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.

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.

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.

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.

Comparative proteomic analysis provides insight into the molecular mechanism of vegetative growth advantage in allotriploid Populus.

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.

Transcriptome comparison of different ploidy reveals the mechanism of photosynthetic efficiency superiority of triploid poplar.

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.

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.

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.

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.

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.

Genotype-environment interaction and stability of fiber properties and growth traits in triploid hybrid clones of Populus tomentosa.

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.

Integrated transcriptome and miRNA sequencing approaches provide insights into salt tolerance in allotriploid Populus cathayana.

MAIN CONCLUSION: Some salt-stress responsive DEGs, mainly involved in ion transmembrane transport, hormone regulation, antioxidant system, osmotic regulation, and some miRNA jointly regulated the salt response process in allotriploid Populus cathayana. The molecular mechanism of plant polyploid stress resistance has been a hot topic in biological research. In this study, Populus diploids and first division restitution (FDR) and second division restitution (SDR) triploids were selected as research materials. All materials were treated with 70 mM NaCl solutions for 30 days in the same pot environment. We observed the growth state of triploids and diploids and determined the ratio of potassium and sodium ions, peroxidase (POD) activity, proline content, and ABA and jasmonic acid (JA) hormone content in leaves in the same culture environment with the same concentration of NaCl solution treatment. In addition, RNA-seq technology was used to study the differential expression of mRNA and miRNA. The results showed that triploid Populus grew well and the K+ content and the K+/Na+ ratio in the salt treatment were significantly lower than those in the control. The contents of ABA, JA, POD, and proline were increased compared with contents in diploid under salt stress. The salt-stress responsive DEGs were mainly involved in ion transport, cell homeostasis, the MAPK signaling pathway, peroxisome, citric acid cycle, and other salt response and growth pathways. The transcription factors mainly included NAC, MYB, MYB_related and AP2/ERF. Moreover, the differentially expressed miRNAs involved 32 families, including 743 miRNAs related to predicted target genes, among which 22 miRNAs were significantly correlated with salt-stress response genes and related to the regulation of hormones, ion transport, reactive oxygen species (ROS) and other biological processes. Our results provided insights into the physiological and molecular aspects for further research into the response mechanisms of allotriploid Populus cathayana to salt stress. This study provided valuable information for the salt tolerance mechanism of allopolyploids.

Growth-regulating factor 5 (GRF5)-mediated gene regulatory network promotes leaf growth and expansion in poplar.

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.

Molecular Mechanism of Vegetative Growth Advantage in Allotriploid Populus.

Allotriploid poplar has a prominent vegetative growth advantage that impacts dramatically on lumber yield. The growth regulation is complex which involves abundant genes, metabolic and signaling pathways, while the information about the functional control process is very little. We used high-throughput sequencing and physiological index measurement to obtain a global overview of differences between allotriploid and diploid Populus. The genes related to plant growth advantage show a higher expression compared to diploid, and most of them are revolved around hormones, photosynthesis and product accumulation. Thus, allotriploid Populus showed more efficient photosynthesis, carbon fixation, sucrose and starch synthesis, and metabolism as well as augmented biosynthesis of auxin, cytokinin, and gibberellin. These data enable the connection of metabolic processes, signaling pathways, and specific gene activity, which will underpin the development of network models to elucidate the process of triploid Populus advantage growth.

Induction, identification and characterization of tetraploidy in Lycium ruthenicum.

Lycium ruthenicum of Solanaceae was widely used as healthy vegetables and natural medicine foods for containing numerous functional components in leaves, roots and fruits. In the present study, tetraploid plants of L. ruthenicum were obtained efficiently by treating their leaves with colchicine in vitro. The highest induction frequency of the tetraploids was 31.4%, which was obtained by preculturing the leaves for 10 days and then treating them with 100 mg/L of colchicine concentration for 48 h. The ploidy levels of the regenerated plants were determined by flow cytometry and chromosome counting methods. Cytological, morphological, and histological characterization validated the results of flow cytometry, revealing the differences between the two kinds of ploidy plants in their tissue culture stage and field production stages. Morphological indexes also provide a simple and intuitionistic method for distinguishing tetraploid from diploid plants. As the chromosome number increased, the stomatal size and number of the chloroplasts in the stomata also increased, but the stomatal density decreased. The results indicate that the chromosome number is correlated with the stomatal index. The generated tetraploid is a potentially useful cultivated variety and will be beneficial for producing triploid progeny in the future.

Analysis of genetic composition and transmitted parental heterozygosity of natural 2n gametes in Populus tomentosa based on SSR markers.

MAIN CONCLUSION: Natural 2n female gametes and transmission of parental heterozygosity by natural 2n gametes in Populus tomentosa are reported for the first time, which provides a new approach to polyploid breeding. Naturally occurring 2n pollen is widespread in Populus tomentosa and plays an important role in polyploid breeding. However, the competitiveness of 2n pollen is lower than that of haploid pollen during pollination and fertilization, so 2n pollen is less efficient at fertilizing haploid female gametes to produce polyploids. In theory, polyploids can also be obtained when 2n female gametes are fertilized by haploid pollen. Thus, the question becomes whether natural 2n female gametes exist in P. tomentosa, which can be answered by examining the genetic composition of natural 2n gametes. In this study, the origin of 87 triploids from the hybrid combination "X-2 × Z-5" was identified by SSR markers and 21% of natural 2n gametes were found to originate from female parents. Four SSR loci with low recombination rates were used to identify the genetic composition of natural 2n gametes. The results showed that the genetic composition of 2n female gametes was mainly characterized by SDR, while 2n male gametes were mainly produced by FDR. Moreover, the transmission of parental heterozygosity by natural 2n gametes, which is significantly different between female and male parents in FDR and SDR types, was analysed using 42 SSR primers. Here, we report naturally occurring 2n female gametes for the first time in P. tomentosa and reveal the genetic constitution and transmitted parental heterozygosity of these gametes. Our results provide a foundation for theoretical research into 2n gametes and their application in new polyploid breeding strategies.

High temperature exposure did not affect induced 2n pollen viability in Populus.

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.

MicroRNA expression changes following synthesis of three full-sib Populus triploid populations with different heterozygosities.

KEY MESSAGE: Through high-throughput sequencing, we compared the relative expression levels of miRNA in three full-sib Populus triploid populations with that in their parents and one diploid hybrid population. We found similar numbers of miRNAs differentially expressed between the parents and the four progeny hybrid populations. In addition, unbalanced parental expression level dominance of miRNAs were found in the three allotriploid and interspecific hybrid populations, which may reprogram gene expression networks and contribute to the growth of Populus hybrids. These results indicated that hybridization has a great impact on the miRNA expression variation in the newly synthesized Populus triploid and diploid hybrid populations. However, we also found no significant differences in miRNA expression among one diploid and three triploid hybrid populations, hinting that miRNA abundances do not increase with the genome content. No dosage effect of miRNA expression could lead to dosage-dependent negative effects on target genes and their downstream pathway in polyploids. We speculate that polyploids may gain advantages from the slight decrease in miRNA regulation, suggesting an important molecular mechanism of polyploid advantage. Hybridization with three types of induced 2n gametes transmitted different parental heterozygosities has been proven as an efficient method for Populus triploid production. Several researches have shown that miRNA could be non-additively expressed in allopolyploids. However, it is still unclear whether the non-additively expressed miRNAs result from the effect of hybridization or polyploidization, and whether a dose response to the additional genomic content exists for the expression of miRNA. Toward this end, through high-throughput sequencing, we compared the expression levels of miRNA in three full-sib Populus triploid populations with that in their parents and one interspecific hybrid population. We found similar numbers of miRNAs differentially expressed between the parents and the four progeny hybrid populations. Unbalanced parental expression level dominance of miRNAs were found in the three triploid and diploid hybrid populations, which may reprogram gene expression networks and affect the growth of Populus hybrids. These results indicated that hybridization has a great impact on the miRNA expression variation in the newly synthesized Populus triploid and diploid hybrid populations. However, we also found no significant differences in miRNA expression among the three triploid populations and the diploid hybrid population. No dosage effect of miRNA expression could lead to dosage-dependent negative effects on target genes and their downstream pathway in polyploids. We speculate that polyploids may gain advantages from the decrease in miRNA negative regulation, suggesting an important molecular mechanism of polyploid advantage.

Proteomic Changes Between Populus Allotriploids and Diploids Revealed Using an iTRAQ-based Quantitative Approach.

BACKGROUND: Polyploid breeding is a powerful approach for Populus genetic improve-ment because polyploid trees have valuable characteristics, including better timber quality and a higher degree of stress resistance compared with their full-sib diploids. However, the genetic mech-anism underlying this phenomenon remains unknown. OBJECTIVE: To better understand the proteomic changes between Populus allotriploids and diploids, we examined the proteomic profiles of allotriploid and diploid Populus by iTRAQ labeling coupled with two-dimensional liquid chromatography and MALDI-TOF/TOF mass spectrometry (MS). METHOD: iTRAQ labeling coupled with two-dimensional liquid chromatography and MALDI-TOF/TOF mass spectrometry (MS). RESULTS: Between the Populus allotriploid and the full-sib diploid, 932 differentially expressed proteins (DEPs) were identified. These DEPs were primarily involved in stress, defense, transportation, transcriptional and/or translational modification, and energy production. The pathway analysis indi-cated that most of the DEPs were implicated in carbohydrate transport and metabolism, nitrogen me-tabolism and glycolysis, and the ribosome assembly pathway. These data suggest high protein di-vergence between Populus allotriploids and diploids, and rapid changes during hybridization. CONCLUSION: The results provide new data for further understanding of the mechanisms of polyploid trees that generally display increased height growth compared with their full-sib diploids.

A strategy for characterization of persistent heteroduplex DNA in higher plants.

Heteroduplex DNA (hDNA) generated during homologous recombination (HR) is an important component that shapes genetic diversity in sexually reproducing organisms. However, studies of this process in higher plants are limited. This is because hDNAs are difficult to capture in higher plants as their reproductive developmental model only produces normal gametes and does not preserve the mitotic products of the post-meiotic segregation (PMS) process which is crucial for studying hDNAs. In this study, using the model system for tree and woody perennial plant biology (Populus), we propose a strategy for characterizing hDNAs in higher plants. We captured hDNAs by constructing triploid hybrids originating from a cross between unreduced 2n eggs (containing hDNA information as a result of inhibition chromosome segregation at the PMS stage) with normal male gametes. These triploid hybrids allowed us to detect the frequency and location of persistent hDNAs resulting from HR at the molecular level. We found that the frequency of persistent hDNAs, which ranged from 5.3 to 76.6%, was related to locations of the simple sequence repeat markers at the chromosomes, such as the locus-centromere distance, the surrounding DNA sequence and epigenetic information, and the richness of protein-coding transcripts at these loci. In summary, this study provides a method for characterizing persistent hDNAs in higher plants. When high-throughput sequencing techniques can be incorporated, genome-wide persistent hDNA assays for higher plants can be easily carried out using the strategy presented in this study.