A genome-wide study of the lipoxygenase gene families in Medicago truncatula and Medicago sativa reveals that MtLOX24 participates in the methyl jasmonate response.

BACKGROUND: Lipoxygenase (LOX) is a multifunctional enzyme that is primarily related to plant organ growth and development, biotic and abiotic stress responses, and production of flavor-associated metabolites. In higher plants, the LOX family encompasses several isozymes with varying expression patterns between tissues and developmental stages. These affect processes including seed germination, seed storage, seedling growth, fruit ripening, and leaf senescence. LOX family genes have multiple functions in response to hormones such as methyl jasmonate (MeJA) and salicylic acid. RESULTS: In this study, we identified 30 and 95 LOX homologs in Medicago truncatula and Medicago sativa, respectively. These genes were characterized with analyses of their basic physical and chemical properties, structures, chromosomal distributions, and phylogenetic relationships to understand structural variations and their physical locations. Phylogenetic analysis was conducted for members of the three LOX subfamilies (9-LOX, type I 13-LOX, and type II 13-LOX) in Arabidopsis thaliana, Glycine max, M. truncatula, and M. sativa. Analysis of predicted promoter elements revealed several relevant cis-acting elements in MtLOX and MsLOX genes, including abscisic acid (ABA) response elements (ABREs), MeJA response elements (CGTCA-motifs), and antioxidant response elements (AREs). Cis-element data combined with transcriptomic data demonstrated that LOX gene family members in these species were most likely related to abiotic stress responses, hormone responses, and plant development. Gene expression patterns were confirmed via quantitative reverse transcription PCR. Several MtLOX genes (namely MtLOX15, MtLOX16, MtLOX20, and MtLOX24) belonging to the type I 13-LOX subfamily and other LOX genes (MtLOX7, MtLOX11, MsLOX23, MsLOX87, MsLOX90, and MsLOX94) showed significantly different expression levels in the flower tissue, suggesting roles in reproductive growth. Type I 13-LOXs (MtLOX16, MtLOX20, MtLOX21, MtLOX24, MsLOX57, MsLOX84, MsLOX85, and MsLOX94) and type II 13-LOXs (MtLOX5, MtLOX6, MtLOX9, MtLOX10, MsLOX18, MsLOX23, and MsLOX30) were MeJA-inducible and were predicted to function in the jasmonic acid signaling pathway. Furthermore, exogenous MtLOX24 expression in Arabidopsis verified that MtLOX24 was involved in MeJA responses, which may be related to insect-induced abiotic stress. CONCLUSIONS: We identified six and four LOX genes specifically expressed in the flowers of M. truncatula and M. sativa, respectively. Eight and seven LOX genes were induced by MeJA in M. truncatula and M. sativa, and the LOX genes identified were mainly distributed in the type I and type II 13-LOX subfamilies. MtLOX24 was up-regulated at 8 h after MeJA induction, and exogenous expression in Arabidopsis demonstrated that MtLOX24 promoted resistance to MeJA-induced stress. This study provides valuable new information regarding the evolutionary history and functions of LOX genes in the genus Medicago.

Warming in combination with increased precipitation mediate the sexual and clonal reproduction in the desert steppe dominant species Stipa breviflora.

BACKGROUND: Clonal plants can successfully adapt to various ecosystems. A trade-off between sexual and clonal reproduction is generally assumed in clonal plants, which may be influenced both by the characteristics of the plant itself and environmental conditions. Currently, it is unclear how climate change, and specifically warming and increased precipitation, might affect sexual and clonal reproduction in clonal plants. Therefore, this study aimed to investigate both the sexual and clonal reproduction responses of Stipa breviflora to warming and increased precipitation. A controlled experiment was conducted by inducing increases in precipitation (ambient condition, 25% and 50% increases) and warming (ambient temperature, 1.5 °C and 3.0 °C increases). RESULTS: Warming significantly influenced both the ratio of reproductive ramet shoot biomass to total shoot biomass, and the ratio of reproductive ramet number to total ramet number. Additionally, the ratio of reproductive ramet shoot biomass to total shoot biomass was also significantly affected by increased precipitation. Increased precipitation benefited sexual reproduction, while effects of warming on reproductive and/or vegetative ramets varied from negative to positive depending on precipitation conditions. There was no relationship between the number or shoot biomass of reproductive ramets and vegetative ramets. Reproductive ramets displayed greater sensitivity to climate change than vegetative ramets. CONCLUSIONS: The findings of our study suggest that there was no trade-off between sexual and clonal reproduction in S. breviflora. The combined impact of warming and increased precipitation promoted sexual reproduction but did not inhibit clonal reproduction. Clonal plants with the capacity for both sexual and clonal reproduction, may cope with climate change well via clonal reproduction, ensuring their survival.

Identification and characterization of stress responsive homeodomain leucine zipper transcription factors in Medicago truncatula.

BACKGROUND: Homeodomain leucine zipper (HD-ZIP) transcription factors play roles in regulating plant development and responses to abiotic stresses; however, how HD-ZIP genes in Medicago truncatula are involved in abiotic stress response remains elusive. METHODS AND RESULTS: The HD-ZIP I genes in Medicago truncatula were identified and characterized, and their expression patterns in different tissues and under different abiotic stresses were analyzed. A total of 15 Medicago truncatula HD-ZIP I genes were identified and a phylogenetic analysis of HD-ZIP I proteins in Arabidopsis thaliana and Medicago truncatula was conducted. Fifteen HD-ZIP I genes showed diverse tissue preferences. Among them, expressions of MtHB22 and MtHB51 were specially detected in vegetative buds. In addition, they responded to various abiotic stresses, including salinity and osmotic stress and abscisic acid (ABA). For instance, MtHB7 and MtHB12 expression levels were found to be positively associated with salt, osmotic stress and ABA in both shoots and roots, while MtHB13 and MtHB23 were negatively associated with these stresses in Medicago truncatula. CONCLUSION: The HD-ZIP I genes in Medicago truncatula are evolutionarily conserved, but also exhibit gene duplication and gene loss events. Differential expression analysis of Medicago truncatula HD-ZIP I genes indicated their crucial roles in abiotic stress responses. Our genome-wide analysis of the HD-ZIP I transcription factor family in Medicago truncatula provided a valuable reference for further research.

Gibberellins Inhibit Flavonoid Biosynthesis and Promote Nitrogen Metabolism in Medicago truncatula.

Bioactive gibberellic acids (GAs) are diterpenoid plant hormones that are biosynthesized through complex pathways and control various aspects of growth and development. Although GA biosynthesis has been intensively studied, the downstream metabolic pathways regulated by GAs have remained largely unexplored. We investigated Tnt1 retrotransposon insertion mutant lines of Medicago truncatula with a dwarf phenotype by forward and reverse genetics screening and phylogenetic, molecular, biochemical, proteomic and metabolomic analyses. Three Tnt1 retrotransposon insertion mutant lines of the gibberellin 3-beta-dioxygenase 1 gene (GA3ox1) with a dwarf phenotype were identified, in which the synthesis of GAs (GA3 and GA4) was inhibited. Phenotypic analysis revealed that plant height, root and petiole length of ga3ox1 mutants were shorter than those of the wild type (Medicago truncatula ecotype R108). Leaf size was also much smaller in ga3ox1 mutants than that in wild-type R108, which is probably due to cell-size diminution instead of a decrease in cell number. Proteomic and metabolomic analyses of ga3ox1/R108 leaves revealed that in the ga3ox1 mutant, flavonoid isoflavonoid biosynthesis was significantly up-regulated, while nitrogen metabolism was down-regulated. Additionally, we further demonstrated that flavonoid and isoflavonoid biosynthesis was induced by prohexadione calcium, an inhibitor of GA3ox enzyme, and inhibited by exogenous GA3. In contrast, nitrogen metabolism was promoted by exogenous GA3 but inhibited by prohexadione calcium. The results of this study further demonstrated that GAs play critical roles in positively regulating nitrogen metabolism and transport and negatively regulating flavonoid biosynthesis through GA-mediated signaling pathways in leaves.

Overexpression of MtRAV3 enhances osmotic and salt tolerance and inhibits growth of Medicago truncatula.

Related to ABI3/VP1 (RAV) transcription factors play important roles in regulating plant growth and stress tolerance, which have been studied in many plant species, but have remained largely unidentified in legumes. To functionally characterize RAV in legumes, MtRAV3 from legume model plant Medicago truncatula was isolated and its function was investigated by overexpressing MtRAV3 in M. truncatula. Expression analysis demonstrated that MtRAV3 was markedly induced by NaCl and polyethylene glycol (PEG). MtRAV3 overexpression enhanced tolerance of transgenic M. truncatula to mannitol, drought and salt stresses, and induced the expression of adversity-related genes, including MtWRKY76, MtMYB61, cold-acclimation specific protein 31 (MtCAS31), alternative oxidase 1 (MtAOX1) and ethylene response factor 1 (MtERF1). There were lower relative electrolyte leakage and higher chlorophyll content of leaves in the MtRAV3 overexpression plants than in wild type plants under both salt and drought stress. MtRAV3 overexpression M. truncatula were featured by some phenotypes of dwarfing, late flowering, more branches, smaller flower and leaf organs. Further investigations showed that the expression levels of DWARF14 (MtD14), CAROTENOID CLEAVAGE DIOXYGENASES 7 (MtCCD7) and GA3-oxidase1 (MtGA3ox1), which related to dwarf and branch phenotype, were obviously reduced, as well as MtGA3ox1' (MTR_1g011580), GA20-oxidase1 (MtGA20ox1), MtGA20ox1' (MTR_1g102070) and GA20-oxidase2 (MtGA20ox2) involved in gibberellins (GAs) pathway. Overall, our results revealed that MtRAV3 exerted an important role in adversity response and plant growth, was a multifunctional gene in M. truncatula, which provided reference for genetic improvement of alfalfa (Medicago sativa).

Transcriptomic and chemical analyses to identify candidate genes involved in color variation of sainfoin flowers.

BACKGROUND: Sainfoin (Onobrychis viciifolia Scop) is not only a high-quality legume forage, but also a nectar-producing plant. Therefore, the flower color of sainfoin is an important agronomic trait, but the factors affecting its flower phenotype are still unclear. To gain insights into the regulatory networks associated with metabolic pathways of coloration compounds (flavonoids or anthocyanins) and identify the key genes, we conducted a comprehensive analysis of the phenotype, metabolome and transcriptome of WF and AF of sainfoin. RESULTS: Delphinidin, petunidin and malvidin derivatives were the main anthocyanin compounds in the AF of sainfoin. These substances were not detected in the WF of sainfoin. The transcriptomes of WF and AF in sainfoin at the S1 and S3 stages were obtained using the Illumina HiSeq4000 platform. Overall, 10,166 (4273 upregulated and 5893 downregulated) and 15,334 (8174 upregulated and 7160 downregulated) DEGs were identified in flowers at S1 and S3 stages, respectively (WF-VS-AF). KEGG pathway annotations showed that 6396 unigenes were annotated to 120 pathways and contained 866 DEGs at S1 stages, and 6396 unigenes were annotated to 131 pathways and included 1546 DEGs at the S3 stage. Nine DEGs belonging to the "flavonoid biosynthesis"and "phenylpropanoid biosynthesis" pathways involved in flower color formation were identified and verified by RT-qPCR analyses. Among these DEGs, 4CL3, FLS, ANS, CHS, DFR and CHI2 exhibited downregulated expression, and F3H exhibited upregulated expression in the WF compared to the AF, resulting in a decrease in anthocyanin synthesis and the formation of WF in sainfoin. CONCLUSIONS: This study is the first to use transcriptome technology to study the mechanism of white flower formation in sainfoin. Our transcriptome data will be a great enrichment of the genetic information for sainfoin. In addition, the data presented herein will provide valuable molecular information for genetic breeding and provide insight into the future study of flower color polymorphisms in sainfoin.

Physiological responses and transcriptome analysis of the Kochia prostrata (L.) Schrad. to seedling drought stress.

Kochia prostrata is a good forage plant, which has important economic and ecological value in arid and semi-arid regions of China. Drought is one of the main factors affecting its productivity. At present, there are few studies on the mechanism of drought resistance. In order to reveal the changes of physiological and biochemical indexes, stomatal structure and gene expression profiles of Kochia prostrata under drought treatment, the classical determination method and high-throughput Illumina Hiseq sequencing platform were applied to the control group (CK) and drought treatment group of Kochia prostrata. The results showed that under the condition of moderate to mild drought stress, the SOD activity reached the maximum value of 350.68 U/g min on the 5th day of stress, and under the condition of severe drought stress, the SOD activity reached the maximum on the 2nd day of stress. The accumulation of Proline remained at a high level on the 5th day of stress, and there was at least one epidermal cell interval between the two adult stomatal of the leaf epidermis, so that the evaporation shell of each stomatal did not overlap, it ensures the efficient gas exchange of the stomatal, indicating that the Kochia prostrata has strong drought resistance. A total of 1,177.46 M reads were obtained by sequencing, with a total of 352.25 Gbp data and Q30 of 85%. In the differential gene annotation to the biological process (BP), a total of 261 GO terms were enriched in the up-regulated genes, and a total of 231 GO terms were enriched in the down-regulated genes. The differentially expressed genes (DEGs) were obtained in 27 KEGG metabolic pathways, which laid a foundation for revealing the molecular mechanism of drought tolerance.

Seed germination responses to seasonal temperature and drought stress are species-specific but not related to seed size in a desert steppe: Implications for effect of climate change on community structure.

Investigating how seed germination of multiple species in an ecosystem responds to environmental conditions is crucial for understanding the mechanisms for community structure and biodiversity maintenance. However, knowledge of seed germination response of species to environmental conditions is still scarce at the community level. We hypothesized that responses of seed germination to environmental conditions differ among species at the community level, and that germination response is not correlated with seed size. To test this hypothesis, we determined the response of seed germination of 20 common species in the Siziwang Desert Steppe, China, to seasonal temperature regimes (representing April, May, June, and July) and drought stress (0, -0.003, -0.027, -0.155, and -0.87 MPa). Seed germination percentage increased with increasing temperature regime, but Allium ramosum, Allium tenuissimum, Artemisia annua, Artemisia mongolica, Artemisia scoparia, Artemisia sieversiana, Bassia dasyphylla, Kochia prastrata, and Neopallasia pectinata germinated to >60% in the lowest temperature regime (April). Germination decreased with increasing water stress, but Allium ramosum, Artemisia annua, Artemisia scoparia, Bassia dasyphylla, Heteropappus altaicus, Kochia prastrata, Neopallasia pectinata, and Potentilla tanacetifolia germinated to near 60% at -0.87 MPa. Among these eight species, germination of six was tolerant to both temperature and water stress. Mean germination percentage in the four temperature regimes and the five water potentials was not significantly correlated with seed mass or seed area, which were highly correlated. Our results suggest that the species-specific germination responses to environmental conditions are important in structuring the desert steppe community and have implications for predicting community structure under climate change. Thus, the predicted warmer and dryer climate will favor germination of drought-tolerant species, resulting in altered proportions of germinants of different species and subsequently change in community composition of the desert steppe.

Adaptive Grouping Cloud Model Shuffled Frog Leaping Algorithm for Solving Continuous Optimization Problems.

The shuffled frog leaping algorithm (SFLA) easily falls into local optimum when it solves multioptimum function optimization problem, which impacts the accuracy and convergence speed. Therefore this paper presents grouped SFLA for solving continuous optimization problems combined with the excellent characteristics of cloud model transformation between qualitative and quantitative research. The algorithm divides the definition domain into several groups and gives each group a set of frogs. Frogs of each region search in their memeplex, and in the search process the algorithm uses the "elite strategy" to update the location information of existing elite frogs through cloud model algorithm. This method narrows the searching space and it can effectively improve the situation of a local optimum; thus convergence speed and accuracy can be significantly improved. The results of computer simulation confirm this conclusion.