**5. Examples of studies performed with the microvine**

#### **5.1 Impacts of temperature on carbon fluxes and fruiting**

The impact of elevated temperature on growth and carbon distribution between vegetative and reproductive organs was investigated. Two contrasting thermal regimes with a difference of 8° C (30/20°C vs. 22/12°C day/night temperature) were imposed during a period of 450°C GDD. The VPD was 1 kPa and the PAR 19 mol. m<sup>−</sup><sup>2</sup> .d<sup>−</sup><sup>1</sup> for the two thermal regimes. The biomass, size, and carbon contents of the leaves, internodes, and berries were characterized from spatial observations at harvest and converted into temporal profiles according to the method described in Section 4. Only the organs that developed during heat treatments, i.e., vegetative phytomers younger than 450°C GDD at harvest and the reproductive phytomers, which were at pre-flowering stage at the beginning of experiments, were retained for analysis. Luchaire et al. [20, 36] have shown that high temperature accelerates the growth and the accumulation of biomass in vegetative organs (leaves and internodes) in thermal time, at the expense of the accumulation of sugars in internodes and the surface area to mass of the leaves (thinner leaves).

Under high temperature, the growth and accumulation of biomass of the fruit slowed down on a thermal time basis. Sugar loading of proximal phytomers (from the post-flowering stage to onset of heat treatment) was also delayed by ca. 400°C GDD at high temperatures. High temperatures increased inflorescence abortion rate (+ 20%) at pre-flowering stages, concomitantly with the beginning of sugar loading in the proximal clusters ripening [20, 36, 38]. These results suggest that

*Advances in Grape and Wine Biotechnology*

development from a single spatial observation of the axis at a given stage. The flow of biomass or metabolites within the organs and their responses to environmental constraints were then addressed using those calculated temporal profiles (Section 5.1).

*Conversion of leaf and young berry growth data collected from the position along the microvine main shoot* 

The spatiotemporal conversion approach presented above can also be used to characterize the evolution of winter bud development along the proleptic axis of the microvine [12]. Bud development was analyzed on microvines grown under standard environmental conditions (25/15°C day/night temperature, VPD 1 kPa, photoperiod 12 h), as explained in Section 3.2.1. The number of preformed phytomers initiated by primary axes within buds increases linearly as a function of the plastochron index (PI) of the proleptic axis in the non-lignified zone (PI < 25). The temporal dynamics of bud development were calculated from the spatial profiles using the proleptic axis PI x phyllochron. The primary axis of the bud displayed a maximum of six phytomers from IP 25 (lignified zone), i.e., 625°C or 57 days after its initiation (phyllochron of 24°C). A maximum of two inflorescence primordia was observed in this zone. The primordia of the first and second inflorescences, located between the preformed phytomers n°4 and n°6 of the primary axis, were initiated from IP 13 and 26 of the proleptic axis, respectively, corresponding to 325°C (or 30 days) and 650°C (or 60 days) after bud initiation. The timing of inflorescence primordium development in winter buds in non-dwarf vines [32] is similar to our observations on microvines. This pattern of winter bud development parameterized for the microvine can be used to evaluate, and potentially predict, the environmental stress impacts during the season 1 on the fructification potential of the season 2.

**4.3 Dynamics of fruit development deriving from neo-formed inflorescence**

The primary characterization of fruit development along a microvine axis showed that the microvine berry displays the two classical growth phases as

observed for berries of macrovines [32, 33]. Microvine berry growth and metabolite

**4.2 Dynamics of inflorescence differentiation within winter buds**

*(plastochron index) into cumulated thermal time after phytomer emergence.*

**14**

**Figure 7.**

high temperature decouples vegetative and reproductive development, increasing the total biomass of vegetative organs while reducing the accumulation of carbon reserves and hampering continuous fruiting.

#### **5.2 Circadian variations of the grape transcriptome**

Transcriptomic studies are difficult to run with macrovines grown outdoor because of the seasonality of fruiting and the day-to-day environment fluctuations. Thus, while transcriptomics is a very common approach today to understand the genetic mechanisms regulating grape development, no work has attempted to describe the circadian evolution of the grape transcriptome. The results published by Rienth et al. [39] were the first for a perennial fleshly fruit that addressed this topic. For this experiment microvines were grown in climatic growth chambers [40] under controlled environments (30/20°C day/night temperature, photoperiod 14 h, VPD 1kPA) for 3 months to encompass a complete reproductive cycle from flowering to ripening. When most proximal grapes reached physiological maturity, berry samples from two green and two ripening developmental stages were collected at different periods of the photo and nyctiperiod, and a whole genome transcriptomic analysis was carried out by Nimblegen® Vitis 12x microarrays.

All genes modulated during the day also showed some variation of expression at night, with 1843 genes that are only regulated at night. The detection of this very large number of specifically regulated genes during the night emphasized the importance of the regulatory mechanisms associated with the nocturnal fruit development. The comparison of differentially modulated transcripts between day and night at different stages showed that circadian regulation was very specific to the stage of development with only nine commonly deregulated genes between day and night at all stages. With respect to activated or deactivated functional categories, genes related to photosynthesis appear strongly repressed at night, in particular in young green berry, and several functional categories related to secondary metabolism (phenylalanine) and abiotic stress have shown strong overexpression at night at all developmental stages.

#### **5.3 Effect of temperature on grape development**

Until recently, the studies on the effect of temperature on grape development have only been performed using non-dwarf varieties, with the experimental limits associated with this model. Rienth et al. [41, 42] were the first to perform temperature experiments using microvines grown under tightly controlled environmental factors (photoperiod, light intensity, temperature, VPD, water, and mineral supply). This study was carried out with the ML1 microvine applying temperature gradients ranging from 12 to 35°C during 2 h to 4 weeks.

A first series of experiments focused on short-term stress effects (2 h, 35°C) of microvine fruits at different stages between green growth and ripening sampled during day and night. Nimblegen® Vitis 12x microarray assays revealed that a large number of genes (5653) respond to the increase in temperature, at all stages of development (**Figure 8**). Temperature effect was time and mainly development stage specific, with berries close to *veraison* being the most reactive to temperature elevation, especially for some categories such as anthocyanin synthesis which was specifically heat repressed at this stage. Furthermore, various genes of secondary metabolism (phenylalanine, anthocyanins) are repressed at the *veraison*, by high temperature with a larger number of genes regulated during the nocturnal phase.

Long-term thermal stresses (> 30 days) were also experimented using various temperature charts to several stages of grape development, taking into account

**17**

**Figure 8.**

*during the grapevine fruit development.*

*The Microvine: A Versatile Plant Model to Boost Grapevine Studies in Physiology and Genetics*

circadian variations of the transcriptome [41]. In these studies, we used highthroughput transcriptomic analysis through RNA-seq (Illumina technology). A total of 10,788 genes could be detected as a function of stage, temperature regime, and photoperiod. The importance of "heat shock"-type genes with highly variable expression patterns as a function of the duration of the stress, the circadian cycle, and the stage of development of the fruit has been highlighted. The rise in temperature led to an acceleration of fruit growth during the green growth phase. In fruit at the onset of ripening, the temperature increased the respiration of malic acid and delayed the accumulation of sugars and downregulating key genes of the flavonoid pathway. For the first time, a decoupling of sugar accumulation and malic acid respiration during ripening could be observed and related to the change in carbohy-

*Schema of the expression changes induced by temperature elevation for some genes of the central metabolism* 

A number of genes known to display an induction at veraison and thereafter were confirmed in microvines displaying a remarkably stable expression pattern with respect to temperature (SPS1, sucrose phosphate synthase 1; XET, xyloglucanendotransglucosidase; thaumatin; MRIP, ripening-induced protein1-like precursor (proline-rich cell wall). However, other well-known ripening-induced proteins were induced in the cold in green stage (GRIP3/4, grape ripening-induced protein ¾, ethylene-responsive 1B, putative extensin proline-rich, cell wall chitinase). During the long-term low T° treatment, fruit transcriptomic analyses showed an overexpression of key enzymes linked to both glycolysis (PK, pyruvate kinase) and malic acid synthesis (PEPce, phosphoenolpyruvate carboxylase; MDH, malate dehydrogenase). Temperature variation also impacted posttranscriptional regulation mechanism such as the PPCK (phosphoenol pyruvate carboxylase kinase) which is overexpressed under heat. This gene expression pattern confirmed physiological observations of sugar-acid decoupling and suggests that under cool condition, where the plant energetic status is more comfortable due to lower vegetative

drate status of the plant as a function of temperature [9].

*DOI: http://dx.doi.org/10.5772/intechopen.86166*

*The Microvine: A Versatile Plant Model to Boost Grapevine Studies in Physiology and Genetics DOI: http://dx.doi.org/10.5772/intechopen.86166*

**Figure 8.**

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at all developmental stages.

**5.3 Effect of temperature on grape development**

gradients ranging from 12 to 35°C during 2 h to 4 weeks.

reserves and hampering continuous fruiting.

**5.2 Circadian variations of the grape transcriptome**

analysis was carried out by Nimblegen® Vitis 12x microarrays.

high temperature decouples vegetative and reproductive development, increasing the total biomass of vegetative organs while reducing the accumulation of carbon

Transcriptomic studies are difficult to run with macrovines grown outdoor because of the seasonality of fruiting and the day-to-day environment fluctuations. Thus, while transcriptomics is a very common approach today to understand the genetic mechanisms regulating grape development, no work has attempted to describe the circadian evolution of the grape transcriptome. The results published by Rienth et al. [39] were the first for a perennial fleshly fruit that addressed this topic. For this experiment microvines were grown in climatic growth chambers [40] under controlled environments (30/20°C day/night temperature, photoperiod 14 h, VPD 1kPA) for 3 months to encompass a complete reproductive cycle from flowering to ripening. When most proximal grapes reached physiological maturity, berry samples from two green and two ripening developmental stages were collected at different periods of the photo and nyctiperiod, and a whole genome transcriptomic

All genes modulated during the day also showed some variation of expression at night, with 1843 genes that are only regulated at night. The detection of this very large number of specifically regulated genes during the night emphasized the importance of the regulatory mechanisms associated with the nocturnal fruit development. The comparison of differentially modulated transcripts between day and night at different stages showed that circadian regulation was very specific to the stage of development with only nine commonly deregulated genes between day and night at all stages. With respect to activated or deactivated functional categories, genes related to photosynthesis appear strongly repressed at night, in particular in young green berry, and several functional categories related to secondary metabolism (phenylalanine) and abiotic stress have shown strong overexpression at night

Until recently, the studies on the effect of temperature on grape development have only been performed using non-dwarf varieties, with the experimental limits associated with this model. Rienth et al. [41, 42] were the first to perform temperature experiments using microvines grown under tightly controlled environmental factors (photoperiod, light intensity, temperature, VPD, water, and mineral supply). This study was carried out with the ML1 microvine applying temperature

A first series of experiments focused on short-term stress effects (2 h, 35°C) of microvine fruits at different stages between green growth and ripening sampled during day and night. Nimblegen® Vitis 12x microarray assays revealed that a large number of genes (5653) respond to the increase in temperature, at all stages of development (**Figure 8**). Temperature effect was time and mainly development stage specific, with berries close to *veraison* being the most reactive to temperature elevation, especially for some categories such as anthocyanin synthesis which was specifically heat repressed at this stage. Furthermore, various genes of secondary metabolism (phenylalanine, anthocyanins) are repressed at the *veraison*, by high temperature with a larger number of genes regulated during the nocturnal phase. Long-term thermal stresses (> 30 days) were also experimented using various temperature charts to several stages of grape development, taking into account

**16**

*Schema of the expression changes induced by temperature elevation for some genes of the central metabolism during the grapevine fruit development.*

circadian variations of the transcriptome [41]. In these studies, we used highthroughput transcriptomic analysis through RNA-seq (Illumina technology). A total of 10,788 genes could be detected as a function of stage, temperature regime, and photoperiod. The importance of "heat shock"-type genes with highly variable expression patterns as a function of the duration of the stress, the circadian cycle, and the stage of development of the fruit has been highlighted. The rise in temperature led to an acceleration of fruit growth during the green growth phase. In fruit at the onset of ripening, the temperature increased the respiration of malic acid and delayed the accumulation of sugars and downregulating key genes of the flavonoid pathway. For the first time, a decoupling of sugar accumulation and malic acid respiration during ripening could be observed and related to the change in carbohydrate status of the plant as a function of temperature [9].

A number of genes known to display an induction at veraison and thereafter were confirmed in microvines displaying a remarkably stable expression pattern with respect to temperature (SPS1, sucrose phosphate synthase 1; XET, xyloglucanendotransglucosidase; thaumatin; MRIP, ripening-induced protein1-like precursor (proline-rich cell wall). However, other well-known ripening-induced proteins were induced in the cold in green stage (GRIP3/4, grape ripening-induced protein ¾, ethylene-responsive 1B, putative extensin proline-rich, cell wall chitinase). During the long-term low T° treatment, fruit transcriptomic analyses showed an overexpression of key enzymes linked to both glycolysis (PK, pyruvate kinase) and malic acid synthesis (PEPce, phosphoenolpyruvate carboxylase; MDH, malate dehydrogenase). Temperature variation also impacted posttranscriptional regulation mechanism such as the PPCK (phosphoenol pyruvate carboxylase kinase) which is overexpressed under heat. This gene expression pattern confirmed physiological observations of sugar-acid decoupling and suggests that under cool condition, where the plant energetic status is more comfortable due to lower vegetative

growth and cellular respiration rate, malic acid respiration, as a supplemental energy source in the fruit, is not compulsory. In cool climate, the allocation of carbon to the fruit can support glycolysis, malate synthesis, and sugar accumulation into the vacuole. Conversely, under hot climate, cytoplasmic sugars could be limiting when the cell starts to accumulate sugar in the vacuole at the onset of ripening. Thus, the malate would be drained from the vacuole to supply energy through respiration and/or through H<sup>+</sup> /sugar exchange at the tonoplast.
