Brassinosteroids affect wood development and properties of Fraxinus mandshurica.

Introduction: Xylem development plays a crucial role in wood formation in woody plants. In recent years, there has been growing attention towards the impact of brassinosteroids (BRs) on this xylem development. In the present study, we evaluated the dynamic variation of xylem development in Fraxinus mandshurica (female parent, M8) and a novel interspecific hybrid F. mandshurica × Fraxinus sogdiana (1601) from May to August 2020. Methods: We obtained RNA-Seq transcriptomes of three tissue types (xylem, phloem, and leaf) to identify the differences in xylem-differentially expressed genes (X-DEGs) and xylem-specifically expressed genes (X-SEGs) in M8 and 1601 variants. We then further evaluated these genes via weighted gene co-expression network analysis (WGCNA) alongside overexpressing FmCPD, a BR biosynthesis enzyme gene, in transient transgenic F. mandshurica. Results: Our results indicated that the xylem development cycle of 1601 was extended by 2 weeks compared to that of M8. In addition, during the later wood development stages (secondary wall thickening) of 1601, an increased cellulose content (14%) and a reduced lignin content (11%) was observed. Furthermore, vessel length and width increased by 67% and 37%, respectively, in 1601 compared with those of M8. A total of 4589 X-DEGs were identified, including enzymes related to phenylpropane metabolism, galactose metabolism, BR synthesis, and signal transduction pathways. WGCNA identified hub X-SEGs involved in cellulose synthesis and BR signaling in the 1601 wood formation-related module (CESA8, COR1, C3H14, and C3H15); in contrast, genes involved in phenylpropane metabolism were significantly enriched in the M8 wood formation-related module (CCoAOMT and CCR). Moreover, overexpression of FmCPD in transient transgenic F. mandshurica affected the expression of genes associated with lignin and cellulose biosynthesis signal transduction. Finally, BR content was determined to be approximately 20% lower in the M8 xylem than in the 1601 xylem, and the exogenous application of BRs (24-epi brassinolide) significantly increased the number of xylem cell layers and altered the composition of the secondary cell walls in F. mandshurica. Discussion: Our findings suggest that BR biosynthesis and signaling play a critical role in the differing wood development and properties observed between M8 and 1601 F. mandshurica.

Basic helix-loop-helix transcription factor PxbHLH02 enhances drought tolerance in Populus (Populus simonii × P. nigra).

The basic helix-loop-helix (bHLH) transcription factors (TFs) are involved in plant morphogenesis and various abiotic and biotic stress responses. However, further exploration is required of drought-responsive bHLH family members and their detailed regulatory mechanisms in Populus. Two bHLH TF genes, PxbHLH01/02, were identified in Populus simonii × P. nigra and cloned. The aim of this study was to examine the role of bHLH TFs in drought tolerance in P. simonii × P. nigra. The results showed that the amino acid sequences of the two genes were homologous to Arabidopsis thaliana UPBEAT1 (AtUPB1) and overexpression of PxbHLH01/02 restored normal root length in the AtUPB1 insertional mutant (upb1-1). The PxbHLH01/02 gene promoter activity analysis suggested that they were involved in stress responses and hormone signaling. Furthermore, Arabidopsis transgenic lines overexpressing PxbHLH01/02 exhibited higher stress tolerance compared with the wild-type. Populus simonii × P. nigra overexpressing PxbHLH02 increased drought tolerance and exhibited higher superoxide dismutase and peroxidase activities, lower H2O2 and malondialdehyde content, and lower relative conductivity. The results of transcriptome sequencing (RNA-seq) and quantitative real-time PCR suggested that the response of PxbHLH02 to drought stress was related to abscisic acid (ABA) signal transduction. Overall, the findings of this study suggest that PxbHLH02 from P. simonii × P. nigra functions as a positive regulator of drought stress responses by regulating stomatal aperture and promoting ABA signal transduction.

Genome-wide analysis and molecular dissection of the SPL gene family in Fraxinus mandshurica.

BACKGROUND: SQUAMOSA promoter binding protein-like (SPL) is a unique family of transcription factors in plants, which is engaged in regulating plant growth and development, physiological and biochemical processes. Fraxinus mandshurica is an excellent timber species with a wide range of uses in northeastern China and enjoys a high reputation in the international market. SPL family analysis has been reported in some plants while SPL family analysis of Fraxinus mandshurica has not been reported. RESULTS: We used phylogeny, conserved motifs, gene structure, secondary structure prediction, miR156 binding sites, promoter cis elements and GO annotation to systematically analyze the FmSPLs family. This was followed by expression analysis by subcellular localization, expression patterns at various tissue sites, abiotic stress and hormone induction. Because FmSPL2 is highly expressed in flowers it was selected to describe the SPL gene family of Fraxinus mandshurica by ectopic expression. Among them, 10 FmSPL genes that were highly expressed at different loci were selected for expression analysis under abiotic stress (NaCl and Cold) and hormone induction (IAA and ABA). These 10 FmSPL genes showed corresponding trends in response to both abiotic stress and hormone induction. We showed that overexpression of FmSPL2 in transgenic Nicotiana tabacum L. resulted in taller plants, shorter root length, increased root number, rounded leaves, and earlier flowering time. CONCLUSIONS: We identified 36 SPL genes, which were classified into seven subfamilies based on sequence analysis. FmSPL2 was selected for subsequent heterologous expression by analysis of expression patterns in various tissues and under abiotic stress and hormone induction, and significant phenotypic changes were observed in the transgenic Nicotiana tabacum L. These results provide insight into the evolutionary origin and biological significance of plant SPL. The aim of this study was to lay the foundation for the genetic improvement of Fraxinus mandshurica and the subsequent functional analysis of FmSPL2.

Fraxinus mandshurica 4-coumarate-CoA ligase 2 enhances drought and osmotic stress tolerance of tobacco by increasing coniferyl alcohol content.

4-Coumarate-CoA ligase (4CL) is an important branch point in the phenylpropane pathway and plays important roles in plant growth and development. In this study, the 4CL2 gene from Fraxinus mandshurica (designated Fm4CL2) was identified and isolated. Sequence analysis revealed that Fm4CL2 is a type I 4CL gene involved in lignin biosynthesis. Analysis of cell wall components revealed that Fm4CL2-overexpressing (OE-Fm4CL2) tobacco showed increased lignin content (by 58.9%) and decreased hemicellulose content (by 41.2%). Detection of small-molecule metabolites in the lignin pathway revealed that coumaric acid content decreased by 48% and coniferyl alcohol content increased by 250% compared with the control values. Compared with wild type, OE-Fm4CL2 tobacco showed increased xylem cell layer number (by 120%) and cell wall thickness (by 54.5%). Under osmotic stress, transgenic tobacco showed higher growth than wild-type tobacco. The germination rate of transgenic tobacco was higher than that of wild type. Reactive oxygen species accumulation and malondialdehyde content were significantly lower in transgenic tobacco than in wild type. Under drought, the expression of stress-related genes was higher in 35S-Fm4CL2-infected Fraxinus mandshurica plants than in control plants. These results indicate that Fm4CL2 overexpression can enhance drought and osmotic stress tolerance of plants.

DNA Methylation Silences Exogenous Gene Expression in Transgenic Birch Progeny.

The genetic stability of exogenous genes in the progeny of transgenic trees is extremely important in forest breeding; however, it remains largely unclear. We selected transgenic birch (Betula platyphylla) and its hybrid F1 progeny to investigate the expression stability and silencing mechanism of exogenous genes. We found that the exogenous genes of transgenic birch could be transmitted to their offspring through sexual reproduction. The exogenous genes were segregated during genetic transmission. The hybrid progeny of transgenic birch WT1×TP22 (184) and WT1×TP23 (212) showed higher Bgt expression and greater insect resistance than their parents. However, the hybrid progeny of transgenic birch TP23×TP49 (196) showed much lower Bgt expression, which was only 13.5% of the expression in its parents. To elucidate the mechanism underlying the variation in gene expression between the parents and progeny, we analyzed the methylation rates of Bgt in its promoter and coding regions. The hybrid progeny with normally expressed exogenous genes showed much lower methylation rates (0-29%) than the hybrid progeny with silenced exogenous genes (32.35-45.95%). These results suggest that transgene silencing in the progeny is mainly due to DNA methylation at cytosine residues. We further demonstrated that methylation in the promoter region, rather than in the coding region, leads to gene silencing. We also investigated the relative expression levels of three methyltransferase genes: BpCMT, BpDRM, and BpMET. The transgenic birch line 196 with a silenced Gus gene showed, respectively, 2.54, 9.92, and 4.54 times higher expression levels of BpCMT, BpDRM, and BpMET than its parents. These trends are consistent with and corroborate the high methylation levels of exogenous genes in the transgenic birch line 196. Therefore, our study suggests that DNA methylation in the promoter region leads to silencing of exogenous genes in transgenic progeny of birch.

Molecular cloning and functional analysis of 4-Coumarate:CoA ligase 4(4CL-like 1)from Fraxinus mandshurica and its role in abiotic stress tolerance and cell wall synthesis.

BACKGROUND: Four-Coumarate:CoA ligase gene (4CL) plays multiple important roles in plant growth and development by catalyzing the formation of CoA ester. 4CL belongs to the plant phenylpropane derivative, which is related to the synthesis of flavonoids and lignin and is a key enzyme in the biosynthetic pathway. RESULTS: In this study, 12 4CL genes of Fraxinus mandschurica were identified and named Fm4CL1-Fm4CL12, respectively. The analysis of the expression pattern of Fm4CL genes indicate that Fm4CL-like 1 gene may play a role in the lignin synthesis pathway. Our study indicate that overexpression of Fm4CL-like 1 increases the lignin content of transgenic tobacco by 39.5% compared to WT, and the S/G ratio of transgenic tobacco increased by 19.7% compared with WT. The xylem cell layer of transgenic line is increased by 40% compared to WT, the xylem cell wall thickness increased by 21.6% compared to the WT. Under mannitol-simulated drought stress, the root length of transgenic tobacco is 64% longer than WT, and the seed germination rate of the transgenic lines is 47% higher than that of WT. In addition, the H2O2 content in the transgenic tobacco was 22% lower than that of WT, while the POD and SOD content was higher than WT by 30 and 24% respectively, which showed Fm4CL-like 1 affect the accumulation of reactive oxygen species (ROS). The MDA content and relative conductivity was 25 and 15% lower than WT, respectively. The water loss rate is 16.7% lower than that of WT. The relative expression levels of stress-related genes NtHAK, NtAPX, NtCAT, NtABF2, and NtZFP were higher than those of WT under stress treatment. The stomatal apertures of OE (Overexpression) were 30% smaller than those of WT, and the photosynthetic rate of OE was 48% higher than that of WT. These results showed that the overexpression line exhibited stronger adaptability to osmotic stress than WT. CONCLUSIONS: Our results indicate that Fm4CL-like 1 is involved in secondary cell wall development and lignin synthesis. Fm4CL-like 1 play an important role in osmotic stress by affecting cell wall and stomatal development.

Arabinose biosynthesis is critical for salt stress tolerance in Arabidopsis.

The capability to maintain cell wall integrity is critical for plants to adapt to unfavourable conditions. l-Arabinose (Ara) is a constituent of several cell wall polysaccharides and many cell wall-localised glycoproteins, but so far the contribution of Ara metabolism to abiotic stress tolerance is still poorly understood. Here, we report that mutations in the MUR4 (also known as HSR8) gene, which is required for the biosynthesis of UDP-Arap in Arabidopsis, led to reduced root elongation under high concentrations of NaCl, KCl, NaNO3 , or KNO3 . The short root phenotype of the mur4/hsr8 mutants under high salinity is rescued by exogenous Ara or gum arabic, a commercial product of arabinogalactan proteins (AGPs) from Acacia senegal. Mutation of the MUR4 gene led to abnormal cell-cell adhesion under salt stress. MUR4 forms either a homodimer or heterodimers with its isoforms. Analysis of the higher order mutants of MUR4 with its three paralogues, MURL, DUR, MEE25, reveals that the paralogues of MUR4 also contribute to the biosynthesis of UDP-Ara and are critical for root elongation. Taken together, our work revealed the importance of the Ara metabolism in salt stress tolerance and also provides new insights into the enzymes involved in the UDP-Ara biosynthesis in plants.

Molecular cloning and functional analysis of a UV-B photoreceptor gene, BpUVR8 (UV Resistance Locus 8), from birch and its role in ABA response.

As a photoreceptor specifically for UV-B light, UVR8 gene plays an important role in the photomorphogenesis and developmental growth of plants. In this research, we isolated the UVR8 gene from birch, named BpUVR8 (AHY02156). BpUVR8 overexpression rescued the uvr8 mutant phenotype using functional complementation assay of BpUVR8 in Arabidopsis uvr8 mutants, which showed that the function of UVR8 is conserved between Arabidopsis and birch. The expression analysis of BpUVR8 indicated that this gene is expressed in various tissues, but its expression levels in leaves are higher than in other organs. Moreover, abiotic stress factors, such as UV-B, salinity, and abscisic acid (ABA) can induce the expression of BpUVR8 gene. Interestingly, the analysis of promoter activity indicated that BpUVR8 promoter not only has the promoting activity but can also respond to the induction of abiotic stress and ABA signal. So, we analyzed its function in ABA response via transgenic UVR8 overexpression in Arabidopsis. The BpUVR8 enhances the susceptibility to ABA, which indicates that BpUVR8 is regulated by ABA and can inhibit seed germination. The root length of 20-day-old 35S::BpUVR8/WT transgenic plants was 18% reduced as compared to the wild-type under the ABA treatment. The membrane of the BpUVR8-overexpressing in Arabidopsis thaliana was the most damaged after ABA treatment and 35S::BpUVR8/WT transgenic plant was more sensitive to ABA than the wild type. These results showed that BpUVR8 is a positive regulator in the ABA signal transduction pathway. In the presence of low dose of UV-B, the sensitivity of wild-type and 35S::BpUVR8/WT plants to ABA was reduced. Moreover, BpUVR8 regulates the expression of a subset of ABA-responsive genes, both in Arabidopsis and Betula platyphylla, under the ABA treatment. Our data provide evidence that BpUVR8 is a positive regulator in the UV-B-induced photomorphogenesis in plants. Moreover, we propose from this research that BpUVR8 might have an important role in integrating plant growth and ABA signaling pathway.

Crosstalk among nitric oxide, calcium and reactive oxygen species during triterpenoid biosynthesis in Betula platyphylla.

We analysed NO, reactive oxygen species (ROS) and Ca2+ crosstalk during triterpenoid biosynthesis in white birch (Betula platyphylla Suk.) cells. Cells were pretreated with diphenyleneiodonium, sodium diethyldithiocarbamate (DDTC) or catalase (CAT), or a Ca2+ channel blocker or chelator before sodium nitroprusside treatment. Changes in triterpenoid, malondialdehyde and proline levels, cell viability, and CAT, ascorbate peroxidase and peroxidase activity were recorded. Furthermore, enzyme gene expression levels related to triterpene biosynthesis, endogenous signalling and antioxidase activity, and cell apoptosis and death rates were measured. Sodium nitroprusside elevated ROS and Ca2+ levels. Oleanolic acid levels in cells pretreated with diphenyleneiodonium and CAT reduced significantly, but it increased with DDTC pretreatment. ROS inhibition downregulated BpDXR, BpCALM and BpNIA expression. Oleanolic acid, BpMnSOD expression, and CAT, ascorbate peroxidase and peroxidase activities reduced when the Ca2+ signalling pathway was blocked. The apoptosis rates of cells pretreated with DDTC and CAT decreased significantly; cell death rates also reduced in groups Ca2+ pretreated with channel blocker and chelator . Thus ROS and Ca2+ participate in triterpenoid biosynthesis, cell apoptosis and death induced by exogenous NO application. Further, NO causes oxidative stress and restricts the level of intracellular ROS through the Ca2+ signalling pathway.

Genome-scale transcriptome analysis in response to nitric oxide in birch cells: implications of the triterpene biosynthetic pathway.

Evidence supporting nitric oxide (NO) as a mediator of plant biochemistry continues to grow, but its functions at the molecular level remains poorly understood and, in some cases, controversial. To study the role of NO at the transcriptional level in Betula platyphylla cells, we conducted a genome-scale transcriptome analysis of these cells. The transcriptome of untreated birch cells and those treated by sodium nitroprusside (SNP) were analyzed using the Solexa sequencing. Data were collected by sequencing cDNA libraries of birch cells, which had a long period to adapt to the suspension culture conditions before SNP-treated cells and untreated cells were sampled. Among the 34,100 UniGenes detected, BLASTX search revealed that 20,631 genes showed significant (E-values≤10-5) sequence similarity with proteins from the NR-database. Numerous expressed sequence tags (i.e., 1374) were identified as differentially expressed between the 12 h SNP-treated cells and control cells samples: 403 up-regulated and 971 down-regulated. From this, we specifically examined a core set of NO-related transcripts. The altered expression levels of several transcripts, as determined by transcriptome analysis, was confirmed by qRT-PCR. The results of transcriptome analysis, gene expression quantification, the content of triterpenoid and activities of defensive enzymes elucidated NO has a significant effect on many processes including triterpenoid production, carbohydrate metabolism and cell wall biosynthesis.

Stability of transgenes in long-term micropropagation of plants of transgenic birch (Betula platyphylla).

The stability of integration and expression level of transgenes in long-term micropropagation clones of transgenic birch (Betula platyphylla Suk.) was examined. Multiplexed PCR and reverse primer PCR demonstrated stable integration of transgenes into regenerated plants. Expression levels of the bgt and gus genes among shoot plantlets, subcultured 4, 7, 9 and 15 times, were significantly different. The transcriptional expression level of extraneous genes in regenerated plants decreased with increasing subculture number. Transcriptional gene silencing (TGS) occured in regenerated transgenic lines. The silencing rate of GUS in the 5th subculture plants was 22-65%. TGS in regenerated plants could be reactivated with 5-azacytidine (Azac) at 50-200 microM. GUS and BGT protein expression was reactivated in the micropropagated transgenic birch plants when treated with Azac. A decrease in expression level with increasing number of subcultures is thus associated with DNA methylation.

Progress on molecular mechanism of T-DNA transport and integration.

Agrobacterium-mediated transformation is probably the most widely used method to introduce genes into plants. Great progress has been made in recent years in studies on the mechanism of Agrobacterium-mediated transformation. Agrobacterium genetically transforms plants by transferring a portion of the resident Ti-plasmid, the T-DNA, to the plant. VirD2 and VirE2 accompany the T-DNA into the plant cell. Both proteins may aid in T-DNA transfer, nuclear targeting and integration into the plant genome. In recent years, some Arabidopsis rat (resistant to transformation) mutants are deficient in T-DNA integration according to some studies. These results showed that plant genes participate in the T-DNA transport and integration process. This paper discusses our current knowledge about the functions of virulence protein, namely VirD2 and VirE2, and plant genes in several aspect of Agrobacterium transformation. The paper discusses two different classes of integration patterns in detail: one is T-DNA right border being linked to genomic sequences by the VirD2 protein, the other is T-DNA integration via SDSA (synthesis-dependent strand-annealing). According to the latest studies we elaborated the T-DNA integration model based on genomic DSB (double-strand breaks) and proposed a new opinion about the mechanism of T-DNA integration.