Integrated transcriptome and proteome analysis reveals brassinosteroid-mediated regulation of cambium initiation and patterning in woody stem.

Wood formation involves sequential developmental events requiring the coordination of multiple hormones. Brassinosteroids (BRs) play a key role in wood development, but little is known about the cellular and molecular processes that underlie wood formation in tree species. Here, we generated transgenic poplar lines with edited PdBRI1 genes, which are orthologs of Arabidopsis vascular-enriched BR receptors, and showed how inhibition of BR signaling influences wood development at the mRNA and/or proteome level. Six Populus PdBRI1 genes formed three gene pairs, each of which was highly expressed in basal stems. Simultaneous mutation of PdBRI1–1, −2, −3 and − 6, which are orthologs of the Arabidopsis vascular-enriched BR receptors BRI1, BRL1 and BRL3, resulted in severe growth defects. In particular, the stems of these mutant lines displayed a discontinuous cambial ring and patterning defects in derived secondary vascular tissues. Abnormal cambial formation within the cortical parenchyma was also observed in the stems of pdbri1–1;2;3;6. Transgenic poplar plants expressing edited versions of PdBRI1–1 or PdBRI1–1;2;6 exhibited phenotypic alterations in stem development at 4.5 months of growth, indicating that there is functional redundancy among these PdBRI1 genes. Integrated analysis of the transcriptome and proteome of pdbri1–1;2;3;6 stems revealed differential expression of a number of genes/proteins associated with wood development and hormones. Concordant (16%) and discordant (84%) regulation of mRNA and protein expression, including wood-associated mRNA/protein expression, was found in pdbri1–1;2;3;6 stems. This study found a dual role of BRs in procambial cell division and xylem differentiation and provides insights into the multiple layers of gene regulation that contribute to wood formation in Populus.

Transgenic poplar gene flow monitoring in China.

Poplar is cultivated widely for pulpwood, firewood, and timber. Transgenic poplar may be part of a solution for wood demand in China. Because transgene escape is an important part of ecological security evaluation of transgenic plants, in this chapter we discuss a real transgenic poplar case study. In this case study, mature transgenic male Populus nigra plants harbored a Bacillus thuringiensis toxin gene (i.e. Bt poplar). A plantation of these plants served as a testbed for a relevant example for gene flow monitoring in China. Furthermore, we discuss environmental risk assessment (ERA) of these transgenic plants. While transgenes can drift to related species through natural and controlled pollination, the probability of transgene drift appears to be very low in the field. The resultantBt poplar seeds occurred at a frequency from about 0.15% at 0 m to about 0.02% at 500 m away from the Bt poplar. The Bt poplar progeny seeds had decreased germination within 3 weeks in the field (from 68% to 0%), compared with the 48% germination rate after 3 weeks at 4°C. The survival rate of seedlings in the field was 0% without any treatments, but increased to 1.7% under four combined treatments (clean and trim, watering, weeding, and cover with plastic to retain moisture) after being seeded in the field for 8 weeks. Hybrid offspring appeared to possess segregated traits following artificially controlled pollination. While hybrids of transgenic poplar and non-transgenic poplar can be excellent germplasm, gene flow should be monitored. Transgene expression in grafted scion and rootstock of transgenic poplar is reviewed. The transgenic poplar studied appears to be safe; no ecological or environmental harm has been observed in China.

Effects of Bt-Cry1Ah1 Transgenic Poplar on Target and Non-Target Pests and Their Parasitic Natural Enemy in Field and Laboratory Trials

Increasing areas of artificial afforestation and poplar monoculture in China have led to serious problems with insect pests. The development of genetic engineering technology, such as transgenic modification with Bacillus thuringiensis (Bt) genes, provides novel solutions to the pest problem. We generated a Bt-Cry1Ah1 gene incorporating codon optimization and transferred it into Populus deltoides × P. euramericana cv “Nanlin895” using an Agrobacterium-mediated method. The resulting Bt-Cry1Ah1 transgenic poplars were planted in the field with permission from the State Forestry Administration in 2017. Field and laboratory studies were conducted in Jiangsu, China, to investigate the effects of these transgenic poplars expressing the Cry1Ah1 protein on target and non-target pests and their parasitic natural enemy. Target pests included Hyphantria cunea (Lepidoptera, Arctiidae), Micromelalopha troglodyta (Lepidoptera, Notodontidae), and Clostera anachoreta (Lepidoptera, Notodontidae). Plagiodera versicolora (Coleoptera, Chrysomelidae) served as the non-target pest. Laboratory trials showed that the six transgenic poplar lines exhibited resistance against the target insects. The corrected mortality rates of the target pest larvae fed leaves from the six lines were as high as 87.0%, significantly higher than that of the control. However, the corrected mortality rate of the non-target pest larvae was markedly lower and did not differ significantly from that of the control. Field experiments showed that transgenic poplar exhibited resistance against H. cunea and M. troglodyta. Field mortality rates were slightly higher than laboratory mortality rates. In addition, we investigated Chouioia cunea (Hymenoptera, Eulophidae) as a parasitoid of H. cunea pupae that had been fed transgenic poplar leaves. The emergence time, parasitism rate, and abundance of C. cunea did not differ significantly from those of the control. Therefore, Bt-Cry1Ah1 transgenic poplar can be used to effectively control damage by target insect pests without negatively affecting non-target insects and parasitoids.

Whole-Genome Resequencing using Next-Generation and Nanopore Sequencing for Molecular Characterization of T-DNA Integration in Transgenic Poplar 741

BackgroundWith the rapid development of transgenic technology, transgenic plants have been planted all over the world, and transgenic forest trees have also been commercialized. At the same time, the potential threat of transgenic plants to human health and the natural environment has aroused widespread concern. Therefore, safety assessments before field release and commercial planting of transgenic plants are necessary. By determining the copy number and integration sites of T-DNA in transgenic plants, the safety of transgenic plants at the genomic level can be assessed.ResultsIn this study, we performed genome resequencing of Pb29, a transgenic high-resistance poplar 741 line that has been commercialized, using next-generation and Nanopore sequencing. The results revealed that there are two T-DNA insertion sites, located at 9,283,905–9,283,937 bp on chromosome 3 (Chr03) and 10,868,777–10,868,803 bp on Chr10. The accuracy of the T-DNA insertion locations and directions was verified using polymerase chain reaction amplification. Through sequence alignment, different degrees of base deletions were detected on the T-DNA left and right border sequences, and in the flanking sequences of the insertion sites. An unknown fragment was inserted between the Chr03 insertion site and the right flanking sequence, but the Pb29 genome did not undergo chromosomal rearrangement. It is worth noting that we did not detect the API gene in the Pb29 genome, indicating that Pb29 is a transgenic line containing only the BtCry1AC gene. On Chr03, the insertion of T-DNA disrupted a gene encoding TAF12 protein, but the transcriptional abundance of this gene did not change significantly in the leaves of Pb29. Additionally, except for the gene located closest to the T-DNA integration site, the expression levels of four other neighboring genes did not change significantly in the leaves of Pb29. ConclusionsThis study provides important molecular information for safety assessments and management of transgenic poplar 741. Our findings also provide a theoretical basis for safety assessments of other transgenic poplar.