Changxun Fang

Characterization of metaproteomics in crop rhizospheric soil.

Soil rhizospheric metaproteomics is a powerful scientific tool to uncover the interactions between plants and microorganisms in the soil ecosystem. The present study established an extraction method suitable for different soils that could increase the extracted protein content. Close to 1000 separate spots with high reproducibility could be identified in the stained 2-DE gels. Among the spots, 189 spots representing 122 proteins on a 2-DE gel of rice soil samples were successfully identified by MALDI-TOF/TOF-MS. These proteins mainly originated from rice and microorganisms. They were involved in protein, energy, nucleotide, and secondary metabolisms, as well as signal transduction and resistance. Three characteristics of the crop rhizospheric metaproteomics seemed apparent: (1) approximately one-third of the protein spots could not be identified by MALDI-TOF/TOF/MS, (2) the conservative proteins from plants formed a feature distribution of crop rhizospheric metaproteome, and (3) there were very complex interactions between plants and microorganisms existing in a crop rhizospheric soil. Further functional analysis on the identified proteins unveiled various metabolic pathways and signal transductions involved in the soil biotic community. This study provides a paradigm for metaproteomic research on soil biology.

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.

Allelopathic enhancement and differential gene expression in rice under low nitrogen treatment.

The allelopathy-competition separation (ACS) based approach was used to explore the biointerference relationship between rice accessions and barnyardgrass exposed to different nitrogen (N) supplies in hydroponics. Rice accession PI312777 exhibited high allelopathic potential to suppress the growth of accompanying weeds, especially when the culture solution had low N content. The non-allelopathic rice Lemont showed an opposite result. Additionally, subtractive hybridization suppression (SSH) was used to construct a forward SSH-cDNA library of PI312777 to investigate gene expression profiles under low N treatment. A total of 35 positive clones from the SSH-cDNA library were sequenced and annotated. According to the function category, 24 genes were classified into five groups related to primary metabolism, phenolic allelochemical synthesis, plant growth/cell cycle regulation, stress response/signal transduction, and protein synthesis/degradation. Among them, two up-regulated genes that encode PAL and cytochrome P450 were selected. Their transcript abundance at low N level was compared further between the allelopathic rice and its counterpart by utilizing real-time quantitative polymerase chain reaction (qRT-PCR). The transcription levels of the two genes increased in both rice accessions when exposed to low N supply, but PI312777 at a higher magnitude than Lemont. At 1, 3, and 7 days of the treatments, the corresponding relative expression levels of PAL were 11.38, 4.83, and 3.57 fold higher in PI312777 root, but there were 1.15, 2.74, and 2.94 fold increases for Lemont, compared with the control plants fed with regular nutrient. The same trend was found for cytochrome P450. These findings suggest that the stronger ability of PI312777 to suppress target weeds, especially in low N nutrient conditions, might be attributed to the stronger activation of the genes that function in de novo synthesis of allelochemicals.

Analysis of gene expressions associated with increased allelopathy in rice (Oryza sativa L.) induced by exogenous salicylic acid.

The defense characteristics of allelopathic rice accession PI312777 and its counterpart Lemont induced by exogenous salicylic acid (SA) to suppress troublesome weed barnyardgrass (BYG) were investigated using the methods of suppression subtractive hybridization (SSH) and real-time fluorescence quantitative PCR (qRT-PCR). The results showed that exogenous SA could induce the allelopathic effect of rice on BYG and this inducible defense was SA dose-respondent and treatment time-dependent. PI312777 exhibited higher inhibitory effect than Lemont on BYG after treated with different concentrations of SA. The activities of cell protective enzymes including SOD, POD and CAT in the BYG plants co-cultured with PI312777 treated by SA were highly depressed compared with the control (co-cultured with rice without SA-treatment). Similar but lower depression on these enzymes except for CAT was also observed in the BYG plants when co-cultured with Lemont treated by SA. It is therefore suggested that allelopathic rice should be more sensitive than non-allelopathic rice to exogenous SA. Seventeen genes induced by SA were obtained by SSH analysis from PI312777. These genes encode receptor-kinase proteins, ubiquitin carrier proteins, proteins related to phenylpropanoid metabolism, antioxidant related proteins and some growth-mediating proteins. The differential expressions of these genes were validated in part by qRT-PCR in the two rice accessions. Our work elucidated that allelopathic rice possesses an active chemical defense and auto-detoxifying enzyme system such as the up-regulated enzymes involved in de novo biosynthesis of phenolic allelochemicals and the glutathione-S-transferase (GST) associated with xenobiotic detoxification.

Proteomic and phosphoproteomic determination of ABA's effects on grain-filling of Oryza sativa L. inferior spikelets

Cultivars of rice (Oryza sativa L.), especially the large-spikelet-type, often fail to achieve the high yield potential due to poor grain-filling of their inferior (late-flowering) spikelets. The superior (early-flowering) spikelets normally contain more abscisic acid (ABA) than the inferior spikelets. It was speculated that ABA might play a pivotal role in the grain-filling of inferior spikelets. To understand the molecular regulation involved in this process, we employed the 2-D gel-based comparative proteomic and phosphoproteomic analyses to search for differentially expressed proteins in the inferior spikelets under exogenous ABA treatment. A total of 111 significantly differential proteins and 31 phosphoproteins were found in the inferior spikelets after treatment. Among them, 100 proteins and 23 phosphoproteins were identified by using MALDI-TOF/TOF MS. In addition, the gene expression patterns of the inferior spikelets were confirmed with RT-PCR. These differentially expressed proteins are active in defense response, carbohydrate, protein, amino acid, energy and secondary metabolisms, as well as cell development and photosynthesis. The results suggest that the grain-filling of rice inferior spikelets is regulated by ABA through some proteins and phosphoproteins participating in carbon, nitrogen and energy metabolisms.

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.

Barnyard grass stress up regulates the biosynthesis of phenolic compounds in allelopathic rice.

Allelopathic rice cultivar PI312777 (PI) and non-allelopathic rice cultivar Lemont (Le) were mixed with barnyard grass (Echinochloa crus-galli L., BYG) at various ratios (rice:weed ratios of 4:1, 2:1, and 1:1) in hydroponic cultures. The expression of four genes, i.e. phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), ferulic acid 5-hydroxylase (F5H), and caffeic acid O-methyltransferases (COMT), which are involved in the biosynthesis of the phenolic compounds in rice, were evaluated by a quantitative real-time polymerase chain reaction (qRT-PCR). The contents of phenolic compounds in leaves, roots, and culture solutions of the two rice cultivars were determined using high performance liquid chromatography (HPLC). The results showed that all of the four genes were up-regulated in leaves and roots of the allelopathic rice PI at all rice:weed ratios. However, three of the four genes, C4H, F5H, and COMT, were down-regulated in the leaves and roots of the non-allelopathic rice Le. The degree to which PAL was up-regulated in leaves and roots was much higher in PI than in Le. The contents of phenolic compounds in PI leaves, roots, and culture solutions were higher than that in Le leaves, roots, and culture solutions. The higher expression of the genes involved in the phenylpropanoid metabolism and the higher contents of phenolic compounds in PI are consistent with the higher inhibitory rates of PI on BYG. These results indicate that the PAL gene in PI is more sensitive to BYG stress than in Le, and barnyard grass up regulates the biosynthesis of phenolic compound in allelopathic rice.

Genomic analysis of allelopathic response to low nitrogen and barnyardgrass competition in rice ( Oryza sativa L.)

To explore the molecular mechanism of allelopathic rice in response to low nitrogen (N) supply or accompanying weed stress, allelopathic rice PI 312777 and its counterpart Lemont were grown under low N supply or co-cultured with barnyardgrass [Echinochloa crus-galli (L.) Beauv.] in hydroponics. The suppression subtractive hybridization (SSH) technique was employed to isolate the up-regulated genes in the treated rice accession. The results indicated that the expression of the genes associated with N utilization was significantly up-regulated in allelopathic rice PI 312777, and the higher efficiency of N uptake and its utilization were also detected in PI 312777 than that in Lemont when the two rice accessions were exposed to low N supply. This result suggested that the allelopathic rice had higher ability to adapt to low N stress than its non-allelopathic counterpart. However, a different response was observed when the allelopathic rice was exposed to accompanying weed (barnyardgrass) co-cultured in full Hoagland solution (normal N supply). It showed that the expression of the genes associated with allelochemical synthesis and its detoxification were all up-regulated in the allelopathic rice when co-cultured with the target weed under normal N supply. The results suggested that the allelopathic rice should be a better competitor in the rice-weed co-culture system, which could be attributed to increasing de novo biosynthesis and detoxification of allelochemicals in rice, consequently resulting in enhanced allelopathic effect on the target and preventing the autotoxicity in this process. These findings suggested that the accompanying weed, barnyardgrass is not only the stressful factor, but also one of the triggers in activating allelopathy in rice. This implies that the allelopathic rice is sensible of the existing target in chemical communication.

Suppression and overexpression of Lsi1 induce differential gene expression in rice under ultraviolet radiation

Low silicon rice gene 1 (Lsi1) belongs to a member of Nod26-like major intrinsic protein (NIP) subfamily and is thought to control silicon (Si) accumulation in rice. In order to further elucidate its regulatory mechanisms in the defense of rice plants to enhanced ultraviolet-B (UV-B) radiation stress, Lsi1 was subjected to suppressed and overexpressed treatments in a UV-B tolerance rice accession Lemont as well as to overexpressed treatment in a UV-B sensitive rice accession Dular. The results showed that transcript levels of Lsi1 increased in Lsi1-overexpressed transgenic lines of rice accessions Lemont and Dular, but down-regulated in Lsi1-RNAi transgenic line of Lemont in comparison with their wild types (WT). A similar tendency was found in the root Si uptake and the Si concentrations in leaves. Further, the different transgenic rice lines and their WT were exposed to enhance UV-B radiation, study by suppression subtractive hybridization (SSH) found that Lsi1 not only could regulate the expression of phenylalanine ammonia-lyase (PAL), 4-coumarate-CoA ligase (4CL) and photolyase (PL), but also could induce other signal transduction-, detoxification-, resistance-, and photosynthesis-related genes. The findings suggested that the regulation of silicon nutrient by enhancing/inhibiting expression of Lsi1 could effectively induce the transcription of the mRNAs relative to tolerance in rice.

Cadmium-stress mitigation through gene expression of rice and silicon addition

Cadmium (Cd) pollution is one of the major concerns in the development of sustainable agriculture, particularly for rice production. Silicon (Si) was recently recognized for its ability to mitigate a variety of abiotic stresses including that caused by Cd on rice. However, mechanism of the complex process is still not fully understood. Under Cd-stress, the low Si-influx 1-RNAi transgenic Lemont rice (Lsi1-RNAi line) exhibited an increased Cd-uptake over its counterparts, the wild type and the Lsi1-overexpression transgenic rice (Lsi1-OE line). In contrast, the Lsi1 expression-enhanced Lsi1-OE line showed the greatest Si-uptake among the three lines, with the highest activities on anti-oxidants (such as, superoxide dismutase) and the lowest content of malondialdehyde. Lsi1 also displayed a negative regulation on Low Cd gene and the natural resistance-associated macrophage proteins, Nramp5, indicating its capacity to alleviate Cd-stress on rice. The results obtained by this study suggested that the mitigation of Cd-toxicity on rice by Si might involve functions, such as the inhibition on Cd-uptake and transport and the enhancement on anti-oxidative enzyme activities, as well as the Lsi1-related expression on regulation of Si-uptake in rice. A new avenue might become available for overcoming the rampant pollution that threatens the rice production in China.