Transformation of poplar (Populus alba) plastids and expression of foreign proteins in tree chloroplasts

Plastid transformation offers several unique advantages compared with nuclear genome transformation, such as high level of transgene expression within plastids, expressing multiple transgenes as operons, lack of position effect due to site-specific transgene integration, and reducing risks of gene flow via pollen due to maternal inheritance of the plastid genome. Plastid transformation has been applied to several herbal species, but as yet there are no applications to tree species. We report here the first successful plastid transformation in a tree species, Populus alba. A vector for plastid transformation of poplar (Populus alba) was constructed, which carried the spectinomycin resistance gene and the green fluorescence protein gene as marker genes. In the regenerated shoots, the site-specific integration of foreign genes and the establishment of a high homoplastomic state were confirmed. Immunoblot analysis and histological observations corroborated the accumulation of green fluorescence protein in chloroplasts. The establishment of a plastid transformation system in poplar provides a novel tool for tree biotechnology.     

Genome sequence and genetic transformation of a widely distributed and cultivated poplar

Populus alba is widely distributed and cultivated in Europe and Asia. This species has been used for diverse studies. In this study, we assembled a de novo genome sequence of P. alba var. pyramidalis (= P. bolleana) and confirmed its high transformation efficiency and short transformation time by experiments. Through a process of hybrid genome assembly, a total of 464 M of the genome was assembled. Annotation analyses predicted 37 901 protein‐coding genes. This genome is highly collinear to that of P. trichocarpa, with most genes having orthologs in the two species. We found a marked expansion of gene families related to histone and the hormone auxin but loss of disease resistance genes in P. alba if compared with the closely related P. trichocarpa. The genome sequence presented here represents a valuable resource for further molecular functional analyses of this species as a new tree model, poplar breeding practices and comparative genomic analyses across different poplars.     

Thaumatin-like proteins are differentially expressed and localized in phloem tissues of hybrid poplar

Background  

Two thaumatin-like proteins (TLPs) were previously identified in phloem exudate of hybrid poplar (Populus trichocarpa × P. deltoides) using proteomics methods, and their sieve element localization confirmed by immunofluorescence. In the current study, we analyzed different tissues to further understand TLP expression and localization in poplar, and used immunogold labelling to determine intracellular localization.     

Anaerobic induction of the maize<Emphasis Type="Italic"> GapC4</Emphasis> promoter in poplar leaves requires light and high CO<Subscript>2</Subscript>

The maize (Zea mays L.) glyceraldehyde-3-phosphate dehydrogenase gene 4 (GapC4) promoter confers anaerobic gene expression in tobacco (Nicotiana tabacum L.), potato (Solanum tuberosum L.) and Arabidopsis thaliana (L.) Heynh. Here we have investigated its expression in hybrid poplar (Populus tremula × P. alba). Our results show that the promoter is not expressed in leaves and stems under normoxic conditions while anaerobiosis induces reporter gene expression in leaves up to a level observed for the STLS-1 promoter from potato that is shown to confer leaf-specific gene expression in transgenic poplar. Anaerobic induction is cell autonomous and requires a CO₂ atmosphere and light. As in tobacco, the GapC4 promoter in poplar is wound inducible. The induction by CO₂ and light may reflect a natural situation because flooding, a natural cause of anaerobiosis, is often accompanied by high CO₂ concentrations in the floodwater. Our results show that the GapC4 promoter is suitable as an anaerobic reporter and as an inducible gene expression system in poplar.Abbreviations CaMV cauliflower mosaic virus - GapC4 glyceraldehyde-3-phosphate dehydrogenase gene 4 - GUS -glucuronidase - 4-MU methylumbelliferone - STLS-1 stem- and leaf-specific promoter 1     

Wound-induced and developmental activation of a poplar tree chitinase gene promoter in transgenic tobacco

Wounding hybrid poplar (Populus trichocarpa × P. deltoides) trees results in the expression of novel wound-inducible (win) mRNAs thought to encode proteins involved in defense against pests and pathogens. Members of thewin6 gene family encode acidic multi-domain chitinases, with combined structure and charge characteristics that differ from previously described chitinases.Win6 expression has been shown to occur in pooled unwounded leaves of a wounded (on multiple leaves) poplar plant. Here we demonstrate that wounding a single leaf induceswin6 expression locally, in the wounded leaf, and remotely, in specific unwounded leaves with strong vascular connections to the wounded leaf. We also demonstrate that awin6 promoter--glucuronidase (GUS) gene fusion (win6-GUS) responds to wounding locally and remotely in transgenic tobacco. These data indicate that the poplarwin6 promoter has regulatory elements that are responsive to wound signals in the heterologous host. In addition,win6-GUS is developmentally activated in unwounded young leaves and floral tissues of transgenic tobacco. Similar developmental expression patterns are found to occur forwin6 in poplar trees, demonstrating that a herbaceous plant can serve as a host for woody tree transgene analysis and can accurately predict expression patterns in tree tissues (e.g. flowers) that would be difficult to study in free-living trees.     

Transformation of white poplar (Populus alba L.) with a novel Arabidopsis thaliana cysteine proteinase inhibitor and analysis of insect pest resistance

Transgenic white poplar (Populus alba L.) plants expressing a novel Arabidopsis thaliana cysteine proteinase inhibitor (Atcys) gene have been produced using Agrobacterium tumefaciens-mediated gene transfer. Internodal stem segments of cv. Villafranca were co-cultivated with the EHA105 pBI-Atcys A. tumefaciens strain. Sixteen putative transgenic plant lines were regenerated from different calli with a transformation efficiency of 11%. The integration and expression of the cysteine proteinase inhibitor (Atcys) gene into the plant genome was confirmed by Southern and northern blot analyses. Papain inhibitory activity was detected in poplar transgenic tissues by means of a specific in vitro assay. Such activity was sufficient to inhibit most of the digestive proteinase activity of chrysomelid beetle (Chrysomela populi L.) and confer resistance to C. populi larvae on selected transgenic plants. A close correspondence between the inhibition of papain and resistance to poplar leaf beetle was observed in all tested transgenic lines. Our results indicate that Atcys could be succesfully employed in breeding programmes aimed at the selection of new poplar genotypes resistant to major insect pests.     

Chromosome-level genome assembly of a triploid poplar Populus alba ‘Berolinensis’

Many recent studies have provided significant insights into polyploid breeding, but limited research has been carried out on trees. The genomic information needed to understand growth and response to abiotic stress in polyploidy trees is largely unknown, but has become critical due to the threats to forests imposed by climate change. Populus alba ‘Berolinensis,’ also known “Yinzhong poplar,” is a triploid poplar from northeast China. This hybrid triploid poplar is widely used as a landscape ornamental and in urban forestry owing to its adaptation to adverse environments and faster growth than its parental diploid. It is an artificially synthesized male allotriploid hybrid, with three haploid genomes of P. alba ‘Berolinensis’ originating from different poplar species, so it is attractive for studying polyploidy genomic mechanisms in heterosis. In this study, we focused on the allelic genomic interactions in P. alba ‘Berolinensis,’ and generated a high-quality chromosome-level genome assembly consisting of 19 allelic chromosomes. Its three haploid chromosome sets are polymorphic with an average of 25.73 nucleotide polymorphism sites per kilobase. We found that some stress-related genes such as RD22 and LEA7 exhibited sequence differences between different haploid genomes. The genome assembly has been deposited in our polyploid genome online analysis website TreeGenomes ( https://www.treegenomes.com ). These polyploid genome-related resources will provide a critical foundation for the molecular breeding of P. alba ‘Berolinensis’ and help us uncover the allopolyploidization effects of heterosis and abiotic stress resistance and traits of polyploidy species in the future.     

Polyphenol oxidase overexpression in transgenic<Emphasis Type="Italic"> Populus</Emphasis> enhances resistance to herbivory by forest tent caterpillar (<Emphasis Type="Italic">Malacosoma disstria</Emphasis>)

In order to functionally analyze the predicted defensive role of leaf polyphenol oxidase (PPO; EC 1.10.3.1) in Populus, transgenic hybrid aspen (Populus tremula × P. alba) plants overexpressing a hybrid poplar (Populus trichocarpa × P. deltoides) PtdPPO1 gene were constructed. Regenerated transgenic plants showed high PPO enzyme activity, PtdPPO1 mRNA levels and PPO protein accumulation. In leaf disk bioassays, forest tent caterpillar (Malacosoma disstria) larvae feeding on PPO-overexpressing transgenics experienced significantly higher mortality and reduced average weight gain compared to larvae feeding on control leaves. However, this effect was observed only when older egg masses were used and the resulting larvae showed reduced growth and vigor. In choice tests, no effect of PPO overexpression was detected. Although PPO in poplar leaves is latent and requires activation with detergents or trypsin for full enzymatic activity, in caterpillar frass the enzyme was extracted in the fully activated form. This activation correlated with partial proteolytic cleavage, suggesting that PPO latency and activation during digestion could be an adaptive and defense-related feature of poplar PPO.     

A preliminary study on the seasonal body temperature rhythms of the captive mountain hare (Lepus timidus)

The mountain hare (Lepus timidus) is a year-round active herbivore adapted to survive the boreal winter. Captive mountain hares (N = 4) were implanted with intraabdominal thermosensitive loggers to record their core body temperature (T_b) for a year and during food deprivation (8–48 h) in summer and winter. The average T_b was 38.7 ± 0.01 °C in summer and 38.3 ± 0.01 °C in winter. The yearly T_b correlated positively with the ambient temperature. The 24-h T_b was the highest from late scotophase to early photophase in summer and winter and the lowest during middle-late photophase in summer or during early-middle scotophase in winter. The range of the 24-h oscillations in T_b increased in three animals in winter. Food deprivation did not induce hypothermia in summer or winter. These preliminary data suggest that the mountain hare can spare a modest amount of energy with the wintertime reduction in T_b.     

An alternative approach for gene transfer in trees using wild-typeAgrobacterium strains

Micropropagated shoots of three forest tree species, poplar (Populus tremula × P.alba), wild cherry (Prunus avium L.) and walnut (Juglans nigra × J. regia), were inoculated each with six different wild-typeAgrobacterium strains. Poplar and wild cherry developed tumors that grew hormone-independently, whereas on walnut, gall formation was weak. On poplar and wild cherry, tumors induced by nopaline strains developed spontaneously shoots that had a normal phenotype and did not carry oncogenic T-DNA. From these observations, we have established a co-inoculation method to transform plants, using poplar as an experimental model. The method is based on inoculation of stem internodes with anAgrobacterium suspension containing both an oncogenic strain that induces shoot differentiation and a disarmed strain that provides the suitable genes in a binary vector. We used the vector pBI121 carryingneo (kanamycin resistance) anduidA (-glucuronidase) genes to facilitate early selection and screening. Poplar plants derived from kanamycin-resistant shoots that did not carry oncogenic T-DNA, were shown to contain and to expressneo anduidA genes. These results suggest that wild-typeAgrobacterium strains that induce shoot formation directly from tumors can be used as a general tool for gene transfer, avoiding difficult regeneration procedures.This work is dedicated to the late Marie-France Michel who initiated the poplar biotechnology project at INRA.     

Computational identification and analysis of immune-associated nucleotide gene family in Arabidopsis thaliana

GTP-binding proteins represent a ubiquitous regulatory mechanism in controlling growth and development in eukaryotes under normal and stress conditions. The IAN/GIMAP proteins belong to a novel family of functionally uncharacterized GTP-binding proteins expressed in both plant and vertebrate cells during anti-pathogenic responses. To gain novel insights into their roles in plants, we did genome-wide analysis of the IAN/GIMAP gene family. We identified 13 Arabidopsis IAN/GIMAP genes, which share similar gene structures and mostly reside in a tandem cluster on chromosomes. Sequence comparison reveals that these genes encode 26–52 kDa proteins with one GTP-binding domain and a conserved box unique to the family. Phylogenetic analysis suggests that the IAN/GIMAP genes of angiosperms and vertebrates may have evolved by independent gene duplication events. GENEVESTIGATOR sources were mined for comprehensive and comparative Arabidopsis IAN/GIMAP gene family expression analysis. These data reveal that IAN/GIMAPs exhibit diverse expression patterns during development and in response to external stimuli, indicating that these paralogous genes are likely involved in complex biological processes in Arabidopsis. Our present findings provide a basis for elucidating the novel GTPase family protein-mediated regulatory mechanisms in the future.     

An efficient Agrobacterium-mediated transformation and regeneration system for leaf explants of two elite aspen hybrid clones Populus alba?��?P. berolinensis and Populus davidiana?��?P. bolleana

Transgenic technology has been successfully used for gene function analyses and trait improvement in cereal plants. However, its usage is limited in woody plants, especially in the difficult-to-transform but commercially viable hybrid poplar. In this work, an efficient regeneration and transformation system was established for the production of two hybrid aspen clones: Populus alba × P. berolinensis and Populus davidiana × P. bolleana. A plant transformation vector designed to express the reporter gene uidA, encoding β-glucuronidase (GUS), driven by the cauliflower mosaic virus 35S promoter, was used to detect transformation event at early stages of plant regeneration, and to optimize the parameters that may affect poplar transformation efficiency. Bacterium strain and age of leaf explant are two major factors that affect transformation efficiency. Addition of thidiazuron (TDZ) improved both regeneration and transformation efficiency. The transformation efficiency is approximately 9.3% for P. alba × P. berolinensis and 16.4% for P. davidiana × P. bolleana. Using this system, transgenic plants were usually produced in less than 1 month after co-cultivation. The growth characteristics and morphology of transgenic plants were identical to the untransformed wild type plants, and the transgenes could be inherited by vegetative propagation, as confirmed by PCR, Southern blotting, RT–PCR and β-glucuronidase staining analyses. The establishment of this system will help to facilitate the studies of gene functions in tree growth and development at a genome level, and as well as the introduction of some valuable traits in aspen breeding.     

Cryptosphaeria Canker of Populus nigra Caused by Cryptosphaeria pullmanensis,a New Threat to Poplar Industry in Iran

Trunk diseases are potential threats to the poplar industry worldwide, including Iran. A survey on trunk diseases of Populus nigra in north‐western Iran revealed a new canker disease associated with dieback and decline of this host in West Azarbaijan Province of Iran. Wood samples were collected from poplar trees showing canker, dieback and decline symptoms and taken to the laboratory. A total of 173 fungal isolates were recovered from symptomatic tissues, of those 147 isolates had similar cultural and morphological features on potato dextrose agar. Based on a combination of morphological characteristics and phylogenetic inferences including DNA sequence data from the internal transcribed spacer regions (ITS1, 5.8S rDNA, and ITS2), all 147 isolates were identified as Cryptosphaeria pullmanensis. The remaining 26 isolates were identified as Cytospora chrysosperma. Pathogenicity of Cr. pullmanensis on two‐year‐old P. nigra and Populus alba saplings under glasshouse conditions confirmed that Cr. pullmanensis is pathogenic on P. nigra and P. alba. Cryptosphaeria pullmanensis is here reported from Iran causing Cryptosphaeria canker on poplar trees for the first time. However, its host range, the extent of geographical distribution and management strategies remain to be examined.