Integrative physiological, metabolomic, and transcriptomic analysis reveals the drought responses of two apple rootstock cultivars.

BACKGROUND: Drought is considered the main environmental factor restricting apple production and thus the development of the apple industry. Rootstocks play an important role in enhancing the drought tolerance of apple plants. Studies of the physiology have demonstrated that 'ZC9-3' is a strong drought-resistant rootstock, whereas 'Jizhen-2' is a weak drought-resistant rootstock. However, the metabolites in these two apple rootstock varieties that respond to drought stress have not yet been characterized, and the molecular mechanisms underlying their responses to drought stress remain unclear. RESULTS: In this study, the physiological and molecular mechanisms underlying differences in the drought resistance of 'Jizhen-2' (drought-sensitive) and 'ZC9-3' (drought-resistant) apple rootstocks were explored. Under drought stress, the relative water content of the leaves was maintained at higher levels in 'ZC9-3' than in 'Jizhen-2', and the photosynthetic, antioxidant, and osmoregulatory capacities of 'ZC9-3' were stronger than those of 'Jizhen-2'. Metabolome analysis revealed a total of 95 and 156 differentially accumulated metabolites in 'Jizhen-2' and 'ZC9-3' under drought stress, respectively. The up-regulated metabolites in the two cultivars were mainly amino acids and derivatives. Transcriptome analysis revealed that there were more differentially expressed genes and transcription factors in 'ZC9-3' than in 'Jizhen-2' throughout the drought treatment. Metabolomic and transcriptomic analysis revealed that amino acid biosynthesis pathways play key roles in mediating drought resistance in apple rootstocks. A total of 13 metabolites, including L-α-aminoadipate, L-homoserine, L-threonine, L-isoleucine, L-valine, L-leucine, (2S)-2-isopropylmalate, anthranilate, L-tryptophan, L-phenylalanine, L-tyrosine, L-glutamate, and L-proline, play an important role in the difference in drought resistance between 'ZC9-3' and 'Jizhen-2'. In addition, 13 genes encoding O-acetylserine-(thiol)-lyase, S-adenosylmethionine synthetase, ketol-acid isomeroreductase, dihydroxyacid dehydratase, isopropylmalate isomerase, branched-chain aminotransferase, pyruvate kinase, 3-dehydroquinate dehydratase/shikimate 5-dehydrogenase, N-acetylglutamate-5-P-reductase, and pyrroline-5-carboxylate synthetase positively regulate the response of 'ZC9-3' to drought stress. CONCLUSIONS: This study enhances our understanding of the response of apple rootstocks to drought stress at the physiological, metabolic, and transcriptional levels and provides key insights that will aid the cultivation of drought-resistant apple rootstock cultivars. Especially, it identifies key metabolites and genes underlying the drought resistance of apple rootstocks.

Exogenous melatonin alleviates apple replant disease by regulating rhizosphere soil microbial community structure and nitrogen metabolism.

Apple replant disease (ARD) is a common soil-borne disease afflicting apple plants. Melatonin is a broad-spectrum oxygen scavenger that plays a key role in alleviating stress-induced damage in plants. In this study, we aimed to determine whether adding melatonin to replant soil can promote plant growth by improving the rhizosphere soil environment and nitrogen metabolism. In replant soil, chlorophyll synthesis was blocked, reactive oxygen species (ROS) accumulated in large quantities, and membrane lipid peroxidation was aggravated; this eventually resulted in slow plant growth. However, the application of 200 µM exogenous melatonin enhanced the tolerance of plants to ARD by up-regulating the expression of antioxidant enzyme-related genes and increasing ROS scavenging enzyme activity. Exogenous melatonin also increased the absorption and utilization of 15N by increasing the expression of nitrogen absorption genes and the activity of nitrogen metabolism enzymes. Exogenous melatonin enhanced the soil microbial environment by promoting soil enzyme activity and bacterial richness and decreasing the abundance of several harmful fungi in rhizosphere soil. Mantel test results showed that soil properties (except for AP) and growth indexes were positively correlated with the rate of 15N absorption and utilization. Spearman correlation analysis showed that the above factors were closely related to the richness and diversity of bacteria and fungi, indicating that the composition of microbial communities might play a key role in mediating change in the soil environment and thus affect nutrient absorption and growth. These findings provide new insights into how melatonin enhances ARD tolerance.

Ectopic expression of HIOMT improves tolerance and nitrogen utilization efficiency in transgenic apple under drought stress.

Melatonin enhances plant tolerance to various environmental stressors. Although exogenous application of melatonin has been investigated, the role of endogenous melatonin metabolism in the response of apples to drought stress and nutrient utilization remains unclear. Here, we investigated the effects of ectopically expressing the human melatonin synthase gene HIOMT on transgenic apple plants under drought stress conditions. The tolerance of transgenic apple lines that ectopically expressed HIOMT improved significantly under drought conditions. After 10 days of natural drought stress treatment, the transgenic apple plants showed higher relative water content, chlorophyll levels and Fv/Fm, and lower relative electrolyte leakage and hydrogen peroxide accumulation, than wild-type plants. The activities of peroxidase, superoxide dismutase and catalase, as well as genes in the ascorbate-glutathione cycle, increased more in transgenic apple plants than in the wild-type. The ectopic expression of HIOMT also markedly alleviated the inhibitory effects of long-term drought stress on plant growth, photosynthetic rate and chlorophyll concentrations in apple plants. The uptake and utilization of 15N increased markedly in the transgenic lines under long-term moderate drought stress. Drought stress sharply reduced the activity of enzymes involved in nitrogen metabolism, but ectopic expression of HIOMT largely reversed that response. The expression levels of genes of nitrogen metabolism and uptake were more upregulated in transgenic apple plants than the wild-type. Overall, our study demonstrates that ectopic expression of HIOMT enhanced the tolerance of apple plants to drought stress, and transgenic apple plants showed improved growth due to higher nutrient utilization efficiency under drought conditions.

Improved tolerance of apple plants to drought stress and nitrogen utilization by modulating the rhizosphere microbiome via melatonin and dopamine.

This study explored the contributions of melatonin and dopamine to the uptake and utilization of nitrogen and the formation of rhizosphere microbial communities in 'Tianhong 2'/M. hupehensis, with the goal improving plant resistance to drought stress. Drought stress was formed by artificially controlling soil moisture content. And melatonin or dopamine solutions were applied to the soil at regular intervals for experimental treatment. After 60 days of treatment, plant indices were determined and the structure of the rhizosphere microbial community was evaluated using high-throughput sequencing technology. The findings revealed two ways through which melatonin and dopamine alleviate the inhibition of growth and development caused by drought stress by promoting nitrogen uptake and utilization in plants. First, melatonin and dopamine promote the absorption and utilization of nitrogen under drought stress by directly activating nitrogen transporters and nitrogen metabolism-related enzymes in the plant. Second, they promote the absorption of nitrogen by regulating the abundances of specific microbial populations, thereby accelerating the transformation of the soil nitrogen pool to available nitrogen that can be absorbed directly by plant roots and utilized by plants. These findings provide a new framework for understanding how melatonin and dopamine regulate the uptake and utilization of nitrogen in plants and improve their ability to cope with environmental disturbances.

Melatonin and dopamine mediate the regulation of nitrogen uptake and metabolism at low ammonium levels in Malus hupehensis.

In plants, ammonium (NH4+) is the main nitrogen source and acts as a physiological and morphological response signaling molecule. Melatonin and dopamine are associated with plant responses to abiotic stress. However, previous studies have rarely focused on nutrient stress, and the roles of melatonin and dopamine in the uptake and metabolism of nitrogen in plants remain unclear. In this study, we investigated the regulatory effects of melatonin and dopamine on nitrogen utilization efficiency in apple seedlings under two NH4+ concentrations (2 and 0.1 mM) by measuring plant growth, root system architecture, 15NH4+ content, and related enzyme activity and gene expression. Under low nitrogen supply, apple seedling growth slowed and showed marked reductions in biomass accumulation, chlorophyll content, and nutrient uptake. However, both melatonin and dopamine significantly improved plant growth, chlorophyll content, and root development and enhanced antioxidant enzyme activity. Exogenous application of melatonin or dopamine also promoted the absorption and accumulation of 15NH4+ and enhanced nitrogen metabolism-related enzyme activity. At the molecular level, melatonin and dopamine significantly increased the expression levels of nitrogen metabolism genes and transporter genes. Overall, these results suggest that melatonin and dopamine can relieve nutrient stress caused by low concentrations of NH4+ through regulating the absorption and metabolism of nitrogen.

miR-615 facilitates porcine epidemic diarrhea virus replication by targeting IRAK1 to inhibit type III interferon expression.

Porcine epidemic diarrhea virus (PEDV) in the Coronavirus family is a highly contagious enteric pathogen in the swine industry, which has evolved mechanisms to evade host innate immune responses. The PEDV-mediated inhibition of interferons (IFNs) has been linked to the nuclear factor-kappa B (NF-κB) pathway. MicroRNAs (miRNAs) are involved in virus-host interactions and IFN-I regulation. However, the mechanism by which the PEDV regulates IFN during PEDV infection has not yet been investigated in its natural target cells. We here report a novel mechanism of viral immune escape involving miR-615, which was screened from a high-throughput sequencing library of porcine intestinal epithelial cells (IECs) infected with PEDV. PEDV infection altered the profiles of miRNAs and the activities of several pathways involved in innate immunity. Overexpression of miR-615 increased PEDV replication, inhibited IFN expression, downregulated the NF-κB pathway, and blocked p65 nuclear translocation. In contrast, knockdown of miR-615 enhanced IFN expression, suppressed PEDV replication, and activated the NF-κB pathway. We further determined that IRAK1 is the target gene of miR-615 in IECs. Our findings show that miR-615 suppresses activation of the NF-κB pathway by suppressing the IRAK1 protein and reducing the generation of IFN-IIIs, which in turn facilitates PEDV infection in IECs. Moreover, miR-615 inhibited PEDV replication and NF-κB pathway activation in both IECs and MARC-145 cells. These findings support an important role for miR-615 in the innate immune regulation of PEDV infections and provide a novel perspective for developing new treatments.

Beneficial Effects of Exogenous Melatonin and Dopamine on Low Nitrate Stress in Malus hupehensis.

Malus hupehensis, as an apple rootstock, is an economically important tree species popular due to its excellent fruit yield and stress resistance. Nitrogen is one of the critical limiting factors of plant growth and fruit yield, so it is crucial to explore new methods to improve nitrogen use efficiency. Melatonin and dopamine, as multifunctional metabolites, play numerous physiological roles in plants. We analyzed the effects of exogenous melatonin and dopamine treatments on the growth, root system architecture, nitrogen absorption, and metabolism of M. hupehensis when seedlings were exposed to nitrate-deficient conditions. Under low nitrate stress, plant growth slowed, and chlorophyll contents and 15NO3 - accumulation decreased significantly. However, the application of 0.1 µmol/L melatonin or 100 µmol/L exogenous dopamine significantly reduced the inhibition attributable to low nitrate levels during the ensuing period of stress treatment, and the effect of dopamine was more obvious. In addition to modifying the root system architecture of nitrate-deficient plants, exogenous melatonin and dopamine also changed the uptake, transport, and distribution of 15NO3 -. Furthermore, both exogenous melatonin and dopamine enhanced tolerance to low nitrate stress by maintaining the activity of enzymes (NR, NiR, GS, Fd-GOGAT, and NADH-GOGAT) and the transcription levels of related genes involved in leaf and root nitrogen metabolism. We also found that exogenous melatonin and dopamine promoted the expression of nitrate transporter genes (NRT1.1, NRT2.4, NRT2.5, and NRT2.7) in nitrate-deficient plant leaves and roots. Our results suggest that both exogenous melatonin and dopamine can mitigate low nitrate stress by changing the root system architecture, promoting the absorption of nitrate, and regulating the expression of genes related to nitrogen transport and metabolism. However, according to a comprehensive analysis of the results, exogenous dopamine plays a more significant role than melatonin in improving plant nitrogen use efficiency.

Crop Load Influences Growth and Hormone Changes in the Roots of "Red Fuji" Apple.

Crop load has a substantial impact on growth of the aerial and belowground parts of apple trees. Here, we examined the effects of different crop loads on growth and hormone levels in apple roots. A crop load of 1.5 (T1.5) fruits per cm2 trunk cross-sectional area (TCSA) treatment resulted in lower root growth vigor, while non-fruiting (T0) and T0.4 conditions showed higher root growth vigor. In all treatments, dead roots increased in length 90 days after full bloom (DAFB), whereas live roots were more abundant at about 50 and 170 DAFB, showing a bimodal curve. During each root growth peak, levels of cytokinins (CTKs), indole acetic acid (IAA), and gibberellic acid (GA3) were higher. Moreover, hormone levels gradually decreased with increasing crop load within each peak. Root turnover tended to decrease with decreasing crop load. These findings indicate that root growth and hormone contents were positively correlated during the fruit growth phase, and that the negative impact of crop load on root growth may have been caused by hormone level decreases.

Detrimental effects of antibiotics on mouse embryos in chromatin integrity, apoptosis and expression of zygotically activated genes.

The effects of specific components in culture medium on embryo physiology have been extensively investigated to optimize in vitro culture systems; however, little attention has been paid to antibiotics, the reagents used most commonly in culture systems to prevent contamination. To investigate the potential effects of routine use of antibiotics on cultured embryos, mouse zygotes were cultured with or without antibiotics. In both groups, the developmental rate and cell number of blastocysts appear to be normal. The proportion of embryos with blastomere fragmentation increased slightly when embryos were cultured with antibiotics. In contrast, the presence of antibiotics in the embryo culture system significantly disturbs expression of zygotically activated genes, damages chromatin integrity and increases apoptosis of cultured embryos. These results provide evidence that, when cultured with antibiotics, embryos with normal appearance may possess intrinsic physiological and genetic abnormalities. We demonstrate that the adverse effects of antibiotics on mammalian embryos are more severe than we previously presumed and that antibiotics are not essential for sterility of embryo culture system therefore abolishing antibiotic supplementation during embryo culture.

Isolation and culture of bovine mammary epithelial stem cells.

Bovine mammary epithelial stem cells (MESCs) are very important in agricultural production and bioengineering. In the present study, we compared different isolation and culture methods for MESCs and observed their growth and differentiation characteristics. MESCs have an extremely weak proliferation capacity, and it is very difficult to obtain and prolong subculture of a bovine mammary epithelial stem cell line. We obtained some multipotent MESC aggregates that looked like spherical colonies. These colonies were only derived from suspension culture and were induced to differentiate into epithelial-like cells, myoepithelial-like cells and secretory cells and to establish a ductal-like structure. In contrast, MESCs cultured in adherent culture displayed low morphogenetic competence and only differentiated into epithelial-like cells. MESCs are often identified by testing their differentiation in vivo; however, herein, we have demonstrated the in vitro differentiation potential of bovine MESCs. In our study, beta 1-integrin and alpha 6-integrin which are expressed by human epidermal stem cells, were found in bovine, which shows that bovine MESCs share the same molecular signature as human MESCs.

Reprogramming donor cells with oocyte extracts improves in vitro development of nuclear transfer embryos.

This study investigated the effects of donor cells pretreated with oocyte extracts on in vitro development of cloned embryos. Bovine fibroblasts were exposed to immature, mature and parthenogenetic oocyte extracts respectively before nuclear transfer. The detectable expression of Oct4 and global deacetylation in the treated cells showed that extracts could reprogram fibroblasts. Although all three groups of extracts exhibited reprogramming capacity, embryo development was not compliant with reprogramming effect. Improved quality and development of blastocysts were observed only in the mature extract treated group. We demonstrated that pretreatment of donor cells with mature oocyte extract improved in vitro development of cloned embryos. Our results suggested that reprogramming donor nuclei to a state synchronized with recipient cytoplasm before nuclear transfer would be beneficial for the development of cloned embryos.