Fengling Shi

The light-harvesting complex (LHC) is the antenna system of photosystem that affects the photosynthetic efficiency of plants. In this study, MsWRKY41 was demonstrated to directly activate the expression of MsAlb3 and MsDVR1 , which involved in LHC protein insertion into thylakoid membrane and chlorophyll biosynthesis, respectively. Knockdown of MsWRKY41 in alfalfa led to yellow-green leaves and abnormal thylakoid structure. MsWRKY41 was the downstream target gene of MsWRKY40, which also directly and positively regulated the expression of four LHC protein genes MsLHCB1/4.3/5/7 . Overexpression of MsWRKY40 increased the accumulation of PSII-LHCII supercomplexes. MsWRKY40 physically interacted with zinc finger protein MsZFP1, which partly inhibited the expression of MsWRKY40-targeted genes. This interaction was weakened by physiologically high levels of ABA, leading to up-regulated expression of MsWRKY40-targeted genes. These results revealed a regulatory module centered on MsWRKY40-MsWRKY41, which integrates the biosynthetic processes of LHCB subunits and chlorophyll into a gene regulatory network, efficiently regulating LHCII biogenesis.

Background: Cold resistance in alfalfa (Medicago sativa L.) is significantly influenced by root system type. The root system plays a crucial role in water absorption and soil stress response. This study investigates the physiological and biochemical responses of creeping-rooted and taprooted alfalfa to cold stress during early winter and mid-winter periods. Methods: Samples were collected on November 3,2023 and January 7,2024, from the experimental plot. Roots at a depth of 20 cm were cleaned with distilled water and stored in cryopreservation tubes at ultra-low temperatures. Free proline, soluble sugars, soluble proteins, malondialdehyde (MDA), superoxide dismutase (SOD) activity and catalase (CAT) activity were measured using standard biochemical methods. Result: Results indicated that with decreasing temperatures, the contents of soluble sugars, soluble proteins, free proline, MDA and CAT activity increased, whereas SOD activity decreased. In the early overwintering stage, creeping-rooted alfalfa exhibited higher soluble sugar content and SOD activity compared to taprooted alfalfa. During the overwintering period, creeping-rooted alfalfa maintained higher levels of soluble sugars, soluble proteins, MDA and CAT and SOD activities. Principal component analysis identified CAT, MDA, soluble sugars and soluble proteins as key indicators for evaluating cold resistance in alfalfa.

The forage quality of alfalfa (Medicago sativa) stems is greater than the leaves. Sucrose hydrolysis provides energy for stem development, with starch being enzymatically converted into sucrose to maintain energy homeostasis. To understand the physiological and molecular networks controlling stem development, morphological characteristics and transcriptome profiles in the stems of two alfalfa cultivars (Zhungeer and WL168) were investigated. Based on transcriptome data, we analysed starch and sugar contents, and enzyme activity related to starch-sugar interconversion. Zhungeer stems were shorter and sturdier than WL168, resulting in significantly higher mechanical strength. Transcriptome analysis showed that starch and sucrose metabolism were significant enriched in the differentially expressed genes of stems development in both cultivars. Genes encoding INV, bglX, HK, TPS and glgC downregulated with the development of stems, while the gene encoding was AMY upregulated. Weighted gene co-expression network analysis revealed that the gene encoding glgC was pivotal in determining the variations in starch and sucrose contents between the two cultivars. Soluble carbohydrate, sucrose, and starch content of WL168 were higher than Zhungeer. Enzyme activities related to sucrose synthesis and hydrolysis (INV, bglX, HK, TPS) showed a downward trend. The change trend of enzyme activity was consistent with gene expression. WL168 stems had higher carbohydrate content than Zhungeer, which accounted for more rapid growth and taller plants. WL168 formed hollow stems were formed during rapid growth, which may be related to the redistribution of carbohydrates in the pith tissue. These results indicated that starch and sucrose metabolism play important roles in the stem development in alfalfa.

Background: The study investigated codon usage bias in Medicago ruthenica transcriptome coding sequences, aiming to lay the foundation for optimizing codon composition and enhancing heterologous gene expression in Medicago ruthenica. Methods: In this research, Medicago ruthenica was used as the research material and 11,581 high-quality transcript gene sequences were selected from transcriptome data. Codon usage patterns and preferences were analyzed using software such as CodonW, R and Excel. Result: The study revealed that the effective number of codons (ENC) ranged from 28.8 to 61.0. The average GC content of codons in expressed genes of Medicago ruthenica was 0.40 and the average GC content of the third nucleotide position of synonymous codons (GC3s) was 0.33. Analysis through ENC-plot, neutrality plot and bias analysis suggested that codon usage bias in the Medicago ruthenica transcriptome may be the result of a combination of factors including selection and mutation. Fifteen optimal codons were selected, with ten ending in ‘A’ and five ending in ‘U’, indicating a preference for ‘A/U’ ending codons in the Medicago ruthenica transcriptome. The frequency of codon usage in Medicago ruthenica was compared to five other organisms, including Arabidopsis thaliana, Glycine max, Nicotiana tabacum, yeast and Escherichia coli, revealing significant differences with E. coli and relatively smaller differences with Nicotiana tabacum.

Background: Pod splitting is the main cause of the low seed yield of legume forage grasses. In the present study, the flowering pattern, seed maturity characteristics and the optimum seed harvesting time of Trifolium lupinaster L. were investigated to improve seed yield and quality. Methods: All the required observations for this study were completed in the new line nursery from July to October 2022. Flowering habits of populations, individual plants, inflorescences and florets were observed for two consecutive months from July 1 to September 1 and daily temperatures and humidity were recorded.Observations on the state of seed and pod development were made by sampling and measuring in three batches every three days from July 25 to August 23 starting from the 12th day after the first flower and continuing until the first day of the season. Starting from July 15, seed yield and constitutive factors were measured every 10 d until September 5th. Result: The results showed that the flowering period of T. lupinaster L. population lasted for 60 days and the peak opening period was on the 11th-20th day after heading began, the florets opened most vigorously at 11:00-13:00 every day and inflorescences were opened more vigorously at 15:00 every day. The opening pattern of florets and inflorescences was greatly affected by temperature and humidity. The fresh weight of pod skin and seeds reached the maximum on the 15th day after flowering; the length and width of pods and seeds reached the maximum on the 18th day; the water content of pod skin and seeds began to decrease sharply on the 21st day, while the pods began to crack and the surface of the seeds dried and hardened; the water content of seeds decreased to below the safe storage moisture at 27 days. Our resuts showed that mid-August was the optimum seed harvesting period for T. lupinaster L.

Background: Medicago falcata L. (M. falcata L.) is widely distributed in Xinjiang and Inner Mongolia, which is a significant source of resistance genes. Its seeds have a high level of physical dormancy (PD). The current study aimed to study the dormancy characteristics, water absorption characteristics and methods for releasing hard seeds of M. falcata L., to reveal the the relationship between seed coat structure and water absorption and to understand the dormancy characteristics, which can provide theoretical basis for seed dormancy and seed biology research. Methods: Seed germination rate (GR), absorption, germination inhibitor activity and initial site of water entry into seeds were measured to investigate the causes of seed dormancy. Treatments to break dormancy included: (1) sandpaper sanding, (2) ultrasonic treatment for 30 min after sandpaper sanding, (3) 98% sulfuric acid immersion (5,10, 20, 40, 60 and 120 min) and (4) hot water immersion at different temperatures (50, 60, 70, 80 and 90oC) for 10 min, 20 min and 30 min. Result: The results showed that the hard rate of untreated seeds was 99.33% and the hard rate after the seed coat was scratched was 1.33%. Based on the inhibitory activity test of M. falcata L. seeds, high concentration seed extract has an inhibitory effect on the growth of other seeds. The toludine staining test showed that the hilum and microscope are the initial and main parts for seed water absorption. The germination rate of seeds after soaking in 98% sulfuric acid for 40 minutes and ultrasonic treatment with sandpaper for 30 minutes is higher than 94%, which is an effective method to relieve the PY of M. falcata L. seeds.

Soil salinization is a global environmental issue and a significant abiotic stress that threatens crop production. Root-associated rhizosphere microbiota play a pivotal role in enhancing plant tolerance to abiotic stresses. However, limited information is available concerning the specific variations in rhizosphere microbiota driven by different plant genotypes (varieties) in response to varying levels of salinity stress. In this study, we compared the growth performance of three alfalfa varieties with varying salt tolerance levels in soils with different degrees of salinization. High-throughput 16S rRNA and ITS sequencing were employed to analyze the rhizosphere microbial communities. Undoubtedly, the increasing salinity significantly inhibited alfalfa growth and reduced rhizosphere microbial diversity. However, intriguingly, salt-tolerant varieties exhibited relatively lower susceptibility to salinity, maintaining more stable rhizosphere bacterial community structure, whereas the reverse was observed for salt-sensitive varieties. Bacillus emerged as the dominant species in alfalfa's adaptation to salinity stress, constituting 21.20% of the shared bacterial genera among the three varieties. The higher abundance of Bacillus, Ensifer, and Pseudomonas in the rhizosphere of salt-tolerant alfalfa varieties is crucial in determining their elevated salt tolerance. As salinity levels increased, salt-sensitive varieties gradually accumulated a substantial population of pathogenic fungi, such as Fusarium and Rhizoctonia. Furthermore, rhizosphere bacteria of salt-tolerant varieties exhibited increased activity in various metabolic pathways, including biosynthesis of secondary metabolites, carbon metabolism, and biosynthesis of amino acids. It is suggested that salt-tolerant alfalfa varieties can provide more carbon sources to the rhizosphere, enriching more effective plant growth-promoting bacteria (PGPB) such as Pseudomonas to mitigate salinity stress. In conclusion, our results highlight the variety-mediated enrichment of rhizosphere microbiota in response to salinity stress, confirming that the high-abundance enrichment of specific dominant rhizosphere microbes and their vital roles play a significant role in conferring high salt adaptability to these varieties.

Land salinization considerably limits crop production. Biological improvement of saline and alkaline land is an important way to achieve efficient land use. It is crucial to study the salt tolerance of halophyte resources in order to explore and improve plant resources through biological improvement. Glaux maritima is a mesophyte halophyte with strong salt tolerance. In this study, we conducted research on the salt tolerance mechanism of G. maritima through phenotypic, physiological, and transcriptomic aspects. The results indicate that leaf cross-sections revealed that G. maritima has a salt gland tissue composed of stalk, collecting, and secretory cells, which are trapped in epidermal cells. At the physiological level, the maximum salt tolerance threshold of G. maritima leaves was 600 mM/L. At this concentration, proline content, relative conductivity, and superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) enzyme activities were maximum. At the transcriptional level, transcriptome data of three experimental groups (N0: 0 mM/L, N3: 600 mM/L, and N4: 800 mM/L) were analyzed, and six essential genes related to proline synthesis and five essential genes related to SOD and CAT enzyme activities were identified. Two genes involved in CAT enzyme activity were also found to play an important role in the MAPK signaling pathway. Trend analysis revealed that the MAPK signaling regulation (37 differentially expressed genes (DEGs)), phytohormone regulation (48 DEGs), glutathione metabolism (8 DEGs), flavonoid and flavonoid biosynthesis (2DEGs), and flavonoid biosynthesis (24 DEGs) pathways played important roles in regulating the salt tolerance of G. maritima. These findings provide valuable information for further studies on the functional characteristics of G. maritima in response to abiotic stress and may contribute to salt resistance breeding of fodder crops for cultivation in saline alkali land.

The reduction of crop yield caused by soil salinization has become a global problem. Halophytes improve saline alkali soil, and the halophyte transcription factors that regulate salt stress are crucial for improving salt tolerance. In this study, 1466 transcription factors were identified by transcriptome sequencing analysis of Glaux maritima leaves after salt stress (0, 600, and 800 mM/L NaCl). Their genes were distributed across 57 transcription factor families. KEGG and GO analyses showed significant enrichment in 14 pathways, with a total of 54 functions annotated. Gene expression analysis showed 820 differentially expressed genes distributed in 11 transcription factor families, including ERF, bHLH, WRKY, and NAC, and 8 expression modules. KEGG analysis revealed four genes with significant positive regulation: ABF2 (Unigene0078257) in the ABA signaling pathway, EIN3 (Unigene0000457 and Unigene0012139), and EIL1 (Unigene0042139) involved in ethylene signal transduction, and two with negative regulation, MYC1/2 (Unigene0009899 and Unigene0027167) in the main regulator of Jasmonic acid signal transduction. Protein–protein interaction networks suggested ABF2 and MYC1/2 as important transcription factors regulating G. maritima salt tolerance. Overall, the salt-tolerant transcription factors discovered in this study provide genetic resources for plant salt tolerance inheritance, and lay a theoretical foundation for the study of the salt-tolerant molecular mechanism of the halophyte Glaux maritima.

Background: In a genetic breeding program, understanding the viability of pollen germination and pollen tube growth is an essential factor for artificial hybridization. Currently, little is known about the influence of sucrose, boric acid concentrations and calcium chloride on pollen viability in alfalfa. This study is valuable for providing information about pollen germination requirements to support further improvement of artificial pollination and breeding programs in alfalfa. Methods: The single-factor experimental and an orthogonal design method (L16 [4]3) was used to compare the effectiveness of key factors (e.g., concentrations of sucrose, boric acid and calcium chloride) to determine the pollen vigor of Medicago varia Martin. ‘Caoyuan No.1’and M. ruthenica L. Trautv. ‘zhilixing’ and to optimize the culture medium for in vitro pollen germination. Four different varieties were chosen to compare the diurnal variation of pollen vigor. Result: The four factors that influenced pollen germination in Medicago varia Martin. ‘Caoyuan No.1’and M. ruthenica L. Trautv. ‘zhilixing’ in order of significance are as follows: sucrose greater than H3BO3 greater than CaCl2. These variables influenced pollen tube growth in the following order: sucrose greater than H3BO3 greater than CaCl2. The optimal culture medium for promoting pollen vigor of Medicago varia Martin.‘Caoyuan No.1’ was 10% sucrose, 300 mon/L H3BO3 and 100 mon/L CaCl2; that of M. ruthenica L. Trautv. ‘zhilixing’ was 10% sucrose, 200 mon/L H3BO3 and 50 mon/L CaCl2. Among several species of alfalfa, ‘Caoyuan No.1’ shows the highest pollen vigor and the daily pollen activity curves of all varieties showed a single peak type.