Insight Into the Multiple Branches Traits of a Mutant in Larix olgensis by Morphological, Cytological, and Transcriptional Analyses.
ABSTRACT: Larix olgensis is a tall deciduous tree species that has many applications in the wood fiber industry. Bud mutations are somatic mutations in plants and are considered an ideal material to identify and describe the molecular mechanism of plant mutation. However, the molecular regulatory mechanisms of bud mutations in L. olgensis remain unknown. In this study, dwarfed (or stunted), short-leaved, and multi-branched mutants of L. olgensis were found and utilized to identify crucial genes and regulatory networks controlling the multiple branch structure of L. olgensis. The physiological data showed that the branch number, bud number, fresh and dry weight, tracheid length, tracheid length-width ratio, inner tracheid diameter, and epidermal cell area of mutant plants were higher than that of wild-type plants. Hormone concentration measurements found that auxin, gibberellin, and abscisic acid in the mutant leaves were higher than that in wild-type plants. Moreover, the transcriptome sequencing of all samples using the Illumina Hiseq sequencing platform. Transcriptome analysis identified, respectively, 632, 157, and 199 differentially expressed genes (DEGs) in buds, leaves, and stems between mutant plants and wild type. DEGs were found to be involved in cell division and differentiation, shoot apical meristem activity, plant hormone biosynthesis, and sugar metabolism. Furthermore, bZIP, WRKY, and AP2/ERF family transcription factors play a role in bud formation. This study provides new insights into the molecular mechanisms of L. olgensis bud and branch formation and establishes a fundamental understanding of the breeding of new varieties in L. olgensis.
Project description:Larix olgensis Henry is an important coniferous species found in plantation forests in northeastern China, but it is vulnerable to pathogens. Nitric oxide (NO) is an important molecule involved in plant resistance to pathogens. To study the regulatory role of NO at the transcriptional level, we characterized the transcriptomic response of L. olgensis seedlings to sodium nitroprusside (SNP, NO donor) using Illumina sequencing and de novo transcriptome assembly. A significant number of putative metabolic pathways and functions associated with the unique sequences were identified. Genes related to plant pathogen infection (FLS2, WRKY33, MAPKKK, and PR1) were upregulated with SNP treatment. This report describes the potential contribution of NO to disease resistance in L. olgensis as induced by biotic stress. Our results provide a substantial contribution to the genomic and transcriptomic resources for L. olgensis, as well as expanding our understanding of the involvement of NO in defense responses at the transcriptional level.
Project description:Drought stress in trees limits their growth, survival, and productivity and it negatively affects the afforestation survival rate. Our study focused on the molecular responses to drought stress in a coniferous species Larix olgensis A. Henry. Drought stress was simulated in one-year-old seedlings using 25% polyethylene glycol 6000. The drought stress response in these seedlings was assessed by analyzing select biochemical parameters, along with gene expression and metabolite profiles. The soluble protein content, peroxidase activity, and malondialdehyde content of L. olgensis were significantly changed during drought stress. Quantitative gene expression analysis identified a total of 8172 differentially expressed genes in seedlings processed after 24 h, 48 h, and 96 h of drought stress treatment. Compared with the gene expression profile of the untreated control, the number of up-regulated genes was higher than that of down-regulated genes, indicating that L. olgensis mainly responded to drought stress through positive regulation. Metabolite analysis of the control and stress-treated samples showed that under drought stress, the increased abundance of linoleic acid was the highest among up-regulated metabolites, which also included some saccharides. A combined analysis of the transcriptome and metabolome revealed that genes dominating the differential expression profile were involved in glutathione metabolism, galactose metabolism, and starch and sucrose metabolism. Moreover, the relative abundance of specific metabolites of these pathways was also altered. Thus, our results indicated that L. olgensis prevented free radical-induced damage through glutathione metabolism and responded to drought through sugar accumulation.
Project description:In a previous study we identified EARLY BUD BREAK 1 (EBB1), an ERF transcription factor, in peach (Prunus persica var. nectarina cultivar Zhongyou 4); however, little is known of how PpEBB1 may regulate bud break. To verify the function of PpEBB1 in bud break, PpEBB1 was transiently transformed into peach buds, resulting in early bud break. Bud break occurred earlier in PpEBB1-oe poplar (Populus trichocarpa) obtained by heterologous transformation than in wild type (WT), consistent with the peach bud results, indicating that PpEBB1 can promote bud break. To explore how PpEBB1 affects bud break, differentially expressed genes (DEGs) between WT and PpEBB1-oe poplar plants were identified by RNA-sequencing. The expression of DEGs associated with hormone metabolism, cell cycle, and cell wall modifications changed substantially according to qRT-PCR. Auxin, ABA, and total trans-zeatin-type cytokinin levels were higher in the PpEBB1-oe plants than in WT plants, while the total N6-(??2-isopentenyl)-adenine-type cytokinins was lower. Yeast two-hybrid and bimolecular fluorescence complementation assays verified that a cell wall modification-related protein (PpEXBL1) interacted with PpEBB1 suggesting that PpEBB1 could interact with these cell wall modification proteins directly. Overall, our study proposed a multifaceted explanation for how PpEBB1 regulates bud break and showed that PpEBB1 promotes bud break by regulating hormone metabolism, the cell cycle, and cell wall modifications.
Project description:The complete chloroplast (cp) genome sequence of <i>Larix kaempferi</i> and <i>L. olgensis var. koreana</i> were determined by Ion torrent Platform sequencing in this study. The <i>L. kaempferi</i> cp genome was 122,158bp consists of two inverted repeat (IR) regions of 436bp each, a large single-copy (LSC) region of 65,394bp, and a small single-copy (SSC) region of 55,892bp. The chloroplast genome sequence of <i>L. olgensis</i> var. <i>koreana</i> was 122,573bp in length, consisting of two IRs (436bp), one LSC (65,597bp), and one SSC (56,104bp), and is longer than that of <i>L. kaempferi</i>. The genome contained 110 genes, including 71 protein-coding genes, 35 tRNA genes, and 4 ribosomal RNA genes. The 13 genes contain introns, including 12 genes with a single intron each and <i>ycf3</i> gene with two introns. And, the rps12 gene is a trans-spliced gene. The phylogenetic analysis revealed that all sampled species in Pinaceae formed a monophyletic clade with high bootstrap value. The genus <i>Larix</i> is closely related to <i>Pseudotsuga</i>.
Project description:BACKGROUND:MiRNAs (microRNA) are 18-24?nt endogenous noncoding RNAs that regulate gene expression at the post-transcriptional level, including tissue-specific, developmental timing and evolutionary conservation gene expression. RESULTS:This study used high-throughput sequencing technology for the first time in Larix olgensis, predicted 78 miRNAs, including 12,229,003 reads sRNA, screened differentially expressed miRNAs. Predicting target genes was helpful for understanding the miRNA regulation function and obtained 333 corresponding target genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation were analysed, mostly including nucleic acid binding, plant hormone signal transduction, pantothenate and CoA biosynthesis, and cellulose synthase. This study will lay the foundation for clarifying the complex miRNA-mediated regulatory network for growth and development. In view of this, spatio-temporal expression of miR396, miR950, miR164, miR166 and miR160 were analysed in Larix olgensis during the growth stages of not lignified, beginning of lignification, and completely lignified in different tissues (root, stem, and leaf) by quantitative real-time PCR (qRT-PCR). There were differences in the expression of miRNAs in roots, stems and leaves in the same growth period. At 60?days, miR160, miR166 and miR396-2 exhibited the highest expression in leaves. At 120?days, most miRNAs in roots and stems decreased significantly. At 180?days, miRNAs were abundantly expressed in roots and stems. Meanwhile, analysis of the expression of miRNAs in leaves revealed that miR396-2 was reduced as time went on, whereas other miRNAs increased initially and then decreased. On the other hand, in the stems, miR166-1 was increase, whereas other miRNAs, especially miR160, miR164, miR396 and miR950-1, first decreased and then increased. Similarly, in the roots, miR950-2 first decreased and then increased, whereas other miRNAs exhibited a trend of continuous increase. CONCLUSIONS:The present investigation included rapid isolation and identification of miRNAs in Larix olgensis through construction of a sRNA library using Solexa and predicted 78 novel miRNAs, which showed differential expression levels in different tissues and stages. These results provided a theoretical basis for further revealing the genetic regulation mechanism of miRNA in the growth and development of conifers and the verification of function in target genes.
Project description:<h4>Background</h4>The axillary bud is an important index of cotton plant-type traits, and the molecular mechanism of axillary bud development in upland cotton has not yet been reported. We obtained a mutant (designated mZ571) with a high-budding phenotype in axillary bud development from the low-budding phenotype variety G. hirsutum Z571 (CCRI 9A02), which provided ideal materials for the study of complex regulatory networks of axillary bud development. In this study, RNA sequencing was carried out to detect gene expression levels during three stages of axillary buds in Z571 (LB, low budding) and mZ571 mutant (HB, high budding).<h4>Results</h4>A total of 7162 DEGs were identified in the three groups (HB-E vs. LB-E, HB-G1 vs. LB-G1, HB-G2 vs. LB-G2), including 4014 downregulated and 3184 upregulated DEGs. Additionally, 221 DEGs were commonly identified in all three groups, accounting for approximately 3.09% of the total DEGs. These DEGs were identified, annotated and classified. A significant number of DEGs were related to hormone metabolism, hormone signal transduction, and starch and sucrose metabolism. In addition, 45, 22 and 9 DEGs involved in hormone metabolic pathways and 67, 22 and 19 DEGs involved in hormone signal transduction pathwayspathway were identified in HB-E vs. LB-E, HB-G1 vs. LB-G1, and HB-G2 vs. LB-G2, respectively, suggesting that endogenous hormones are the primary factors influencing cotton axillary bud growth. Hormone and soluble sugar content measurements revealed that mZ571 exhibited higher concentrations of zeatin, gibberellins and soluble sugar in all three stages, which confirmed that these hormone metabolism-, hormone signal transduction- and starch metabolism-related genes showed interaction effects contributing to the divergence of axillary bud growth between mZ571 and Z571.<h4>Conclusions</h4>Our results confirmed the importance of endogenous hormones and sugars in the development of axillary buds, and we found that mZ571 plants, with a high-budding phenotype of axillary buds, exhibited higher endogenous hormone and sugar concentrations. Overall, we present a model for the emergence and development of cotton axillary buds that provides insights into the complexity and dynamic nature of the regulatory network during axillary bud emergence and development.
Project description:To study the function of LoHDZ2 in larch, we first constructed a VB191103-LoHDZ2::GUS overexpression vector. Through Agrobacterium-mediated infection, the expression vector was transferred into a larch embryogenic cell line. A stable resistant cell line was subsequently screened, and mature embryos were induced to grow until they developed into seedlings. Antagonistic cell lines were identified at both the DNA and RNA levels. The transgenic cell lines were then subjected to GUS staining, and transgenic cell lines were ultimately identified and obtained. These transgenic cell lines were sequenced to identify differentially expressed genes, and a cluster analysis was performed. The resistant cell lines were cultured under stress conditions involving 20% PEG<sub>6000</sub> and 200 mM NaCl proliferation media (1/10-BM). After the stress treatment, the contents of peroxidase (POD), malondialdehyde (MDA) and superoxide dismutase (SOD) in both wild-type and transgenic cell lines were measured. The results are summarized below: (1) When the specific fragment of the target gene in the genome of the resistant cell line was amplified. At the RNA level, the expression of the fragment in four resistant lines increased. In addition, GUS staining showed a blue reaction, indicating that LoHDZ2 was successfully integrated into the larch embryonic cell lines. (2) To verify the accuracy and reliability of the transcriptome data, 10 differentially expressed genes (5 upregulated and 5 down regulated genes) were subjected to qRT-PCR verification. The results showed that the expression trend of the 10 differentially expressed genes was the same as that revealed by RNA-Seq, indicating that the transcriptome data were reliable. (3) The transcriptome sequencing showed that 176 genes were upregulated and that 140 genes were down regulated. Through GO enrichment analysis and KEGG metabolic pathway analysis, the screened differentially expressed genes were related to biological processes such as larch metabolism and response to stimuli, indicating that these genes may be closely involved in the regulation of the larch response to external stimuli, including heat stress, drought stress, metal ion stress and bacterial infection, and may participate in the growth process. (4) After 20% PEG<sub>6000</sub> treatment, the POD enzyme activity of the transgenic cell line was greater than that of the wild-type; this activity could effectively remove the amount of peroxide produced. The MDA content of the transgenic cell lines was lower than that of the wild-type cell lines, and the accumulation degree of harmful substances was low, indicating that the degree of oxidative damage of the transgenic cell lines was lower than that of the wild-type cell lines. The SOD content of the transgenic cell lines was lower than that of the wild-type cell lines, indicating that the drought resistance of the transgenic cell lines was enhanced. After 200 mM NaCl treatment, although the increase in SOD content was not obvious, the same trend was detected, indicating that the resistance of the transgenic cell lines was indeed stronger than that of the wild-type cell lines. According to the results of previous experiments, after this gene was overexpressed in tobacco, the transformed plants showed obvious dwarfing, which may indicate that the stress resistance of the plant was enhanced. In conclusion, a transgenic larch cell line was successfully obtained, and transgenic larch seedlings were successfully induced. LoHDZ2 may participate in the response of plants to the external environment, and may participate in the growth and development of Larix olgensis by affecting plant metabolic pathways.
Project description:Bud mutation (bud sport) is mutation occurred in one branch in a tree and the mutant branch shows different phenotype from normal branches. Because bud mutation occurs in a part of plant body, genomic backgrounds of mutant branch and normal branches are the same, except some mutations. Therefore bud mutants are ideal material to identify key genes for important traits of crops. We studied M-bM-^@M-^XGiant LafM-bM-^@M-^Y, a bud mutant setting large fruit, which generated spontaneously in European pear M-bM-^@M-^XLa FranceM-bM-^@M-^Y tree. In M-bM-^@M-^XGiant LafM-bM-^@M-^Y, increase of cell size and DNA reduplication occurred specifically in fruit flesh. The DNA reduplication in M-bM-^@M-^XGiant LafM-bM-^@M-^Y was observed at 1 week before full bloom stage. This suggested that DNA reduplication observed in M-bM-^@M-^XGiant LafM-bM-^@M-^Y fruit is not endoreduplication, but endomitosis or nuclear fusion. To identify genes expressed differentially between in M-bM-^@M-^XGiant LafM-bM-^@M-^Y and in M-bM-^@M-^XLa FranceM-bM-^@M-^Y, microarray analysis was performed with RNA from receptacle (fruit flesh) at 1 week before full bloom stage. To isolate the receptacle, laser microdissection was applied. Genes encoding proteins localized in nucleus and cytoskeleton were up-regulated in M-bM-^@M-^XGiant LafM-bM-^@M-^Y. Among these genes, we found several genes which shared homology with previously described DNA reduplication related genes. These are candidates of responsible gene of M-bM-^@M-^XGiant LafM-bM-^@M-^Y mutation. The gene expression profiles between European pear (Pyrus communis) M-bM-^@M-^XLa FranceM-bM-^@M-^Y and its bud mutant M-bM-^@M-^XGiant LafM-bM-^@M-^Y were compared. RNA was extracted from receptacle which was isolated from flower bud at 1 week before full bloom stage by using laser microdissection. Three biological replications were analyzed.
Project description:Hybrids of larch (Larix kaempferi × Larix olgensis) are important afforestation species in northeastern China. They are routinely propagated via rooted stem cuttings. Despite the importance of rooting, little is known about the regulation of adventitious root development in larch hybrids. 454 GS FLX Titanium technology represents a new method for characterizing the transcriptomes of non-model species. This method can be used to identify differentially expressed genes, and then two-dimensional difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) analyses can be used to analyze their corresponding proteins. In this study, we analyzed semi-lignified cuttings of two clones of L. kaempferi × L. olgensis with different rooting capacities to study the molecular basis of adventitious root development.We analyzed two clones; clone 25-5, with strong rooting capacity, and clone 23-12, with weak rooting capacity. We constructed four cDNA libraries from 25-5 and 23-12 at two development stages. Sequencing was conducted using the 454 pyrosequencing platform. A total of 957832 raw reads was produced; 95.07% were high-quality reads, and were assembled into 45137 contigs and 61647 singletons. The functions of the unigenes, as indicated by their Gene Ontology annotation, included diverse roles in the molecular functions, biological processes, and cellular component categories. We analyzed 75 protein spots (-fold change ? 2, P ? 0.05) by 2D-DIGE, and identified the differentially expressed proteins using MALDI-TOF/TOF MS. A joint analysis of transcriptome and proteome showed genes related to two pathways, polyamine synthesis and stress response, might play an important role on adventitious root development.These results provide fundamental and important information for research on the molecular mechanism of adventitious root development. We also demonstrated for the first time the combined use of two important technologies as a powerful approach to advance research on non-model, but otherwise important, larch species.
Project description:The aerial architecture of flowering plants is determined to a large extent by shoot growth and shoot branching arising from the initiation and growth of axillary meristems. We have identified an Arabidopsis mutant, supershoot (sps), which is characterized by a massive overproliferation of shoots, such that a single plant can generate 500 or more inflorescences. Analysis of the mutant plants shows that the primary defect is because of an increase in the number of meristems formed in leaf axils, together with release of bud arrest, resulting in reiterative branch formation from rosette and cauline leaves. The SPS gene is shown here to encode a cytochrome P450, and together with a 3- to 9-fold increase in levels of Z-type cytokinins in sps mutant plants, indicate a role for SPS in modulating hormone levels. The expression pattern of SPS, with strong expression at the leaf axils, correlates well with the phenotypic defects. Our results indicate that control of shoot branching in Arabidopsis may be accomplished in part by suppression of axillary meristem initiation and growth through the localized attenuation of cytokinin levels at sites of bud initiation.