OsRLR4 binds to the OsAUX1 promoter to negatively regulate primary root development in rice.

Root architecture is one of the most important agronomic traits that determines rice crop yield. The primary root (PR) absorbs mineral nutrients and provides mechanical support; however, the molecular mechanisms of PR elongation remain unclear in rice. Here, the two loss-of-function T-DNA insertion mutants of root length regulator 4 (OsRLR4), osrlr4-1 and osrlr4-2 with longer PR, and three OsRLR4 overexpression lines, OE-OsRLR4-1/-2/-3 with shorter PR compared to the wild type/Hwayoung (WT/HY), were identified. OsRLR4 is one of five members of the PRAF subfamily of the regulator chromosome condensation 1 (RCC1) family. Phylogenetic analysis of OsRLR4 from wild and cultivated rice indicated that it is under selective sweeps, suggesting its potential role in domestication. OsRLR4 controls PR development by regulating auxin accumulation in the PR tip and thus the root apical meristem activity. A series of biochemical and genetic analyses demonstrated that OsRLR4 functions directly upstream of the auxin transporter OsAUX1. Moreover, OsRLR4 interacts with the TRITHORAX-like protein OsTrx1 to promote H3K4me3 deposition at the OsAUX1 promoter, thus altering its transcription level. This work provides insight into the cooperation of auxin and epigenetic modifications in regulating root architecture and provides a genetic resource for plant architecture breeding.

The essential roles of OsFtsH2 in developing the chloroplast of rice.

BACKGROUND: Filamentation temperature-sensitive H (FtsH) is an ATP-dependent zinc metalloprotease with ATPase activity, proteolysis activity and molecular chaperone-like activity. For now, a total of nine FtsH proteins have been encoded in rice, but their functions have not revealed in detail. In order to investigate the molecular mechanism of OsFtsH2 here, several osftsh2 knockout mutants were successfully generated by the CRISPR/Cas9 gene editing technology. RESULTS: All the mutants exhibited a phenotype of striking albino leaf and could not survive through the stage of three leaves. OsFtsH2 was located in the chloroplast and preferentially expressed in green tissues. In addition, osftsh2 mutants could not form normal chloroplasts and had lost photosynthetic autotrophic capacity. RNA sequencing analysis indicated that many biological processes such as photosynthesis-related pathways and plant hormone signal transduction were significantly affected in osftsh2 mutants. CONCLUSIONS: Overall, the results suggested OsFtsH2 to be essential for chloroplast development in rice.

OsCpn60ß1 is Essential for Chloroplast Development in Rice (Oryza sativa L.).

The chaperonin 60 (Cpn60) protein is of great importance to plants due to its involvement in modulating the folding of numerous chloroplast protein polypeptides. In chloroplasts, Cpn60 is differentiated into two subunit types-Cpn60α and Cpn60ß and the rice genome encodes three α and three ß plastid chaperonin subunits. However, the functions of Cpn60 family members in rice were poorly understood. In order to investigate the molecular mechanism of OsCpn60ß1, we attempted to disrupt the OsCpn60ß1 gene by CRISPR/Cas9-mediated targeted mutagenesis in this study. We succeeded in the production of homozygous OsCpn60ß1 knockout rice plants. The OsCpn60ß1 mutant displayed a striking albino leaf phenotype and was seedling lethal. Electron microscopy observation demonstrated that chloroplasts were severely disrupted in the OsCpn60ß1 mutant. In addition, OsCpn60ß1 was located in the chloroplast and OsCpn60ß1 is constitutively expressed in various tissues particularly in the green tissues. The label-free qualitative proteomics showed that photosynthesis-related pathways and ribosomal pathways were significantly inhibited in OsCpn60ß1 mutants. These results indicate that OsCpn60ß1 is essential for chloroplast development in rice.

Hyperspectral imaging combined with machine learning as a tool to obtain high-throughput plant salt-stress phenotyping.

The rapid selection of salinity-tolerant crops to increase food production in salinized lands is important for sustainable agriculture. Recently, high-throughput plant phenotyping technologies have been adopted that use plant morphological and physiological measurements in a non-destructive manner to accelerate plant breeding processes. Here, a hyperspectral imaging (HSI) technique was implemented to monitor the plant phenotypes of 13 okra (Abelmoschus esculentus L.) genotypes after 2 and 7 days of salt treatment. Physiological and biochemical traits, such as fresh weight, SPAD, elemental contents and photosynthesis-related parameters, which require laborious, time-consuming measurements, were also investigated. Traditional laboratory-based methods indicated the diverse performance levels of different okra genotypes in response to salinity stress. We introduced improved plant and leaf segmentation approaches to RGB images extracted from HSI imaging based on deep learning. The state-of-the-art performance of the deep-learning approach for segmentation resulted in an intersection over union score of 0.94 for plant segmentation and a symmetric best dice score of 85.4 for leaf segmentation. Moreover, deleterious effects of salinity affected the physiological and biochemical processes of okra, which resulted in substantial changes in the spectral information. Four sample predictions were constructed based on the spectral data, with correlation coefficients of 0.835, 0.704, 0.609 and 0.588 for SPAD, sodium concentration, photosynthetic rate and transpiration rate, respectively. The results confirmed the usefulness of high-throughput phenotyping for studying plant salinity stress using a combination of HSI and deep-learning approaches.

Highly Efficient Nanoscale Analysis of Plant Stomata and Cell Surface Using Polyaddition Silicone Rubber.

Stomata control gas exchange and water transpiration and are one of the most important physiological apparatuses in higher plants. The regulation of stomatal aperture is closely coordinated with photosynthesis, nutrient uptake, plant growth, development, and so on. With advances in scanning electron microscopy (SEM), high-resolution images of plant stomata and cell surfaces can be obtained from detached plant tissues. However, this method does not allow for rapid analysis of the dynamic variation of plant stomata and cell surfaces in situ under nondestructive conditions. In this study, we demonstrated a novel plant surface impression technique (PSIT, Silagum-Light as correction impression material based on A-silicones for all two-phase impression techniques) that allows for precise analysis of plant stomata aperture and cell surfaces. Using this method, we successfully monitored the dynamic variation of stomata and observed the nanoscale microstructure of soybean leaf trichomes and dragonfly wings. Additionally, compared with the analytical precision and the time used for preparing the observation samples between PSIT and traditional SEM, the results suggested that the analytical precision of PSIT was the same to traditional SEM, but the PSIT was more easy to operate. Thus, our results indicated that PSIT can be widely applied to the plant science field.

Improving enzymatic digestibility of wheat straw pretreated by a cellulase-free xylanase-secreting Pseudomonas boreopolis G22 with simultaneous production of bioflocculants.

BACKGROUND: Xylan removal by bacterial pretreatments has been confirmed to increase the digestibility of biomass. Here, an effective xylan removal technique has been developed to enhance the digestibility of wheat straw and simultaneously produce bioflocculants by a cellulase-free xylanase-secreting strain, Pseudomonas boreopolis G22. RESULTS: The results indicated that P. boreopolis G22 is an alkaliphilic strain which can secrete abundant amounts of xylanase. This xylanase had activity levels of 2.67-1.75 U mL-1 after an incubation period of 5-25 days. The xylanase showed peak activity levels at pH 8.6, and retained more than 85% relative activity in the pH range of 7.2-9.8. After 15 days of cultivation, the hemicellulose contents of the wheat straw were significantly decreased by 32.5%, while its cellulose contents were increased by 27.3%, compared to that of the control. The maximum reducing sugars released from the 15-day-pretreated wheat straw were 1.8-fold higher than that of the untreated wheat straw, under optimal enzymatic hydrolysis conditions. In addition, a maximum bioflocculant yield of 2.08 g L-1 was extracted from the fermentation broth after 15 days of incubation. The aforementioned bioflocculants could be used to efficiently decolorize a dye solution. CONCLUSIONS: The results indicate that the cellulase-free xylanase-secreting P. boreopolis G22 may be a potential strain for wheat straw pretreatments. The strain G22 does not only enhance the enzymatic digestibility of wheat straw, but also simultaneously produces a number of bioflocculants that can be used for various industrial applications.

Active photosynthetic inhibition mediated by MPK3/MPK6 is critical to effector-triggered immunity.

Extensive research revealed tremendous details about how plants sense pathogen effectors during effector-triggered immunity (ETI). However, less is known about downstream signaling events. In this report, we demonstrate that prolonged activation of MPK3 and MPK6, two Arabidopsis pathogen-responsive mitogen-activated protein kinases (MPKs), is essential to ETI mediated by both coiled coil-nucleotide binding site-leucine rich repeats (CNLs) and toll/interleukin-1 receptor nucleotide binding site-leucine rich repeats (TNLs) types of R proteins. MPK3/MPK6 activation rapidly alters the expression of photosynthesis-related genes and inhibits photosynthesis, which promotes the accumulation of superoxide ([Formula: see text]) and hydrogen peroxide (H2O2), two major reactive oxygen species (ROS), in chloroplasts under light. In the chemical-genetically rescued mpk3 mpk6 double mutants, ETI-induced photosynthetic inhibition and chloroplastic ROS accumulation are compromised, which correlates with delayed hypersensitive response (HR) cell death and compromised resistance. Furthermore, protection of chloroplasts by expressing a plastid-targeted cyanobacterial flavodoxin (pFLD) delays photosynthetic inhibition and compromises ETI. Collectively, this study highlights a critical role of MPK3/MPK6 in manipulating plant photosynthetic activities to promote ROS accumulation in chloroplasts and HR cell death, which contributes to the robustness of ETI. Furthermore, the dual functionality of MPK3/MPK6 cascade in promoting defense and inhibiting photosynthesis potentially allow it to orchestrate the trade-off between plant growth and defense in plant immunity.

Bioflocculants' production from a cellulase-free xylanase-producing Pseudomonas boreopolis G22 by degrading biomass and its application in cost-effective harvest of microalgae.

The major problem for industrial application of bioflocculants is its high production cost. Here, a novel bacterium Pseudomonas boreopolis G22, which can secret a cellulase-free xylanase and simultaneously produce bioflocculants (MBF-G22) through directly converting untreated biomass, was isolated. The bioflocculants' production of G22 was closely related to its xylanase activity, hydrolysis ability of biomass and the hemicellulose loss caused by G22. The optimal fermentation conditions with the highest bioflocculants' yield (3.75 mg g-1 dry biomass) were obtained at the fermentation time of 96 h, incubation temperature of 30 °C, inoculum concentration of 1.0% and biomass concentration of 1.0% in an initial pH value of 7.0. MBF-G22 mainly consisted of polysaccharides (63.3%) with a molecular weight of 3.982 × 106 Da and showed the highest flocculating efficiency of 97.1% at a dosage of 3.5 mg L-1. In addition, MBF-G22 showed high flocculating efficiency of microalgae (95.7%) at a dosage of 80 mg L-1.

Bioflocculants' production in a biomass-degrading bacterium using untreated corn stover as carbon source and use of bioflocculants for microalgae harvest.

BACKGROUND: Bioflocculation has been developed as a cost-effective and environment-friendly method to harvest multiple microalgae. However, the high production cost of bioflocculants makes it difficult to scale up. In the current study, low-cost bioflocculants were produced from untreated corn stover by a biomass-degrading bacterium Pseudomonas sp. GO2. RESULTS: Pseudomonas sp. GO2 showed excellent production ability of bioflocculants through directly hydrolyzing various biomasses. The untreated corn stover was selected as carbon source for bioflocculants' production due to its highest flocculating efficiency compared to that when using other biomasses as carbon source. The effects of fermentation parameters on bioflocculants' production were optimized via response surface methodology. According to the optimal model, an ideal flocculating efficiency of 99.8% was obtained with the fermentation time of 130.46 h, initial pH of 7.46, and biomass content of 0.64%. The relative importance of carboxymethyl cellulase and xylanase accounted for 51.8% in the process of bioflocculants' production by boosted regression tree analysis, further indicating that the bioflocculants were mainly from the hydrolysates of biomass. Biochemical analysis showed that it contained 59.0% polysaccharides with uronic acid (34.2%), 32.1% protein, and 6.1% nucleic acid in the bioflocculants, which had an average molecular weight as 1.33 × 106 Da. In addition, the bioflocculants showed the highest flocculating efficiency at a concentration of 12.5 mg L-1 and were stable over broad ranges of pH and temperature. The highest flocculating efficiencies obtained for Chlorella zofingiensis and Neochloris oleoabundans were 77.9 and 88.9%, respectively. CONCLUSIONS: The results indicated that Pseudomonas sp. GO2 can directly utilize various untreated lignocellulolytic biomasses to produce low-cost bioflocculants, which showed the high efficiency to harvest two green microalgae in a low GO2 fermentation broth/algal culture ratio.

Overexpression of a Laccase with Dye Decolorization Activity from Bacillus sp. Induced in Escherichia coli.

Laccases from bacteria have been widely studied in the past 2 decades due to the higher growth rate of bacteria and their excellent thermal and alkaline pH stability. In this study, a novel laccase gene was cloned from Bacillus sp., analyzed, and functionally expressed in Escherichia coli. The laccase was highly induced in the E. coli expression system with a maximum intracellular activity of 16 U mg-1 protein. The optimal temperature and pH of the purified laccase were 40°C and 4.6, respectively, when ABTS (2,2'-azino-bis[3-ethylbenzothiazoline-6-sulfonate]) was used as the substrate. The purified laccase showed high stability in the pH range of 3.0-9.0, and retained more than 70% of its activity after 24 h of incubation at 40°C with a pH value of 9.0. Furthermore, the enzyme exhibited extremely high temperature and ion metal tolerance. The half-life of the purified laccase at 70°C was 15.9 h. The purified laccase could efficiently decolorize 3 chemical dyes, especially in the presence of ABTS as a mediator. The high production of this laccase in E. coli and exceptional characteristics of the recombinant enzyme protein make it a promising candidate for industrial applications.

Enhancing digestibility of Miscanthus using lignocellulolytic enzyme produced by Bacillus.

In this study an effective bacterial pretreatment method was developed to improve digestibility of Miscanthus. Seven new bacterial isolates, which showed excellent xylanase production ability using Miscanthus as carbon source, were used to perform the pretreatment experiments. After pretreatment, the hemicellulose content and crystallinity index of Miscanthus were decreased, while the reducing sugars released from Miscanthus were significantly increased by 30.8-87.8% after enzymatic hydrolysis. Bacillus sp. G0 was selected to optimize the pretreatment parameters via response surface methodology due to its high reducing sugars released from Miscanthus. According to the optimal model, the pretreatment parameters were set as citrate buffer/G0 fermentation broth ratio at 0.34, pretreatment time at 100h and Tween-20 concentration at 1.73%. The reducing sugars released from Miscanthus pretreated by optimal parameters were 305mgg-1 dry biomass. The results suggested our bacterial pretreatment approaches have great potential to increase digestibility of bioenergy crops.

Physiological effects of autotoxicity due to DHAP stress on Picea schrenkiana regeneration.

Picea Schrenkiana as one of the most important zonal vegetation was an endemic species in Middle Asia. Natural regeneration of P. Schrenkiana is a long existing problem troubling scientists. The autotoxicity of 3,4-dihydroxy-acetophenone (DHAP) was found to be a causative factor causing the failure of P. Schrenkiana natural regeneration. The effects of concentrations of DHAP treatment on the viability of root cell, activities of antioxidant enzymes and levels of P. Schrenkiana phytohormones were performed to disclose the physiological mechanism of DHAP autotoxicity. It was observed that high concentration of DHAP could inhibit the seed germination and seedling growth, but had a hormesis at low concentrations. Analyses showed that the root cells significantly lost their viability treated with high DHAP. The enzymes activities of seedlings were significantly stimulated by the treatment of 0.5 mM DHAP to give a transient increase and then decrease as DHAP concentration increased to 1.0 mM except for GR (glutathione reductase) in which DHAP treatment had little effect on its activity. Comparing with the control, an increase in the levels of phytohormones ZT (zeatin), GA3 (gibberellic acid) and IAA (indole acetic acid) was induced by the treatment of DHAP at low concentrations (0.1-0.25 mM), but the significant deficiency was found treated by high concentrations (0.5-1.0 mM). In addition, the ABA (abscisic acid) level increased in all experimental observations. These results suggested that DHAP significantly affected indices of growth and physiology, and provided some new information about different effect in P. Schrenkiana treated with DHAP.

Identification of two transcription factors activating the expression of OsXIP in rice defence response.

BACKGROUND: Xylanase inhibitors have been confirmed to be involved in plant defence. OsXIP is a XIP-type rice xylanase inhibitor, yet its transcriptional regulation remains unknown. RESULTS: Herbivore infestation, wounding and methyl jasmonate (MeJA) treatment enhanced mRNA levels and protein levels of OsXIP. By analyzing different 5' deletion mutants of OsXIP promoter exposed to rice brown planthopper Nilaparvata lugens stress, a 562 bp region (-1451 - -889) was finally identified as the key sequence for the herbivores stress response. Using yeast one-hybrid screening, coupled with chromatin immunoprecipitation analysis, a basic helix-loop-helix protein (OsbHLH59) and an APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor OsERF71 directly binding to the 562 bp key sequence to activate the expression of OsXIP were identified, which is further supported by transient expression assay. Moreover, transcriptional analysis revealed that mechanical wounding and treatment with MeJA resulted in an obvious increase in transcript levels of OsbHLH59 and OsERF71 in root and shoot tissues. CONCLUSIONS: Our data shows that two proteins as direct transcriptional activators of OsXIP responding to stress were identified. These results reveal a coordinated regulatory mechanism of OsXIP, which may probably be involved in defence responses via a JA-mediated signaling pathway.

The small auxin-up RNA OsSAUR45 affects auxin synthesis and transport in rice.

KEY MESSAGE: This research is the first to demonstrate that OsSAUR45 is involved in plant growth though affecting auxin synthesis and transport by repressing OsYUCCA and OsPIN gene expression in rice. Small auxin-up RNAs (SAURs) comprise a large multigene family and are rapidly activated as part of the primary auxin response in plants. However, little is known about the role of SAURs in plant growth and development, especially in monocots. Here, we report the biological function of OsSAUR45 in the model plant rice (Oryza sativa). OsSAUR45 is expressed in a tissue-specific pattern and is localized to the cytoplasm. Rice lines overexpressing OsSAUR45 displayed pleiotropic developmental defects including reduced plant height and primary root length, fewer adventitious roots, narrower leaves, and reduced seed setting. Auxin levels and transport were reduced in the OsSAUR45 overexpression lines, potentially because of decreased expression of Flavin-binding monooxygenase family proteins (OsYUCCAs) and PIN-FORMED family proteins (OsPINs). Exogenous auxin application rapidly induced OsSAUR45 expression and partially restored the phenotype of rice lines overexpressing OsSAUR45. These results demonstrate that OsSAUR45 is involved in plant growth by affecting auxin synthesis and transport through the repression of OsYUCCA and OsPIN gene expression in rice.

Exogenous malic acid alleviates cadmium toxicity in Miscanthus sacchariflorus through enhancing photosynthetic capacity and restraining ROS accumulation.

Malic acid (MA) plays an important role in the regulation of plant growth, stomatal aperture, nutrition elements homeostasis and toxic metals tolerance. However, little is known about the effects of exogenous MA on physiological and biochemical responses to toxic metals in plants. To measure the alleviation roles of exogenous MA against cadmium (Cd), we determined the effects of MA on plant growth, net photosynthetic rate (Pn), reactive oxygen species (ROS) accumulation and the activities of anti-oxidant enzymes in the leaves of Miscanthus sacchariflorus (M. sacchariflorus) under Cd stress. The Cd exposure alone significantly inhibited plant growth and Pn, but increased the accumulation of ROS even though the anti-oxidant enzymes were markedly activated in the leaves of M. sacchariflorus. Treatment with MA significantly enhanced plant growth and decreased Cd accumulation accompanied by increasing Pn under Cd stress as compared to Cd stress alone, especially when treatment with high concentration of MA (200µM) was used. In addition, Cd and MA indicated synergistic effects by further increasing the activities and genes expression of partial anti-oxidant enzymes, thus resulting in higher glutathione accumulation and reduction of ROS production. The results showed that application of MA alleviated Cd-induced phytotoxicity and oxidant damage through the regulation of both enzymatic and non-enzymatic anti-oxidants under Cd stress in M. sacchariflorus.

Malate secretion from the root system is an important reason for higher resistance of Miscanthus sacchariflorus to cadmium.

Miscanthus is a vigorous perennial Gramineae genus grown throughout the world as a promising bioenergy crop and generally regarded as heavy metal tolerant due to its ability to absorb heavy metals. However, little is known about the mechanism for heavy metal tolerance in Miscanthus. In this study, two Miscanthus species (Miscanthus sacchariflorus and Miscanthus floridulus) exhibiting different cadmium (Cd) sensitivity were used to address the mechanisms of Cd tolerance. Under the same Cd stress, M. sacchariflorus showed higher Cd tolerance with better growth and lower Cd accumulation in both shoots and roots than M. floridulus. The malate (MA) content significantly increased in root exudates of M. sacchariflorus following Cd treatment while it was almost unchanged in M. floridulus. Cellular Cd analysis and flux data showed that exogenous MA application markedly restricted Cd influx and accumulation while an anion-channel inhibitor (phenylglyoxal) effectively blocked Cd-induced MA secretion and increased Cd influx in M. sacchariflorus, indicating that MA secretion could alleviate Cd toxicity by reducing Cd uptake. The genes of malate dehydrogenases (MsMDHs) and Al-activated malate transporter 1 (MsALMT1) in M. sacchariflorus were highly upregulated under Cd stress, compared with that in M. floridulus. The results indicate that Cd-induced MA synthesis and secretion efficiently alleviate Cd toxicity by reducing Cd influx in M. sacchariflorus.

Seasonal Dynamics of Metabolites in Needles of Taxus wallichiana var. mairei.

Seasonal variations of the phytochemicals contents in needles of T. wallichiana var. mairei due to the effects of growth meteorological parameters were investigated in this study. The needles of T. wallichiana var. mairei were collected from different months and the contents of taxoids (paclitaxel, 10-deacetylbaccatin III (10-DAB), baccatin III, cephalomannine, 10-deacetyltaxol (10-DAT)), flavones (ginkgetin, amentoflavone, quercetin) and polysaccharides were quantified by ultra performance liquid chromatography (UPLC) and the resonance light scattering (RIL) method. The content of taxoids gave the highest level of 1.77 ± 0.38 mg·g-1 in January, and the lowest value of 0.61 ± 0.08 mg·g-1 in September. Unlike taxoids, the content of flavonoids was the highest in August. The content of polysaccharides reached peak value of 28.52 ± 0.57 mg·g-1 in September, which was two times higher than the lowest content of 9.39 ± 0.17 mg·g-1 in January. The contents of paclitaxel, 10-DAB, 10-DAT and polysaccharides significantly depended on meteorological parameters. The mean of minimum temperature (R = -0.61) and length of daylight (R = -0.60) were significantly correlated to 10-DAB content, while 10-DAT level showed significant correlation with length of daylight (R = -0.70) and relative humidity (R = 0.70). In addition, temperature had significantly negative effect on the content of paclitaxel and a significantly positive effect on that of polysaccharides. This study enriched the knowledge on the accumulation pattern of metabolites and could help us to determine the collecting time of T. wallichiana var. mairei for medicinal use.

Effects of climate warming on plant autotoxicity in forest evolution: a case simulation analysis for Picea schrenkiana regeneration.

In order to explore how plant autotoxicity changes with climate warming, the autotoxicity of P. schrenkiana needles' water extract, organic extract fractions, and key allelochemical DHAP was systemically investigated at the temperature rising 2 and 4°C based on the data-monitored soil temperature during the last decade in the stage of Schrenk spruce regeneration (seed germination and seedling growth). The results showed that the criterion day and night temperatures were 12°C and 4°C for seed germination, and 14°C and 6°C for seedling growth, respectively. In the presence of water extract, the temperature rise of 2°C significantly inhibited the germination vigor and rate of P. Schrenkiana seed, and a temperature rise of 4°C significantly increased the inhibition to the seedling growth (P < 0.05). Among the three organic fractions, the low-polar fraction showed to be more phytotoxic than the other two fractions, causing significant inhibitory effects on the seed germination and growth even at low concentration of 0.1 mg/mL, and the inhibition effect was enhanced as temperature increased. The temperature rise significantly enhanced the promotion effect of DHAP, while the inhibition effect of temperature rise became less important with increasing concentration of DHAP. This investigation revealed that autotoxicity of P. schrenkiana was affected by the climate warming. As expected, it provided an insight into the mechanism and effectiveness of allelopathy in bridging the causal relationship between forest evolution and climate warming.

Real-time kinetics of cadmium transport and transcriptomic analysis in low cadmium accumulator Miscanthus sacchariflorus.

MAIN CONCLUSION: The molecular mechanism of low Cd influxes and accumulation in Miscanthus sacchariflorus is revealed by RNA sequencing technique. Soil cadmium (Cd) pollution has posed a serious threat to our soil quality and food security as well as to human health. Some wild plants exhibit high tolerance to heavy metals stress. However, mechanisms of Cd tolerance of wild plants remain to be fully clarified. In this study, we found that two Miscanthus species, Miscanthus (M.) sacchariflorus and M. floridulus, showed different Cd-tolerant mechanisms. M. sacchariflorus accumulated less Cd in both root and leaf by limiting Cd uptake from root and showed superior Cd tolerance, while M. floridulus not only absorbs more Cd from root but also transports more Cd to shoot. To investigate the molecular mechanism of different Cd uptake patterns in the two Miscanthus species, we analyzed the transcriptome of M. sacchariflorus and identified transcriptional changes in response to Cd in roots by high-throughput RNA-sequencing technology. A total of 92,985 unigenes were obtained from M. sacchariflorus root cDNA samples. Based on the assembled de novo transcriptome, 681 DEGs which included 345 upregulated and 336 downregulated genes were detected between two libraries of untreated and Cd-treated roots. Gene ontology (GO) and pathway enrichment analysis revealed that upregulated DEGs under Cd stress are predominately involved in metabolic pathway, starch and sucrose and biosynthesis of secondary metabolites and metal ion transporters. Quantitative RT-PCR was employed to compare the expression levels of some metal transport genes in roots of two Miscanthus species, and the genes involved in Cd uptake from root and transfer from root to shoot were extremely different. The results not only enrich genomic resource but also help to better understand the molecular mechanisms of Cd accumulation and tolerance in wild plants.

Different Growth and Physiological Responses to Cadmium of the Three Miscanthus Species.

Miscanthus has been proposed as a promising crop for phytoremediation due to its high biomass yield and remarkable adaptability to different environments. However, little is known about the resistance of Miscanthus spp. to cadmium (Cd). To determine any differences in resistance of Miscanthus to Cd, we examined plant growth, net photosynthetic rate (Pn), activities of anti-oxidant and C4 photosynthetic enzymes, concentrations of Cd in leaves and roots, and observed the chloroplast structure in three Miscanthus species treated with 0, 10, 50, 100 or 200 µM Cd in solutions. Miscanthus sinensis showed more sensitivity to Cd, including sharp decreases in growth, Pn, PEPC activity and damage to chloroplast structure, and the highest H2O2 and Cd concentrations in leaves and roots after Cd treatments. Miscanthus sacchariflorus showed higher resistance to Cd and better growth, had the highest Pn and phosphoenolpyruvate carboxylase (PEPC) activities and integrative chloroplast structure and the lowest hydrogen peroxide (H2O2) and leaf and root Cd concentrations. The results could play an important role in understanding the mechanisms of Cd tolerance in plants and in application of phytoremediation.