Proline Metabolism Process and Antioxidant Potential of Lycium ruthenicum Murr. in Response to NaCl Treatments.

Salinity influences the level of antioxidants and proline content, which are both involved in the regulation of stress responses in plants. To examine the interplay between the antioxidant system and proline metabolism in plant stress acclimation, explants of Lycium ruthenicum were subjected to NaCl treatments, and the growth characteristics, antioxidant enzyme activities, proline accumulation, and metabolic enzyme content were analyzed. The results revealed that NaCl concentrations between 50 to 150 mM have a positive effect on the growth of L. ruthenicum explants. Increasing NaCl concentrations elevated the activities of superoxide dismutase (SOD) and catalase (CAT), while hydrogen peroxide (H2O2) content was inhibited, suggesting that the elevated antioxidants play a central protective role in superoxide anion (O2•-) and H2O2 scavenging processes in response to NaCl treatments. Also, high proline levels also protect antioxidant enzyme machinery, thus protecting the plants from oxidative damage and enhancing osmotic adjustment. Increasing levels of pyrroline-5-carboxylate synthetase (P5CS), pyrroline-5-carboxylate reductase (P5CR), and ornithine-δ-aminotransferase (δ-OAT) were observed, resulting in elevated level of proline. In addition, the expression levels of LrP5CS1, -2, -3, LrOAT-1, and -2 were promoted in NaCl treatments. According to the combined analysis of metabolic enzyme activities and their relative expression, it is confirmed that the glutamate (Glu) pathway is activated in L. ruthenicum faced with different levels of NaCl concentrations. However, Glu supplied by δ-OAT is fed back into the main pathway for proline metabolism.

Transcriptome-wide identification and expression analysis of the KT/HAK/KUP family in Salicornia europaea L. under varied NaCl and KCl treatments.

Background: The KT/HAK/KUP (KUP) transporters play important roles in potassium (K+) uptake and translocation, regulation of osmotic potential, salt tolerance, root morphogenesis and plant development. However, the KUP family has not been systematically studied in the typical halophyte Salicornia europaea L., and the specific expression patterns of SeKUPs under NaCl condition and K+ deficiency are unknown. Methods: In this study, SeKUPs were screened from PacBio transcriptome data of Salicornia europaea L. using bioinformatics. The identification, phylogenetic analysis and prediction of conserved motifs of SeKUPs were extensively explored. Moreover, the expression levels of 24 selected SeKUPs were assayed by real-time quantitative polymerase chain reaction (RT-qPCR). Results: In this study, a total of 24 putative SeKUPs were identified in S. europaea. Nineteen SeKUPs with the fixed domain EA[ML]FADL were used to construct the phylogenetic tree, and they were divided into four clusters (clusters I-IV). MEME analysis identified 10 motifs in S. europaea, and the motif analysis suggested that 19 of the identified SeKUPs had at least four K+ transporter motifs existed in all SeKUPs (with the exception of SeKUP-2). The RT-qPCR analysis showed that the expression levels of most SeKUPs were significantly up-regulated in S. europaea when they were exposed to K+ deficiency and high salinity, implying that these SeKUPs may play a key role in the absorption and transport of K+ and Na+ in S. europaea. Discussions: Our results laid the foundation for revealing the salt tolerance mechanism of SeKUPs, and provided key candidate genes for further studies on the function of KUP family in S. europaea.

Metabolomics analysis unveils important changes involved in the salt tolerance of Salicornia europaea.

Salicornia europaea is one of the world's salt-tolerant plant species and is recognized as a model plant for studying the metabolism and molecular mechanisms of halophytes under salinity. To investigate the metabolic responses to salinity stress in S. europaea, this study performed a widely targeted metabolomic analysis after analyzing the physiological characteristics of plants exposed to various NaCl treatments. S. europaea exhibited excellent salt tolerance and could withstand extremely high NaCl concentrations, while lower NaCl conditions (50 and 100 mM) significantly promoted growth by increasing tissue succulence and maintaining a relatively stable K+ concentration. A total of 552 metabolites were detected, 500 of which were differently accumulated, mainly consisting of lipids, organic acids, saccharides, alcohols, amino acids, flavonoids, phenolic acids, and alkaloids. Sucrose, glucose, p-proline, quercetin and its derivatives, and kaempferol derivatives represented core metabolites that are responsive to salinity stress. Glycolysis, flavone and flavonol biosynthesis, and phenylpropanoid biosynthesis were considered as the most important pathways responsible for salt stress response by increasing the osmotic tolerance and antioxidant activities. The high accumulation of some saccharides, flavonoids, and phenolic acids under 50 mM NaCl compared with 300 mM NaCl might contribute to the improved salt tolerance under the 50 mM NaCl treatment. Furthermore, quercetin, quercetin derivatives, and kaempferol derivatives showed varied change patterns in the roots and shoots, while coumaric, caffeic, and ferulic acids increased significantly in the roots, implying that the coping strategies in the shoots and roots varied under salinity stress. These findings lay the foundation for further analysis of the mechanism underlying the response of S. europaea to salinity.

Complete chloroplast genome of Hordeum brevisubulatum: Genome organization, synonymous codon usage, phylogenetic relationships, and comparative structure analysis.

BACKGROUND: Hordeum brevisubulatum, known as fine perennial forage, is used for soil salinity improvement in northern China. Chloroplast (cp) genome is an ideal model for assessing its genome evolution and the phylogenetic relationships. We de novo sequenced and analyzed the cp genome of H. brevisubulatum, providing a fundamental reference for further studies in genetics and molecular breeding. RESULTS: The cp genome of H. brevisubulatum was 137,155 bp in length with a typical quadripartite structure. A total of 130 functional genes were annotated and the gene of accD was lost in the process of evolution. Among all the annotated genes, 16 different genes harbored introns and the genes of ycf3 and rps12 contained two introns. Parity rule 2 (PR2) plot analysis showed that majority of genes had a bias toward T over A in the coding strand in all five Hordeum species, and a slight G over C in the other four Hordeum species except for H. bogdanil. Additionally, 52 dispersed repeat sequences and 182 simple sequence repeats were identified. Moreover, some unique SSRs of each species could be used as molecular markers for further study. Compared to the other four Hordeum species, H. brevisubulatum was most closely related to H. bogdanii and its cp genome was relatively conserved. Moreover, inverted repeat regions (IRa and IRb) were less divergent than other parts and coding regions were relatively conserved compared to non-coding regions. Main divergence was presented at the SSC/IR border. CONCLUSIONS: This research comprehensively describes the architecture of the H. brevisubulatum cp genome and improves our understanding of its cp biology and genetic diversity, which will facilitate biological discoveries and cp genome engineering.

Transcriptome-wide characterization and functional analysis of Xyloglucan endo-transglycosylase/hydrolase (XTH) gene family of Salicornia europaea L. under salinity and drought stress.

BACKGROUND: Salicornia europaea is a halophyte that has a very pronounced salt tolerance. As a cell wall manipulating enzyme, xyloglucan endotransglycosylase/hydrolase (XTH) plays an important role in plant resistance to abiotic stress. However, no systematic study of the XTH gene family in S. europaea is well known. PacBio Iso-Seq transcriptome sequence data were used for bioinformatics and gene expression analysis using real-time quantitative polymerase chain reaction (RT-qPCR). RESULTS: Transcriptome sequencing (PacBio Iso-Seq system) generated 16,465,671 sub-reads and after quality control of Iso-Seq, 29,520 isoforms were obtained with an average length of 2112 bp. A total of 24,869 unigenes, with 98% of which were obtained using coding sequences (CDSs), and 6398 possible transcription factors (TFs) were identified. Thirty-five (35) non-redundant potential SeXTH proteins were identified in S. europaea and categorized into group I/II and group III based on their genetic relatedness. Prediction of the conserved motif revealed that the DE(I/L/F/V)DF(I)EFLG domain was conserved in the S. europaea proteins and a potential N-linked glycosylation domain N(T)V(R/L/T/I)T(S/K/R/F/P)G was also located near the catalytic residues. All SeXTH genes exhibited discrete expression patterns in different tissues, at different times, and under different stresses. For example, 27 and 15 SeXTH genes were positively expressed under salt stress in shoots and roots at 200 mM NaCl in 24 h, and 34 SeXTH genes were also positively regulated under 48 h of drought stress in shoots and roots. This indicates their function in adaptation to salt and drought stress. CONCLUSION: The present study discovered SeXTH gene family traits that are potential stress resistance regulators in S. europaea, and this provides a basis for future functional diversity research.

Analysis of codon usage patterns of the chloroplast genome in Delphinium grandiflorum L. reveals a preference for AT-ending codons as a result of major selection constraints.

BACKGROUND: Codon usage bias analysis is a suitable strategy for identifying the principal evolutionary driving forces in different organisms. Delphinium grandiflorum L. is a perennial herb with high economic value and typical biological characteristics. Evolutionary analysis of D. grandiflorum can provide a rich resource of genetic information for developing hybridization resources of the genus Delphinium. METHODS: Synonymous codon usage (SCU) and related indices of 51 coding sequences from the D. grandiflorum chloroplast (cp) genome were calculated using Codon W, Cups of EMBOSS, SPSS and Microsoft Excel. Multivariate statistical analysis combined by principal component analysis (PCA), correspondence analysis (COA), PR2-plot mapping analysis and ENC plot analysis was then conducted to explore the factors affecting the usage of synonymous codons. RESULTS: The SCU bias of D. grandiflorum was weak and codons preferred A/T ending. A SCU imbalance between A/T and G/C at the third base position was revealed by PR2-plot mapping analysis. A total of eight codons were identified as the optimal codons. The PCA and COA results indicated that base composition (GC content, GC3 content) and gene expression were important for SCU bias. A majority of genes were distributed below the expected curve from the ENC plot analysis and up the standard curve by neutrality plot analysis. Our results showed that with the exception of notable mutation pressure effects, the majority of genetic evolution in the D. grandiflorum cp genome might be driven by natural selection. DISCUSSIONS: Our results provide a theoretical foundation for elucidating the genetic architecture and mechanisms of D. grandiflorum, and contribute to enriching D. grandiflorum genetic resources.

Clinical characteristics and risk factors for in-hospital mortality of lung cancer patients with COVID-19: A multicenter, retrospective, cohort study.

BACKGROUND: Data on clinical, laboratory, and radiographic characteristics and risk factors for in-hospital mortality of lung cancer patients with COVID-19 are scarce. Here, we aimed to characterize the early clinical features of lung cancer patients with COVID-19 and identify risk factors associated with in-hospital mortality. METHODS: All consecutive lung cancer patients with laboratory-confirmed COVID-19 admitted to 12 hospitals in Hubei province, China, from 3 January to 6 May 2020 were included in the study. Patients without definite clinical outcomes during the period were excluded. Data on initial clinical, laboratory and radiographic findings were compared between survivors and nonsurvivors. Univariable and multivariable logistic regression analyses were used to explore the risk factors associated with in-hospital mortality. RESULTS: Of the 45 lung cancer patients (median [interquartile range] age, 66 [58-74] years; 68.9% males) included, 34 (75.6%) discharged and 11 (24.4%) died. Fever (73.3%) and cough (53.3%) were the dominant initial symptoms, and respiratory symptoms were common. Lung cancer patients also presented atypical appearances of COVID-19. In the multivariable analysis, prolonged prolongation prothrombin time (PT) (OR = 2.1, 95% CI: 1.00-4.41, P = 0.0497) and elevated high sensitivity cardiac troponin I (hs-TNI) (OR = 7.65, 95% CI: 1.24-47.39, P = 0.0287) were associated with an increased risk of in-hospital mortality. CONCLUSIONS: Lung cancer patients with COVID-19 have high in-hospital mortality. Prolonged PT and elevated hs-TNI are independent risk factors for in-hospital mortality of lung cancer patients with COVID-19. KEY POINTS: SIGNIFICANT FINDINGS OF THE STUDY: Lung cancer patients with COVID-19 have atypical early symptoms and imaging features. The prolonged prothrombin time and elevated high sensitivity cardiac troponin I are independent risk factors for in-hospital mortality of lung cancer patients with COVID-19. WHAT THIS STUDY ADDS: This study characterizes the early clinical features of lung cancer patients with COVID-19 in China, and identifies the risk factors associated with in-hospital mortality of lung cancer patients with COVID-19.

Identification and expression analysis of the WRKY gene family during different developmental stages in Lycium ruthenicum Murr. fruit.

BACKGROUND: The WRKY gene family, one of the major transcription factor families in plants, plays crucial regulatory roles in physiological and biological developmental processes, and the adaptation of plants to the environment. However, the systematic study of WRKY structure, expression profiling, and regulatory functions has not been extensively reported in Lycium ruthenicum, although these aspects have been comprehensively studied in most plant species. METHODS: In this study, the WRKY genes were identified from a L. ruthenicum transcriptome database by using bioinformatics. The identification, phylogenetic analysis, zinc-finger structures, and conserved motif prediction were extensively explored. Moreover, the expression levels of 23 selected genes with fragments per kilobase of exons per million mapped reads (FPKM) >5 were assayed during different fruit developmental stages with real-time quantitative polymerase chain reaction (RT-qPCR). RESULTS: A total of 73 putative WRKY proteins in the L. ruthenicum transcriptome database were identified and examined. Forty-four proteins with the WRKY domain were identified and divided into three major groups with several subgroups, in accordance with those in other plant species. All 44 LrWRKY proteins contained one or two conserved WRKY domains and a zinc-finger structure. Conserved motif prediction revealed conservation of the WRKY DNA-binding domain in L. ruthenicum proteins. The selected LrWRKY genes exhibited discrete expression patterns during different fruit developmental stages. Interestingly, five LrWRKYs (-20, -21, -28, -30, and -31) were expressed remarkably throughout the fruit developmental stages. DISCUSSION: Our results reveal the characteristics of the LrWRKY gene family, thus laying a foundation for further functional analysis of the WRKY family in L. ruthenicum.

Identification of the regulatory networks and hub genes controlling alfalfa floral pigmentation variation using RNA-sequencing analysis.

BACKGROUND: To understand the gene expression networks controlling flower color formation in alfalfa, flowers anthocyanins were identified using two materials with contrasting flower colors, namely Defu and Zhongtian No. 3, and transcriptome analyses of PacBio full-length sequencing combined with RNA sequencing were performed, across four flower developmental stages. RESULTS: Malvidin and petunidin glycoside derivatives were the major anthocyanins in the flowers of Defu, which were lacking in the flowers of Zhongtian No. 3. The two transcriptomic datasets provided a comprehensive and systems-level view on the dynamic gene expression networks underpinning alfalfa flower color formation. By weighted gene coexpression network analyses, we identified candidate genes and hub genes from the modules closely related to floral developmental stages. PAL, 4CL, CHS, CHR, F3'H, DFR, and UFGT were enriched in the important modules. Additionally, PAL6, PAL9, 4CL18, CHS2, 4 and 8 were identified as hub genes. Thus, a hypothesis explaining the lack of purple color in the flower of Zhongtian No. 3 was proposed. CONCLUSIONS: These analyses identified a large number of potential key regulators controlling flower color pigmentation, thereby providing new insights into the molecular networks underlying alfalfa flower development.

HEADLESS, a WUSCHEL homolog, uncovers novel aspects of shoot meristem regulation and leaf blade development in Medicago truncatula.

The formation and maintenance of the shoot apical meristem (SAM) are critical for plant development. However, the underlying molecular mechanism of regulating meristematic cell activity is poorly understood in the model legume Medicago truncatula. Using forward genetic approaches, we identified HEADLESS (HDL), a homolog of Arabidopsis WUSCHEL, required for SAM maintenance and leaf development in M. truncatula. Disruption of HDL led to disorganized specification and arrest of the SAM and axillary meristems, resulting in the hdl mutant being locked in the vegetative phase without apparent stem elongation. hdl mutant leaves are shorter in the proximal-distal axis due to reduced leaf length elongation, which resulted in a higher blade width/length ratio and altered leaf shape, uncovering novel phenotypes undescribed in the Arabidopsis wus mutant. HDL functions as a transcriptional repressor by recruiting MtTPL through its conserved WUS-box and EAR-like motif. Further genetic analysis revealed that HDL and STENOFOLIA (STF), a key regulator of M. truncatula lamina outgrowth, act independently in leaf development although HDL could recruit MtTPL in the same manner as STF does. Our results indicate that HDL has conserved and novel functions in regulating shoot meristems and leaf shape in M. truncatula, providing new avenues for understanding meristem biology and plant development.

Characterization of the complete chloroplast genome of Delphinium grandiflorum L.

Delphinium grandiflorum L. is a perennial herb, and has very high medicinal value. However, the evolutionary relationship analysis of D. grandiflorum is limited. Its cp genome was 157,339 bp in length, containing a pair of inverted repeated regions (52,304 bp), separated by a large single copy region of 88,098 bp, and a small single copy region of 16,937 bp. Moreover, a total of 117 functional genes were annotated, including 79 mRNA, 30 tRNA genes, and 8 rRNA genes. The phylogenetic relationships inferred that D. grandiflorum was closely related to Gymnaconitum gymnandrum. This study will provide a theoretical basis for species identification and biological research.

Transcriptomic analysis of Lycium ruthenicum Murr. during fruit ripening provides insight into structural and regulatory genes in the anthocyanin biosynthetic pathway.

Fruit development in Lycium ruthenicum Murr. involves a succession of physiological and biochemical changes reflecting the transcriptional modulation of thousands of genes. Although recent studies have investigated the dynamic transcriptomic responses during fruit ripening in L. ruthenicum, most have been limited in scope, and thus systematic data representing the structural genes and transcription factors involved in anthocyanin biosynthesis are lacking. In this study, the transcriptomes of three ripening stages associated with anthocyanin accumulation, including S1 (green ripeness stage), S2 (skin color change) and S3 (complete ripeness stage) in L. ruthenicum were investigated using Illumina sequencing. Of a total of 43,573 assembled unigenes, 12,734 were differentially expressed during fruit ripening in L. ruthenicum. Twenty-five significantly differentially expressed structural genes (including PAL, C4H, 4CL, CHS, CHI, F3H, F3'H, F3'5'H, DFR, ANS and UFGT) were identified that might be associated with anthocyanin biosynthesis. Additionally, several transcription factors, including MYB, bHLH, WD40, NAC, WRKY, bZIP and MADS, were correlated with the structural genes, implying their important interaction with anthocyanin biosynthesis-related genes. Our findings provide insight into anthocyanin biosynthesis and regulation patterns in L. ruthenicum and offer a systematic basis for elucidating the molecular mechanisms governing anthocyanin biosynthesis in L. ruthenicum.