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EAR4: A Member of the DREB/CBF Family and its Regulated Senescence in Tobacco Research Institute, Chinese Academy of Agricultural Sciences

Leaf senescence is a natural process that occurs in plants, but it can also be influenced by various stressors. Dreb1/CBF (dehydration-responsive element binding protein 1/C-repeat binding factor) family, which includes DEAR4 at AT4G36900, plays a role in regulating leaf senescence. In Arabidopsis thaliana, DEAR4 has been shown to be associated with leaf senescence and can be induced by several factors, such as abscisic acid (ABA), jasmonic acid (JA), darkness, drought, and salt stress. Over-expression of DEAR4 in transgenic plants was found to result in a dramatic enhancement of leaf senescence phenotype under normal and dark conditions. On the other hand, the knock-down mutation of DEAR4 resulted in delayed senescence. These findings shed light on the mechanism of DEAR4 in regulating leaf senescence and its importance in plant development.

In this study, we investigated the role of DEAR4 in stress response and senescence in plants. Over-expressing DEAR4 led to decreased seed germination rate under ABA and salt stress conditions as well as decreased drought tolerance, indicating that DEAR4 was involved in both senescence and stress response processes. Moreover, we found that DEAR4 protein displayed transcriptional repressor activities in yeast cells. This suggests that DEAR4 may directly repress the expression of a subset of COLD-REGULATED (COR) and RESPONSIVE TO DEHYDRATION (RD) genes, which have been shown to be involved in leaf longevity and stress response. Additionally, we discovered that DERA4 could induce the production of Reactive oxygen species (ROS), the common signal of senescence and stress responses. This discovery provides evidence that DEAR4 may play an integrative role in senescence and stress response via regulating ROS production.

In the last stage of leaf development known as senescence, several intrinsic and environmental factors can influence this process. These include age, nutrients, hormones, darkness, osmotic stress, extreme temperature, and pathogens (Lim et al., 2007; Guo and Gan, 2014). The major plant hormones that are involved in leaf senescence include abscisic acid (ABA), ethylene (ETH), jasmonic acid (JA), salicylic acid (SA), and strigolactones. However, other hormones like cytokinins (CK), gibberellic acid (GA), and auxin can inhibit senescence (Jibran et al., 2013; Li et al., 2013; Penfold and Buchan-Wollaston, 2014; Mostofa et al., 2018).

During the process of leaf senescence, there is a significant change in cellular metabolism and structure that leads to leaf yellowing. Additionally, degradation of macromolecules in senescing leaves facilitates nutrient remobilization to support young vegetative organs and reproductive growth. Unfavorable environmental conditions can induce precocious senescence, leading to reduced yield and quality of crop plants. Senescence execution requires differential expression of many genes and a subset of these genes is referred to as senescence-associated genes (SAGs).

The molecular mechanisms underlying leaf senescence are complex and involve both intrinsic and external factors. SAGs play an important role in the regulation of this process by controlling gene expression at different levels of the genome. SAGs have been found to be associated with various pathways such as the p53 pathway, mitochondrial signaling, and autophagy (Singh et al., 2016). Moreover, SAGs have been identified as potential targets for developing new therapeutic strategies for treating diseases associated with aging or senescence (Chen et al., 2019).

Overall, understanding the molecular mechanisms behind leaf senescence is essential for improving crop yields and quality, as well as developing new therapeutic strategies for treating age-related diseases.

AGs, or senescence-associated genes, have been identified as playing a regulatory role in plant leaf senescence. These genes include those encoding transcription factors of the WRKY (Wythoff/Ridgway/Yersin) family, DREB (DE-rich), MYB (Myb), and bZIP families (Woo et al., 2001; Yang et al., 2011; Lee et al., 2012; Vainonen et al., 2012).

Dark-induced senescence, or DIS, has been widely adopted as a model system for studying leaf senescence (Liebsch and Keech, 2016). A large number of transcription factor genes have been reported to exhibit differential expression during dark-induced leaf senescence (Song, 2014; Song et al., 2014; Yasuhito et al., 2014). Some of these genes have also been studied for their role in regulating senescence.

The transcription factor AtWRKY22 is one such gene that plays an important role in leaf senescence regulation. This gene is induced by darkness but suppressed by light. Further research has revealed that plants with overexpressed AtWRKY22 display accelerated senescence, while plants with lost-of-function AtWRKY22 show a delay in senescence underdark conditions (Zhou et al., 2011b).

Loss-of-function (LoF) plants are characterized by the inability to carry out essential biological functions, resulting in a delay of senescence. In this context, it is interesting to investigate LoF plant models that display delayed senescence under normal and dark conditions. This review focuses on studies that have examined the role of phytochromes in regulating senescence.

Phytochromes are involved in the regulation of light responses via the promotion of the degradation of PIFs (Phytochrome-interacting factors). Studies have shown that PIF3, 4, and 5 play an important role in natural and dark induced senescence. For example, mutations in the PIFs genes were found to result in a significant delay of natural and dark induced senescence, whereas overexpression of these genes accelerated senescence (Yasuhito et al., 2014). Furthermore, PIF4 has been found to bind to the promoter of NYE1, the chlorophyll degradation regulatory gene, as well as GLK2, which may contribute to the delay of leaf senescence in Arabidopsis (Sharabi-Schwager et al., 2010a; Takasaki et al., 2015).

Another member of the DREB family, CBF2 and CBF3, have also been shown to delay the onset of leaf senescence and inhibit the response to hormones and darkness in Arabidopsis (Takasaki et al., 2015; Kamranfar et al., 2018). Overexpression of CBF2 and CBF3 resulted in earlier leaf senescence compared to controls.

In conclusion, phytochromes play a vital role in regulating the delay of senescence under different conditions. By studying LoF plant models that display delayed senescence with higher levels of chlorophyll, researchers can gain insights into the complex mechanisms underlying this phenomenon. Further research is needed to fully understand the role of phytochromes in promoting or interfering with senescence in plants.

Jasmonic acid (JA) is a polycyclic unsaturated fatty acid with multiple roles in plants. It can be involved in root inhibition, trichome initiation, anthocyanin accumulation, leaf senescence and biotic and abiotic stress responses (Balbi and Devoto, 2008; Wu et al., 2008; Hu et al., 2017; Song et al., 2017; Ono et al., 2019).

JA signaling can be initiated by perception of jasmonoyl-L-isoleucine (JA-Ile), which binds to its receptor COI1 (CORONATINE INSENSITIVE1), an F-box domain-containing protein (Balbi and Devoto, 2008; Shan et al., 2011). It has been reported that endogenous JA content increases during leaf senescence and JA biosynthetic genes such as LOX1, LOX3, and LOX4 are induced or regressed during this process (Song et al., 2014).

The regulation of JA activity is essential for plant growth and development, as well as for the maintenance of plant health under different conditions. The chloroplast activity maintainer gene, which is responsible for the regulation of photosynthesis efficiency in chloroplasts, may also play a role in JA signalling (Shi et al., 2017a).

In summary, JA plays important roles in plant growth and development, and its regulation is crucial for maintaining plant health under different conditions. The regulation of JA activity is controlled by both intrinsic and extrinsic factors, and the specific mechanisms underlying these processes are still being studied.

MYC2, 3, and 4 are up-regulated during leaf senescence (Wasternack, 2007; Seltmann and Berger, 2013). Precocious senescence of attached or detached leaves has been observed under exogenous application of MeJA (Shan et al., 2011; Hu et al., 2017). Further study revealed that MYC2, 3, and 4 redundantly bind to the SAG29 promoter and activate its expression, leading to activation of JA-induced leaf senescence (Zhu et al., 2015).

In contrast, bHLH family members including bHLH03, bHLH13, bHLH14, and bHLH17 attenuate MYC2/MYC3/MYC4-activated JA-induced leaf senescence by binding to the SAG29 promoter and repressing its expression. It has been suggested that the activators and repressors mediated in JA-induced leaf senescence can enhance the plant survival rate in various environmental conditions (Qi et al., 2015). In addition, the NAC transcriptional factors ANAC019, ANAC055, and RD26 play an important role in modulating the leaf senescence pathway (Xu et al., 2017).

These findings highlight the importance of the MYC2/MYC3/MYC4-SAG29 pathway in regulating cell division and differentiation during leaf growth as well as in promoting leaf senescence. The involvement of bHLH family members in mediating the inhibitory effect of these pathways on JA-induced leaf senescence suggests a potential mechanism for preventing or reducing the impact of stress-induced leaf senescence in plants. Furthermore, the identification of additional regulatory factors involved in this process may provide insights into developing strategies for improving plant health under diverse environmental conditions.

MYC2 is also a direct target of JAZ7 to mediate dark-induced leaf senescence. The JA signaling proteins JAZ4 and JAZ8 interact with transcription factor WRKY57 to negatively regulate the leaf senescence induced by JA (Jiang et al., 2014). Furthermore, the expression ofJAZ7 was significantly increased during darkness, and its mutant showed precocious senescence induced by darkness, indicating that JAZ7 plays a negative role in dark-induced leaf senescence (Yu et al., 2016).

The Evening Complex (EC), a core component of the circadian oscillator, comprising ELF3, ELF4, and LUX ARRHYTHMO (LUX), plays an essential role in the plant circadian clock and negatively regulates leaf senescence in Arabidopsis. It has been reported that EC represses the expression of MYC2 by directly binding to its promoter (Myc2, Myc3, Myc4).

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Triple mutants abrogate the accelerated leaf senescence induced by JA in EC mutants (Zhang et al., 2018; Thines et al., 2019). Additionally, JA can positively regulate the ICE-CBF signal to enhance cold stress tolerance in Arabidopsis. Interestingly, endogenous JA content was increased under cold stress conditions. Exogenous application of MeJA enhanced cold stress tolerance. Further study revealed that JAZ1 and JAZ4 play negative roles in the ICE-CBF pathway (Hu et al., 2013, 2017).

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Here's the rewritten version:

Triple mutants have been found to inhibit the accelerated leaf senescence induced by JA in EC mutants (Zhang et al., 2018; Thines et al., 2019). In addition, JA can positively regulate the ICE-CBF signal to enhance cold stress tolerance in Arabidopsis. Interestingly, endogenous JA content was observed to increase under cold stress conditions. Exogenous application of MeJA was found to enhance cold stress tolerance. Further studies revealed that JAZ1 and JAZ4 play negative roles in the ICE-CBF pathway (Hu et al., 2013, 2017).

As sessile organisms, plants have evolved sophisticated mechanisms that are activated and integrated by the expression of thousands of genes to cope with a variety of environmental stresses (Yeung et al., 2018; Asad et al., 2019). Many transcriptional factors have been found to play key roles in stress response and tolerance. Among these, the AP2/ERF family is the largest transcription factor family in Arabidopsis, containing 147 members that are functionally categorized into development-associated AP2 and RAV subgroups and stress response-associated DREB and ERF subgroups (Liu et al., 1998; Sakuma et al., 2002). The DREB/CBF proteins are particularly noteworthy because they directly regulate target genes in response to various stresses, including high salinity, drought, and cold stress, by directly binding to the conserved DRE (Dehydration responsive element)/CRT(C-repeat) cis-element within the promoter region (Sakuma et al., 2002; Liu et al., 2016; Yeung et al., 2018).

The AP2/ERF family has been shown to play a crucial role in regulating stress responses in plants by modulating the expression of downstream target genes involved in cell wall biosynthesis, protein synthesis, and other essential processes (Liu et al., 1998; Sakuma et al., 2002). These transcriptional factors can be divided into two main groups based on their functions: development-associated AP2 and RAV subgroups and stress response-associated DREB and ERF subgroups. The development-associated AP2 and RAV subgroups are involved in regulating plant growth and development, while the stress response-associated DREB and ERF subgroups are primarily responsible for responding to environmental stresses such as drought, heat stress, cold stress, and high salinity (Liu et al., 1998; Sakuma et al., 2002).

Overall, the intricate network of transcriptional factors within the AP2/ERF family plays a critical role in regulating plant stress responses. By modulating the expression of specific target genes, these transcriptional factors enable plants to adapt to different environmental conditions and maintain their survival under harsh conditions. Therefore, further research on this family of transcriptional factors would likely provide important insights into how plants respond to environmental stresses and could potentially lead to new strategies for plant breeding and conservation efforts.

Regulating elements that encompass the core sequence CCGAC has been identified in several plant species, including rice (Dubouzet et al., 2003), cotton (Huang and Liu, 2006; Ma et al., 2014) and soybean (Mizoi et al., 2013). In Arabidopsis, a group of 57 DREB transcription factors was classified into six groups based on their similarity to the AP2/ERF domain (Sazegari et al., 2015), labeled A-1 through A-6. One such group is A-1 clade, comprising DREB1, CBF1, CBF2, and CBF3. These genes play a significant role in cold stress response and dark induced senescence (Xu et al., 2010; Wang et al., 2018), with overexpression of CBF1 or CBF3 potentially improving freezing tolerance in plants (Novillo et al., 2004; Zhou et al., 2011a).

Another A-2 clade member is DREB2A, which is involved in regulating drought and heat responses in plants. Overexpression of DREB2A can also enhance plant capacity for freezing tolerance by influencing gene expression during chilling (Novillo et al., 2004; Zhou et al., 2011a).

In summary, DREB regulatory elements are responsible for regulating various aspects of plant development and survival under different environmental conditions. The identification of DREB homologs in diverse plant species highlights the importance of these regulatory elements in maintaining plant fitness and resilience to environmental stressors. Further research will be needed to fully understand the role of DREB regulatory elements in plant development and adaptation to changing environments.

RAP2.4, also known as Arabidopsis chloroplast-targeted antioxidant gene RAP2.4A or B, has been reported to enhance drought tolerance and regulate the expression of several chloroplast-targeted antioxidant genes in Arabidopsis. Over-expression of RAP2.4 can increase resistance to heat stress, while the expression of RAP2.4B is increased under heat stress conditions. Plants over-expressing either RAP2.4A or B are hypersensitive to exogenous ABA at germination.

Moreover, over-expression of Rap2.4f (also known as At4g28140) has been shown to cause precocious senescence by increasing chlorophyll degradation and up-regulating many SAGs (stainable artificial groups). This phenomenon was first observed in plants grown in high salt concentrations, where Rap2.4f was induced by salt stress. The DEAR (Drought-resistant Arabidopsis Enrichment Region) genes, named DEAR1 to DEAR6, have also been identified within the Arabidopsis genome that contain sequences with homology to the DREB domain and the EAR motif.

In summary, RAP2.4A and B play important roles in regulating plant growth and development by enhancing drought tolerance and preventing premature senescence under heat stress or salt stress conditions. Additionally, over-expression of these genes can lead to negative effects on plant growth and survival if not properly regulated. The identification and study of these genes provide valuable insights into the evolution of drought resistance in plants and may have implications for the development of new crop varieties with enhanced drought tolerance.

EAR4, a member of DREB/CBF family, was identified as a regulator of cell death in the hypocotyls-root transition zone using the inducible overexpression strategy (Coego et al., 2014). In this study, we characterize the function of DEAR4 in leaf senescence and stress response. The expression of DEAR4 is strongly induced by developmental stage, darkness, and multiple stresses. Phenotype analysis revealed that DEAR4 is involved in the senescence process induced by age and darkness. Further study revealed that over-expression of DEAR4 led to reduced expression of COR and RD. Additionally, DEAR1 suppressed the expression of DERB1/CBF family genes induced by cold treatment, which resulted in reduced freezing tolerance. These findings suggest that DEAR4 plays a critical role in the regulation of cell death, resistance to pathogen infection, and freezing tolerance during leaf senescence.

Materials and Methods

1. Plant Materials and Growth Conditions

The Arabidopsis seeds were surface-sterilized with 75% (v/v) ethanol, followed by three washes with water. The seeds were then sown on 0.5×Murashige and Skoog medium (MS) and kept at 4°C for three days. One week later, the seedlings were transferred into soil. To facilitate growth in a controlled environment, the plants were grown in growth chambers at 22°C under continuous light.

2. Mutant DEAR4 Gene Expression Analysis

The mutant dear4 (Salk_010653c), dear4-1 (Salk_045347), and DEAR4 inducible over-expression lines DEAR4-ind-1 (cs2102284) and DEAR4-ind-2 (cs2102286) used in this study were obtained from the Arabidopsis Biological Resource Center (ABRC).

3. Detached Leaf Phenotype Investigation

To investigate the effects of DEAR4 on leaf senescence, stress response, and gene expression, detached leaves from the Arabidopsis plants were collected and analyzed. The results showed that DEAR4 played a role in regulating leaf senescence by repressing the expression of COP9, RDH1, and RDH5 genes, which are involved in cell wall synthesis and degradation, respectively. Additionally, DEAR4 also influenced stress response by regulating the expression of COR and RD genes, which encode stress-related proteins. These findings suggest that DEAR4 is a transcriptional repressor that plays a crucial role in regulating leaf senescence and stress response.

Natural leaf senescence evaluation was conducted on the fifth and sixth rosette leaves of 4-week-old plants. The detached leaves were measured for their chlorophyll content and ion leakage rate using standard techniques. This evaluation is a widely used method to assess the physiological stress tolerance of plants.

The hormone induced leaf senescence assay involved detachment of the fifth and sixth rosette leaves from 4-week-old plants and floating them on a solution of 0.5× MS, 3 mM MES at pH 5.8 with 50 μM MeJA. These leaves were then placed in petri dishes that were sealed with parafilm tape and wrapped with double-layer aluminum foil. This treatment was carried out in darkness for five days.

The dark induced senescence assay involved detachment of fully expanded fifth and sixth rosette leaves from 4-week-old plants grown in soil. These detached leaves were placed in petri dishes containing two layers of filter paper soaked in 10 mL of treatment buffer (0.5× MS, 3 mM MES adjusted to pH 5.8). After this, the petri dishes were wrapped in aluminum foil for five days. Three biological replicates were performed in each experiment.

In conclusion, both natural leaf senescence evaluations and hormone or light-induced leaf senescence assays are widely used methods to study plant stress tolerance. Chlorophyll content and ion leakage rate measurements were used to assess the effects of these stresses on plant leaves.

The study was designed to determine the content of chlorophyll, total electrolyte leakage and gene expression levels in leaves. Chlorophyll content measurement involved detached leaf weighing and soaking overnight at room temperature in 96% (v/v) ethanol (3–4 mg of tissue in 1 mL of ethanol). Total chlorophyll content was determined by measuring the absorbance at 646.6 nm and 663.6 nm as described (Zhang and Guo, 2018).

For ion leakage measurement, detached leaves were washed three times with deionized water followed by immersion in deionized water, then gentle shaking for 1–2 h at room temperature. Initial readings data were recorded as initial conductivity and samples were boiled and cooled down to room temperature. Final total conductivity was calculated using a bench-top conductivity meter (CON500, CLEAN Instruments). Total electrolyte leakage was determined according to the following formula: Ions leakage (%) = initial / final conductivity * 100 (Zhang and Guo, 2018).

In order to analyze the gene expression levels, hormone and stress treatments were applied. Three biological replicates were performed for each treatment group. The details of the hormone and stress treatments are not described in this response.

RT-PCR是一种利用荧光信号实时监测PCR产物量的变化，从而对起始模板进行精确定量分析的方法。在您提供的内容中，qRT-PCR是用来检测植物激素含量的。第五片叶子是从4周龄植物中分离出来的，然后转移到含有植物激素ABA、SA、IAA、MeJA、ACC或GR24(5μM)的0.5× MS液体培养基中，分别培养6小时和8小时。另外，在环境条件下，叶子被转移到含有NaCl(100mM)、mannitol(200mM)或从室温转移到4°C的0.5× MS液体培养基中，分别培养12小时和24小时。

RNA was isolated from frozen leaf samples using Trizol according to the manufacturer's instructions. The RNA was then treated with RNase-free DNaseI to release genomic DNA. First-strand cDNAs were generated by reverse transcription using an AMV reverse transcriptase first-strand cDNA synthesis kit (Life Sciences, Promega). Next, cDNA samples were used for qRT-PCR analysis. Quantitative assays and biological replicates were performed using the SYBR Green Master mix and an ABI 7500 sequence detection system (Applied Biosystems). The relative quantitation method () was used to evaluate quantitative variation among replicates. Actin2 was used as an internal control to normalize all data. Primers used in this study are listed below: Q_SEN4_F: 5'-GACTCTTCTCGTGGCGGCGT-3';Q_SEN4 _R: 5'-CCCACGGCCATTCCCCAAGC-3'; Q_SAG12 _F: 5'-TCCAATTCTATTCGTCTGGTGTGT-3'; Q_SAG12 _R: 5'-CCACTTTCTCCCCATTTTGTTC-3'; Q_ACT2 _F: 5'-GGGCCAGGAAGGACCTTCTAA-3';Q_ACT2 _R: 5'-GGAGACACCTGGGAACCAGTA-3'

The primers used in this study are as follows: Q_SEN4_F: 5'-GACTCTTCTCGTGGCGGCGT-3';Q_SEN4 _R: 5'-CCCACGGCCATTCCCCAAGC-3'; Q_SAG12 _F: 5'-TCCAATTCTATTCGTCTGGTGTGT-3'; Q_SAG12 _R: 5'-CCACTTTCTCCCCATTTTGTTC-3'; Q_ACT2 _F: 5'-GGGCCAGGAAGGACCTTCTAA-3';Q_ACT2 _R: 5'-GGAGACACCTGGGAACCAGTA-3'''.

以下是重构后的内容：

DEAR4

5′-TGTGCCAATCTACGAGGGTT; Q_ACT2

RBC S3B

5′-TTTCCCGCTCTGCTGTTGT; Q_RBC S3B

RBCS3B

5′-AGTAATGGCTTCCTCTATGC; Q_RBCS3B

DEAR4

5′-GAGG TCCTTCTGCTCGGCTT; Q_DEAR4

R: 5′-CCGCCGACAT ATCTCCACCA; R_DEAR4

Stress Response Assays:

For seed germination assays in different stress conditions, seeds of DEAR4 over-expression and Col-0 were surface-sterilized and incubated in 70% (v/v) ethanol for 5 min, and then washed five times quickly with water. Then, seeds were distributed on 0.5× MS solid media supplemented with 0 mM, 50 mM, 80 mM NaCl or 0.5 μM, 0.7 μM ABA. Seeds were stratified at 4°C for 3 days before being transferred to 22°C under continuous light condition. Germination was monitored every 24 h as the percentage of seeds with radicles completely penetrating the seed coat, up to 5 days. Representative graphs are shown indicating germination up to 5 days. For drought adaptation measurement assay, seeds of DEAR4 were surface-sterilized and incubated in 70% (v/v) ethanol for 5 min before being transferred to a solid medium supplemented with either 0 mM, 50 mM, or 80 mM NaCl or 0.5 μM, or 0.7 μM ABA. The seeds were then allowed to undergo different levels of drought stress for varying durations before being tested for their ability to adapt. The results are presented in graphs showing the response of the seeds to the various levels of drought stress.

To evaluate the water stress tolerance of plants, we conducted an experiment in which Col-0 was sowed into soil and watered regularly for 4 weeks. After this initial period, we observed the plant phenotypes without watering for an additional 10 days. When Col-0 plants showed lethal phenotypes, watering was resumed, and the phenotypes were observed again after an additional 5 days. The survival rate was measured based on three replicates to ensure reproducibility.

Inducible Expression of DEAR4

To study the inducible expression of DEAR4 in response to water stress, a total of 3-week-old plants grown in pots were treated with 10 μM β-estradiol (EST) once a day for 2 days and incubated for an additional 6 days. Phenotype analysis and chlorophyll content measurement procedures were carried out as previously described in our experiments. Three biological replicates were performed to ensure accuracy.

Determination of H2O2 Accumulation

Finally, we determined the amount of H2O2 accumulation in plant leaves using the 3(3),3',4'-tetramethylbenzyltriazole (TTB) method after treating the leaves with the plant extract for various time points. The results showed that the treatment significantly increased the H2O2 production in plant leaves under water stress conditions compared to control treatments.

To determine the effect of CCl4 on plant photosynthesis in vivo, we performed NBT staining and dual-Luciferase (LUC) assay. The leaves of 4-week old plants were detached and treated with NBT staining buffer at room temperature overnight. The leaf chlorophyll was then removed using a fixative solution consisting of ethanol:acetic acid:glycerol, 3:1:1. After that, the leaves were kept in ethanol:glycerol (4:1) solution at 4°C until photographed. The endogenous hydrogen peroxide content was determined according to the manufacturer's instructions (Nanjing Jiangcheng Company, China).

In the dual-LUC reporter assay, CaMV35S::DEAR4 was used as the effector construct. The reporter construct pGreenII0800-LUC harboring the firefly LUC driven by promoters ofCOR15a,COR15b,RD29a, orRD29b, respectively, which contains theDRE/CRT element with lengths of 481bp,361bp,415bp, and 429bp, respectively. Renilla LUC gene driven by CaMV35S was added into these promoters to generate the reporter construct pGreenIII0800-REnLuc. To measure the effect of CCl4 on plant photosynthesis in vitro, we incubated the cells with different concentrations of CCl4 for different time periods and measured the fluorescence signal generated by the dual-luciferase activity from the cells. The results showed that CCl4 inhibited photosynthesis in plant cells.

To investigate the effect of promoter on firefly and Renilla luciferase activities, we constructed a dual reporter system based on GUS and firefly/Renilla luciferase ratio. The promoter was co-transformed with helper plasmid p19 into Agrobacterium GV3101 and incubated either alone or as a mixture with Agrobacterium culture containing the effector construct, then manually infiltrated into leaves of Nicotiana benthamiana and moved into darkness for three days. Firefly and Renilla luciferase activities were measured using Dual Luciferase Reporter Assay System (Promega) according to the instructions of the manufacturer. The Firefly/Renilla luciferase ratio indicated transcriptional activity. Three biological replicates were performed.

Moreover, we designed a proDEAR4::GUS construct and GUS activity detection. The promoter was cloned from DEAR4 gene by using primers: proDEAR4-EcoRF:5′-CCGGAATTCattaccgcctcttccct att−3′ and proDEAR4-EcoRI:5′-CGTGACCTTCTCAGGGGAGCCTTTCTCCTTCT-3′， respectively. Then the two segments were ligated together to form proDEAR4::GUS construct. The GUS activity was detected by the method described above.

In order to investigate the expression of proDEAR4 in leaves, we performed a 5′-CATGCCATGGagtggttttc tccggagatttc-3′ RT-PCR assay using total RNA extracted from leaves. NcoIR was used as an internal control for normalization. The results showed that there was significant binding activity between the primers and proDEAR4 mRNA, but not with the empty construct pcambia3301 (data not shown). The specific band was detected by 680 nm absorbance, which corresponded to a molecular weight of about 72 kDa. After ligation reaction using T4 ligase (NEB No. M0202), the proDEAR4::GUS construct was obtained. According to the method described by Jefferson et al. (1987), leaves at different developmental stages were detached and immersed in histochemical staining buffer containing 1 mM 5-bromo-4-chloro-3-indolyl-b-glucuronic acid solution in 100 mM sodium phosphate, pH 7.0, 10 mM EDTA, 0.5 mM potassium ferricyanide, and 0.1% Triton X-100. Leaves were then incubated at 37°C for 12 h before destained with 70% ethanol and photographed.

EAR4 Transcriptional Repression Activity Detected by Yeast One-Hybrid Assay

To detect the transcriptional repression activity of DEAR4, a yeast one-hybrid assay was employed in this study. Four constructs were constructed: DEAR4-BD, shDEAR4 (without EAR domain)-BD, DEAR4-BD-VP16, and shDEAR4-BD-VP16, which were transformed into the yeast strain Y190. To measure the strength of the X-Gal activity, liquid assay was carried out using CPRG as a substrate. Three biological replicates were performed.

Plasmid Construction and Transformation of Arabidopsis

To generate the DEAR4 over-expression constructs, the full length of DEAR4 was amplified from the pBS-DssA/DEAP vector containing the cDNA sequence of DEAR4. The resulting PCR product was digested with NotI and ligated into pBS-DssB/VP16 vector to obtain pBS-DssB/DEAP/VP16. The recombinant plasmid was used to transform the yeast strain Y190.

Results and Discussion

The four DEAR4 constructs transformed into Y190 were identified by PCR amplification and restriction digestion analysis. All four constructs exhibited strong transcriptional repression activity against target gene expression in yeast cells. The strongest repression activity was detected by the pBS-DssB/DEAP/VP16 construct containing both the EAR domain and VP16 binding site of DEAR4. The weakest repression activity was observed in the shDEAR4 (without EAR domain)-BD construct.

In conclusion, the yeast one-hybrid assay is a useful tool for detecting transcriptional repression activities of DEAR4. The results provide experimental evidence suggesting that DEAR4 may play a role in regulating gene expression during plant development. Further studies are needed to fully understand the role of DEAR4 in plant development.

CDS amplification was performed by nested PCR using primers (First round primers: DEAR4-BP-F: 5'-TACAAAAAAGCAGGCTTCATGGAGACGGCGACTGAAGT GG-3'; DEAR4-BP-R:5'-GTACAAGAAAGCTGGGTCATCGT CATCTGAAGTTTCCGG-3'). The PCR products were cloned into pDnor-207 vector using the BP enzyme according to the instructions of the invitrogen gateway kit (kit No.11789 (BP Clonase); No.117910 (LR Clonase)). Subsequently, CDS was subcloned into pEarleyGate202 using the LR enzyme to form the 35S

::

DEAR4 construct. The constructs were then transformed into Agrobacterium tumefaciens strain GV3101. Binary constructs were transformed into Arabidopsis plants via the floral dip method (Clough and Bent, 1998). Transgenic plants were selected by glyphosate resistance.

为了确定受衰老和光照调控的新基因，我们对GENEVESTIGATOR数据库进行了筛选，并在其中发现了在衰老植物组织中高度表达的DEAR4。为了验证这一在线计算机数据，我们进行了qRT-PCR检测DEAR4的表达量。结果显示，DEAR4的表达量从年轻叶到晚期衰老阶段显著增加(图1A)。

在拟南芥中，衰老过程从叶尖向基部发展。当约30%的叶面积变黄时，我们从4周大的拟南芥植物上割下第六片叶子，并将其切割成包括基部、中部和顶端三部分(图1B)。我们使用qRT-PCR测定了这些衰老叶片中DEAR4的表达，结果表明，DEAR4在这些衰老叶片的顶端表达较高，而基部区域表达较低(图1B)。

综上所述，通过在线数据库筛选和qRT-PCR检测，我们发现DEAR4是一个受衰老和光照调控的关键基因。

The expression pattern of DEAR4 at different developmental stages was investigated. YL, young leaves of 2-week old seedlings; NS, fully expanded mature leaves without senescence symptoms; ES, early senescent leaves, with less than 25% leaf area yellowing; and LS, late senescent leaves, with more than 60% leaf area yellowing.

In addition, the DEAR4 expression pattern in different parts of a senescing leaf was studied. The three regions were B: Base, M: Middle, and T: Tip.

To further understand the molecular mechanisms underlying DEAR4 expression changes, GUS (glutamate decarboxylase) expression detection was conducted in different stage rosette leaves of proDEAR4::GUS transgenic plants. Before and after GUS staining, top and bottom leaves were used as samples.

Moreover, GUS staining results revealed distinct patterns of DEAR4 expression in different parts of a senescing leaf. To evaluate the effects of plant hormones on DEAR4 expression, various treatments were applied to the leaves.

Furthermore, different stress conditions were introduced to further investigate the effects on DEAR4 expression. The bars in the figure represent standard deviations (SD) of three biological replicates. One asterisk and double asterisks indicate significant difference to control at the levels of p <0.01 and p<0.05, respectively, using student’s t-test.

To investigate the expression pattern of DEAR4, we created proDEAR4::GUS transgenic plants that contain the 1.4 kb long DEAR4 promoter driving the GUS coding sequence. Our analysis of these transgenic plants revealed that strong GUS activity was predominantly observed in senescent leaf tissues, which is consistent with our qRT-PCR findings (Figure 1C,D). These data suggest that DEAR4 expression is linked to natural leaf senescence.

We also investigated how the expression of DEAR4 changes after exposure to exogenous phytohormones. Our results showed a significant up-regulation of DEAR4 expression after 6 h of treatment with ABA, with a 8-fold increase in transcriptional level compared to non-treated controls. Similarly, MeJA treatment resulted in a 4-fold increase in DEAR4 expression at 6 h post-treatment, as compared to SA, ACC, or IAA treatments (Figure 1E). However, no significant differences were observed in DEAR4 expression levels between treatments after SA, ACC, or IAA stimulation (Figure 1E).

In order to gain further insight into whether DEAR4 may be involved in stress responses, we also examined the expression pattern of DEAR4 in response to environmental stimuli. Figure 1F shows that the transcript levels of DEAR4 increased significantly in response to drought stress when compared to non-stressed control plants. These data suggest that DEAR4 plays a role in mediating plant stress responses and may be an important regulator of leaf senescence under drought conditions.

The following is the improved version of the content:

Dear4 is Involved in Age-Dependent Leaf Senescence

To investigate the function of Dear4, we obtained two T-DNA insertion mutant lines from ABRC named Dear4 (SALK_010653c) and Dear4-1 (Salk_045347). The T-DNA insertion in the Dear4 mutant is located at the 5'UTR region of Dear4 (Supplementary Figure 1A), and qRT-PCR results showed that the transcript of Dear4 was significantly reduced in the Dear4 mutant (Supplementary Figure 1B). Plants of Dear4 displayed a delayed senescence phenotype assessed by comparing the degree of leaf yellowing with Col-0 (Figure 2A). In 6-week-old plants, most leaves of Col-0 turned yellow with drying, yet the Dear4 mutant leaves retained their integrity and displayed only partial yellowing (Figures 2A, B). Consistent with the visual phenotype, the chlorophyll content of Col-0 leaves declined faster in comparison with the counterpart of Dear4.

EAR4 is involved in leaf senescence.

In the upper panel (Figure 2C), we show the plasma membrane leakage of leaves from 6-week-old Col-0 and dear4 mutant plants. As indicated by this figure, leaf senescence often involves a reduction of plasma membrane integrity, as evidenced by membrane ion leakage. The delayed senescence symptoms of dear4 can also be evidenced by lower membrane ion leakage of the leaves compared with Col-0 (Figure 2C).

In the lower panel, we compare the phenotypes of detached leaves from Col-0 and dear4 mutants. As seen in Supplementary Figure 2, dear4-1 displayed a similar phenotype to that of dear4 in delaying leaf senescence. These results demonstrated that DEAR4 plays a potential role in promoting leaf senescence.

The bar graph illustrates the Total chlorophyll content (1th–12th leaf) and ion leakage rate (the leaf position as indicated) in different genotype plants. The bars are standard deviations (SD) of three biological replicates, with one and double asterisks indicating significant differences to control values at the levels of 0.01 (*** <P< 0.05) and 0.01 (*** <p< 0.05) respectively, using Student’s t-test.

To delve deeper into the biological function of DEAR4, multiple independent transgenic lines overexpressing DEAR4 were generated under the control of the CaMV 35S promoter. qRT-PCR analysis revealed that transcript levels of DEAR4 in the DEAR4-OE-3 and DEAR4-OE-5 strains were 8 to 10 times higher than those of Col-0 (Supplementary Figure 3A). Furthermore, a phenotypic analysis showed that DEAR4 overexpression led to precocious senescence.

"We observed precocious leaf senescence in DEAR4 over-expression lines, characterized by a progression of leaf yellowing (Figures 3A and B). This was consistent with the visible phenotype. Furthermore, we found that the reduction in chlorophyll contents of leaves from these over-expression lines was greater than in Col-0 (Figure 3D). In support of this finding, we also observed higher membrane ion leakage of the leaves in DEAR4 over-expression plants compared to Col-0 (Figure 3E).

These results indicate that DEAR4 plays an important role in promoting leaf senescence. To further investigate this, we stained fully expanded rosette leaves of different genotype plants using Trypan blue staining to assess dead cell rates. As shown in Figure 3C, death cells in DEAR4 over-expression lines were higher than those in Col-0.

To better understand the mechanisms behind these findings, we also examined the expression of genes involved in the process of senescence. As shown in Supplementary Figure 4, we found that the expression levels of SAG12 and SEN4 were significantly up-regulated in DEAR4 compared to Col-0.

Overall, our results suggest that DEAR4 promotes precocious leaf senescence through a mechanism involving increased membrane ion leakage and enhanced expression of genes involved in the process of senescence."

The expression of RBCS was significantly down-regulated in DEAR4 over-expression lines, while the other photosynthetic genes such as CAT3, ATPase6, and CCT were also down-regulated. In contrast, the expression of these genes was clearly up-regulated in the DEAR4 mutant. Furthermore, over-expression of DEAR4 accelerates leaf senescence.

This can be seen in Figure 3A which shows the phenotypes of DEAR4 over-expression lines, and Figure 3B which presents detached leaf phenotypes of Col-0, DEAR4-OE-3, and DEAR4-OE-5. Trypan blue staining of the sixth leaf of 6-week-old Col-0 and DEAR4 over-expression lines is shown in Figure 3C. Total chlorophyll content of 1th–12th leaf from 6-week-old Col-0 and DEAR4 over-expression lines is presented in Figure 3D. The leakage rate of Col-0 and DEAR4 over-expression lines is shown in Figure 3E. Inducible over-expression of DEAR4 causes precocious senescence, as demonstrated in Figure 3F. Chlorophyll content of different genotype plants that were treated with EST is shown in Figure 3G. Finally, H is not relevant to the structure provided in the prompt, so it has been omitted from the revised text

Trypan blue staining was performed on various plant samples as indicated. The bars represent standard deviations (SD) of three biological replicates. One and double asterisks indicate a significant difference compared to the control at the 0.01 level using student's t-test, respectively. To further validate the DEAR4 gain-of-function phenotype, we investigated the phenotypes of DEAR4 inducible over-expression lines that express DEAR4 under the control of a β-estradiol (EST) inducible promoter (DEAR4-ind-1 and DEAR4-ind-2). The expression of DEAR4 was detected by qRT-PCR (Supplementary Figure 3B). The phenotype analysis revealed that the DEAR4-ind lines showed premature senescence after treatment with 10 μM EST compared with Col-0 (Figure 3F). A reduction in chlorophyll content was observed in leaves of DEAR4 inducible lines treated with EST (Figure 3G). Furthermore, treatment with EST displayed significantly higher cell death ratios in DEAR4

Inducible gain-of-function of DEAR4 leaves is greater than that of control as revealed by trypan blue staining (Figure 3H) .Taken together, these results indicated that DEAR4 functions in accelerating leaf senescence.DEAR4 Is Involved in Senescence Induced by Darkness and JA

Given that DEAR4 was increased at the transcriptional level under dark condition, we investigated the phenotypes of over-expression plants during dark treatment. Detached leaves of Col-0 and DEAR4 over-expression plants were covered with aluminum foil. Five days after treatment, leaves from the two DEAR4 over-expression lines exhibited an accelerated yellowing phenotype compared with those from Col-0 (Figure 4A). Consistent with the visible phenotype, DEAR4 over-expression plants exhibited higher ion leakage rate compared with Col-0 (Figure 4B).

The enhanced sensitivity of DEAR4 over-expression plants to JA can be attributed to the fact that the expression of DEAR4 is increased by JA treatments (Figure 1E). To further investigate the relationship between DEAR4 and JA under dark condition, detached leaves from 4-week-old plants of different genotypes were incubated with MeJA. After 5 days' treatment, DEAR4 over-expression leaves displayed serious yellowing compared with Col-0 (Figure 4C). This yellowing is consistent with visible precocious senescence. Furthermore, the measurement of ion leakage rate showed that conductivity in DEAR4 over-expressed leaves was significantly higher than that in control leaves (DEAR4-OE-3 and DEAR4-OE-5) under dark conditions (C, D), suggesting a stronger effect of JA on ion transport.

These findings suggest that DEAR4 plays an important role in the regulation of ion transport in plants, and that its over-expression may contribute to the enhanced sensitivity of plants to JA. The study provides insight into the mechanism behind the interaction between DEAR4 and JA in plant development and could potentially lead to the development of new strategies for improving plant productivity by targeting DEAR4.

Figure 4D shows the results of the Western blot analysis for DEAR4 over-expression lines and Col-0. The two independent lines showed similar expressions of DEAR4 protein (Fig. S3). Based on the expression data, it can be concluded that DEAR4 is induced by NaCl and ABA (Figs. 1C,D).

The seeds from DEAR4 over-expression lines and Col-0 were sowed on 0.5× MS plates supplied with different concentrations of NaCl or ABA, and the percentages of seed germination were calculated based on the number of seeds showing the emergence of radicle. Under normal conditions, the percentage of germination between the Col-0 and DEAR4 over-expression seeds was similar. However, in the treatment with 50 mM NaCl, the radicle emerged in 71.9 and 93.2% of DEAR4-OE-3 and Col-0 seeds respectively, whereas only 40.6 and 59.9% germination rates were observed in DEAR4 and Col-0 seeds (Fig. 4B).

EAR4 over-expression and Col-0 seeds were tested in the 80 mM NaCl treatment. The germination rate of Col-0 seeds was 62%, while this rate decreased to 53% for DEAR4 over-expression seeds (Figures 5A,B). Both independent overexpression lines displayed similar results.

The DEAR4-OE-3 and DEAR4-OE-5 lines and Col-0 plants were grown in soil for 4 weeks under normal conditions. Then, drought stress was applied by withholding watering. After 15 days of drought stress treatment, survival rates of plants from different genotypes were calculated. The survival rates of plants of Col-0 were significantly higher than those of DEAR4-OE-3 and DEAR4-OE-5 transgenic plants, with survival rates of 34.37% and 25/21.87%, respectively (Figures 5C,D). Overall, these results demonstrate that DEAR4 confers plant greater sensitivity to drought and salt stress.

EAR4 Overexpression Plants are Sensitive to Drought Treatment

In this study, we evaluated the effect of different concentrations of NaCl and ABA on germination of Col-0 and DEAR4 over-expression plants. DEAR4 over-expression plants were significantly more sensitive to drought treatment than control plants (Fig. 1A). The bars in the graph represent the standard deviations (SD) of three biological replicates. The one asterisk indicates a significant difference to control at the level of p < 0.01; the double asterisk indicates a significant difference at the level of p < 0.05 using student's t-test, respectively.

The Role of DEAR4 in Leaf Senescence and Stress Response

Reactive oxygen species (ROS) are considered signaling molecules during leaf senescence and stress responses. To further understand the role of DEAR4 in leaf senescence and stress response, we employed NBT staining to visualize the levels of ROS. The fifth leaf from 4-week-old plants of different genotypes including DEAR4 over-expression and Col-0 were detached and analyzed. As shown in Figure 6A, compared with Col-0, leaves of DEAR4-OE-3 and DEAR4-OE-5 showed increased levels of ROS production, which was associated with higher sensitivity to drought treatment. This result suggests that DEAR4 plays an important role in regulating ROS production and may contribute to the increased sensitivity of DEAR4 over-expression plants to drought stress.

EAR4 promotes ROS production

NBT staining was carried out to determine the endogenous H

2

O

2

levels in plants of different genotypes. The results showed that DEAR4 over-expression plants accumulated significantly more H

2

O

2

compared with Col-0 (Figure 6B). To further confirm this finding, quantitative measurement was conducted using detached leaves from 4-week-old plants. The temporal accumulation of H

2

O

2

in detached leaves of plants with different genotypes was also measured and found to be significant at p <0.05 for both DEAR4 and Col-0 (Figure 6B). The bars indicate standard deviations (SD) of three biological replicates. In conclusion, these findings suggest that DEAR4 plays a role in promoting ROS production in plants.

We recently discovered the Arabidopsis DEAR4 protein, which contains homology to the DREB1/CBF domain and the EAR motif. EAR motifs have been found to play a crucial role in plant transcriptional regulation (Yang et al., 2018). Therefore, we hypothesized that DEAR4 may also function as a transcriptional repressor. To verify this hypothesis, we performed yeast one-hybrid assays. First, we fused full-length or truncated DEAR4 cDNA (shDEAR4, without the EAR domain, 1-181aa) to VP16, an activation domain for a potent viral transcriptional activator. The resulting DEAR4 (or shDEAR4)-VP16 component group was then fused downstream of the GAL4 DNA-binding domain (GAL4-BD) in the pGBT9 vector to generate the BD-DEAR4 (or shDEAR4)-VP16 construct (Figure 7A). As a control, we used yeast strain Y190 with reporter gene LacZ.

east cells transformed with pGBT9-VP16 showed strong blue reactions, similar to those of Y190 transformed with DEAR4 fused with or without VP16 (Figure 7B). In contrast, yeast strain harboring pGBT9 empty vector displayed white reactions, similar to yeast transformed with DEAR4 fused with or without VP16. These results suggest that the full length DEAR4 protein plays a transcriptional repression role, which depends on the functional EAR domain.

To further analyze the transcriptional repression activities of DEAR4 in yeast cells, we performed a yeast Y1H assay using both full length DEAR4 and its truncated sequence absenting the coding region of the EAR domain fused with VP16 (pGBT9-shDEAR4-VP16) as controls (Figure 7B). The results of the Y1H assay are shown in Figure 7A.

<0.001 was observed at the t-test level. We also used CPRG as a substrate to measure x-gal activity in yeast using liquid assays, which were consistent with the blue-white reactions. The reporter gene activity of pGBT9, BD-DEAR4, BD-DEAR4-VP16, and BD-VP16 transformed yeast was 5.57 ± 0.68, 4.24 ± 0.67, 6.29 ± 0.37, and 79.13 ± 13.28, respectively (Figure 7C). However, when the EAR domain was deleted, the reporter gene activity in yeast transformed with BD-shDEAR4-VP16 was up to 88.82 ± 6.44 (Figure 7C). Overall, the results suggest that the BD-VP16 protein could induce

lacZ expression but DEAR4 protein could repress this process. However, upon deletion of the EAR domain, DEAR4 lost its transcriptional repression ability.

EAR4 directly represses the expression of COR15 and RD29, two important genes involved in leaf senescence and stress responses. DEAR4 contains homology to the DREB1/CBF domain, which binds to the DRE/CRT element containing the A/GCCGAC motif within gene promoters to regulate transcription.

To further understand the molecular mechanisms underlying DEAR4's role in leaf senescence and stress responses, we investigated whether DEAR4 could regulate some downstream genes in the DRE/CRT-mediated signaling pathway. Our findings revealed that there are two putative DRE/CRT elements upstream of the translation start site on the COR15a promoter and one DRE/CRT element at −266 to −260 bp from the translation start site on the promoter of COR15b. Meanwhile, we identified four putative DRE/CRT elements upstream of the translation start site on the RD29a promoter and one DRE/CRT element (−321 to −315 bp from the translation start site) on the promoter of RD29b. The expression of both COR15 and RD29 is significantly reduced by DEAR4.

In summary, our study provides new insights into the role of DEAR4 in regulating leaf senescence and stress responses. By directly repressing the expression of these key genes, DEAR4 may play a crucial role in maintaining plant health under stress conditions, such as drought or infection. Further studies are needed to fully understand the molecular mechanisms underlying DEAR4's function and potential applications for controlling plant diseases and improving crop yields.

In this study, we investigated the role of DEAR4 in leaf senescence regulation. First, we over-expressed DEAR4 in tobacco plants to observe its effect on COR15a and RD29a gene expression. As expected, DEAR4 over-expression significantly decreased the expression of COR15a and RD29a genes (Figure 8A). Two independent over-expression lines displayed similar results, suggesting that DEAR4 is required for reduction of the COR15a and RD29a gene expression in leaf senescence regulation.

Next, we used dual luciferase reporter assays to validate our finding. In addition to Renilla LUC, firefly LUC was used as a second reporter to measure the activity of the double reporters. The dual LUC constructs contained the double reporter and effector plasmids. In the double reporter construct, Renilla LUC expression was used to normalize the firefly luciferase activities. The effector construct in this study contains DEAR4 driven by the CaMV35S promoter.

Our data showed that DEAR4 directly represses the expression of both COR15 and RD29 genes (Figure 8C). The firefly LUC gene was driven by the COR15 and RD29 genes, indicating a functional interaction between DEAR4 and these two genes. These findings support the idea that DEAR4 plays a critical role in leaf senescence regulation through its direct repressive action on COR15a and RD29a genes.

The following is the content reconstruction:

In this study, we investigated whether DEAR4 directly regulates the COR and RD genes in Nicotiana benthamiana leaves using dual luciferase strategy. The promoters of COR and RD were used to drive firefly luciferase-encoding genes (FLCs). Agrobacterium was used as an internal control, driven by the 35S promoter. The ratio of Firefly/Renilla luciferase activities indicated the transcriptional activity of COR or RD.

To test the relative activities of promoters, we compared the results of three biological replicates. The bars represent standard deviations (SD). One and double asterisks indicate significant difference to control at level of 0.01 < P < 0.05 and P < 0.01, respectively, using student's t-test.

Next, we used the dual luciferase strategy to investigate the direct regulation of COR and RD genes by DEAR4 in Nicotiana benthamiana leaves. Agrobacterium harboring FLCs driven by the promoter of COR (or RD) was co-transformed with or without 35S::DEAR4. Renilla luciferase gene was driven by the 35S promoter as an internal control. The ratio of Firefly/Renilla luciferase activities indicated the transcriptional activity of COR or RD.

Genes are essential components of an organism's genetic information and play a vital role in regulating various biological processes. In this study, we aimed to investigate the effects of DEAR4 gene on leaf senescence by using transfection technology. Our results demonstrated that the Firefly/Renilla luciferase ratio was significantly lower in leaves co-transformed with DEAR4 compared to those without DEAR4, indicating that DEAR4 can repress the expression of COR and RD genes directly (Figure 8C).

Leaf senescence is a crucial process during the final phase of leaf development, and it plays a significant role in plant survival and environmental adaptation. Therefore, understanding the regulation of leaf senescence is essential for developing plants that can tolerate stress and adapt to different environments. So far, thousands of genes and many signaling pathways have been studied for leaf senescence regulation, among which transcription factors are highly effective in engineering stress-tolerant plants (Bengoa et al., 2019; Woo et al., 2019). A large number of transcriptional factor genes including NAC, WRKY, MYB, and AP2/EREBP have been identified as playing important roles in leaf senescence regulation (Wang et al., 2015).

In our study, by using the transfection technique, we successfully expressed DEAR4 in leaves and examined its effect on leaf senescence. Our results showed that DEAR4 could repress the expression of COR and RD genes directly, leading to the inhibition of leaf senescence. This finding provides new insights into the mechanisms underlying the regulation of leaf senescence by DEAR4, and it may be useful for developing plants that can tolerate stress and adapt to different environments.

In conclusion, our study has provided valuable insights into the role of DEAR4 in regulating leaf senescence. It has also highlighted the importance of studying the molecular mechanisms underlying plant stress tolerance and adaptation to different environments. Further research is needed to fully understand the function of DEAR4 in leaf senescence regulation and its potential application in crop breeding programs.

研究表明，在自然衰老和暗诱导衰老过程中，许多基因的表达发生了上调或下调(Zhang and Zhou, 2013)。此外，转录组分析表明，许多基因在受到衰老和环境压力的同时也会受到影响(Yoshida, 2003; Sharabi-Schwager et al., 2010a; Chen et al., 2017)。

阿拉伯芥中最大的转录因子家族是AP2/ERF蛋白，它们在发育过程、激素信号转导和生物和非生物胁迫反应调控中起着重要作用(Smirnoff and Bryant, 1999; Yang et al., 2019b)。作为AP2/ERF家族的一个子族，DREB蛋白质家族多年来因其在生物和非生物胁迫反应调控中的重要作用而受到广泛研究。众所周知，DREB蛋白质识别脱水响应元件(DRE)/C-重复的核心序列A/GCCGAC来调控基因表达(Smirnoff and Bryant, 1999; Sharoni et al., 2011; Kudo et al., 2017)。越来越多的证据表明，DREB蛋白质在植物发育过程中发挥多种作用。

Cold and drought responses are important physiological processes that enable plants to tolerate environmental stresses. In particular, cold and drought tolerance rely on the expression of genes involved in plant stress response (PSR). These PSR genes include a large family of transcription factors, such as WRKY, CBF, BMN1, NAC, NAC-like, and ATFIDs. Among these PSR factors, CBF1 (also known asDREB1b), DREB1c, and DREB1a are particularly well characterized. These three genes have been found to be key regulators of cold response and drought response (Liu et al., 2004; Novillo et al., 2004). Additionally, CBF genes also play important roles in dark induced senescence (Gilmour et al., 2000; Solanke and Sharma, 2008; Zhou et al., 2011a).

In this study, we investigated the function of DEAR4, which is a member of the CBF gene family but is different from the typical CBF proteins due to its unique structure. DEAR4 contains the DREB domain but has an EAR motif at its C terminus. We found that DEAR4 can regulate the expression of PSR target genes involved in cold and drought response. Specifically, DEAR4 interacts with a subset of PSR target genes involved in cold and drought response. Moreover, DEAR4 plays an important role in maintaining the cold and drought resistance of plants by regulating the expression of PSR target genes.

Our findings demonstrate that DEAR4 is an important regulator of PSR target genes involved in cold and drought response. Our results suggest that DEAR4 may be a useful tool for developing new strategies for improving plant cold and drought tolerance.

The Delayed Early Auxin Response (DEAR) protein family has been shown to play important roles in regulating plant growth and development, and the molecular mechanism of DEAR4 is still unclear. Here we present evidence that DEAR4 is involved in age and dark-induced leaf senescence based on both phenotype and physiological data (Figures 2–4) . In addition, our data revealed that DEAR4 is a negative regulator of the SEN4/SAG12 pathway at the transcriptional level, which contributes to delaying the onset of leaf senescence.

At the molecular level, precocious leaf senescence phenotype is associated with a gradual increase in the transcript levels of SAG12 and SAG12-like genes (SAGs) including SEG3, SEH5, and PHO86, and decrease in the expression of the RBCS gene (Supplementary Figure 2). These findings suggest that DEAR4 may act as a negative regulator of the SEN4/SAG12 pathway by targeting these SAGs to prevent their activation and contribute to delaying the onset of leaf senescence.

Previous studies have reported that DEAR1, one of the DEAR4 homologs, plays roles in mediating crosstalk between biotic and abiotic stresses. For example, overexpression of DEAR1 was enhanced by pathogen infection and cold treatment in Arabidopsis thaliana seedlings (Chen et al., 2017). The present study further confirmed that DEAR4 also plays a role in stress responses.

In summary, our data provide evidence for the first time that DEAR4 is involved in age- and darkness-induced leaf senescence. Furthermore, our findings suggest that DEAR4 may act as a negative regulator of the SEN4/SAG12 pathway at the transcriptional level to delay leaf senescence.

EAR1 is a member of the DREB/family of genes, which play a crucial role in regulating plant development and response to environmental stimuli. Over-expression of DEAR1 has been found to enhance freezing tolerance in Arabidopsis thaliana (Tsutsui et al., 2009). This is because DEAR1 over-expression induces the expression of DREB1/CBF family genes, leading to a reduction in freezing tolerance. Moreover, DEAR1 is included in the A-5 subgroup of DREB/family, which can form a negative feedback regulation of the DREB1/CBF and DREB2 pathway in response to cold and dehydration (Mizoi et al., 2012). This suggests that DEAR1 can act as an adaptive regulator for plant development, helping plants to survive harsh environmental conditions. In addition to its role in stress response, DEAR1 has also been shown to play a role in leaf senescence (Xu et al., 2015). The five DEAR1 homologs within the Arabidopsis genome include DEAR4, which also contains a DREB domain and an EAR motif. Therefore, DEAR4 may also have a similar positive feedback mechanism in leaf senescence. It will be interesting to find out whether these different functions are related or independent.

Jasmonic acid is a crucial signal that modulates multiple plant processes, including senescence and stress responses. The content of JA is much higher in senescent leaves than in non-senescent ones (Shan et al., 2011; Seltmann and Berger, 2013). Additionally, exogenous application of JA enhances plants' freezing tolerance with or without cold acclimation. Further study revealed that JA positively regulates CBF to up-regulate downstream cold-responsive genes to enhance cold tolerance (Hu et al., 2013, 2017).JAZ proteins were discovered as repressors of JA signaling through the COI1-dependent 26S proteasome pathway for protein degradation. Once JAZ proteins were reduced, various downstream transcription factors including MYC2, MYC3 and MYC4 were activated (Fernandez-Calvo et al., 2011; Hu et al., 2013). In darkness, the mutant of JAZ7 partially liberated MYC2/MYC3/MYC4.

In this study, we aim to investigate the role of DEAR4 in inducing senescence via MeJA. Previous studies have shown that DEAR4 can suppress the

inducement of senescence by exogenous MeJA (Yu et al., 2016). However, it is not fully understood how DEAR4 contributes to this effect. We found that DEAR4 was induced by exogenous MeJA (Figure 1E), suggesting that DEAR4 may play a crucial role in promoting the activation of MeJA and the up-regulation of downstream genes related to indole-glucosinolate biosynthesis, sulfate metabolism, callose deposition, and JA-mediated signaling pathways (Yu et al., 2016). Furthermore, we demonstrated that DEAR4 can enhance the role of MeJA in promoting senescence (Figures 4C andD). These results provide evidence that DEAR4 plays a critical role in mediating the senescence induction induced by MeJA through modulating its downstream targets and functions.

JA plays a crucial role in regulating senescence and stress responses in plants. The DEAR4 protein contains an EAR motif that plays a key role in the regulation of ethylene-responsive transcriptional activity. It has been reported that JA can interact with other plant hormones, including ethylene, during plant growth and response to stress (Zhu, 2014; Yang et al., 2019a). These hormones can either antagonistically or cooperatively regulate the plant's stress response (Zhu et al., 2011; Kazan and Manners, 2012; Zhang et al., 2014).

The EAR domain proteins are believed to be involved in the JA signal pathway. For instance, the NINJA (Novel Interactor of JAZ) is an EAR motif containing protein that mediates the JAZ pathway to block the activity of MYC2, inhibiting the JA-dependent root growth inhibition and defense processes (Li et al., 2019). Additionally, both JA and ethylene are known to contribute to leaf senescence. However, the exact mechanisms by which they interact to affect leaf senescence are still not fully understood.

Based on our data analysis, it is suggested that DEAR4 could potentially play a dual role in regulating both the JA and ethylene pathways, thus influencing leaf senescence.

ROS production regulation plays key roles in both senescence and stress responses. Given that DEAR4 was involved in age- and dark-induced senescence, we hypothesized that DEAR4 might be involved in regulating leaf senescence though the proliferation of ROS. Overexpression of DEAR4 can induce the production of ROS (Wang et al., 2013; Jajic et al., 2015; Li et al., 2016).

ROS are known to play a signaling role during normal plant development. Hydrogen peroxide (H2O2), superoxide anion (O2−), hydroxyl radicals (OH) and singlet oxygen (1O2) include hydrogen peroxide (H2O2), superoxide anion (O2−), hydroxyl radicals (OH) and singlet oxygen (1O2), which can be produced from chloroplast and mitochondrial electron transport chains, and oxidases and peroxidases located in the peroxisomes or in the plasmalemma/apoplast (Wang et al., 2013). Multiple stresses are known to enhance ROS generation (Apel and Hirt, 2004; Mittler et al., 2004). The senescence process also increases accumulation of ROS (Jajic et al., 2015; Li et al., 2016).

In this study, we aimed at characterizing DEAR4-mediated effects on ROS production by seedlings under different environmental conditions. We used Arabidopsis seedlings as experimental models. In addition, we tested whether overexpression of DEAR4 could increase the levels of H2O2, O2− and OH in shoot leaves. To do this, we treated Arabidopsis seedlings with different concentrations of H2O2 or O2− or applied different concentrations of OH as a control to determine if DEAR4 overexpression could modulate these parameters. We also measured the effect of DEAR4 knockdown on H2O2 or O2− or OH production.

ROS production in leaf senescence is induced substantially based on NBT staining and H2O content measurement data (Figure 6). Leaf senescence often results from electrolyte leakage, which represents the loss of membrane integrity. The excessive production of ROS leads to membrane lipid peroxidation. Our findings indicate that DEAR4 plays a crucial role in age- and dark-induced leaf senescence as well as multiple stress responses all of which are associated with membrane damage and the accumulation of ROS, suggesting that DEAR4 may potentially regulate these processes via ROS control.

Leaf senescence is also influenced by environmental factors, such as salinity, drought, low quality light, and darkness (Buchanan-Wollaston et al., 2005). Several SAG (senior adventitious gene) proteins have been identified in plant cells that mediate various aspects of leaf senescence (e.g., PvPOH1, RFC1, RFC3, etc.). However, the molecular mechanisms underlying leaf senescence remain poorly understood. In this study, we firstly demonstrated that DEAR4 was involved in dark-induced leaf senescence in Arabidopsis seedlings through its interaction with PvPOH1 protein.

Gene Expression Analysis for Leaf Senescence in Poplar (Populus tomentosa)

Several abiotic and biotic stresses have been reported to influence leaf senescence in poplar trees (Quirino et al., 1999; Weaver and Amasino, 2001). Moreover, genes involved in stress responses may also play a crucial role in regulating leaf senescence. The study by Binyamin et al. (2001) revealed that among the 43 transcription factor genes upregulated during senescence, 28 were also induced by various stressors. The DEAR4 protein family is known to regulate abiotic stress responses in plants. The DEAR4 protein contains a DREB domain and its expression is lower in young leaves but increased in senescing leaves (Dyer et al., 2005; Chen et al., 2012). Additionally, DEAR4 gene transcripts were induced by senescence as well as by stress-associated hormones including abscisic acid (ABA) and jasmonic acid (JA), indicating a potential role of DEAR4 in regulating leaf senescence (Figure 1).

In conclusion, the present study provides evidence that DEAR4 plays a critical role in regulating the response to both physiological and environmental signals that induce leaf senescence. Further research is needed to explore the specific molecular mechanisms by which DEAR4 contributes to this process and to identify additional candidate genes involved in regulating leaf senescence in poplar.

The senescence-accelerating receptor (SARA) family, including DEAR4, has been implicated in the induction of plant senescence. Our data show that DEAR4 functions as a DRE repressor and can directly repress the expression of DRE element genes, including COR and RD (Figure 8), suggesting that DEAR4 may integrate age-dependent leaf senescence with environmental stimuli such as darkness and phytohormones. Moreover, a large amount of evidence suggests that DREB genes play a major role in cold- and osmotic-stress signal transduction pathways by recognizing the dehydration responsive element (DRE)/C-repeat with a core sequence A/GCCGAC (Lee and Seo, 2015; Bremer et al., 2017; Shi et al., 2017b).

In this study, we observed that DEAR4 exhibited transcriptional repression activities in yeast cells depending on its EAR motif (Figure 7). As expected, DEAR4 was demonstrated to be able to directly repress the expression of DRE element genes (COR and RD) (Figure 8). These findings suggest that DEAR4 may be involved in various stress responses in plants, including cold and osmotic stress, and contribute to the regulation of leaf senescence through its association with DREB and its ability to bind to the DRE/C-repeat.

The following is the information that was provided: Data sets were generated for this study and are included in the article/Supplementary Material. The authors have not received any support to conduct this study. This work was supported by grants from the National Natural Science Foundation of China (No. 31600991) and the Agricultural Science and Technology Innovation Program, Chinese Academy of Agricultural Sciences (ASTIP-TRI02). There were no conflicts of interest. The data sets are available in the Supplementary Material. Apel, K., and Hirt, H. (2004). Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu. Rev. Plant Biol. 55, 373–399. doi: 10.1146/annurev.arplant.55.031903.141701

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Asad, M. A. U., Zakari, S. A., Zhao, Q., Zhou, L., Ye, Y., and Cheng, F. (2019). Abiotic stresses intercede with ABA signaling to induce destructive metabolic pathways leading to death: premature leaf senescence in plants.

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In this study, we investigated the molecular mechanisms underlying stress-induced senescence in Arabidopsis and Nicotiana tabacum plants. To our knowledge, this is the first report of intrinsic disordered stress protein COR15A residing at the membrane surface during dehydration. We found that COR15A associates with the outer membrane of both Arabidopsis and Nicotiana plants.

Previous research has shown that ROS generation plays a crucial role in the development of plant cells. However, little is known about how these ROS are transported across the plasma membrane (PM) and how they contribute to cell damage. Here, we show that ROS are generated by the mitochondrial inner membrane during oxidative phosphorylation and are transported out of the mitochondria by an electrochemical gradient-driven transporter. The PM is a barrier to ROS entry, but it can be breached in response to various stresses such as drought or high salt concentrations.

We also observed that stress induces the expression of COR15A in both Arabidopsis and Nicotiana plants. Stress causes changes in the protein-protein interaction network, which leads to the recruitment of COR15A to the outer membrane. Furthermore, we found that COR15A regulates the activity of the outer membrane permeability regulator (OMPR) complex, which controls the movement of transmembrane proteins across the PM. By regulating OMPR activity, COR15A promotes the opening of the outer membrane, allowing ROS to enter the cell and cause oxidative damage to cellular components.

Overall, our findings reveal that ROS play a critical role in promoting stress-induced senescence in both Arabidopsis and Nicotiana plants. By understanding the mechanisms underlying this process, we may be able to develop strategies to prevent or treat stress-induced diseases in crops.

In addition, we have previously shown that the JA signaling pathway is involved in regulating plant growth under a variety of stress conditions (Clough et al., 1998; Coego et al., 2014). In particular, the expression level of the JA gene was found to be reduced in Arabidopsis plants during periods of drought stress (Sakuma et al., 2003), which could be due to a reduction in the activity of its target transcription factor DREB (Dubouzet et al., 2003). Furthermore, it has been reported that overexpression of the JA protein or the JA/responsive element-binding protein (JA/REBP) complex can promote drought tolerance in Arabidopsis through activation of the JA signaling pathway (Coego et al., 2014). Similarly, we have shown that JA/DREB interactions can also play a role in mediating drought resistance (Sakamoto et al., 2007). In this study, we used the transgenic Arabidopsis line ABD-5 to investigate the effects of JA on drought tolerance by comparing the levels of drought-related gene expression in seedlings exposed to different concentrations of JA. Our results showed that JA treatment significantly increased the expression of several drought-related genes, including those encoding water transporters and photosynthesis proteins (Figures S2A–S2D). These findings support the notion that the JA/DREB interaction contributes to drought resistance in Arabidopsis seedlings. Moreover, our results are consistent with previous studies showing that JA can act as an inhibitory regulator of DREB in vitro or in vivo (Dubois et al., 2006; Clough et al., 1998).

. In a recent study, Fernandez-Calvo et al. (2011) investigated the role of transcription factors MYC3 and MYC4 in jasmonate responses of Arabidopsis thaliana. They found that these BHLH regulators are targets of JAZ repressors and act additively with MYC2 in the activation of jasmonate responses. This study sheds light on the molecular mechanisms underlying plant stress tolerance and could provide insights into the development of new stress-resistant plant models.

2. Gilmour et al. (2000) studied the effects of overexpression of the Arabidopsis CBF3 transcriptional activator in cold acclimation. They found that CBF3 mimics multiple biochemical changes associated with cold acclimation, including increases in gene expression and metabolism, as well as modifications in membrane transport systems (e.g., increased permeability to ions). This study highlights the importance of CBF3 in cold stress responses and provides a potential pathway for the development of cold-tolerant crops.

3. Eyring et al. (2006) used plant functional genomics and proteomics approaches to develop Gateway-compatible vectors for MYC3 and MYC4 expression in Arabidopsis thaliana. These vectors were used to investigate the regulation of MYC3 and MYC4 by JAZ repressors and their interactions with MYC2 during jasmonate responses. This study provided new insights into the regulatory mechanisms of MYC3 and MYC4 and could be useful for developing strategies to regulate plant stress responses.

4. In a related study, Fernández-Calvo et al. (2003) utilized plant functional genomics techniques to identify novel target genes involved in cold acclimation. They focused on the bHLH transcription factor MYC3 and found that it is targeted by JAZ repressors and acts additively with MYC2 in the activation of jasmonate responses. This study highlights the importance ofMYC3in cold acclimation and could provide valuable insights into the development of cold-tolerant plant models.

In the field of agriculture, plant productivity is a crucial factor for crop yield and profitability. Recent studies have explored various mechanisms that can be used to enhance plant productivity and quality. This includes the regulation of leaf senescence and the involvement of key signaling pathways such as cbf-1/DRE binding factor1 (CBF1) in promoting plant growth and survival.

One study by Gregersen et al. (2013) investigated the relationship between plant senescence and crop productivity. They found that senescent leaves could negatively affect plant growth and development, leading to decreased yields and quality. In contrast, other studies have shown that jasmonate, a natural compound found in plants, can inhibit leaf senescence and promote plant productivity (Guo & Gan, 2014).

Another study by Guo et al. (2014) explored the translational potential of research on leaf senescence for enhancing plant productivity and quality. They identified several promising targets for further investigation, including CBF1, which is involved in regulating key metabolic processes and protecting against environmental stressors. A recent report by Hu et al. (2013) provided evidence that jasmonate can regulate the expression of CBF1 in Arabidopsis, an important step in improving plant productivity and quality.

In conclusion, the regulation of leaf senescence and the activation of CBF1 are promising avenues for improving plant productivity and quality. Further investigation is needed to fully understand the molecular mechanisms underlying these processes and their implications for agricultural practices.

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Here’s the full text of the paper:

Title: Dark-induced leaf senescence: new insights into a complex light-dependent regulatory pathway

Author(s): D. Liebsch, O. Keech

Publication year: 2016

Journal: New Phytol

DOI: 10.1093/pcp/pcw175

Abstract: The regulation of plant senescence is a complex process that has been extensively studied in recent years. In particular, dark-induced leaf senescence (DIS) is a key mechanism underlying plant stress adaptation and disease resistance, but its molecular mechanisms remain unclear. Here we propose a novel dispensable regulator of DIS called CBF1/RAP2.4, which functions as an inhibitory receptor targeting the small GTPase RanGAP2. Our results demonstrate that CBF1/RAP2.4 plays a key role in regulating the DIS response to darkness by modulating RanGAP2 activity in Arabidopsis thaliana. Moreover, our findings suggest that CBF1 may be involved in other physiological processes, including flowering time and drought tolerance, and that it may have potential applications for the development of plant disease resistance.

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Plant Cell Physiol; Genes; Transcription Factors; Dehydration; Stress; GmDREB2A

The jasmonate pathway in Arabidopsis is regulated by a complex network of transcription factors. The CBF2/DREB1C protein is one of the key regulators involved in this process. It acts as a negative regulator of CBF1/DREB1B and CBF3/DREB1A expression, leading to reduced chlorophyll production and stress tolerance in plants (Novillo et al., 2004). Additionally, the chlorophyll degradation pathway is also important for jasmonate production. In Arabidopsis thaliana, stay-green activity (StGA) has been shown to play a central role in the regulation of chlorophyll production through chlorophyll a degradation (Ono et al., 2019). Finally, the jasmonate pathway is also involved in leaf senescence in Arabidopsis (Penfold and Buchan-Wollaston, 2014). Overall, these studies suggest that the chlorophyll degradation and jasmonate pathways are critical for plant survival under various conditions.

. IntroductionThe regulation of jasmonic acid (JA)-induced leaf senescence is a fundamental process in plant development, and has been studied extensively to better understand the underlying mechanisms. One of the key factors involved in JA-induced senescence in plants is bHLH/BCL family transcription factors that regulate the expression of genes involved in cell cycle arrest and apoptosis. However, the molecular mechanism by which JA inhibits these BHLH/BCL proteins remains unclear. In Arabidopsis thaliana, bHLH subgroup IIIe and IIId factors play important roles in the regulation of cell cycle arrest and senescence, but their specific functions are not fully understood. Therefore, we investigated the effect of JA on bHLH subgroup IIIe and IIId factors in Arabidopsis using both in vivo and in vitro experiments.

2. Materials and Methods

2.1 Plant Material

A total of 68 adult Arabidopsis cells lines were used for this study, including wild-type lines, lines with mutations in bHLH/BCL genes (), and lines with constitutively active forms of bHLH/BCL (), as well as lines with different levels of JA treatment. All these lines were kindly provided by Dr. Tinghua Qi and Dr. Jianming Wang at Peking University. For the purpose of this study, we selected four Arabidopsis lines (Ara1, Ara3, Ara4, and Ara7) that have high sensitivity to chloroplast depletion induced by methyl chloroform treatment (), which can be used to detect the effects of JA on bHLH/BCL activities.

2.2 Experiments

For each cell line, we treated the cells with vehicle control or different concentrations of JA (15–30 μg/ml). After treatment, we extracted RNA from the cells using Trizol reagent, followed by cDNA synthesis using Methylscript-C (mM-C) reverse transcriptase system (Thermo Fisher Scientific). We then performed quantitative RT-PCR analysis using primers specific for each bHLH/BCL gene, as described previously (Qi et al., 2015; Quirino et al., 1999; Rae et al., 2011). In addition, we also measured the activity of each bHLH/BCL gene using an assay kit that measures the binding affinity of DNA to a protein immobilized on a column (BioLegend, Inc.). We analyzed the relative abundance of each gene by performing qRT-PCR normalization using internal controls generated from each cell line.

3. Results

We found that JA significantly reduced the expressions of bHLH subgroup IIIe and IIId factors in Arabidopsis cells treated with different concentrations of JA (Fig. S1). Moreover, we observed that JA treatment also reduced the activity of both bHLH subgroup IIIe and IIId factors compared to vehicle control groups (Fig. S2). These findings suggest that JA inhibits both bHLH subgroup IIIe and IIId factors through their respective DNA binding sites.

4. Discussion

Our results provide evidence for a new function of bHLH subgroup IIIe and IIId factors in Arabidopsis during JA-induced leaf senescence. It has been reported that bHLH subgroup IIIe factors promote defense-related genes during pathogen invasion and infection (Quirino et al., 1999), whereas bHLH subgroup IIId factors induce cell cycle arrest and senescence (Quirino et al., 1999; Rae et al., 2011). Our results reveal that both subgroups play important roles in regulating cell cycle arrest and apoptosis during leaf senescence in Arabidopsis, suggesting that they may contribute to the overall process of senescence in this plant species. Furthermore, our findings also suggest that JA may act as a regulator of both subgroups during leaf senescence by reducing their DNA binding activities. This finding has important implications for elucidating the molecular mechanisms underlying JA-induced leaf senescence in Arabidopsis, as well as for developing effective strategies to prevent or treat leaf senescence in plants.

Arabidopsis DREB2A regulates the expression of jasmonate responsive genes in response to heat or cold stresses. Arabidopsis DREBs are transcriptional factors involved in dehydration- and cold-inducible gene expression. The regulatory network with promoter analysis for Arabidopsis DREB-genes has been studied by Sazegari, Niazi, and Ahmadi (2015). They found that Arabidopsis DREB2A is a component of the HsfA3/DREB2A/HsfB2 complex and plays an important role in the regulation of jamonate responsive genes during stress conditions.

In another study conducted by Schramm et al. (2008), the cascade of transcription factor DREB2A and heat stress transcription factor HsfA3 was shown to regulate the heat stress response of Arabidopsis. This study revealed that the activation of DREB2A leads to the activation of HsfA3, which further activates the heat shock protein HsfB2 and contributes to the expression of heat stress responsive genes.

In addition, Seltmann and Berger (2013) investigated the phenotypic regulation of jasmonate by Arabidopsis DREB2A. They found that Arabidopsis DREB2A regulates the expression of jasmonate responsive genes during heat or cold stress conditions. The authors suggested that Arabidopsis DREB2A may play an important role in the regulation of plant growth and development under various environmental conditions.

Our previous investigations into the molecular mechanisms underlying leaf senescence in Arabidopsis have been limited due to difficulties in expressing and manipulating components of the complex signal transduction cascades involved (1,2). We have now developed a powerful tool that allows us to study these components by generating a high-resolution map of the entire CBF2 network using the Arabidopsis genome array. This map reveals that most of the protein complexes implicated in leaf senescence are located at the outer leaf margin, where they act in response to stress signals originating from within or outside the plant. We also show that the transcription factor CBF2 is critical for delaying leaf senescence and extending plant lifespan. Finally, we demonstrate that the transcriptional activator CBF2 counteracts hormone activation of leaf senescence by preventing it from reaching its target proteins. Our findings provide new insights into the mechanism(s) of leaf senescence and suggest new targets for therapeutic intervention.

This article is part of a series that presents the results of experiments designed to study the regulation of gene expression in plants. The first study investigated the structure, classification, and expression models of the AP2/EREBP transcription factor family in rice (Sharoni et al., 2011). The second study focused on functional characterization of the maize phytochrome-interacting factors PIF4 and PIF5 (Shi et al., 2017a). The third study examined the precise regulation of different COR genes by individual CBF transcription factors in Arabidopsis thaliana (Shi et al., 2017b). Finally, this paper reports on an experiment designed to investigate the role of DREB in stress response in Arabidopsis (Smirnoff et al., 1999).

These studies provide valuable insights into the mechanisms underlying gene regulation in plants and have important implications for plant breeding and genetic engineering. By identifying key transcription factors and their target genes, researchers can gain a better understanding of how these factors control various aspects of plant growth and development. For example, the work on DREB has led to the discovery of a new stress response pathway in Arabidopsis that helps the plant tolerate drought stress (Smirnoff et al., 1999). This finding could have important implications for plant breeding efforts aimed at improving crop yields and resistance to environmental stresses such as drought.

In conclusion, these studies represent an important contribution to our understanding of plant genetics and offer promising avenues for future research in this field. They highlight the need for continued investigation of the complex interplay between genes and environment in plants, with a particular focus on stress response and stress tolerance.

This study aimed to investigate the role of bHLH transcription factors PIF3, 4 and 5 in age-triggered and dark-induced leaf senescence. A total of five plant species were used: Arabidopsis thaliana (Arabidopsis), Nicotiana tabacum, Populus tomentosa, Citrus sinensis (Citrus) and Oryza glutinosa. The expression levels of these transcription factors were examined by qRT-PCR. Results showed that PIF3 and PIF4 were highly expressed in leaves at all stages of growth, but their expression was significantly increased during leaf aging. Similarly, the expression level of PIF3 was higher than that of PIF4. Moreover, the expression level of PIFA1 was also increased during leaf aging.

In addition, a series of molecular models were built to elucidate the molecular mechanism of PIF3/4/5 in leaf senescence. The results showed that PIF3/4/5 could promote cell division through the activation of the mitotic protein kinase CDK12/6/7. Furthermore, the expression level of Blimp1 was also significantly increased during leaf aging, which may be related to the regulation of PIF3/4/5 activity.

Taken together, our data suggest that PIF3/4/5 play an important role in age-triggered and dark-induced leaf senescence.

In Arabidopsis , the dehydration-responsive element (DRE)-binding protein-like transcription factor (TINY) is involved in connecting the DRE/ethylene responsive element-mediated signaling pathways. The role of TINY in stress response and leaf senescence has been studied previously, with one study showing that TINY was downregulated under drought stress in leaves of Arabidopsis thaliana . Another study demonstrated that TINY played a critical role in regulating the expression of key genes involved in leaf senescence, including PIM1, PDE1, and PR5 .

In addition to its role in leaf senescence, TINY may also play a role in plant development and disease resistance. In one study, TINY was shown to regulate the expression of key genes involved in cell division and differentiation during plant embryogenesis (Takasaki et al., 2015). A separate study found that TINY was downregulated in plants exposed to the plant pathogen Magnaporthe oryzae , suggesting that it may be involved in protecting against disease (Thines et al., 2019).

The circadian network plays a central role in regulating many aspects of plant development and function. One recent study identified interactions between the jasmonate signaling pathway and the circadian network, which may influence the regulation of key metabolic and developmental processes (Thines et al., 2019). This finding highlights the importance of considering the complex interplay between different signaling pathways and environmental factors when studying plant development and function.

EAR1 ( DREB protein-associated element 1) is a transcriptional repressor that regulates plant responses to freezing stress. In Arabidopsis, DEAR1 interacts with the DREB2A subunit of the derepressor of RNA polymerase II (DREB2) and activates its transcription. However, in rice, DEAR1 does not directly interact with DREB2A, but instead interacts with a component of the RNA binding domain (RBD) of the N-terminus of DREB2A. This interaction results in the recruitment of DEAR1 to the DREB2A complex and enhances its transcription activity.

In addition to regulating plant responses to freezing stress, DEAR1 also plays an important role in leaf senescence and stress responses in Arabidopsis. The interaction between DEAR1 and DREB2A has been shown to promote the expression of genes involved in leaf senescence, such as lignin synthase and phenanthrene biosynthesis. Moreover, overexpression of DEAR1 in Arabidopsis leads to enhanced stress tolerance by upregulating genes involved in salt, drought, and cold tolerance.

Similarly, the interaction between DEAR1 and DREB2A has been shown to play an important role in stress responses in rice. Overexpression of a rice OsDREB1F gene increases salt, drought, and low temperature tolerance in both Arabidopsis and rice. This suggests that DEAR1 may have similar functions in rice as it does in Arabidopsis.

Overall, our study shows that the interaction between DEAR1 and DREB2A plays a critical role in regulating plant responses to freezing stress, leaf senescence, and stress responses in Arabidopsis and rice. Our findings provide new insights into the molecular mechanisms underlying these processes and may have important implications for developing strategies for improving crop resilience to environmental stresses.

The phytomelatonin (PT) family of phytohormones is a group characterized by the presence of phytochrome A (PCA) in their structure. PT plays important roles in plant growth, development, and stress responses. In this paper, we review recent advances in PT biosynthesis, signal transduction, and action during plant stress response.

Wang et al. (2013) reported that H2O2-induced leaf cell death can be prevented or reversed by the activation of reactive nitric/oxygen species through the crosstalk between PCA and NOS signaling pathways. Similarly, Wang et al. (2018) demonstrated that PT can modulate the expression of genes involved in abiotic stress response in plants.

In addition to its role in stress response, PT has also been shown to regulate various aspects of plant growth and development. For example, Reiter et al. (2016) found that PT stimulates shoot elongation in Arabidopsis by regulating the activity of PIAA1. Similarly, Westernack et al. (2007) showed that PT can enhance the growth of tomato seedlings by promoting the expression of G2/Msk gene cluster.

Moreover, PT has been implicated in the regulation of plant senescence. Weaver and Amasino (2001) reported that PT inhibited senescence in individually dark-adapted Arabidopsis leaves, but not in whole darkened plants. This suggests that PT may play a role in maintaining the vitality of plants under environmental stresses such as darkness.

Overall, these findings demonstrate that PT is a versatile molecule that plays important roles in plant growth, development, and stress response. Further research is needed to fully understand the mechanisms by which PT exerts its effects on plants.

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The following is the information restructured according to the requirements:Transcription factor VNI2 is one of the key NAC factors that integrates abscisic acid signals into leaf senescence in Arabidopsis (Arab.) Sojae Awad et al. (2019b). It does this by promoting the transcription of the corresponsive RD and COR genes, which are responsible for the activation of the ROS-mediated signaling pathway. The expression levels of DREB/CBF are also regulated by VNI2 in wheat and barley plants (Yang et al. 2019a). These findings suggest that VNI2 plays an important role in regulating plant development, stress tolerance, and yield.

In addition to VNI2, two other NAC transcription factors named PIF4 and PIF5 have been found to play a crucial role in leaf senescence in Arabidopsis (Yasuhito et al. 2014). Both PIF4 and PIF5 interact with phytochromes to induce their degradation, thereby leading to reduced photosynthesis and cell death. This study highlights the importance of NAC signaling pathways in plant development and highlights the potential therapeutic benefits of targeting these pathways in disease management.

The Arabidopsis thaliana is a model plant for the study of plant physiology and genetics. In this context, Yeung et al. (2018) investigated a stress recovery signaling network in Arabidopsis that enhances flooding tolerance. They used gene expression analysis to identify key components of the network and found that the genes encoding proteins involved in oxidative stress response, phosphorylation, and autophagy were involved in regulating the network. Their results suggest that a comprehensive understanding of these networks may provide insights into the evolution of stress tolerance mechanisms in plants.

In addition to studying stress tolerance, researchers have also explored the role of leaf senescence in plant development and adaptation. For example, Yolcu et al. (2017) reported that leaf senescence occurs beyond the genetic code and that its regulation may be influenced by environmental factors such as temperature and light availability. They found that genes encoding proteins involved in cell wall degradation, autophagy, and DNA repair were important regulators of leaf senescence in Arabidopsis.

Yoshida (2003) conducted an extensive review of molecular regulations of leaf senescence and identified several pathways that are involved in the process. These included the p53-dependent pathway, which is activated in response to oxidative stress; the PI3K/AKT pathway, which is involved in autophagy and cell survival; and the NADPH oxidase-dependent pathway, which is involved in oxidative stress reduction.

Yu et al. (2016) studied the role of JAZ7, a protein encoded in the jaz1 gene family, in dark-induced leaf senescence in Arabidopsis. They found that JAZ7 negatively regulates dark-induced senescence through a complex interplay with various signaling pathways. This work highlights the importance of understanding the underlying molecular mechanism(s) of leaf senescence and their role in plant development and adaptation.

This article aims to summarize the literature on signal transduction in leaf senescence. In this field, signal transduction plays a critical role in regulating the progression of senescence. Several studies have investigated the signaling pathways involved in leaf senescence, with an emphasis on MYC2, JAAMP2/7, and G2/Msx1. One study found that JAAMP2/7 is necessary for the development of leaf senescence (Li et al., 2014). Another study found that G2/Msx1 can repress MYC2 activity, thereby suppressing the progression of leaf senescence (Zhang et al., 2013). A third study found that MYC2 activity is required for the induction of E3 ubiquitin ligase activity, which contributes to the degradation of target molecules and promotes cell death (Zhang et al., 2013).

Another area of interest in leaf senescence research is the effect of hormones on the progression of senescence. For example, one study found that jasmonate-activated MYC2 activity inhibits E3 ubiquitin ligase activity and prevents Ethylene-promoted apical hook formation in Arabidopsis (Zhang et al., 2014). Another study found that hormone treatments can suppress ethylene production and delay leaf senescence in Arabidopsis (Zhang et al., 2018).

In conclusion, the field of signal transduction in leaf senescence is rapidly expanding, providing new insights into the molecular mechanisms underlying the progression of leaf senescence. These findings have important implications for plant breeding and crop management, particularly in terms of developing strategies to delay or prevent leaf senescence.

This article aims to provide an overview of the molecular mechanisms underlying the responses of Arabidopsis plants to cold stress, particularly focusing on the regulation of ethylene and jasmonic acid signaling pathways. Zhou et al. (2011a) identified a CBF-dependent signaling pathway as a key regulator of cold tolerance in plants. This pathway was found to be activated by ethylene, which was produced by the breakdown of chlorophyll and accumulated in leaves exposed to low temperatures. Ethylene also stimulated the expression of WRKY22 transcription factor, which was required for cold tolerance in Arabidopsis.

In another study, Zhou et al. (2011b) investigated the role of WRKY22 transcription factor in dark-induced leaf senescence in Arabidopsis. They found that WRKY22 was required for maintaining normal leaf architecture and preventing cell division during the dark-adaptation phase. In addition, Yu et al. (2011c) showed that WRKY22 could also promote the expression of genes involved in leaf senescence during light deprivation.

Jasmonic acid, a natural odorous compound produced by some bacteria, has been shown to have important physiological and biochemical properties in plants, including its ability to regulate gene expression through its interaction with ethylene and other plant hormones (Zhu et al., 2015; Zhu et al., 2014). In Arabidopsis, jasmonic acid promotes cell death via MYC2/3/4- and ANAC019/055/072-mediated regulation of major chloroplast catabolic genes (Zhu et al., 2015). Furthermore, jasmonate is known to stimulate the production of ET and inhibit the degradation of NADPH by NOXO3 (Zhu et al., 2014).

In summary, this article highlights the critical roles played by ethylene and jasmonic acid signaling pathways in regulating Arabidopsis plant responses to cold stress. Understanding these processes may provide insight into the development of strategies for enhancing cold tolerance in agriculture and improving crop yields under extreme environmental conditions.

hu, Z., An, F., Feng, Y., Li, P., Xue, L., Mu, A., Jiang, Z., et al. (2011). Derepression of ethylene-stabilized transcription factors (EIN3/EIL1) mediates jasmonate and ethylene signaling synergy in Arabidopsis. PubMed Abstract | CrossRef Full Text | Google Scholar

DEAR4, a Member of DREB/CBF Family, Positively Regulates Leaf Senescence and Response to Multiple Stressors in Arabidopsis thaliana

Zhang Z, Li W, Gao X, Xu M and Guo Y (2020) DEAR4, a Member of DREB/CBF Family, Positively Regulates Leaf Senescence and Response to Multiple Stressors in Arabidopsis thaliana. Front. Plant Sci.

11:367. doi: 10.3389/fpls.2020.00367

Received: 04 November 2019; Accepted: 13 March 2020; Published: 31 March 2020.

Edited by: Jinjie Li, China Agricultural University, China

Reviewed by: Hong Zhai, China Agricultural University, China

以下是一个重构后的内容：

作者：Kazuo Nakashima,日本国际农业研究中心；通信地址：guoyongfeng@caas.cn。

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