UV-B perception and signalling grape homologues.
Grapevine is one of the most abundant crops worldwide, with varieties destined for fresh and dry consumption, as well as wine production. Unfortunately, grapevine plants are affected by both biotic and abiotic stresses, generating significant economic losses. These conditions can negatively impact grape cultivation at different stages: plant and berry development during pre- and post-harvest, production, fresh fruit processing and export, along with wine quality. Most of the grapevine varieties are susceptible to several pathogens and within this chapter, particular attention is given to fungi (Botrytis cinerea and Erysiphe necator) and viruses, since they are a worldwide concern. Within the latter, special focus is given to the grapevine leafroll disease, a complex and destructive infection. On the other hand, abiotic stress is also relevant in grapevine, and in this chapter it will be exemplified by UV-B radiation and its impact on growth and fruit development, plant adaptive responses and its relationship with the quality of grape berries for winemaking. The main biotic and abiotic grapevine stress factors are reviewed in this chapter, considering a special focus on biotechnological approaches carried out in order to address them and minimize their detrimental consequences.
- grapevine fungal diseases
- Erysiphe necator
- Botrytis cinerea
- grapevine viruses
- UV-B radiation
- grapevine biotechnology
Most of the grapevine varieties are susceptible to several biotic agents, such as phytoplasma, bacteria, fungi, oomycetes, viruses and nematodes, which dramatically reduce plant yield and fruit quality, and negatively impact plant development. In vineyards, the most important diseases are caused by microorganisms such as fungi, oomycetes and viruses. Within pathogenic fungi,
On the other hand, nearly 60 viruses can infect grapevine plants, a much higher number than the ones affecting other perennial crops. Under natural conditions, grapevine viruses are transmitted by insect or nematode vectors. However, since grapevine is usually propagated by grafting, viruses can also disseminate within plants through these cuttings . It is noteworthy to mention that unlike to other pathogens, grapevine plants present no virus resistance, meaning that they can establish compatible interactions where viral pathogens can spread throughout all tissues, generating a global cellular stress and developmental defects [4–6]. Regarding these infections, the leafroll disease is one of the most complex viral diseases known and is considered one of the most destructive in grapevines. In addition to their economic detriment to grapevine cultures, all viruses are relevant when the sanitary status of the vineyard is considered.
Abiotic stress factors, particularly water availability, temperature and light are also relevant in grapevine. Among them, ultraviolet (UV)-B radiation impacts on grapevine plants growth and normal fruit development.
Biotechnological approaches aimed to solve grapevine stress affections, in areas regarding grapevine physiology and genetics, are a main requirement for optimizing and improving quality of this species through biotechnological tools.
2. Grapevine biotic stress
As mentioned above, biotic stress is related to infection caused by phytopathogenic organisms such as bacteria, nematodes, fungi, oomycetes and viruses, among others. These pathogens get the necessary elements for growth and reproduction from its hosts. According to their infection strategies, plant pathogens can be classified as necrotrophics, biotrophics and hemibiotrophics. Necrotrophic pathogens on one hand, extract nutrients from dead cells during colonization, secreting lytic enzymes and phytotoxins in order to promote necrosis in the host plant. Biotrophic pathogens, on the other hand, feed on living tissue maintaining the viability of the host in order to obtain metabolism products. Finally, hemibiotrophic pathogens start with a biotrophic infection phase, followed by a late necrotrophic one .
2.1. Fungal diseases in grapevine: a biotrophic and necrotrophic model
Nowadays, most of the wine, table grape and dried fruit cultivars have the Eurasian grape species
The most common fungal grape diseases are the powdery mildew and grey mould caused by the biotrophic pathogen
2.1.1. Powdery mildew:
The powdery mildew disease is associated with large production losses as it reduces yield and fruit quality, mainly affecting the sugar content and acidity of the berries, although it can also infect other green tissues. This pathogen can be found in all grape-growing regions, especially in dry and warm weathers .
Being an obligated biotrophic pathogen,
When environmental or nutritional conditions become unfavourable,
2.1.2. Grey mould:
The necrotrophic fungus
Grey mould disease causes heavy yield losses in table and wine grapes all around the world. As a consequence of the increase of the international trade of cold-stored products, this fungus has gained great importance because it can grow effectively over long periods of time at just above freezing temperatures . In the field, it can spread to other grapes by insects which can carry viable conidia and generate mechanical damage .
Once the conidium attaches to its host, it can germinate and develop to an infective structure called appressorium, which is able to breach the cuticle by means of a penetration peg (Figure 2). The underlying cells are killed by the fungus, and the primary lesion is established. After the skin barrier is damaged,
In some tissues,
2.2. Grapevine responses to fungal diseases
Plants are considered to have two types of immunity: a general one against a broad spectrum of microorganisms, and other specific one against a particular pathogen. Both responses are characterized by their ability to recognize pathogen components, transduce the stress signal and induce a defence response. However, the main difference between the both is considered to be the robustness and duration of the response .
The first type of immunity is known as PTI (pathogen-associated molecular patterns (PAMP) triggered immunity) and is activated by PAMP recognition receptors (PRR) that detect structural pathogen components and transduce the signal for the induction of a basal response. This type of immunity is mainly related to the prevention of pathogen entry into plant cells ; however, it is not completely effective against biotrophic and necrotrophic fungi. On the latter case, the response is activated by damage-associated molecular patterns (DAMP) recognition mainly derived from the host cell wall fragments generated by CWDE .
The second line of defence is known as ETI or effector triggered immunity, capable of directly or indirectly recognizing specific pathogen effectors through the expression of resistance proteins (R proteins). This recognition induces a more robust and efficient response, mainly against biotrophic pathogens, by preventing them to complete their life cycle in the host, interrupting nutrient uptake and eliminating the infected cells along with the pathogen . Since this response against biotrophic pathogens (and hemibiotrophic too) generally ends with programmed cell death (PCD) of infected tissue, some necrotrophic pathogens induce this mechanism during infection in order to bypass plant defences and rapidly kill tissue for nutritional benefits .
Plant defence mechanisms are finely regulated by plant hormones, mainly jasmonic acid (JA), ethylene (Et) and salicylic acid (SA), which communicate synergistically or antagonistically depending on the type of pathogen. Generally speaking, the defence against necrotrophic pathogens are considered to be mediated by JA and Et, while the defence against biotrophic pathogens by SA . However,
2.2.1. Grapevine defences against
Resistance to powdery mildew in the Vitaceae family is closely related to its evolutionary history.
Ontogenic, or age-related resistance, also has a role in the defence against
2.2.2. Grapevine defences against
Low or no resistant phenotypes to grey mould have been described in most common table grape
Structural barriers are related to the fungal primary infection process (i.e. appressoria formation and plant tissue penetration), while inducible responses are associated with subsequent infection ones . In this case, PTI is mainly activated by DAMPs, host cell wall fragments generated by fungal CWDE and PAMPs such as chitin fragments of fungal cell walls, among others. These are identified by specific PRR receptors, such as cell-wall-associated kinases, which in turn activate the defence signalling cascade, culminating in hormones and transcription factors biosynthesis [12, 24]. This response induces protease inhibitors generation and secondary metabolite biosynthesis (i.e. anthocyanins and phytoalexins). The flavonoid phytoalexin plays an important role in the defence response of grapes. The rapid production of resveratrol, major compound of the stilbene family, and its transformation into Viniferins enhance resistance to fungal pathogens in grapevine cultivars . Resveratrol and pterostilbene (two grapevine phytoalexins) produce malformation or growth inhibition of germ tubes, cytoplasmic granulation of the cellular content and the disruption of the plasma membrane in
2.3. Biotechnological strategies for fungal control in grapevine
Regarding control of fungal pathogens, major improvement efforts have been directed towards enhancing fungal-disease resistance in table and wine grape cultivars. Development and optimization of alternative strategies to reduce the use of classic chemical inputs for protection against diseases in vineyard is becoming a necessity. Nowadays, fungal-related diseases are controlled through fungicide applications of organic and inorganic composition. The most used compounds are sulphurs, petroleum-based oils, inorganic salts, benzimidazoles and ergosterol biosynthesis inhibitors, among others . However, these management practices usually generate negative impacts on the environment and have elevated health and safety hazards. Various sources have speculated that sulphur, the most heavily used agricultural chemical, can cause respiratory illnesses and other adverse health effects . In soil, sulphur is slowly converted by bacteria to sulphate, which generally does not cause harm. Other synthetic compounds used for treatment and prevention, such as sterol inhibitors have not been reported as having negative environmental or human health effects.
2.3.1. Genetic improvement
Genetic improvement is an agronomic practice widely used to confer interest features to a crop through hybridization between different cultivars or even species, in order to obtain new varieties. In
Many North American
Genetic knowledge of the resistance trait is crucial to achieve a significant improvement of grapevine through breeding. Several powdery mildew resistance loci have been identified and mapped to date. The Run1 locus was described in
One of the main concerns about using pathogen resistance genes in plant breeding is the potential appearance of new pathogen strains that could breakdown the resistance. To overcome this latent problem, actual breeding efforts are focusing on stacking or pyramiding two or more resistance genes within a single cultivar to increase the durability of the resistance in the field. In this scenario, pathogen reproduction will be restricted even if infection by a new pathogen strain with a modified or lost effector molecule occurs. Thus, biotechnological tools have become essential for the development of new resistant cultivars. Marker-assisted gene pyramiding is one of the main applications of DNA markers in plant breeding. The use of molecular marker-assisted selection allows the identification of segregants that may exhibit the same phenotype but carry multiple resistant genes . A grapevine progeny with individuals carrying both Run1 and Ren1 loci was developed in 2010, where Run1 was introgressed from a
Unlike to what happens with
2.3.2. Genetic manipulation
The development of highly reproducible genetic engineering protocols for grapevine cultivars and rootstocks now allows the identiﬁcation, screening and/or introduction of grapevine-derived genes related to desirable traits, such as disease resistance.
Pathogenesis-related (PR) proteins were screened for their response to fungal pathogen infection. Genetically modiﬁed (GM) grapevines constitutively expressing rice chitinase genes exhibited enhanced resistance to powdery mildew [49, 50]; however, no resistance was observed when plants expressed barley chitinase genes . Other non-grapevine-derived genes, such as the polygalacturonase inhibiting protein (PGIP) and other lytic peptides, were demonstrated to improve fungal disease resistance .
Two endochitinase (
Genetic manipulation of phytoalexins has been done in order to increase disease resistance of plants. Use of modern molecular biology tools for elucidating the control mechanisms of phytoalexin synthesis and for engineering disease-resistant plants is based on the expression of stress- or disease-related genes. Few reports attempting the manipulation of phytoalexins biosynthesis by genetic engineering have been published, with most of them related to resveratrol, the major phytoalexin from Vitaceae. STS, the key enzyme in resveratrol synthesis, uses as substrates precursor molecules that are present throughout the plant kingdom. Therefore, the introduction of a single gene is sufficient to synthesize resveratrol in heterologous plant species .
The grapevine rootstock 41-B, overexpressing the grapevine
All the aforementioned results demonstrate that improved fungal tolerance can be accomplished through transgene expression. In addition, they support the use of iterative molecular and physiological phenotyping in order to select tolerant individuals from GM grapevine populations.
2.3.3. Biological control
Biological control of fungal pathogens is based on the use of microorganisms to prevent or reduce the damage produced during infection. Among the best studied biocontrol agents we can find are the filamentous fungi of the
Another biocontrol mechanism is the activation of the induced systemic resistance (ISR) in plants; this mechanism can be induced by elicitors released by the biocontrol agent (ranging a wide variety of molecules), and it has been attributed to non-pathogenic microorganisms associated to plants, such as saprophytes . Generally speaking, microorganisms exhibit a combination of the mentioned mechanisms, thus reducing the risk of pathogen resistance .
Among the bacteria able to synthesize and secrete anti-fungal molecules, those belonging to the genus
Another control strategy consists in the stimulation and/or potentiation of the grapevine defence responses by the means of elicitors . Elicitors are defined as a more specific class of purified molecules originated from microorganisms or plants which are able to stimulate an innate immune response in plants .
Elicitor perception also increases the level of plant resistance against future pathogen attack . Induced resistance is often related to the ‘priming’ or potentiation phenomenon, and some molecules perceived by plants have also been shown to induce these effects [66, 68]. The definition of priming is related to the physiological state of the plant after an initial biotic or abiotic stimulus. This priming allows the plant to respond in a faster and/or stronger way to following biotic and/or abiotic challenges, often resulting in an improved tolerance in comparison to non-primed plants . The mechanism of this phenomenon remains relatively unknown to date, but recent hypotheses suggest that accumulation of dormant MAPKs, chromatin modifications and alterations of primary metabolism could be involved in the process [66, 68].
Bacterial elicitors were recently shown to stimulate innate immunity in grapevine cultivars through cytoskeleton re-organization, early signalling event activation and defence gene induction . Fungal elicitors have also been proved to be very efficient in stimulating innate immunity in grapevines. The deacetylated derivative of chitin (chitosan) elicitor triggered defence responses and protection against
Few of these products have shown acceptable effectiveness against biotrophic pathogens. Therefore, until now, there is not an elicitor-based product that can be used instead of conventional agrochemicals in order to successfully fight
2.4. Viral infections in grapevine: an example of compatible host-pathogen interaction
Viral diseases in grapevine are highly complex. This complexity is due to the large amount of different viruses that can infect grapevine plants, occurring most of the time as multiple infections, and because of the nature of the compatible pathogen-host interactions that is established. Viral infections in grapevine plants affect vegetative organs inducing foliar deformations, alterations in leaf colour and, in some cases, graft rejection (Figure 3) .
Severe infections also reduce berry setting and cause irregular and delayed ripening [72, 73]. Currently, more than 60 viruses have been described in grapevine , which together with viroids, phytoplasmas and insect-transmitted xylematic bacteria, correspond to the highest number of intracellular pathogen described for a single crop. Grapevine infecting viruses are classified according to several parameters, including size particle, genome structure, replication strategies, transmission vector and serological information . In general, grapevine infecting viruses exhibit single-stranded RNA (ssRNA) genomes, and the most relevant belong to the Nepovirus, Ampelovirus, Closterovirus and Vitivirus genera.
Viruses belonging to the Nepovirus genus are widely disseminated and are responsible for the degeneration disease. The most representative are the GFLV (Grapevine Fanleaf Virus), ArMV (Arabis Mosaic Virus), SLRSV (Strawberry Latent Ringspot Virus), ToRSV (Tomato Ringspot Virus) and TRSV (Tobacco Ringspot Virus) [72–75]. Most viruses of these groups are not serologically related but share physical and biological attributes . Regarding their infection vectors, Nepoviruses can be transmitted by one or more nematodes species [76, 77]. Moreover, it has been established that GFLV is transmitted by
Grapevine leafroll disease (GLD) is one of the most important viral diseases affecting grapevines worldwide [3, 81–83]. It is generally accepted that this disease is caused by 11 viral agents, named GLRaV-1 to GLRaV-11 , and according to specific genome sequences, their taxonomic classification includes members of the Ampelovirus genus (GLRaV-1, -3, -4, -5, -6 and -9), the Closterovirus genus (GLRaV-2) and the Velarivirus genus (GLRaV-7). Besides the diversity of viral agents associated with GLD, it is widely assumed that GLRaV-3 is the main etiological factor contributing to the disease. Viral agents responsible for GLD are flexuous filaments, 1800 × 12 nm in size with the unique Closterovirus architecture . These particles are responsible for the characteristic GLD symptom, expressed as red colour leaves with green vein pattern, often curled downwards and brittle . In red cultivars, GLD symptomatology is much more evident in comparison to white cultivars, where the disease can be asymptomatic ; nevertheless, white cultivars can show inter-veinal yellowing of leaves and leaf rolling .
Grapevine virus A and B (GVA and GVB), which belong to the Vitivirus genus, are also relevant . GVA is related with the Kober stem grooving symptom, where severe grooving on the grafted stems occurs [86, 87], while GVB is associated with the corky bark syndrome consisting of soft, rubbery and abnormal swelling of the basal internodes of the canes, longitudinal cracks and cork forming, typical of the rugose wood complex . Vitivirus genus has other species less ubiquitous, named GVD, GVE, and the most recently discovered GVF , causing similar symptoms to the corky rugose wood but its role is still unclear .
Interestingly, in many cases viruses are present in grapevine as multiple infections [91, 92], where the symptomatology can be a combination of those triggered by individual viral agents. This situation is exacerbated by the fact that grapevine is propagated through cuttings. Asexual propagation is the predominant method to generate clones which are genetically identical to the parental plants, allowing worldwide distribution since centuries, together with the dissemination of infectious agents across the grapevine-growing regions, spreading their detrimental consequences to grape production .
It is noteworthy to mention that, unlike to other pathogens, grapevine plants show no resistance to viruses, meaning that plants and viruses establish compatible interactions where pathogens can spread throughout all tissues without any active resistance response, generating a global cellular stress and developmental defects. It is well known that susceptible hosts are not completely passive against a pathogen, and can set up a defence response that could be less intense and not strong enough to stop viral replication and dissemination [4, 6]. Within the latter, the emergence of visible plant symptoms is none other than the sum of different molecular, cellular and physiological variations of the plant defence processes in response to viral infections. Moreover, as seen in compatible interactions, several changes in gene expression occur which determine the disease symptom development and the viral levels in the infected tissues . The dynamics of compatible interactions can be even more complex, considering that the infections could be chronic, and that there are variables to take into account, such as cultivars, species and environmental clues, among others . All of these aspects modify the manner the infection is phenotypically expressed.
2.5. Molecular and physiological changes in grapevine in response to viral diseases
Current understanding of host-virus interactions derives mostly from studies in leaves of red-berry
Transcript profiles of leaves from the red cultivars Cabernet Sauvignon and Carménère naturally infected with GLRaV-3, were characterized using the
It has been proposed that some overlap exists between leaf-senescence and pathogen-defence programs, with transcript profiling in red cultivars further supporting this concept [5, 95]. Several marker genes of the leaf senescence process are expressed during natural viral infection in grapevines. Genes induced during viral disease in grapevine plants are also induced during leaf senescence triggered by natural factors, showing a clear correspondence between the senescence program and plant responses during viral compatible disease. The generation of ROS could be responsible for the partial activation of the senescence program during viral diseases, since ROS are necessary for the expression of defence-related genes and also act as promoters of senescence . This relationship may represent a strategy used by plants in order to adapt to viral pathogens, recycle nutrients from infected leaves and mobilize them to distant tissues, and allow a plant-pathogen relationship to be established, even for long periods of time .
A different study characterized the expression of flavonoid biosynthetic pathway genes in GLRaV-3 infected symptomatic leaves in a red-fruited wine grape cultivar (cv. Merlot) . Based on the accumulation of specific flavonoids in GLRaV-3 infected plants, these authors suggest that the expression of the flavonoid biosynthetic pathway is activated during the infection, and is responsible for the characteristic changes in leaf colour. These molecules could confer protection from oxidative stress and opportunistic pathogens during the infection.
Even though berries are the most valuable part of grapevine plants, little attention has been given to the effect of viruses during fruit development and ripening. Evidence suggest that autotrophic leaves located near berry clusters serve as the main source of photoassimilates to ripening berries . Photoassimilates are normally transported via phloem, as well as viruses such as GLRaV-3. Therefore, it is reasonable to think that the infection may alter the molecules flow towards the berries, and that this effect may vary according to the asymptomatic or symptomatic phases of the infection and grapevine phenological stages [3, 83].
The effects of a chronical infection with GLRaV-3 during berry ripening in grapevine have been studied in the red cultivar Cabernet Sauvignon . Interestingly, this virus affects the normal fruit ripening process, resulting in incomplete berry ripening in terms of gene expression patterns. Genes associated with anthocyanin biosynthesis and sugar metabolism are down-regulated in berries from infected plants, consistent with a decrease in up to 40% in total anthocyanin content. These changes are observed specifically at ripening, where the infection has a greater impact in comparison with other stages of berries development. These authors also suggest the presence of viral particles in berries, probably colonizing the organ through the vasculature during fruit development.
Lately, the effect of GLRaV-3 on the chemical properties of fruit, juice and wine from
2.6. Diagnostic and control methods for grapevine viruses
2.6.1. Diagnostic methods
Since grapevine viruses can show detrimental effects on plant physiology, it is necessary to have appropriate and reliable diagnosis methods to achieve an efficient control of pathogens propagation. So far, several techniques have been applied to identify infected plant material, including biological indexing, serology and molecular assays [3, 83, 99].
Biological indexing, mostly performed as part of certification programs, refers to grafting of candidate vine on woody indicators of the Vitis genus. Later on, the indicator plant is observed for the development of virus disease symptoms. However, this approach is time-consuming, labour intensive and dependent on virus titer, the success of the viral inoculation, strain variations and skilled personnel . Serological methods are based on the recognition of viral proteins by specific antibodies. Of these, the enzyme-linked immunosorbent assay (ELISA) is the most widely applied [83, 99], and commercial kits are available. Serological approaches are robust and scalable, although less sensitive than nucleic acid-based techniques. Special attention must be given to sampling, considering differences in virus accumulation through plant tissues and seasonal variations in virus titer, and it has been described that genetic variants can affect the robustness of these methods [3, 83]. Nucleic acid-based methods, on the other hand, detect the genomic components of the viruses. These methods are commonly used due to their high sensitivity, in comparison with other diagnostic approaches. They can detect the presence of viral genomes even at low viral titer, are rapid, allow the scaling and the simultaneous analysis of a high number of samples or several viruses at once . Since most of grapevine viruses have RNA genomes, reverse transcription-polymerase chain reaction (RT-PCR) is the selected molecular assay for the detection of these pathogens [83, 101]. Several techniques have been developed based on PCR variants [83, 102], but the use of real-time PCR allows quantification of virus titer . Recently, new generation sequencing (NGS) has been used for rapid identification and sequencing of all putative viruses present in a candidate sample, allowing the identification of new viral agents as well [3, 81, 99, 104]. The use of NGS technologies as diagnostic tool requires no prior knowledge of the pathogens present in the sample, but is still expensive in order to be used as a routine procedure.
2.6.2. Multiplex PCR to detect complex viral infections
As it was mentioned before, viral diseases in grapevine often occurs as multiple infections, where several viral agents are present simultaneously and can contribute to the overall symptoms development. Several papers describe viral detection by molecular approaches, which are reviewed in [83, 99]. However, a simple and efficient commercial method for the detection of several grapevine viruses at once is currently not available. A method for virus detection must fulfil several criteria, such as sensitivity, specificity, accuracy, number of samples that can be tested simultaneously and cost, among others. Therefore, to have reliable diagnosis methods is a permanent challenge for grapevine growers.
In our laboratory (unpublished work), we have designed a system for simultaneous virus detection in vines, consisting in a multiplex PCR that can detect up to seven RNA viral genomes, in addition to the detection of the gene coding for the small sub-unit of grapevine Rubisco enzyme as a plant positive control. Using bioinformatics tools, specific primers against different viruses were designed to generate products of different sizes. Then, primers were labelled at the 5’ end with 6-FAM fluorophore, in order to be detected by capillary electrophoresis. This method allows the specific and simultaneous detection of GFKV, GVB, GLRaV-1, GLRaV-2, GLRaV-3, GLRaV-4 and GLRaV-7, in a quick, efficient and single PCR (Figure 5).
This type of multiplex PCR can be used to generate commercial kits that can serve to detect viral agents present in a vineyard or to test the plant material that will be later used in clonal propagation. With the proper bioinformatics analysis, more viruses can be added to the system, allowing a much more versatile detection kit.
2.6.3. Control methods
There are several control methods that are routinely applied in order to prevent virus dissemination. For instance, sanitary selection and certification of propagation material helps to reduce potential virus dispersion . Since viruses are transmitted by vectors, control of viral diseases can be achieved by the restriction of such vectors with the use of agrochemicals . However, agrochemicals utilization increases production costs, and additionally are associated with detrimental effects to the environment and human health, while most of modern agronomical practices tend to reduce its use. Sanitation techniques, on the other hand, are aimed to treat infected material and eliminate viral titer. Among these techniques, thermotherapy is the most frequently applied although it is not effective for all grapevine viruses . The
2.6.4. Inducing virus resistance in grapevine by transgenesis
Biotechnology arises as an alternative to allow the generation of virus-resistant grapevine plants by transgenesis, mainly involving the expression of viral components and exploiting the naturally occurring gene silencing [107–117]. This strategy requires plant transformation with a short sequence of the pathogen genome in a way that a double-strand RNA structure is formed during transcription, initiating gene silencing in the host. In our lab, induction of virus silencing was accomplished in grapevine rootstocks in order to be used for grafting . It is expected that the mobile signal-inducing virus silencing in the rootstock will also be able to reach the scion, and as a consequence, trigger virus silencing in the non-transgenic scion. This approach is very versatile, since the resistance against a specific virus can be obtained in all the varieties used as scion with a particular virus-resistant transgenic rootstock. We have transformed rootstocks plants (110 Richter and Harmony) by co-culture of embryogenic and organogenic tissues with
Therefore, a viral infection of a non-transgenic scion could be silenced if it is grafted on a transgenic rootstock carrying sequences that triggers PTGS. This strategy is an interesting alternative to considerate in virus-free breeding programs because the infection in non-transgenic grapevines from any cultivar could be abolished using a transgenic rootstock, keeping cultivars and, more important, the fruit produced non-transgenic.
3. Grapevine abiotic stress
Grapevine crops are often exposed to sub-optimal growing conditions which cause several abiotic stresses, as they are constantly exposed to different water regimes, nutrient deficiency or excess, extreme heat or low temperatures and deficit or excess of light . All plants, including grapevine, need Sun energy in order to produce organic compounds through photosynthesis, but sunlight is a sum of different wavelengths. Among them, the ultraviolet radiation (UVR) plays an important role, however, the main problem with UV light is that as the wavelength declines, its energy content increases, mainly as UV-B radiation, and therefore its potential to cause photo-biological damage increases. UV-B is not only potentially harmful, but it also serves as an environmental information source, though information about it is still scarce. As general abiotic stresses have been extensively reviewed [119–125], we will have special focus on UV-B-mediated perception and signalling responses of grapevine and photo-biotechnological approaches to improve fruit quality for winemaking.
3.1. Solar ultraviolet B levels, ozone layer depletion and increase of UV-B radiation
Solar energy is the primary source of energy for all surface phenomena, especially autotrophic organisms. Among them, plants use solar radiation not only as an energy source, but also as a key signal containing vital information about the environment in which they live [126, 127]. Solar radiation not only includes the visible spectrum (400–700 nm) necessary for photosynthesis, but also other types of radiation. Near 7% of the electromagnetic radiation emitted by the Sun is within the ultraviolet radiation (UVR) spectrum (200–400 nm) [128–130]. UVR has been divided into three different bands: UV-A (315–400 nm), UV-B (280–315 nm) and UV-C (200–280 nm) [130, 131]. As it passes through the atmosphere, the total transmitted radiation flux is considerably reduced, and the composition of UVR is modified. Shortwave UV-C is completely absorbed by atmospheric gases, while UV-B is partially absorbed by the stratospheric ozone (O3), leaving only a small fraction (<0.5% of total sunlight energy) transmitted to the Earth surface. UV-A, on the other hand, is not absorbed by ozone [130, 132]. Over the last 50 years, the ozone concentration has diminished by 5%, mainly due to the release of anthropogenic pollutants, such as chlorofluorocarbons (CFCs) and other halogenated ozone-depleting substances [126, 133]. As a direct consequence of the ozone reduction, an increase in the flux of UV-B radiation has been registered during the last years [126, 128, 132]. Although, UV-B radiation is only a minor component of solar radiation, due to its high energy, its potential for causing biological damage is exceptionally high . Besides the regulation of solar UV-B by the ozone layer, there are several factors influencing UV-B radiation levels, such as latitude, altitude, season, time of the day, weather conditions, surface reflection, atmospheric pollution and shading by plant canopies [126, 133]. From a wine producer’s point of view, the establishment, planning and vineyard management are additional factors to take into account that can influence UV-B levels on plants. These factors including climate, presence and slope aspect, site elevation, trellis and training system and vine vigour, among others, could be directly influencing both intercepted light in canopies and fruit zone .
3.2. Effects of UV-B in plants
Due to the sessile lifestyle, plants are forced to adapt to changes in environmental conditions while achieving an equilibrium between optimal photosynthetically active radiation (PAR) capture and UV-B protection [131, 132]. The UV-B radiation has several detrimental effects but it also serves as a key regulator of plant morphology and physiological, biochemical and genetic mechanisms [127, 129, 133, 134]. Plants actively respond to irradiation with high or low UV-B doses, either by the activation of repair mechanisms or by stimulation of photomorphogenic processes [128, 129]. In general, low UV-B doses reduces growth and expansion of leaves, produces leaf thickness, increases epicuticular waxes, trichomes number and axillary branching, reduces stem elongation, suppresses both hypocotyl extension and root growth and enhances flavonoid biosynthesis, mostly flavonol [127–129, 132, 134]. Plants under UV-B radiation present a compact architecture, although different phenotypes have been reported. This may relate to UV-induced morphological changes being underpinned by different mechanisms at high and low UV-B doses .
In grapevine, the high UV-B doses reduce shoot length and leaf area, increase both leaf thickness  and accumulation of terpenes with antioxidant properties . On the other hand, flavonol biosynthesis is dramatically activated under both high and low UV-B exposures in the berry skin [9, 10]. Also, membrane-related terpenes are increased in low fluence of UV-B in grapevine leaves .
3.3. UVR8-mediated photomorphogenic mechanisms in response to UV-B in plants
In order to maximize its growth and survival, plants detect, respond and adapt to UV-B rays. This type of radiation is a key environmental cue, which initiates diverse pathways affecting metabolism, development and viability. Many of the UV-B radiation effects involve differential regulation of gene expression. This response depends on the exposition nature (high or low UV-B doses), the degree of adaptation and acclimation to the radiation, and the interaction with other environmental factors. UV-B radiation responses are mediated by two signalling pathways in
3.4. Elucidating the grapevine UV-B signalling pathway
|VvUVR1||VIT_07s0031g02560||No change||Carbonell-Bejerano et al. (2014)|
Loyola et al. (2016)
3.5. Manipulation of UV-B perception and signalling components to improve plant shape and fruit quality in grapevine
It is known that grapevine is a vigorous growing plant; hence, one of the main objectives for viticulture practices is to reduce the size of the canopies and alter the shape of the vine, in order to increase field plant density and improve fruit organoleptic qualities, among others [8, 156]. Moreover, a higher plant density means greater productivity per area unit. To meet these objectives, conventional genetic improvement of most fruit crops, including grapevine, has been extensively done, with several obstacles in the way. Among the latter we can find long juvenility periods, seedlessness, self-incompatibility, high heterozygosity and sterility. Therefore, conventional breeding techniques are difficult, expensive and time consuming . Because of this, genetic improvement through genetic engineering techniques offers an attractive alternative in order to overcome these problems.
|Gene name||Role in UV-B signallinga||Grape homologue||Experimental approach||Phenotype or trait of|
|AtUVR8||Photoreceptor||VvUVR1||Over-expression||UV-B tolerance, dwarfing, increased flavonoids levels, enhanced ||Rizzini et al. ; Demkura and Ballaré |
|Over-expression||UV-B tolerance, dwarfing, increased flavonoids levels.||Oravecz et al. |
|AtHY5||Positive regulator||VvHY5||Over-expression||UV-B tolerance, dwarfing, increased flavonoids levels.||Ulm et al. |
|AtHYH||Positive regulator||VvHYH||Over-expression||Moderated UV-B tolerance, increased flavonoids levels.||Brown and Jenkins |
|AtRUP2||Negative regulator||VvRUP||Silencing or|
|UV-B tolerance, extreme dwarfing, increased|
|Heijde and Ulm |
In vine growing, the production of dwarf and semi-dwarf canopies with short and numerous shoots, in order to increase field vine density, are normally used for both dwarfing rootstocks and spur varieties [8, 156]. However, rootstocks and spur varieties are available for only a few species and graft compatibility is often a problem. Therefore, an alternative to this is the use of photo-biotechnology techniques which may contribute to the creation of dwarf varieties by genetic engineering, modifying, for example, UV-B perception and/or signalling components (see Table 2).
Photo-biotechnology refers to over- or down-expressing genes with photo-biological relevance . Since photoreceptors and/or light signalling cascade components regulate the expression of critical development and plant growth genes, genetic manipulation of these is viewed as a promising strategy to develop fruit crops with improved agronomic traits . Therefore, photo-biotechnology offers a promising approach for studying the influence of UV-B signal transduction components on plant development and may be used to improve crop yield, shade tolerance, growth and fruit ripening, canopies shape, hormone synthesis and biosynthesis of metabolites and pigments. For example, a promising study in tomato showed that down-regulation of
The quality of grape berries for winemaking integrates various aspects, but as for red wines, the accumulation of phenolic compounds by UV-B is highly necessary . Wines with the highest phenolic concentrations are generally considered of excellence, therefore, these molecules are said to play a significant role in winemaking since they are key determinants of wine quality . All of the aforementioned evidence suggests that UV-B protective mechanisms may potentially lead to important industrial applications, relevant to the wine industry. UVR8 may prove to be an attractive and suitable target to manipulate plant growth and/or plant tolerance to abiotic stress, generating UV-B-resistant grapevines with enhanced secondary metabolites levels (i.e. phenolic compounds).
In summary, the elucidation of the UV-B signalling pathway and the role of photomorphogenesis, in addition to advances in genetic manipulation of grapes, are unique biotechnological tools that could be used to improve grapevines in order to meet and surpass market expectations.
This work was supported by the National Commission for Science and Technology CONICYT (FONDECYT grant number 1150220) and the Millennium Nucleus for Plant Synthetic Biology and Systems Biology NC130030. G. Armijo, R. Loyola and F. Restovic were supported by FONDECYT postdoctoral grant number 3140324, 3150578 and 3150259, respectively.