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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Plant Sciences".

Deadline for manuscript submissions: closed (30 April 2022) | Viewed by 28482

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Special Issue Editors

Prof. Dr. Matthias Fladung

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Guest Editor

Thuenen-Institute of Forest Genetics, Sieker Landstr. 2, 22927 Grosshansdorf, Germany
Interests: genetic engineering; genome editing; genomics of different tree species

Dr. Birgit Kersten

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Guest Editor

Thünen Institute of Forest Genetics, Sieker Landstrasse 2, D-22927 Grosshansdorf, Germany
Interests: plant molecular biology; bioinformatics; functional genome research; tree genomics and transcriptomics
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Special Issue Information

Dear Colleagues,

The improvement of forest trees is necessary to meet the growing demand for renewable wood resources in the near future. However, genetic improvement of forest trees by conventional breeding is limited by both long generation times and the space required to maintain large populations of forest trees. Biotechnology offers numerous possibilities for tree improvement, potentially greater than for agricultural crops.

The Special Issue “Tree Genetic Engineering, Genome Editing, and Genomics” aims to summarize all ongoing recent activities around the world on accelerated tree improvement for the production of more wood on less land, improving wood quality, increasing resistance to pests and pathogens, and increasing stress tolerance in the face of climate change, a major challenge to biodiversity.

As the Guest Editors of this Special Issue, we would like to invite you to participate in this venture by contributing a paper on the topic of your choice within the broad area covered by “Tree Genetic Engineering, Genome Editing, and Genomics”. You can contribute on more than one topic, and are free to have co-authors of your choice.

We are look forward receiving your contribution.

Prof. Dr. Matthias Fladung
Dr. Birgit Kersten
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. International Journal of Molecular Sciences is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. There is an Article Processing Charge (APC) for publication in this open access journal. For details about the APC please see here. Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

forest tree
wood production
wood quality
tree growth
pest resistance
climate change
short rotation coppice
photosynthesis
mycorrhiza
gene technology
genome editing
CRISPR/Cas
molecular marker
next-generation sequencing

Published Papers (10 papers)

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Editorial

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3 pages, 184 KiB

Open AccessEditorial

Tree Genetic Engineering, Genome Editing and Genomics

by Matthias Fladung and Birgit Kersten

Int. J. Mol. Sci. 2022, 23(22), 13980; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms232213980 - 12 Nov 2022

Viewed by 1361

Abstract

In this Special Issue [...] Full article

(This article belongs to the Special Issue Tree Genetic Engineering, Genome Editing and Genomics)

Research

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17 pages, 13164 KiB

Open AccessArticle

Transcriptomic Complexity of Culm Growth and Development in Different Types of Moso Bamboo

by Long Li, Binao Zhou, Dong Liu, Hongyu Wu, Qianqian Shi, Shuyan Lin and Wenjing Yao

Int. J. Mol. Sci. 2023, 24(8), 7425; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms24087425 - 18 Apr 2023

Cited by 2 | Viewed by 1298

Abstract

Moso bamboo is capable of both sexual and asexual reproduction during natural growth, resulting in four distinct types of culms: the bamboo shoot-culm, the seedling stem, the leptomorph rhizome, and a long-ignored culm—the outward-rhizome. Sometimes, when the outward rhizomes break through the soil, they continue to grow longitudinally and develop into a new individual. However, the roles of alternative transcription start sites (aTSS) or termination sites (aTTS) as well as alternative splicing (AS) have not been comprehensively studied for their development. To re-annotate the moso bamboo genome and identify genome-wide aTSS, aTTS, and AS in growing culms, we utilized single-molecule long-read sequencing technology. In total, 169,433 non-redundant isoforms and 14,840 new gene loci were identified. Among 1311 lncRNAs, most of which showed a positive correlation with their target mRNAs, one-third of these IncRNAs were preferentially expressed in winter bamboo shoots. In addition, the predominant AS type observed in moso bamboo was intron retention, while aTSS and aTTS events occurred more frequently than AS. Notably, most genes with AS events were also accompanied by aTSS and aTTS events. Outward rhizome growth in moso bamboo was associated with a significant increase in intron retention, possibly due to changes in the growth environment. As different types of moso bamboo culms grow and develop, a significant number of isoforms undergo changes in their conserved domains due to the regulation of aTSS, aTTS, and AS. As a result, these isoforms may play different roles than their original functions. These isoforms then performed different functions from their original roles, contributing to the transcriptomic complexity of moso bamboo. Overall, this study provided a comprehensive overview of the transcriptomic changes underlying different types of moso bamboo culm growth and development. Full article

(This article belongs to the Special Issue Tree Genetic Engineering, Genome Editing and Genomics)

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12 pages, 3148 KiB

Open AccessArticle

A Constitutively Active Cytokinin Receptor Variant Increases Cambial Activity and Stem Growth in Poplar

by Michael Riefler, Tobias Brügmann, Matthias Fladung and Thomas Schmülling

Int. J. Mol. Sci. 2022, 23(15), 8321; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23158321 - 28 Jul 2022

Cited by 2 | Viewed by 1434

Abstract

The cambial meristem is responsible for bark and wood formation in woody plants. The activity of the cambial meristem is controlled by various factors; one of them is the plant hormone cytokinin. Here, we have explored different approaches to genetically engineering cambial activity in poplar plants by the ectopic expression of a cytokinin biosynthesis gene with enhanced activity (named ROCK4) or of a gene encoding a constitutively active cytokinin receptor variant (ROCK3). Both genes are derived from Arabidopsis thaliana and were expressed in poplar trees under the control of their own promoter or the cambium-specific pHB8 promoter. pIPT3:ROCK4- and pHB8:ROCK4-expressing plants were smaller than wild-type plants and formed more lateral branches; pHB8:ROCK4 transgenic plants additionally showed an increased stem diameter. In contrast, pAHK3:ROCK3- and pHB8:ROCK3-expressing plants grew taller than wild type without an altered branching pattern and formed more cambial cells, leading to increased radial stem growth. The effectivity of ROCK3 when expressed in either secondary phloem cells or in cambial cells is consistent with a dual, tissue-autonomous and non-autonomous activity of cytokinin in regulating cambial activity. We propose ROCK3 as a novel gene to enhance biomass formation in woody plants. Full article

(This article belongs to the Special Issue Tree Genetic Engineering, Genome Editing and Genomics)

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12 pages, 3078 KiB

Open AccessArticle

Investigation of PtSGT1 and PtSGT4 Function in Cellulose Biosynthesis in Populus tomentosa Using CRISPR/Cas9 Technology

by Yinxuan Xue, Siyan Li, Deyu Miao, Sai Huang, Bin Guo, Shanwen Li and Xin-Min An

Int. J. Mol. Sci. 2021, 22(24), 13200; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms222413200 - 07 Dec 2021

Cited by 3 | Viewed by 2355

Abstract

Cellulose synthesis is a complex process in plant cells that is important for wood processing, pulping, and papermaking. Cellulose synthesis begins with the glycosylation of sitosterol by sitosterol glycosyltransferase (SGT) to produce sitosterol-glucoside (SG), which acts as the guiding primer for cellulose production. However, the biological functions of SGTs in Populus tomentosa(P. tomentosa) remain largely unknown. Two full-length PtSGT genes (PtSGT1 and PtSGT4) were previously isolated from P. tomentosa and characterized. In the present study, CRISPR/Cas9 gene-editing technology was used to construct PtSGT1-sgRNA and PtSGT4-sgRNA expression vectors, which were genetically transformed into P. tomentosa using the Agrobacterium-mediated method to obtain transgenic lines. Nucleic acid and amino acid sequencing analysis revealed both base insertions and deletions, in addition to reading frame shifts and early termination of translation in the transgenic lines. Sugar metabolism analysis indicated that sucrose and fructose were significantly downregulated in stems and leaves of mutant PtSGT1-1 and PtSGT4-1. Glucose levels did not change significantly in roots and stems of PtSGT1-1 mutants; however, glucose was significantly upregulated in stems and downregulated in leaves of the PtSGT4-1 mutants. Dissection of the plants revealed disordered and loosely arranged xylem cells in the PtSGT4-1 mutant, which were larger and thinner than those of the wild-type. This work will enhance our understanding of cellulose synthesis in the cell walls of woody plants. Full article

(This article belongs to the Special Issue Tree Genetic Engineering, Genome Editing and Genomics)

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17 pages, 2907 KiB

Open AccessArticle

Tracing CRISPR/Cas12a Mediated Genome Editing Events in Apple Using High-Throughput Genotyping by PCR Capillary Gel Electrophoresis

by Susan Schröpfer and Henryk Flachowsky

Int. J. Mol. Sci. 2021, 22(22), 12611; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms222212611 - 22 Nov 2021

Cited by 7 | Viewed by 2844

Abstract

The use of the novel CRISPR/Cas12a system is advantageous, as it expands the possibilities for genome editing (GE) applications due to its different features compared to the commonly used CRISPR/Cas9 system. In this work, the CRISPR/Cas12a system was applied for the first time to apple to investigate its general usability for GE applications. Efficient guide RNAs targeting different exons of the endogenous reporter gene MdPDS, whose disruption leads to the albino phenotype, were pre-selected by in vitro cleavage assays. A construct was transferred to apple encoding for a CRISPR/Cas12a system that simultaneously targets two loci in MdPDS. Using fluorescent PCR capillary electrophoresis and amplicon deep sequencing, all identified GE events of regenerated albino shoots were characterized as deletions. Large deletions between the two neighboring target sites were not observed. Furthermore, a chimeric composition of regenerates and shoots that exhibited multiple GE events was observed frequently. By comparing both analytical methods, it was shown that fluorescent PCR capillary gel electrophoresis is a sensitive high-throughput genotyping method that allows accurate predictions of the size and proportion of indel mutations for multiple loci simultaneously. Especially for species exhibiting high frequencies of chimerism, it can be recommended as a cost-effective method for efficient selection of homohistont GE lines. Full article

(This article belongs to the Special Issue Tree Genetic Engineering, Genome Editing and Genomics)

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15 pages, 3649 KiB

Open AccessArticle

Selection and Validation of Candidate Reference Genes for Gene Expression Analysis by RT-qPCR in Rubus

by Yaqiong Wu, Chunhong Zhang, Haiyan Yang, Lianfei Lyu, Weilin Li and Wenlong Wu

Int. J. Mol. Sci. 2021, 22(19), 10533; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms221910533 - 29 Sep 2021

Cited by 17 | Viewed by 1895

Abstract

Due to the lack of effective and stable reference genes, studies on functional genes in Rubus, a genus of economically important small berry crops, have been greatly limited. To select the best internal reference genes of different types, we selected four representative cultivars of blackberry and raspberry (red raspberry, yellow raspberry, and black raspberry) as the research material and used RT-qPCR technology combined with three internal stability analysis software programs (geNorm, NormFinder, and BestKeeper) to analyze 12 candidate reference genes for the stability of their expression. The number of most suitable internal reference genes for different cultivars, tissues, and fruit developmental stages of Rubus was calculated by geNorm software to be two. Based on the results obtained with the three software programs, the most stable genes in the different cultivars were RuEEF1A and Ru18S. Finally, to validate the reliability of selected reference genes, the expression pattern of the RuCYP73A gene was analyzed, and the results highlighted the importance of appropriate reference gene selection. RuEEF1A and Ru18S were screened as reference genes for their relatively stable expression, providing a reference for the further study of key functional genes in blackberry and raspberry and an effective tool for the analysis of differential gene expression. Full article

(This article belongs to the Special Issue Tree Genetic Engineering, Genome Editing and Genomics)

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16 pages, 2686 KiB

Open AccessArticle

CRISPR-Knockout of CSE Gene Improves Saccharification Efficiency by Reducing Lignin Content in Hybrid Poplar

by Hyun-A Jang, Eun-Kyung Bae, Min-Ha Kim, Su-Jin Park, Na-Young Choi, Seung-Won Pyo, Chanhui Lee, Ho-Young Jeong, Hyoshin Lee, Young-Im Choi and Jae-Heung Ko

Int. J. Mol. Sci. 2021, 22(18), 9750; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22189750 - 09 Sep 2021

Cited by 22 | Viewed by 2878

Abstract

Caffeoyl shikimate esterase (CSE) has been shown to play an important role in lignin biosynthesis in plants and is, therefore, a promising target for generating improved lignocellulosic biomass crops for sustainable biofuel production. Populus spp. has two CSE genes (CSE1 and CSE2) and, thus, the hybrid poplar (Populus alba × P. glandulosa) investigated in this study has four CSE genes. Here, we present transgenic hybrid poplars with knockouts of each CSE gene achieved by CRISPR/Cas9. To knockout the CSE genes of the hybrid poplar, we designed three single guide RNAs (sg1–sg3), and produced three different transgenic poplars with either CSE1 (CSE1-sg2), CSE2 (CSE2-sg3), or both genes (CSE1/2-sg1) mutated. CSE1-sg2 and CSE2-sg3 poplars showed up to 29.1% reduction in lignin deposition with irregularly shaped xylem vessels. However, CSE1-sg2 and CSE2-sg3 poplars were morphologically indistinguishable from WT and showed no significant differences in growth in a long-term living modified organism (LMO) field-test covering four seasons. Gene expression analysis revealed that many lignin biosynthetic genes were downregulated in CSE1-sg2 and CSE2-sg3 poplars. Indeed, the CSE1-sg2 and CSE2-sg3 poplars had up to 25% higher saccharification efficiency than the WT control. Our results demonstrate that precise editing of CSE by CRISPR/Cas9 technology can improve lignocellulosic biomass without a growth penalty. Full article

(This article belongs to the Special Issue Tree Genetic Engineering, Genome Editing and Genomics)

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30 pages, 3753 KiB

Open AccessArticle

Genome-Wide Identification, Structure Characterization, and Expression Pattern Profiling of the Aquaporin Gene Family in Betula pendula

by Jean-Stéphane Venisse, Eele Õunapuu-Pikas, Maxime Dupont, Aurélie Gousset-Dupont, Mouadh Saadaoui, Mohamed Faize, Song Chen, Su Chen, Gilles Petel, Boris Fumanal, Patricia Roeckel-Drevet, Arne Sellin and Philippe Label

Int. J. Mol. Sci. 2021, 22(14), 7269; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22147269 - 06 Jul 2021

Cited by 4 | Viewed by 3154

Abstract

Aquaporin water channels (AQPs) constitute a large family of transmembrane proteins present throughout all kingdoms of life. They play key roles in the flux of water and many solutes across the membranes. The AQP diversity, protein features, and biological functions of silver birch are still unknown. A genome analysis of Betula pendula identified 33 putative genes encoding full-length AQP sequences (BpeAQPs). They are grouped into five subfamilies, representing ten plasma membrane intrinsic proteins (PIPs), eight tonoplast intrinsic proteins (TIPs), eight NOD26-like intrinsic proteins (NIPs), four X intrinsic proteins (XIPs), and three small basic intrinsic proteins (SIPs). The BpeAQP gene structure is conserved within each subfamily, with exon numbers ranging from one to five. The predictions of the aromatic/arginine selectivity filter (ar/R), Froger’s positions, specificity-determining positions, and 2D and 3D biochemical properties indicate noticeable transport specificities to various non-aqueous substrates between members and/or subfamilies. Nevertheless, overall, the BpePIPs display mostly hydrophilic ar/R selective filter and lining-pore residues, whereas the BpeTIP, BpeNIP, BpeSIP, and BpeXIP subfamilies mostly contain hydrophobic permeation signatures. Transcriptional expression analyses indicate that 23 BpeAQP genes are transcribed, including five organ-related expressions. Surprisingly, no significant transcriptional expression is monitored in leaves in response to cold stress (6 °C), although interesting trends can be distinguished and will be discussed, notably in relation to the plasticity of this pioneer species, B. pendula. The current study presents the first detailed genome-wide analysis of the AQP gene family in a Betulaceae species, and our results lay a foundation for a better understanding of the specific functions of the BpeAQP genes in the responses of the silver birch trees to cold stress. Full article

(This article belongs to the Special Issue Tree Genetic Engineering, Genome Editing and Genomics)

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Review

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26 pages, 964 KiB

Open AccessReview

From Genome Sequencing to CRISPR-Based Genome Editing for Climate-Resilient Forest Trees

by Hieu Xuan Cao, Giang Thi Ha Vu and Oliver Gailing

Int. J. Mol. Sci. 2022, 23(2), 966; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23020966 - 16 Jan 2022

Cited by 15 | Viewed by 4166

Abstract

Due to the economic and ecological importance of forest trees, modern breeding and genetic manipulation of forest trees have become increasingly prevalent. The CRISPR-based technology provides a versatile, powerful, and widely accepted tool for analyzing gene function and precise genetic modification in virtually any species but remains largely unexplored in forest species. Rapidly accumulating genetic and genomic resources for forest trees enabled the identification of numerous genes and biological processes that are associated with important traits such as wood quality, drought, or pest resistance, facilitating the selection of suitable gene editing targets. Here, we introduce and discuss the latest progress, opportunities, and challenges of genome sequencing and editing for improving forest sustainability. Full article

(This article belongs to the Special Issue Tree Genetic Engineering, Genome Editing and Genomics)

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29 pages, 1464 KiB

Open AccessReview

Achievements and Challenges of Genomics-Assisted Breeding in Forest Trees: From Marker-Assisted Selection to Genome Editing

by Sunny Ahmar, Paulina Ballesta, Mohsin Ali and Freddy Mora-Poblete

Int. J. Mol. Sci. 2021, 22(19), 10583; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms221910583 - 30 Sep 2021

Cited by 19 | Viewed by 5252

Abstract

Forest tree breeding efforts have focused mainly on improving traits of economic importance, selecting trees suited to new environments or generating trees that are more resilient to biotic and abiotic stressors. This review describes various methods of forest tree selection assisted by genomics and the main technological challenges and achievements in research at the genomic level. Due to the long rotation time of a forest plantation and the resulting long generation times necessary to complete a breeding cycle, the use of advanced techniques with traditional breeding have been necessary, allowing the use of more precise methods for determining the genetic architecture of traits of interest, such as genome-wide association studies (GWASs) and genomic selection (GS). In this sense, main factors that determine the accuracy of genomic prediction models are also addressed. In turn, the introduction of genome editing opens the door to new possibilities in forest trees and especially clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9). It is a highly efficient and effective genome editing technique that has been used to effectively implement targetable changes at specific places in the genome of a forest tree. In this sense, forest trees still lack a transformation method and an inefficient number of genotypes for CRISPR/Cas9. This challenge could be addressed with the use of the newly developing technique GRF-GIF with speed breeding. Full article

(This article belongs to the Special Issue Tree Genetic Engineering, Genome Editing and Genomics)

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