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Identification and Expression Analysis of E2F/DP Gene Family of Rubus idaeus
YANGMingjiao, ZHAOZhilei, LIMing, LIHan, ZHANGYumeng, FANXiaowei, ZHENGYichen, ZHANGRuijie, QINGMengyao, GUYuhong
Chin Agric Sci Bull ›› 2026, Vol. 42 ›› Issue (16) : 61-70.
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Abbreviation (ISO4): Chin Agric Sci Bull
Editor in chief: Yulong YIN
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Identification and Expression Analysis of E2F/DP Gene Family of Rubus idaeus
The E2F/DP gene family plays a significant regulatory role in the growth, development and stress response of plants. The bioinformatics analysis of the E2F/DP gene family in red raspberry (Rubus idaeus) and the expression pattern analysis in different tissues and after exogenous spraying of melatonin lay the foundation for in-depth exploration of the function of E2F/DP genes in red raspberry. In this study, three members of the E2F/DP gene family were obtained from the transcriptome database of ‘Heritage’ red raspberry. The E2F/DP gene family of red raspberry was analyzed by bioinformatics methods. Subsequently, the expression levels of E2F/DP in different tissues and organs, fruits at different developmental stages and fruits after exogenous spraying of melatonin were analyzed by qRT-PCR. The proteins encoded by RuE2F/DPs have a minimum of 243 amino acids and a maximum of 761 amino acids, with a minimum relative molecular mass of 26.88 kDa and a maximum of 82.55 kDa. Their isoelectric points range from 5.69 to 9.07. RuE2F/DP-1 is a stable hydrophilic protein, while RuE2F/DP2-3 is unstable hydrophilic protein. All RuE2F/DPs-encoded proteins are located in the cell nucleus, have no signal peptides, are non-secretory proteins, non-transmembrane proteins, and contain the conserved E2F_TDP domain. Their secondary structures are all composed of four types of structures, including α-helices, β-turns, random coils, and extended strands. The prediction results of their tertiary structures are consistent with their secondary structures. RuE2F/DP-1 and RuE2F/DP-3 have expression correlations. The RuE2F/DP gene family contains 9 to 14 exons and 4 to 10 conserved motifs; phylogenetic analysis indicates that the RuE2F/DP family members belong to different subgroups, and red raspberry is more closely related to strawberry; the cis-acting elements in the promoter suggest that the RuE2F/DP gene family mainly participates in plant growth and development and responses to drought, low temperature and other stress environments; qRT-PCR analysis shows that the expression levels of three members of the RuE2F/DP gene family in young roots and green fruits are higher than those in other tissues; melatonin treatment has a positive regulatory function on RuE2F/DP1-2 and a negative regulatory function on RuE2F/DP-3. The research results reveal that the RuE2F/DP gene family may play a role in the growth of red raspberry roots, the growth and development of fruits, the response to adverse stress during the growth process, and the hormone response to melatonin.
red raspberry (Rubus idaeus) / E2F/DP gene / bioinformatics analysis / expression analysis / RT-qPCR / melatonin / tissue specificity
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Blackberry and raspberry have high nutritional, health value, and are popular with consumers for their unique flavors. To explore the relationships between nutrient accumulation, antioxidant substance contents in blackberry and raspberry fruits, and fruit growth and development, seven Rubus cultivars were selected, and contents of the main active substance were determined. “Clode Summit” had the highest soluble sugar and fructose contents, “Chester”—the highest total phenol content, and “Bristol’—the highest anthocyanin content. Generally, the contents of flavonoids and total phenols showed a downward trend with the development of fruit in seven Rubus cultivars, and the content of anthocyanins increased rapidly in the later stage of development. Pearson correlation analysis showed extremely significant correlation between antioxidant activity and the contents of vitamin E, total phenols, and flavonoids. Flavonoids were extremely significantly positively correlated with the content of total phenols, and the contents of flavonoids and anthocyanins in various cultivars were highly negatively correlated. Considering the different nutritional ingredients and active antioxidant substance contents, “Clode Summit”, “Bristol”, and “Chester” are recommended for raw consumption, processing, and medicinal purposes, respectively. These results provide a reference for comparing the main active substance contents in different Rubus cultivars and their changes across fruit development stages.
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Red raspberries (Rubus idaeus L.) are increasingly popular foods in contemporary diets due to their freshness, nutritional value and health claims. Among the existing cultivars, “Kweli” is one of the most productive and widely cultivated. In this study, the nutritional value and chemical composition of “Kweli” red raspberry were characterized by the official method of food analysis and chromatographic techniques, and its antioxidant and antimicrobial activities were tested against biological/biochemical oxidizable substrates and foodborne bacteria and fungi strains, respectively. Carbohydrates (including fructose and glucose, 14.3 and 12.6 g/100 g dw, respectively), proteins (6.8 g/100 g dw), and ashes (3.90 g/100 g dw) were major constituents. The fat content was quite low and constituted mainly by unsaturated fatty acids (58.3%), with a predominance of oleic acid. Fresh red raspberry also contained high levels of citric (2.7 g/100 g) and ascorbic (17 mg/100 g) acids. The anthocyanins (4.51 mg/g extract) cyanidin-O-hexoside and mostly cyanidin-O-sophoroside were identified in the red raspberry hydroethanolic extract, which was able to inhibit thiobarbituric acid reactive substances (TBARS) formation (EC50 of 122 µg/mL), oxidative hemolysis (IC50 of 298 µg/mL), and β-carotene bleaching (EC50 of 18.7 µg/mL). In turn, the extract was more effective than the food additive E224 against Bacillus cereus. All these results highlighted the nutritional quality of “Kweli” red raspberry and showed some compositional differences in relation to other cultivars. Therefore, its inclusion in a daily diet can be helpful to obtain nutrients and antioxidants and bring health benefits.
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Anthocyanins are natural products that give color to plants. As natural plant pigments, anthocyanins also have a series of health-promoting benefits. Many researchers have proved that anthocyanins have therapeutic effects on diseases, such as circulatory, nervous, endocrine, digestive, sensory, urinary and immune systems. Additionally, a large number of studies have reported that anthocyanins have an anticancer effect through a wide range of anti-inflammatory and antioxidant effects. The anti-disease impact and mechanism of anthocyanins are diverse, so they have high research value. This review summarizes the research progress of anthocyanins on the pharmacological agents of different diseases to provide references for subsequent research.
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采用液态深层快速发酵法发酵红树莓果醋，分析红树莓酿造过程中总多酚、总黄酮、花色苷的含量以及1,1-二苯基-2-三硝基苯肼（1,1-diphenyl-2-picrylhydrazyl，DPPH）自由基、羟自由基（·OH）、超氧阴离子自由基（O2－·）清除率的变化。结果表明：总多酚、总黄酮、花色苷的含量先升高后降低，3 种自由基清除率随着醋酸发酵明显升高，醋酸发酵10 d后，总多酚、总黄酮、花色苷含量分别保留了68%、85%、38%，DPPH自由基、O2－·、·OH清除率分别为98.63%、83.82%、78.36%。相关性分析显示总多酚、总黄酮、花色苷含量互为极显著正相关，且与主成分1成很高的负相关；·OH清除率、O2－·清除率、DPPH自由基清除率互为极显著正相关，且与主成分1成很高的正相关。聚类分析将酿造过程分为3 个集群，代表发酵过程中抗氧化性能不同的变化趋势。
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Raspberry fruit pomace, a byproduct of juice production, was studied as a potential source of antioxidant compounds. Target high-performance liquid chromatography analysis of important polyphenolic compounds (gallic, p-coumaric, caffeic, quercitrin, chlorogenic, and ellagic acid) was performed together with analysis of total phenolic content (TPC), total flavonoid content (TFC), total anthocyanins content (TAC), and antioxidant capacity (via ferric reducing antioxidant power (FRAP) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assays). The differences in polyphenolic content of Rubus idaeus L. pomace were evaluated following ultrasound-assisted extraction and conventional maceration with different organic solvents. Additionally, the yield of free phenolics was measured in hydrolyzed pomace extracts. The results obtained show that the ultrasound method maximizes the quantity of antioxidant compounds in terms of TPC (27.79 mg/L gallic acid equivalent (GAE)), TFC (8.02 mg/g quercetin equivalent (QE)), TAC (7.13 mg/L cyanidine-3-glucoside equivalent (C3G Eq)), caffeic (19.17 µg/g), chlorogenic (3.56 µg/g), ellagic (105.52 µg/g), and gallic acids (8.75 µg/g), as well as FRAP (1002.72 µmol/L) and DPPH assays (969.71 µmol/mL vitamin C equivalent (vit C Eq); 567.00 µmol/100 g Trolox equivalent (TE)). On the other hand, conventional maceration maximizes the yield of quercetin and p-coumaric acid. In terms of biowaste valorization, raspberry fruit pomace has a promising industrial potential and may prove to be useful in the development of antioxidant dietary supplements.
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As major regulators of the cell cycle, apoptosis and differentiation, E2F transcription factors have been studied extensively in a broad range of organisms. The recent identification of atypical E2F family members further expands our structural, functional and molecular view of the cellular E2F activity. Unlike other family members, atypical E2Fs have a duplicated DNA-binding domain and control gene expression without heterodimerization with dimerization partner proteins. Recently, knockout strategies in plants and mammals have pinpointed that atypical E2Fs have a crucial role in plant cell size control, endocycle regulation, proliferation and apoptotic response upon DNA stress. Their position at the crossroads of proliferation and DNA stress response marks these novel E2F proteins as interesting study objects in the field of tumor biology.
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Cell cycle entry and quiescence are regulated by the E2F transcription factors in association with RETINOBLASTOMA-RELATED (RBR). E2FB is considered to be a transcriptional activator of cell cycle genes, but its function during development remains poorly understood. Here, by studying E2FB-RBR interaction, E2F target gene expression, and epidermal cell number and shape in mutant and overexpression lines during leaf development in Arabidopsis (), we show that E2FB in association with RBR plays a role in the inhibition of cell proliferation to establish quiescence. In young leaves, both RBR and E2FB are abundant and form a repressor complex that is reinforced by an autoregulatory loop. Increased E2FB levels, either by expression driven by its own promoter or ectopically together with DIMERIZATION PARTNER A, further elevate the amount of this repressor complex, leading to reduced leaf cell number. Cell overproliferation in mutants and in plants overexpressing a truncated form of E2FB lacking the RBR binding domain strongly suggested that RBR repression specifically acts through E2FB. The increased number of small cells below the guard cells and of fully developed stomata indicated that meristemoids preferentially hyperproliferate. As leaf development progresses and cells differentiate, the amount of RBR and E2FB gradually declined. At this stage, elevation of E2FB level can overcome RBR repression, leading to reactivation of cell division in pavement cells. In summary, E2FB in association with RBR is central to regulating cell proliferation during organ development to determine final leaf cell number.© 2020 The authors. All Rights Reserved.
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Organogenesis in plants is almost entirely a postembryonic process. This unique feature implies a strict coupling of cell proliferation and differentiation, including cell division, arrest, cell cycle reactivation, endoreplication, and differentiation. The plant retinoblastoma-related (RBR) protein modulates the activity of E2F transcription factors to restrict cell proliferation. Arabidopsis contains a single RBR gene, and its loss of function precludes gamete formation and early development. To determine the relevance of the RBR/E2F pathway during organogenesis, outside its involvement in cell division, we have used an inducible system to inactivate RBR function and release E2F activity. Here, we have focused on leaves where cell proliferation and differentiation are temporally and developmentally regulated. Our results reveal that RBR restricts cell division early during leaf development when cell proliferation predominates, while it regulates endocycle occurrence at later stages. Moreover, shortly after leaving the cell cycle, most of leaf epidermal pavement cells retain the ability to reenter the cell cycle and proliferate, but maintain epidermal cell fate. On the contrary, mesophyll cells in the inner layers do not respond in this way to RBR loss of activity. We conclude that there exists a distinct response of different cells to RBR inactivation in terms of maintaining the balance between cell division and endoreplication during Arabidopsis (Arabidopsis thaliana) leaf development.
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Post-embryonic growth in plants depends on the continuous supply of undifferentiated cells within meristems. Proliferating cells maintain their competence for division by active repression of differentiation and the associated endocycle entry. We show by upregulation and downregulation of E2FA that it is required for maintaining proliferation, as well as for endocycle entry. While E2FB-RBR1 (retinoblastoma-related protein 1) complexes are reduced after sucrose addition or at elevated CYCD3;1 levels, E2FA maintains a stable complex with RBR1 in proliferating cells. Chromatin immunoprecipitation shows that RBR1 binds in the proximity of E2F promoter elements in CCS52A1 and CSS52A2 genes, central regulators for the switch from proliferation to endocycles. Overexpression of a truncated E2FA mutant (E2FA(ΔRB)) lacking the RBR1-binding domain interferes with RBR1 recruitment to promoters through E2FA, leading to decreased meristem size in roots, premature cell expansion and hyperactivated endocycle in leaves. E2F target genes, including CCS52A1 and CCS52A2, are upregulated in E2FA(ΔRB) and e2fa knockout lines. These data suggest that E2FA in complex with RBR1 forms a repressor complex in proliferating cells to inhibit premature differentiation and endocycle entry. Thus, E2FA regulates organ growth via two distinct, sequentially operating pathways.
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Eukaryotic E2Fs are conserved transcription factors playing crucial and antagonistic roles in several pathways related to cell division, DNA repair, and differentiation. In plants, these processes are strictly intermingled at the growing zone to produce postembryonic development in response to internal signals and environmental cues. Of the six AtE2F proteins found in Arabidopsis (Arabidopsis thaliana), only AtE2Fa and AtE2Fb have been clearly indicated as activators of E2F-responsive genes. AtE2Fa activity was shown to induce S phase and endoreduplication, whereas the function of AtE2Fb and the interrelationship between these two transcription factors was unclear. We have investigated the role played by the AtE2Fb gene during cell cycle and development performing in situ RNA hybridization, immunolocalization of the AtE2Fb protein in planta, and analysis of AtE2Fb promoter activity in transgenic plants. Overexpression of AtE2Fb in transgenic Arabidopsis plants led to striking modifications of the morphology of roots, cotyledons, and leaves that can be ascribed to stimulation of cell division. The accumulation of the AtE2Fb protein in these lines was paralleled by an increased expression of E2F-responsive G1/S and G2/M marker genes. These results suggest that AtE2Fa and AtE2Fb have specific expression patterns and play similar but distinct roles during cell cycle progression.
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E2F transcription factors are key components of the RB/E2F pathway that, through the action of cyclin-dependent kinases, regulates cell cycle progression in both plants and animals. Moreover, plant and animal E2Fs have also been shown to regulate other cellular functions in addition to cell proliferation. Based on structural and functional features, they can be divided into different classes that have been shown to act as activators or repressors of E2F-dependent genes. Among the first plant E2F factors to be reported, we previously described DcE2F1, an activating E2F which is expressed in cycling carrot (Daucus carota) cells. In this study, we describe the identification of the additional members of the E2F/DP family of D. carota, which includes four typical E2Fs, three atypical E2F/DEL genes, and three related DP genes. Expression analyses of the carrot E2F and DP genes reveal distinctive patterns and suggest that the functions of some of them are not necessarily linked to cell proliferation. DcE2F1 was previously shown to transactivate an E2F-responsive promoter in transient assays but the functional role of this protein in planta was not defined. Sequence comparisons indicate that DcE2F1 could be an ortholog of the AtE2FA factor of Arabidopsis thaliana. Moreover, ectopic expression of the DcE2F1 cDNA in transgenic Arabidopsis plants is able to upregulate AtE2FB and promotes cell proliferation, giving rise to polycotyly with low frequency, effects that are highly similar to those observed when over-expressing AtE2FA. These results indicate that DcE2F1 is involved in the control of cell proliferation and plays important roles in the regulation of embryo and plant development.
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李平平. CaRAP2-12介导褪黑素调控辣椒采后类胡萝卜素合成机理的研究[D]. 昆明: 云南农业大学, 2023.
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孙惠莉. 外源褪黑素对梨果皮花青素合成的影响及其调控机制研究[D]. 沈阳: 沈阳农业大学, 2023.
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陈强, 黄馨慧, 张峥, 等. 褪黑素对薄皮甜瓜采后软化和乙烯合成的影响[J]. 生物技术通报, 2024, 40(4):139-147.
【目的】褪黑素在采后果实保鲜中发挥重要作用，探究褪黑素对薄皮甜瓜采后软化和乙烯合成的影响。【方法】以0、100、200、300、400 μmol/L褪黑素溶液浸泡薄皮甜瓜‘花姑娘’果实，测定果实的硬度、失重率、腐烂率以及内源乙烯生成量，筛选出最适采后保鲜的浓度。比较褪黑素与对照果实的ACC含量、ACS以及ACO酶活性，并通过转录组测序技术，筛选出与果实软化和乙烯合成相关的差异表达基因，并通过实时荧光定量技术进行验证。【结果】在贮藏期间，200 μmol/L的褪黑素处理可明显地降低薄皮甜瓜‘花姑娘’果实的失重率和腐烂率，更好地维持果实的硬度和外观。褪黑素处理可降低果实内源乙烯和ACC含量，但并未延迟高峰期的出现。转录组测序表明，褪黑素和对照在果实软化和乙烯合成途径中存在差异表达基因，褪黑素显著抑制CmPG1、CmPLB1、CmACO1、CmACO2、CmACS1、CmEXP等结构基因的表达，并且部分ERF和LBD转录因子的表达也受到了抑制。【结论】应用200 μmol/L的褪黑素处理‘花姑娘’可以有效延缓果实软化、重量损失，降低内源乙烯生成量，通过抑制果实软化及乙烯合成相关基因的表达，延长果实的货架期。
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张瑞杰, 李明, 李迎超, 等. 红树莓GeBP基因家族鉴定及表达分析[J]. 植物生理学报, 2024, 60(6):1007-1015.
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E2F/DP proteins have been shown to regulate genes implicated in cell cycle control and DNA repair. However, to date, research into the potential role of the Moso bamboo E2F/DP family has been limited.Here, we identified 23 E2F/DPs in the Moso bamboo genome, including nine E2F genes, six DP genes, eight DEL genes and one gene with a partial E2F domain. An estimation of the divergence time of the paralogous gene pairs suggested that the E2F/DP family expansion primarily occurred through a whole-genome duplication event. A regulatory element and coexpression network analysis indicated that E2F/DP regulated the expression of cell cycle-related genes. A yeast two-hybrid assay and expression analysis based on transcriptome data and in situ hybridization indicated that the PheE2F-PheDP complex played important roles in winter Moso bamboo shoot growth. The qRT-PCR results showed that the PheE2F/DPs exhibited diverse expression patterns in response to drought and salt treatment and diurnal cycles.Our findings provide novel insights into the Moso bamboo E2F/DP family and partial experimental evidence for further functional verification of the PheE2F/DPs.
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\n This review deals with important issues in regulation of terminal alternative oxidase (AOX) of plant mitochondria, which are currently investigated and discussed. AOX ensures electron transport along the alternative nonphosphorylating cyanide-resistant pathway in mitochondrial ETC and plays an important role in the maintenance of redox balance in the cell and defense against stress. Principal results concerning AOX signaling were obtained in the works dealing with expression of the\n АОХ1а\n gene of\n Arabidopsis thaliana\n (\n AtAOX1a\n ). Reports about\n АОХ\n expression in different plant species were analyzed. Mechanisms of transcriptional and posttranscriptional control over expression of\n АОХ\n genes were described. The role of some\n cis\n -regulatory elements of\n AtАОХ1а\n promoter and related transcription factors was considered. It was shown that transduction of the signal causing expression of\n АОХ\n genes may follow anterograde and retrograde pathways. Information about mitochondrial retrograde regulation of AOX and ROS-dependent and ROS-independent signal pathways of induction of\n АОХ\n expression is given. A hypothetic sequence of signal events of light induction of\n АОХ1а\n expression in wheat leaf cell in the course of greening was worked out. Interactions between AOX signaling and hormonal signal pathways, as well as induction of\n АОХ\n by the signals of plastid origin, were described. In conclusion, it was highlighted that AOX is integrated into the common signal pathway of the cell and acts as a target and a regulator of signal events.\n
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Members of the ERF transcription-factor family participate in a number of biological processes, viz., responses to hormones, adaptation to biotic and abiotic stress, metabolism regulation, beneficial symbiotic interactions, cell differentiation and developmental processes. So far, no tissue-expression profile of any cucumber ERF protein has been reported in detail. Recent completion of the cucumber full-genome sequence has come to facilitate, not only genome-wide analysis of ERF family members in cucumbers themselves, but also a comparative analysis with those in Arabidopsis and rice. In this study, 103 hypothetical ERF family genes in the cucumber genome were identified, phylogenetic analysis indicating their classification into 10 groups, designated I to X. Motif analysis further indicated that most of the conserved motifs outside the AP2/ERF domain, are selectively distributed among the specific clades in the phylogenetic tree. From chromosomal localization and genome distribution analysis, it appears that tandem-duplication may have contributed to CsERF gene expansion. Intron/exon structure analysis indicated that a few CsERFs still conserved the former intron-position patterns existent in the common ancestor of monocots and eudicots. Expression analysis revealed the widespread distribution of the cucumber ERF gene family within plant tissues, thereby implying the probability of their performing various roles therein. Furthermore, members of some groups presented mutually similar expression patterns that might be related to their phylogenetic groups.
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The identification and characterization of cis-regulatory DNA sequences and how they function to coordinate responses to developmental and environmental cues is of paramount importance to plant biology. Key to these regulatory processes are cis-regulatory modules (CRMs), which include enhancers and silencers. Despite the extraordinary advances in high-quality sequence assemblies and genome annotations, the identification and understanding of CRMs, and how they regulate gene expression, lag significantly behind. This is especially true for their distinguishing characteristics and activity states. Here, we review the current knowledge on CRMs and breakthrough technologies enabling identification, characterization, and validation of CRMs; we compare the genomic distributions of CRMs with respect to their target genes between different plant species, and discuss the role of transposable elements harboring CRMs in the evolution of gene expression. This is an exciting time to study cis-regulomes in plants; however, significant existing challenges need to be overcome to fully understand and appreciate the role of CRMs in plant biology and in crop improvement.
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