Abstract
The aim of this chapter is to provide a description of the latest scientific advances in the field of gene functional analysis in grapevine. It provides general information about the studies conducted during the past decade to understand the natural variation of this plant and how this information has been exploited for the understanding of traits of interest. Likewise, it is exposed how the use of biotechnology tools have helped to characterize the mechanisms of gene expression and its regulation, as well as the subcellular localization of proteins and their interactions with other molecules. Finally, an approximation to the new technologies of gene editing and their potential application in the functional study of grapevine has been carried out.
Keywords
- Vitis vinifera
- QTL
- gene transfer
- genome editing
- CRISPR/Cas system
1. Introduction
In recent years, studies of plant functional analysis are becoming increasingly relevant. Genome sequencing of a number of organisms is providing the scientific community with a vast resource of DNA sequence information that is revolutionizing the way science is being done. Thereby, progress has been made in the functional genomics of grapevine following the whole genome sequencing and assembling of
Nowadays, there are several tools used by reverse genetics to induce variation into a gene and then used to infer its function. Genome editing is one of them [4]. This novel technique employs engineered nucleases that cut the DNA specifically generating targeted double‐strand breaks (DSBs) [5]. Starting from the already known nucleases, such as zinc finger proteins and transcription activator‐like effectors, and the recent discovered technology, the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system, a precise manipulation of gene sequences as well as the addition or deletion of DNA fragments on specific locus has recently become possible for some areas. For functional analysis, this means the possibility of making specific mutations in order to discover new gene functions, different relations between them, or even a synthetic pathway.
Another interesting challenge in grapevine investigation is the identification of those genes that contribute to the natural genetic variation of specific traits as well as understanding their main functions. The frequent quantitative nature of genetic variation requires the use of quantitative trait loci (QTLs) mapping approaches to understand the genetic structure of traits [6]. Interval mapping based on DNA markers could be used to genetically localize QTLs in natural and experimental populations [7, 8]. The development of new QTL mapping strategies and DNA research tools [9, 10], as well as the successful identification of few genes responsible of simple Mendelian traits [11, 12], have reinvigorated interest in grapevine QTL analysis. The present chapter summarizes the latest scientific advances in the field of grapevine functional analysis through the development and use of different biotechnological tools. It also describes diverse methods for the discovery and modification of genes that contribute to the finding of new sources of variability.
2. From QTL mapping to gene function
In model organisms, induced mutagenesis provides a powerful alternative for gene function discovery strategies derived from the knowledge of the phenotypic variation in plant (i.e., forward genetics). Because the prospects of gene identification are high and every gene affecting a trait is potentially a target, mutagenesis may present advantages with respect to natural, polygenic variation (i.e., quantitative trait loci) for identifying functional pathways and complex traits [13]. However, mutagenesis has not been extensively used in grapevine, although physical and chemical mutagens have been investigated [14, 15]. So far, the only published report on chemical mutagenesis of
On the other hand, the extensive development of genetic resources including Amplified fragment length polymorphism (AFLP) [17], Random amplification of polymorphic DNA (RAPD) [18], Simple Sequence Repeat (SSR) [19], and single nucleotide polymorphism (SNP) [20] markers for grapevine genetics in the era before the availability of the grapevine genome [1] allowed the characterization of the genetic determinants for several grapevine traits by means of QTL mapping. The study of the genetic control of major agronomic traits in grapevine using QTL analysis allowed the elucidation of traits such as seedlessness and berry weight [12, 18, 21–23]; berry phenolic composition [24–28]; aroma [29, 30]; berry firmness [31, 32]; fertility [9, 33]; flower hermaphroditism [34]; cluster architecture [35]; pathogen resistance [34, 36–38]; plant phenology [39, 40]; and adaptation to abiotic stresses [41] and to climate change [42]. Despite several of the reports allowed the identification of candidate sequences, just few of them have finally characterized the genes responsible for a particular function or trait. Fortunately, those few paradigmatic examples are relate to two major grapevine quality traits: seedlessness and aroma.
Seedlessness, the absence of seeds in the berry [43], has been largely studied since the first genetic reports on the trait [18, 21, 44]. Those studies were mainly performed by the analysis of seed and berry size inheritance. In these works, the phenotypes segregate in experimental populations with a continuous distribution, an indicative of the quantitate nature of the trait. To date, the most accepted model proposed that seedlessness is under the control of a dominant regulator gene named seed development inhibitor (
The functional characterization of
On the other hand, it has been widely studied that aromas in grapevine arise from volatile compounds, such as terpenes, norisoprenoids, and thiols [50]. Particularly, the aroma of Muscat grapes is linked to the presence of the monoterpenes geraniol, linalool, nerol, and α‐terpineol [51]. In plants, all isoprenoids are formed through two different and partially independent pathways, the mevalonic acid pathway (MEP, in the cytoplasm) and the methylerythritol phosphate pathway (in plastids). However, experimental reports have shown that most species mainly use one of the two pathways. Particularly in grapevine, the dominant pathway for monoterpene biosynthesis in leaves and berries is MEP route [52].
The route to the functional characterization of the genetic basis of aroma in grapevine starts with two simultaneous reports demonstrating the colocalization of a grapevine 1‐deoxy‐d‐xylulose 5‐phosphate synthase (
Despite the robustness of the two presented examples for seedlessness and aroma, forthcoming gene functions determination in grapevine will be boosted by the use of new and more efficient tools. Recently, the availability of next‐generation sequencing and whole‐genome sequence information allowed the generation of a SNP‐based genotyping array [55]. This array, developed for the grapevine international community, was built after the resequencing of 43
3. Gene transfer technologies as a tool for functional analysis
In the plant biotechnology community, the term transformation is used to describe the insertion of engineered gene sequences into a plant cell, leading to a change in the genetic makeup of the target cell and its derivatives (i.e., reverse genetics). The foreign molecule can function for a short time in the nucleus as an extrachromosomal entity (transient transformation) [57], or the integration into the genetic material of the target cell can be necessary for long‐term functionality and expression (stable transformation) [58, 59]. Therefore, transient expression has been used to evaluate factors that influence the stability or consistency of gene expression [60]. The final determination of factors that modulate transgene expression must ultimately be made only following introduction to plant cells for stable transformation. Methods for molecule transfer in grapevine involve biological methods (
3.1. Indirect gene transfer methods: Agrobacterium ‐mediated transformation of grapevine
Indirect transformation methods introduce plasmids, that is, independent circular molecules of DNA that are found in bacteria, into the target cell by means of bacteria capable of transferring genes to higher plant species [63]. The most popular used microorganisms are
Although other methods are suitable for plant transformation, such as protoplast or biolistic transformation, the
The most significant applications of transformation with
3.1.1. Agroinjection and agroinfiltration
Transformation and regeneration of grapevine plants have been achieved via organogenesis [84], embryogenesis [83, 93], or from nonembryogenic cell cultures [86, 94], demonstrating that grapevine is not recalcitrant to
Similarly, the agroinfiltration method consists of plunging detached leaves [69, 79] or whole plants [81] into the bacterial suspension. As the flow of bacterial suspensions through stomata and across the epidermis is impeded by the high surface tension of aqueous solutions, transient transformation is obtained through the rapid release of a vacuum to introduce the bacterial suspension into the mesophyll cells. Nowadays, this method represents an easy and noninvasive technique that allows gene expression in the whole leaf [67]. Agroinfiltration is usually performed on tissues of young plantlets grown
3.1.2. Cocultivation
This method is used to introduce bacterial suspension into intercellular spaces within plant tissue, simply by submerging above‐ground parts of the plant into an
Cocultivation with
3.1.3. Agroinoculation
Agroinfiltration was classically used for introducing gene constructs driven by a Ti plasmid. Additionally, this technique has enabled the introduction of virus‐derived vectors into several
3.1.4. Sonication‐assisted Agrobacterium ‐mediated transformation (SAAT)
A number of grapevine cultivars have been stably transformed using
3.1.5. Hairy roots
Hairy root technology has already been used in several functional studies relative to grapevine. Secondary metabolism investigation associated to flavonoid analysis of hairy roots overexpressing
3.2. Direct gene transfer methods in grapevine
Due to the difficulty of transforming plants through the use of
3.2.1. Polyethylene glycol (PEG) treatment and electroporation of protoplasts
Due to the rigid cellulose wall, it has been relatively difficult to handle plant cells. Several methods based on mechanical removal of cell walls and on the use of solvents have been used to degrade cell wall for the obtention of protoplasts. However, the methods involving the use of hydrolytic enzymes have been the most popular ones [106]. The protoplasts used for transformation are usually isolated by enzymatic digestion of mesophyll cells from leaves [107–109], berry mesocarp [106, 110], roots [111], stems [112], embryogenic tissue [108, 113, 114], and from fast‐grown suspension‐cultured cells [115–117].
Although the generation of transgenic lines represents a powerful research tool for characterizing plant gene function, protoplast‐based protocols for grapevine stable transformation have some drawbacks. The production of polyphenols and phytoalexins is induced at a high level during the digestion process and the corresponding genes remain activated during the culture of
PEG‐mediated transient assay utilizing protoplasts has become a powerful tool for rapid gene functional analysis that can be readily carried out using ordinary lab supplies and usually has high transformation efficiency. Wang et al. [117] described a rapid and efficient transient expression system for PEG‐mediated transformation of protoplasts derived from grape berry suspension‐cultured cells. The system was applied for subcellular localization studies of flavonoid biosynthesis enzymes using GFP as a reporter gene. In the same way, Zhao et al. [109] reported a simplified and highly efficient method for the isolation of mesophyll protoplasts from grapevine leaves and a modified transfection protocol using PEG. This transient transformation of protoplasts was developed to characterize the function of a heterologous plant defense gene through its gene expression, and was regarded as suitable for the study of protein expression, protein subcellular localization, and protein‐protein interaction.
In electroporation, cells are permeabilized by the application of very short, high‐voltage electric pulses to introduce DNA into cells. As mentioned before, the use of electroporation is restricted to stable transformation in species whose protoplasts are regenerable. Thus, the primary application of electroporation to plants has been for DNA uptake for studies of transient gene expression. As an example, protoplast electroporation has been used for virus inoculation [107, 108]. Valat et al. [107] used the mesophyll protoplast electroporation as a rapid screening technique of transgenic grapevine clones expressing the viral capsid gene or the movement protein gene of grapevine fanleaf virus (GFLV) to identify material that reduces or inhibit the accumulation of viral proteins at the cell level.
3.2.2. Particle bombardment (biolistics)
The most commonly used method for direct DNA uptake (or naked DNA introduction) is particle bombardment. Also known as biolistics, the technique consists on the acceleration of high‐density carrier particles covered with genes that pass through the cells, leaving the DNA inside. Although it is mainly reported as a stable transformation method, it is also convenient for transient expression assays. However, it requires expensive equipment and causes severe tissue damage, and usually yields low transformation efficiency. The advantages of this technique are that it has no limitation on species ranges, genotypes, or subcellular organelles. In grapevine, it has been assayed with the use of circular plasmids [89, 91, 104, 120] and minimal cassettes [121, 122]. Moreover, it has let cotransformation with multiple genes [61, 121]. Likewise, particle bombardment has allowed the manipulation of organ sections such as leaves [91], embryos [91], and suspension‐cultured cells [89, 104, 120–122] as targets for grape transformation.
Due to its versatility, the technique has been applied for several functional studies such as promoter analysis [89, 91, 120] and the regulatory function of some transcription factors [104]. In their work, Höll et al. [104] demonstrated via transient gene reporter assays that the cotransformation of cell suspensions with transcription factors (MYB14 and MYB15) and promoter sequences specifically activate the promoters of
4. New tools for genome editing
The dominant genome editing tools before 2013 were zinc finger nucleases (ZFNs) and transcription activator‐like effector nucleases (TALENs) [4]. Both are artificial fusion proteins comprising an engineered DNA‐binding domain fused to the nonspecific nuclease domain of the restriction enzyme
4.1. The CRISPR/Cas9 system
Clustered regularly interspaced short palindromic repeat‐associated Cas is an adaptive bacterial and archaeal immune system that uses antisense RNAs to control invasions of phages and plasmids [127]. CRISPR loci are short variable spacers separated by short repeats that are transcribed into noncoding RNAs. The noncoding RNAs form a functional complex with CRISPR‐associated Cas proteins and guide the complex to cleave complementary invading DNA [128] (Figure 3).
There are three CRISPR/Cas system types (I, II, and III) that uses distinct molecular mechanisms to achieve nucleic acid recognition and cleavage [129]. In genome editing, type II CRISPR/Cas system has been developed as a new gene‐targeting tool. The Cas9 endonuclease, from
4.2. Applications in plants
Although CRISPR/Cas system has not been applied on
With CRISPR/Cas9 system, efficient NHEJ‐mediated targeted mutagenesis was detected in
The fact that the CRISPR/Cas system allows multiplexing gene editing is particularly valuable [141, 148]. Xing et al. [149] developed a system where several sgRNA could be generated from one construct. Subsequently, simultaneous multiplex mutageneses were analyzed in maize protoplasts, transgenic maize lines, and transgenic
The CRISPR/Cas9 system can be used for several purposes in addition to genome editing. Disabled nucleases (catalytically inactive version of Cas9, dCas9) can still bind to their target DNA sequence, so they can be expressed as a fusion protein with the transactivation or transrepression domain of a transcription factor and be used to regulate gene expression [152, 153].
Besides all the great applications of this system, a major concern when using an RNA‐guided Cas9 is the off‐site target activity. Off‐site targeting is defined by the tolerance of Cas9 to mismatch in the RNA guide sequence and it is dependent on the number, position, and distribution of mismatches throughout the entire guide sequence [154–156]. The technical decision of using a 20‐nucleotide motif complementary to the target DNA in the sgRNA may leads to the possibility that it cross‐hybridizes to highly similar DNA sequences in other genomic regions. So, the use of genome‐specific designing tools for guide RNAs [157] in CRISPR‐Cas experiments is a strongly desirable prerequisite.
Although the CRISPR/Cas9 system has been thoroughly investigated in the past three years, there are no experiments made on grapevine yet. The first advances need to mention that Wang et al. [157] computationally identified and characterized five different types of CRISPR/Cas9 target sites and developed a user‐friendly database for upcoming editing projects of grapevine genomes. These novelties provide an encouraging future perspective for genome editing by the use of the CRISPR/Cas9 system.
5. Conclusions
A major challenge for grapevine research is the identification of genes and gene variants responsible for important agronomic traits and to assign biological function to annotated sequences. Demonstration of biological function requires genetic approaches that deal with genetic variation. During the last two decades, different molecular techniques have allowed the fine characterization of the natural genetic variation underlying QTLs for traits of agronomic interest, and in few cases, identified the responsible genes. Along with QTLs, the development of new methodologies of gene editing such as CRISPR/Cas and gene transfer methods, the detailed genomic, transcriptomic, and proteomic studies have been performed. Nowadays, these techniques are constantly evolving and becoming more and more simple, efficient, and precise. These set of tools will soon help promoting the progress in knowledge, both in functional genomics and biotechnology, for its subsequent application.
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