ERF Transcription Factors Research Articles

High temperature reduces peel color in eggplant (Solanum melongena) as revealed by RNA-seq analysis.

To acquire the eggplant transcriptome under high temperature stress, 18 cDNA libraries were constructed and sequenced. A total of 136.31 Gb of clean data was obtained, and 88.86%-92.35% of the clean reads were mapped to the eggplant reference genome. Under high temperature, the number of down-regulated genes was more than that of up-regulated genes and there were more differentially expressed genes on the 10th day after flowering than on the 15th and 20th days after flowering. On the 10th day after flowering, the key genes CHI, 3GT, F3'5'H, DFR2, ANS, and F3H in anthocyanin synthetic pathway and most ERF, WRKY, bHLH, and MYB transcription factors were all down-regulated. High temperature significantly decreased the total anthocyanin content in peels. The results showed that at the early stage of peel coloring, high temperature inhibited the expressions of key genes in anthocyanin biosynthetic pathways through the regulation of transcription factors, leading to a significant decrease in total anthocyanin content, which might reduce the peel color in eggplant.

OsERF101, an ERF family transcription factor, regulates drought stress response in reproductive tissues.

An ERF transcription factor OsERF101 is predominantly expressed in rice reproductive tissues and plays an important role in improving rice seed setting rate under drought stress. Drought reduces grain yield due to the cumulative damage effects to plant vegetative and reproductive developmental processes. However, the genes involved in these processes are still not completely understood. In this study, we identified a gene named OsERF101 as an important positive regulator in the adaptive responses to dehydration stress during the reproductive and vegetative stages. This gene encodes a member of APETALA2/Ethylene-Responsive Element Binding Protein (AP2/EREBP) family. OsERF101 was predominantly expressed in flowers, particularly in the tapetum and microspores under normal growth conditions. It was induced by drought, PEG6000 and abscisic acid (ABA) in leaves. During the vegetative stage, OsERF101-overexpression plants were more resistant to osmotic stress caused by PEG6000 compared to the control plants. They also had higher survival and seed setting rates than wild type when subjected to reproductive-stage drought stress. Further physiological analysis revealed that the pollen fertility was improved in the overexpression lines, while the knockout mutant and RNAi lines showed reduced pollen fertility and compromised drought tolerance during the reproductive stage. The increased proline content and peroxidase activity in OsERF101-overexpression plants might contribute to the improved drought-tolerance of plants. In addition, OsERF101-overexpression plants displayed ABA susceptible phenotype, in which the expression levels of ABA-responsive genes RD22, LEA3, and PODs were up-regulated. Taken together, our results indicate that OsERF101 is a gene that regulates dehydration responses during the vegetative and reproductive stages.

Comprehensive panicle phenotyping reveals that qSrn7/FZP influences higher-order branching

Rice grain number directly affects crop yield. Identifying alleles that improve panicle architecture would greatly aid the development of high-yield varieties. Here, we show that the quantitative trait locus qSrn7 contains rice FRIZZY PANICLE (FZP), a previously reported gene encoding an ERF transcription factor that promotes floral transition. Reduced expression of FZP in the reproductive stage increases the extent of higher order branching of the panicle, resulting in increased grain number. Genotype analysis of this gene in cultivars from the publicly available National Institute of Agrobiological Sciences (NIAS) Core Collection demonstrated that the extent of higher order branching, especially in the upper panicle, was increased in those cultivars carrying the FZP allele associated with qSrn7. Furthermore, chromosome segment substitution lines resulting from a cross between Koshihikari and Kasalath, the latter of which carries qSrn7/FZP, also showed that upper panicle higher order branching and grain yield were increased by qSrn7/FZP. Our findings indicate that qSrn7/FZP influences panicle branching pattern and is thus useful in the breeding of high-yield rice varieties.

A tomato ERF transcription factor, SlERF84, confers enhanced tolerance to drought and salt stress but negatively regulates immunity against Pseudomonas syringae pv. tomato DC3000

ERF proteins are plant-specific transcription factors that play significant roles in plant defense against various stresses. However, only little information regarding stress-related ERF genes is available in tomato (Solanum lycopersicum, Sl). In this study, a tomato ERF gene, SlERF84, was cloned and functionally characterized. The nucleus localization of SlERF84-sGFP was confirmed through a transient expression assay. Transactivation assays in yeast demonstrated that SlERF84 functions as a transcriptional activator. Real-time PCR analysis revealed that SlERF84 could be markedly induced by drought, salt and by several phytohormones (ABA, MeJA and ACC). Overexpression of SlERF84 in Arabidopsis endows transgenic plants with ABA hypersensitivity and enhanced tolerance to drought and salt stress. Histochemical staining assay showed that SlERF84 renders transgenic plants better ROS-scavenging capability. Pathogen inoculation assay revealed that SlERF84 might negatively modulate plant defense response to Pseudomonas syringae pv. tomato DC3000. Moreover, the transcript levels of pathogenesis-related genes AtPR1 and AtPR3 were compromised in transgenic Arabidopsis, as compared to that in Col-0 plants when inoculated with Pseudomonas syringae pv. tomato DC3000. These results suggest that SlERF84 functions as a stress-responsive transcription factor in differentially modulation of abiotic and biotic stress tolerance, and may have applications in the engineering of economically important crops.

Comparative Analysis of Transcriptomes to Identify Genes Associated with Fruit Size in the Early Stage of Fruit Development in Pyrus pyrifolia.

Pear (Pyrus L.) is an important commercial fruit in the world. The fruit size is one of the important characters in fruit quality. The previous research reported that the fruit size of pear was mainly caused by the number of cell in about 40 days after blossom (DAB) in nature. However, studies about the mechanisms underlying cell division in young fruit development are very limited in pear. Two pear accessions codenamed ‘GH59B’ with big fruit and ‘GH81S’ with small fruit in three stages were sampled and the RNA-seq high-throughput sequencing was used to evaluate changes of gene transcription levels in the early stage of fruit development. The difference of cell size among two samples was little in 40 DAB, implying that the difference of the fruit size was caused by the number of the cell. More than 274,517,982 high quality reads from six libraries of fruit development were sequenced. A total of 797 differentially expressed genes (DEGs) were identified. Three cytokinin dehydrogenase genes and two gibberellin 2-beta-dioxygenase gene were identified in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to zeatin and gibberellin. Their expression was upregulated at 20 DAB in ‘GH81S’ and at 30 DAB in ‘GH59B’, suggesting that the small fruit size might be related to the early degradation of cytokinin and gibberellin inducing a short period of cell division. A total of 38 DEGs of transcription factors were found and 23 DEGs including NAC, ERF and bHLH transcription factors were highly related with cytokinin dehydrogenase and gibberellin dioxygenase genes. Altogether, the results of the present study provide information from a comprehensive gene expression analysis and insight into the molecular mechanism underlying the difference of fruit size in Pyrus pyrifolia.

Expression of grape ACS1 in tomato decreases ethylene and alters the balance between auxin and ethylene during shoot and root formation

Ethylene plays an important role in the grape rachis, where its production can be 10 times higher than in the berry. VvACS1 is the only rachis-specific ACC synthase (ACS) gene, and its expression is coincident with ethylene production in the rachis of Vitis vinifera ‘Thompson seedless’. VvACS1 was cloned and ectopically expressed in tomato (Solanum lycopersicum ‘Moneymaker’). Lateral buds were increased in two- or four-week-old 35s∷VvACS1 transgenic tomato plants after transplanting. Compared with wild-type (WT) plants, the transgenic tomato plants showed higher expression of the VvACS1 gene in the flowers, leaves, rachis, and fruits. There was no obvious difference of ACS activity in the fruit of tomato, and only increased ACS activity in the rachis of tomato. Ethylene production was decreased in flowers, leaves, and fruits (seven weeks after full bloom), while the relative expression of endogenous tomato ACS1 and ACS6 genes was not down-regulated by the ectopic expression of VvACS1. These results imply that post-transcriptional or post-translational regulation of ACS may occur, resulting in lower ethylene production in the transgenic tomato plants. Moreover, expression of VvACS1 in tomato resulted in decreased auxin and increased zeatin contents in the lateral buds, as well as reduced or delayed formation of adventitious roots in lateral bud cuttings. RNA-Seq and qRT-PCR analyses of rooted lateral bud cuttings indicated that the relative expression levels of the genes for zeatin O-glucosyltransferase-like, auxin repressed/dormancy-associated protein, and ERF transcription factors were higher in transgenic tomatoes than in WT, suggesting that ethylene may regulate auxin transport and distribution in shoots and that adventitious root formation employs coordination between auxin and ethylene.

PsnERF75 Transcription Factor from Populus simonii × P. nigra Confers Salt Tolerance in Transgenic Arabidopsis

ERF transcription factor involves in many aspects of plant development and response to both biotic and abiotic environmental stimuli. In this study, we provided evidence that PsnERF75 can improve the salt tolerance ability of transgenic Arabidopsis. RT-qPCR analysis described the spatio-temporal expression patterns of PsnERF75 in different poplar tissues. Under salt stress condition, root and leaf tissues were more sensitive than stem tissue. In addition, seed germination rates of PsnERF75 transgenic Arabidopsis were significantly enhanced. Growth status of PsnERF75 transgenic Arabidopsis seedlings was much better than that of the controls. Physiological and histochemical assays indicated PsnERF75 transgenic Arabidopsis have a greater capacity to eliminate reactive oxygen species and lighten the damages to plants than the controls. Using RNA-seq analysis, 100 DEGs that were significant differentially expressed between PsnERF75 transgenic Arabidopsis and the controls under salt stress conditions were identified. Seventeen upregulated and 10 down-regulated DEGs participate in plant salt stress response process. In addition, subcellular localization showed that PsnERF75 is a nuclear-localized protein. All of the results will provide novel insights into the functions of ERF75 in plants under abiotic stress condition.

Screening of proteins interacting with ERF transcriptional factor from Populus simonii × P.nigra by yeast two-hybrid method

ABSTRACTTo investigate the biology functions of ERF transcriptional factor in Populus simonii × P.nigra, the proteins interacting with ERF were screened by the yeast two-hybrid method. The bait vector harbouring ERF and cDNA library of Populus simonii × P.nigra were constructed. The bait had no self-activation and toxicity to yeast cells in the yeast two-hybrid system. The library titering experiment indicated that it could meet the requirement for the yeast two-hybrid library screen. Five proteins that interacted with ERF were obtained, including galactinol-sucrose galactosyl transferase 2, phosphoinositide-specific phospholipase C family protein, MLP-like protein, senescence-associated protein and an unknown protein. Real-time polymerase chain reaction showed that the expression levels of ERF and five interacting protein genes increased after salt treatment. Gene ontology analysis associated most of the interacting proteins with stress response. These results showed that ERF might share similar function with these proteins and they act together to regulate the salt response of Populus simonii × P.nigra. This study provided experimental evidence for further revealing the biological function of the ERF transcription factor in poplar.

Ethylene-Related Gene Expression Networks in Wood Formation.

Thickening of tree stems is the result of secondary growth, accomplished by the meristematic activity of the vascular cambium. Secondary growth of the stem entails developmental cascades resulting in the formation of secondary phloem outwards and secondary xylem (i.e., wood) inwards of the stem. Signaling and transcriptional reprogramming by the phytohormone ethylene modifies cambial growth and cell differentiation, but the molecular link between ethylene and secondary growth remains unknown. We addressed this shortcoming by analyzing expression profiles and co-expression networks of ethylene pathway genes using the AspWood transcriptome database which covers all stages of secondary growth in aspen (Populus tremula) stems. ACC synthase expression suggests that the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) is synthesized during xylem expansion and xylem cell maturation. Ethylene-mediated transcriptional reprogramming occurs during all stages of secondary growth, as deduced from AspWood expression profiles of ethylene-responsive genes. A network centrality analysis of the AspWood dataset identified EIN3D and 11 ERFs as hubs. No overlap was found between the co-expressed genes of the EIN3 and ERF hubs, suggesting target diversification and hence independent roles for these transcription factor families during normal wood formation. The EIN3D hub was part of a large co-expression gene module, which contained 16 transcription factors, among them several new candidates that have not been earlier connected to wood formation and a VND-INTERACTING 2 (VNI2) homolog. We experimentally demonstrated Populus EIN3D function in ethylene signaling in Arabidopsis thaliana. The ERF hubs ERF118 and ERF119 were connected on the basis of their expression pattern and gene co-expression module composition to xylem cell expansion and secondary cell wall formation, respectively. We hereby establish data resources for ethylene-responsive genes and potential targets for EIN3D and ERF transcription factors in Populus stem tissues, which can help to understand the range of ethylene targeted biological processes during secondary growth.

Hydrogen Peroxide-Induced Oxidative Stress in Sugarcane and Response Expression Pattern of Stress-Responsive Genes Through Quantitative RT-PCR

Economic production of sugarcane is highly affected by oxidative stress due to biotic and abiotic stress factors. The main objective of this study was to determine which concentration of hydrogen peroxide can induce the oxidative stress conditions in sugarcane and determine whether several key gene expression responses reported in other species are triggered in sugarcane. Sixty-day-old sugarcane plants were sprayed with 30% H2O2 (300, 500 and 1000 ppm) for three consecutive days. Controls were maintained by spraying with water. Quantitative RT-PCR was performed to quantify the gene expression levels. The PCR products were cloned and sequenced to confirm the identity of the gene amplified. The representative sequences of the stress-responsive genes were deposited in GenBank (Acc. No KX828698 for HSP gene, KX828699 for APX gene, KX828700 for NAC transcription factor and KX828701 for ERF transcription factor). The expression pattern of stress-responsive key genes (HSP, NAC, ERF, GST, MYBAS and CAT) showed higher expression levels within 48 h of H2O2 treatment at 500 and 1000 ppm concentration. However, MYBAS transcription factor and catalase genes showed higher expression levels at a higher concentration of H2O2 (1000 ppm) of H2O2 at 48 h of treatment. Ascorbate peroxidase gene was up-regulated at 1000 ppm of H2O2 at 72 h of treatment. NAC transcription factor, heat shock proteins, ethylene-responsive factor and glutathione S-transferase showed a significant increase in expression at 500 ppm concentration of H2O2 at 48 h. Based on the expression pattern levels, 500 ppm of hydrogen peroxide at 48 h can induce the oxidative stress in sugarcane. The expression levels indicated that HSP, NAC, ERF, GST and MYBAS genes might play a role in signal transduction pathways, and APX and CAT are involved in scavenging reactive oxygen species.

Transcriptome analysis of flax (Linum usitatissimum L.) undergoing osmotic stress

Flax (Linum usitatissimum L.), an important industrial crop, is frequently decimated by pathogens, insect pests and adverse environmental conditions. Identification of flax resistance genes would facilitate development of varieties resistant to such diverse insults. Recently, a powerful method, high-throughput RNA sequencing (RNA-seq) technology, has been used effectively for transcript analysis and resistance gene discovery. In order to discover genes important for osmotic stress responses in flax, we performed transcriptome sequencing and screened differentially expressed unigenes (DEUs) of seedlings grown under normal and PEG 6000-induced stress treatments. Eight flax cDNA libraries were constructed and randomly sequenced using an Illumina technique and included one control sample and three treatment samples, each studied in duplicate. A total of 471,208,288 clean reads were obtained, of which 385,803,673 (81.88%) unique mapped reads and 23,042,410 multiple mapped reads were aligned to the Arabidopsis thaliana reference genome. Ultimately, 2533 out of 3922 unigenes were annotated using homology searches of KEGG Orthology (KO), PFAM, Gene Ontology (GO) and COG/KOG databases. Of these, 239 unigenes were differentially expressed (with at least 2-fold expression changes, FDR < 0.01) among three different PEG 6000-treatment durations compared with untreated control. Eight, five, four, three and one unigenes were assigned to NAC, LEA, WRKY, ERF and BLIP transcription factor families, respectively. For functional classification and pathway assignments, the 239 unigenes were mapped to three Gene Ontology (GO) categories including cellular component, molecular function and biological process, with 9, 10 and 19 functional terms for each category, respectively. In addition, these unigenes were assigned to 10 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Furthermore, we validated the expression profiles of six DEUs using qRT-PCR to further confirm the accuracy of the RNA-seq data. To our knowledge, this study is the first systematic transcriptome analysis of flax osmotic stress response genes. The results reported here should aid identification of functional genes and putative pathways involved in flax osmotic resistance. Ultimately, this work should provide a reference map for discovery of additional stress tolerance genes useful for molecular breeding of flax varieties tolerant to a variety of stresses.

ERF transcription factors involved in salt response in tomato

ERF transcription factors are important regulatory components of ethylene signaling, known to be involved in plant development and stress responses by regulating the expression of ethylene responsive genes. However, relatively less is known about the function of ERF gene family in wild tomato response to salt stress. Previously, we have carried out a microarray analysis on the salt-responsive genes in salt tolerant wild tomato Solanum pimpinellifolium ‘PI365967’. Five ERF genes (SpERFs) with induced expression upon salt treatment were subsequently selected for further characterization. Sequence analysis revealed that the seven amino acid residues in ERF proteins which are critical for their binding to the GCC-box in the promoter of ethylene responsive genes were conserved in five SpERFs. Two of five SpERFs (SpERF.B7 and SpERF.C11) belong to B3 sub-group as predicted by phylogenetic analysis. Expression analysis indicated that two SpERFs of B3 sub-group were more strongly induced in salt-tolerant genotypes than in salt-sensitive genotypes, in the order of PI365967 > LA2711 > Moneymaker, suggesting a positive role of B3 sub-group SpERFs in salt stress in tomato. To further confirm the function of B3 SpERFs in salt response, SpERF.B7 (one of two B3 SpERFs) was over-expressed in Arabidopsis. Consistent with our expectation, transgenic lines exhibited enhanced salt tolerance. These results will provide useful information for improving salt tolerance in tomato breeding in the future.

Co-expression of PeDREB2a and KcERF Improves Drought and Salt Tolerance in Transgenic Lotus corniculatus

DREB and ERF transcription factors play important roles in plant adaptation to abiotic stress. This study explores the function of co-overexpression of PeDREB2a and KcERF in transgenic Lotus corniculatus under abiotic stress. The two genes PeDREB2a and KcERF were expressed in L. corniculatus to study the effect on phenotype, physiology, and biochemical aspects of transgenic plants under drought and salt stress. The results indicated that transgenic L. corniculatus plants show better growth as compared to wild type under drought and salt conditions. Transgenic plants also showed elevated expression of stress-related genes under normal conditions, salt, and drought stress. The results revealed that co-overexpression of the two transcription factors, PeDREB2a, and KcERF, improved drought and salt tolerance in transgenic L. corniculatus.

Jasmonate-induced biosynthesis of steroidal glycoalkaloids depends on COI1 proteins in tomato

In tomato, perception of jasmonates by a receptor complex, which includes the F-box protein CORONATINE INSENSITIVE 1 (COI1), elicits biosynthesis of defensive steroidal glycoalkaloids (SGAs) via a jasmonate-responsive ERF transcription factor, JRE4/GAME9. Although JRE4 is upregulated by jasmonate and induces the expression of many metabolic genes involved in SGA biosynthesis, it is not known whether JRE4 alone is sufficient for increased SGA biosynthesis upon activation of jasmonate signaling. Here, we show that application of methyl jasmonate induces the expression of JRE4 and SGA biosynthesis genes in leaves and hairy roots of wild-type tomato, but not in jasmonic acid insensitive 1 (jai1), a loss-of-function mutant allele of the tomato COI1 gene. Induced overexpression of JRE4 increased the expression of SGA biosynthesis genes in transgenic hairy roots of both wild-type tomato and the jai1 mutant, suggesting that JRE4 is the primary transcription factor that functions downstream of the jasmonate signaling pathway.

Metabolomic and Proteomic Analysis of Maize Embryonic Callus induced from immature embryo

The low ratio of embryonic callus (EC) induction has inhibited the rapid development of maize genetic engineering. Still, little is known to explain the genotype-dependence of EC induction. Here, we performed a large-scale, quantitative analysis of the maize EC metabolome and proteome at three typical induction stages in two inbred lines with a range of EC induction capabilities. Comparison of the metabolomes and proteomes suggests that the differential molecular responses begin at an early stage of development and continue throughout the process of EC formation. The two inbred lines show different responses under various conditions, such as metal ion binding, cell enlargement, stem cell formation, meristematic activity maintenance, somatic embryogenesis, cell wall synthesis, and hormone signal transduction. Furthermore, the differences in hormone (auxin, cytokinin, gibberellin, salicylic acid, jasmonic acid, brassinosteroid and ethylene) synthesis and transduction ability could partially explain the higher EC induction ratio in the inbred line 18-599R. During EC formation, repression of the “histone deacetylase 2 and ERF transcription factors” complex in 18-599R activated the expression of downstream genes, which further promoted EC induction. Together, our data provide new insights into the molecular regulatory mechanism responsible for efficient EC induction in maize.

Understanding Host-Pathogen Interactions with Expression Profiling of NILs Carrying Rice-Blast Resistance Pi9 Gene.

Magnaporthe oryzae infection causes rice blast, a destructive disease that is responsible for considerable decrease in rice yield. Development of resistant varieties via introgressing resistance genes with marker-assisted breeding can eliminate pesticide use and minimize crop losses. Here, resistant near-isogenic line (NIL) of Pusa Basmati-1(PB1) carrying broad spectrum rice blast resistance gene Pi9 was used to investigate Pi9-mediated resistance response. Infected and uninfected resistant NIL and susceptible control line were subjected to RNA-Seq. With the exception of one gene (Pi9), transcriptional signatures between the two lines were alike, reflecting basal similarities in their profiles. Resistant and susceptible lines possessed 1043 (727 up-regulated and 316 down-regulated) and 568 (341 up-regulated and 227 down-regulated) unique and significant differentially expressed loci (SDEL), respectively. Pathway analysis revealed higher transcriptional activation of kinases, WRKY, MYB, and ERF transcription factors, JA-ET hormones, chitinases, glycosyl hydrolases, lipid biosynthesis, pathogenesis and secondary metabolism related genes in resistant NIL than susceptible line. Singular enrichment analysis demonstrated that blast resistant NIL is significantly enriched with genes for primary and secondary metabolism, response to biotic stimulus and transcriptional regulation. The co-expression network showed proteins of genes in response to biotic stimulus interacted in a manner unique to resistant NIL upon M. oryzae infection. These data suggest that Pi9 modulates genome-wide transcriptional regulation in resistant NIL but not in susceptible PB1. We successfully used transcriptome profiling to understand the molecular basis of Pi9-mediated resistance mechanisms, identified potential candidate genes involved in early pathogen response and revealed the sophisticated transcriptional reprogramming during rice-M. oryzae interactions.

Gene expression analysis in Musa acuminata during compatible interactions with Meloidogyne incognita.

Endoparasitic root-knot nematodes (RKNs) ( Meloidogyne spp.) cause considerable losses in banana ( Musa spp.), with Meloidogyne incognita a predominant species in Cavendish sub-group bananas. This study investigates the root transcriptome in Musa acuminata genotypes 4297-06 (AA) and Cavendish Grande Naine (CAV; AAA) during early compatible interactions with M. incognita . Roots were analysed by brightfield light microscopy over a 35 d period to examine nematode penetration and morphological cell transformation. RNA samples were extracted 3, 7 and 10 days after inoculation (DAI) with nematode J2 juveniles, and cDNA libraries were sequenced using lllumina HiSeq technology. Sequences were mapped to the M. acuminata ssp. malaccensis var. Pahang genome sequence, differentially expressed genes (DEGs) identified and transcript representation determined by gene set enrichment and pathway mapping. Microscopic analysis revealed a life cycle of M. incognita completing in 24 d in CAV and 27 d in 4279-06. Comparable numbers of DEGs were up- and downregulated in each genotype, with potential involvement of many in early host defence responses involving reactive oxygen species and jasmonate/ethylene signalling. DEGs revealed concomitant auxin metabolism and cell wall modification processes likely to be involved in giant cell formation. Notable transcripts related to host defence included those coding for leucine-rich repeat receptor-like serine/threonine-protein kinases, peroxidases, thaumatin-like pathogenesis-related proteins, and DREB, ERF, MYB, NAC and WRKY transcription factors. Transcripts related to giant cell development included indole acetic acid-amido synthetase GH3.8 genes, involved in auxin metabolism, as well as genes encoding expansins and hydrolases, involved in cell wall modification. Expression analysis in M. acuminata during compatible interactions with RKNs provides insights into genes modulated during infection and giant cell formation. Increased understanding of both defence responses to limit parasitism during compatible interactions and effector-targeted host genes in this complex interaction will facilitate the development of genetic improvement measures for RKNs.

Identification, characterization and expression analysis of ERF transcription factor VviERF073 and standardization of stable reference gene under salt stress in grape.

Salinity is one of the several abiotic factors affecting grape productivity and quality. Transcription factors belonging to AP2/ERF family play an important role in abiotic stress response. Based on the in silico analysis of salt stress libraries of grape ESTs, a transcription factor VviERF073 , having a single AP2/ERF conserved domain was identified. The sequence of VviERF073 was analyzed in 'Thompson Seedless'. VviERF073 belonged to the B2 subfamily of ERF transcription factors. Under salt stress, the relative expression of VviERF073 increased 2.7 fold in the leaves of grafted grapevine within six hours of salt stress. The expression increased to 8.7 fold at 15 days of salt stress, thus indicating its role in early as well as late response to salt stress. However, expression of VviERF073 did not change significantly in grapevines raised on own roots. The expression varied in the roots of 'Thompson Seedless' but not in the roots of '110R'. Under salt stress conditions, GAPDH and EF1α for grafted vines and Actin and Tubulin for own root vines, were identified as the stable reference genes for qPCR data normalization.

Rice aleurone layer specific OsNF-YB1 regulates grain filling and endosperm development by interacting with an ERF transcription factor.

Grain yield and quality of rice mainly depend on grain filling and endosperm development. Here we report that a rice NUCLEAR FACTOR Y (NF-Y) transcription factor, OsNF-YB1, is specifically expressed in the aleurone layer of developing endosperm and regulates grain filling and endosperm development. Knockdown of OsNF-YB1 expression by RNAi significantly retarded grain filling, leading to small grains with chalky endosperm as well as altered starch quality. Whereas OsNF-YB1 shows subcellular localization in both the cytosol and the nucleus in roots, it was specifically targeted to the nucleus of aleurone layer cells, which was facilitated by interacting with OsNF-YC proteins preferentially expressed in the aleurone layer. RNA sequencing analysis revealed that genes related to membrane transport and ATP biosynthesis were enriched in the down-regulated category in OsNF-YB1 RNAi plants, which is consistent with the crucial role of OsNF-YB1 in rice grain filling and endosperm development. Identification of the genome-wide targets of OsNF-YB1 by ChIP sequencing showed that OsNF-YB1 directly regulates genes involved in the transport of nutrients such as sugar and amino acids. Interestingly, different from the binding sites reported for other NF-Y complexes, the GCC box, the binding motif of ERF transcription factors, was enriched in the binding peaks of OsNF-YB1. Indeed, further analyses confirmed the interaction of OsERF#115 with OsNF-YB1, and OsERF#115 directly binds to the GCC box. It is proposed that OsNF-YB1 specifically regulate the transcription of downstream genes during rice endosperm development by forming protein complexes consisting of OsNF-YB1, OsNF-YC and ERF, providing informative insights into the molecular functional mechanisms of the NF-Y factor.

Arg156 in the AP2-Domain Exhibits the Highest Binding Activity among the 20 Individuals to the GCC Box in BnaERF-B3-hy15, a Mutant ERF Transcription Factor from Brassica napus.

To develop mutants of the ERF factor with more binding activities to the GCC box, we performed in vitro directed evolution by using DNA shuffling and screened mutants through yeast one-hybrid assay. Here, a series of mutants were obtained and used to reveal key amino acids that induce changes in the DNA binding activity of the BnaERF-B3 protein. With the BnaERF-B3-hy15 as the template, we produced 12 mutants which host individual mutation of potential key residues. We found that amino acid 156 is the key site, and the other 18 mutants host the 18 corresponding individual amino acid residues at site 156. Among the 20 individuals comprising WT (Gly156), Mu3 (Arg156), and 18 mutants with other 18 amino acid residues, Arg156 in the AP2-domain is the amino acid residue with the highest binding activity to the GCC box. The structure of the α-helix in the AP2-domain affects the binding activity. Other residues within AP2-domain modulated binding activity of ERF protein, suggesting that these positions are important for binding activity. Comparison of the mutant and wild-type transcription factors revealed the relationship of protein function and sequence modification. Our result provides a potential useful resource for understanding the trans-activation of ERF proteins.