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The seed is the starting point of the plant life cycle, and seed germination is a crucial stage that influences crop yield and quality. Under a changing climate, seeds will be more frequently exposed to various harsh environments (heat, drought, floods, and salinity) and soil-borne diseases, which may result in reduced germination and loss of vigor, threatening seed development and compromising crop yield. As such, seeking strategies to enhance the stress resistance capability of seeds (rapid seed germination and seedling establishment under stress conditions) is critically important to maintain crop yield.
Actually, plants have evolved sophisticated defense systems to cope with diverse abiotic and biotic stresses. Under stress, a series of molecular events such as stress perception, signal transduction, transcriptome/metabolome changes occur in plants, and these molecular changes can be "remembered" by plants to respond faster and/or stronger in the face of stress in the future, the so-called "stress memory". Based on this, our team hypothesized that stimulating seeds at the germination stage, inducing stress memory, is likely to enhance the seedlings’ stress resistance. Reactive oxygen species (ROS) are important signaling molecules that can act as "danger" or "warning" signals to trigger immune responses. However, ROS are chemically unstable molecules with relatively short lifetimes. Nanozymes are nanomaterials with enzymatic properties. Our previous studies have shown that AgNPs can trigger ROS generation in algae/plants. Besides, the small size enables AgNPs to easily penetrate the biological barriers of the seed coat. As such, AgNPs could be a model nanobiostimulant to be used as seed treatment agent. Taking the staple crop rice as the test plant and salt as the representative of abiotic stress, our team explored the performance of AgNPs seed priming on seed germination under stress. The results showed that seeds primed with AgNPs (40 mg/L) for 24 h exhibited accelerated germination speed, significantly increased seedling vigor, biomass and root length compared to seeds with hydropriming treatment under stress. These results verified our hypothesis that AgNPs-priming can stimulate rice seeds to form stress memory and enhance salt tolerance. Importantly, this "stress memory" can last to the seedling stage, enhancing the resistance of 37-day-old rice seedlings to both salt and rice blast disease, indicating that seed defense-priming is able to increase the resistance of seedlings to abiotic and biotic stresses simultaneously (Figure 1).
Figure 1: (A) Representative picture of 7-day-old rice seedlings. (B) Germination speed. (C) Germination rate. (D) Seed vigor. (E) Biomass. (F) Root length.
The underlying mechanisms for the enhanced resistance were elucidated by single particle-inductively coupled plasma–mass spectrometry (SP-ICP-MS), electron paramagnetic resonance (EPR). The results showed that AgNPs can penetrate the seed coat and enter the seed tissues and all the Ag in seeds was in nanoparticulate form instead of ionized Ag. We next visualized and quantified ROS in rice seeds and confirmed that ROS species generated by AgNPs are OH• by using electron paramagnetic resonance (EPR) spectroscopy, which mainly located in embryo (Figure 2).
Figure 2. (A) Ag distribution in different tissues (seed coat, embryo, and endosperm) of rice seeds after priming with AgNPs for 24 h. (B) Size distribution of nanoparticulate Ag in rice seeds primed with AgNPs for 24 h. (C) Images of water- or AgNPs-primed rice seeds captured using fluorescence (left) and brightfield (right) microscopy. Primed rice seeds were stained with DCFH-DA for 30 min before microscopic observation. (D) POD-like activities of AgNPs. (E) EPR results showing the generation of hydroxyl radicals (OH•).
Multiomics techniques (metabolomics combined with transcriptomics) were further used to explore the molecular mechanisms behind stress memory. GC-MS-based untargeted metabolomics results showed that AgNPs-priming resulted in metabolic reprogramming in rice seeds. Several metabolites with ROS-scavenging capacities were significantly decreased upon AgNPs-priming, including ascorbic acid, alpha-tocopherol, and four phenolics (dehydrocholesterol, 4-hydroxycinnamic acid, catechol, and 1,2,4-benzenetriol). The alteration of these metabolic imprints suggests that AgNPs triggered tentative disturbance of ROS-homeostasis during AgNPs-priming, but AgNPs-boosted OH• are within the threshold value, which mainly aids redox biology, instead of inducing cytotoxicity. Besides, AgNPs-priming also increased the level of several metabolites involved in stress signal transduction, including salicylic acid, niacinamide and glycerol-3-phosphate. The upregulation of these signaling molecules might be a strong indicator that AgNPs-priming triggered immune responses in rice seeds. Transcriptome results also showed that AgNPs-priming activated pathways involved in stress signal transduction and defense responses, including plant hormone signal transduction, glutathione metabolism, flavone and flavonol biosynthesis, MAPK signaling pathway, and plant–pathogen interactions. Taken together, these omics results revealed molecular events inside AgNPs-primed seeds, elucidating the molecular mechanisms behind enhanced resistance (Figure 3).
Figure 3. (A) OPLS-DA score plots based on metabolomics data set of rice seeds primed with water (hydropriming) or AgNPs. (B) OPLS-DA VIP score plot showing the responsible metabolites leading to the separation. (C) Boxplot representing the relative abundance of significantly changed metabolites (screened by t-test) in rice seeds treated with 40 mg/L of AgNPs. Blue, pink, yellow, and purple mean sugars, antioxidants, shikimate pathway intermediates, and signaling metabolites.
This study proposed a simple nanopriming seed treatment strategy that can enhance crop stress resistance, which is of great significance for improving crop climate resilience, reducing the use of pesticides and fertilizers, and promoting sustainable agricultural development. The research results were published in ACS Nano on December 16, 2022 under the title "AgNPs-triggered Seed Metabolic and Transcriptional Reprogramming Enhanced Rice Salt Tolerance and Blast Resistance". Xin Yan, postgraduate student at Nanjing University, is the first author of the paper, and Professor Lijuan Zhao is the corresponding author of the paper. The co-authors of this paper also include Si Chen, postgraduate student at Nanjing University, Zhengyan Pan, associate researcher at the Rice Institute of Liaoning Province, Weichen Zhao, postgraduate student at China Agricultural University, and Professor Yukui Rui from China Agricultural University. We would like to thank the National Natural Science Foundation of China (21876081, 21906081) for their funding of the above research work.
Article link: //doi.org/10.1021/acsnano.2c09181
Key words: seed treatment, nanoparticle, biostimulant, stress resilience, stress memory
