The interplay of growth-regulating factor 5 and BZR1 in coregulating chlorophyll degradation in poplar.

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.

Positive regulation of the Eucommia rubber biosynthesis-related gene EuFPS1 by EuWRKY30 in Eucommia ulmoides.

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.

Stomatal density suppressor PagSDD1 is a "generalist" gene that promotes plant growth and improves water use efficiency.

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.

MYC2 regulates stomatal density and water use efficiency via targeting EPF2/EPFL4/EPFL9 in poplar.

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.

Genome-wide identification, characterization, and expression analysis of CCT transcription factors in poplar.

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.

Spatiotemporal miRNA and transcriptomic network dynamically regulate the developmental and senescence processes of poplar leaves.

Poplar is an important afforestation and urban greening species. Poplar leaf development occurs in stages, from young to mature and then from mature to senescent; these are accompanied by various phenotypic and physiological changes. However, the associated transcriptional regulatory network is relatively unexplored. We first used principal component analysis to classify poplar leaves at different leaf positions into two stages: developmental maturity (the stage of maximum photosynthetic capacity); and the stage when photosynthetic capacity started to decline and gradually changed to senescence. The two stages were then further subdivided into five intervals by gene expression clustering analysis: young leaves, the period of cell genesis and functional differentiation (L1); young leaves, the period of development and initial formation of photosynthetic capacity (L3-L7); the period of maximum photosynthetic capacity of functional leaves (L9-L13); the period of decreasing photosynthetic capacity of functional leaves (L15-L27); and the period of senescent leaves (L29). Using a weighted co-expression gene network analysis of regulatory genes, high-resolution spatiotemporal transcriptional regulatory networks were constructed to reveal the core regulators that regulate leaf development. Spatiotemporal transcriptome data of poplar leaves revealed dynamic changes in genes and miRNAs during leaf development and identified several core regulators of leaf development, such as GRF5 and MYB5. This in-depth analysis of transcriptional regulation during leaf development provides a theoretical basis for exploring the biological basis of the transcriptional regulation of leaf development and the molecular design of breeding for delaying leaf senescence.

Integrated transcriptomic and metabolomic analysis reveals the effects of polyploidization on the lignin content and metabolic pathway in Eucalyptus.

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.

Regional testing of triploid hybrid clones of populus tomentosa.

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.

The NAC transcription factor family in Eucommia ulmoides: Genome-wide identification, characterization, and network analysis in relation to the rubber biosynthetic genes.

The NAC transcription factor family is a large plant gene family, participating in plant growth and development, secondary metabolite synthesis, biotic and abiotic stresses responses, and hormone signaling. Eucommia ulmoides is a widely planted economic tree species in China that can produce trans-polyisoprene: Eucommia rubber (Eu-rubber). However, genome-wide identification of the NAC gene family has not been reported in E. ulmoides. In this study, 71 NAC proteins were identified based on genomic database of E. ulmoides. Phylogenetic analysis showed that the EuNAC proteins were distributed in 17 subgroups based on homology with NAC proteins in Arabidopsis, including the E. ulmoides-specific subgroup Eu_NAC. Gene structure analysis suggested that the number of exons varied from 1 to 7, and multitudinous EuNAC genes contained two or three exons. Chromosomal location analysis revealed that the EuNAC genes were unevenly distributed on 16 chromosomes. Three pairs of genes of tandem duplicates genes and 12 segmental duplications were detected, which indicated that segmental duplications may provide the primary driving force of expansion of EuNAC. Prediction of cis-regulatory elements indicated that the EuNAC genes were involved in development, light response, stress response and hormone response. For the gene expression analysis, the expression levels of EuNAC genes in various tissues were quite different. To explore the effect of EuNAC genes on Eu-rubber biosynthesis, a co-expression regulatory network between Eu-rubber biosynthesis genes and EuNAC genes was constructed, which indicated that six EuNAC genes may play an important role in the regulation of Eu-rubber biosynthesis. In addition, this six EuNAC genes expression profiles in E. ulmoides different tissues were consistent with the trend in Eu-rubber content. Quantitative real-time PCR analysis showed that EuNAC genes were responsive to different hormone treatment. These results will provide a useful reference for further studies addressing the functional characteristics of the NAC genes and its potential role in Eu-rubber biosynthesis.

Effect of a suitable treatment period on the genetic transformation efficiency of the plant leaf disc method.

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-2'-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.

Overexpression of PagSTOMAGEN, a Positive Regulator of Stomatal Density, Promotes Vegetative Growth in Poplar.

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.

Morphological, Transcriptome, and Hormone Analysis of Dwarfism in Tetraploids of Populus alba × P. glandulosa.

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.

Genome-wide characterization and analysis of Golden 2-Like transcription factors related to leaf chlorophyll synthesis in diploid and triploid Eucalyptus urophylla.

Golden 2-Like (GLK) transcription factors play a crucial role in chloroplast development and chlorophyll synthesis in many plant taxa. To date, no systematic analysis of GLK transcription factors in tree species has been conducted. In this study, 40 EgrGLK genes in the Eucalyptus grandis genome were identified and divided into seven groups based on the gene structure and motif composition. The EgrGLK genes were mapped to 11 chromosomes and the distribution of genes on chromosome was uneven. Phylogenetic analysis of GLK proteins between E. grandis and other species provided information for the high evolutionary conservation of GLK genes among different species. Prediction of cis-regulatory elements indicated that the EgrGLK genes were involved in development, light response, and hormone response. Based on the finding that the content of chlorophyll in mature leaves was the highest, and leaf chlorophyll content of triploid Eucalyptus urophylla was higher than that of the diploid control, EgrGLK expression pattern in leaves of triploid and diploid E. urophylla was examined by means of transcriptome analysis. Differential expression of EgrGLK genes in leaves of E. urophylla of different ploidies was consistent with the trend in chlorophyll content. To further explore the relationship between EgrGLK expression and chlorophyll synthesis, co-expression networks were generated, which indicated that EgrGLK genes may have a positive regulatory relationship with chlorophyll synthesis. In addition, three EgrGLK genes that may play an important role in chlorophyll synthesis were identified in the co-expression networks. And the prediction of miRNAs targeting EgrGLK genes showed that miRNAs might play an important role in the regulation of EgrGLK gene expression. This research provides valuable information for further functional characterization of GLK genes in Eucalyptus.

Induction and Characterization of Tetraploid Through Zygotic Chromosome Doubling in Eucalyptus urophylla.

Improvements in plant growth can bring great benefits to the forest industry. Eucalyptus urophylla is an important plantation species worldwide, and given that ploidy increases are often associated with plant phenotype changes, it was reasoned that its polyploidization may have good prospects and great significance toward its cultivation. In this study, the zygotic development period of E. urophylla was observed through paraffin sections, and a correlation between the development time of flower buds after pollination and the zygotic development period was established. On this basis, it was determined that the 25th day after pollination was the appropriate time for a high temperature to induce zygotic chromosome doubling. Then tetraploid E. urophylla was successfully obtained for the first time through zygotic chromosome doubling induced by high temperature, and the appropriate conditions were treating flower branches at 44°C for 6 h. The characterization of tetraploid E. urophylla was performed. Chromosome duplication brought about slower growing trees with thicker leaves, larger cells, higher net photosynthetic rates, and a higher content of certain secondary metabolites. Additionally, the molecular mechanisms for the variation in the tetraploid's characteristics were studied. The qRT-PCR results showed that genes mediating the tetraploid characteristics showed the same change trend as those of the characteristics, which verified that tetraploid trait variation was mainly caused by gene expression changes. Furthermore, although the tetraploid had no growth advantage compared with the diploid, it can provide important germplasm resources for future breeding, especially for the creation of triploids.

Genome-Wide Identification of the Eucalyptus urophylla GATA Gene Family and Its Diverse Roles in Chlorophyll Biosynthesis.

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.

Insights into the Molecular Regulation of Lignin Content in Triploid Poplar Leaves.

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.

Transcriptome analysis uncovering regulatory networks and hub genes of Populus photosynthesis and chlorophyll content.

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.

Transcriptome analysis of gibberellins and abscisic acid during the flooding response in Fokienia hodginsii.

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.

Gibberellins as a novel mutagen for inducing 2n gametes in plants.

The plant hormone gibberellin (GA) regulates many physiological processes, such as cell differentiation, cell elongation, seed germination, and the response to abiotic stress. Here, we found that injecting male flower buds with exogenous gibberellic acid (GA3) caused defects in meiotic cytokinesis by interfering with radial microtubule array formation resulting in meiotic restitution and 2n pollen production in Populus. A protocol for inducing 2n pollen in Populus with GA3 was established by investigating the effects of the dominant meiotic stage, GA3 concentration, and injection time. The dominant meiotic stage (F = 41.882, P < 0.001) and GA3 injection time (F = 172.466, P < 0.001) had significant effects on the frequency of induced 2n pollen. However, the GA3 concentration (F = 1.391, P = 0.253) did not have a significant effect on the frequency of induced 2n pollen. The highest frequency of GA3-induced 2n pollen (21.37%) was observed when the dominant meiotic stage of the pollen mother cells was prophase II and seven injections of 10 µM GA3 were given. Eighteen triploids were generated from GA3-induced 2n pollen. Thus, GA3 can be exploited as a novel mutagen to induce flowering plants to generate diploid male gametes. Our findings provide some new insight into the function of GAs in plants.

High-Frequency Homologous Recombination Occurred Preferentially in Populus.

Homologous recombination (HR), the most significant event in meiosis, has important implications for genetic diversity and evolution in organisms. Heteroduplex DNA (hDNA), the product of HR, can be captured by artificially induced chromosome doubling during the development of the embryo sac to inhibit postmeiotic segregation, subsequently, and hDNAs are directly detected using codominant simple sequence repeat (SSR) markers. In the present study, two hybrid triploid populations derived from doubling the chromosomes of the embryo sac induced by high temperature in Populus tomentosa served as starting materials. Eighty-seven, 62, and 79 SSR markers on chromosomes 01, 04, and 19, respectively, that were heterozygous in the maternal parent and different from the paternal parent were screened to detect and characterize the hDNA in P. tomentosa. The results showed that the hDNA frequency patterns on chromosomes changed slightly when the number of SSR primers increased. The highest hDNA frequency occurred at the adjacent terminal on chromosomes, which was slightly higher than those at the terminals in the two genotypic individuals, and the hDNA frequency gradually decreased as the locus-centromere distance decreased. With the increase in the number of SSR markers employed for detection, the number of recombination events (REs) detected significantly increased. In regions with high methylation or long terminal repeat (LTR) retrotransposon enrichment, the frequency of hDNA was low, and high frequencies were observed in regions with low sequence complexity and high gene density. High-frequency recombination occurring at high gene density regions strongly affected the association between molecular markers and quantitative trait loci (QTLs), which was an important factor contributing to the difficulty encountered by MAS in achieving the expected breeding results.