Promoter of COR2-like gene is a stress inducible regulatory region in banana.

A promoter is a crucial component in driving the expression of a transgene of interest for biotechnological applications in crop improvement and thus characterization of varied regulatory regions is essential. Here, we identified the promoter of COR2-like (codeinone reductase-like) from banana and characterized its tissue specific and stress inducible nature. MusaCOR2-like of banana is closely related to COR2 and CHR (chalcone reductase) sequences from different plant species and contains signature sequences including a catalytic tetrad typical of proteins with aldo-keto reductase activity. Transcript level of MusaCOR2-like was strongly induced in response to drought, salinity and exposure of signaling molecules such as abscisic acid, methyl-jasmonate and salicylic acid. Induction of MusaCOR2-like under stress strongly correlated with the presence of multiple cis-elements associated with stress responses in the PMusaCOR2-like sequence isolated from Musa cultivar Rasthali. Transgenic tobacco lines harbouring PMusaCOR2-like-GUS displayed visible GUS expression in vascular tissue of leaves and stem while its expression was undetectable in roots under control conditions. Exposure to drought, salinity and cold strongly induced GUS expression from PMusaCOR2-like-GUS in transgenic tobacco shoots in a window period of 3H to 12H. Applications of salicylic acid, methyl-jasmonate, abscisic acid and ethephon also activate GUS in transgenic shoots at different period, with salicylic acid and abscisic acid being the stronger stimulants of PMusaCOR2-like. Using PMusaCOR2-like-GUS fusion and expression profiling, the current study sheds insights into a complex regulation of COR2-like, one of the least studied genes of secondary metabolite pathway in plants.

Analysis of plant pararetrovirus promoter sequence(s) for developing a useful synthetic promoter with enhanced activity in rice, pearl millet, and tobacco plants.

Promoters are one of the most important components for many gene-based research as they can fine-tune precise gene expression. Many unique plant promoters have been characterized, but strong promoters with dual expression in both monocot and dicot systems are still lacking. In this study, we attempted to make such a promoter by combining specific domains from monocot-infecting pararetroviral-based promoters sugarcane bacilliform virus (SCBV) and banana streak virus (BSV) to a strong dicot-infecting pararetroviral-based promoter mirabilis mosaic virus (MMV). The generated chimeric promoters, MS, SM, MB, and BM, were tested in monocot and dicot systems and further validated in transgenic tobacco plants. We found that the developed chimeric promoters were species-specific (monocot or dicot), which depended on their respective core promoter (CP) region. Furthermore, with this knowledge, deletion-hybrid promoters were developed and evaluated, which led to the development of a unique dual-expressing promoter, MSD3, with high gene expression efficiency (GUS and GFP reporter genes) in rice, pearl millet, and tobacco plants. We conclude that the MSD3 promoter can be an important genetic tool and will be valuable in plant biology research and application.

Study on the expression regulation of the CTR1 gene in the ethylene signaling pathway.

The CONSTITUTIVE TRIPLERESPONSE1 (CTR1) is a crucial component in the ethylene signaling pathway. CTR1 transmits signals perceived by ethylene receptors to downstream EIN2 proteins through phosphorylation/dephosphorylation. Although some studies have explored the functions and mechanisms of CTR1, research on its expression and regulation remains relatively limited. This study investigates the tissue-specific expression of the Arabidopsis CTR1 gene and its expression and regulatory mechanisms under ethylene induction. Arabidopsis was treated with ethylene, and changes in CTR1 gene expression were detected using real-time quantitative PCR. The experimental results show that in rosette leaves of 28-day-old Arabidopsis, CTR1 expression is induced by ethylene. To investigate its molecular mechanism, the promoter sequence of the CTR1 was cloned and vectors were constructed by linking the promoter sequence with luciferase and GUS genes. Stable transgenic Arabidopsis lines were obtained, and promoter activity in these materials was analyzed. Promoter activity analysis confirmed that CTR1 promoter activity is ethylene-inducible and that this induction is dependent on the functions of proteins such as EIN2, EIN3, and EILs. Additionally, the study found that CTR1 expression is higher during seed germination and maintained at lower levels in mature leaves and plants. This study provides a detailed observation of CTR1 gene expression and, for the first time, identifies that the CTR1 promoter is regulated by ethylene induction, offering new options for designing ethylene signaling pathway reporter systems.

Physiological roles of Arabidopsis MCA1 and MCA2 based on their dynamic expression patterns.

Determining the mechanisms by which plants sense and respond to mechanical stimuli is crucial for unraveling the detailed processes by which plants grow and develop. Mechanosensitive (MS) channels, including MCA1 and its paralog MCA2 in Arabidopsis thaliana, may be essential for these processes. Although significant progress has been made in elucidating the physiological roles of MS channels, comprehensive insights into their expression dynamics remain elusive. Here, we summarize recent advancements and new data on the spatiotemporal expression patterns of the MCA1 and MCA2 genes, revealing their involvement in various developmental processes. Then, we describe findings from our study, in which the expression profiles of MCA1 and MCA2 were characterized in different plant organs at various developmental stages through histochemical analyses and semiquantitative RT‒PCR. Our findings revealed that MCA1 and MCA2 are preferentially expressed in young tissues, suggesting their pivotal roles in processes such as cell division, expansion, and mechanosensing. Lastly, we discuss the differential expression patterns observed in reproductive organs and trichomes, hinting at their specialized functions in response to mechanical cues. Overall, this review provides valuable insights into the dynamic expression patterns of MCA1 and MCA2, paving the way for future research on the precise roles of these genes in planta.

Promoter of Cassava MeAHL31 Responds to Diverse Abiotic Stresses and Hormone Signals in Transgenic Arabidopsis.

The AT-hook motif nuclear-localized (AHL) family is pivotal for the abiotic stress response in plants. However, the function of the cassava AHL genes has not been elucidated. Promoters, as important regulatory elements of gene expression, play a crucial role in stress resistance. In this study, the promoter of the cassava MeAHL31 gene was cloned. The MeAHL31 protein was localized to the cytoplasm and the nucleus. qRT-PCR analysis revealed that the MeAHL31 gene was expressed in almost all tissues tested, and the expression in tuber roots was 321.3 times higher than that in petioles. Promoter analysis showed that the MeAHL31 promoter contains drought, methyl jasmonate (MeJA), abscisic acid (ABA), and gibberellin (GA) cis-acting elements. Expression analysis indicated that the MeAHL31 gene is dramatically affected by treatments with salt, drought, MeJA, ABA, and GA3. Histochemical staining in the proMeAHL31-GUS transgenic Arabidopsis corroborated that the GUS staining was found in most tissues and organs, excluding seeds. Beta-glucuronidase (GUS) activity assays showed that the activities in the proMeAHL31-GUS transgenic Arabidopsis were enhanced by different concentrations of NaCl, mannitol (for simulating drought), and MeJA treatments. The integrated findings suggest that the MeAHL31 promoter responds to the abiotic stresses of salt and drought, and its activity is regulated by the MeJA hormone signal.

Sorption and mobility assessment of tembotrione in soils of upper, trans and middle Gangetic plain zones of India.

The presence of undesired agrochemicals residues in soil and water poses risks to both human health and the environment. The behavior of pesticides in soil depends both on the physico-chemical properties of pesticides and soil type. This study examined the adsorption-desorption and leaching behavior of the maize herbicide tembotrione in soils of the upper (UGPZ), trans (TGPZ) and middle Gangetic plain zones of India. Soil samples were extracted using acetone followed by partitioning with dichloromethane, whereas liquid-liquid extraction using dichloromethane was used for aqueous samples. Residues of tembotrione and its metabolite TCMBA, {2-chloro-4-(methylsulfonyl)-3-[(2,2,2-trifluoroethoxy) methyl] benzoic acid}, were quantified using liquid chromatography-tandem mass spectrometry. The data revealed that tembotrione adsorption decreased with increasing pH and dissolved organic matter but increased with salinity. The maximum adsorption occurred at pH 4, 0.01 m sodium citrate and 4 g/L NaCl, with corresponding Freundlich constants of 1.83, 2.28 and 3.32, respectively. The hysteresis index <1 indicated faster adsorption than desorption. Leaching studies under different flow conditions revealed least mobility in UGPZ soil and high mobility in TGPZ soil, consistent with groundwater ubiquity scores of 4.27 and 4.81, respectively. Soil amendments decreased tembotrione mobility in the order: unamended > wheat straw ash > wheat straw > farm yard manure > compost. The transformation of tembotrione to TCMBA and its mobility in soil columns were also assessed.

Concomitant determination of pesticides in soil and drainage water over a potato cropping season reveal dissipations largely in accordance with respective models.

Pesticides are widely used in agriculture where they do not only reach their targets but also distribute to other environmental compartments and negatively affect non-target organisms. To prospectively assess their environmental risk, several tools and models using pesticide persistence (DT50) and leaching potential (groundwater ubiquity score (GUS), EXPOSIT) have been developed. Here, we simultaneously quantified 18 pesticides in soil and drainage water during a conventionally grown potato culture at field scale with high temporal resolution and compared our findings with predictions of the above models. Overall dissipations of all freshly applied compounds in soil were in line with published DT50 field values and their occurrences in drainage water were generally consistent with GUS and EXPOSIT models, respectively. In contrast, soil concentrations of the legacy pesticide atrazine and one of its transformation products (atrazine-2-hydroxy) were constant during the entire sampling campaign. Moreover, during peak discharge atrazine concentrations in drainage water were diluted whereas those of freshly applied pesticides were maximal. This difference demonstrates that the applied risk assessment tools were capable of predicting environmental concentrations and dissipation of pesticides at the short and medium time scale of a few half-lives after application, but fell short of capturing long-term trace residues.

Artificially designed synthetic promoter for a high level of salt induction using a cis-engineering approach.

This work aimed to design a synthetic salt-inducible promoter using a cis-engineering approach. The designed promoter (PS) comprises a minimal promoter sequence for basal-level expression and upstream cis-regulatory elements (CREs) from promoters of salinity-stress-induced genes. The copy number, spacer lengths, and locations of CREs were manually determined based on their occurrence within native promoters. The initial activity profile of the synthesized PS promoter in transiently transformed N. tabacum leaves shows a seven-fold, five-fold, and four-fold increase in reporter GUS activity under salt, drought, and abscisic acid stress, respectively, at the 24-h interval, compared to the constitutive CaMV35S promoter. Analysis of gus expression in stable Arabidopsis transformants showed that the PS promoter induces over a two-fold increase in expression under drought or abscisic acid stress and a five-fold increase under salt stress at 24- and 48-h intervals, compared to the CaMV35S promoter. The promoter PS exhibits higher and more sustained activity under salt, drought, and abscisic acid stress compared to the constitutive CaMV35S.

Functional Characterization and Localization of Plant-Growth Promoting Bacteria Grown Under Stressful Conditions.

Various bacterial species are associated with plant roots. However, symbiotic and free-living plant growth-promoting bacteria (PGPB) can only help plants to grow and develop under normal and stressful conditions. Several biochemical and in vitro assays were previously designed to differentiate between the PGPB and other plant-associated bacterial strains. This chapter describes and summarizes some of these assays and proposes a strategy to screen for PGPB. To determine the involvement of the PGPB in abiotic stress tolerance, assays for the ability to produce 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, ammonium, gibberellic acid (GA), indole acetic acid (IAA), and microbial volatile organic compounds (mVOCs) are described in this chapter. Additionally, assays to show the capacity to solubilize micronutrients such as potassium, phosphorus, and zinc by bacteria were also summarized in this chapter. To determine the contribution of the PGPB in biotic stress tolerance in plants, Fe-siderophore, hydrogen cyanide, and antibiotic and antifungal metabolites production assays were described. Moreover, assays to investigate the growth-promotion activities of a bacterium strain on plants, using the gnotobiotic root elongation, in vitro, and pots assays, were explained. Finally, an assay for the localization of endophytic bacterium in plant tissues was also presented in this chapter. Although the assays described in this chapter can give evidence of the nature of the mechanism behind the PGPB actions, other unknown growth-promoting means are yet to decipher, and until then, new methodologies will be developed.

Quantitative Estimation of Promoter Activity in Cannabis sativa Using Agroinfiltration-Based Transient Gene Expression.

To properly assess promoter activity, which is critical for understanding biosynthetic pathways in different plant species, we use agroinfiltration-based transient gene expression assay. We compare the activity of several known promoters in Nicotiana benthamiana with their activity in Cannabis sativa (both hemp and medicinal cannabis), which has attracted much attention in recent years for its industrial, medicinal, and recreational properties. Here we describe an optimized protocol for transient expression in Cannabis combined with a ratiometric GUS reporter system that allows more accurate evaluation of promoter activity and reduces the effects of variable infiltration efficiency.

Evaluation of novel promoters for vascular tissue-specific gene expression in Populus.

Due to the extended generation cycle of trees, the breeding process for forest trees tends to be time-consuming. Genetic engineering has emerged as a viable approach to expedite the genetic breeding of forest trees. However, current genetic engineering techniques employed in forest trees often utilize continuous expression promoters such as CaMV 35S, which may result in unintended consequences by introducing genes into non-target tissues. Therefore, it is imperative to develop specific promoters for forest trees to facilitate targeted and precise design and breeding. In this study, we utilized single-cell RNA-Seq data and co-expression network analysis during wood formation to identify three vascular tissue-specific genes in poplar, PP2-A10, PXY, and VNS07, which are expressed in the phloem, cambium/expanding xylem, and mature xylem, respectively. Subsequently, we cloned the promoters of these three genes from '84K' poplar and constructed them into a vector containing the eyGFPuv visual selection marker, along with the 35S mini enhancer to drive GUS gene expression. Transgenic poplars expressing the ProPagPP2-A10::GUS, ProPagPXY::GUS, and ProPagVNS07::GUS constructs were obtained. To further elucidate the tissue specificity of these promoters, we employed qPCR, histochemical staining, and GUS enzyme activity. Our findings not only establish a solid foundation for the future utilization of these promoters to precisely express of specific functional genes in stems but also provide a novel perspective for the modular breeding of forest trees.

Characterization of an African Swine Fever Virus Field Isolate from Vietnam with Deletions in the Left Variable Multigene Family Region.

In this paper, we report the characterization of a genetically modified live-attenuated African swine fever virus (ASFV) field strain isolated from Vietnam. The isolate, ASFV-GUS-Vietnam, belongs to p72 genotype II, has six multi-gene family (MGF) genes deleted, and an Escherichia coli GusA gene (GUS) inserted. When six 6-8-week-old pigs were inoculated with ASFV-GUS-Vietnam oro-nasally (2 × 105 TCID50/pig), they developed viremia, mild fever, lethargy, and inappetence, and shed the virus in their oral and nasal secretions and feces. One of the pigs developed severe clinical signs and was euthanized 12 days post-infection, while the remaining five pigs recovered. When ASFV-GUS-Vietnam was inoculated intramuscularly (2 × 103 TCID50/pig) into four 6-8 weeks old pigs, they also developed viremia, mild fever, lethargy, inappetence, and shed the virus in their oral and nasal secretions and feces. Two contact pigs housed together with the four intramuscularly inoculated pigs, started to develop fever, viremia, loss of appetite, and lethargy 12 days post-contact, confirming horizontal transmission of ASFV-GUS-Vietnam. One of the contact pigs died of ASF on day 23 post-contact, while the other one recovered. The pigs that survived the exposure to ASFV-GUS-Vietnam via the mucosal or parenteral route were fully protected against the highly virulent ASFV Georgia 2007/1 challenge. This study showed that ASFV-GUS-Vietnam field isolate is able to induce complete protection in the majority of the pigs against highly virulent homologous ASFV challenge, but has the potential for horizontal transmission, and can be fatal in some animals. This study highlights the need for proper monitoring and surveillance when ASFV live-attenuated virus-based vaccines are used in the field for ASF control in endemic countries.

Humic acid improves wheat growth by modulating auxin and cytokinin biosynthesis pathways.

Humic acids have been widely used for centuries to enhance plant growth and productivity. The beneficial effects of humic acids have been attributed to different functional groups and phytohormone-like compounds enclosed in macrostructure. However, the mechanisms underlying the plant growth-promoting effects of humic acids are only partially understood. We hypothesize that the bio-stimulatory effect of humic acids is mainly due to the modulation of innate pathways of auxin and cytokinin biosynthesis in treated plants. A physiological investigation along with molecular characterization was carried out to understand the mechanism of bio-stimulatory effects of humic acid. A gene expression analysis was performed for the genes involved in auxin and cytokinin biosynthesis pathways in wheat seedlings. Furthermore, Arabidopsis thaliana transgenic lines generated by fusing the auxin-responsive DR5 and cytokinin-responsive ARR5 promoter to ß-glucuronidase (GUS) reporter were used to study the GUS expression analysis in humic acid treated seedlings. This study demonstrates that humic acid treatment improved the shoot and root growth of wheat seedlings. The expression of several genes involved in auxin (Tryptophan Aminotransferase of Arabidopsis and Gretchen Hagen 3.2) and cytokinin (Lonely Guy3) biosynthesis pathways were up-regulated in humic acid-treated seedlings compared to the control. Furthermore, GUS expression analysis showed that bioactive compounds of humic acid stimulate endogenous auxin and cytokinin-like activities. This study is the first report in which using ARR5:GUS lines we demonstrate the biostimulants activity of humic acid.

Discovery of a botanical compound as a broad-spectrum inhibitor against gut microbial ß-glucuronidases from the Tibetan medicine Rhodiola crenulata.

Gut microbial ß-glucuronidases (gmß-GUS) played crucial roles in regulating a variety of endogenous substances and xenobiotics on the circulating level, thus had been recognized as key modulators of drug toxicity and human diseases. Inhibition or inactivation of gmß-GUS enzymes has become a promising therapeutic strategy to alleviate drug-induced intestinal toxicity. Herein, the Rhodiola crenulata extract (RCE) was found with potent and broad-spectrum inhibition on multiple gmß-GUS enzymes. Subsequently, the anti-gmß-GUS activities of the major constituents in RCE were tested and the results showed that 1,2,3,4,6-penta-O-galloyl-ß-d-glucopyranose (PGG) acted as a strong and broad-spectrum inhibitor on multiple gmß-GUS (including EcGUS, CpGUS, SaGUS, and EeGUS). Inhibition kinetic assays demonstrated that PGG effectively inhibited four gmß-GUS in a non-competitive manner, with the Ki values ranging from 0.12 µM to 1.29 µM. Docking simulations showed that PGG could tightly bound to the non-catalytic sites of various gmß-GUS, mainly via hydrogen bonding and aromatic interactions. It was also found that PGG could strongly inhibit the total gmß-GUS activity in mice feces, with the IC50 value of 1.24 µM. Collectively, our findings revealed that RCE and its constituent PGG could strongly inhibit multiple gmß-GUS enzymes, suggesting that RCE and PGG could be used for alleviating gmß-GUS associated enterotoxicity.

Evaluation of Parameters Affecting Agrobacterium-Mediated Transient Gene Expression in Industrial Hemp (Cannabis sativa L.).

Industrial hemp Cannabis sativa L. is an economically important crop mostly grown for its fiber, oil, and seeds. Due to its increasing applications in the pharmaceutical industry and a lack of knowledge of gene functions in cannabinoid biosynthesis pathways, developing an efficient transformation platform for the genetic engineering of industrial hemp has become necessary to enable functional genomic and industrial application studies. A critical step in the development of Agrobacterium tumefaciens-mediated transformation in the hemp genus is the establishment of optimal conditions for T-DNA gene delivery into different explants from which whole plantlets can be regenerated. As a first step in the development of a successful Agrobacterium tumefaciens-mediated transformation method for hemp gene editing, the factors influencing the successful T-DNA integration and expression (as measured by transient ß-glucuronidase (GUS) and Green Florescent Protein (GFP) expression) were investigated. In this study, the parameters for an agroinfiltration system in hemp, which applies to the stable transformation method, were optimized. In the present study, we tested different explants, such as 1- to 3-week-old leaves, cotyledons, hypocotyls, root segments, nodal parts, and 2- to 3-week-old leaf-derived calli. We observed that the 3-week-old leaves were the best explant for transient gene expression. Fully expanded 2- to 3-week-old leaf explants, in combination with 30 min of immersion time, 60 µM silver nitrate, 0.5 µM calcium chloride, 150 µM natural phenolic compound acetosyringone, and a bacterial density of OD600nm = 0.4 resulted in the highest GUS and GFP expression. The improved method of genetic transformation established in the present study will be useful for the introduction of foreign genes of interest, using the latest technologies such as genome editing, and studying gene functions that regulate secondary metabolites in hemp.

Isolation and functional characterization of cold-induced gene (AmCIP) promoter from Ammopiptanthus mongolicus.

AmCIP is a dehydrin-like protein which involved in abiotic stress tolerance in xerophytes evergreen woody plant A. mongolicus. AmCIP could be induced in the cotyledon and radicle during cold acclimation. To further elucidate the regulation of the upstream region of the gene, we isolated and characterized the promoter of AmCIP. Herein, a 1115 bp 5'-flanking region of AmCIP genomic DNA was isolated and cloned by genome walking from A. mongolicus and the segment sequence was identified as "PrAmCIP" promoter. Analysis of the promoter sequence revealed the presences of some basic cis-acting elements, which were related to various environmental stresses and plant hormones. GUS histochemical staining of transgene tobacco showed that PrAmCIP was induced by 4℃, 55℃, NaCl, mannitol and ABA, whereas it could hardly drive GUS gene expression under normal conditions. Furthermore, we constructed three deletion fragments and genetically transformed them into Arabidopsis thaliana. GUS histochemical staining showed that the MYCATERD1 element of the CP7 fragment (-189 âˆ¼ -1) may be a key element in response to drought. In conclusion, we provide an inducible promoter, PrAmCIP, which can be applied to the development of transgenic plants for abiotic stresse tolerance.

Isolation and Functional Characterization of a Constitutive Promoter in Upland Cotton (Gossypium hirsutum L.).

Multiple cis-acting elements are present in promoter sequences that play critical regulatory roles in gene transcription and expression. In this study, we isolated the cotton FDH (Fiddlehead) gene promoter (pGhFDH) using a real-time reverse transcription-PCR (qRT-PCR) expression analysis and performed a cis-acting elements prediction analysis. The plant expression vector pGhFDH::GUS was constructed using the Gateway approach and was used for the genetic transformation of Arabidopsis and upland cotton plants to obtain transgenic lines. Histochemical staining and a ß-glucuronidase (GUS) activity assay showed that the GUS protein was detected in the roots, stems, leaves, inflorescences, and pods of transgenic Arabidopsis thaliana lines. Notably, high GUS activity was observed in different tissues. In the transgenic lines, high GUS activity was detected in different tissues such as leaves, stalks, buds, petals, androecium, endosperm, and fibers, where the pGhFDH-driven GUS expression levels were 3-10-fold higher compared to those under the CaMV 35S promoter at 10-30 days post-anthesis (DPA) during fiber development. The results indicate that pGhFDH can be used as an endogenous constitutive promoter to drive the expression of target genes in various cotton tissues to facilitate functional genomic studies and accelerate cotton molecular breeding.

A rapid and efficient Agrobacterium-mediated transient transformation system in grape berries.

Transient transformation is extremely useful for rapid in vivo assessment of gene function, especially for fruit-related genes. Grape berry, while an important fruit crop, is recalcitrant to transient transformation, due to the high turgor pressure in its mesocarp cells that limits the ability of Agrobacterium to penetrate into the tissue. It is urgent to establish a simple transient transformation system for rapid analysis of gene function. In this study, different injection methods, grape genotypes, and developmental stages were tested in order to develop a rapid and efficient Agrobacterium-mediated transient transformation methodology for grape berries. Two injection methods, namely punch injection and direct injection, were evaluated using the ß-glucuronidase (GUS) gene and by x-gluc tissue staining and 4-methylumbelliferyl-ß-D-glucuronide fluorescence analysis. The results indicated that there were no significant differences on transformation effects between the two methods, but the latter was more suitable because of its simplicity and convenience. Six grape cultivars ('Hanxiangmi', 'Moldova', 'Zijixin', 'Jumeigui', 'Shine-Muscat', and 'A17') were tested for transient transformation. 'Hanxiangmi', 'Moldova', and 'Zijixin' grape berries were not suitable for agroinfiltration due to frequently fruit cracking, browning, and formation of scar skin. The fruit integrity rates of 'Jumeigui', 'Shine-Muscat', and 'A17' berries were all above 80%, and GUS activity was detected in the berries of the three cultivars 3-14 days after injection with the Agrobacterium culture, while higher GUS activities were observed in the 'Jumeigui' berries. The levels of GUS activity in injected berries at 7-8 weeks after full blooming (WAFB) were more than twice at 6 WAFB. In subsequent assays, the over-expression of MYB transcription factor VvMYB44 via transient transformation accelerated the anthocyanin accumulation and fruit coloring through raising the expression levels of VvLAR1, VvUFGT, VvLDOX, VvANS, and VvDFR, which verified the effectiveness of this transformation system. These experiments finally identified the reliable grape cultivars and suitable operational approach for transient transformation and further indicated that this Agrobacterium-mediated transient transformation system was efficient and suitable for the elucidation of gene function in grape berries.

Functional Characterization of CsF3Ha and Its Promoter in Response to Visible Light and Plant Growth Regulators in the Tea Plant.

Flavanone 3-hydroxylase (F3H) catalyzes trihydroxyflavanone formation into dihydroflavonols in the anthocyanin biosynthesis pathway, serving as precursors for anthocyanin synthesis. To investigate the CsF3Ha promoter's regulation in the 'Zijuan' tea plant, we cloned the CsF3Ha gene from this plant. It was up-regulated under various visible light conditions (blue, red, and ultraviolet (UV)) and using plant growth regulators (PGRs), including abscisic acid (ABA), gibberellic acid (GA3), salicylic acid (SA), ethephon, and methyl jasmonate (MeJA). The 1691 bp promoter sequence was cloned. The full-length promoter P1 (1691 bp) and its two deletion derivatives, P2 (890 bp) and P3 (467 bp), were fused with the ß-glucuronidase (GUS) reporter gene, and were introduced into tobacco via Agrobacterium-mediated transformation. GUS staining, activity analysis, and relative expression showed that visible light and PGRs responded to promoter fragments. The anthocyanin content analysis revealed a significant increase due to visible light and PGRs. These findings suggest that diverse treatments indirectly enhance anthocyanin accumulation in 'Zijuan' tea plant leaves, establishing a foundation for further research on CsF3Ha promoter activity and its regulatory role in anthocyanin accumulation.