Inhibition effects of A. argyi water extract (AAWE) on rice seedlings
In our previous study, we found that AAWE strongly inhibited the seed germination and growth of Brassica pekinensis, Lactuca sativa, Oryza sativa, Portulaca oleracea, Oxalis corniculata, and Setaria viridis [19]. To further explore the mechanisms underlying the allelopathic inhibition of AAWE, we first investigated the site of action using rice as a model plant. Our results showed that AAWE had a concentration-dependent inhibitory effect on rice (Fig. 1A). Plant growth could be significantly inhibited at a very low extract concentration (0.01 g/mL), while the rice seeds could not germinate when the AAWE treatment concentration was up to 0.15 mg/mL (Fig. 1A). Therefore, to investigate the influence of AAWE treatment on plants, we chose the lower treatment concentration range (0.01–0.05 mg/mL) at which rice could germinate and grow. As shown in Figs. 1B and 1C, rice growth was significantly (P < 0.01) inhibited under the 0.01 g/mL AAWE treatment, and the inhibitory effect was enhanced as the treatment concentration increased. In addition, the AAWE-treated rice leaves yellowed. Further statistical analysis showed that the plant height, root length, and number of roots under AAWE treatment significantly decreased compared to the control (Fig. 1D, E, F). These results indicate that AAWE dramatically inhibited the root and leaf system of rice. Therefore, we further investigated the mechanisms of AAWE inhibitory effects on the development of rice roots and leaves.
AAWE destroyed the growth and development of the root system
As shown in Supplemental Fig. 1A, the rice root number decreased, and the structure significantly changed after AAWE treatment compared to the control. We performed microscopic observation of transverse sections of the control and AAWE-treated rice root tip cells to investigate the influence of AAWE. In the control, the cells in the transected surface of the root tip of rice seedlings were regularly and uniformly arranged and were composed of clearly distinguishable cell layers. In order from the outside to the inside, these were the epidermal layer, cortical parenchyma, endothecium, and pericycle. However, after AAWE treatment, the number of cell layers in the rice root tips decreased, and the arrangement was disordered. Moreover, most of the cortical parenchyma cells expanded more than twice, indicating that the root tip cells probably lost their ability to divide (Fig. 2A). This effect was enhanced as AAWE treatment concentrations increased, and epidermal cells began to shed at 0.03 g/mL (Fig. 2A). The results of longitudinal section observation were consistent with transverse section observations, the root tip cells of the control seedlings were arranged regularly and clearly, and the cell classification in each functional area was obvious. Nevertheless, epidermal cells in the rice root tip became wrinkled, shrunken, and even ruptured when exposed to 0.01 g/mL AAWE. The cells in the shoot apical meristem, quiescent center, and root cap were also disordered (Fig. 2B). In addition, scanning electron microscope (SEM) analysis demonstrated that aberrant alterations occurred along the surface of the roots upon introduction to AAWE compared to the control (Supplemental Fig. 1B).
We further performed transmission electron microscopy (TEM) observations to compare the subcellular structure of control and AAWE-treated rice plants. The results showed that cells in the blank group were regularly and closely arranged, while cells in the treatment group (0.03 g/mL) were enlarged and disordered (Fig. 2C). Magnified observation of a single cell revealed that the membrane system of the blank group was complete, with clearly visible endoplasmic reticulum (Fig. 2C-1), Golgi body (Fig. 2C-2), vacuoles (Fig. 2C-3), and other organelles. However, organelles could hardly be found, and many black deposits appeared inside the cell under AAWE treatment, indicating that the organelles probably degraded. We further compared the nuclear structure in the blank and AAWE treatment groups to detect whether the control center of the whole cell was affected. The results showed that the nucleus in the blank group was intact, but the nuclear membrane of the treated group was degraded, and the nucleoli diffused (Fig. 2C-4). Fungi were also observed in the AAWE treatment group, including the nucleus (Fig. 2C-5), which could be due to very weak antifungal activity after AAWE treatment. These results demonstrated that AAWE had a strong destructive influence on the integrity of organelles and nuclei in rice cells.
AAWE altered phytohormone homeostasis in rice roots
Phytohormones, including auxin, cytokinin, gibberellins, and abscisic acid, play vital roles in plant root development [27]. As rice roots were destroyed after AAWE treatment, we explored whether the regulation of endogenous phytohormones was out of balance. Therefore, we detected the content of endogenous phytohormones in the control and AAWE-treated roots. For auxins, 0.05 g/mL AAWE treatment promoted the production of the main auxins (IAA), and the IAA content in rice roots reached 5.83-fold that of the control. Meanwhile, as a low molecular weight binding state of IAA, IAA-ASP content increased remarkably under low AAWE concentration treatment. Although the IAA-ASP content showed a decreasing trend as the AAWE treatment concentration increased, it still presented a significant enhancement compared to the blank control group. These results suggest that AAWE promotes auxin production and accumulation of recipient plants (Fig. 3A).
For cytokinins, one free cytokinin (isopentenyladenosine, IP) and two tRNA cytokinins (iP Riboside, IPR and trans-zeatin riboside, t-ZR) were determined. The results showed that AAWE had different effects on different cytokinins (Fig. 3B). The IP and t-ZR contents first increased and then decreased as the AAWE treatment concentration increased. The IP content was highest at 0.03 g/mL, while that of t-ZR was at 0.01 g/mL, which were 2.90 and 2.47 times those of the control, respectively. In addition, the IPR content in rice roots was significantly greater than that in the blank control, while the IPR content increased in an AAWE dose-dependent manner. At the maximum concentration of AAWE (0.05 g/mL), IPR accumulation reached 0.53 ng/g, which was approximately 2.66 times greater than the level of IPR in the blank control.
We further measured three physiologically active gibberellins to evaluate the effect of AAWE on gibberellin content. The results revealed that gibberellin showed an increasing trend under AAWE stress (Fig. 3C). GA1 and GA3 significantly increased when the treated AAWE concentration reached 0.03 g/mL. GA7 began to be synthesized and detected under the 0.03 g/mL AAWE treatment, although it was barely synthesized by plants under natural conditions and low AAWE treatment concentrations. In addition, the level of ABA in 0.05 g/mL AAWE-treated rice roots was 3.10 times higher than that in the control, but low-concentration AAWE treatment did not significantly impact ABA content (Fig. 3D). This suggests that the ABA pathway was not particularly sensitive to AAWE. In summary, rice hormones in roots showed an upward trend after AAWE treatment, and the contents of some hormones reached several times of those in the blank group, leading to negative effects on normal plant growth.
AAWE inhibited the absorption and transportation of photosynthesis-essential mineral elements
The root is the main organ of plants that absorbs mineral nutrients, which are transported to the aboveground parts, especially the leaves. The uptake and transportation of mineral nutrients are essential for plant life activities. We next measured essential elements in rice roots to investigate the impact of AAWE on mineral nutrient uptake and transportation. The N, P, and K contents in both leaves and roots were up-regulated slightly under AAWE treatment (Fig. 4A, B, C). These results indicated that A. argyi allelopathy did not affect the absorption of macroelements and likely exhibited a promotion effect. Ca and Mg play significant roles in maintaining cell integrity [28]. Mg participates in the synthesis of chlorophyll and maintains the stability of the chloroplast structure [29]. Our results showed that the contents of Ca and Mg in AAWE-treated rice plants were significantly enriched in roots but decreased in leaves (Fig. 4D, E), indicating that the transport of these two nutrient elements from roots to leaves was mostly blocked. More importantly, the absorption of Fe and Mn was significantly inhibited in roots and leaves, and Mn was barely absorbed by the plants under high concentrations of AAWE treatment (Fig. 4F, G). Due to the crucial roles of Fe and Mn in photosynthesis [30, 31], the sharp decrease of these two elements in leaves and roots indicated that the photosynthetic efficiency of rice treated with AAWE likely decreased. Finally, Cu, which promotes photosynthesis and protein accumulation in plants [32], had no significant difference between control and AAWE-treated rice plants in leaves but significantly decreased in AAWE-treated roots compared to the control (Fig. 4H). Altogether, AAWE treatment mainly affected the absorption and transport of trace elements, which likely led to defects in photosynthesis and the primary metabolism of rice.
AAWE destroyed the growth and development of leaves
In addition to abnormalities in roots, we found that AAWE treatment also seriously inhibited the growth of rice leaves. The AAWE-treated rice leaves exhibited abnormal symptoms, such as leaf rolling, yellowing, and withering (Supplemental Fig. 2). We used TEM to observe ultrastructural changes in AAWE-treated rice leaf cells. In the control group, mesophyll cells had an obviously intact structure and chloroplasts with abundant starch granules attached to the cell wall (Fig. 5A). However, in the AAWE-treated group, anomalous black sediment was observed in the cells, the number of chloroplasts was significantly reduced, and no starch grains were observed (Fig. 5A). Moreover, chloroplasts in AAWE-treated rice were significantly different from those in the control. In the control, chloroplasts presented a long fusiform structure with a clear and complete membrane structure. The matrix in the chloroplasts was uniform and dense, and grana thylakoids in the control were orderly stacked. Compared to the control, the membrane structure of AAWE-treated chloroplasts was obviously damaged, the arrangement of the internal grana and thylakoid was disordered or even disappeared, and the lamellae were blurred and loosely arranged, which probably led to the absence of starch grains (Fig. 5A). Further analysis showed that the chlorophyll content in 0.02 g/mL, 0.03 g/mL, 0.04 g/mL, and 0.05 g/mL AAWE-treated rice leaves significantly decreased compared to the control. And the inhibitory effect of AAWE on chlorophyll content showed a dose-dependent relationship with AAWE concentration. The chlorophyll content in 0.05 g/mL AAWE-treated rice leaves only accounted for 15.34% of that in the blank control, indicating that photosynthesis was almost completely lost (Fig. 5B). Moreover, the soluble sugar content decreased under A. argyi allelopathy (Fig. 5C). These results suggest that AAWE treatment affects chlorophyll content and photosynthesis, leading to a decrease in photosynthetic products.
Another interesting phenomenon is that almost the entire membrane structure was significantly damaged in AAWE-treated mesophyll cells. The nuclear membrane of the blank group was clear, and the chromatin was evenly distributed in the nucleus, while the membrane structure of the AAWE-treated group was dissolved (Fig. 5A-1). Mitochondria in the blank group had obvious membrane boundaries, while the boundary between mitochondria and other organelles was blurred in the AAWE-treated group (Fig. 5A-2). In addition, the cell membrane was damaged, scattered in the center of the cell, and no longer close to the cell wall (Fig. 5A). These results demonstrate that AAWE treatment seriously destroys the membrane structure and the integrity of organelles in rice mesophyll cells.
AAWE caused oxidative damage in rice leaves
Previous studies have shown that the intracellular level of reactive oxygen species (ROS) dramatically increases in harsh environments, which seriously damages the cell structure. Considering the severely damaged cell structure, we speculated that an ROS burst likely occurred in AAWE-treated rice mesophyll cells. Therefore, we detected ROS levels in both the control and AAWE-treated groups. Consistent with our expectations, AAWE treatment induced an ROS burst in rice. ROS levels increased as the AAWE concentration raised. The ROS content reached 8280 pg/g in 0.05 g/mL AAWE-treated rice, which was much higher than the 5145 pg/g in the control (Fig. 5E). The MDA content is usually used as an indicator to assess the extent of lipid peroxidation and membrane system damage. Therefore, we further measured the MDA content in the control and AAWE-treated groups. The results showed that the MDA content in rice seedlings also continuously increased as the AAWE concentration increased (Fig. 5D). The MDA content in rice under the 0.05 g/mL AAWE treatment was 5.68 times that of the control. These results suggest that AAWE greatly induced ROS bursts and MDA accumulation in rice.
Plants have evolved many antioxidative enzymes to eliminate ROS, including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), which can protect cells from damage by scavenging or weakening the toxic effects of free radicals [33]. We found that the activities of these three enzymes were greatly affected by AAWE treatment. All AAWE-treated samples displayed higher SOD activities than the control samples, which were approximately 37.82% ~ 41.94% (P < 0 0.01) higher than those of the corresponding controls (Fig. 5F). In contrast, POD activity showed a concentration-dependent decrease from 0.01 to 0.05 g/mL (P < 0.001). POD activity in 0.05 g/mL AAWE-treated samples was only 48.09% that of the control group (Fig. 5G). Similarly, CAT activity also decreased after AAWE treatment (Fig. 5H). This suggests that under low-stress conditions, cells can activate their own antioxidant defense system and quickly clear their own ROS. However, when the stress concentration exceeds its own tolerance, oxidative damage will be very serious.
AAWE strongly influenced the transcription of key driver genes related to primary metabolism
To further understand the molecular mechanisms of allelopathic effects of AAWE, genome-wide gene expression profiles were compared between water- and 0.03 g/mL AAWE- treated rice roots and leaves. After further screening, 689 genes with |Log2FC|≥ 1 and Q-value < 0.05 were selected for further investigation (Supplemental Fig. 3A). The clustering heat map is shown in Fig. 6A. There were 311 DEGs in AAWE-treated leaves, of which 245 DEGs were up-regulated, and 66 DEGs were down-regulated. In AAWE-treated root samples, there were 418 DEGs, among which 254 were up-regulated, and 164 were down-regulated. KEGG classification of DEGs showed that most genes were involved in metabolism (Supplemental Fig. 3B). To elucidate DEG functions in response to AAWE stress, the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) databases were used to enrich DEGs into the corresponding pathways and categories. In leaves, the DEGs were enriched in some important KEGG pathways, such as nitrogen metabolism, photosynthesis, porphyrin, and chlorophyll metabolism (Fig. 6B). Meanwhile, the results of the GO enrichment demonstrated that DEGs were involved in oxidation–reduction processes, transmembrane transport, photosynthesis, and iron ion transport (Fig. 6C). We also analyzed the DEGs between water- and AAWE-treated roots. KEGG pathways related to the biosynthesis of secondary metabolites and starch and sucrose metabolism were identified (Fig. 6D). The GO categories of hydrogen peroxide catabolic process, reactive oxygen species metabolic process, gibberellic acid homeostasis, and iron ion transmembrane transport were also identified (Fig. 6E).
Key driver gene analysis (KDA) was performed to systematically investigate the influence of AAWE on rice plants (Fig. 6F), and ten key driver genes related to the primary metabolism of plants were significantly affected (Supplemental Table 1). Interestingly, all of these genes were involved in starch and sucrose metabolism or galactose metabolism pathways, and some of these genes were linked to chloroplasts. Therefore, we speculated that A. argyi allelopathy influenced the primary metabolic processes of rice, such as photosynthesis, and subsequently drove the abnormal expression of other pathways in rice. Furthermore, we performed functional annotation analysis on the related genes linked to the key driver genes (Fig. 6G). Our results demonstrated that genes involved in the starch and sucrose metabolism pathway accounted for 39% of the total, followed by the biosynthesis of secondary metabolites pathway, which accounted for 16%. These results indicated that these related genes were not only related to primary metabolism but were also closely related to secondary metabolism, thus affecting the growth and development of the whole plant.
To validate the transcriptome reliability, RT–qPCR was used to confirm the expression levels of 15 genes involved in the rice response to A. argyi allelochemicals. First, the key genes involved in photosynthesis and chlorophyll synthesis pathways in leaves were verified. In our previous study [19], the expression levels of related genes in rice leaves were significantly inhibited after a long treatment time (21 days) with AAWE. In this study, we analyzed the key driver genes of all DEGs between the control and AAWE-treated rice and found that the primary metabolic pathway was the key driver pathway. Therefore, we measured the expression levels of genes related to photosynthesis and chlorophyll synthesis in leaves at an earlier period (13 days) after A. argyi stress to detect whether the plant's primary metabolism process was affected during early stages. In the chlorophyll synthesis pathway, HEML gene expression was not significantly changed (Supplemental Fig. 4A), and CHLD expression was significantly inhibited at low AAWE concentrations but slightly promoted at 0.05 g/mL (Supplemental Fig. 4B). In addition, the expression of other chlorophyll biosynthesis-related genes gradually decreased as AAWE treatment concentrations increased, including HEMA, CHLH, CRD, CHLG, and CAO, which indicated that chlorophyll biosynthesis was significantly blocked (Fig. 6H, I, J, K, L). The PsbY gene is a member of photosystem II, and Os04g38410 encodes the subunit of the LHCII complex, which plays a crucial role in photosynthesis. The expression levels of these two genes were significantly down-regulated under AAWE treatment (Fig. 6M, N).
Moreover, the contents of various hormones changed after A. argyi treatment (Fig. 3). As such, we also analyzed the expression levels of hormone-related genes in roots. YUCCA and IAA1 were the key genes in the auxin synthesis and signalling pathways, respectively, and their changing trends were consistent. The expression levels of YUCCA and IAA1 in rice treated with low and medium AAWE concentrations gradually decreased, while those in rice treated with high concentrations increased (Supplemental Fig. 4C, 4D). Compared with the blank treatment, the expression level of the auxin signalling pathway-related gene IAA1 significantly increased under 0.05 g/mL treatment (Supplemental Fig. 4C, 4D). Moreover, GA20, a crucial gene in gibberellin synthesis, first decreased and then increased (Supplemental Fig. 4E), while the expression of DELLA, a signalling pathway gene decreased with increasing AAWE concentrations, indicating that the signalling function of gibberellin might be continuously inhibited (Supplemental Fig. 4F). In addition, the expression levels of two genes related to abscisic acid signalling pathway NCED4 and PYR were similar to genes participating in the auxin signalling pathway (Fig. 6O, Supplemental Fig. 4G). Among them, the expression of NCED4 in rice treated with high AAWE concentrations was significantly higher than that in the blank group (Fig. 6O). These results demonstrate that the expression levels of crucial genes participating in hormone signalling pathways dramatically changed under AAWE treatment.