BnXTH1 regulates cadmium tolerance by modulating vacuolar compartmentalization and the cadmium binding capacity of cell walls in ramie (Boehmeria nivea).

Xyloglucan endotransglucosylase/hydrolases (XTH) are cell wall-modifying enzymes important in plant response to abiotic stress. However, the role of XTH in cadmium (Cd) tolerance in ramie remains largely unknown. Here, we identified and cloned BnXTH1, a member of the XTH family, in response to Cd stress in ramie. The BnXTH1 promoter (BnXTH1p) demonstrated that MeJA induces the response of BnXTH1p to Cd stress. Moreover, overexpressing BnXTH1 in Boehmeria nivea increased Cd tolerance by significantly increasing the Cd content in the cell wall and decreasing Cd inside ramie cells. Cadmium stress induced BnXTH1-expression and consequently increased xyloglucan endotransglucosylase (XET) activity, leading to high xyloglucan contents and increased hemicellulose contents in ramie. The elevated hemicellulose content increased Cd chelation onto the cell walls and reduced the level of intracellular Cd. Interestingly, overexpressing BnXTH1 significantly increased the content of Cd in vacuoles of ramie and vacuolar compartmentalization genes. Altogether, these results evidence that Cd stress induced MeJA accumulation in ramie, thus, activating BnXTH1 expression and increasing the content of xyloglucan to enhance the hemicellulose binding capacity and increase Cd chelation onto cell walls. BnXTH1 also enhances the vacuolar Cd compartmentalization and reduces the level of Cd entering the organelles and soluble solution.

BnAP2-12 overexpression delays ramie flowering: evidence from AP2/ERF gene expression.

Introduction: The APETALA2/ethylene response factor (AP2/ERF) superfamily plays a significant role in regulating plant gene expression in response to growth and development. To date, there have been no studies into whether the ramie AP2/ERF genes are involved in the regulation of flower development. Methods: Here, 84 BnAP2/ERF members were identified from the ramie genome database, and various bioinformatics data on the AP2/ERF gene family, structure, replication, promoters and regulatory networks were analysed. BnAP2-12 was transferred into Arabidopsis through the flower-dipping method. Results: Phylogenetic analysis classified the 84 BnAP2/ERF members into four subfamilies: AP2 (18), RAV (3), ERF (42), and DREB (21). The functional domain analysis of genes revealed 10 conserved motifs. Genetic mapping localised the 84 members on 14 chromosomes, among which chromosomes 1, 3, 5, and 8 had more members. Collinearity analysis revealed that 43.37% possibly resulted from replication events during the evolution of the ramie genome. Promoter sequence analysis identified classified cis-acting elements associated with plant growth and development, and responses to stress, hormones, and light. Transcriptomic comparison identified 3,635 differentially expressed genes (DEGs) between male and female flowers (1,803 and 1,832 upregulated and downregulated genes, respectively). Kyoto Encyclopaedia of Genes and Genomes pathway analysis categorised DEGs involved in metabolic pathways and biosynthesis of secondary metabolites. Gene Ontology enrichment analysis further identified enriched genes associated with pollen and female gamete formations. Of the 84 BnAP2/ERFs genes, 22 and 8 upregulated and downregulated genes, respectively, were present in female flowers. Co-expression network analysis identified AP2/ERF members associated with flower development, including BnAP2-12. Subcellular localisation analysis showed that the BnAP2-12 protein is localised in the nucleus and cell membrane. Overexpression BnAP2-12 delayed the flowering time of Arabidopsis thaliana. Conclusion: These findings provide insights into the mechanism of ramie flower development.

Waterlogging increases greenhouse gas release and decreases yield in winter rapeseed (Brassica napus L.) seedlings.

A sustainable future depends on increasing agricultural carbon (C) and nitrogen (N) sequestration. Winter rapeseeds are facing severe yield loss after waterlogging due to the effects of extreme rainfall, especially in the seedling stage, where rainfall is most sensitive. Uncertainty exists over the farming greenhouse gas (GHG) release of rapeseed seedlings following the onset of waterlogging. The effect of waterlogging on GHG release and leaf gas exchange in winter rapeseed was examined in a pot experiment. The experiment included waterlogging treatments lasting 7-day and 21-day and normal irrigation as a control treatment. According to our findings, (1) The ecosystem of rapeseed seedlings released methane (CH4) and nitrous oxide (N2O) in a clear up change that was impacted by ongoing waterlogging. Among them, N2O release had a transient rise during the early stages under the effect of seedling fertilizer. (2) The net photosynthetic rate, transpiration rate, stomatal conductance, plant height, soil moisture, and soil oxidation-reduction potential of rapeseed all significantly decreased due to the ongoing waterlogging. However, rapeseed leaves showed a significant increase in intercellular carbon dioxide (CO2) concentration and leaf chlorophyll content values after waterlogging. Additionally, the findings demonstrated an extremely significant increase in the sustained-flux global warming potential of the sum CO2-eq of CH4 and N2O throughout the entire waterlogging stress period. Therefore, continuous waterlogging can increase C and N release from rapeseed seedlings ecosystem and decrease yield. Therefore, we suggest increasing drainage techniques to decrease the release of agricultural GHGs and promote sustainable crop production.

Exogenous plant growth regulator and foliar fertilizers for phytoextraction of cadmium with Boehmeria nivea [L.] Gaudich from contaminated field soil.

As a enrichment plant, ramie can be used for the phytoremediation of cadmium (Cd)-contaminated soil. However, it is worth exploring the role of plant growth regulators and foliar fertilizers in the process of plant growth and development and Cd adsorption. By measuring the agronomic traits, Cd content of aboveground and underground ramie, calculating the Cd transfer coefficient (TF) and Cd bioconcentration factors (BCF), and the correlation between various indicators. This study examined the effects of plant growth regulators and foliar fertilizers on ramie's capacity for Cd accumulation and transportation. Plant growth regulators and foliar fertilizers increased the Cd content of the aboveground ramie, reduced the Cd content of the underground ramie, and increased the TF. Among them, GA-1 increased the Cd content of the aboveground ramie to 3 times more than that of the control and reduced the Cd content of the underground ramie by 54.76%. Salicylic acid (SA) increased the Cd content of the aboveground ramie to three times more than that of the control. The combination of GA and foliar fertilizer reduced the Cd content of the aboveground and underground ramie and the TF and BCF of the underground ramie. After the hormones were sprayed, the TF of ramie had a significant positive correlation with the Cd content of the aboveground ramie; the BCF of the aboveground ramie had a significant positive correlation with the Cd content and TF of the aboveground ramie. The results indicate that Brassinolide (BR), gibberellin (GA), ethephon (ETH), polyamines (PAs), and salicylic acid (SA) have different effects on the enrichment and transport of Cd in ramie. This study provided an effective method to improve the capacity for ramie to adsorb heavy metals during cultivation.

Identification of the Xyloglucan Endotransglycosylase/Hydrolase (XTH) Gene Family Members Expressed in Boehmeria nivea in Response to Cadmium Stress.

Xyloglucan endotransglycosylase/hydrolase (XTH) genes play an important role in plant resistance to abiotic stress. However, systematic studies of the response of Boehmeria nivea (ramie) XTH genes (BnXTHs) to cadmium (Cd) stress are lacking. We sought to identify the BnXTH-family genes in ramie through bioinformatics analyses and to investigate their responses to Cd stress. We identified 19 members of the BnXTH gene family from the ramie genome, referred to as BnXTH1-19, among which BnXTH18 and BnXTH19 were located on no chromosomes and the remaining genes were unevenly distributed across 11 chromosomes. The 19 members were divided into four groups, Groups I/II/IIIA/IIIB, according to their phylogenetic relationships, and these groups were supported by analyses of intron-exon structure and conserved motif composition. A highly conserved catalytic site (HDEIDFEFLG) was observed in all BnXTH proteins. Additionally, three gene pairs (BnXTH6-BnXTH16, BnXTH8-BnXTH9, and BnXTH17-BnXTH18) were obtained with a fragment and tandem-repeat event analysis of the ramie genome. An analysis of cisregulatory elements revealed that BnXTH expression might be regulated by multiple hormones and abiotic and biotic stress responses. In particular, 17 cisregulatory elements related to abiotic and biotic stress responses and 11 cisregulatory elements related to hormone responses were identified. We also found that most BnXTH genes responded to Cd stress, and BnXTH1, BnXTH3, BnXTH6, and BnXTH15 were most likely to contribute to the Cd tolerance of ramie, as evidenced by the substantial increases in expression under Cd treatment. Heterologous expression of BnXTH1, BnXTH6, and BnXTH15 significantly enhanced the Cd tolerance of transgenic yeast cells. These results suggest that the BnXTH gene family is involved in Cd stress responses, laying a theoretical foundation for functional studies of BnXTH genes and the innovative breeding of Cd-tolerant ramie.

Molecular Tools and Their Applications in Developing Salt-Tolerant Soybean (Glycine max L.) Cultivars.

Abiotic stresses are one of the significant threats to soybean (Glycine max L.) growth and yields worldwide. Soybean has a crucial role in the global food supply chain and food security and contributes the main protein share compared to other crops. Hence, there is a vast scientific saddle on soybean researchers to develop tolerant genotypes to meet the growing need of food for the huge population. A large portion of cultivated land is damaged by salinity stress, and the situation worsens yearly. In past years, many attempts have increased soybean resilience to salinity stress. Different molecular techniques such as quantitative trait loci mapping (QTL), genetic engineering, transcriptome, transcription factor analysis (TFs), CRISPR/Cas9, as well as other conventional methods are used for the breeding of salt-tolerant cultivars of soybean to safeguard its yield under changing environments. These powerful genetic tools ensure sustainable soybean yields, preserving genetic variability for future use. Only a few reports about a detailed overview of soybean salinity tolerance have been published. Therefore, this review focuses on a detailed overview of several molecular techniques for soybean salinity tolerance and draws a future research direction. Thus, the updated review will provide complete guidelines for researchers working on the genetic mechanism of salinity tolerance in soybean.

The Role of Hemicellulose in Cadmium Tolerance in Ramie (Boehmeria nivea (L.) Gaud.).

Ramie cell walls play an important role in cadmium (Cd) detoxification. However, the Cd binding capacity of the cell wall components and the cell wall compositions among ramie species remains unclear. Therefore, this study compared two ramie populations ('Dazhuhuangbaima' (low-Cd-accumulating population) and 'Zhongzhu 1' (high-Cd-accumulating population)) with different Cd enrichment characteristics. The two ramie populations were treated with 0, 25, and 75 mg kg-1 Cd for 30 days; then, their root length, plant height, biomass, Cd enrichment in the organs, subcellular Cd distribution, Cd content in the cell wall polysaccharides, and hemicellulose content were determined. The root length, plant height, biomass, and Cd enrichment in all organs were significantly higher (p ≤ 0.05) in 'Zhongzhu 1' than in 'Dazhuhuangbaima' under Cd stress. In addition, the subcellular Cd distribution analysis revealed that Cd was mainly found in the cell wall in both ramie populations. Among the cell wall fractions, Cd was mainly bound to the hemicelluloses, with 60.38-73.10% and 50.05-64.45% Cd accumulating in the 'Zhongzhu 1' and 'Dazhuhuangbaima' cell wall hemicelluloses, respectively. However, the Cd concentration in the 'Zhongzhu 1' hemicellulose was significantly higher (p ≤ 0.05) than that in the 'Dazhuhuangbaima' hemicellulose. Hemicellulose content analysis further revealed that the hemicellulose concentration increased with the Cd concentration in both populations, but it was significantly higher (p ≤ 0.05) in 'Zhongzhu 1' than in 'Dazhuhuangbaima' across all Cd treatments. Thus, ramie copes under Cd stress by increasing the hemicellulose content in the cell wall. The findings in this study confirm that hemicellulose is the main enrichment site for Cd in ramie. It also provides a theoretical basis for Cd enrichment breeding in ramie.

A de novo regulation design shows an effectiveness in altering plant secondary metabolism.

Introduction: Transcription factors (TFs) and cis-regulatory elements (CREs) control gene transcripts involved in various biological processes. We hypothesize that TFs and CREs can be effective molecular tools for De Novo regulation designs to engineer plants. Objectives: We selected two Arabidopsis TF types and two tobacco CRE types to design a De Novo regulation and evaluated its effectiveness in plant engineering. Methods: G-box and MYB recognition elements (MREs) were identified in four Nicotiana tabacum JAZs (NtJAZs) promoters. MRE-like and G-box like elements were identified in one nicotine pathway gene promoter. TF screening led to select Arabidopsis Production of Anthocyanin Pigment 1 (PAP1/MYB) and Transparent Testa 8 (TT8/bHLH). Two NtJAZ and two nicotine pathway gene promoters were cloned from commercial Narrow Leaf Madole (NL) and KY171 (KY) tobacco cultivars. Electrophoretic mobility shift assay (EMSA), cross-linked chromatin immunoprecipitation (ChIP), and dual-luciferase assays were performed to test the promoter binding and activation by PAP1 (P), TT8 (T), PAP1/TT8 together, and the PAP1/TT8/Transparent Testa Glabra 1 (TTG1) complex. A DNA cassette was designed and then synthesized for stacking and expressing PAP1 and TT8 together. Three years of field trials were performed by following industrial and GMO protocols. Gene expression and metabolic profiling were completed to characterize plant secondary metabolism. Results: PAP1, TT8, PAP1/TT8, and the PAP1/TT8/TTG1 complex bound to and activated NtJAZ promoters but did not bind to nicotine pathway gene promoters. The engineered red P + T plants significantly upregulated four NtJAZs but downregulated the tobacco alkaloid biosynthesis. Field trials showed significant reduction of five tobacco alkaloids and four carcinogenic tobacco specific nitrosamines in most or all cured leaves of engineered P + T and PAP1 genotypes. Conclusion: G-boxes, MREs, and two TF types are appropriate molecular tools for a De Novo regulation design to create a novel distant-pathway cross regulation for altering plant secondary metabolism.

Seasonal Nutrient Cycling and Enrichment of Nutrient-Related Soil Microbes Aid in the Adaptation of Ramie (Boehmeria nivea L.) to Nutrient-Deficient Conditions.

The breeding for varieties tolerant of adverse growing conditions is critical for sustainable agriculture, especially for ramie (Boehmeria nivea L.). However, a lack of information on the tolerance of ramie to nutrient-deficient conditions has hindered efforts to breed ramie varieties tolerant of such conditions. The main objective of this study was to explore the tolerance strategies of ramie plants under poor soil conditions using long-term (8-9 years) field trials. Genotypes of Duobeiti 1 and Xiangzhu XB were highly tolerant of poor soil conditions. The contributions of seasonal nutrient cycling and rhizobacteria to the ability of ramie to tolerate poor soil were tested. Nitrogen and phosphorus retranslocation to the root at the end of the growing season helped ramie adapt to poor soil conditions. The contribution of the microbial community was analyzed using high-throughput Illumina MiSeq sequencing technology. The enrichment of beneficial bacteria (mainly Bradyrhizobium, Gaiella, and norank_o_Gaiellales) and the reduction of harmful fungi (mainly Cladosporium and Aspergillus) also contributed to the ability of ramie to tolerate poor soils. The results of this study provide new insight into the ability of ramie to tolerate adverse conditions and aid future efforts to breed and cultivate ramie tolerant of adverse conditions.

Correlation Analysis of Lignin Accumulation and Expression of Key Genes Involved in Lignin Biosynthesis of Ramie (Boehmeria nivea).

The phloem of the stem of ramie (Boehmeria nivea) is an important source of natural fiber for the textile industry. However, the lignin content in the phloem affects the quality of ramie phloem fiber. In this study, the lignin content and related key gene expression levels were analyzed in the phloem and xylem at different developmental periods. The results showed that the relative expression levels of lignin synthesis-related key genes in the xylem and phloem of the stem gradually decreased from the fast-growing period to the late maturation period, but the corresponding lignin content increased significantly. However, the relative expression levels of a few genes were the highest during the maturation period. During all three periods, the lignin content in ramie stems was positively correlated with the expression of genes, including PAL, C4H and 4CL1 in the phenylpropanoid pathway, F5H and CCoAOMT in the lignin-specific synthetic pathway, and CAD in the downstream pathway of lignin synthesis, but the lignin content was negatively correlated with the expression of genes including 4CL3 in the phenylpropanoid pathway and UDP-GT in the shunt pathway of lignin monomer synthesis. The ramie 4CL3 recombinant protein prefers cinnamic acid as a substrate during catalysis, and it negatively regulates lignin synthesis. It is speculated that ramie 4CL3 is mainly involved in the synthesis of ramie flavonoid compounds, and that 4CL1 is mainly involved in lignin synthesis.

A Ramie bZIP Transcription Factor BnbZIP2 Is Involved in Drought, Salt, and Heavy Metal Stress Response.

bZIP transcription factors play key roles in plant growth, development, and stress signaling. A bZIP gene BnbZIP2 (GenBank accession number: KP642148) was cloned from ramie. BnbZIP2 has a 1416 base pair open reading frame, encoding a 471 amino acid protein containing a characteristic bZIP domain and a leucine zipper. BnbZIP2 shares high sequence similarity with bZIP factors from other plants. The BnbZIP2 protein is localized to both nuclei and cytoplasm. Transcripts of BnbZIP2 were found in various tissues in ramie, with significantly higher levels in female and male flowers. Its expression was induced by drought, high salinity, and abscisic acid treatments. Analysis of the cis-elements in promoters of BnbZIP2 identified cis-acting elements involved in growth, developmental processes, and a variety of stress responses. Transgenic Arabidopsis plants' overexpression of BnbZIP2 exhibited more sensitivity to drought and heavy metal Cd stress during seed germination, whereas more tolerance to high-salinity stress than the wild type during both seed germination and plant development. Thus, BnbZIP2 may act as a positive regulator in plants' response to high-salinity stress and be an important candidate gene for molecular breeding of salt-tolerant plants.

Expression profiling of cadmium response genes in ramie (Boehmeria nivea L.) root.

Ramie (Boehmeria nivea), a perennial herb belongs to Urticaceae family, is a rapid growth and high biomass crop with highly tolerant and accumulative to heavy metals. However, the gene expression and regulation caused by cadmium (Cd) in ramie has not been well studied. In the present study, a gene expression database of ramie root in the absence (control) or presence of 100 µM Cd was established. Solexa high-throughput sequencing technology showed that 3,654,395 and 3,572,333 tags have been obtained from control and Cd treatment respectively. In total, 3887 genes were detected with significant differential expression levels, in which 2883 genes were up-regulated and 1004 genes were down-regulated. Gene ontology and pathway-based analyses were performed to determine and further to understand the biological functions of those differentially expressed genes. Fifteen genes were selected and their expression levels were confirmed by quantitative RT-PCR, and twelve of them showed consistent expression patterns with the digital gene expression data. Results on these expression profiling of genes lay the basis for biotechnological modification of new transgenic plants with improved phytoremediation capacity.