Transgenic Calli Research Articles

Development of an inducible excision system of a visual marker Ipomoea batatas Myb gene from the genome of transgenic cells.

In the plant genetic transformation process, single selection by a chemical-resistant marker gene occasionally allows the proliferation of non-transgenic cells, escaping selection pressure. The additional use of a visual marker gene is effective for accurate selection. For instance, R2R3-MYB genes are used for regulating anthocyanin biosynthesis; however, constitutive Myb expression in transgenic plants is not always desirable and may cause developmental abnormalities due to excess anthocyanin accumulation. To overcome the remaining problems in the use of Myb as a visible marker, we developed T-DNA. Ipomoea batatas Myb (IbMyb) and Cre expression cassettes were inserted between two loxP sequences, and the hygromycin phosphotransferase (HPT) and green fluorescent protein (GFP) expression cassettes were located outside of the loxP-IbMyb-Cre-loxP region. In the developed system, IbMyb and Cre were excised from the genomes of transgenic cells using heat-inducible Cre-loxP recombination. Upon heat treatment in a general incubator, green shoots emerged from purple tobacco transgenic calli that were pigmented with IbMyb expression. The excision of IbMyb from the genome of green shoots was confirmed using polymerase chain reaction (PCR) and sequencing. GFP expression was observed in the roots of the obtained green transgenic plants. We report that the system developed here operated successfully in tobacco, showing the potential to provide an easier and cheaper visual selection of transgenic cells in the genetic transformation process.

Screening Nos-bar gene of Transgenic Rice Callus (Oryza Sativa L.) Containing Antihypertensive Gene Encoding Gg-7pAH

Peptides derived from food protein have the potential to become antihypertensive agents with relatively few adverse side effects. The Gg-7pAH gene encoding antihypertensive is a gene containing the Cys-Met-Try-Leu-Ala-Ser-Gly (CMYLASG) heptapeptide, angiotensin-converting enzyme (ACE) inhibitor peptide from Melinjo (Gnetum gnemon L) seeds. This study aimed to investigate the expression of the tandem repeat heptapeptide (10x peptides, Gg7-pAH) and Nos-bar genes through callus resistance to phosphinothricin (PPT) in 4 different phases of callus: co-cultivation, elimination, screening, and regeneration. The Gg7-pAH gene was constructed in the pGreen II 0229 vector containing the Nos-bar gene and  Gg7-pAH gene under the control of the Glu4 promoter and transformed into rice callus using an Agrobacterium tumefacient vector. PPT was used in the screening at precise concentrations of 0, 20, 50, and 100 ppm. The screening efficiency was then evaluated by calculating the putative callus. As a result, 37 putative callus were developed with a concentration of 0 ppm PPT, 36 putative callus with a concentration of 20 ppm PPT, and none at 50 and 100 ppm PPT. The Gg-7pAH gene was identified by PCR analysis in putatively transgenic rice callus. According to PCR data, a positive callus with an antihypertensive gene insertion was detected at four phases: media, co-cultivation, elimination, screening, and regeneration. These findings suggest that the recombinant Gg-7pAH gene, which is transformed into rice plants to produce rice plants that can treat hypertension, has some potential.

Establishment of an efficient transformation system and its application in regulatory mechanism analysis of biological macromolecules in tea plants

Tea (Camellia sinensis), one of the most important beverage crops originated from China and is now cultivated worldwide, provides numerous secondary metabolites that account for its health benefits and rich flavor. However, the lack of an efficient and reliable genetic transformation system has seriously hindered the gene function investigation and precise breeding of C. sinensis. In this study, we established a highly efficient, labor-saving, and cost-effective Agrobacterium rhizogenes-mediated hairy roots genetic transformation system for C. sinensis, which can be used for gene overexpression and genome editing. The established transformation system was simple to operate, bypassing tissue culture and antibiotic screening, and only took two months to complete. We used this system to conduct function analysis of transcription factor CsMYB73 and found that CsMYB73 negatively regulates L-theanine synthesis in tea plant. Additionally, callus formation was successfully induced using transgenic roots, and the transgenic callus exhibited normal chlorophyll production, enabling the study of the corresponding biological functions. Furthermore, this genetic transformation system was effective for multiple C. sinensis varieties and other woody plant species. By overcoming technical obstacles such as low efficiency, long experimental periods, and high costs, this genetic transformation will be a valuable tool for routine gene investigation and precise breeding in tea plants.

VvSnRK1-VvSS3 regulates sugar accumulation during grape berry ripening in response to abscisic acid

Sugar is the primary product of photosynthesis and is mainly transported in plants as sucrose. Sucrose synthase 3 (VvSS3) and SNF1-related protein kinase 1 (VvSnRK1) are both critical enzymes in the metabolism of sugar in plants but are not clear about the relationship between VvSS3 and VvSnRK1 in the regulation of sugar metabolism. In this study, the spatiotemporal expression of VvSS3 and VvSnRK1 was measured during three stages of grape berry development from hard nucleolus phase to maturation phase. Our results indicated that VvSS3 and VvSnRK1 isoforms overexpressed at the beginning veraison and sugar accumulation occurred at ripening stage. We also provided transgenic calli of overexpressed VvSS3 using Agrobacterium GV3101-mediated transformation and found VvSS3 is localized in cell membrane. Furthermore, up-regulation of VvSnRK1s occurred with overexpression of VvSS3 but abscisic acid (ABA) exogenous down-regulated VvSnRK1s expression. VvSS3-overexpressed transgenic calli also increased the accumulation of soluble sugar and anthocyanin content by increasing the expression of genes related to anthocyanin and ABA synthesis. Furthermore, the interaction of VvSS3 with VvSnRK1β was confirmed by yeast two-hybrid (Y2H) and Bimolecular fluorescence complementation (BiFC). These results suggest that the VvSS3 gene has an important role in sugar accumulation during fruit ripening in grapevines.

Transcriptional repression of MdMa1 by MdMYB21 in Ma locus decreases malic acid content in apple fruit.

Malic acid is a major organic acid component of apples and a crucial determinant of fruit organoleptic quality. A candidate gene for malic acid content, designated MdMa1, was previously identified in the Ma locus, which is a major quantitative trait locus (QTL) for apple fruit acidity located on the linkage group 16. Region-based association mapping to detect candidate genes in the Ma locus identified MdMa1 and an additional MdMYB21 gene putatively associated with malic acid. MdMYB21 was significantly associated with fruit malic acid content, accounting for ~7.48% of the observed phenotypic variation in the apple germplasm collection. Analyses of transgenic apple calli, fruits and tomatoes demonstrated that MdMYB21 negatively regulated malic acid accumulation. The apple fruit acidity-related MdMa1 and its tomato ortholog, SlALMT9, exhibited lower expression profiles in apple calli, mature fruits and tomatoes in which MdMYB21 was overexpressed, compared with their corresponding wild-type variety. MdMYB21 directly binds to the MdMa1 promoter and represses its expression. Interestingly, a 2-bp variation in the MdMYB21 promoter region altered its expression and regulation of its target gene, MdMa1, expression. Our findings not only demonstrate the efficiency of integrating QTL and association mapping in the identification of candidate genes controlling complex traits in apples, but also provide insights into the complex regulatory mechanism of fruit malic acid accumulation.

Effect of BBM gene on callus growth and ginsenoside content in Panax quinquefolius

Baby Boom(BBM) gene is a key regulatory factor in embryonic development and regeneration, cell proliferation, callus growth, and differentiation promotion. Since the genetic transformation system of Panax quinquefolius is unstable with low efficiency and long period, this study attempted to transfer BBM gene of Zea mays to P. quinquefolius callus by gene gunship to investigate its effect on the callus growth and ginsenoside content, laying a foundation for establishing efficient genetic transformation system of P. quinquefolius. Four transgenic callus of P. quinquefolius with different transformation events were obtained by screening for glufosinate ammonium resistance and molecular identification by PCR. The growth state and growth rate of wild-type and transgenic callus were compared in the same growth period. The content of ginsenoside in transgenic callus was determined by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry(UPLC-MS/MS). The results showed that transgenic callus growth rate was significantly higher than that of wild-type callus. In addition, the content of ginsenoside Rb_1, Rg_1, Ro, and Re was significantly higher than that in wild-type callus. The paper preliminarily proved the function of BBM gene in promoting growth rate and increasing ginsenoside content, which provided a scientific basis to establish a stable and efficient genetic transformation system for Panax plants in the future.

Synthesis of betanin by expression of the core betalain biosynthetic pathway in carrot

Betalain has received increased attention because of its high nutritional value and crucial physiological functions. Based on the elucidation of its core biosynthetic pathway, betalain can be produced in additional plants by metabolic engineering. Synthesis of betalain in carrot (Daucus carota L.) can improve its nutritional quality and economic value by extracting betalain from the fleshy root, non-edible part, and processing residue of carrot. In this study, two different constructs, namely, pYB:mCD (AomelOS, BvCYP76AD1S, and BvDODA1S) and pYB:CDD (BvCYP76AD1S, BvDODA1S, and MjcDOPA5GTS), were introduced into carrot for betanin synthesis by Agrobacterium-mediated transformation. Betanin can be synthetized in both transgenic calli, and pYB:mCD-transgenic callus can be used to produce betacyanin by suspension culture. However, pYB:mCD-transgenic seedlings can synthetize betanin only by tyrosine feeding. The pYB:CDD-transgenic lines can synthetize betanin in whole plants. The betanin content in fleshy root of pYB:CDD-transgenic carrot was (63.4 ± 9) μg ·g−1 fresh weight according to quantitative analysis. These betanin-producing carrot plant materials can be used to synthesize betanin for industrial application or consumption as dietary sources.

Rho of plant GTPase MxROP1 regulates the responses to Fe deficiency by targeting Zinc Ribbon 3 in apple rootstocks

The Rho-related GTPase of plants (ROPs) is a multifunctional molecular switch that can regulate plant growth, development, and responses to the environment. However, how the ROPs regulate the response to Fe deficiency has not been thoroughly clarified. In this study, we found that Fe deficiency induced MxROP1 in apple (Malus xiaojinensis) at both the transcriptional and translational levels. The overexpression of MxROP1, MxROP1DN (inactive form), and MxROP1CA (active form) in apple roots increased the activity of ferric chelate reductase and the ability to acidify the rhizosphere, and the lines that overexpressed MxROP1DN exhibited the strongest reaction to enhanced Fe uptake. Yeast two-hybrid library screening indicated that MxROP1 interacted with MxZR3.1, a DNL zinc finger protein that negatively regulates the responses to Fe deficiency. We identified their interaction in vitro and in vivo in more detail using pull-down and bimolecular fluorescence complementation assays, respectively, and MxROP1DN-MxZR3.1 interacted the most strongly. Furthermore, MxROP1 negatively affected the stability of MxZR3.1 protein in vitro as shown by a cell semi-degradation assay, and the application of MG132 inhibited the degradation of MxZR3.1-HIS proteins. This indicated that MxROP1 caused the degradation of MxZR3.1 protein through the 26 S proteasome pathway. Similar results were found in OE-MxROP1+OE-MxZR3.1 transgenic apple calli compared with those in the OE-MxZR3.1 calli. We also demonstrated that MxZR3.1 interacted with MxbHLH39, a known positive transcription factor and core component of Fe deficiency, and MxROP1 affected the interaction of MxZR3.1-MxbHLH39 as shown in a competitive binding assay. Τhis clarified one MxROP1-MxZR3.1-MxbHLH39 pathway that maintains Fe homeostasis in M. xiaojinensis.

Apple WRKY transcription factor MdWRKY56 positively modulates drought stress tolerance

Drought seriously threatens the growth, development, and yield of crops. WRKY transcription factors (TFs) play important roles in abiotic stress responses. However, the study of WRKY TFs in woody plant apple (Malus domestica) in terms of drought tolerance regulation is limited. This study aimed to functionally characterize an apple group IIc WRKY gene MdWRKY56. The transcription abundance of MdWRKY56 was significantly upregulated under drought stress. The subcellular localization analysis revealed that the MdWRKY56 protein localized to the nucleus. Transcriptional activation experiments proved that MdWRKY56 had transcriptional activity in yeast, and its N-terminal played a decisive role in this activity. The functional analysis using MdWRKY56 transgenic Arabidopsis, apple calli, and apple plants indicated that the MdWRKY56 TF functioned positively in the modulation of osmotic and drought stress tolerance, with the MdWRKY56-overexpressed transgenic materials showing lower electrolyte leakage, malondialdehyde (MDA) content, and reactive oxygen species (ROS) accumulation, and higher fresh weight, proline content, and antioxidant enzyme activities. In conclusion, our findings indicated that MdWRKY56 served as an important positive regulator of drought stress tolerance in apple.

Establishment of Highly Efficient Plant Regeneration, Callus Transformation and Analysis of Botrytis cinerea-Responsive PR Promoters in Lilium brownii var. viridulum.

Lilium brownii var. viridulum, commonly called Longya lily, is a well-known flower and vegetable plant in China that has poor tolerance to Botrytis fungal disease. The molecularimprovement has mainly been restricted to an efficient regeneration and transformation system. In this study, the highly efficient regeneration of Longya lily was established through the optimization of embryogenic callus, adventitious shoot and rooting induction. The major factors influencing transformation (antibiotics, Agrobacterium concentration, infection time, suspension solution and coculture medium) were examined. The expression responses of PR promoters (ZmPR4 and BjCHI1) to B. cinerea were assessed in transgenic calli. The results showed that Murashige and Skoog (MS) medium with 1.0 mg·L-1 picloram (PIC) and 0.2 mg·L-1 1-naphthaleneacetic acid (NAA) under light conditions and MS with 0.5 mg·L-1 6-benzylaminopurine (6-BA) and 1.0 mg·L-1 NAA under darkness were optimal for embryogenic callus induction (64.67% rate) and proliferation (3.96 coefficient). Callus inoculation into MS containing 2.0 mg·L-1 thidiazuron (TDZ), 0.4 mg·L-1 NAA, 1.0 mg·L-1 TDZ and 0.5 mg·L-1 NAA led to shooting induction (92.22 of rate) and proliferation (3.28 of coefficient) promotion, respectively. The rooting rate reached 99.00% on MS with 0.3 mg·L-1 NAA. Moreover, a transformation rate of 65.56% was achieved by soaking the callus in Agrobacterium at an OD600 of 0.4 for 10 min in modified MS without NH4NO3 as the suspension solution and coculture medium before selecting 75 mg·L-1 hygromycin and 300 mg·L-1 cefotaxime. Only the BjCHI1 promoter was obviously expressed in transgenic calli. These results could facilitate the generation of Longya lily transgenic plants with improved B. cinerea resistance.

MdIPT1, an adenylate isopentenyltransferase coding gene from Malus domestica, is involved in branching and flowering regulation

Flowering and shoot branching are significant agricultural traits for apple tree breeding. Cytokinin metabolism and signaling pathways play a crucial role in plant development. However, little is known about cytokinin biosynthetic molecular mechanism and function involved in apple flowering and branching. In this study, an adenylate isopentenyl transferase coding gene MdIPT1, homologous to AtIPT3/AtIPT5 in Arabidopsis thaliana, was identified. MdIPT1 was highly expressed in apple floral and axillary buds and was dramatically up-regulated during floral induction and axillary bud outgrowth. The promoter of MdIPT1 showed high activity in multiple tissues and responded to different hormone treatments. The MdIPT1-overexpressing Arabidopsis showed a multi-branching and early-flowering phenotype, with elevated endogenous cytokinin levels and altered expression of genes related to branching and flower formation. Overexpression of MdIPT1 confers the growth vigor of transgenic apple callus on a CKs-deficient medium. Our findings suggest that MdIPT1 is a positive regulator involved in branching and flowering. The data presented herein provide extensive research results on MdIPT1 and will contribute to molecular breeding for new apple varieties.

Overexpression of type II rice metacaspase, OsMC4, increases endoplasmic reticulum stress tolerance in transgenic rice calli

The endoplasmic reticulum (ER) is an organelle responsible for regulating protein synthesis in plants. High salinity can lead to the accumulation of misfolded proteins, resulting in an ER stress response mechanism known as the unfolded protein response (UPR). Failure of the UPR to reverse the effect of protein misfolding will activate programmed cell death (PCD). Metacaspase genes are known regulate PCD in plants. This study aims to provide a comprehensive analysis of the expression patterns of type II rice metacaspase (OsMC) genes in response to ER and salinity stress in rice leaf. Among five type II metacaspases in the rice genome, OsMC4, OsMC5, and OsMC8 expressions were found to be upregulated during treatment with tunicamycin (ER stress) and sodium chloride, NaCl (salinity stress). A construct of taqRFP::OsMC4, controlled by the CaMV35S promoter, was generated and transformed into rice calli. The transgenic rice calli overexpressing taqRFP::OsMC4 demonstrated significant changes in the expression of the ER stress-marker genes, protein disulfide isomerase (OsPDI), and binding immunoglobulin protein (OsBiP). The results from this analysis provide preliminary evidence that at least one of the type II metacaspases, OsMC4, is be able to reduce ER and salinity stresses in rice. Further functional analysis of OsMC genes in ER and salinity stress tolerance could be carried out in transgenic rice overexpressing OsMCs in the future to improve stress tolerance. Simple summaryOryza sativa, commonly known as rice, is one of the most consumed crops in the world. In response to multiple biotic and abiotic factors, a series of endoplasmic reticulum (ER) stress response regulators are activated. There is evidence that high salinity triggers ER stress in plants. This study aims to determine the level of gene expression among type II metacaspases in rice in response to ER and salinity stress and to assess how they may be linked to PCD in rice calli. Three metacaspase genes, OsMC4, OsMC5, and OsMC8, have been observed to have significant expression post-treatment with tunicamycin in rice leaf. Overexpression of taqRFP::OsMC4 in rice calli significantly reduces the expression level of the stress markers, OsBiP and OsPDI, indicating that the stress level is relatively lower in the transgenic calli compared to the wild-type calli. Therefore, overexpression of taqRFP::OsMC4 in rice may increase rice tolerance towards ER and salinity stress. These expression analyses of the OsMCs family provide valuable information for further functional studies on the biological roles of OsMCs in ER and salinity stress responses.

Identification and functional characterization of AcMYB113 in anthocyanin metabolism of Aesculus chinensis Bunge var. chinensis leaves

Anthocyanins can be induced by environmental factors such as low-temperature and play essential roles in plant color formation. In this study, leaves of Aesculus chinensis Bunge var. chinensis with different colors under natural low-temperature in autumn were collected and grouped into green leaf (GL) and red leaf (RL). To reveal the underlying mechanism of color formation in RL, a combined analysis of the metabolome and transcriptome was conducted with GL and RL. Metabolic analyses revealed that total anthocyanin content and primary anthocyanin components were increased RL relative to GL and cyanidin was the main anthocyanin compound in RL. Transcriptome analysis provided a total of 18720 differentially expressed genes (DEGs), of which 9150 DEGs were upregulated and 9570 DEGs were downregulated in RL relative to GL. KEGG analysis showed that DEGs were mainly enriched in flavonoid biosynthesis, phenylalanine metabolism, and phenylpropanoid biosynthesis. Furthermore, co-expression network analysis indicated that 56 AcMYB transcription factors were highly expressed in RL compared with GL, among which AcMYB113 (an R2R3-MYB TF) had a strong correlation with anthocyanins. Overexpression of AcMYB113 in apple resulted in dark-purple transgenic calluses. In addition, the transient expression experiment showed that AcMYB113 enhanced anthocyanin synthesis by activating pathways of anthocyanin biosynthesis in leaves of Aesculus chinensis Bunge var. chinensis. Taken together, our findings reveal new insights into the molecular mechanism of anthocyanin accumulation in RL and provide candidate genes for the breeding of anthocyanin-rich cultivars.

Peach DELLA Protein PpeDGYLA Is Not Degraded in the Presence of Active GA and Causes Dwarfism When Overexpressed in Poplar and Arabidopsis

Controlling the tree size of fruit species such as peach can reduce the amount of labor and input needed for orchard management. The phytohormone gibberellin (GA) positively regulates tree size by inducing degradation of the GA signaling repressor DELLA. The N-terminal DELLA domain in this protein is critical for its GA-dependent interaction with the GA receptor GID1 and the resulting degradation of the DELLA protein, which allows for growth-promoting GA signaling. In this study, a DELLA family member, PpeDGYLA, contains a DELLA domain but has amino acid changes in three conserved motifs (DELLA into DGYLA, LEQLE into LERLE, and TVHYNP into AVLYNP). In the absence or presence of GA3, the PpeDGYLA protein did not interact with PpeGID1c and was stable in 35S-PpeDGYLA peach transgenic callus. The overexpression of PpeDGYLA in both polar and Arabidopsis showed an extremely dwarfed phenotype, and these transgenic plants were insensitive to GA3 treatment. PpeDGYLA could interact with PpeARF6-1 and -2, supposed growth-promoting factors. It is suggested that the changes in the DELLA domain of PpeDGYLA may, to some extent, account for the severe dwarf phenotype of poplar and Arabidopsis transgenic plants. In addition, our study showed that the DELLA family contained three clades (DELLA-like, DELLA, and DGLLA). PpeDGYLA clustered into the DGLLA clade and was expressed in all of the analyzed tissues. These results lay the foundation for the further study of the repression of tree size by PpeDGYLA.

The apple 14-3-3 gene MdGRF6 negatively regulates salt tolerance.

The 14-3-3 (GRF, general regulatory factor) regulatory proteins are highly conserved and are widely distributed throughout the eukaryotes. They are involved in the growth and development of organisms via target protein interactions. Although many plant 14-3-3 proteins were identified in response to stresses, little is known about their involvement in salt tolerance in apples. In our study, nineteen apple 14-3-3 proteins were cloned and identified. The transcript levels of Md14-3-3 genes were either up or down-regulated in response to salinity treatments. Specifically, the transcript level of MdGRF6 (a member of the Md14-3-3 genes family) decreased due to salt stress treatment. The phenotypes of transgenic tobacco lines and wild-type (WT) did not affect plant growth under normal conditions. However, the germination rate and salt tolerance of transgenic tobacco was lower compared to the WT. Transgenic tobacco demonstrated decreased salt tolerance. The transgenic apple calli overexpressing MdGRF6 exhibited greater sensitivity to salt stress compared to the WT plants, whereas the MdGRF6-RNAi transgenic apple calli improved salt stress tolerance. Moreover, the salt stress-related genes (MdSOS2, MdSOS3, MdNHX1, MdATK2/3, MdCBL-1, MdMYB46, MdWRKY30, and MdHB-7) were more strongly down-regulated in MdGRF6-OE transgenic apple calli lines than in the WT when subjected to salt stress treatment. Taken together, these results provide new insights into the roles of 14-3-3 protein MdGRF6 in modulating salt responses in plants.

Introducing sorghum DREB2 gene in maize (Zea mays L.) to improve drought and salinity tolerance.

Salinity and drought are significant abiotic stresses causing a considerable loss of seed and biomass yield in most commercial crops. Some of the most critical players in the abscisic acid pathway are drought responsive element binding (DREB) proteins that are a part of AP2/ethylene response factor transcription factors that bind to promoters of some family genes needed to be expressed under abiotic stresses. In this study, salt- and drought-tolerant maize plants were produced from immature maize embryos bombarded by the sorghum (Sorghum bicolor L.) DREB2 gene that is linked to hygromycin resistance (hpt) genes. The putative transgenic calli were transferred to an N6 medium containing 1mg/L benzylaminopurine and 50mg/L hygromycin. Regeneration was completed after 4 weeks on selective media under a 16/8h light/dark condition at 25°C. Polymerase chain reaction (PCR) and reverse transcription-PCR approved the existence of upstream promoter (rd29a), hpt gene, and the expression of the DREB2 in transgenes up to the third generation (T2). It was found that the K+/Na+ ratio and the amount of proline as a screening indicator were higher in transgenic plants compared to their wild types. This result is a promising model to enhance maize tolerance to abiotic stressors.

The MdmiR156n Regulates Drought Tolerance and Flavonoid Synthesis in Apple Calli and Arabidopsis.

Drought is the major abiotic stress that limits apple productivity and quality. To date, many important and divergent regulatory functions of miR156/SBP genes in plant growth and development have been well understood. However, little is known about the role of apple miR156 in response to abiotic stress. To better understand the functions of MdmiR156 in abiotic stress tolerance, we constructed the overexpression (OE) and short tandem target mimic (STTM) vector of MdmiR156n and performed its functional analysis through the characterization of transgenic apple calli and Arabidopsis thaliana plants. In this study, MdmiR156n overexpression significantly increased the length of primary roots and the number of lateral roots in transgenic Arabidopsis plants under drought stress. In addition, MdmiR156n transgenic Arabidopsis and apple calli had a lower electrolyte leakage rate and less cell membrane damage than WT and STTM156 after drought stress. Further studies showed that MdmiR156n overexpression promoted the accumulation of flavonoids and scavenging of reactive oxygen species (ROS) under drought conditions in transgenic apple calli and A. thaliana plants. Taken together, overexpression MdmiR156n enhances drought tolerance by regulating flavonoid synthesis and ROS signaling cascades in apple calli and A. thaliana.

Sufficient coumarin accumulation improves apple resistance to Cytospora mali under high potassium status.

Cytospora canker, caused by Cytospora mali, is the most destructive disease in production of apples (Malus domestica). Adding potassium (K) to apple trees can effectively control this disease. However, the underlying mechanisms of apple resistance to C. mali under high-K status remain unknown. Here, we found that high K (HK, 9.30 g/kg) apple tissues exhibited high disease resistance. The resistance was impeded when blocking K channels, leading to susceptibility even under HK conditions. We detected a suite of resistance events in HK apple tissues, including upregulation of resistance genes, callose deposition, and formation of ligno-suberized tissues. Further multi-omics revealed that the phenylpropanoid pathway was reprogrammed by increasing K content from low K (LK, 4.30 g/kg) status, leading to increases of 18 antifungal chemicals. Among them, the physiological concentration of coumarin (1,2-benzopyrone) became sufficient to inhibit C. mali growth in HK tissues, and exogenous application could improve the C. mali resistance of LK apple branches. Transgenic apple calli overexpressing beta-glucosidase 40 (MdBGLU40), which encodes enzyme for coumarin synthesis, contained higher levels of coumarin and exhibited high resistance to C. mali even under LK conditions. Conversely, suppression of MdBGLU40 through RNAi reduced coumarin content and resistance in HK apple calli, supporting the importance of coumarin accumulation in vivo for apple resistance. Moreover, we found that the upregulation of transcription factor MdMYB1r1 directly activated MdBGLU40, and the binding affinity of MdMYB1r1 to the MdBGLU40 promoter increased in HK apple tissue, leading to high levels of coumarin and resistance in HK apple. Overall, we found that accumulation of defensive metabolites strengthened resistance in apple when raising K from insufficient to optimal status, and these results highlight the optimization of K content in fertilization practices as a disease management strategy.

γ-Aminobutyric acid plays a key role in alleviating Glomerella leaf spot in apples.

The fungal disease Glomerella leaf spot (GLS) seriously impacts apple production. As a nonprotein amino acid, γ-aminobutyric acid (GABA) is widely involved in biotic and abiotic stresses. However, it is not clear whether GABA is involved in a plant's response to GLS, nor is its molecular mechanism understood. Here, we found that exogenous GABA could significantly alleviate GLS, reduce lesion lengths, and increase antioxidant capacity. MdGAD1 was identified as a possible key gene for GABA synthesis in apple. Further analysis indicated that MdGAD1 promoted antioxidant capacity to improve apple GLS resistance in transgenic apple calli and leaves. Yeast one-hybrid analysis identified the transcription factor MdWRKY33 upstream of MdGAD1. Electrophoretic mobility shift assay, β-glucuronidase activity, and luciferase activity further supported that MdWRKY33 bound directly to the promoter of MdGAD1. The content of GABA and the transcription level of MdGAD1 in the MdWRKY33 transgenic calli were higher than that of the wild type. When MdWRKY33 transgenic calli and leaves were inoculated with GLS, MdWKRY33 positively regulated resistance to GLS. These results explained the positive regulatory effects of GABA on apple GLS and provided insight into the metabolic regulatory network of GABA.

Ectopic expression of MmSERT, a mouse serotonin transporter gene, regulates salt tolerance and ABA sensitivity in apple and Arabidopsis

5-hydroxytryptamine (5-HT) is ubiquitously present in animals and plants, playing a vital regulatory role. SERT, a conserved serotonin reuptake transporter in animals, regulates intracellular and extracellular concentrations of 5-HT. Few studies have reported 5-HT transporters in plants. Hence, we cloned MmSERT, a serotonin reuptake transporter, from Mus musculus. Ectopic expression of MmSERT into apple calli, apple roots and Arabidopsis. Because 5-HT plays a momentous role in plant stress tolerance, we used MmSERT transgenic materials for stress treatment. We found that MmSERT transgenic materials, including apple calli, apple roots and Arabidopsis, exhibited a stronger salt tolerance phenotype. The reactive oxygen species (ROS) produced were significantly lower in MmSERT transgenic materials compared with controls under salt stress. Meanwhile, MmSERT induced the expression of SOS1, SOS3, NHX1, LEA5 and LTP1 in response to salt stress. 5-HT is the precursor of melatonin, which regulates plant growth under adversity and effectively scavenges ROS. Detection of MmSERT transgenic apple calli and Arabidopsis revealed higher melatonin levels than controls. Besides, MmSERT decreased the sensitivity of apple calli and Arabidopsis to abscisic acid (ABA). In summary, these results demonstrated that MmSERT plays a vital role in plant stress resistances, which perhaps serves as a reference for the application of transgenic technology to improve crops in the future.