Glutathione S-transferase activity facilitates rice tolerance to the barnyard grass root exudate DIMBOA.

BACKGROUND: In paddy fields, the noxious weed barnyard grass secretes 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) to interfere with rice growth. Rice is unable to synthesize DIMBOA. Rice cultivars with high or low levels of allelopathy may respond differently to DIMBOA. RESULTS: In this study, we found that low concentrations of DIMBOA (≤ 0.06 mM) promoted seedling growth in allelopathic rice PI312777, while DIMBOA (≤ 0.08 mM) had no significant influence on the nonallelopathic rice Lemont. DIMBOA treatment caused changes in the expression of a large number of glutathione S-transferase (GST) proteins, which resulting in enrichment of the glutathione metabolic pathway. This pathway facilitates plant detoxification of heterologous substances. The basal levels of GST activity in Lemont were significantly higher than those in PI312777, while GST activity in PI312777 was slightly induced by increasing DIMBOA concentrations. Overexpression of GST genes (Os09g0367700 and Os01g0949800) in these two cultivars enhanced rice resistance to DIMBOA. CONCLUSIONS: Taken together, our results indicated that different rice accessions with different levels of allelopathy have variable tolerance to DIMBOA. Lemont had higher GST activity, which helped it tolerate DIMBOA, while PI312777 had lower GST activity that was more inducible. The enhancement of GST expression facilitates rice tolerance to DIMBOA toxins from barnyard grass root exudates.

Intercropping with Achyranthes bidentata alleviates Rehmannia glutinosa consecutive monoculture problem by reestablishing rhizosphere microenvironment.

Background: The consecutive monoculture of Rehmannia glutinosa leads to a serious decrease in its production and quality. Previous studies have demonstrated that intercropping altered species diversity and rhizosphere microbial diversity. However, it remained unknown whether the impaired growth of monocultured plants could be restored by enhanced belowground interspecific interactions. Method: In the present research, a continuous cropping facilitator Achyranthes bidentata was intercropped with R. glutinosa under pot conditions, and three different types of root barrier treatments were set, including that complete belowground interaction (N), partial belowground interaction (S), and no belowground interspecies interaction (M), with the aims to investigate belowground interaction and the underlying mechanism of alleviated replanting disease of R. glutinosa by intercropping with A. bidentata. Results: The results showed that the land equivalent ratio (LER) of the two years was 1.17, and the system productivity index (SPI) increased by 16.92 % under S treatment, whereas no significant difference was found in N and M regimes. In the rhizosphere soil, intercropping systems had significantly increased the contents of sugars and malic acid in the soil of R. glutinosa, together with the content of organic matter and the invertase and urease activities. Meanwhile, intercropping increased the community diversity of fungi and bacteria, and the relative abundance of potential beneficial bacteria, such as Bacillus, Nitrospira, and Sphingomonas, despite the pathogenic Fusarium oxysporum was still the dominant genus in the rhizospheric soil of R. glutinosa under various treatments. The results of antagonism experiments and exogenous addition of specific bacteria showed that Bacillus spp. isolated from rhizosphere soil had a significant antagonistic effect on the pathogen of R. glutinosa. Conlusion: Taken together, our study indicated that the R. glutinosa//A. bidentata intercropping systems alleviate the consecutive monoculture problem of R. glutinosa by recruiting beneficial bacteria. The studies we have conducted have a positive effect on sustainable agricultural development.

Spatial-Temporal Distribution of Allelopathic Rice Roots in Paddy Soil and Its Impact on Weed-Suppressive Activity at the Seedling Stages.

Background: Allelochemicals secreted by allelopathic rice roots are transmitted to the receptor rhizosphere through the soil medium to inhibit the growth of the surrounding weeds. This research aimed to explore the relationships between the spatial-temporal distribution of rice roots in soil and weed-suppression ability at its seedling stage. Results: This study first examined the root distribution of three rice cultivars in paddy soil in both vertical and horizontal directions at 3-6 leaf stage. Then, an experiment using rice-barnyardgrass mixed culture was conducted to analyze the allelopathic potential and allelochemical content secreted by rice roots in different lateral soil layers. The results showed that allelopathic rice had a smaller root diameter and larger root length density, root surface area density, and root dry weight density than those of non-allelopathic rice, in the top 5 cm at 5- and 6-leaf stages. In particular, there were significant differences in root distribution at the horizontal distance of 6-12 cm. Besides, allelopathic rice significantly inhibited the above-ground growth of barnyardgrass co-cultured at 12 cm lateral distance in situ, and the content of benzoic acid derivatives in allelopathic rice in a 6-12 cm soil circle was higher than that observed at 0-6 cm distance. Moreover, correlation analysis confirmed that the distribution of roots in the horizontal distance was significantly correlated with weed inhibition effect and allelochemical content. Conclusion: These results implied that spatial distribution of allelopathic rice roots in paddy soil, particularly at the lateral distance, appears to have important impact on its weed-suppressive activity at the seedling stage, suggesting that modifying root distribution in soil may be a novel method to strengthen the ability of rice seedlings to resist paddy weeds.

Deciphering the Molecular Mechanisms of Chilling Tolerance in Lsi1-Overexpressing Rice.

Improving tolerance to low-temperature stress during the rice seedling stage is of great significance in agricultural science. In this study, using the low silicon gene 1 (Lsi1)-overexpressing (Dular-OE) and wild-type rice (Dular-WT), we showed that Lsi1 overexpression enhances chilling tolerance in Dular-OE. The overexpression of the Lsi1 increases silicon absorption, but it was not the main reason for chilling tolerance in Dular-OE. Instead, our data suggest that the overexpression of a Lsi1-encoding NIP and its interaction with key proteins lead to chilling tolerance in Dular-OE. Additionally, we show that the high-mobility group protein (HMG1) binds to the promoter of Lsi1, positively regulating its expression. Moreover, Nod26-like major intrinsic protein (NIP)'s interaction with α and ß subunits of ATP synthase and the 14-3-3f protein was validated by co-immunoprecipitation (Co-IP), bimolecular fluorescent complementary (BiFC), and GST-pulldown assays. Western blotting revealed that the overexpression of NIP positively regulates the ATP-synthase subunits that subsequently upregulate calcineurin B-like interacting protein kinases (CIPK) negatively regulating 14-3-3f. Overall, these NIP-mediated changes trigger corresponding pathways in an orderly manner, enhancing chilling tolerance in Dular-OE.

Comparison of Silicon-Evoked Responses on Arsenic Stress between Different Dular Rice Genotypes.

Arsenic is one of the most hazardous metalloids in nature, and due to its high water solubility, it is one of the most important causes of pollution. However, silicon reduces the uptake and transport of arsenic in rice. This study investigates the interaction of different arsenic and silicon levels on dry weight, protein content, and concentrations of arsenic and silicon in two different rice shoots and roots of Dular wild-type (DU-WT) and Dular Lsi1-overexpressed (DU-OE) rice. It should be noted that all seedlings were subjected to four different treatments. For RNA-seq and qPCR, the DU-WT genotype was selected as the control and DU-OE as the treatment. With the addition of silicone treatment, dry weight and protein content in the shoots and roots of both rice lines were increased, while the concentration of arsenic in these two organs was decreased. When seedlings were exposed to arsenic treatments, protein content, silicon concentration, and dry weight were decreased in both roots and shoots, while arsenic concentration was increased in both rice genotypes. The RNA-seq in DU-OE showed 5823 differentially expressed genes (DEGs), of which 2604 were up-regulated and 3219 down-regulated. Treatment of rice by arsenic and silicon has changed the expression of genes encoding cytokinin-responsive GATA transcription factor 1, protein IN2-1 homolog B, calcium-binding EGF domain-containing protein, Os01g0369700 protein, probable glutathione S-transferase GSTU1, glutathione S-transferase protein, Os09g0367700 protein, isocitrate dehydrogenase (NADP), and Os08g0522400 protein in the root of DU-OE. The present study's findings showed that in the presence of silicon, the transgenic genotype is much more resistant to arsenic than the wild genotype of Dular rice.

Serine hydroxymethyltransferase localised in the endoplasmic reticulum plays a role in scavenging H2O2 to enhance rice chilling tolerance.

BACKGROUND: Rice is a chilling-sensitive crop that would suffer serious damage from low temperatures. Overexpression of the Lsi1 gene (Lsi1-OX) in rice enhances its chilling tolerance. This study revealed that a serine hydroxymethyltransferase (OsSHMT) mainly localised in the endoplasmic reticulum (ER) is involved in increasing tolerance to chilling. RESULTS: A higher transcription level of OsSHMT was detected in Lsi1-OX rice than in the wild type. Histone H1 and nucleic acid binding protein were found to bind to the promoter region of OsSHMT and regulate its expression, and the transcription levels of these proteins were also up-regulated in the Lsi1-OX rice. Moreover, OsSHMT interacts with ATP synthase subunit α, heat shock protein Hsp70, mitochondrial substrate carrier family protein, ascorbate peroxidase 1 and ATP synthase subunit ß. Lsi1-encoded protein OsNIP2;1 also interacts with ATP synthase subunit ß, and the coordination of these proteins appears to function in reducing reactive oxygen species, as the H2O2 content of transgenic OsSHMT Arabidopsis thaliana was lower than that of the non-transgenic line under chilling treatment. CONCLUSIONS: Our results indicate that ER-localised OsSHMT plays a role in scavenging H2O2 to enhance the chilling tolerance of Lsi1-OX rice and that ATP synthase subunit ß is an intermediate junction between OsNIP2;1 and OsSHMT.

MYB57 transcriptionally regulates MAPK11 to interact with PAL2;3 and modulate rice allelopathy.

Rice allelopathy is a natural method of weed control that is regarded as an eco-friendly practice in agroecology. The allelopathic potential of rice is regulated by various genes, including those that encode transcription factors. Our study characterized a MYB transcription factor, OsMYB57, to explore its role in the regulation of rice allelopathy. Increasing the expression of OsMYB57 in rice using the transcription activator VP64 resulted in increased inhibitory ratios against barnyardgrass. The gene expression levels of OsPAL, OsC4H, OsOMT, and OsCAD from the phenylpropanoid pathway were also up-regulated, and the content of l-phenylalanine increased. Chromatin immunoprecipitation incorporated with HiSeq demonstrated that OsMYB57 transcriptionally regulated a mitogen-activated protein kinase (OsMAPK11); in addition, OsMAPK11 interacted with OsPAL2;3. The expression of OsPAL2;3was higher in the allelopathic rice PI312777 than in the non-allelopathic rice Lemont, and OsPAL2;3 was negatively regulated by Whirly transcription factors. Moreover, microbes with weed-suppression potential, including Penicillium spp. and Bacillus spp., were assembled in the rhizosphere of the rice accession Kitaake with increased expression of OsMYB57, and were responsible for phenolic acid induction. Our findings suggest that OsMYB57 positively regulates rice allelopathy, providing an option for the improvement of rice allelopathic traits through genetic modification.

Transcriptome analysis reveals that barnyard grass exudates increase the allelopathic potential of allelopathic and non-allelopathic rice (Oryza sativa) accessions.

BACKGROUND: Allelopathy in rice (Oryza sativa) is a chemically induced response that is elevated by the exogenous application of chemical compounds and barnyard grass root exudates. An in-depth understanding of the response mechanisms of rice to chemical induction is necessary for the identification of target genes for increasing the allelopathic potential of rice. However, no previous studies have evaluated the transcriptomic changes associated with allelopathy in rice in response to barnyard grass exudates treatment. Thus, the aim of the present study was to reveal differentially expressed genes (DEGs) in allelopathic and non-allelopathic rice seedlings treated with barnyard grass exudates to identify target allelopathy genes. RESULTS: The inhibitory effect of the culture solutions on the allelopathic rice accession PI312777 (PI) and the non-allelopathic rice accession Lemont (LE) significantly increased (P < 0.05) after treatment with barnyard grass root exudates. The RNA sequencing results revealed that 14,891 genes in PI(+B) vs. LE(+B), 12,505 genes in PI(+B) vs. PI(-B), and 5857 genes in LE(+B) vs. LE(-B) were differentially expressed following root exudates treatment. These DEGs were classified into three categories and 32 functional groups, i.e., 12 groups in the biological process category, 12 groups in the cellular component category, and eight groups in the molecular function category. There were 5857 and 2846 upregulated genes and 135 and 50 upregulated Gene Ontology terms (P < 0.05) in the biological process category in PI(+B) vs. PI(-B) and LE(+B) vs. LE(-B), respectively. These results indicated that the allelopathic accession PI is more sensitive than the non-allelopathic accession LE to exogenous root exudates treatment. Genes related to rice allelochemical-related biosynthesis pathways, particularly the shikimic acid and acetic acid pathways, were significantly differentially expressed in both rice accessions. These findings suggested that phenolic acids, fatty acids, and flavonoids, which constitute the downstream metabolites of the shikimic acid and acetic acid pathways, are significantly expressed in response to root exudates of barnyard grass. CONCLUSIONS: The allelopathic potential of both rice accessions could be significantly enhanced by barnyard grass root exudates application. Furthermore, genes related to the biosynthesis pathways of reported rice allelochemicals were significantly differentially expressed in both accessions. Phenylalanine ammonia lyase was determined to be a potential target for the regulation of chemical induction.

Lsi1 modulates the antioxidant capacity of rice and protects against ultraviolet-B radiation.

Silicon (Si) enhances the resistance of rice to biotic and abiotic stress. In rice, the accumulation of Si is controlled by the low silicon rice 1 (Lsi1) gene; overexpression of Lsi1 (Lsi1-OX) increases Si uptake and accumulation, while the reverse is observed in Lsi1-RNA interference (Lsi1-RNAi) transgenic rice. When the two transgenic rice lines and wild-type (WT) rice were exposed to ultraviolet (UV)-B radiation, the Lsi1-OX or Lsi1-RNAi rice showed differential microRNA (miRNA) expression, compared to WT rice. These miRNAs were predicted to target genes involved in light signal transduction and cell detoxification. The greatest capacities of ascorbate peroxidase, superoxide dismutase, peroxidase, and phenylalanine ammonia lyase (PAL) and highest contents of phenolics, flavonoids, and proline were found in Lsi1-OX rice, followed by WT rice and Lsi1-RNAi transgenic rice. A further comparison of the transcript levels of individual PAL genes revealed that the expression of PAL2-2 (Os02g0626400) was positively regulated by Lsi1. Our results demonstrate that Lsi1 overexpression or interference causes changes in both miRNA expression and antioxidant capacity in rice, and therefore modulates rice tolerance to UV-B radiation. Furthermore, we demonstrated that PAL2-2 was positively regulated by Lsi1 during this process.

Protein Phosphatase (PP2C9) Induces Protein Expression Differentially to Mediate Nitrogen Utilization Efficiency in Rice under Nitrogen-Deficient Condition.

Nitrogen (N) is an essential element usually limiting in plant growth and a basic factor for increasing the input cost in agriculture. To ensure the food security and environmental sustainability it is urgently required to manage the N fertilizer. The identification or development of genotypes with high nitrogen utilization efficiency (NUE) which can grow efficiently and sustain yield in low N conditions is a possible solution. In this study, two isogenic rice genotypes i.e., wild-type rice kitaake and its transgenic line PP2C9TL overexpressed protein phosphatase gene (PP2C9) were used for comparative proteomics analysis at control and low level of N to identify specific proteins and encoding genes related to high NUE. 2D gel electrophoresis was used to perform the differential proteome analysis. In the leaf proteome, 30 protein spots were differentially expressed between the two isogenic lines under low N level which were involved in the process of energy, photosynthesis, N metabolism, signaling, and defense mechanisms. In addition, we have found that protein phosphatase enhances nitrate reductase activation by downregulation of SnRK1 and 14-3-3 proteins. Furthermore, we showed that PP2C9TL exhibits higher NUE than WT due to higher activity of nitrate reductase. This study provides new insights on the rice proteome which would be useful in the development of new strategies to increase NUE in cereal crops.

Overexpression of Lsi1 in cold-sensitive rice mediates transcriptional regulatory networks and enhances resistance to chilling stress.

Frequent cold spells in late spring can damage early rice seedlings. However, overexpression of the silicon-uptake gene Lsi1 (Lsi1-OX) in cold-sensitive rice (Oryza sativa L., accession: Dular) notably enhances its chilling resistance. In this study, we found that continual chilling led to chlorophyll and RNA degradation in wild-type Dular leaves, whereas leaves from a Lsi1-OX line exhibited no obvious changes. A comparison of the global mRNA expression between the two rice lines showed that genes encoding photosynthesis-antenna proteins were downregulated and those encoding the proteasome were upregulated in the wild-type organism. Moreover, the differential responses of the two rice lines to chilling stress were found to correlate with the transcription factor OsWRKY53, which was predicted target of the respective microRNA (miRNA) novel-m0586-5p. In addition, miRNAs that targeted genes involved in the process of reactive oxygen species (ROS) metabolism were differentially expressed in the two rice lines after chilling stress, when comparative analysis of the outcomes of RNA sequencing on the two rice lines. Our results suggest that when overexpressed Lsi1 in cold-sensitive rice, it possibility regulates the transcription factor OsWRKY53 in addition to the genes involved in the ROS metabolism, thus mediating resistance to chilling stress.

Methyl-CpG binding domain protein acts to regulate the repair of cyclobutane pyrimidine dimers on rice DNA.

UVB radiation causes cyclobutane pyrimidine dimers (CPDs) to form on the DNA of living organisms. This study found that overexpression of the silicon absorbance gene Lsi1 reduced the accumulation of CPDs in rice, which profited from the reactivation by photolyase. The transcript abundance of deoxyribodipyrimidine photolyase (Os10g0167600) was generally correlated with the silicon content of the rice, and the up-regulation of Os10g0167600 was found to be highest in the UVB-treated Lsi1-overexpressed (Lsi1-OX) rice. A trans-acting factor, methyl-CpG binding domain protein (OsMeCP), was found to interact with the cis-element of Os10g0167600. The nucleic location of OsMeCP effectively enabled the transcriptional regulation. Compared with the WT, the level of OsMeCP was lower in the Lsi1-OX rice but higher in the Lsi1-RNAi line. Rice cultured in a high silicate-concentration solution also exhibited less OsMeCP abundance. Overexpression of OsMeCP led to lower Os10g0167600 transcript levels and a higher CPD content than in the WT, but the reverse was true in the OsMeCP-RNAi line. These findings indicate that OsMeCP acts as a negative regulator of silicon, and can mediate the repression of the transcription from Os10g0167600, which inhibits the photoreactivation of the photolyase involved in the repair of CPDs.

Mixed Phenolic Acids Mediated Proliferation of Pathogens Talaromyces helicus and Kosakonia sacchari in Continuously Monocultured Radix pseudostellariae Rhizosphere Soil.

Radix pseudostellariae L. is a common and popular Chinese medication. However, continuous monoculture has increased its susceptibility to severe diseases. We identified two pathogenic microorganisms, Talaromyces helicus M. (KU355274) and Kosakonia sacchari W. (KU324465), and their antagonistic bacterium, Bacillus pumilus Z. in rhizosphere soil of continuously monocultured R. pseudostellariae. Nine types of phenolic acids were identified both in the rhizosphere soil and in culture medium under sterile conditions. A syringic acid and phenolic acid mixture significantly promoted the growth of T. helicus and K. sacchari. T. helicus could utilize eight types of phenolic acids, whereas K. sacchari could only use four phenolic acids. K. sacchari produced protocatechuic acid when consuming vanillin. Protocatechuic acid negatively affected the growth of B. pumilus. The 3A-DON toxin produced by T. helicus promoted the growth of K. sacchari and inhibited growth of B. pumilus at low concentrations. These data help explain why phenolic exudates mediate a microflora shift and structure disorder in the rhizosphere soil of continuously monocultured R. pseudostellariae and lead to increased replanting disease incidence.

Soil Microbial Community Structure and Metabolic Activity of Pinus elliottii Plantations across Different Stand Ages in a Subtropical Area.

Soil microbes play an essential role in the forest ecosystem as an active component. This study examined the hypothesis that soil microbial community structure and metabolic activity would vary with the increasing stand ages in long-term pure plantations of Pinus elliottii. The phospholipid fatty acids (PLFA) combined with community level physiological profiles (CLPP) method was used to assess these characteristics in the rhizospheric soils of P. elliottii. We found that the soil microbial communities were significantly different among different stand ages of P. elliottii plantations. The PLFA analysis indicated that the bacterial biomass was higher than the actinomycic and fungal biomass in all stand ages. However, the bacterial biomass decreased with the increasing stand ages, while the fungal biomass increased. The four maximum biomarker concentrations in rhizospheric soils of P. elliottii for all stand ages were 18:1ω9c, 16:1ω7c, 18:3ω6c (6,9,12) and cy19:0, representing measures of fungal and gram negative bacterial biomass. In addition, CLPP analysis revealed that the utilization rate of amino acids, polymers, phenolic acids, and carbohydrates of soil microbial community gradually decreased with increasing stand ages, though this pattern was not observed for carboxylic acids and amines. Microbial community diversity, as determined by the Simpson index, Shannon-Wiener index, Richness index and McIntosh index, significantly decreased as stand age increased. Overall, both the PLFA and CLPP illustrated that the long-term pure plantation pattern exacerbated the microecological imbalance previously described in the rhizospheric soils of P. elliottii, and markedly decreased the soil microbial community diversity and metabolic activity. Based on the correlation analysis, we concluded that the soil nutrient and C/N ratio most significantly contributed to the variation of soil microbial community structure and metabolic activity in different stand ages of P. elliottii plantations.

Terminal Restriction Fragment Length Polymorphism Analysis of Soil Bacterial Communities under Different Vegetation Types in Subtropical Area.

Soil microbes are active players in energy flow and material exchange of the forest ecosystems, but the research on the relationship between the microbial diversity and the vegetation types is less conducted, especially in the subtropical area of China. In this present study, the rhizosphere soils of evergreen broad-leaf forest (EBF), coniferous forest (CF), subalpine dwarf forest (SDF) and alpine meadow (AM) were chosen as test sites. Terminal-restriction fragment length polymorphisms (T-RFLP) analysis was used to detect the composition and diversity of soil bacterial communities under different vegetation types in the National Natural Reserve of Wuyi Mountains. Our results revealed distinct differences in soil microbial composition under different vegetation types. Total 73 microbes were identified in soil samples of the four vegetation types, and 56, 49, 46 and 36 clones were obtained from the soils of EBF, CF, SDF and AM, respectively, and subsequently sequenced. The Actinobacteria, Fusobacterium, Bacteroidetes and Proteobacteria were the most predominant in all soil samples. The order of Shannon-Wiener index (H) of all soil samples was in the order of EBF>CF>SDF>AM, whereas bacterial species richness as estimated by four restriction enzymes indicated no significant difference. Principal component analysis (PCA) revealed that the soil bacterial communities' structures of EBF, CF, SDF and AM were clearly separated along the first and second principal components, which explained 62.17% and 31.58% of the total variance, respectively. The soil physical-chemical properties such as total organic carbon (TOC), total nitrogen (TN), total phosphorus (TP) and total potassium (TK) were positively correlated with the diversity of bacterial communities.

Interaction of Pseudostellaria heterophylla with Fusarium oxysporum f.sp. heterophylla mediated by its root exudates in a consecutive monoculture system.

In this study, quantitative real-time PCR (qPCR) was used to determine the amount of Fusarium oxysporum, an important replant disease pathogen in Pseudostellaria heterophylla rhizospheric soil. Moreover, HPLC was used to identify phenolic acids in root exudates then it was further to explore the effects of the phenolic acid allelochemicals on the growth of F. oxysporum f.sp. heterophylla. The amount of F. oxysporum increased significantly in P. heterophylla rhizosphere soil under a consecutive replant system as monitored through qPCR analysis. Furthermore, the growth of F. oxysporum f.sp. heterophylla mycelium was enhanced by root exudates with a maximum increase of 23.8%. In addition, the number of spores increased to a maximum of 12.5-fold. Some phenolic acids promoted the growth of F. oxysporum f.sp. heterophylla mycelium and spore production. Our study revealed that phenolic acids in the root secretion of P. heterophylla increased long with its development, which was closely related to changes in rhizospheric microorganisms. The population of pathogenic microorganisms such as F. oxysporum in the rhizosphere soil of P. heterophylla also sharply increased. Our results on plant-microbe communication will help to better clarify the cause of problems associated with P. heterophylla under consecutive monoculture treatment.

Identification and comparative analysis of microRNAs in barnyardgrass (Echinochloa crus-galli) in response to rice allelopathy.

Rice allelopathy is a hot topic in the field of allelopathy, and behaviour of donor allelopathic rice has been well documented. However, few study addresses response of receiver barnyardgrass (BYG). We found that expression of miRNAs relevant to plant hormone signal transduction, nucleotide excision repair and the peroxisome proliferator-activated receptor and p53 signalling pathways was enhanced in BYG co-cultured with the allelopathic rice cultivar PI312777, the expression levels of these miRNAs in BYG plants were positively correlated with allelopathic potential of the co-cultured rice varieties. Treatment of BYG plants with rice-produced phenolic acids also increased miRNA expression in BYG, while treatment with rice-produced terpenoids had no obvious effect on miRNA expression. In the hydroponic system, the largest number of Myxococcus sp. was found in the growth medium containing rice with the highest allelopathic potential. The addition of phenolic acids in the hydroponic medium also increased the number of Myxococcus sp. More interestingly, inoculation with Myxococcus xanthus significantly increased miRNA expression in the treated BYG. Jointed treatments of ferulic acid and M. xanthus led to strongest growth inhibition of BYG. The results suggest that there exist involvement of Myxococcus sp. and mediation of miRNA expression in rice allelopathy against BYG.

Method for RNA extraction and cDNA library construction from microbes in crop rhizosphere soil.

Techniques to analyze the transcriptome of the soil rhizosphere are essential to reveal the interactions and communications between plants and microorganisms in the soil ecosystem. In this study, different volumes of Al2(SO4)3 were added to rhizosphere soil samples to precipitate humic substances, which interfere with most procedures of RNA and DNA analyses. After humic substances were precipitated, cells of soil microorganisms were broken by vortexing with glass beads, and then DNA and RNA were recovered using Tris-HCl buffer with LiCl, SDS, and EDTA. The crude extract was precipitated and dissolved in RNAse-free water, and then separated by agarose gel electrophoresis. We determined the optimum volume of Al2(SO4)3 for treating rhizosphere soil of rice, tobacco, sugarcane, Rehmannia glutinosa, and Pseudostellaria heterophylla. The crude nucleic acids extract from rice soil was treated with DNase I and then RNA was purified using a gel filtration column. The purified RNA was reverse-transcribed into single-strand cDNA and then ligated with an adaptor at each end before amplifying ds cDNA. The ds cDNA was sub-cloned for subsequent gene sequence analysis. We conducted qPCR to amplify 16S ribosomal DNA and observed highly efficient amplification. These results show that the extraction method can be optimized to isolate and obtain high-quality nucleic acids from microbes in different rhizosphere soils, suitable for genomic and post-genomic analyses.

Changes in rice allelopathy and rhizosphere microflora by inhibiting rice phenylalanine ammonia-lyase gene expression.

Gene expression of phenylalanine ammonia-lyase (PAL) in allelopathic rice PI312777 was inhibited by RNA interference (RNAi). Transgenic rice showed lower levels of PAL gene expression and PAL activity than wild type rice (WT). The concentrations of phenolic compounds were lower in the root tissues and root exudates of transgenic rice than in those of wild type plants. When barndyardgrass (BYG) was used as the receiver plant, the allelopathic potential of transgenic rice was reduced. The sizes of the bacterial and fungal populations in rice rhizospheric soil at the 3-, 5-, and 7-leaf stages were estimated by using quantitative PCR (qPCR), which showed a decrease in both populations at all stages of leaf development analyzed. However, PI312777 had a larger microbial population than transgenic rice. In addition, in T-RFLP studies, 14 different groups of bacteria were detected in WT and only 6 were detected in transgenic rice. This indicates that there was less rhizospheric bacterial diversity associated with transgenic rice than with WT. These findings collectively suggest that PAL functions as a positive regulator of rice allelopathic potential.