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 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.

Photosynthesis, fluorescence, and nutrition of Zhonglan No. 2, a new alfalfa cultivar.

BACKGROUND: The years after planting play an important role in the above-ground biomass and nutritive value of alfalfa. Zhonglan No. 2 (Medicago sativa L. cv. Zhonglan No. 2) is a new breeding alfalfa cultivar characterized by high drought tolerance and high yield. To determine the optimum time for utilization of Zhonglan No. 2, we examined growth traits, chlorophyll content, photosynthetic and fluorescence parameters, and composition and nutritive values at the late vegetative and early flowering stages of the first stubble in the second, third, fourth, sixth, and eleventh years after planting. RESULTS: In general, the height and leaf area decreased with increasing number of years after planting. At the late vegetative stage, the fourth-year alfalfa exhibited higher stomatal conductance (Gs) and intercellular CO2 concentration (Ci), and better water use efficiency, and at the early flowering stage, the fourth-year alfalfa had the highest (P < 0.05) leaf net photosynthetic rate (Pn) and carboxylation efficiency (CE). Total digestible nutrients did not differ among years, but, in the early flowering stage, crude protein content decreased with years (P < 0.05). Malondialdehyde (MDA) content and total antioxidant capacity did not differ among years after planting, suggesting aging did not impose oxidative stress on this alfalfa cultivar. CONCLUSIONS: Based on height, chlorophyll content, crude protein (CP) content, and photosynthetic and fluorescence parameters, the fourth year after planting, at the early flowering stage, was the best for using Zhonglan No. 2. © 2021 Society of Chemical Industry.

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.

Phosphorus and naphthalene acetic acid increased the seed yield by regulating carbon and nitrogen assimilation of flax.

To evaluate the impact of phosphorus (P) combined with exogenous NAA on flax yield, enhance flax P utilization efficiency and productivity, minimize resource inputs and mitigate negative environmental and human effects. Therefore, it is crucial to comprehend the physiological and biochemical responses of flax to P and naphthylacetic acid (NAA) in order to guide future agronomic management strategies for increasing seed yield. A randomized complete block design trial was conducted under semi-arid conditions in Northwest China, using a factorial split-plot to investigate the effects of three P (0, 67.5, and 135.0 kg P2O5 ha-1) and three exogenous spray NAA levels (0, 20, and 40 mg NAA L-1) on sucrose phosphate synthase (SPS) and diphosphoribulose carboxylase (Rubisco) activities as well as nitrogen (N) and P accumulation and translocation in flax. Results indicated that the SPS and Rubisco activities, N and P accumulation at flowering and maturity along with assimilation and translocation post-flowering, fruiting branches per plant, tillers per plant, capsules per plant, and seed yield were 95, 105, 14, 27, 55, 15, 13, 110, 103, 82, 16, 61, 8, and 13% greater in the P treatments compared to those in the zero P treatment, respectively. Moreover, those characteristics were observed to be greater with exogenous spray NAA treatments compared to that no spray NAA treatment. Additionally, the maximum SPS and Rubisco activities, N and P accumulation, assimilation post-flowering and translocation, capsules per plant, and seed yield were achieved with the application of 67.5 kg P2O5 ha-1 with 20 mg NAA L-1. Therefore, these findings demonstrate that the appropriate combination of P fertilizer and spray NAA is an effective agronomic management strategy for regulating carbon and nitrogen assimilation by maintaining photosynthetic efficiency in plants to increase flax productivity.

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.

Transcriptome analysis of halophyte Nitraria tangutorum reveals multiple mechanisms to enhance salt resistance.

As a typical halophyte, Nitraria tangutorum Bobr. has attracted the interest of many researchers with the excellent salt tolerance. Elucidation of the mechanism of N. tangutorum salinity tolerance will facilitate the genetic improvement of productive plants faced with salinity. To reveal the molecular response to gradually accumulated salt stress in N. tangutorum, RNA-sequencing and analysis of gradually accumulated NaCl treated samples and control samples were performed, and a total of 1419 differentially expressed genes were identified, including 949 down-regulated genes and 470 up-regulated genes. Detailed analysis uncovered that the catabolism of organic compounds mainly based on oxidative phosphorylation genes was up-regulated. Additionally, various antioxidant genes, especially anthocyanin-related genes, were found to help N. tangutorum remove reactive oxygen species. Moreover, the Mitogen activated protein kinase signaling pathway and other signaling pathways co-regulated various salt tolerance activities. Additionally, intracellular ion homeostasis was maintained via regulation of osmotic regulator-related genes, cutin-related genes, and cell elongation-related genes to retain cellular water and reduce ion concentration. In particularly, simultaneous up-regulation in cytoskeleton-related genes, cell wall-related genes, and auxin-related genes, provided evidence of important role of cell expansion in plant salt tolerance. In conclusion, complex regulatory mechanisms modulated by multiple genes might contribute to the salt tolerance by N. tangutorum.

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.

Improved salt tolerance of Chenopodium quinoa Willd. contributed by Pseudomonas sp. strain M30-35.

BACKGROUND: Plant-growth-promoting rhizobacteria (PGPR) can promote plant growth and enhance plant tolerance to salt stress. Pseudomonas sp. strain M30-35 might confer abiotic stress tolerance to its host plants. We evaluated the effects of M30-35 inoculation on the growth and metabolite accumulation of Chenopodium quinoa Willd. during salt stress growth conditions. METHODS: The effects of M30-35 on the growth of C. quinoa seedlings were tested under salt stress. Seedling growth parameters measured included chlorophyll content, root activity, levels of plant- phosphorus (P), and saponin content. RESULTS: M30-35 increased biomass production and root activity compared to non-inoculated plants fertilized with rhizobia and plants grown under severe salt stress conditions. The photosynthetic pigment content of chlorophyll a and b were higher in M30-35-inoculated C. quinoa seedlings under high salt stress conditions compared to non-inoculated seedlings. The stability of P content was also maintained. The content of saponin, an important secondary metabolite in C. quinoa, was increased by the inoculation of M30-35 under 300 mM NaCl conditions. CONCLUSION: Inoculation of M30-35 rescues the growth diminution of C. quinoa seedlings under salt stress.

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.

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.

Systematic selection and validation of suitable reference genes for quantitative real-time PCR normalization studies of gene expression in Nitraria tangutorum.

Suitable reference genes can be used to calibrate the error in quantitative real-time PCR (qPCR) experiments, making the results more credible. However, there are no reference genes suitable for multiple species and under different experimental conditions. Nitraria tangutorum Bobr. is a typical plant native to desert areas. It is drought-resistant, saline-alkali resistant, extreme temperatures-resistant, and has strong adaptability. To date, the importance of this germplasm has not been sufficiently understood; therefore, it is still unclear which genes can be used as reference genes to calibrate qPCR data of N. tangutorum. In this study we analyzed the expression levels of 10 candidate reference genes (ACT, GAPDH, TUA, TUB, CYP, UBC, His, PP2A, HSP, and EF1-α) in N. tangutorum seedlings under a series of experimental conditions, including in different organs (root, stem, and leaf) and under abiotic stresses (salt, drought, heat, and cold) and hormone stimuli (abscisic acid) by qPCR. Three software programs (geNorm, NormFinder, and BestKeeper) were used to evaluate the expression stability of the ten genes. Comprehensive analysis showed that EF1-α and His had the best expression stability, whereas HSP was the least suitable as a reference gene. The expression profile of NtCER7, a gene related to the regulation of cuticular wax biosynthesis in N. tangutorum, verified the accuracy of the experimental results. Based on this study, we recommend EF1-α and His as suitable reference genes for N. tangutorum. This paper provides the first data on stable reference genes in N. tangutorum, which will be beneficial to studying the gene expression of N. tangutorum and other Nitraria species in the future.

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.

Characterization of the complete chloroplast genome of Phleum pratense L. cv. Minshan.

Severe seed degradation of Phleum pratense L. cv. Minshan restricts its productivity and promotion, the chloroplast genome and evolutionary relationship analysis of Minshan could provide inheritance reasons on seed degradation and fundamental genetic reference for its molecular breeding and biological research. Its chloroplast genome was 134,973 bp in length, containing a pair of inverted repeated regions (42,726 bp) which were separated by a large single copy region of 79,473 bp, and a small single copy region of 12,774 bp. Moreover, a total of 114 functional genes were annotated, including 79 mRNA, 32 tRNA genes, and 5 rRNA genes. The phylogenetic relationships of 25 species indicated that Minshan was closely related to Avena damascene.

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.