Ectopic expression of Camellia oleifera Abel. gibberellin 20-oxidase gene increased plant height and promoted secondary cell walls deposition in Arabidopsis.

MAIN CONCLUSION: Ectopic expression of Camellia oleifera Abel. gibberellin 20-oxidase 1 caused a taller phenotype, promoted secondary cell wall deposition, leaf enlargement, and early flowering, and reduced chlorophyll and anthocyanin accumulation and seed enlargement phenotype in Arabidopsis. Plant height and secondary cell wall (SCW) deposition are important plant traits. Gibberellins (GAs) play important roles in regulating plant height and SCWs deposition. Gibberellin 20-oxidase (GA20ox) is an important enzyme involved in GA biosynthesis. In the present study, we identified a GA synthesis gene in Camellia oleifera. The total length of the CoGA20ox1 gene sequence was 1146 bp, encoding 381 amino acids. Transgenic plants with CoGA20ox1 had a taller phenotype; a seed enlargement phenotype; promoted SCWs deposition, leaf enlargement, and early flowering; and reduced chlorophyll and anthocyanin accumulation. Genetic analysis showed that the mutant ga20ox1-3 Arabidopsis partially rescued the phenotype of CoGA20ox1 overexpression plants. The results showed that CoGA20ox1 participates in the growth and development of C. oleifera. The morphological changes in CoGA20ox1 overexpressed plants provide a theoretical basis for further exploration of GA biosynthesis and analysis of the molecular mechanism in C. oleifera.

Variations in Essential Oils from the Leaves of Cinnamomum bodinieri in China.

Cinnamomum plants are rich in natural essential oils, which are widely used as materials in the fragrance, insecticidal, antibacterial agent, pharmaceutical, and food industries; however, few studies have investigated the essential oil components of Cinnamomum bodinieri. Therefore, this study investigated the diversity of essential oils from the leaves of 885 individual C. bodinieri plants across 32 populations in five provinces. Essential oils were extracted by hydrodistillation, and then qualitative and quantitative analyses of the compounds were performed by GC-MS and GC-FID. A total of 87 chemical constituents were identified in the essential oils, including 33 monoterpenes, 48 sesquiterpenes, and six other compounds. The average oil yield was 0.75%, and individual oil yields ranged from 0.01% to 4.28%. A total of 16 chemotypes were classified according to variations in the essential oil chemical constituents of C. bodinieri, among which the camphor-type, citral-type, and eucalyptol-type were dominant. Moreover, the borneol-type, cymol-type, elemol-type, methylisoeugenol-type, and selina-6-en-4-ol-type were reported in C. bodinieri for the first time. The yield and principal components of the essential oils were mainly affected by altitude, temperature, and sunshine duration, among which altitude had the most significant effect; thus, low-altitude areas are more suitable for the synthesis and accumulation of essential oils. Based on the different characteristics of the essential oils in the leaves of C. bodinieri, several excellent populations and individuals were identified in this study. Moreover, the findings provide a foundation for breeding superior varieties and studying essential oil biosynthesis mechanisms in the future.

Analysis of Camellia oleifera transcriptome reveals key pathways and hub genes involved during different photoperiods.

BACKGROUND: Camellia oleifera Abel. (C. oleifera) is an important traditional woody species in China that produces edible oil. However, the current literature lacks a proper understanding of C. oleifera's ability to adapt to different photoperiods. RESULTS: Our results indicate that the photoperiod can significantly impact flowering time in C. oleifera. We grew a total of nine samples under the short day condition (SD), middle day condition (MD) and long day condition (LD). Transcriptome analysis yielded 66.94 Gb of high-quality clean reads, with an average of over 6.73 Gb of reads for per sample. Following assembly, a total of 120,080 transcripts were obtained and 94,979 unigenes annotated. A total of 3475 differentially expressed genes (DEGs) were identified between the SD_MD, SD_LD, and MD_LD gene sets. Moreover, WGCNA identified ten gene modules. Genes in pink module (92 genes) were positively correlated with SD, and negatively correlated with both MD and LD. Genes in the magenta module (42 genes) were positively correlated with MD and negatively correlated with both LD and SD. Finally, genes in the yellow module (1758 genes) were positively correlated with both SD and MD, but negatively correlated with LD. KEGG enrichment analysis revealed that genes in the pink, magenta, and yellow modules were involved in flavonoid biosynthesis, amino sugar and nucleotide sugar metabolism and circadian rhythm pathways. Additionally, eight hub genes (GI, AP2, WRKY65, SCR, SHR, PHR1, ERF106, and SCL3) were obtained through network analysis. The hub genes had high connectivity with other photoperiod-sensitive DEGs. The expression levels of hub genes were verified by qRT-PCR analysis. CONCLUSION: An increase in light duration promotes earlier flowering of C. oleifera. Flavonoid biosynthesis, amino sugar and nucleotide sugar metabolism, and circadian rhythm pathways may function in the photoperiodic flowering pathway of C. oleifera. We also identified eight hub genes that may play a role in this pathway. Ultimately, this work contributes to our understanding of the photoperiodic flowering pathway of C. oleifera and further informs molecular breeding programs on the plant's photoperiodic sensitivity.

Characterization and fine mapping of a new dwarf mutant in Brassica napus.

BACKGROUND: Plant height is an important plant characteristic closely related to yield performance of many crops. Reasonable reduction of plant height of crops is beneficial for improving yield and enhancing lodging resistance. RESULTS: In the present study, we described the Brassica napus dwarf mutant bnd2 that was isolated using ethyl methanesulfonate (EMS) mutagenesis. Compared to wild type (WT), bnd2 exhibited reduced height and shorter hypocotyl and petiole leaves. By crossing the bnd2 mutant with the WT strain, we found that the ratio of the mutant to the WT in the F2 population was close to 1:3, indicating that bnd2 is a recessive mutation of a single locus. Following bulked segregant analysis (BSA) by resequencing, BND2 was found to be located in the 13.77-18.08 Mb interval of chromosome A08, with a length of 4.31 Mb. After fine mapping with single nucleotide polymorphism (SNP) and insertion/deletion (InDel) markers, the gene was narrowed to a 140-Kb interval ranging from 15.62 Mb to 15.76 Mb. According to reference genome annotation, there were 27 genes in the interval, of which BnaA08g20960D had an SNP type variation in the intron between the mutant and its parent, which may be the candidate gene corresponding to BND2. The hybrid line derived from a cross between the mutant bnd2 and the commercial cultivar L329 had similar plant height but higher grain yield compared to the commercial cultivar, suggesting that the allele bnd2 is beneficial for hybrid breeding of lodging resistant and high yield rapeseed. CONCLUSION: In this study, we identified a novel dwarf mutant of rapeseed with a new locus, which may be useful for functional analyses of genetic mechanisms of plant architecture and grain yield in rapeseed.

FKF1 F-box protein promotes flowering in part by negatively regulating DELLA protein stability under long-day photoperiod in Arabidopsis.

FLAVIN-BINDING KELCH REPEAT F-BOX 1 (FKF1) encodes an F-box protein that regulates photoperiod flowering in Arabidopsis under long-day conditions (LDs). Gibberellin (GA) is also important for regulating flowering under LDs. However, how FKF1 and the GA pathway work in concert in regulating flowering is not fully understood. Here, we showed that the mutation of FKF1 could cause accumulation of DELLA proteins, which are crucial repressors in GA signaling pathway, thereby reducing plant sensitivity to GA in flowering. Both in vitro and in vivo biochemical analyses demonstrated that FKF1 directly interacted with DELLA proteins. Furthermore, we showed that FKF1 promoted ubiquitination and degradation of DELLA proteins. Analysis of genetic data revealed that FKF1 acted partially through DELLAs to regulate flowering under LDs. In addition, DELLAs exerted a negative feedback on FKF1 expression. Collectively, these findings demonstrate that FKF1 promotes flowering partially by negatively regulating DELLA protein stability under LDs, and suggesting a potential mechanism linking the FKF1 to the GA signaling DELLA proteins.

A photo-responsive F-box protein FOF2 regulates floral initiation by promoting FLC expression in Arabidopsis.

Floral initiation is regulated by various genetic pathways in response to light, temperature, hormones and developmental status; however, the molecular mechanisms underlying the interactions between different genetic pathways are not fully understood. Here, we show that the photoresponsive gene FOF2 (F-box of flowering 2) negatively regulates flowering. FOF2 encodes a putative F-box protein that interacts specifically with ASK14, and its overexpression results in later flowering under both long-day and short-day photoperiods. Conversely, transgenic plants expressing the F-box domain deletion mutant of FOF2 (FOF2ΔF), or double loss of function mutant of FOF2 and FOL1 (FOF2-LIKE 1) present early flowering phenotypes. The late flowering phenotype of the FOF2 overexpression lines is suppressed by the flc-3 loss-of-function mutation. Furthermore, FOF2 mRNA expression is regulated by autonomous pathway gene FCA, and the repressive effect of FOF2 in flowering can be overcome by vernalization. Interestingly, FOF2 expression is regulated by light. The protein level of FOF2 accumulates in response to light, whereas it is degraded under dark conditions via the 26S proteasome pathway. Our findings suggest a possible mechanistic link between light conditions and the autonomous floral promotion pathway in Arabidopsis.

The Role of Iron Competition in the Antagonistic Action of the Rice Endophyte Streptomyces sporocinereus OsiSh-2 Against the Pathogen Magnaporthe oryzae.

Rice blast caused by Magnaporthe oryzae severely impacts global rice yield stability. The rice endophyte Streptomyces sporocinereus OsiSh-2, with strong antagonistic activity towards M. oryzae, has been reported in our previous study. To decipher the model of the antagonistic action of OsiSh-2 towards M. oryzae, we compared the iron-capturing abilities of these two strains. The cultivation of OsiSh-2 and a M. oryzae strain under iron-rich and iron-starved conditions showed that M. oryzae depended more on iron supplementation for growth and development than did OsiSh-2. Genomic analysis of the S. sporocinereus and M. oryzae species strains revealed that they might possess different iron acquisition strategies. The actinobacterium OsiSh-2 is likely to favor siderophore utilization compared to the fungus M. oryzae. In addition, protein annotations found that OsiSh-2 contains the highest number of the siderophore biosynthetic gene clusters among the 13 endophytic actinomycete strains and 13 antifungal actinomycete strains that we compared, indicating the prominent siderophore production potential of OsiSh-2. Additionally, we verified that OsiSh-2 could excrete considerably more siderophores than Guy11 under iron-restricted conditions and displayed greater Fe3+-reducing activity during iron-supplemental conditions. Measurements of the iron mobilization between the antagonistic OsiSh-2 and Guy11 showed that the iron concentration is higher around OsiSh-2 than around Guy11. In addition, adding iron near OsiSh-2 could decrease the antagonism of OsiSh-2 towards Guy11. Our study revealed that the antagonistic capacity displayed by OsiSh-2 towards M. oryzae was related to the competition for iron. The highly efficient iron acquisition system of OsiSh-2 may offer valuable insight for the biocontrol of rice blast.

The blue light receptor CRY1 interacts with GID1 and DELLA proteins to repress GA signaling during photomorphogenesis in Arabidopsis.

Light is a critical environmental cue that regulates a variety of diverse plant developmental processes. Cryptochrome 1 (CRY1) is the major photoreceptor that mediates blue light-dependent photomorphogenic responses such as the inhibition of hypocotyl elongation. Gibberellin (GA) participates in the repression of photomorphogenesis and promotes hypocotyl elongation. However, the antagonistic interaction between blue light and GA is not well understood. Here, we report that blue light represses GA-induced degradation of the DELLA proteins (DELLAs), which are key negative regulators in the GA signaling pathway, via CRY1, thereby inhibiting the GA response during hypocotyl elongation. Both in vitro and in vivo biochemical analyses demonstrated that CRY1 physically interacts with GA receptors-GA-INSENSITIVE DWARF 1 proteins (GID1s)-and DELLAs in a blue light-dependent manner. Furthermore, we showed that CRY1 inhibits the association between GID1s and DELLAs. Genetically, CRY1 antagonizes the function of GID1s to repress the expression of cell elongation-related genes and thus hypocotyl elongation. Taken together, our findings demonstrate that CRY1 coordinates blue light and GA signaling for plant photomorphogenesis by stabilizing DELLAs through the binding and inactivation of GID1s, providing new insights into the mechanism by which blue light antagonizes the function of GA in photomorphogenesis.

Phenotype and TMT-based quantitative proteomics analysis of Brassica napus reveals new insight into chlorophyll synthesis and chloroplast structure.

The conversion of light energy into chemical energy in leaves is very important for plant growth and development. During this process, chlorophylls and their derivatives are indispensable as their fundamental role in the energy absorption and transduction activities. Chlorophyll variation mutants are important materials for studying chlorophyll metabolism, chloroplast biogenesis, photosynthesis and related physiological processes. Here, a chlorophyll-reduced mutant (crm1) was isolated from ethyl methanesulfonate (EMS) mutagenized Brassica napus. Compared to wild type, crm1 showed yellow leaves, reduced chlorophyll content, fewer thylakoid stacks and retarded growth. Quantitative mass spectrometry analysis with Tandem Mass Tag (TMT) isobaric labeling showed that totally 4575 proteins were identified from the chloroplast of Brassica napus leaves, and 466 of which displayed differential accumulations between wild type and crm1. The differential abundance proteins were found to be involved in chlorophyll metabolism, photosynthesis, phagosome and proteasome. Our results suggest that the decreased abundance of chlorophyll biosynthetic enzymes, proteins involved in photosynthesis might account for the reduced chlorophyll content, impaired thylakoid structure, and reduction of plant productivity. The increased abundance of proteins involved in phagosome and proteasome pathways might allow plants to adapt the proteome to environmental conditions to ensure growth and survival due to chlorophyll reduction. BIOLOGICAL SIGNIFICANCE: Photosynthesis, which consists of light and dark reactions, is fundamental to biomass production. Chloroplast is regarded as the main site for photosynthesis. During photosynthesis, the pigment chlorophyll is essential for light harvesting and energy transfer. This work provides new insights into protein expression patterns, and enables the identification of many attractive candidates for investigation of chlorophyll biosynthesis, chloroplast structure and photosynthesis in Brassica napus. These findings may be applied to improve the photosynthetic efficiency by genetic engineering in crops.

Ectopic expression of GA 2-oxidase 6 from rapeseed (Brassica napus L.) causes dwarfism, late flowering and enhanced chlorophyll accumulation in Arabidopsis thaliana.

Gibberellins (GAs) are endogenous hormones that play an important role in higher plant growth and development. GA2-oxidase (GA2ox) promotes catabolism and inactivation of bioactive GAs or their precursors. In this study, we identified the GA2-oxidase gene, BnGA2ox6, and found it to be highly expressed in the silique and flower. Overexpression of BnGA2ox6 in Arabidopsis resulted in GA-deficiency symptoms, including inhibited elongation of the hypocotyl and stem, delayed seed germination, and late flowering. BnGA2ox6 overexpression reduced silique growth, but had no effect on seed development. Additionally, BnGA2ox6 overexpression enhanced chlorophyll b and total chlorophyll accumulation, and downregulated mRNA expression levels of the CHL1 and RCCR genes, which are involved in the chlorophyll degradation. These findings suggest that BnGA2ox6 regulates plant hight, silique development, flowering and chlorophyll accumulation in transgenic Arabidopsis.

Construction and Validation of a Dual-Transgene Vector System for Stable Transformation in Plants.

In this study, we constructed dual-transgene vectors (pDT1, pDT7, and pDT7G) that simultaneously co-expressed two genes in plants. ACTIN2 and UBQ10 promoters were used to control the expression of these two genes. The 4×Myc, 3×HA, and 3×Flag reporter genes allowed for the convenient identification of a tunable co-expression system in plants, whereas the dexamethasone (Dex) inducible reporter gene C-terminus of the glucocorticoid receptor (cGR) provided Dex-dependent translocation of the fusion gene between the nucleus and cytoplasm. The function of pDT vectors was validated using four pairwise genes in Nicotiana benthamiana or Arabidopsis thaliana. The co-expression efficiency of two genes from the pDT1 and pDT7G vectors was 35% and 42%, respectively, which ensured the generation of sufficient transgenic materials. These pDT vectors are simple, reliable, efficient, and time-saving tools for the co-expression of two genes through a single transformation event and can be used in the study of protein-protein interactions or multi-component complexes.