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Chlorophyll (Chl) is essential for plants to carry out photosynthesis, growth and development processes. Growth‐regulating factors (GRFs) play a vital role in regulating Chl degradation in plants. However, the molecular mechanism by which GRF5 regulates Chl degradation in poplar remains unknown. Here we found that overexpression of PpnGRF5‐1 increased Chl content in leaves and promoted chloroplast development in poplar. Overexpression of PpnGRF5‐1 in poplar delayed Chl degradation induced by external factors, such as hormones, darkness and salt stress. PpnGRF5‐1 responded to brassinosteroid (BR) signalling during BR‐induced Chl degradation and reduced the expression levels of Chl degradation and senescence‐related genes. PpnGRF5‐1 inhibited the expression of Chl b reductases PagNYC1 and PagNOL. PpnGRF5‐1 could interact with PagBZR1 in the nucleus. PagBZR1 also inhibited the expression of PagNYC1. In addition, we found that the protein–protein interaction between PagBZR1 and PpnGRF5‐1 enhanced the inhibitory effect of PpnGRF5‐1 on the Chl b reductases PagNYC1 and PagNOL. BZR1 and GRF5‐1 were upregulated, and NOL and NYC1 were downregulated in triploid poplars compared to diploids. This study revealed a new mechanism by which PpnGRF5‐1 regulates Chl degradation in poplars and lays the foundation for comprehensively analysing the molecular mechanism of Chl metabolism in triploid poplars. [ABSTRACT FROM AUTHOR]

Summary: Although polyploid plants have lower stomatal density than their diploid counterparts, the molecular mechanisms underlying this difference remain elusive.Here, we constructed a network based on the triploid poplar transcriptome data and triple‐gene mutual interaction algorithm and found that PpnMYC2 was related to stomatal development‐related genes PpnEPF2, PpnEPFL4, and PpnEPFL9. The interactions between PpnMYC2 and PagJAZs were experimentally validated. PpnMYC2‐overexpressing poplar and Arabidopsis thaliana had reduced stomatal density. Poplar overexpressing PpnMYC2 had higher water use efficiency and drought resistance.RNA‐sequencing data of poplars overexpressing PpnMYC2 showed that PpnMYC2 promotes the expression of stomatal density inhibitors PagEPF2 and PagEPFL4 and inhibits the expression of the stomatal density‐positive regulator PagEPFL9. Yeast one‐hybrid system, electrophoretic mobility shift assay, ChIP‐qPCR, and dual‐luciferase assay were employed to substantiate that PpnMYC2 directly regulated PagEPF2, PagEPFL4, and PagEPFL9. PpnMYC2, PpnEPF2, and PpnEPFL4 were significantly upregulated, whereas PpnEPFL9 was downregulated during stomatal formation in triploid poplar.Our results are of great significance for revealing the regulation mechanism of plant stomatal occurrence and polyploid stomatal density, as well as reducing stomatal density and improving plant water use efficiency by overexpressing MYC2. [ABSTRACT FROM AUTHOR]

Background: Lignin is a major restriction factor for the industrial production of biomass resources, such as pulp and bioenergy. Eucalyptus is one of the most important sources of pulp and bioenergy. After polyploidization, the lignin content of forest trees is generally reduced, which is considered a beneficial genetic improvement. However, the differences in the lignin content between triploid and diploid Eucalyptus and the underlying regulatory mechanism are still unclear. Results: We conducted a comprehensive analysis at the phenotypic, transcriptional and metabolite levels between Eucalyptus urophylla triploids and diploids to reveal the effects of polyploidization on the lignin content and lignin metabolic pathway. The results showed that the lignin content of Eucalyptus urophylla triploid stems was significantly lower than that of diploids. Lignin-related metabolites were differentially accumulated between triploids and diploids, among which coniferaldehyde, p-coumaryl alcohol, sinapaldehyde and coniferyl alcohol had significant positive correlations with lignin content, indicating that they might be primarily contributing metabolites. Most lignin biosynthetic genes were significantly downregulated, among which 11 genes were significantly positively correlated with the lignin content and above metabolites. Furthermore, we constructed a co-expression network between lignin biosynthetic genes and transcription factors based on weighted gene co-expression network analysis. The network identified some putative orthologues of secondary cell wall (SCW)-related transcription factors, among which MYB52, MYB42, NAC076, and LBD15 were significantly downregulated in Eucalyptus urophylla triploids. In addition, potential important transcription factors, including HSL1, BEE3, HHO3, and NAC046, also had high degrees of connectivity and high edge weights with lignin biosynthetic genes, indicating that they might also be involved in the variation of lignin accumulation between triploid and diploid Eucalyptus urophylla. Conclusions: The results demonstrated that some lignin-related metabolites, lignin biosynthetic genes and transcription factors in Eucalyptus urophylla triploids may be relatively sensitive in response to the polyploidization effect, significantly changing their expression levels, which ultimately correlated with the varied lignin content. The analysis of the underlying formation mechanism could provide beneficial information for the development and utilization of polyploid biomass resources, which will be also valuable for genetic improvement in other bioenergy plants. [ABSTRACT FROM AUTHOR]

Background: Triploid Populus tomentosa, a timber tree species, has been widely planted in northern China owing to its potential high yields and high wood quality. Though genetic variances in growth traits and wood properties have been reported across several planting sites, regional testing of triploid hybrid clones of P. tomentosa has not been conducted on a large scale. Results: Ten 5-year clonal trials were used to evaluate the inheritance of growth traits, to determine suitable deployment zones, and to identify optimal triploid clones at each experimental site to determine the clones that would be suitable at all sites. A total of 2,430 trees from nine triploid hybrid clones were sampled during the ten trials. The clonal and site effects and clone × site interactions were highly significant (P < 0.001) for all the studied growth and yield traits. The estimated repeatability of means for diameter at breast height (DBH) and tree height (H) was 0.83, which was slightly higher than for stem volume (SV) and estimated stand volume (ESV) (0.78). The Weixian (WX), Gaotang (GT), and Yanzhou (YZ) sites were each considered to be suitable deployment zones, and the Zhengzhou (ZZ), Taiyuan (TY), Pinggu (PG), and Xiangfen (XF) sites were found to be the optimal deployment zones. The TY and ZZ sites were the best discriminative environments, and the GT and XF sites were the best representative environments. GGE pilot analysis revealed that yield performance and stability were significantly different among all the studied triploid hybrid clones across the ten test sites. It was therefore necessary to develop a suitable triploid hybrid clone that could do well at each site. Taking into account both yield performance and stability, the triploid hybrid clone S2 was determined to be an ideal genotype. Conclusions: For triploid hybrid clones, the WX, GT, and YZ sites represented suitable deployment zones and the ZZ, TY, PG, and XF sites represented optimal deployment zones. Yield performance and stability were significantly different among all the studied triploid hybrid clones across the ten test sites. Developing a suitable triploid hybrid clone that could do well at all sites was therefore desirable. [ABSTRACT FROM AUTHOR]

Background: Agrobacterium tumefaciens-mediated leaf disc genetic transformation is an important way to achieve transgenics or gene editing. Ensuring stable and efficient genetic transformation is still an important problem in modern biology. It is assumed that the difference in the development status of genetic transformation cells of receptor materials is the main reason for the difference and instability of genetic transformation efficiency; the stable and efficient genetic transformation rate can be obtained by defining the appropriate treatment period of the receptor material and applying genetic transformation in a timely manner. Results: Based on these assumptions, we studied and established an efficient and stable Agrobacterium-mediated plant transformation system with hybrid poplar (Populus alba × Populus glandulosa, 84 K) leaves, stem segments and tobacco leaves as the research objects. There were differences in the development process of leaf bud primordial cells from different explants, and the genetic transformation efficiency was significantly related to the cell development stage of the in vitro cultured materials. Among them, the genetic transformation rate of poplar and tobacco leaves was the highest on the 3rd and 2nd day of culture, reaching 86.6% and 57.3%, respectively. The genetic transformation rate of poplar stem segments was the highest on the 4th day of culture, reaching 77.8%. The best treatment period was from the development of leaf bud primordial cells to the S phase of the cell cycle. The number of cells detected using flow cytometry and 5-ethynyl-2ʹ-deoxyuridine (EdU) staining, the expression of cell cycle-related protein CDKB1; 2, CDKD1; 1, CYCA3; 4, CYCD1; 1, CYCD3; 2, CYCD6; 1, and CYCH; 1 of explants, and morphological changes of explants can be used as indicators to determine the appropriate treatment period for genetic transformation. Conclusions: Our study provides a new and universal set of methods and characteristics to identify the S phase of the cell cycle and apply genetic transformation treatments at the appropriate time. Our results are of great significance for improving the efficiency and stability of plant leaf disc genetic transformation. [ABSTRACT FROM AUTHOR]

Poplar is an important fast-growing tree, and its photosynthetic capacity directly affects its vegetative growth. Stomatal density is closely related to photosynthetic capacity and growth characteristics in plants. Here, we isolated PagSTOMAGEN from the hybrid poplar (Populus alba × Populus glandulosa) clone 84K and investigated its biological function in vegetative growth. PagSTOMAGEN was expressed predominantly in young tissues and localized in the plasma membrane. Compared with wild-type 84K poplars, PagSTOMAGEN-overexpressing plants displayed an increased plant height, leaf area, internode number, basal diameter, biomass, IAA content, IPR content, and stomatal density. Higher stomatal density improved the net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, and transpiration rate in transgenic poplar. The differential expression of genes related to stomatal development showed a diverged influence of PagSTOMAGEN at different stages of stomatal development. Finally, transcriptomic analysis showed that PagSTOMAGEN affected vegetative growth by affecting the expression of photosynthesis and plant hormone-related genes (such as SAUR75, PQL2, PSBX, ERF1, GNC, GRF5, and ARF11). Taken together, our data indicate that PagSTOMAGEN could positively regulate stomatal density and increase the photosynthetic rate and plant hormone content, thereby promoting vegetative growth in poplar. Our study is of great significance for understanding the relationship between stoma, photosynthesis, and yield breeding in poplar. [ABSTRACT FROM AUTHOR]

Breeding for dwarfism is an important approach to improve lodging resistance. Here, we performed comparative analysis of the phenotype, transcriptome, and hormone contents between diploids and tetraploids of poplar 84K (Populus alba × P. glandulosa). Compared with diploids, the indole-3-acetic acid (IAA) and gibberellin (GA3) contents were increased, whereas the jasmonic acid (JA) and abscisic acid (ABA) contents were decreased in tetraploids. RNA-sequencing revealed that differentially expressed genes (DEGs) in leaves of tetraploids were mainly involved in plant hormone pathways. Most DEGs associated with IAA and GA promotion of plant growth and development were downregulated, whereas most DEGs associated with ABA and JA promotion of plant senescence were upregulated. Weighted gene co-expression network analysis indicated that certain transcription factors may be involved in the regulation of genes involved in plant hormone pathways. Thus, the altered expression of some genes in the plant hormone pathways may lead to a reduction in IAA and GA contents, as well as an elevation in ABA and JA contents, resulting in the dwarfing of tetraploids. The results show that polyploidization is a complex biological process affected by multiple plant hormone signals, and it provides a foundation for further exploration of the mechanism of tetraploids dwarfing in forest trees. [ABSTRACT FROM AUTHOR]

Woody biomass feedstock produced from hybrid poplar can be converted into bioenergy via thermochemical and biochemical processes. To better understand the genetic control of key properties (biomass production, extractives, holocellulose, lignin, and higher heating value) that relate to the quality of biomass feedstock in triploid hybrid clones of Populus tomentosa, genetic relationships among selected traits were examined in 5-year-old clonal field trials located in Yanzhou, Gaotang, Xiangfen, Zhengzhou, and Taiyuan, northern China. In total, 270 trees from nine clones were sampled from the five sites. Site had a highly significant ( P < 0.001) effect on biomass production, extractives, lignin, and higher heating value, and it had a moderate effect on holocellulose. Clonal effects were also significant ( P < 0.05) for all studied traits except for holocellulose. Clone × site interactions were significant for all studied traits except for holocellulose. The estimated repeatability of clonal means for biomass production (0.81) was higher than for lignin (0.75), extractives (0.68), higher heating value (0.58), and holocellulose (0.54). A negative or significantly negative correlation between holocellulose, extractives, lignin, and higher heating value was observed at each site. This suggests that selection for holocellulose might lead to a reduction in extractives, lignin, and higher heating value in triploid breeding of P. tomentosa. [ABSTRACT FROM AUTHOR]

Eucommia ulmoides is a temperate gum source plant that produces trans-polyisoprene (TPI), also known as Eucommia rubber. The structural configuration and function of TPI offer a new material with important potential for industrial development. In this study, we detected the TPI content in the leaves of diploid and triploid E. ulmoides plants. The average TPI content in the leaves of triploid E. ulmoides was significantly higher than that of diploid. Transcriptome data and weighted gene co-expression network analyses identified a significant positive correlation between the EuFPS1 gene and TPI content. Overexpression of EuFPS1 increased the density of rubber particles and TPI content, indicating its crucial role in TPI biosynthesis. In addition, the expression of EuHDZ25 in E. ulmoides was significantly positively correlated with EuFPS1 expression. Yeast one-hybrid and dual-luciferase assays demonstrated that EuHDZ25 mainly promotes TPI biosynthesis through positive regulation of EuFPS1 expression. The significantly up-regulated expression of EuHDZ25 and its consequent upregulation of EuFPS1 during the biosynthesis of TPI may partially explain the increased TPI content of triploids. This study provides an important theoretical foundation for further exploring the molecular mechanism of secondary metabolites content variation in polyploids and can help to promote the development and utilization of rubber resources. [ABSTRACT FROM AUTHOR]

Eucommia rubber is a secondary metabolite from Eucommia ulmoides that has attracted much attention because of its unique properties and enormous potential for application. However, the transcriptional mechanism regulating its biosynthesis has not yet been determined. Farnesyl pyrophosphate synthase is a key enzyme in the Eucommia rubber biosynthesis. In this study, the promoter of EuFPS1 was used as bait, EuWRKY30 was screened from the cDNA library of EuFPS1 via a yeast one-hybrid system. EuWRKY30 belongs to the WRKY IIa subfamily and contains a WRKY domain and a C2H2 zinc finger motif, and the expressed protein is located in the nucleus. EuWRKY30 and EuFPS1 exhibited similar tissue expression patterns, and yeast one-hybrid and dual-luciferase experiments confirmed that EuWRKY30 directly binds to the W-box element in the EuFPS1 promoter and activates its expression. Moreover, the overexpression of EuWRKY30 significantly upregulated the expression level of EuFPS1 , further increasing the density of the rubber particles and Eucommia rubber content. The results of this study indicated that EuWRKY30 positively regulates EuFPS1 , which plays a critical role in the synthesis of Eucommia rubber, provided a basis for further analysis of the underlying transcriptional regulatory mechanisms. [ABSTRACT FROM AUTHOR]

GATA transcription factors have been demonstrated to play key regulatory roles in plant growth, development, and hormonal response. However, the knowledge concerning the evolution of GATA genes in Eucalyptus urophylla and their trans-regulatory interaction is indistinct. Phylogenetic analysis and study of conserved motifs, exon structures, and expression patterns resolved the evolutionary relationships of these GATA proteins. Phylogenetic analysis showed that EgrGATAs are broadly distributed in four subfamilies. Cis-element analysis of promoters revealed that EgrGATA genes respond to light and are influenced by multiple hormones and abiotic stresses. Transcriptome analysis revealed distinct temporal and spatial expression patterns of EgrGATA genes in various tissues of E. urophylla S.T.Blake, which was confirmed by real-time quantitative PCR (RT-qPCR). Further research revealed that EurGNC and EurCGA1 were localized in the nucleus, and EurGNC directly binds to the cis-element of the EurGUN5 promoter, implying its potential roles in the regulation of chlorophyll synthesis. This comprehensive study provides new insights into the evolution of GATAs and could help to improve the photosynthetic assimilation and vegetative growth of E. urophylla at the genetic level. [ABSTRACT FROM AUTHOR]

After polyploidization, plants usually undergo some morphological and physiological changes, including the lignin content of polyploids usually becoming lower than that of diploids. However, the regulatory mechanism of the variation of lignin content in polyploid plants remains unclear. Therefore, in this research, we used full-sib poplar triploids and diploids to explore the molecular regulatory basis of lignin content in poplar triploid leaves through the determination of lignin content, the observation of xylem cells, and transcriptome sequencing. The results showed that the lignin content of triploid leaves was significantly lower than that of diploid leaves. The xylem cells of triploid leaves were significantly larger than those of diploids. Transcriptome sequencing data show that most lignin biosynthesis genes were significantly downregulated, and genes related to cell growth were mostly upregulated in triploid leaves compared with diploid leaves. In addition, co-expression network analysis showed that several transcription factors might be involved in the regulation of lignin biosynthesis. Consequently, the altered expression of genes related to lignin might lead to the reduced lignin content in triploids. These results provide a theoretical basis for further exploring the molecular mechanism of the variation of polyploid lignin content and the utilization of polyploid lignocellulosic resources. [ABSTRACT FROM AUTHOR]

DNA methylation is an epigenetic mechanism with the potential to regulate gene expression and affect plant phenotypes. Both hybridization and genome doubling may affect the DNA methylation status of newly formed allopolyploid plants. Previous studies demonstrated that changes in cytosine methylation levels and patterns were different among individual hybrid plant, therefore, studies investigating the characteristics of variation in cytosine methylation status must be conducted at the population level to avoid sampling error. In the present study, an F1 hybrid diploid population and three allotriploid populations with different heterozygosity [originating from first-division restitution (FDR), second-division restitution (SDR), and post-meiotic restitution (PMR) 2n eggs of the same female parent] were used to investigate cytosine methylation inheritance and variation relative to their common parents using methylation-sensitive amplification polymorphism (MSAP). The variation in cytosine methylation in individuals in each population exhibited substantial differences, confirming the necessity of population epigenetics. The total methylation levels of the diploid population were significantly higher than in the parents, but those of the three allotriploid populations were significantly lower than in the parents, indicating that both hybridization and polyploidization contributed to cytosine methylation variation. The vast majority of methylated status could be inherited from the parents, and the average percentages of non-additive variation were 6.29, 3.27, 5.49 and 5.07% in the diploid, FDR, SDR and PMR progeny populations, respectively. This study lays a foundation for further research on population epigenetics in allopolyploids. [ABSTRACT FROM AUTHOR]

Flooding is one of the main abiotic stresses suffered by plants. Plants respond to flooding stress through regulating their morphological structure, endogenous hormone biosynthesis, and genetic signaling transduction. We previously found that Fokienia hodginsii varieties originating from Gutian exhibited typical flooding tolerance traits compared to three other provenances (Yongzhou, Sanming, Nanping), expressed as increased height, longer diameter at breast height (DBH), and smaller branch angle. Herein, the changes in endogenous gibberellins (GA) and abscisic acid (ABA) contents were measured under flooding stress in F. hodginsii, and ABA was found to decrease, whereas GA increased with time. Furthermore, the GA and ABA contents of the varieties originating from Gutian and the three other provenances were measured, and the results indicated that F. hodginsii from Gutian could respond more rapidly to flooding stress. The transcriptomes of the varieties originating from Gutian and the other three provenances were compared using RNA sequencing to explore the underlying genetic mechanisms of the flood-resistant phenotypes in F. hodginsii. The results indicated that two flood-stress response genes (TRINITY_DN142_c0_g2 and TRINITY_DN7657_c0_g1) were highly related to both the ABA and GA response in F. hodginsii. [ABSTRACT FROM AUTHOR]

To better understand the genetic control of fiber properties (fiber length [FL], fiber width [FW] and coarseness [C]) and growth traits (tree height [H], diameter at breast height [DBH] and stem volume [SV]) in triploid hybrid clones ofPopulus tomentosacarr., genetic relationships among selected fiber properties with growth traits were examined in five-year-old clonal field trials located in Yanzhou, Gaotang and Xiangfen, China. In total, 180 trees from 10 clones were sampled from the three sites. The site had a moderate effect on FW, C and tree growth, and a highly significant (P<0.001) effect on FL and fiber length/width (FL/W). Clonal effects were also significant (P<0.05) for all studied traits except for the DBH and SV. Clone×site interactions were significant for all studied traits (except for FL). The estimated repeatability of clonal mean for FL (0.91) was higher than for FW (0.88), C (0.77), FL/W (0.74), H (0.62), DBH (0.61) and SV (0.55). Intersite genetic correlation estimates indicated that fiber properties were more stable than growth traits. Positively insignificant genetic correlation estimates between FL and growth trait and negatively insignificant genetic correlation estimates between C and growth traits were observed. This suggests that in the triploid hybrid poplar, clones selection for growth traits will not result in a significant increase or reduction in the fiber properties. [ABSTRACT FROM AUTHOR]

The serine protease SDD1 regulates stomatal density, but its potential impact on plant vegetative growth is unclear. Our study reveals a substantial upregulation of SDD1 in triploid poplar apical buds and leaves, suggesting its possible role in their growth regulation. We cloned PagSDD1 from poplar 84 K (Populus alba × P. glandulosa) and found that overexpression in poplar, soybean, and lettuce led to decreased leaf stomatal density. Furthermore, PagSDD1 represses PagEPF1 , PagEPF2 , PagEPFL9 , PagSPCH , PagMUTE , and PagFAMA expression. In contrast, PagSDD1 promotes the expression of its receptors, PagTMM and PagERECTA. PagSDD1 -OE poplars showed stronger drought tolerance than wild-type poplars. Simultaneously, PagSDD1 -OE poplar, soybean, and lettuce had vegetative growth advantages. RNA sequencing revealed a significant upregulation of genes PagLHCB2.1 and PagGRF5 , correlating positively with photosynthetic rate, and PagCYCA3;4 and PagEXPA8 linked to cell division and differentiation in PagSDD1 -OE poplars. This increase promoted leaf photosynthesis, boosted auxin and cytokinin accumulation, and enhanced vegetative growth. SDD1 overexpression can increase the biomass of poplar, soybean, and lettuce by approximately 70, 176, and 155 %, respectively, and increase the water use efficiency of poplar leaves by over 52 %, which is of great value for the molecular design and breeding of plants with growth and water-saving target traits. • The serine protease PagSDD1 is highly expressed in triploid poplar. • PagSDD1 reduces stomatal density and enhances drought tolerance. • PagSDD1 promotes leaf photosynthesis and boosts auxin and cytokinin accumulation. • PagSDD1 promotes plant growth by increasing PagGRF5 and PagCYCA3;4 gene expression. [ABSTRACT FROM AUTHOR]

Background: Clones provide a sensitive method for evaluating genotypic stability and detecting genotype-environment (G × E) interactions because of non-additive genetic effects among clones and there being no genetic effect among ramets of an ortet. With this study, we aimed to confirm and expand earlier findings, estimate stability parameters, and provide accurate estimates of clonal repeatabilities and genetic gains for a triploid breeding program of P. tomentosa Carr. Results: Six 5-year-old clonal trials established in Northern China were used to determine the clonal variation, clone × site interactions, and the stability parameters of fiber properties of wood and growth traits. 360 trees from ten hybrid clones were collected from six sites. The clonal and site effects had a highly significant effect (P < 0.001) for all studied traits. While the clone × site interactions had a highly significant effect (P < 0.001) on fiber length (FL), coarseness (C), and tree growth (tree height [H], diameter at breast height [DBH] and stem volume [SV]), and a moderate effect (P < 0.05) on fiber width (FW) and fiber length/width (FL/W). For FL and SV, most of the triploid hybrid clones had higher reaction norms to the improvement in growth conditions and higher phenotypic plasticity. The estimated clonal repeatability of FW (0.93) was slightly higher than for FL (0.89), FL/W (0.83), C (0.91), DBH (0.76), H (0.85), and SV (0.80). Three clonal testing sites were sufficient to estimate quantitative parameters of fiber properties, however, more clonal testing sites would help improve the accuracy of quantitative parameters of the growth traits. Conclusions: Our results highlight that accurate estimation of quantitative parameters for growth traits in triploid hybrid clones of P. tomentosa requires more clonal testing sites than the fiber properties. [ABSTRACT FROM AUTHOR]

Triploid poplars have obvious growth advantages, especially in leaf development and photosynthetic characteristics, but the molecular mechanism has not been revealed yet. In order to better understand the regulation mechanisms of leaf and chlorophyll development in the triploid poplars, we combined the leaf phenotypic data with the transcriptomic data of the 5th, 10th, and 25th leaves from triploid and diploid poplars, using weighted gene co-expression network analysis (WGCNA), and revealed that PpnGRF5-1 had a strong correlation with leaf development and net photosynthetic rate (Pn). PpnGRF5-1 overexpression transgenic plants showed that the leaf area, Pn, and chlorophyll concentration were significantly increased. Transcriptomic data analysis of the third leaf from PpnGRF5-1 overexpression transgenic plants showed that PpnGRF5-1 could up-regulate the expression levels of chlorophyll synthesis genes and down-regulate the transcription of chlorophyll degradation enzymes. Overall, our studies have greatly expanded our understanding of the molecular mechanisms regulating triploid growth dominance. • The gene cluster related to net photosynthetic rate in the triploid and diploid poplars. • PpnGRF5-1 has the function of regulating leaf development and chlorophyll development. • PpnGRF5-1 can increase the transcription of chlorophyll synthesis genes and reduce the expression of chlorophyll degradation genes. [ABSTRACT FROM AUTHOR]

Though allotriploid poplar shows a salient vegetative growth advantage that impacts biomass and lumber yield, the proteomic data of Populus allotriploids have not been scrutinized for identifying the underlying molecular mechanisms. We conducted a large-scale label-free proteomics profiling of the 5th, 10th, and 25th leaves of allotriploids and diploids, and identified 4587 protein groups. Among 932 differentially expressed proteins (DEPs), 22 are transcription factors (TFs) that could regulate vegetative growth advantage in allotriploids. The DEPs involved in light reaction, Calvin cycle, and photorespiration, protein synthesis, sucrose synthesis, starch synthesis, and starch decomposition displayed elevated expression in Populus allotriploids. However, the DEPs functioning in sucrose decomposition, tricarboxylic acid (TCA) cycle, and protein degradation exhibited significantly downregulated expression. The alternations of these DEPs augmented efficiency of photosynthesis, carbon fixation, sucrose and starch accumulation, and decreased capacity of carbohydrate consumption, leading to larger volume of biomass and vigorous growth in Populus allotriploids. • DEPs involved in vegetative growth advantage were characterized in Populus allotriploids. • DEPs involved in different biological processes exhibited varied differential expression patterns in triploids. • The cooperation of DEPs together contributed to larger volume of biomass and vigorous growth in Populus allotriploids. [ABSTRACT FROM AUTHOR]

Summary: Although polyploid plants have larger leaves than their diploid counterparts, the molecular mechanisms underlying this difference (or trait) remain elusive.Differentially expressed genes (DEGs) between triploid and full‐sib diploid poplar trees were identified from two transcriptomic data sets followed by a gene association study among DEGs to identify key leaf growth regulators. Yeast one‐hybrid system, electrophoretic mobility shift assay, and dual‐luciferase assay were employed to substantiate that PpnGRF5‐1 directly regulated PpnCKX1. The interactions between PpnGRF5‐1 and growth‐regulating factor (GRF)‐interacting factors (GIFs) were experimentally validated and a multilayered hierarchical regulatory network (ML‐hGRN)‐mediated by PpnGRF5‐1 was constructed with top‐down graphic Gaussian model (GGM) algorithm by combining RNA‐sequencing data from its overexpression lines and DAP‐sequencing data.PpnGRF5‐1 is a negative regulator of PpnCKX1. Overexpression of PpnGRF5‐1 in diploid transgenic lines resulted in larger leaves resembling those of triploids, and significantly increased zeatin and isopentenyladenine in the apical buds and third leaves. PpnGRF5‐1 also interacted with GIFs to increase its regulatory diversity and capacity. An ML‐hGRN‐mediated by PpnGRF5‐1 was obtained and could largely elucidate larger leaves.PpnGRF5‐1 and the ML‐hGRN‐mediated by PpnGRF5‐1 were underlying the leaf growth and development. [ABSTRACT FROM AUTHOR]

The CCT [CONSTANS (CO), CO-like, and TIMING OF CAB EXPRESSION1 (TOC1)] gene family is involved in photoperiodic flowering and adaptation to different environments. In this study, 39 CCT family genes from the poplar genome were identified and characterized, including 18 COL, 7 PRR, and 14 CMF TFs. Phylogenetics analysis showed that the PtrCCT gene family could be classified into five classes (Classes I–V) that have close relationships with Arabidopsis thaliana. Eight pairs of PtrCCTs had collinear relationships through interchromosomal synteny analysis in poplar, suggesting segmental duplication played a vital role in the expansion of the poplar CCT gene family. Besides, synteny analyses of the CCT members among poplar and different species provided more clues for PtrCCT gene family evolution. Cis -acting elements in the promoters of PtrCCTs predicted their involvement in light responses, hormone responses, biotic/abiotic stress responses, and plant growth and development. Eight members of the PpnCCT gene family were differentially expressed in the apical buds and leaves of triploid poplar compared to diploids. We then focused on PpnCCT39 upregulated in triploid poplars and showed that PpnCCT39 was localized in the nucleus, chloroplast, and cytoplasm and could interact with CLPP1 in the chloroplast. Overexpression of PpnCCT39 in poplar increased chlorophyll contents and enhanced photosynthetic rate. This study provided comprehensive information for the CCT gene family and set up a basis for its function identification in poplar. • Genowe-wide identification and analysis of the CCT gene family were first performed in poplar. • Expression profiles of PtrCCTs in diploid and triploid poplar were analyzed. • PpnCCT39 was localized in the nucleus, chloroplast, and cytoplasm and could interact with CLPP1 in the chloroplast. • PpnCCT39 increased chlorophyll contents and enhanced photosynthetic rate in poplar. [ABSTRACT FROM AUTHOR]

The induction of unreduced gametes via chromosome doubling is an effective way to produce triploids. During this process, the transmitted parental heterozygosity varies by different origins of unreduced gametes. In this study, a total of 110 allotriploid individuals from two cross combinations who shared only one male parent were obtained by chromosome doubling of megaspore via high temperature treatment in Populus tomentosa. Twenty-six SSR loci, at which the allelic configurations of the female parents 'MC1' and 'MC2' were heterozygous and different from male parent 'YX1', were screened. Among them, five pairs of pericentromeric SSR primers were selected to identify the origins of unreduced female gametes. Our results showed that 30 individuals from 110 allotriploids derived from the first division restitution (FDR), and 80 allotriploids from the second division restitution (SDR) in these two cross combinations. The transmitted parental heterozygosity of unreduced female gametes via FDR were 0.824 and 0.769 in two cross combinations, respectively, which was significantly higher than that of SDR (0.395 and 0.396, respectively). The FDR type unreduced gametes with higher heterozygosity transmitted from parents may be more desirable in triploid breeding. However, no significant difference in transmitted parental heterozygosity was found between the two FDR groups and two SDR groups. Correct elucidation of the origins of unreduced gametes may be conducive to their effective utilization for further sexual hybridization in triploid poplar breeding. [ABSTRACT FROM AUTHOR]

Hybridization and introgression have resulted in reticulate evolution within the genus Populus. Consequently, the origin and evolutionary history of some hybrids has become blurred. P. hopeiensis and P. tomentosa are endemic to China, and there is still controversy about their origin. We employ phylogeny, Bayesian estimation of admixture, and approximate Bayesian computation to investigate their origin with 10 nuclear DNA and 6 cpDNA regions. The combined evidences firmly support the hypothesis that they are hybrids and dominated by F1s. P. hopeiensis was generated via hybridization between the paternal species P. alba and maternal species P. davidiana. Surprisingly, P. tomentosa was divided into two genetic types with different maternal parents. P. adenopoda hybridized with P. alba directly to generate the first genetic type (mb1) and hybridized with P. davidiana followed by P. alba to generate the second (mb2). In both genetic types, P. alba acted as the male parent. The maternal parent was P. adenopoda and P. davidiana for mb1 and mb2, respectively. Hybridization not only generated these hybrids but also resulted in a unidirectional gene flow from P. davidiana to P. adenopoda. The Populus species have maintained a delicate balance between their genetic integrity and gene exchange. [ABSTRACT FROM AUTHOR]

High temperature exposure is widely used as a physical mutagenic agent to induce 2n gametes in Populus. However, whether high temperature exposure affects induced 2n pollen viability remains unknown. To clarify whether high temperature exposure affected the induced 2n pollen viability, 2n pollen induced by 38 and 41 °C temperatures, pollen morphology, 2n pollen germination in vitro, and crossing induced 2n pollen with normal gametes to produce a triploid was, based on observations of meiosis, conducted in Populus canescens. We found that the dominant meiotic stages (F = 56.6, p < .001) and the treatment duration (F = 21.4, p < .001) significantly affected the occurrence rate of induced 2n pollen. A significant decrease in pollen production and an increase in aborted pollen were observed (p < .001). High temperature sometimes affected in ectexine deposition and some narrow furrows were also analysed via details of ectexine structure. However, no significant difference in 2n pollen germination rate was observed between natural 2n pollen (26.7%) and high-temperature-induced 2n pollen (26.2%), and 42 triploids were created by crossing high-temperature-induced 2n pollen, suggesting that 38 and 41 °C temperatures exposure will not result in dysfunctional induced 2n pollen. [ABSTRACT FROM AUTHOR]

When hundreds of cross-pollinated trees planted in The National Gene Bank of Populus tomentosa blossom and bear fruit, in theory, half-sib progeny should originate from hundreds of combinations. Paternity testing was performed on the superior half-sib progeny with outstanding performance using microsatellite markers, a process that can be used to rapidly screen for superior cross combinations and significantly improve the efficiency and effectiveness of genetic improvements. Based on the comprehensive evaluation of the eight main growth traits of 2292 plants of 30 half-sib families, 77 plants consisting of 43 triploid and 34 diploid plants were selected as superior. We used 14 pairs of highly polymorphic SSR primers to identify the male parents of the 77 superior plants. Our results identified 29 male trees as the pollen parents of 59 superior plants from 49 cross combinations. The probabilities of 2-14, 3-48-1, 1903, 2-1, 4234, 5099, and 4114 as male parents were significantly higher than those of other males, suggesting that these male parents have potential for applications in breeding strategies of P. tomentosa. Thus, this study used SSR molecular markers to identify the male parents of superior individuals in a P. tomentosa population, laying a foundation for the application of maternal efficiency and construction of optimal hybrid parent groups. [ABSTRACT FROM AUTHOR]

Eucommia ulmoides Oliver is an important temperate species because it contains Eucommia rubber, iridoids, and other medicinal substances. Triploid breeding may be an effective way to improve the contents of gutta percha and other secondary metabolites in Eucommia leaves. To obtain triploid trees, 2n female gametes were induced by exposing female buds to high temperatures. The results showed that although the Eucommia female flower bud development is not synchronous, it still exhibits regularity. We estimated the period of megaspore mother cell meiosis based on the number of days since the adjacent male flower produced its pollen.Using female buds at 1-4 days after pollination, we obtained triploid seedlings after female buds were treated with different temperatures and durations to induce megaspore chromosome doubling. Among the offspring, 23 of Eucommia triploid seedlings were detected. Our findings indicate that the most suitable condition for inducing megaspore chromosome doubling occurred at 3 days after adjacent male tree produce pollen when the meiosis of the megasporocytes was between pachytene and diplotene. The best treatment of 45 °C for 4 h gave triploid induction of 5.74 %. [ABSTRACT FROM AUTHOR]

Artificially inducing 2n gametes through chromosome doubling is an effective way to obtain polyploids. In this study, Eucalyptus urophylla microsporogenesis and flower development were investigated to guide 2n pollen induction. We also investigated suitable conditions for colchicine treatment. Our results showed that E. urophylla 2n pollen was spherical and had a large volume (mean diameter 28.57 ± 0.46 μm), while normal untreated pollen (mean diameter 19.68 ± 0.11 μm) was tetrahedron. The highest rate of 2n pollen production was 28.71 % when the flower buds, which ranged in size from 3.5 to 4.0 mm, underwent treatment with 0.5 % colchicine solution for 6 h. Further studies suggested that diplotene to diakinesis and metaphase I to telophase I were suitable meiotic stages for chromosomes doubling, due to asynchronous development of microsporogenesis between the anthers in a single flower bud. These data help to illuminate research in other areas, such as triploid eucalyptus production by chromosome doubling of female gametes. [ABSTRACT FROM AUTHOR]

In this report, we compared transcriptomic differences between a synthetic Populus section Tacamahaca triploid driven by second-division restitution and its parents using a high-throughput RNA-seq method. A total of 4,080 genes were differentially expressed between the high-growth vigor allotriploids (SDR-H) and their parents, and 719 genes were non-additively expressed in SDR-H. Differences in gene expression between the allotriploid and male parent were more significant than those between the allotriploid and female parent, whichmay be caused by maternal effects. We observed 3,559 differentially expressed genes (DEGs) between the SDR-H and male parent. Notably, the genes were mainly involved in metabolic process, cell proliferation, DNA methylation, cell division, and meristem and developmental growth. Among the 1,056 DEGs between SDR-H and female parent, many genes were associated with metabolic process and carbon utilization. In addition, 1,789 DEGs between high- and low-growth vigor allotriploid were mainly associated with metabolic process, auxin poplar transport, and regulation of meristem growth. Our results indicated that the higher poplar ploidy level can generate extensive transcriptomic diversity compared with its parents. Overall, these results increased our understanding of the driving force for phenotypic variation and adaptation in allopolyploids driven by second-division restitution. [ABSTRACT FROM AUTHOR]

Photosynthesis plays vital role in plant growth and development throughout its life, and it is influenced by environmental signals and circadian rhythms. We analyzed the transcriptome landscape of the two poplars progeny with contrasting photosynthesis rates at three times point (ZT4, ZT16, ZT22), constructed gene regulatory network that related to circadian rhythm and photosynthesis. We suggest that the differences in photosynthetic rate between the progenies may originate from plant endogenous circadian oscillators prepare poplar plants for photosynthesis by regulating photosynthesis-associated nuclear genes and carotenoid metabolism genes before dawn, genes associated with plant hormone signal transduction and transcription factor increase leaf size and stomatal movement, the influence of other core regulatory factors on chlorophyll accumulation. Furthermore, overexpression of candidate regulatory gene, AP3 (Potri.007G017000), induced leaf senescence and reduced the content of chlorophyll. These results demonstrated that many potential key regulators are integrated closely with chlorophyll content and photosynthesis. • The diurnal changes of differentially expressed genes between two poplar populations with contrasting photosynthesis rates. • The circadian oscillating regulated physiological processes to prepare for photosynthesis before lights. • Overexpression of a core gene (AP3) in network decreased chlorophyll content. [ABSTRACT FROM AUTHOR]

Polyploidy has played an important role in Populus breeding because allopolyploid trees generally display characteristics of increased height growth over diploids, and tree growth is determined by cell division and expansion in the apical region. However, the transcriptome characteristics of the shoot apical meristem involved in stem segment elongation and plant height growth remain unclear. In this study, apical buds were sampled from high and low growth vigor plants from a single full-sib cross between the parental clones, and their gene expression profiles were compared using high-throughput sequencing. In total, 4,870 genes were identified with differential expression between the high growth vigor allotriploid and its parents, and 821 genes were nonadditively expressed in the high growth vigor allotriploid. The differences in gene expression between the allotriploid and the male parent were more significant than those between it and its female parent. Many of the differentially expressed genes related to cell proliferation, meristem initiation, transcription factors, DNA and RNA methylation. Our results indicated that Populus allotriploids can generate extensive transcriptomic diversity compared with their parents. In addition, we found that the DEGs between high and low growth vigor Populus allotriploids are involved in important biological processes. Major changes in gene expression probably provide a driving force for phenotypic variation and adaptation in allopolyploids. The DEGs selected in this study provide important insight to obtain a better understanding of height growth heterosis in Populus allotriploids. [ABSTRACT FROM AUTHOR]

Nitrogen (N) is an essential mineral element for plant growth processes, and its availability severely affects the productivity of plants, especially trees. MicroRNAs (miRNAs) are a class of non-coding RNAs approximately 21 nucleotides in length that play important roles in plant growth, development and stress responses. To identify Populus miRNAs and their functions in response to nutrition stress, high-throughput sequencing was performed using Populus tomentosa plantlets treated with or without low concentrations of N. We identified 160 conserved miRNAs, 15 known but non-conserved miRNAs, 2 candidate novel miRNAs and 71 corresponding miRNA*s. Differential expression analysis showed that expression of the 21 conserved miRNA families was significantly altered. Real-time quantitative PCR (qPCR) was used to further validate and analyze the dynamic expression of the identified miRNAs. A total of 218 target genes from the low-N-responsive miRNAs were predicted, and their functions were further annotated in combination with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. These results suggest that miRNAs play important roles in the response of Populus to low N stress. Furthermore, this study provides the first identification and profiles of N stress-responsive miRNAs from trees. [ABSTRACT FROM AUTHOR]

To obtain a better understanding of variability in wood properties along the stem in triploid hybrids ofPopulus tomentosaL., basic wood density (BWD) and fiber traits were measured at four sample heights for each tree sampled from three clonal trials. The BWD showed a similar trend at each site in that density was initially decreased then increased with increasing height position. For fiber traits, the fiber length (FL) decreased linearly with the height of the trunk, whereas fiber width and coarseness changed relatively slightly with increasing height position. In addition, the vertical wood properties distribution patterns were different from clones so that some clones showed more markedly change in height positions than others. The joint analysis indicated significant height position effect for BWD and fiber traits, in accordance with site and clonal effect. Significant site × height interaction was found on wood properties, whereas the significant clone × height interaction was only obtained at fiber width. Moreover, variation in the relationships between FL and BWD or other fiber traits showed no consistent pattern with increasing height position. [ABSTRACT FROM PUBLISHER]

Abstract: Salt is one of the main environmental factors limiting plant growth and a better understanding of mechanisms of salt stress would aid efforts to bolster plant salt tolerance. MicroRNAs are well known for their important regulatory roles in response to abiotic stress in plants. In this study, high-throughput sequencing was employed to identify miRNAs in Populus tomentosa plantlets treated or not with salt (200 mM for 10 h). We found 141 conserved miRNAs belonging to 31 families, 29 non-conserved but previously-known miRNAs belonging to 26 families, and 17 novel miRNAs. Under salt stress, 19 miRNAs belonging to seven conserved miRNA families were significantly downregulated, and two miRNAs belonging to two conserved miRNA families were upregulated. Of seven non-conserved miRNAs with significantly altered expression, five were downregulated and two were upregulated. Furthermore, eight miRNAs were validated by qRT-PCR and their dynamic differential expressions were analyzed. In addition, 269 target genes of identified miRNAs were predicted and categorized by function. These results provide new insights into salt-responsive miRNAs in Populus. [Copyright &y& Elsevier]

MicroRNAs (miRNAs) are a class of small, non-coding RNAs that play important downregulation roles in plants growth, development, and stress responses. To better identify Populus tomentosa miRNAs and understand the functions of miRNAs in response to water stress (drought and flooding), 152 conserved miRNAs belonging to 36 miRNA families, 8 known but non-conserved miRNAs and 64 candidate novel miRNAs belonging to 54 miRNA families were identified and analyzed from three small RNA (sRNA) libraries (drought treatment, flooding treatment, and control) by high-throughput sequencing combined with qRT-PCR. Significant changes in the expression of 17 conserved miRNA families and nine novel miRNAs were observed in response to drought stress, and in seven conserved miRNA families and five novel miRNAs in response to flooding stress. Both miRNA and miRNA*s were involved in the regulation of plant stress responses. The annotation of the potential targets of miRNAs with differential expression indicate that many types of genes encoding transcription factors, enzymes, and signal transduction components are implicated in the abiotic stress response.. [ABSTRACT FROM AUTHOR]

Tetraploid plants often have altered rates of vegetative growth relative to their diploid progenitors. However, the molecular basis for altered growth rates remains a mystery. This study reports microRNA (miRNA) and gene expression differences in Populus tetraploids and counterpart diploids using RNA and miRNA sequencing. The results showed that there was no significant difference between young leaves in the expression of vegetative growth-related miRNAs. However, as leaves aged, the expression of auxin- and gibberellin-related miRNAs was significantly upregulated, while the expression of senescence-related miRNAs was significantly downregulated. The dose effect enhanced the negative regulation of the target genes with ARFs, GA20ox, GA3ox, and GAMYB being downregulated, and TCP and NAC being upregulated. As a result, the chloroplast degradation of tetraploid leaves was accelerated, the photosynthetic rate was decreased, and the synthesis and decomposition ability of carbohydrate was decreased. [ABSTRACT FROM AUTHOR]

Parent selection is the core of hybrid breeding. The breeding strategy involving the parental identification of superior open-pollinated progeny of Populous tomentosa germplasm resources can significantly improve the efficiency of parental matching. However, due to some factors such as loose powdering time and pollen competitiveness, the offspring derived from open-pollination families which do not undergo completely random mating. Although hybrid combinations based on the male identification method have a high combining ability, this method cannot easily cover the mating combinations of all male and female specimens in the germplasm bank. In addition, the performance of superior plants in open-pollinated families also affects the selection result. If the trait performance value is higher than the population average, then the special combining ability of the reconstructed hybrid combination may be overestimated. Obtaining a solution to the above problems is of great significance for improving the efficiency and accuracy of selecting hybrid parents of P. tomentosa. In this study, 24 pairs of SSR (Simple Sequence Repeats) molecular markers were used to analyze the genetic differentiation of P. tomentosa germplasm resources. The results showed that the genetic variation of the P. tomentosa population was derived from individuals within the provenance, indicating that high genetic diversity is preserved in provenances. The correlation analysis showed that there was a significant positive correlation between the special combining ability of planting height and diameter at breast height (dbh) of the 34 full-sib progeny population and the genetic distance between the parents. Then, the genetic distance between 18 female plants with high fertility and 68 male plants with large pollen quantity was analyzed using this correlation. Fifteen female parents and 12 male parents were screened out, and 52 hybrid combinations with high specific combining ability for growth traits were predicted. Furthermore, for the male parent identification of superior individual plants, we constructed the breeding parent population including 10 female parents and 5 male parents, generating 14 hybrid combinations with potentially high combining ability. The results of the hybridization test showed that the specific combining ability of plant height and dbh was significantly higher than the controlled pollination. Moreover, genetic distance and paternal identification can be used to rapidly and efficiently construct hybrid parent combinations and breeding parent populations. [ABSTRACT FROM AUTHOR]

The induction of unreduced gametes via chromosome doubling is an effective way to produce triploids. During this process, the transmitted parental heterozygosity varies by different origins of unreduced gametes. In this study, a total of 110 allotriploid individuals from two cross combinations who shared only one male parent were obtained by chromosome doubling of megaspore via high temperature treatment in Populus tomentosa. Twenty-six SSR loci, at which the allelic configurations of the female parents 'MC1' and 'MC2' were heterozygous and different from male parent 'YX1', were screened. Among them, five pairs of pericentromeric SSR primers were selected to identify the origins of unreduced female gametes. Our results showed that 30 individuals from 110 allotriploids derived from the first division restitution (FDR), and 80 allotriploids from the second division restitution (SDR) in these two cross combinations. The transmitted parental heterozygosity of unreduced female gametes via FDR were 0.824 and 0.769 in two cross combinations, respectively, which was significantly higher than that of SDR (0.395 and 0.396, respectively). The FDR type unreduced gametes with higher heterozygosity transmitted from parents may be more desirable in triploid breeding. However, no significant difference in transmitted parental heterozygosity was found between the two FDR groups and two SDR groups. Correct elucidation of the origins of unreduced gametes may be conducive to their effective utilization for further sexual hybridization in triploid poplar breeding. [ABSTRACT FROM AUTHOR]