arabidopsis.

AtERF73/HRE1 is an AP2/ERF transcription factor in Arabidopsis and has two distinct alternative splicing variants, HRE1α and HRE1β. In this study, we examined the differences between the molecular functions of HRE1α and HRE1β. We found that HRE1α and HRE1β are both involved in hypoxia response and root development and have transactivation activity. Two conserved motifs in the C-terminal region of HRE1α and HRE1β, EELL and LWSY-like, contributed to their transactivation activity, specifically the four E residues in the EELL motif and the MGLWS amino acid sequence at the end of the LWSY-like motif. The N-terminal region of HRE1β also showed transactivation activity, mediated by the VDDG motif, whereas that of HRE1α did not. The transactivation activity of HRE1β was stronger than that of HRE1α in Arabidopsis protoplasts. Both transcription factors transactivated downstream genes via the GCC box. RNA-sequencing analysis further supported that both HRE1α and HRE1β might regulate gene expression associated with the hypoxia stress response, although they may transactivate different subsets of genes in downstream pathways. Our results, together with previous studies, suggested that HRE1α and HRE1β differentially transactivate downstream genes in hypoxia response and root development in Arabidopsis.