High quality haplotype-resolved genome assemblies of Populus tomentosa Carr., a stabilized interspecific hybrid species widespread in Asia.

Populus has a wide ecogeographical range spanning the Northern Hemisphere, and interspecific hybrids are common. Populus tomentosa Carr. is widely distributed and cultivated in the eastern region of Asia, where it plays multiple important roles in forestry, agriculture, conservation, and urban horticulture. Reference genomes are available for several Populus species, however, our goals were to produce a very high quality de novo chromosome-level genome assembly in P. tomentosa genome that could serve as a reference for evolutionary and ecological studies of hybrid speciation throughout the genus. Here, combining long-read sequencing and Hi-C scaffolding, we present a high-quality, haplotype-resolved genome assembly. The genome size was 740.2 Mb, with a contig N50 size of 5.47 Mb and a scaffold N50 size of 46.68 Mb, consisting of 38 chromosomes, as expected with the known diploid chromosome number (2n = 2x = 38). A total of 59,124 protein-coding genes were identified. Phylogenomic analyses revealed that P. tomentosa is comprised of two distinct subgenomes, which we deomonstrate is likely to have resulted from hybridization between Populus adenopoda as the female parent and Populus alba var. pyramidalis as the male parent, with an origin of approximately 3.93 Ma. Although highly colinear, significant structural variation was found between the two subgenomes. Our study provides a valuable resource for ecological genetics and forest biotechnology.

Characterization of two highly similar CBF/DREB1-like genes, PhCBF4a and PhCBF4b, in Populus hopeiensis.

The C-repeat binding factors (CBFs)/dehydration-responsive element-binding protein (DREBs) are a group of conserved transcription factors that play an important role in the response and adaptation to environmental stress in many plants. Two highly similar CBF/DREB1-like genes, PhCBF4a and PhCBF4b, were previously identified in Populus hopeiensis. In this paper, we describe the function of these proteins in detail in terms of abiotic stress tolerance. Phylogenic analysis suggests that PhCBF4a and PhCBF4b are expressed as two distinct alleles. Expression of both genes is induced mainly by dehydration, low-temperature, and high-salinity. Agroinfiltration experiments in tobacco leaves revealed differential transcriptional activation of the genes, likely driven by differences in their C-terminal regions. When constitutively expressed in Arabidopsis, PhCBF4a and PhCBF4b induced elevated expression of the CBF/DREB1 regulons without prior stimulus, resulting in dwarfism, delayed flowering, and greater drought tolerance compared with vector controls. These results demonstrate that PhCBF4a and PhCBF4b are functional transcriptional regulators involved in the response of P. hopeiensis to abiotic stresses.

A Novel Moderate Constitutive Promoter Derived from Poplar (Populus tomentosa Carrière).

A novel sequence that functions as a promoter element for moderate constitutive expression of transgenes, designated as the PtMCP promoter, was isolated from the woody perennial Populus tomentosa. The PtMCP promoter was fused to the GUS reporter gene to characterize its expression pattern in different species. In stable Arabidopsis transformants, transcripts of the GUS reporter gene could be detected by RT-PCR in the root, stem, leaf, flower and silique. Further histochemical and fluorometric GUS activity assays demonstrated that the promoter could direct transgene expression in all tissues and organs, including roots, stems, rosette leaves, cauline leaves and flowers of seedlings and maturing plants. Its constitutive expression pattern was similar to that of the CaMV35S promoter, but the level of GUS activity was significantly lower than in CaMV35S promoter::GUS plants. We also characterized the promoter through transient expression in transgenic tobacco and observed similar expression patterns. Histochemical GUS staining and quantitative analysis detected GUS activity in all tissues and organs of tobacco, including roots, stems, leaves, flower buds and flowers, but GUS activity in PtMCP promoter::GUS plants was significantly lower than in CaMV35S promoter::GUS plants. Our results suggested that the PtMCP promoter from poplar is a constitutive promoter with moderate activity and that its function is presumably conserved in different species. Therefore, the PtMCP promoter may provide a practical choice to direct moderate level constitutive expression of transgenes and could be a valuable new tool in plant genetic engineering.

Ectopic expression of a poplar APETALA3-like gene in tobacco causes early flowering and fast growth.

A MADS-box gene, designated PtAP3, was isolated from a floral bud cDNA library derived from Populus tomentosa. Analysis by multiple alignments of both nucleotide and amino acid sequences, together with phylogenetic analysis, revealed that PtAP3 is an ortholog of Arabidopsis AP3. Analysis of RNA extracts from vegetative and reproductive tissues of P. tomentosa by RT-PCR indicated that PtAP3 is expressed in roots, stems, leaves and vegetative and floral buds. Notably, the expression of PtAP3 fluctuated during floral bud development between September and February with differences between male and female buds. In the former, a gradual down-regulation during this period, interrupted by a slight up-regulation in December, was followed by a sharper up-regulation on February. In developing female floral buds, expression was stable from September to November, sharply up-regulated in December, and then gradually down-regulated until February. The functional role of PtAP3 was investigated in transgenic tobacco plants. Of 25 transformants, nine displayed an earlier flowering phenotype compared with the wild type plants. Furthermore, transgenic tobacco had faster growth and more leaves than untransformed controls. The traits proved to be heritable between the T0 and T1 generations. Our results demonstrate a regulatory role of the PtAP3 gene during plant flowering and growth and suggest that the gene may be an interesting target for genetic modification to induce early flowering in plants.

Isolation of a LEAFY homolog from Populus tomentosa: expression of PtLFY in P. tomentosa floral buds and PtLFY-IR-mediated gene silencing in tobacco (Nicotiana tabacum).

To understand the genetic and molecular mechanisms underlying floral development in Populus tomentosa, we isolated PtLFY, a LEAFY homolog, from a P. tomentosa floral bud cDNA library. DNA gel blot analysis showed that PtLFY is present as a single copy in the genomes of both male and female individuals of P. tomentosa. The genomic copy is composed of three exons and two introns. Relative expression levels of PtLFY in tissues of P. tomentosa were estimated by RT-PCR; our results revealed that PtLFY mRNA is highly abundant in roots and both male and female floral buds. A low level of gene expression was detected in stems and vegetative buds, and no PtLFY-specific transcripts were detected in leaves. PtLFY expression patterns were analyzed during the development of both male and female floral buds in P. tomentosa via real-time quantitative RT-PCR. Continuous, stable and high-level expression of PtLFY-specific mRNA was detected in both male and female floral buds from September 13th to February 25th, but the level of PtLFY transcripts detected in male floral buds was considerably higher than in female floral buds. Our results also showed an inverted repeat PtLFY fragment (PtLFY-IR) effectively blocked flowering of transgenic tobacco plants, and that this effect appeared to be due to post-transcriptional silencing of the endogenous tobacco LFY homologs NFL1 and NFL2.

[Gene and gene engineering of carotenoid biosynthesis].

Carotenoids have a range of diverse biological functions and actions, especially playing an important role in human health with provitamin A activity, anti-cancer activity, enhancing immune ability and so on. Human body can't synthesis carotenoids by itself and must absorb them from outside. However, carotenoid contents in many plant are very low, and many kinds of carotenoid are difficult to produce by chemical ways. With the elucidation of carotenoid biosynthetic pathway and cloning genes of relative enzymes from microorganisms and higher plants, it is possible to regulate carotenoid biosynthesis via genetic engineering. This article reviews gene cloning of carotenoid biosynthetic enzymes in microorganisms and higher plants, and advances in the studies of carotenoid production in heterologous microorganisms and crop plants using gene-manipulated carotenoid biosynthesis.