Dual-transgenic BiFC vector systems for protein-protein interaction analysis in plants.

Protein-protein interaction (PPI) play a pivotal role in cellular signal transduction. The bimolecular fluorescence complementation (BiFC) assay offers a rapid and intuitive means to ascertain the localization and interactions of target proteins within living cells. BiFC is based on fluorescence complementation by reconstitution of a functional fluorescent protein by co-expression of N- and C-terminal fragments of this protein. When fusion proteins interact, the N- and C-terminal fragments come into close proximity, leading to the reconstitution of the fluorescent protein. In the conventional approach, the N-terminal and C-terminal fragments of the fluorescent protein are typically expressed using two separate vectors, which largely relies on the efficiency of the transformation of the two vectors in the same cells. Furthermore, issues of vector incompatibility can often result in loss of one plasmid. To address these challenges, we have developed novel dual-transgenic BiFC vectors, designed as pDTQs, derived from the previously published pDT1 vector. This set of BiFC vectors offers the following advantages: 1) Both fluorescent fusion proteins are expressed sequentially within a single vector, enhancing expression efficiency; 2) Independent promoters and terminators regulate the expression of the two proteins potentially mitigating vector compatibility issues; 3) A long linker is inserted between the fluorescent protein fragment and the gene of interest, facilitating the recombination of the fused fluorescent protein into an active form; 4) Four distinct types of fluorescent proteins, namely, EYFP, mVenus, mRFP1Q66T and mCherry are available for BiFC analysis. We assessed the efficiency of the pDTQs system by investigating the oligomerization of Arabidopsis CRY2 and CRY2-BIC2 interactions in N. benthamiana. Notably, the pDTQs were found to be applicable in rice, underscoring their potential utility across various plant species.

H3K27 demethylase SsJMJ4 negatively regulates drought-stress responses in sugarcane.

Sugarcane (Saccharum spp.), a leading sugar and energy crop, is seriously impacted by drought stress. However, the molecular mechanisms underlying sugarcane drought resistance, especially the functions of epigenetic regulators, remain elusive. Here, we show that a S. spontaneum KDM4/JHDM3 group JmjC protein, SsJMJ4, negatively regulates drought-stress responses through its H3K27me3 demethylase activity. Ectopic overexpression of SsJMJ4 in Arabidopsis reduced drought resistance possibly by promoting expression of AtWRKY54 and AtWRKY70, encoding two negative regulators of drought stress. SsJMJ4 directly bound to AtWRKY54 and AtWRKY70, and reduced H3K27me3 levels at these loci to ensure their proper transcription under normal conditions. Drought stress down-regulated both transcription and protein abundance of SsJMJ4, which was correlated with the reduced occupancy of SsJMJ4 at AtWRKY54 and AtWRKY70 chromatin, increased H3K27me3 levels at these loci, as well as reduced transcription levels of these genes. In S. spontaneum, drought stress-repressed transcription of SsWRKY122, an ortholog of AtWRKY54 and AtWRKY70, was associated with increased H3K27me3 levels at these loci. Transient overexpression of SsJMJ4 in S. spontaneum protoplasts raised transcription of SsWRKY122, paralleled with reduced H3K27me3 levels at its loci. These results suggest that the SsJMJ4-mediated dynamic deposition of H3K27me3 is required for an appropriate response to drought stress.

[Research advances in histone modification-regulated plant stress memory and defense priming.] / ç»„è›‹ç™½ä¿®é¥°è°ƒæŽ§æ¤ç‰©å¯¹èƒè¿«çš„è®°å¿†ä¸Žè­¦å¤‡æŠ—æ€§çš„ç ”ç©¶è¿›å±•.

Plants, grown in the immobile soils, have evolved various strategies in response to environmental stresses, including the "stress memory" and "defense priming" mechanisms. The environmental stresses cannot immediately change the DNA base sequence in plants in the short-term. Therefore, epigenetic inheritance is a key mechanism for stress memory and defense priming. In particular, histone modification is considered to be the most important mechanism, which offers the possibility of stress memory. We summarized research advances in plant histone modifications involved in stress memory and defense priming under biotic and abiotic stresses, and proposed pro-blems in the field and the focus and directions in the future research. In-depth understanding of the relationship between histone modification and environmental stresses would facilitate the quick adaptation of plants to harsh environments, and provide theoretical and technical guidance for plant phenotype shaping, organ regeneration, and crop genetic improvement.

JMJ27-mediated histone H3K9 demethylation positively regulates drought-stress responses in Arabidopsis.

Dimethylation of histone H3 at lysine 9 (H3K9me2) is associated with heterochromatinization and transcriptional gene silencing in plants. The activation of sets of genes by drought stress is correlated with reduced H3K9me2 levels, but the role of H3K9 methylation in the regulation of drought-stress responses remains elusive. Here, we show that the Jumonji domain-containing H3K9 demethylase JMJ27 positively regulates drought-stress responses through its histone demethylase activity. RNA-seq analysis identified JMJ27-regulated genes, including positive regulators of drought stress GALACTINOL SYNTHASE 2 (GOLS2) and RESPONSE TO DESICCATION 20 (RD20). Genetic analysis demonstrated that JMJ27 positively regulates drought-stress responses at least partly through GOLS2 and RD20. JMJ27 directly associated with GOLS2 and RD20, and protected these loci from silencing by reducing H3K9me2 levels under normal conditions. REGULATORY PARTICLE NON-ATPASE 1a (RPN1a), a subunit of the 26S proteasome, interacted with JMJ27 and negatively regulated JMJ27 accumulation. Drought stress diminished RPN1a abundance, resulting in increased JMJ27 abundance. The drought stress-promoted occupancy of JMJ27 at GOLS2 and RD20 chromatin may reinforce their transcriptional induction by locally reducing the H3K9me2 levels. These results indicate that the RPN1a-JMJ27 module precisely regulates dynamic H3K9me2 deposition plasticity, ensuring proper adaptation to drought stress in Arabidopsis.

Genome-Wide Characterization of DNase I-Hypersensitive Sites and Cold Response Regulatory Landscapes in Grasses.

Deep sequencing of DNase-I treated chromatin (DNase-seq) can be used to identify DNase I-hypersensitive sites (DHSs) and facilitates genome-scale mining of de novo cis-regulatory DNA elements. Here, we adapted DNase-seq to generate genome-wide maps of DHSs using control and cold-treated leaf, stem, and root tissues of three widely studied grass species: Brachypodium distachyon, foxtail millet (Setaria italica), and sorghum (Sorghum bicolor). Functional validation demonstrated that 12 of 15 DHSs drove reporter gene expression in transiently transgenic B. distachyon protoplasts. DHSs under both normal and cold treatment substantially differed among tissues and species. Intriguingly, the putative DHS-derived transcription factors (TFs) are largely colocated among tissues and species and include 17 ubiquitous motifs covering all grass taxa and all tissues examined in this study. This feature allowed us to reconstruct a regulatory network that responds to cold stress. Ethylene-responsive TFs SHINE3, ERF2, and ERF9 occurred frequently in cold feedback loops in the tissues examined, pointing to their possible roles in the regulatory network. Overall, we provide experimental annotation of 322,713 DHSs and 93 derived cold-response TF binding motifs in multiple grasses, which could serve as a valuable resource for elucidating the transcriptional networks that function in the cold-stress response and other physiological processes.

Intraspecific variations in cadmium tolerance and phytoaccumulation in giant duckweed (Spirodela polyrhiza).

Duckweeds are widely recognized for the heavy metal phytoremediation. However, the intraspecific variations in biological responses of duckweeds to heavy metal remain largely unknown. Here, the toxicity and phytoaccumulation of cadmium (Cd) were synchronously evaluated in 30 accessions of giant duckweed (Spirodela polyrhiza) collected from different provenances in Southern China. Exposure to 1 µM Cd decreased relative growth rates of dry weight, fronds number and fronds area, as well as photosynthetic pigment contents, while it increased H2O2 accumulation, lipid peroxidation and activities of anti-oxidant enzymes in the majority of accessions. Cd treatment led to remarkable Cd accumulation but little changes in the starch content in giant duckweed. The biological responses to Cd varied among the accessions. Further correlation analysis indicated that growth traits and Cd concentration were positively correlated with Cd accumulation, while the contents of chlorophyll, H2O2 and MDA were negatively associated with Cd accumulation. Our results proved the great intraspecific variation in Cd tolerance of giant duckweed, suggesting a valuable natural resource for Cd phytoremediation. Moreover, different mechanisms may be exploited by S. polyrhiza for phytoaccumulation, but growth maintenance, Cd uptake and antioxidative enzyme-independent ROS-scavenging under Cd exposure are the common mechanisms contributing to Cd accumulation ability.

Optimization of Mature Embryo-Based Tissue Culture and Agrobacterium-Mediated Transformation in Model Grass Brachypodium distachyon.

Agrobacterium-mediated genetic transformation is well established in the model grass Brachypodium distachyon. However, most protocols employ immature embryos because of their better regenerative capacity. A major problem associated with the immature embryo system is that they are available only during a limited time window of growing plants. In this study, we have developed an optimized Agrobacterium-mediated genetic transformation protocol that utilizes mature embryos. We have adopted seed shearing and photoautotrophic rooting (PR) in callus induction and root regeneration, respectively, with evident significant improvement in these aspects. We have also revealed that the newly developed chemical inducer Fipexide (FPX) had the ability to induce callus, shoots, and roots. By comparison, we have demonstrated that FPX shows higher efficiency in shoot generation than other frequently used chemicals in our mature embryo-based system. In addition, we demonstrated that the age of embryogenetic callus severely affects the transformation efficiency (TE), with the seven-week-old embryogenetic callus having the highest TE reaching 52.6%, which is comparable with that in immature embryo transformation. The new methodologies reported here will advance the development and utilization of Brachypodium as a new model system for grass genomics.

Overexpression of the trehalose-6-phosphate phosphatase family gene AtTPPF improves the drought tolerance of Arabidopsis thaliana.

BACKGROUND: Trehalose-6-phosphate phosphatases (TPPs), which are encoded by members of the TPP gene family, can improve the drought tolerance of plants. However, the molecular mechanisms underlying the dynamic regulation of TPP genes during drought stress remain unclear. In this study, we explored the function of an Arabidopsis TPP gene by conducting comparative analyses of a loss-of-function mutant and overexpression lines. RESULTS: The loss-of-function mutation of Arabidopsis thaliana TPPF, a member of the TPP gene family, resulted in a drought-sensitive phenotype, while a line overexpressing TPPF showed significantly increased drought tolerance and trehalose accumulation. Compared with wild-type plants, tppf1 mutants accumulated more H2O2 under drought, while AtTPPF-overexpressing plants accumulated less H2O2 under drought. Overexpression of AtTPPF led to increased contents of trehalose, sucrose, and total soluble sugars under drought conditions; these compounds may play a role in scavenging reactive oxygen species. Yeast one-hybrid and luciferase activity assays revealed that DREB1A could bind to the DRE/CRT element within the AtTPPF promoter and activate the expression of AtTPPF. A transcriptome analysis of the TPPF-overexpressing plants revealed that the expression levels of drought-repressed genes involved in electron transport activity and cell wall modification were upregulated, while those of stress-related transcription factors related to water deprivation were downregulated. These results indicate that, as well as its involvement in regulating trehalose and soluble sugars, AtTPPF is involved in regulating the transcription of stress-responsive genes. CONCLUSION: AtTPPF functions in regulating levels of trehalose, reactive oxygen species, and sucrose levels during drought stress, and the expression of AtTPPF is activated by DREB1A in Arabidopsis. These findings shed light on the molecular mechanism by which AtTPPF regulates the response to drought stress.

Changes in distribution and microstructure of bauxite residue aggregates following amendments addition.

Bauxite residue is a highly alkaline byproduct which is routinely discarded at residue disposal areas. Improving soil formation process to revegetate the special degraded lands is a promising strategy for sustainable management of the refining industry. A laboratory incubation experiment was used to evaluate the effects of gypsum and vermicompost on stable aggregate formation of bauxite residue. Aggregate size distribution was quantified by fractal theory, whilst residue microstructure was determined by scanning electron microscopy and synchrotron-based X-ray micro-computed tomography. Amendments addition increased the content of macro-aggregates (>250 µm) and enhanced aggregate stability of bauxite residue. Following gypsum and vermicompost addition, fractal dimension decreased from 2.84 to 2.77, which indicated a more homogeneous distribution of aggregate particles. Images from scanning electron microscopy and three-dimensional microstructure demonstrated that amendments stimulate the formation of improved structure in residue aggregates. Pore parameters including porosity, pore throat surface area, path length, and path tortuosity increased under amendment additions. Changes in aggregate size distribution and microstructure of bauxite residue indicated that additions of gypsum and vermicompost were beneficial to physical condition of bauxite residue which may enhance the ease of vegetation.

[Risk Analysis of Heavy Metal Contamination in Farmland Soil Around a Bauxite Residue Disposal Area in Guangxi].

Bauxite residue disposal areas (BRDA) appear to result in the heavy metal pollution of the farm fields surrounding them. In total, 194 topsoil samples were collected from the fields surrounding a BRDA in Guangxi in order to comprehensively understand the pollutant characteristics. These characteristics and their ecological risks were assessed by the Nemerow and Harkanson indices, whilst the sources and correlations of eight heavy metals (V, Cr, Ni, Cu, Zn, As, Pb, and Co) were analyzed by means of the spatial interpolation method, correlation analysis, and principal component analysis (PCA). The results demonstrated that the surrounding fields were seriously polluted by heavy metals. Ninety-two percent of samples were polluted, including 36% that showed serious pollution, and As was the dominant contaminant. The ecological risk results showed that the risks of the surrounding fields were medium, and As was responsible for 68% of this. Spatial interpolation suggested that concentrations of heavy metals in the northeastward and southwestward areas were higher, however the southeastward areas were lower. Multivariate statistics indicated that the possible source of As contaminant was different to those of V, Ni, Zn, Pb, and Co; As was primarily influenced by anthropogenic contamination, including atmospheric sedimentation, and agricultural fertilization. Cr was affected by both soil parent material and atmospheric sedimentation, whereas V, Ni, Zn, Pb, And Co levels were mainly affected by soil parent material.

Arabidopsis small ubiquitin-related modifier protease ASP1 positively regulates abscisic acid signaling during early seedling development.

The small ubiquitin-related modifier (SUMO) modification plays an important role in the regulation of abscisic acid (ABA) signaling, but the function of the SUMO protease, in ABA signaling, remains largely unknown. Here, we show that the SUMO protease, ASP1 positively regulates ABA signaling. Mutations in ASP1 resulted in an ABA-insensitive phenotype, during early seedling development. Wild-type ASP1 successfully rescued, whereas an ASP1 mutant (C577S), defective in SUMO protease activity, failed to rescue, the ABA-insensitive phenotype of asp1-1. Expression of ABI5 and MYB30 target genes was attenuated in asp1-1 and our genetic analyses revealed that ASP1 may function upstream of ABI5 and MYB30. Interestingly, ASP1 accumulated upon ABA treatment, and ABA-induced accumulation of ABI5 (a positive regulator of ABA signaling) was abolished, whereas ABA-induced accumulation of MYB30 (a negative regulator of ABA signaling) was increased in asp1-1. These findings support the hypothesis that increased levels of ASP1, upon ABA treatment, tilt the balance between ABI5 and MYB30 towards ABI5-mediated ABA signaling.

Do aeration conditions affect arsenic and phosphate accumulation and phosphate transporter expression in rice (Oryza sativa L.)?

Widespread contamination of rice with arsenic (As) has revealed a major exposure pathway to humans. The present study aimed to investigate the effects of oxygen in the rhizosphere on phosphate (P) transporter (for arsenate transportation) expressions, on As and P accumulation and As speciation in four rice genotypes. Oxygenation marginally increased root and shoot length. Total As concentrations in rice roots were dramatically reduced following aeration compared to stagnant treatments (p < 0.001). Aeration treatments significantly increased arsenate while reducing arsenite concentrations in roots (p < 0.001). Root arsenite concentrations were 1.5-2.5 times greater in stagnant than in aeration treatments. Total P concentrations in rice roots were dramatically increased following aeration compared to stagnant treatments. The relative abundance of phosphate transporter (inorganic phosphate transporter and phosphate/H+ symporter family protein) expressions showed downregulation in aeration treatments, particularly for SY-9586, XWX-17, and XWX-12 in inorganic phosphate transporter expressions and XWX-17 in phosphate/H+ symporter family protein expression (p < 0.05). The relative abundance of phosphate carrier protein expressions were relatively higher than the other phosphate transporters, showing upregulation in aeration treatments.

Tobacco crop substitution: pilot effort in China.

In China, approximately 20 million farmers produce the world's largest share of tobacco. Showing that income from crop substitution can exceed that from tobacco growth is essential to persuading farm families to stop planting tobacco, grown abundantly in Yunnan Province. In the Yuxi Municipality, collaborators from the Yuxi Bureau of Agriculture and the University of California at Los Angeles School of Public Health initiated a tobacco crop substitution project. At 3 sites, 458 farm families volunteered to participate in a new, for-profit cooperative model. This project successfully identified an approach engaging farmers in cooperatives to substitute food crops for tobacco, thereby increasing farmers' annual income between 21% and 110% per acre.