Single-cell RNA-seq of Lotus japonicus provide insights into identification and function of root cell types of legume.

The roots of legume plant play a crucial role in nitrogen fixation. However, the transcriptomes of different cell types of legume root and their functions remain largely unknown. Here, we performed single-cell RNA sequencing and profiled more than 22,000 single cells from root tips of Lotus japonicus, a model species of legume. We identified seven clusters corresponding to seven major cell types, which were validated by in situ hybridization. Further analysis revealed regulatory programs including phytohormone and nodulation associated with specific cell types, and revealed conserved and diverged features for the cell types. Our results represent the first single-cell resolution transcriptome for legume root tips and a valuable resource for studying the developmental and physiological functions of various cell types in legumes.

The key regulator LcERF056 enhances salt tolerance by modulating reactive oxygen species-related genes in Lotus corniculatus.

BACKGROUND: The APETALA2/ethylene response factor (AP2/ERF) family are important regulatory factors involved in plants' response to environmental stimuli. However, their roles in salt tolerance in Lotus corniculatus remain unclear. RESULTS: Here, the key salt-responsive transcription factor LcERF056 was cloned and characterised. LcERF056 belonging to the B3-1 (IX) subfamily of ERFs was considerably upregulated by salt treatment. LcERF056-fused GFP was exclusively localised to nuclei. Furthermore, LcERF056- overexpression (OE) transgenic Arabidopsis and L. corniculatus lines exhibited significantly high tolerance to salt treatment compared with wild-type (WT) or RNA interference expression (RNAi) transgenic lines at the phenotypic and physiological levels. Transcriptome analysis of OE, RNAi, and WT lines showed that LcERF056 regulated the downstream genes involved in several metabolic pathways. Chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) and yeast one-hybrid (Y1H) assay demonstrated that LcERF056 could bind to cis-element GCC box or DRE of reactive oxygen species (ROS)-related genes such as lipid-transfer protein, peroxidase and ribosomal protein. CONCLUSION: Our results suggested that the key regulator LcERF056 plays important roles in salt tolerance in L. corniculatus by modulating ROS-related genes. Therefore, it may be a useful target for engineering salt-tolerant L. corniculatus or other crops.

Shoot-soil ecological stoichiometry of alfalfa under nitrogen and phosphorus fertilization in the Loess Plateau.

Plants and soil interactions greatly affect ecosystems processes and properties. Ecological stoichiometry is an effective means to explore the C, N, P correlation between plants and soil and the relationship between plant growth and nutrient supply. Serious soil erosion on China's Loess Plateau has further barrenness the soil. Fertilization solves the problem of ecosystem degradation by improving soil fertility and regulating the ecological stoichiometric between soil and plants. No fertilization (CK), nitrogen fertilization (N), phosphorus fertilization (P) and N and P combined fertilization (NP) treatments were set in an alfalfa grassland. Organic carbon (C), nitrogen (N) and phosphorus (P) nutrients and their stoichiometry were measured in shoot and soil. P and NP fertilization increased shoot C concentration (3.12%, 0.91%), and all fertilization decreased shoot N concentration (6.96%). The variation of shoot C and N concentrations resulted in a greater increase in shoot C:N under the fertilization treatment than that under CK (8.24%). Most fertilization treatments increased shoot P concentration (4.63%) at each cut, which induced a decrease of shoot C:P. Shoot N:P of most treatments were greater than 23, but it was lower under N and NP fertilization than that under CK. Fertilization only increased soil C in 2014, but had no effect on soil N. Soil P content was significantly higher under P fertilization in 2014 (34.53%), and all fertilization in the second cut of 2015 (124.32%). Shoot and soil C:P and N:P having the opposite changes to shoot and soil P, respectively. Our results suggest that the change of P after fertilization largely drove the changes of stoichiometric. The growth of alfalfa in the Loess Plateau was severely restricted by P. It is an effective method to increase the biomass of alfalfa by increasing the addition of N or NP fertilizer to alleviate P limitation.

Transcriptome analysis reveals potential genes involved in flower pigmentation in a red-flowered mutant of white clover (Trifolium repens L.).

White clover (Trifolium repens L.) has been cultivated for ornamental use because of its flowers, leaf marks and creeping habit. Although a mutation in flower color is very infrequent in this species, the red-flowered mutant of white clover was a novel germplasm for ornamental white clover breeding. The mechanism of flower pigmentation in white clover is still limited because of the rarity of mutation materials and the lack of genomic data. In this study, two cDNA libraries from red-flowered white clover mutant between sunlight-exposed plants and shade-treated plants, respectively, were used for transcriptome sequencing. A total of 157,964 unigenes with an average length of 728bp and a median length of 1346bp were isolated. A large number of differentially expressed genes (6282) that were potentially involved in multiple biological and metabolic pathways, including anthocyanin flavonoid biosynthetic pathway and flavonoid biosynthetic pathway, were obtained, 70 of which could be identified as putative homologues of color-related genes. Furthermore, eight key candidate genes (CHS, F3'H, F3'5'H, UFGT, FLS, LAR, ANS, and DFR) in flavonoid biological synthesis pathway were identified by quantitative real-time PCR (qRT-PCR). Mass sequence data obtained by RNA-Seq of white clover and its red-flowered mutant provided basic sequence information and a platform for future molecular biological research on the red flower trait.

Proteomic analysis of early salt stress responsive proteins in alfalfa roots and shoots.

BACKGROUND: Alfalfa (Medicago sativa) is the most extensively cultivated forage legume in the world, and salinity stress is the most problematic environmental factors limiting alfalfa production. To evaluate alfalfa tissue variations in response to salt stress, comparative physiological and proteomic analyses were made of salt responses in the roots and shoots of the alfalfa. METHOD: A two-dimensional gel electrophoresis (2-DE)-based proteomic technique was employed to identify the differentially abundant proteins (DAPs) from salt-treated alfalfa roots and shoots of the salt tolerance cultivars Zhongmu No 1 cultivar, which was subjected to a range of salt stress concentrations for 9 days. In parallel, REL, MAD and H2O2 contents, and the activities of antioxidant enzymes of shoots and roots were determinand. RESULT: Twenty-seven spots in the shoots and 36 spots in the roots that exhibited showed significant abundance variations were identified by MALDI-TOF-TOF MS. These DAPs are mainly involved in the biological processes of photosynthesis, stress and defense, carbohydrate and energy metabolism, second metabolism, protein metabolism, transcriptional regulation, cell wall and cytoskeleton metabolism, ion transpor, signal transduction. In parallel, physiological data were correlated well with our proteomic results. It is worth emphasizing that some novel salt-responsive proteins were identified, such as CP12, pathogenesis-related protein 2, harvest-induced protein, isoliquiritigenin 2'-O-methyltransferase. qRT-PCR was used to study the gene expression levels of the four above-mentioned proteins; four patterns are consistent with those of induced protein. CONCLUSION: The primary mechanisms underlying the ability of alfalfa seedlings to tolerate salt stress were photosynthesis, detoxifying and antioxidant, secondary metabolism, and ion transport. And it also suggests that the different tissues responded to salt-stress in different ways.

Comparative proteomic analysis of alfalfa revealed new salt and drought stress-related factors involved in seed germination.

Salinity and drought are two major environmental factors that limit the growth and yield of many forage crops in semi-arid and arid regions. Alfalfa (Medicago sativa L.) is one of the most important forage crops in many countries. We aim to investigate the molecular mechanisms of alfalfa in response to salt and drought stresses in this study. Physiological and proteomic analyses were applied to examine the Zhongmu NO.3 alfalfa seed germination stage with 200 mM NaCl and 180 g·L-1 polyethylene glycol (PEG) treatments. The germination ability of the seed and the accumulation of osmotic solutes were quite different between the NaCl and PEG treatments. More than 800 protein spots were detected by proteomics technology on two-dimensional electrophoresis (2-DE) gels. The abundance of twenty-eight proteins were decreased or increased after salt and drought stress. Seventeen of these proteins were identified and classified into six functional categories through mass spectrometry (MS). The six groups involved in salt- and PEG-mediated stress included defense response, energy metabolism, protein synthesis and degradation, oxidative stress, carbohydrate metabolism-associated proteins, and unknown proteins. We discovered that some proteins related to carbohydrate metabolism and energy production increased in abundance under salt- and PEG-mediated drought stress. This demonstrates a common mechanism of energy consumption during abiotic stresses. Further study of these proteins with unknown function will provide insights into the molecular mechanisms of abiotic stress and the discovery of new candidate markers.

Simultaneous isolation of DNA, RNA, and protein from Medicago truncatula L.

We describe a method for the simultaneous extraction of proteins and nucleic acids from Medicago truncatula tissues. Using a modified TRIzol reagent method, we developed a simple and an effective way to simultaneously extract proteins and nucleic acids from a single sample. We verified that this method does not affect the quality or quantitation of the isolated DNA and RNA. Furthermore, we used 2-DE to compare M. truncatula leaf, stem, and root samples processed using this new method with two commonly used methods: phenol extraction/methanol-ammonium acetate precipitation and trichloroacetic acid/acetone precipitation. The results showed that our method was superior to the other methods, based on 2-DE patterns. We also demonstrated that our protocol is compatible with proteomic analysis, as 10 out of 14 selected proteins isolated by the method were identified by MALDI-TOF-MS/MS. The protocol described can be used with sample preparation protocols for proteomic, transcriptomic, and genomic studies.