5-Aminolevulinic acid improves cold resistance through regulation of SlMYB4/SlMYB88-SlGSTU43 module to scavenge reactive oxygen species in tomato.

Cold stress severely affects the growth and quality of tomato. 5-Aminolevulinic acid (ALA) can effectively improve tomato's cold stress tolerance. In this study, a tomato glutathione S-transferase gene, SlGSTU43, was identified. Results showed that ALA strongly induced the expression of SlGSTU43 under cold stress. SlGSTU43-overexpressing lines showed increased resistance to cold stress through an enhanced ability to scavenge reactive oxygen species. On the contrary, slgstu43 mutant lines were sensitive to cold stress, and ALA did not improve their cold stress tolerance. Thus, SlGSTU43 is a key gene in the process of ALA improving tomato cold tolerance. Through yeast library screening, SlMYB4 and SlMYB88 were preliminarily identified as transcription factors that bind to the SlGSTU43 promoter. Electrophoretic mobility shift, yeast one-hybrid, dual luciferase, and chromatin immunoprecipitation assays experiments verified that SlMYB4 and SlMYB88 can bind to the SlGSTU43 promoter. Further experiments showed that SlMYB4 and SlMYB88 are involved in the process of ALA-improving tomato's cold stress tolerance and they positively regulate the expression of SlGSTU43. The findings provide new insights into the mechanism by which ALA improves cold stress tolerance. SlGSTU43, as a valuable gene, could be added to the cold-responsive gene repository. Subsequently, it could be used in genetic engineering to enhance the cold tolerance of tomato.

Crosstalk between 5-Aminolevulinic Acid and Abscisic Acid Adjusted Leaf Iron Accumulation and Chlorophyll Synthesis to Enhance the Cold Tolerance in Solanum lycopersicum Seedlings.

Previous studies found that 5-aminolevulinic acid (ALA) and abscisic acid (ABA) can mitigate damage from adversity by enhancing photosynthesis. However, it is not clear whether they have positive effects on iron utilization and chlorophyll synthesis of tomato seedlings under low-temperature stress. To investigate the possible functional relationship between ABA and ALA and elucidate the possible mechanisms of action of ALA to alleviate low-temperature stress in tomato seedlings, this experiment analyzed the effects of ALA and ABA on chlorophyll synthesis in tomato seedling leaves sprayed with exogenous of ALA (25 mg·L-1) or ABA (100 µM) under low-temperature stress (8-18 °C/8-12 °C, day/night). The results show that exogenous ALA increased the Fv/Fm of tomato leaves by 5.31% and increased the accumulation of iron and chlorophyll by 101.15% and 15.18%, respectively, compared to the low-temperature treatment alone, and tomato resistance of low-temperature stress was enhanced. Meanwhile, exogenous application of ALA increased the ABA content by 39.43%, and subsequent application of exogenous ABA revealed that tomato seedlings showed similar effects to exogenous ALA under low-temperature stress, with increased accumulation of iron and chlorophyll in tomato seedlings, which eventually increased the maximum photochemical efficiency of PS II. Under low-temperature stress, application of exogenous ABA significantly reduced ALA content, but the expression of key enzyme genes (PPGD, HEMB1, HEME1, and HEMF1), precursors of chlorophyll synthesis by ALA, was significantly elevated, presumably because the increased activity of these enzymes after external application of ABA accelerated ALA consumption. In conclusion, ABA may crosstalk with ALA to improve the photochemical efficiency and low temperature resistance of tomatoes by regulating chlorophyll synthesis and iron accumulation.

VqWRKY56 interacts with VqbZIPC22 in grapevine to promote proanthocyanidin biosynthesis and increase resistance to powdery mildew.

Powdery mildew (PM) is a severe fungal disease of cultivated grapevine world-wide. Proanthocyanidins (PAs) play an important role in resistance to fungal pathogens; however, little is known about PA-mediated PM resistance in grapevine. We identified a WRKY transcription factor, VqWRKY56, from Vitis quinquangularis, the expression of which was significantly induced by PM. Overexpression (OE) of VqWRKY56 in Vitis vinifera increased PA content and reduced susceptibility to PM. Furthermore, the transgenic plants showed more cell death and increased accumulation of salicylic acid and reactive oxygen species. Transient silencing of VqWRKY56 in V. quinquangularis and V. vinifera reduced PA accumulation and increased the susceptibility to PM. VqWRKY56 interacted with VqbZIPC22 in vitro and in planta. The protein VqWRKY56 can bind to VvCHS3, VvLAR1, and VvANR promoters, and VqbZIPC22 can bind to VvANR promoter. Co-expression of VqWRKY56 and VqbZIPC22 significantly increased the transcript level of VvCHS3, VvLAR1, and VvANR genes. Finally, transient OE of VqbZIPC22 in V. vinifera promoted PA accumulation and improved resistance to PM, while transient silencing in V. quinquangularis had the opposite effect. Our study provides new insights into the mechanism of PA regulation by VqWRKY56 in grapevine and provides a basis for further metabolic engineering of PA biosynthesis to improve PM resistance.

Exogenous 5-Aminolevulinic acid improved low-temperature tolerance tomato seedling by regulating starch content and phenylalanine metabolism.

Tomato is an important horticultural cash crop, and low-temperature stress has seriously affected the yield and quality of tomato. 5-Aminolevulinic acid (ALA) is widely used in agriculture as an efficient and harmless growth regulator. It is currently unclear whether exogenous ALA can cope with low-temperature stress by regulating tomato starch content and phenylalanine metabolism. In this study, exogenous ALA remarkably improved the low-temperature tolerance of tomato seedlings. RNA-sequencing results showed that exogenous ALA affected starch metabolism and phenylalanine metabolism in tomato seedling leaves under low-temperature stress. Subsequently, we used histochemical staining, observation of chloroplast microstructure, substance content determination, and qRT-PCR analysis to demonstrate that exogenous ALA could improve the low-temperature tolerance of tomato seedlings by regulating starch content and phenylalanine metabolism (SlPAL, SlPOD1, and SlPOD2). Simultaneously, we found that exogenous ALA induced the expression of SlMYBs and SlWRKYs under low-temperature stress. In addition, dual luciferase, yeast one hybrid, and electrophoretic mobility shift assays indicate that SlMYB4 and SlMYB88 could regulate the expression of SlPOD2 in phenylalanine metabolism. We demonstrated that exogenous ALA could improve the low-temperature tolerance of tomato seedlings by regulating starch content and phenylalanine metabolism.

Exogenous 5-aminolevulinic acid alleviates low-temperature injury by regulating glutathione metabolism and ß-alanine metabolism in tomato seedling roots.

Food availability represents a major worldwide concern due to climate change and population growth. Low-temperature stress (LTS) severely restricts the growth of tomato seedlings. Exogenous 5-aminolevulinic acid (ALA) can alleviate the harm of abiotic stress including LTS; however, data on its protective mechanism on tomato seedling roots, the effects of organelle structure, and the regulation of metabolic pathways under LTS are lacking. In this study, we hope to fill the above gaps by exploring the effects of exogenous ALA on morphology, mitochondrial ultrastructure, reactive oxygen species (ROS) enrichment, physiological indicators, related gene expression, and metabolic pathway in tomato seedlings root under LTS. Results showed that ALA pretreatment could increase the activity of antioxidant enzymes and the content of antioxidant substances in tomato seedlings roots under LTS to scavenge the massively accumulated ROS, thereby protecting the mitochondrial structure of roots and promoting root development under LTS. Combined transcriptomic and metabolomic analysis showed that exogenous ALA pretreatment activated the glutathione metabolism and ß-alanine metabolism of tomato seedling roots under LTS, further enhanced the scavenging ability of tomato seedling roots to ROS, and improved the low-temperature tolerance of tomato seedlings. The findings provide a new insight into the regulation of the low-temperature tolerance of tomato by exogenous ALA.

Exogenous 5-aminolevulinic acid alleviates low-temperature damage by modulating the xanthophyll cycle and nutrient uptake in tomato seedlings.

5-Aminolevulinic acid (ALA), an antioxidant existing in plants, has been widely reported to participate in the process of coping with cold stress of plants. In this study, exogenous ALA promoted the growth of tomato plants and alleviated the appearance of purple tomato leaves under low-temperature stress. At the same time, exogenous ALA improved antioxidant enzyme activities, SlSOD gene expression, Fv/Fm, and proline contents and reduced H2O2 contents, SlRBOH gene expression, relative electrical conductivity, and malondialdehyde contents to alleviate the damage caused by low temperature to tomato seedlings. Compared with low-temperature stress, spraying exogenous ALA before low-temperature stress could restore the indicators of photochemical quenching, actual photochemical efficiency, electron transport rate, and nonphotochemical quenching to normal. Exogenous ALA could increase the total contents of the xanthophyll cycle pool, the positive de-epoxidation rate of the xanthophyll cycle and improved the expression levels of key genes in the xanthophyll cycle under low-temperature stress. In addition, we found that exogenous ALA significantly enhanced the absorption of mineral nutrients, promoted the transfer and distribution of mineral nutrients to the leaves, and improved the expression levels of mineral nutrient absorption-related genes, which were all conducive to the improved adaptation of tomato seedlings under low-temperature stress. In summary, the application of exogenous ALA can increase tomato seedlings' tolerance to low-temperature stress by improving the xanthophyll cycle and the ability of the absorption of mineral nutrients in tomato seedlings.

Genome-Wide Identification, Characterization, and Expression Analysis Related to Low-Temperature Stress of the CmGLP Gene Family in Cucumis melo L.

Germin-like protein (GLP) participates in plant growth and development and plays an important role in plant stress. In the present study, 22 CmGLPs belonging to five classes were identified in the melon genome. Each member of the CmGLPs family contains a typical Cupin_1 domain. We conducted a genome-wide analysis of the melon GLP gene family characterization. CmGLPs were randomly distributed in the melon chromosomes, with the largest number on chromosome 8, having eight family members. Gene duplication events drive the evolution and expansion of the melon GLP gene family. Based on the phylogenetic tree analysis of GLP proteins in melon, rice, Arabidopsis, and cucumber, it was found that the GLP gene families of different species have diverged in evolution. Based on qRT-PCR results, all members of the CmGLP gene family could be expressed in different tissues of melon. Most CmGLP genes were up-regulated after low-temperature stress. The relative expression of CmGLP2-5 increased by 157.13 times at 48 h after low-temperature treatment. This finding suggests that the CmGLP2-5 might play an important role in low-temperature stress in melon. Furthermore, quantitative dual LUC assays indicated that CmMYB23 and CmWRKY33 can bind the promoter fragment of the CmGLP2-5. These results were helpful in understanding the functional succession and evolution of the melon GLP gene family and further revealed the response of CmGLPs to low-temperature stress in melon.

Genome-wide identification, characterization, and expression analysis related to autotoxicity of the GST gene family in Cucumis melo L.

Glutathione S-transferase (GST) plays an important role in plant resistance to biotic and abiotic stresses. In this paper, the characteristics of melon GST gene family members were analyzed from a genome-wide perspective. Forty-nine GSTs were identified in melon genome, belonging to eight classes. Through the phylogenetic analysis of GST proteins in melon and other plants, it was found that members from the same subfamily in different species clustered together, indicating that the subfamilies of GST have diversified before the divergence within these species. The results of chromosome mapping showed that GSTs were present in all chromosomes except for chromosome 5. Gene replication events played an important role in the expansion and evolution of melon GST gene family. Ten GSTs with significant differential expression were screened in the transcriptome database related to melon autotoxicity stress. The differential expression of these 10 GSTs was detected in roots and leaves of melon seedlings treated with cinnamic acid. The relative expression level of CmGSTU7, CmGSTU10, CmGSTU18, CmGSTF2 and CmGSTL1 in roots of melon seedlings was significantly higher than that in control group. It suggested that the five GSTs might play an important role in cinnamic acid mediated autotoxicity stress in melon. The results of this paper were helpful to reveal the evolution and functional succession of GST family and further understand the response of GST to autotoxicity stress in melon.

Specific response mechanism to autotoxicity in melon (Cucumis melo L.) root revealed by physiological analyses combined with transcriptome profiling.

Melon is of great value in food, medicine and industry. In recent years, the continuous cropping obstacles of melon is increasingly prominent, which seriously affects the cultivation. Autotoxicity is the key factor for the obstacles. Root is the first line against autotoxicity and main organs for autotoxins secretion. Some physiological responses and differentially expressed genes (DEGs) related to autotoxicity are only limited to root system. Considering the lack of relevant research, physiological researches combined with transcriptome sequencing of melon seedling after autotoxicity stress mediated by root exudates (RE) was performed to help characterize the response mechanism to autotoxicity in melon roots. The results showed that autotoxicity inhibited root morphogenesis of melon seedlings, induced the excessive accumulation of reactive oxygen species (ROS) and lipid peroxidation in roots, and activated most antioxidant enzymes. Compared with the control group, the osmoregulation substance content was always at a high level. DEGs response to autotoxicity in roots were distinguished from that in leaves. Functional annotation of these DEGs suggested that autotoxicity affected biological regulation in a negative manner. DEGs were mainly involved in the synthesis of antioxidants, DNA damage and metabolism, and stress response. These setbacks were associated with the deterioration of root morphogenesis, generation of dwarf and slender roots, and ultimately leading to plant death. The results may provide important information for revealing the response mechanism of root to autotoxicity, and provide theoretical basis for solving the continuous cropping obstacles in melon.