Heritable epigenetic modification of BpPIN1 is associated with leaf shapes in Betula pendula.

The new variety Betula pendula 'Dalecarlica', selected from Betula pendula, shows high ornamental value owing to its lobed leaf shape. In this study, to identify the genetic components of leaf shape formation, we performed bulked segregant analysis and molecular marker-based fine mapping to identify the causal gene responsible for lobed leaves in B. pendula 'Dalecarlica'. The most significant variations associated with leaf shape were identified within the gene BpPIN1 encoding a member of the PIN-FORMED family, responsible for the auxin efflux carrier. We further confirmed the hypomethylation at the promoter region promoting the expression level of BpPIN1, which causes stronger and longer veins and lobed leaf shape in B. pendula 'Dalecarlica'. These results indicated that DNA methylation at the BpPIN1 promoter region is associated with leaf shapes in B. pendula. Our findings revealed an epigenetic mechanism of BpPIN1 in the regulation of leaf shape in Betula  Linn. (birch), which could help in the molecular breeding of ornamental traits.

Analysis of oxidase activity and transcriptomic changes related to cutting propagation of hybrid larch.

Hybrid larch is the main timber and afforestation tree species in Northeast China. To solve the problem of rooting difficulties in larch cutting propagation, enzyme activity determination and transcriptome sequencing were carried out on the rooting tissues at five timepoints after cutting. peroxidase (POD), indole acetic acid oxidase (IAAO) and polyphenol oxidase (PPO) play important roles in the larch rooting process after cutting. A total of 101.20 Gb of clean data was obtained by transcriptome sequencing, and 43,246 unigenes were obtained after further screening and assembly. According to GO analysis and KEGG enrichment analysis, we think that plant hormones play an important role in the rooting process of larch stem cuttings. in the plant hormone signal transduction pathway, a larch gene c141104.graph_c0 that is homologous to the Arabidopsis AUX1 was found to be significantly up-regulated. We suggest that AUX1 may promote IAA transport in larch, thus affecting adventitious root development. According to the results of POD, PPO IAAO indexes and GO analysis, we think s1 and s2 periods may be important periods in the rooting process of larch stem cuttings, so we built a gene regulatory network, a total of 14genes, including LBD, NAC, AP2/ERF, bHLH and etc., may be important in different stages of cutting propagation. As the rooting rate after cutting inhibits the development of larch clone propagation, identifying the genes that regulate rooting could help us to preliminarily understand the molecular mechanism of adventitious root formation and select a better treatment method for cutting propagation.

Anatomical observation and transcriptome analysis of buds reveal the association between the AP2 gene family and reproductive induction in hybrid larch (Larix kaempferi × Larix olgensis).

Hybrid larch is an excellent afforestation species in northern China. The instability of seed yield is an urgent problem to be solved. The biological characteristics related to seed setting in larch are different from those in angiosperms and other gymnosperms. Studying the developmental mechanism of the larch sporophyll can deepen our understanding of conifer reproductive development and help to ensure an adequate supply of seeds in the seed orchard. The results showed that the formation of microstrobilus primordia in hybrid larch could be observed in anatomical sections collected in the middle of July. The contents of endogenous gibberellin 3 (GA3) and abscisic acid (ABA) were higher and the contents of GA4, GA7, jasmonic acid and salicylic acid were lower in multiseeded larch. Transcriptome analysis showed that transcription factors were significantly enriched in the AP2 family. There were 23 differentially expressed genes in the buds of the multiseeded and less-seeded types, and the expression of most of these genes was higher in the buds than in the needles. We conclude that mid-July is the early stage of reproductive organ development in hybrid larch and is suitable for the study of reproductive development. GA3 and ABA may be helpful for improving seed setting in larch, and 23 AP2/EREBP family genes are involved in the regulation of reproductive development in larch.

Molecular mechanism of leaf adaxial upward curling caused by BpPIN3 suppression in Betula pendula.

Leaves are one of the vegetative organs of plants that are essential for plant growth and development. PIN-FORMED (PINs) gene is an indoleacetic acid (IAA) transporter that plays a critical role in leaf development. To determine the function of BpPIN3 in leaf polarity formation in Betula pendula, the transgenic lines with BpPIN3 overexpression (OE) and BpPIN3-reduced expression (RE) were analyzed using the Agrobacterium-mediated method. The RE lines displayed the characteristics of leaf margin adaxial upward curling, with lower expression of BpPIN3 resulting in greater rolling. Tissue localization of IAA in the auxin GUS reporter system proved that auxin in the RE was mainly distributed in the secondary veins, palisade tissues, and epidermal cells in the leaf margin area. The auxin content in the leaf margin area was significantly greater than that in the main vein tissue. The cell density of the palisade tissue and the ratio of palisade tissue to spongy tissue in the curled leaf margin of the RE lines were found to be significantly decreased. RNA-seq analysis revealed that the RE hormone-signaling pathway genes were significantly enriched compared with those of the OE and WT lines; in particular, the auxin response-related genes SAURs (i.e., SAUR23, SAUR24, SAUR28, and SAUR50) and GH3.10 were found to be significantly upregulated. qRT-PCR analysis indicated that BpPIN3 expression at the leaf margin was significantly lower than that near the main vein in the RE lines. In contrast, the expression levels of SAURs and GH3.10 were significantly higher than those near the midrib. In conclusion, BpPIN3 regulates the expression of auxin response-related genes and the polar transport of auxin to change the polar form of the proximal and distal axes of birch leaves.

PsnWRKY70 Negatively Regulates NaHCO3 Tolerance in Populus.

Poplar is an important afforestation and ornamental tree species in Northeast China. The distribution area of saline-alkali land is approximately 765 hm2 in Northeast China. The breeding of saline-alkali-resistant transgenic trees could be an effective method of afforestation in saline-alkali land. WRKY transcription factors play a crucial role in abiotic stress. In this study, we analyzed the genetic stability of the two-year-old PsnWRKY70 transgenic poplars. The results showed that PsnWRKY70 of transgenic poplars had been expressed stably and normally at the mRNA level. The gene interference expression (RE) lines had no significant effect on the growth of PsnWRKY70 under NaHCO3 stress, and the alkali damage index of RE lines was significantly lower than that of WT and overexpression (OE) lines at day 15 under NaHCO3 stress. POD activity was significantly higher in RE lines than in WT. The MDA content of the RE line was lower than that of the WT line. Transcriptome analysis showed that RE lines up-regulated genes enriched in cell wall organization or biogenesis pathway-related genes such as EXPA8, EXPA4, EXPA3, EXPA1, EXPB3, EXP10, PME53, PME34, PME36, XTH9, XTH6, XTH23, CESA1, CESA3, CES9; FLA11, FLA16 and FLA7 genes. These genes play an important role in NaHCO3 stress. Our study showed that the interference expression of the PsnWRKY70 gene can enhance the tolerance of NaHCO3 in poplar.

Role of PsnWRKY70 in Regulatory Network Response to Infection with Alternaria alternata (Fr.) Keissl in Populus.

WRKY is an important complex family of transcription factors involved in plant immune responses. Among them, WRKY70 plays an important role in the process of the plant defense response to the invasion of pathogens. However, the defense mechanism of PsnWRKY70 is not clear in Populus nigra. In this study, we showed that PsnWRKY70-overexpression lines (OE) had fewer leaf blight symptoms than PsnWRKY70-repressing lines (RE). PsnWRKY70 activated MAP kinase cascade genes (PsnM2K4, PsnMPK3, PsnM3K18), calcium channel proteins-related genes (PsnCNG3, PsnCNGC1, PsnCNG4), and calcium-dependent protein kinases genes (PsnCDPKL, PsnCDPKW, PsnCDPKS, PsnCDPKQ). Furthermore, 129 genes of PsnWRKY70 putative genome-wide direct targets (DTGs) were identified by using transcriptome (RNA-seq) and DNA affinity purification sequencing (DAP-seq). PsnWRKY70 directly binds to the promoters of homologous genes and LRR domain proteins to promote the expression of WRKY6, WRKY18, WRKY22, and WRKY22-1, LRR domain proteins LRR8, LRR-RLK, ADR1-like 2, NB-ARC, etc. Our study suggests that PsnWRKY70 enhances the resistance of A. alternata in poplar by activating genes in both pathogen-associated molecular pattern-triggered immunity (PTI) and effector-triggered immunity (ETI).

BpEIN3.1 represses leaf senescence by inhibiting synthesis of ethylene and abscisic acid in Betula platyphylla.

Leaf senescence and abscission play crucial role in annual plant adapting to seasonal alteration and climate changes by shortening life cycle and development process in response to abiotic and/or biotic stressors underlying phytohormones and environmental signals. Ethylene and abscisic acid are the major phytohormones that promotes leaf senescence, involving various transcription factors, such as EIN3 (ethylene-insensitive 3) and EIL (ethylene-insensitive 3-like) gene family, controlling leaf senescence through metabolite biosynthesis and signal transduction pathways. However, the roles of EIN3 regulating leaf senescence responding to environmental changes in perennial plant, especially forestry tree, remain unclear. In this study, we found that BpEIN3.1 from a subordinated to EIL3 subclade, is a transcription repressor and regulated light-dependent premature leaf senescence in birch (Betula platyphylla). BpEIN3.1 might inhibits the transcription of BpATPS1 by binding to its promoter. Shading suppressed premature leaf senescence in birch ein3.1 mutant line. Ethylene and abscisic acid biosynthesis were also reduced. In addition, abscisic acid positively regulated the expression of BpEIN3.1. This was demonstrated by the hormone-response element analysis of BpEIN3.1 promoter and its gene expression after the hormone treatments. Moreover, our results showed that abscisic acid is also involved in maintaining homeostasis. The molecular mechanism of leaf senescence provides a possibility to increasing wood production by delaying of leaf senescence.

Chloroplast (Cp) Transcriptome of P. davidiana Dode×P. bolleana Lauch provides insight into the Cp drought response and Populus Cp phylogeny.

BACKGROUND: Raw second-generation (2G) lignocellulosic biomass materials have the potential for development into a sustainable and renewable source of energy. Poplar is regarded as a promising 2G material (P. davidiana Dode×P. bolleana Lauch, P. bolleana, P. davidiana, P. euphratica, et al). However, their large-scale commercialization still faces many obstacles. For example, drought prevents sufficient irrigation or rainfall, which can reduce soil moisture and eventually destroy the chloroplast, the plant photosynthetic organelle. Heterosis is widely used in the production of drought-tolerant materials, such as the superior clone "Shanxinyang" selected from the offspring of Populus davidiana Dode×Populus bolleana Lauch. Because it produces good wood and is easily genetically transformed, "Shanxinyang" has become a promising material for use in tree genetics. It is also one of the most abundant biofuel plants in northern China. Understanding the genetic features of chloroplasts, the cp transcriptome and physiology is crucial to elucidating the chloroplast drought-response model. RESULTS: In this study, the whole genome of "Shanxinyang" was sequenced. The chloroplast genome was assembled, and chloroplast structure was analysed and compared with that of other popular plants. Chloroplast transcriptome analysis was performed under drought conditions. The total length of the "Shanxinyang" chloroplast genome was 156,190 bp, the GC content was 36.75%, and the genome was composed of four typical areas (LSC, IRa, IRb, and SSC). A total of 114 simple repeats were detected in the chloroplast genome of "Shanxinyang". In cp transcriptome analysis, we found 161 up-regulated and 157 down-regulated genes under drought, and 9 cpDEGs was randomly selected to conduct reverse transcription (RT)-qPCR., in which the Log2 (fold change) was significantly consistent with the qPCR results. The analysis of chloroplast transcription under drought provided clues for understanding chloroplast function under drought. The phylogenetic position of "Shanxinyang" within Populus was analysed by using the chloroplast genome sequences of 23 Populus plants, showing that "Shanxinyang" belongs to Sect. Populus and is sister to Populus davidiana. Further, mVISTA analysis showed that the variation in non-coding (regulatory) regions was greater than that in coding regions, which suggests that further attention should be paid to the chloroplast in order to obtain new evolutionary or functional insights related to aspects of plant biology. CONCLUSIONS: Our findings indicate that complex prokaryotic genome regulation occurs when processing transcripts under drought stress. The results not only offer clues for understanding the chloroplast genome and transcription features in woody plants but also serve as a basis for future molecular studies on poplar species.

Functional Study of BpPP2C1 Revealed Its Role in Salt Stress in Betula platyphylla.

PP2C protein phosphatase family is one of the largest gene families in the plant genome. Many PP2C family members are involved in the regulation of abiotic stress. We found that BpPP2C1 gene has highly up-regulated in root under salt stress in Betula platyphylla. Thus, transgenic plants of Betula platyphylla with overexpression and knockout of BpPP2C1 gene were generated using a zygote transformation system. Under NaCl stress treatment, we measured the phenotypic traits of transgenic plants, chlorophyll-fluorescence parameters, peroxidase (POD) activity, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) content. We found that BpPP2C1 overexpressed lines showed obvious salt tolerance, while BpPP2C1 knocked out plants were sensitive to salt stress. Transcriptome analysis identified significantly amount of differentially expressed genes associated with salt stress in BpPP2C1 transgenic lines, especially genes in abscisic acid signaling pathway, flavonoid biosynthetic pathway, oxidative stress and anion transport. Functional study of BpPP2C1 in Betula platyphylla revealed its role in salt stress.

Characterization and T-DNA insertion sites identification of a multiple-branches mutant br in Betula platyphylla × Betula pendula.

BACKGROUND: Plant architecture, which is mostly determined by shoot branching, plays an important role in plant growth and development. Thus, it is essential to explore the regulatory molecular mechanism of branching patterns based on the economic and ecological importance. In our previous work, a multiple-branches birch mutant br was identified from 19 CINNAMOYL-COENZYME A REDUCTASE 1 (CCR1)-overexpressed transgenic lines, and the expression patterns of differentially expressed genes in br were analyzed. In this study, we further explored some other characteristics of br, including plant architecture, wood properties, photosynthetic characteristics, and IAA and Zeatin contents. Meanwhile, the T-DNA insertion sites caused by the insertion of exogenous BpCCR1 in br were identified to explain the causes of the mutation phenotypes. RESULTS: The mutant br exhibited slower growth, more abundant and weaker branches, and lower wood basic density and lignin content than BpCCR1 transgenic line (OE2) and wild type (WT). Compared to WT and OE2, br had high stomatal conductance (Gs), transpiration rate (Tr), but a low non-photochemical quenching coefficient (NPQ) and chlorophyll content. In addition, br displayed an equal IAA and Zeatin content ratio of main branches' apical buds to lateral branches' apical buds and high ratio of Zeatin to IAA content. Two T-DNA insertion sites caused by the insertion of exogenous BpCCR1 in br genome were found. On one site, chromosome 2 (Chr2), no known gene was detected on the flanking sequence. The other site was on Chr5, with an insertion of 388 bp T-DNA sequence, resulting in deletion of 107 bp 5' untranslated region (UTR) and 264 bp coding sequence (CDS) on CORONATINE INSENSITIVE 1 (BpCOII). In comparison with OE2 and WT, BpCOI1 was down-regulated in br, and the sensitivity of br to Methyl Jasmonate (MeJA) was abnormal. CONCLUSIONS: Plant architecture, wood properties, photosynthetic characteristics, and IAA and Zeatin contents in main and lateral branches' apical buds changed in br over the study's time period. One T-DNA insertion was identified on the first exon of BpCOI1, which resulted in the reduction of BpCOI1 expression and abnormal perception to MeJA in br. These mutation phenotypes might be associated with a partial loss of BpCOI1 in birch.