I

Effects of irrigation frequency on water use efficiency and

grapevine performance – a review

Simone Cavallini

Dissertation to obtain a Master’s Degree in

Viticulture and Oenology Engineering

Supervisor: Prof. Joaquim Miguel Costa

Supervisor: Prof. Claudio Lovisolo

Jury:

PRESIDENT

PhD Carlos Manuel Antunes Lopes, Associate Professor with Habilitation at Instituto

Superior de Agronomia, Universidade de Lisboa.

MEMBERS

PhD Claudio Lovisolo, Full Professor at Università Degli Studi di Torino;

PhD Luísa Cristina dos Mártires Ferreira de Carvalho, Assistant Professor at Instituto Superior

de Agronomia, Universidade de Lisboa.

2020

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III

Abstract

The effects of global warming have led to increased evapotranspiration losses, modified and more

erratic precipitation patterns and increased risks of severe water stress. These events have a negative

impact on viticulture but also stimulate the search for alternative solutions for vineyard management.

Some of these solutions involve novel irrigation strategies and improved knowledge on grapevine

stress physiology. Therefore, this thesis aims to introduce the topic of irrigation frequency as an

additional strategy to develop an adequate irrigation strategy, which can influence distribution of

water and roots in the soil, influence water use efficiency (WUE) as well as the dynamics of berry

ripening while contributing for water savings.

This review summarizes grapevine responses to water stress, examining major changes in physiological

and molecular processes and analyzing the effects on WUE. The tolerance mechanisms implemented

by grapevine to respond to increased leaf temperature and the antioxidant protective systems

activated to counteract adverse conditions, are reviewed. Some of the most important methods to

monitor plant and soil water status are shortly described and explained. In addition, the effect of deficit

irrigation strategies and the role of irrigation frequency on vine’s performance are discussed.

The review of literature showed that irrigation frequency has a major impact on stomatal control,

resulting in a purely hydraulic regulating mechanism in the case of high irrigation frequency and the

grapevine transpiration tends to drop drastically once the effect of irrigation water disappears.

However, under identical growing conditions, low irrigation frequency, associated with a consistent

water volume, could activate hormonal stomatal regulation mechanism, mediated by the ABA signal.

In this case, grapevine’s transpiration tends to decrease moderately, over a longer period, which will

positively affect berry volume and sugar accumulation rate.

Keywords

Abscisic acid - Irrigation frequency - Vine physiology - Water deficit - Water use efficiency

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Resumo

Os efeitos do aquecimento global conduziram a um aumento das perdas de água por

evapotranspiração, a padrões de precipitação alterados e mais erráticos e a um risco crescente de

stress hídrico severo. A combinação destes eventos tem impacto negativo na viticultura, mas estimula

também a procura de soluções alternativas para optimizar a gestão da vinha. Algumas destas soluções

envolvem novas estratégias de rega e um melhor conhecimento sobre a fisiologia do stress hídrico em

videira. Este trabalhovisa, portanto, introduzir o tema da frequência de rega como uma ferramenta

adicional para a implementaçao de uma estratégia de irrigação mais adequada.

Este trabalho revê as respostas da videira ao stress hídrico e analisa os seus efeitos na eficiência do

uso de água. São descritos os mecanismos de tolerância implementados pela videira para controlar a

temperatura das folhas e os sistemas de proteção antioxidante ativados para neutralizar condições

adversas. Alguns dos principais métodos para avaliar o estado hídrico do solo e das plantas são

também revistos e explicados brevemente. Descrevem-se igualmente os efeitos das estratégias de

rega deficitária e o papel da frequência de rega no comportamento da videira .

A revisão da literatura permitiu concluir que a frequência de rega tem grande impacto na regulação

estomática através de um mecanismo de regulação hidráulico no caso de elevada frequência de rega.

A transpiração da videira mostra também ser drasticamente reduzida assim que o efeito da rega

desaparece. No entanto, e sob as mesmas condições de cultivo, uma baixa frequência de rega,

associada a um volume consistente de água, poderá ativar o mecanismo de regulação hormonal

estomática mediado pelo ácido abcissico. Neste caso, o nível de transpiração da videira tende a

diminuir moderadamente, durante um período mais longo, afetando positivamente o volume do bago

e acumulação de açúcar neste.

Palavras-chave

Ácido abscísico - Frequência de rega - Fisiologia da videira – Déficite hídrico - Eficiência no

uso da água

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Resumo alargado

Em regiões particularmente afetadas pelas alterações climáticas, como as do Mediterrâneo, o

desenvolvimento vegetativo da videira e a qualidade dos bagos podem ser influenciados pela maior

frequência de ondas de calor e por períodos mais longos de seca severa. Estes eventos têm impacto

negativo na viticultura, e promovem a procura de soluções alternativas para melhor gestão da vinha.

Entre elas, está a instalação de sistemas de rega, que podem aliviar o stress hídrico da cultura e garantir

produção e qualidade. Todavia, a água é um recurso cada vez mais escasso e que deve ser otimizado

e poupado. Além disso, a qualidade das uvas está sempre dependente da capacidade de adaptação da

videira à secura e o uso de rega pode alterar as respostas fisiológicas da videira.

A regulação estomática depende de mecanismos hidráulicos (mediados pelo potencial hídrico) e de

mecanismos químicos (mediados pelo ácido abcissico - ABA). Uma situação de stress hídrico suave ou

moderado conduz a uma redução da transpiração devido ao fecho dos estomas. A condutância

estomática e a transpiração estão diretamente relacionadas, pelo que uma menor abertura dos

estomas reduz proporcionalmente a transpiração, levando a um aumento da temperatura da folha.

Por outro lado, o fecho estomático e a taxa fotossintética não têm uma proporcionalidade direta, pelo

que reduções da abertura estomática resultam normalmente numa maior eficiência no uso de água

(EUA).

A regulação térmica dos órgãos é influenciada pela quantidade de água transpirada pelas folhas e é

determinada pela regulação estomática. A passagem de água do estado líquido para o gasoso requer

quantidades consideráveis de energia que se perdem sob a forma de calor, garantindo assim o

arrefecimento dos órgãos da planta através do chamado arrefecimento evaporativo. Por isso, a rega

é uma estratégia eficiente de proteger a videira contra o calor excessivo. No entanto, é necessário ter

em conta differenças ao nível do comportamento estomático que resulta em diferentes capacidade e

respostas adaptativas ao stress hídrico entre diferentes castas/genótipos de videira.

É também importante considerar os mecanismos envolvidos na resposta a um aumento da

temperatura. Em condições ditas adversas, a videira ativa uma série de mecanismos para proteger e

reparar o seu aparelho fotossintético e na primeira linha de defesa está a dissipação do excesso de

energia sob a forma de calor, graças principalmente ao ciclo das xantofilas. No entanto, intensidades

elevadas de radiação e a altas temperatura podem levar à formação de ROS (espécies reativas de

oxigénio), e em paralelo ao aumento da concentração de moléculas fotoprotectoras nas folhas. Entre

estas moléculas contam-se os carotenóides, enzimas antioxidantes, flavonoides. Nos tecidos dos bagos

acumulam-se moléculas como a quercetina, canferol e antocianinas, que podem melhorar a

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composição dos bagos e as implicações destes compostos antioxidantes na qualidade dos bagos é bem

conhecidas.

Por conseguinte, é necessário considerar o compromisso teórico entre aliviar o stress hídrico, e

aumentar a condutância estomática/transpiração para promover o arrefecimento evaporativo assim

como os mecanismos de proteção e reparação implementados pela videira para contrariar a

occorrência de fotoinbição, durante períodos de stress abiótico e que pode levar também a uma

melhoria da qualidade dos bagos.

A redução das disponibilidade de água em termos qualitativos e quantitativos incentivou a adoção de

algumas estratégias de rega (ex. rega deficitária) que impôem um stress moderado e controlado. A

frequência de rega é uma técnica adicional que pode complementar às estratégias de rega deficitárias

já consolidadas.

Analisando os efeitos de uma frequência de rega elevada associada a um volume reduzido de água,

observou-se que as vinhas tinham desenvolvido um maior sistema radicular. No entanto, as zonas de

absorção radicular concentraram-se principalmente na parte mais superficial do solo (primeiros 25 cm)

tornando assim o estado hídrico da videira muito mais dependente da variação dos teores de água do

solo. Em particular, uma rega frequente em solos predominantemente argilosos conduz à formação

de bolbos de rega pequenos e superficiais que favorecem a evaporação e diminuem a EUA. Por sua

vez, baixas frequências de rega resultam numa melhor distribuição da água no solo e num maior

desenvolvimento radicular a niveis mais profundos . Ao distribuir uma quantidade substancial de água,

promove-se a infiltração em profundidade durante o período entre regas consecutivas. Desta forma

as raízes mais superficiais terão menos água disponível e começarão a sintetizar o ácido abscisico que

depois será translocado para as folhas promovendo o fecho estomático.. Contudo, este fenómeno em

condições de campo pode ser menos consistente devido ao processo de redistribuição da água e de

raízes molhadas e secas.

Além disso, frequências de rega elevadas podem afetar negativamente a taxa de transpiração. Na

verdade, a transpiração tende a diminuir acentuadamente entre cada evento de rega muito

provavelmente devido ao facto da resposta estomática ser puramente hidráulica pois o curto intervalo

de tempo entre eventos de rega não permite ativar o metabolismo do ABA. Em vez disso, uma

frequência baixa de rega promove a transpiração, que depois tende a diminuir gradualmente devido

ao fecho gradual dos estomas acompanhado por um aumento da biossíntese do ABA.

Por conseguinte, uma baixa frequência de rega parece melhorar a EUA e ao mesmo tempo permite

aliviar o défice hídrico sazonal comparativamente com frequências de rega mais elevadas, com

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repercussões positivas ao nível da maturação dos bagos. De facto, quer o rendimento quer a a

acumulação de açúcar nos bagos aumentaram em resposta a uma diminuição da frequência de rega.

No entanto, é necessário ainda estudar melhor sobre os efeitos da frequência de rega em outros

aspetos fisiológicos da videira e como o efeito combinado da frequência de rega e das características

do solo e dos castas pode influenciar a escolha de das estratégias de rega em viticultura. Também será

importante quantificar melhor até que ponto a frequência de rega ajuda a poupar água e a influenciar

a composição dos bagos.

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INDEX

I. INTRODUCTION ..................................................................................................................................... 1

II. STOMATAL REGULATION AND WATER USE EFFICIENCY ...................................................................... 3

2.1 Water Use Efficiency ...................................................................................................................3

2.2 How does stomatal regulation affect WUE? ...............................................................................4

2.3 How grapevine responds to water stress? ..................................................................................5

2.4 Different stomatal sensitivity to ABA: the iso and anisohydric behaviour .................................8

2.5 Thermal regulation ......................................................................................................................9

III. HOW DOES ABA METABOLISM MODULATES GRAPEVINE PERFORMANCE? ....................................14

3.1 Effect of water application and stress-recovery responses on ABA metabolism .................... 14

3.2 Impact of ABA-induced process on secondary metabolite accumulation ............................... 16

3.3 ABA metabolism and carbon relocation to berries .................................................................. 17

IV. PRECISE AND MORE EFFICIENT IRRIGATION .....................................................................................19

4.1 Deficit irrigation strategies ....................................................................................................... 19

4.1.1 Regulated deficit irrigation ............................................................................................ 19

4.1.2 Sustained deficit irrigation ............................................................................................. 20

4.1.3 Partial rootzone drying .................................................................................................. 20

4.1.4 Some additional considerations..................................................................................... 22

4.2 Sensors for precise soil and plant water status ....................................................................... 23

4.2.1 Indirect methods ............................................................................................................ 23

4.2.1.1 Soil-based methods ............................................................................................ 23

4.2.1.2 Atmosphere-based methods .............................................................................. 24

4.2.2 Direct or plant-based methods ...................................................................................... 24

4.2.2.1 Visual observation .............................................................................................. 24

4.2.2.2 Pressure chamber ............................................................................................... 25

4.2.2.3 Carbon isotope δ13C ............................................................................................ 26

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4.2.2.4 Sap flow sensors ................................................................................................. 27

4.2.2.5 Leaf gas exchange ............................................................................................... 28

4.2.2.6 Thermal data ....................................................................................................... 28

V. THE ROLE OF IRRIGATION FREQUENCY ..............................................................................................30

5.1 Root development and distribution ......................................................................................... 32

5.2 Irrigation volumes and irrigation frequency ............................................................................ 33

5.3 Grape ripening .......................................................................................................................... 37

VI. CONCLUDING REMARKS AND FUTURE PROSPECTS ..........................................................................40

VII. REFERENCES .....................................................................................................................................43

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List of figures

Figure 2.1. Measurement from crop to leaf level of grapevine water use efficiency (WUE)

(adapted from Medrano et al., 2015).

Figure 2.2. Different morpho-physiological responses to drying soil conditions (adapted from

Simonneau et al., 2017).

Figure 2.3. Summary of the line of defence implemented by grapevine under stressful condition

caused by an excess of electrons in the excited state (adapted from Derks et al., 2015).

Figure 3.1. Leaf water potential measured in cv Grenache plants irrigated, subject to water

stress, or re-watered in the two experiments (Perrone et al., 2012b).

Figure 3.2. Time course of foliar ABA content measured during 36 h in re-watered, water

stressed and irrigated grapevines (Perrone et al., 2012b).

Figure 4.1. Implementation of partial rootzone drying (Stoll, 2000).

Figure 4.2. Pressure chamber used to determine water potential in plants (Chavarria and

Santos, 2012).

Figure 4.3. A sap flow sensor installed in the vineyard (Rienth and Scholasch, 2019).

Figure 5.1. Representative scheme of soil water distribution and ABA metabolism.

Figure 5.2. Grapevine transpiration variation in response to different irrigation frequency during

a short period of eleven days (Scholasch, 2018).

Figure 5.3. Variation in grapevine transpiration in response to decreasing irrigation frequency

between year 1 and year 2 (Scholasch, 2018).

Figure 5.4. Variation in grapevine transpiration in response to decreasing irrigation frequency

between year 1 and year 3 (Scholasch, 2018).

Figure 5.5. Effect of different irrigation frequency on berry weight (Scholasch, 2018).

Figure 5.6. Effect of different irrigation frequency on berry sugar accumulation (Scholasch,

2018).

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List of tables

Table 1. Threshold values of water potential, stomatal conductance and carbon isotope (Rienth

and Scholasch, 2019).

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Abbreviations

ABA - Abscisic acid

ABA- - Active form of abscisic acid

ABAH - Protonated form of abscisic acid

An - Net photosynthesis

CWSI - Crop Water Stress Index

DI - Deficit Irrigation

E - Transpiration

e.g. - exampli gratia

ETc - Crop evapotranspiration

FI - Full Irrigated

gs - Stomatal conductance to water vapour

i.e. - id est

IG - Index of relative stomatal conductance

MPa - MegaPascal

PRD - Partial Rootzone Drying

RDI - Regulated Deficit Irrigation

Tc - Canopy temperature

Tleaf - Leaf temperature

VPD - Vapour Pressure Deficit

WDI - Water Deficit Index

WUE - Water Use Efficiency

ΔTcanopy-air - Temperature difference between canopy and the surrounding air

Ѱleaf - Leaf water potential

Ѱpd - Pre-dawn leaf water potential

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I. INTRODUCTION

Grapevine irrigation was considered, until a few years ago, incompatible with berry quality and

was used only in case of severe drought problems. The data updated to 2016 indicate that

less than 10% of vineyards in Europe were irrigated (Costa et al., 2016), but in recent years

the percentage has been rising rapidly because of climate change which led to an increase in

drought, higher air temperature and evaporative demand (IPCC, 2014), causing detrimental

impacts on grapevines growth and berry quality (Bernardo et al., 2018).

Moreover, climate change models predict even more arid conditions for the near future

(Mancosu et al., 2015; Fraga et al, 2019). The Mediterranean territories will be the most

affected, with a possible decrease in rainfall between 30 and 40% (Giorgi and Lionello, 2008),

which explains why water is an increasingly precious and limited resource especially in this

semi-arid climate areas, and in which plants are often subjected to periods of water deficits,

with high impact on plant physiology (EEA, 2012). In addition, water has become a major factor

for competitiveness of Mediterranean viticulture (Costa et al., 2020).

Therefore, irrigation plays a key role in reducing risks and provides external water supplement

to avoid severe losses in berry yield and composition (Chaves et al., 2010; Vadez et al., 2013),

especially when a rapid rise in temperature occurs (Keller, 2010). Nevertheless, it becomes

essential to define the accurate water amount to return to the plant, as this may sustain or

even enhance the yield and berry composition (Costa et al., 2016). On the contrary, excessive

amounts of irrigation water, although promoting berry yield, they may also favour

disproportionately vegetative growth, reduce sugar and anthocyanins content (Medrano et al.,

2015; Bota et al., 2016). In addition, an excessive canopy leaf surface may increase water

losses due to transpiration, resulting in reduced water use efficiency (WUE) (Chaves et al.,

2010). For this reason, vineyards aiming at being irrigated must carefully consider how to

analyse and develop efficient irrigation scheduling and strategies (i.e. deficit irrigation by

application of irrigation at levels below what would be required to sustain 100%

evapotranspiration) in order to create the best vine conditions to produce high quality grapes,

especially for red wines, while minimizing yield losses.

Besides precise monitoring of vine water use, or Deficit Irrigation (DI), other approaches can

help to optimize WUE and save water. This is the case of irrigation frequency, which is a

complex theme that has started to be studied in recent years (Scholasch et al., 2009; Sebastian

et al., 2015) but that is important as developing an adequate irrigation strategy and that can

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be complementary to it. Like DI, also the irrigation frequency could influence WUE, for

example, determining losses of water trough evaporation or deep percolation (Wang et al.,

2006; Sebastian et al., 2015) and/or influencing the vine transpiration level (Scholasch, 2018).

In addition, recent studies have shown that irrigation frequency can induce different effects on

yield and grape composition (Selles et al., 2004; Wang et al., 2006; Myburgh, 2012; Bowen et

al., 2012a,b; Scholasch, 2018) influencing net photosynthesis and stomatal conductance

(Sebastian et al., 2015). However, studies are still limited to fully understand the effects, mainly

because results obtained are generally discordant and influenced by several factors such as

the amount of water returned to the plant, soil hydraulic properties and the response of varieties

to water stress. For these reasons, it remains important to better understand how vines

respond to different irrigation frequency at both physiological and agronomical levels.

In this thesis we will analyse the role of irrigation frequency and the support that it could provide

to achieve the agronomic objective of keeping the berry quality high and increasing WUE and

save water in the vineyard. However, to achieve this, it is essential to find theoretical

compromises between: (i) net photosynthesis and stomatal conductance; and (ii) water deficits

imposed and leaf temperature. Always bearing in mind that relieving the water stress to which

vines are normally subjected via irrigation could reduce the activation of intrinsic vine response

mechanisms that counteract the effects of environmental stress. These drought adaptation

mechanisms, on which we will focus, cause perturbations to physiological and molecular

processes that can be used to balance vine vegetative and reproductive growth and induce an

improvement in the quality and composition of the grapes.

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II. STOMATAL REGULATION AND WATER USE EFFICIENCY

Stomata are microscopic openings present at very high density (150-200/mm2) preferentially

at the abaxial side of leaves. Stomata allow and mainly regulate the loss of water vapour to

the atmosphere and the entry of carbon dioxide inside the leaf. Each stoma has two guard

cells that can change shape and size to close or open the stoma. The driving force of this

process is the wide gradient of water potential that arises between the atmosphere and the

interior of the leaf.

2.1 Water Use Efficiency

Due to climate change, particularly in semi-arid areas, improving WUE has become a major

goal of sustainable vineyard (Medrano et al., 2015). WUE can be defined by instantaneous

leaf measurements or by complementary measurements at plants and crops levels (Fig. 2.1).

However, the comparison between these types of measurements often does not reveal any

relationship, limiting the applicability of the research conducted.

Crop WUE can be described, from an agronomic point of view, as the ratio of water used in

crop production versus biomass or yield (Medrano et al., 2015). The total amount of water

consumed during the growing season is the sum of water lost by transpiration and not. In fact,

it also considers water lost by soil evaporation, runoff, and deep percolation.

Instead, whole-plant WUE considers only the water transpired by the grapevine and the plant

carbon and biomass acquisition. By calculating plant WUE as biomass acquired per used

water, other important physiological factors are to be considered such as respiration losses

and night transpiration (Medrano et al., 2015). Also, carbon isotope (δ13C) is a good indicator

of WUE at plant level (Romero et al., 2014).

From a more plant physiology point of view, the “instantaneous” WUE of the leaf is generally

defined as the ratio of net photosynthesis (An) to transpiration (E) (Flexas et al., 2010; Medina

and Gilbert, 2016). However, the ratio An/E is highly affected by environmental conditions,

namely vapour pressure deficit (VPD) that influences transpiration rate. That means that a

robust comparison of plants under different climatic conditions cannot be made. In order to

avoid ambiguity associated with the effects of VPD, we may use instead the ratio of net

photosynthesis to stomatal conductance (An/gs) referred to as “intrinsic” water use efficiency

(Flexas et al., 2010; Medrano et al., 2009).

4

Figure 2.1. Measurement from crop to leaf level of grapevine water use efficiency (WUE) (adapted from Medrano et al., 2015).

Usually, leaf WUE is estimated based on measurements of portable instruments on fully

expanded leaves, well exposed to light, around mid-morning because the highest values of An,

gs and E are observed at that time. The limitation of this measurement is the large variation in

the values that are obtained during the day and that these daily changes are even higher under

water stress. Moreover, measurements are very dependent on the leaf microclimate

environment, showing a different value of WUE in the same canopy. The leaf WUE

measurements obtained are not in question, even if an integration with samples collected

throughout the day and from different leaves in different positions of the vine could make the

results obtained more accurate (Medrano et al., 2012).

2.2 How does stomatal regulation affect WUE? Regulation of stomatal aperture and closure is a central process to determine plant WUE and

carbon gain (Bertolino et al., 2019). During water stress the first plant response is stomata

closure to reduce transpiration, avoid critical , and consequently, xylem tensions that could

trigger cavitation (Dayer et al., 2019). This phenomenon is possible by adjusting guard cell

turgor pressure. An increase in guard cell turgor pressure led to a greater stomatal aperture

instead decrease in turgor pressure led to stomata closure. Thus, stomatal regulation and

WUE are closely related and if it is true that the gs and E are directly related under a constant

5

air VPD while the gs and the photosynthesis rate have a non-linear relationship it is also true

that reduced stomatal opening may improve WUE (Chaves et al., 2002; Medrano et al., 2015).

In general, plant evolution has permitted to balance the need for carbon assimilation from the

atmosphere with the need to moderate water loss (Sorrentino et al., 2016). This equilibrium is

influenced by several environmental factors such as temperature variations, light intensity,

atmospheric CO2 concentration, air humidity, and soil moisture content (Chaves et al., 2016).

Deficit irrigation may lead to increased WUE because a mild stress condition decreases faster

and more markedly the transpiration process due to stomatal closure, than net photosynthesis

(Fereres and Soriano, 2007; Chaves et al., 2007; 2010). DI strategies emerged as an additional

tool to increase water savings in irrigated agriculture by allowing crops to withstand mild water

stress with no or only marginal decreases of yield and quality, namely in species like grapevine

(Costa et al., 2007). Nevertheless, the response to DI strategies depends on several factors

including genetic characteristics; certain grapevine varieties and rootstocks show different

stomatal sensitivity to drought (Costa et al., 2007; Bauerle et al., 2008).

2.3 How grapevine responds to water stress?

Drop in plant water potential is the first consequence of a drying soil; water becomes less and

less available to the plant, as it is binds to soil particles (Simonneau et al., 2017). This

phenomenon induces different morpho-physiological responses in plants (Fig. 2.2). Stomatal

closure and leaf/shoot growth inhibition are some of the primarily grapevine replies

implemented to avoid excessive water loss (Chaves et al., 2002). Many other responses are

involved in detecting environmental variations such as hydraulic signals, expression and

function of aquaporins, electric signal and abscisic acid (ABA) biosynthesis in roots (Pantin et

al., 2013; Speirs et al., 2013; McAdam et al., 2016). Nevertheless, literature also shows that

ABA can also be synthesized in both shoots (Christmann et al., 2007) and leaves (Endo et al.,

2008; Speirs et al., 2013).

Abscisic acid belongs to a class of isoprenoids, also called terpenoids. ABA has origin in the

cleavage of C40 carotenoids (Nambara and Marion-Poll, 2005). Under normal condition, ABA

is under protonated form (ABAH) in xylem sap, but water stressed plant response involves first

the acidification of the apoplast by protons pumped from the cytosol. The lower protons (H+)

concentration in the apoplast induces the protonated form dissociation (ABA- + H+), allowing

ABA transport in its active form (ABA-) (Wilkinson and Davies, 2002; Lovisolo et al., 2016).

In grapevine, stomatal control under dry conditions has been directly related to ABA signalling

(Rossdeutsch et al., 2016), which interacts with the process of plant adaptation to abiotic stress

6

(Wilkinson and Davies, 2002; Yamaguchi-Shinozaki and Shinozaki, 2006). In particular, the

ABA synthesized in dehydrated roots has been proposed as the pivot of response to drought

in plants (Simonneau et al., 2017). The relation between stomatal response and soil moisture

has been also confirmed at the molecular level by Speirs et al. (2013), who found that, in

response to water deficit, expression of ABA genes VviNCED1 and VviNCED2 were activated

more in the roots than in other organs. The nine-cis-epoxycarotenoid dioxygenase (NCED)

has been suggested to be the regulatory enzyme for ABA biosynthesis, because its

overexpression gives a substantial ABA accumulation (Nambara and Marion-Poll, 2005;

Koyama et al., 2010)

However, Soar et al. (2004) examining the high concentrations of ABA in the leaf, concluded

that ABA in mature leaves and xylem come from local synthesis. Also, Speirs et al. (2013)

demonstrated the importance of local ABA regulation through the expression of Hyd1, a gene

that encodes ABA 8-hydroxylase a protein responsible of ABA catabolism in leaves. Gene

expression in leaves is related to VPD: when VPD is low (lower than 2,5 kPa) the Hyd1

expression is elevated and ABA control disappear, while when VPD is high (related to high air

temperature and low relative humidity) the gene expression is minimal, and ABA remain in the

leaf. It therefore appears that environmental conditions lead to a switch in ABA metabolism

directly from the site of action and not necessarily from the roots, allowing a quick optimization

of gas exchange.

Moreover, ABA metabolism can also be one of the major factors involved in a variable

response of grapevine genotypes to water stress as well as to irrigation. Indeed, Vitis vinifera

genotypes with different levels of drought adaptation were shown to respond differently in

terms of ABA biosynthesis capacity or catabolism, and this has occurred under both well-

watered and dry conditions (Rossdeutsch et al., 2016). Among Vitis genotypes, differences in

stomatal sensitivity to drought were associated with ABA concentration in xylem sap or leaves

and there is variability in stomatal sensitivity to ABA (Hopper et al., 2014; Tramontini et al.,

2014).

7

Figure 1.2. Different morpho-physiological responses to drying soil conditions (adapted from Simonneau et al., 2017).

Additional factors that may interact with and influence stomatal responses are osmotic

adjustment, xylem hydraulic conductivity and environmental factors.

Osmotic adjustment is one of the consequences of grapevine transpiration. Water loss

increases concentration of solutes (particularly potassium) inside the cell by increasing the

osmotic potential, which is a component of total water potential. As a result, when transpiration

is reduced (i.e. during the afternoon and night) the osmotic adjustment allows to take water

from nearby cells. It seems that the plant synthetizes molecules such as proline and sorbitol

to increase osmotic potential (Patakas et al., 2002). Solutes, as well as allowing osmotic

adjustment, may also have other functions such as nutrient or energy storage, membrane

protection or detoxifying activities (Szabados et al., 2011). Osmotic adjustment is a

fundamental strategy, under genetic control (Simonneau et al., 2017) implemented by plants

to ensure the integrity of their tissues even in drought conditions.

Hydraulic conductance also affects the whole plant water use. Under water deficit, hydraulic

conductance decreases in fine roots and leaves (Vandeleur et al., 2009) protecting grapevine

from severe stress.

ABA biosynthesized in the roots, due to water stress, seems to be a mechanism to increase

hydraulic conductance (Pantin et al., 2013) and therefore the amount of water sent to the

leaves. ABA can also stimulate genetic expression of aquaporins that allow easier water

transport from perivascular cells to xylem cells to meet the transpiration demand of the shoots

8

(Vandeleur et al., 2014). Nevertheless, contrasting results in leaf hydraulic conductance were

observed with exogenous ABA applications due to the different sensitivity to ABA of the

different grapevine genotypes (Coupel-Ledru et al., 2017).

On the other hand, in the leaves, the presence of ABA in the xylem leads to a decrease in

hydraulic conductivity by decreasing water permeability in the vascular bundle sheath cells

(Shatil-Cohen et al., 2011) and by inactivating bundle sheath aquaporins (Pantin et al., 2013).

This further indirect mechanism demonstrates how ABA can regulate stomata by reducing

water permeability of leaf vascular tissues (Dayer et al., 2019).

2.4 Different stomatal sensitivity to ABA: the iso and anisohydric behaviour

Vitis genotypes show different stomata sensitivity to decreased soil water content. Grapevines

with a typical conservative or “pessimistic” response to drought (fast stomatal closure) were

classified as “isohydric”. Instead, grapevines that are less susceptible to drought (delayed

stomatal closure) have shown an “optimistic” response and were classified as “anisohydric”

(Soar et al., 2006). In grapevine with isohydric behaviour, ABA signal leads to avoid severe

water stress by closing stomata, allowing vines to conserve water while keeping the leaf water

potential constant.

An anisohydric cultivar, can be less susceptible to ABA and/or synthesizes less ABA or, ABA

signaling can exert less control over stomatal closure (Lovisolo et al., 2010). These cultivars

keep transpiring even under moderate water stress, showing sharp decreases in their leaf

water potential. The lower stomatal sensitivity suggests a hydraulic control of the water that in

this case leads to tolerate water stress and not to avoid it (Lovisolo et al., 2008).

The expression of root aquaporins is strategic for the typical anisohydric behaviour. In fact, in

both iso and aniso varieties roots show increased suberization during soil drying, reducing their

hydraulic conductivity. Only aniso varieties partially compensate with greater expression of

aquaporins (Dayer et al., 2019). Instead, it is hypothesised that in the isohydric V. vinifera such

as cv Grenache, ABA biosynthesis in the roots increases apoplastic concentration due to an

increase of suberisation of apoplastic barriers causes a reduction of water conductivity which

is not compensated by the expression of aquaporins mediating the water transport (Lovisolo

et al., 2010). In fact, different levels of aquaporin expression are induced by genetic factors

and environmental signals, involving ABA metabolism and signalling (Rossdeutsch et al.,

2016).

9

In field conditions, however, it is noted that the behaviour is never corresponding to one

extreme or another but can take on intermediate situations (Chaves et al., 2010). In fact, even

in the same variety different behaviours were observed depending on the growth conditions

(Lovisolo et al., 2010; Charrier et al., 2018). This makes difficult to establish a strict

classification into the two classes (Chaves et al., 2010).

2.5 Thermal regulation

Plant’s temperature depends on the balance between the incoming energy and energy loss

(Jones and Rotenberg, 2011). Plants must keep the rate of energy absorbed and the rate of

energy lost in balance to allow optimal metabolic activity, and ultimately to be able to survive.

Energy exchanges between the plant/leaf and the surrounding environment include radiative

transfer, sensible heat transfer by convection processes, latent heat transfer as a result of

evaporation and transfer to and from storage (by conduction) (Jones and Rotenberg, 2011;

Costa et al., 2013; Lin et al., 2017).

Leaf optics and morphology can affect such energy exchanges (Leigh et al., 2017). For

example, a high leaf surface-to-volume ratio enables the rapid energy exchange (Costa et al.,

2010). In turn, darker coloured leaves heat faster by sunlight than light coloured ones (Sánchez

et al., 2001). Gains and losses of energy by plants/leaves are very important. Indeed, if all

solar energy were absorbed and there was no heat dissipation, a leaf with an actual water

thickness of 300 μm would heat up to 100 degrees, every minute (Taiz and Zeiger, 2002). The

leaf energy equilibrium depends primarily on plant transpiration and therefore on the stomatal

regulation. Leaves cool down mainly via evaporative cooling that is linked to transpiration, via

the stoma and to a minor extent via the cuticle (10-15 % of total transpiration) (Palliotti et al.,

2018). Evaporation of water requires a substantial amount of energy due to the high heat of

water vaporization (about 2,260 kJ/kg) (Tiwari and Tiwari, 2006). In plants, leaf evaporative

cooling occurs due to heat absorption from leaf tissues to support evaporation (endothermic

process).

Under stress conditions, stomatal closure occurs to limit water loss and to re-establish water

potential and avoid embolism (Tombesi et al., 2015). This stomatal response limits

transpiration, and consequently the evaporative cooling, resulting in higher leaf temperature

(Jones et al., 2009; Costa et al., 2013). For example, in poplar, evaporative cooling of

transpiring leaves resulted in reductions in leaf temperature up to 9°C in well-watered poplar

but only 1°C in drought-stressed poplar plants (Urban et al., 2017). Therefore, different

stomatal control/regulation, and consequently, different heat dissipation behaviour can be

10

observed among plant species and genotypes. In the case of grapevine, the anisohydric

variety ‘Touriga Nacional’ exhibited a greater heat dissipation via evaporative cooling due to

higher diurnal stomatal conductance than the variety ‘Syrah’ (Costa et al., 2012) that instead

exhibited a near isohydric response, contrary to earlier reports (Schultz, 2003; Soar et al.,

2006). Indeed, cultivars with a marked anisohydric behaviour maintain a less strict stomatal

regulation or delay stomatal response (Chaves et al., 2010) showing lower leaf temperatures,

under the same growing conditions as compared to isohydric cvs that have a tighter stomatal

regulation, under mild to moderate water stress. Isohydric varieties may present more frequent

supra-optimal leaf temperatures in dry conditions and heatwaves event. Isohydric plants can

thus show higher intrinsic WUE at expenses of an excessive leaf temperature that may lead

to a reduction in photosynthesis activity. However, the results observed by Garcia-Forner et

al. (2017) on models of the isohydric (Q. ilex) and anisohydric (P. latifolia) behavior in

Mediterranean forests caution against making direct links between water potential regulation,

stomatal control and carbon assimilation. In fact, the anisohydric P. latifolia tended to show

lower assimilation rates than Q. ilex under extreme drought, contrary to what was assumed.

Therefore, there is the need of a compromise between the control of water loss via transpiration

and CO2 absorption for photosynthesis (Chaves et al., 2016).

Temperature exerts direct control over the plant physiology through the regulation of all

enzyme activities, the transport of ions, carbohydrates and phytoregulators (Chaves et al.,

2010). Optimal grapevine leaf temperatures for photosynthesis are around 25-30C and

temperatures above such optimum can lead to reduced photosynthetic capacity, and in more

dramatic conditions (e.g. under heatwaves), it can lead ultimately to leaf burn and early

abscission or accelerated leaf senescence (Palliotti et al., 2014). In addition, heat stress can

also negatively affect berry color and flavor development (Greer and Weedon, 2013).

Under stressful conditions, such as high irradiance and high temperature, grapevine, which is

considered drought-tolerant plant, activates a series of mechanisms for protecting, adjusting,

and repairing the photosynthesis apparatus. In fact, leaves may absorb excessive light energy,

which causes photo-oxidative damage, in particular, by compromising the photosystem II

(PSII). Photon energy is excessive when the excited electrons exceed those that can be used

for NADP+ reduction to NADPH and ATP synthesis. Excess electrons in an excited state

originate in chloroplast Reactive Oxygen Species (ROS) that can damage chloroplast tissues,

increasing the hazard of photoinhibition (Dayer et al., 2019).

Photoinhibition can be defined as dynamic, a short-term downregulation of photosynthesis. A

chronic photoinhibition occurs when irreversible loss of functionality of PSII units occurs

11

(Osmond, 1994). Nevertheless, it is known and documented that there are many

photoprotective mechanism related to dynamic photoinhibition, including non-photochemical

quenching (Wang et al., 2009) and photorespiration (Guan and Gu, 2009).

Photorespiration allows to protect the apparatus from inhibition in different ways: (i) consuming

excess excitation energy (Flexas et al., 1999), since carbon oxygenation requires twice as

many electrons as carbon fixation; (ii) providing electron acceptor NADP+ to photoreaction and

provide phosphorus to photophosphorylation (Gao et al., 1989).

However, dissipation of excess energy as heat (Fig. 2.3) is the most important leaf

photoprotective mechanism (Flexas and Medrano, 2002). In leaves of Vitis vinifera, the non-

photochemical quenching photoprotective mechanism is mainly represented by the

xanthophyll cycle, allows to dissipate thermal energy, about 45–64 % of the absorbed light

energy under normal conditions, while under environmental stress this could increase to 75–

92 % (Hendrickson et al., 2004).

Under more severe water stress, more severe stomatal closure leads to a decrease in leaf gas

exchange. Evaporative cooling mechanism due to transpiration is reduced and the capacity of

heat loss is diminished, and leaf temperature increases. In addition, thermal energy dissipation

from the leaf to the atmosphere is limited when air temperature is high. Therefore, the

xanthophyll cycle may become insufficient to dissipate absorbed energy.

Increased light intensity leads to an increase in the photoprotective compound concentration

In V. vinifera leaves (Kolb et al., 2001) which have an ROS scavenging function. Ascorbic acid

(also termed vitamin C), glutathione, flavonoids, and carotenoids (Fini et al., 2011) are

photoprotective responses that act as antioxidant molecules to protect the photosynthetic

apparatus. Several antioxidant enzymes such us superoxide dismutase, catalase,

peroxiredoxins, ascorbate peroxidase, and glutathione reductase also play a role in the ROS

signalling or degradation pathways (Vidigal et al., 2013). Carotenoids, for example, in addition

to being accessory pigments also play a fundamental role in photoprotection of photosynthetic

apparatus. In fact, if the triplet excitation chlorophyll is not rapidly extinguished, the excited

state of oxygen (*O2) may form; highly reactive chemical species with lipids, proteins and

carbohydrates (Triantaphylidès and Havaux, 2009). Photoprotective carotenoids extinguish

the excited state of chlorophyll, releasing energy as heat and protecting pigments and

unsaturated fatty acids from oxidative damage (Carvalho et al., 2015b). The first line of

antioxidative defense in leaf is implemented by carotenoids simultaneously with ascorbate and

glutathione, as observed by Carvalho et al. (2015a) on grapevine variety “Trincadeira”

12

subjected to heat stress. Moreover, flavonoids, in particular dihydroxy B-ring-substituted

flavonoids, also have a crucial antioxidant role in plant responses to several abiotic stresses,

inhibiting and reducing the formation of ROS (Agati et al., 2012).

The leaf antioxidant protection system is triggered by UV-B irradiation and ABA appears to act

downstream in the signalling pathway for most enzymes of the photoprotection response (Berli

et al., 2010, 2011). Other metabolic protection responses can be triggered by the combined

effect of ABA and UV-B irradiation, such as the accumulation of quercetin and kaempferol in

grape tissues. In Arabidopsis thaliana leaves (Page et al., 2012) light intensity was shown to

influence accumulation of anthocyanins also classifying these molecules as photoprotectants.

In addition, leaf carotenoids increased from UV-B treatment and the level of ABA has also

increased because carotenoids are ABA precursors (Nambara and Marion-Poll, 2005).

It can be assumed that grapevine is stimulated to a greater production of photoprotective

molecules in the leaf to counteract adverse environmental conditions and that these molecules

are also addressed to sink organs, such as berry, through phloem increasing the final grape

composition, since the possible implications of these antioxidant compounds at the level of

berry quality is well known (Ferrandino and Lovisolo, 2014). This hypothesis could be

confirmed by a research that has determined, by both DPPH and FRAP tests, a strong

antioxidant ROS-scavenging activity in the berry skin and wine of “Aglianico” grapes subject

to controlled drought and high light levels (De Nisco et al., 2013).

Overall, varieties of V. vinifera showing greater tolerance to stressful environmental conditions

that cause oxidative stress and that hypothetically could benefit from it to increase berry quality

and would be important in terms of economic value (Carvalho et al., 2015b). Therefore, it is

necessary to clarify a further theoretical compromise between stomatal conductance (gs) and

the repair/rescue mechanisms against photoinhibition.

13

Figure 2.3. Summary of the line of defence implemented by grapevine under stressful conditions and due to an excess of electrons in the excited state. The first line of defence is to dissipate excessive energy as heat, which is called thermal

energy dissipation. Moreover, ROS scavenging process of photoprotectors and repair process of protein D1 linked to the PSII reaction centre complex, also play an important role in avoiding sustained

photoinhibition in leaves (adapted from Derks et al., 2015).

14

III. HOW DOES ABA METABOLISM MODULATES GRAPEVINE

PERFORMANCE?

3.1 Effect of water application and stress-recovery responses on ABA metabolism It is important to consider temperature and air evaporative demand condition during recovery

from water stress because the dynamics of leaf water potential (Ѱleaf) are different depending

on the environmental conditions. For example, under high atmospheric evaporative water

demand conditions (high transpiration conditions), recovery of water potential occurs much

faster than under low atmospheric evaporative water demand conditions (low transpiration

conditions), and during the berry ripening period, temperature and VPD are usually high

inducing the plant to a high transpiration.

In general, leaf water potential and leaf transpiration decrease during a gradual imposition of

soil water deficit, whereas either xylem (Rodrigues et al., 2008; Rogiers et al., 2012) or leaf

tissue (Lovisolo et al., 2002; Perrone et al., 2012a,b) ABA content increase. In grapevines

upon rehydration Ѱleaf recovers rather quickly both in pot-grown (Cerqueira et al., 2015) and in

field conditions (Medrano et al., 2003), due to the large size of grape xylem vessels and the

high hydraulic conductivity of grapevine shoots (Lovisolo and Schubert, 1998). Also, Perrone

et al. (2012b) showed, under conditions of low VPD, that Ѱleaf reached similar values of full

irrigated plants, within five hours from re-watering, although it has taken only two hours to

reach the same level under conditions of high evaporative water demand (Fig. 3.1).

Figure 3.1. Leaf water potential measured in Vitis Vinifera cv Grenache plants irrigated (IRR), subject to water stress (WS), or re-watered (REC) in the two experiments (A and B). Exp. A carried out in

spring under low atmospheric evaporative water demand (12 mbar bar−1). Exp. B carried out in summer under high atmospheric evaporative water demand (above 30 mbar bar−1). Arrow shows time

of rehydration. Bars are standard errors of the means (n=6) (Perrone et al., 2012b).

15

Recovery of leaf water potential happens quickly, but the recovery of transpiration does not

take place so quickly (only after an additional 48 hours). It is possible that such a phenomenon

could be induced by ABA root-to-shoot signalling which proved to be the way allowing plants

to withstand water stress, as it modulates the relationship between transpiration and leaf water

potential (Hartung et al., 2002; Heilmeier et al., 2007). In fact, significant peaks of leaf ABA

content (Fig. 3.2) have been measured five hours after rehydration in the first day, and even

in the morning of the second day of recovery, after ten days of water deprivation.

Figure 3.2. Time course of foliar ABA content measured during 36 h in re-watered (diamonds), water stressed (triangles) and irrigated (squares) grapevines. Recovering plants were re-watered at 07:00 h

(arrow) after a 10 days of water deprivation (Perrone et al., 2012b).

It has been reported for several years the phenomenon called ‘after effect’ of foliar ABA content

(Dörffling et al., 1980) on the reduction of transpiration by stomatal control, meaning that ABA

accumulated in roots during drought can cause residual effects after re-watering and during

recovery (Lovisolo et al., 2010), owing to ABA delivery from root to shoot. Accordingly, it has

been found that in both the day of re-watering and the day after, transpiration decreased or not

increased for a few hours after a peak level in leaf ABA concentration (Perrone et al., 2012b).

In this way, foliar ABA can control grapevine transpiration, not only upon water stress, but also

during the early phases of the recovery (Lovisolo et al., 2002) preventing water loss in dry

environments where vines are subjected to periodical summer drought (Tombesi et al., 2015).

Furthermore, it has been observed that the ‘after effect’ of foliar ABA generates the necessary

osmotic gradients that drive the vessel refilling (Salleo et al., 2009; Secchi and Zwieniecki,

2010), favoring faster rehydration and the increase of xylem pressure, thus promoting

embolism recovery (Tombesi et al., 2015).

16

3.2 Impact of ABA-induced process on secondary metabolite accumulation

The effect of water stress on secondary metabolite accumulation in grapevine berries has

widely investigated in the past years (Ojeda et al., 2002; Koundouras et al., 2006; Poni et al.,

2007). A moderate soil water deficit increases the possibility of having quality grapes (Intrigliolo

et al., 2012), in particular, for red cultivars (Van Leeuwen and Seguin, 2006). In turn, excess

of soil water, can reduce berry colour intensity and sugar content (Medrano et al., 2003). This

concept is valid for both isohydric varieties such as ‘Grenache’ (Coipel et al., 2006),

‘Tempranillo’ (Santesteban et al., 2011), ‘Manto negro’ (Medrano et al., 2003), as well as for

anisohydric ones such as ‘Cabernet Sauvignon’ (Bindon et al., 2008), ‘Cabernet Franc’

(Matthews and Anderson, 1988), ‘Muscat of Alexandria’ (dos Santos et al., 2007). However,

there are several factors that can influence the effect of deficit irrigation berry composition and

wine quality such as climatic conditions, soil properties, and amount and timing of irrigation

(Keller, 2005).

Increased ABA concentration in response to water deficit conditions, has also effects at

molecular levels. In grapevine berries, for example, higher ABA content promoted the

anthocyanins, proanthocyanidins and flavonols biosynthetic pathway during ripening

(Castellarin et al., 2007a,b; Deluc et al., 2011; Savoi et al., 2016), although it is unclear whether

the rise in the concentration of ABA at véraison is due to an in loco ABA biosynthesis, or either

to ABA translocation from other organs (Castellarin et al., 2011). Moreover, under water stress

conditions, strongly upregulated gene expression closely related to anthocyanin accumulation

and transport into the vacuole (Ageorges et al., 2006; Deluc et al., 2007) was observed.

Several experiments were carried out testing the effects of exogenous application of ABA at

véraison. Results showed that ABA can improve processes related to berry ripening, such as

the accumulation of phenolic compounds (Sandhu et al., 2011; Xi et al., 2013; Ruiz-Garcia et

al., 2013) leading to improved grape/wine antioxidant capacity. Therefore, ABA seems to act

as a driver of accumulation of some secondary metabolites, polyphenols in particular, and

since berry quality, in particular for the production of red wine, is largely dependent on

secondary metabolites, we may conclude that deficit irrigation, in particular after véraison,

could allow to reach a vegetative-production balance which would improve the final berries

composition (Chaves et al., 2010).

However, very importantly for practical application, Deluc et al. (2009) has observed that the

response to water stress can vary with the cultivar and that berry skin-colour and water stress

enhance different photoprotection mechanisms in the berry. In detail, metabolic responses

17

activated by water stress in ‘Cabernet Sauvignon’ improve biosynthesis of glutamate and

proline and increase anthocyanin concentrations. Instead, in white berries of the cv

‘Chardonnay’, which do not contain significant amounts of anthocyanins (photoprotective

molecules) and therefore, under water stress conditions, compensates by the activation of

parts of the phenylpropanoid, energy, carotenoid and isoprenoid metabolic pathways that lead

to increase of antheraxanthin, flavonols and aroma volatiles concentrations (Deluc et al.,

2009). Since moderate water stress conditions almost doubled ABA concentrations in

‘Cabernet Sauvignon’ berries, whereas it decreased ABA content in ‘Chardonnay’ berries at

véraison and shortly thereafter, supports the hypothesis that ABA improves accumulation of

proline, sugar, and anthocyanin concentrations.

3.3 ABA metabolism and carbon relocation to berries

The mechanisms of grapevine biological response to abiotic stress, especially drought,

involving morphological as well as metabolic changes (e.g. related to ABA) controlled by

modified gene expression can play a major role in plant resistance to adverse conditions

(Swamy and Smith, 1999).

Water stress induces changes in the expression of certain carbon partitioning genes in both

leaves and berries. In previous trials conducted by Pastenes and colleagues (2014), a

molecular response was observed. These authors found that in source organs, it allows an

increase in carbon export and, in the sink organs, an increase in import, in fact:

(i) in leaves an increase in gene expression for sucrose transporters, without affecting

the transcript abundance for the phloem loading protein, was found;

(ii) in berries, the transcripts activity for the cell wall invertase was parallel to water

stress, in fact, for the more stressing irrigation strategy, the activity of invertase

reaching significantly higher levels than in the irrigation strategy with higher

volumes of irrigation water returned to the plant;

(iii) similar behaviour was observed for the hexose transport in the source tissues as a

means for supporting the sugar accumulation in berries.

This mean that, even if leaf carbon assimilation has been reduced, in an extent proportional to

the stressing condition of the vine, source organs are always ready to transport sugar to the

berries, due to higher gene expression that promotes sink strength (Pastenes et al., 2014).

A strong sugar accumulation in berries upon water stress was also observed by Roby et al.,

(2004), and the response is thought to be mediated by ABA (Gambetta et al., 2010), in fact,

18

sugar and ABA signalling pathways interact synergistically in the control of sugar and

anthocyanin transport in grape berries (Conde et al., 2006; Lecourieux et al., 2010). Pirie and

Mullins (1976) were the first to detect this synergic effect of ABA and sucrose.

However, several experiments with different irrigation regimes have found controversial and

variable results on berry sugar concentration, with reports indicating decreases (Wang et al.,

2003; Castellarin et al., 2007a), other indicating increases (Roby et al., 2004; Santesteban and

Royo, 2006) and other indicating no changes (Pastenes et al., 2014). This can be explained

by the fact that under extreme water restrictions (leaf water potential less than -1.4 MPa),

acceleration of berry sugar accumulation can fail because, even though there are high ABA

concentrations in berry, there is a sharp decrease in photosynthetic capacity which does not

allow to reach adequate sugar concentrations (Chaves et al., 2010).

Once again, it is confirmed that low availability of water in the soil does not always improve

final berry quality because induction of anthocyanin synthesis in grape berries is dependent

on a certain concentration threshold of ABA and sugars (Gambetta et al., 2010).

19

IV. PRECISE AND MORE EFFICIENT IRRIGATION

4.1 Deficit irrigation strategies

Reduced water availability in semi-arid agricultural areas, less precipitation and increased

competition for water resources, has forced the adoption of more sustainable irrigation

strategies, with the aim of using water more efficiently (Costa et al., 2016; Fraga et al., 2019).

Water-use-efficient irrigation must be built on a more complete biological knowledge of

grapevine’s physiology as well as on more precise monitoring of soil and vine’s water stress

physiological parameters, to assure than plants are not subjected to severe water stress nor

to excessive water consumption.

The main objective of a deficit irrigation (DI) strategy is to improve the grape quality by

optimizing the use of water without substantially compromising the final yield. Next several DI

strategies are described in detail.

4.1.1 Regulated deficit irrigation

Regulated deficit irrigation (RDI) strategy is one of the most suitable tools in semi-arid areas

to control excessive vigour, improving water-use efficiency and berry quality compared to full

irrigated grapevines (Santos et al., 2005), but this depends on the timing of application

(McCarthy et al., 2002) and on the severity of the stress imposed (Costa el al., 2016). RDI

technique is based on the grapevine sensitivity to water stress not constant during its

phenological phases and on the alternation of the water deficit imposed in specific periods. For

example, after veraison, when the grapevine is less sensitive to water reduction (Marsal et al.,

2002; Kang and Zhang, 2004), calculated water deficit could increase anthocyanin

accumulation (Dry et al., 2001).

In turn, application of RDI before véraison, typically increases the skin to pulp ratio, compared

to well-watered vines (Ojeda et al., 2002; Roby et al., 2004), resulting in larger concentration

of anthocyanins and phenolic compounds at harvest (Matthews and Anderson, 1988).

Irrigation should be applied only when a determined parameter drops below a certain threshold

value. Although leaf water potential has been proposed as an indicative parameter for crop

irrigation as well as for RDI programs in grapevines (Girona et al., 2002) and other extensive

crops (Goldhamer et al., 1999), continuously monitoring of some direct or indirect indicators

20

(e.g. gs or An) could allow to better decide when and how much water must be applied for a

rational use of irrigation water. Moreover, the use of imaging approaches on thermography

could also permit a faster and robust assessment of vine’s water status in vineyards (Jones et

al., 2002; Grant et al., 2007).

4.1.2 Sustained deficit irrigation

RDI is based on the more general concept of sustained deficit irrigation strategy (SDI) which

implies that water is provided at levels below full crop water requirements (ETc)

homogeneously during the growing season.

In Mediterranean areas, SDI may save water via increased transpiration efficiency (Chaves et

al., 2010) and, at the same time, optimize or stabilize yields and quality in several horticultural

crops, namely grapevines, orchard fruit trees and vegetables (Bravdo, 2005; Fereres and

Soriano, 2007; Costa et al., 2007). In the particular of grapevine cultivation, results obtained

from the use of DI strategy have shown that it can favour composition of berries given the

significant increase in the contents of anthocyanins and total phenols as compared to full

irrigation strategy (Medrano et al. 2015). However, the effects can differ according to the

environmental conditions (Bravdo, 2005; Chaves et al., 2007) and the genotypes of rootstock

and cultivar (De la Hera et al., 2007; Fereres and Soriano, 2007; Chaves et al., 2010).

In previous studies of Souza et al. (2003, 2005), it has been found in the cvs Moscatel and

Castelão that SDI (at 50% ETc) as compared to FI grapevines (at 100% ETc) promoted WUE

as both intrinsic (expressed by the A/gs ratio) and in long term, as revealed by the rise in 13C

in grapevine tissues. These positive effects were also found in other V. vinifera L. cvs and/or

other locations (Dry et al., 2000a,b; Stoll et al., 2000; Loveys et al., 2004; Lanari et al., 2014;

Trigo-Córdoba et al., 2015).

4.1.3 Partial rootzone drying

Partial rootzone drying (PRD) is another irrigation technique based on plant physiology

research making use of previous results of Loveys (1992) and based on root signals and their

influence on whole plant. In detail, half of the root system is kept frequently irrigated whereas

the other half is kept dry, and they alternate with a frequency of 10-15 days (Fig. 4.1) (Costa

et al., 2007). The alternation frequency of the irrigated sides must be determined by the type

of soil and other factors such as rainfall (Stoll et al., 2000; Santos et al., 2003).

21

Figure 4.1. Implementation of partial rootzone drying (Stoll, 2000).

The concept is that the “wet-side” sustains turgor in the shoot while the dehydrated roots in the

“dry-side” activate the synthesis of chemical signal that could be transferred to the shoot

enabling the adjustment of stomata aperture (Santos et al., 2003; Kang and Zhang, 2004).

PRD method in grapevines reduces stomatal conductance without modifying leaf water status

(Souza et al., 2003), thereby increasing water use efficiency and reducing vegetative growth

(Santos et al., 2005), which is basically the aim of deficit irrigation in vineyards (Chaves et al.,

2007; Costa et al., 2007).

One possible candidate for root-to-shoot chemical signal could be ABA that plays a role in

regulating plant water use (Hartung and Wilkinson, 2009). It has been demostrated that PRD

strategy stimulates a hormonal response that leads to a content of ABA in the leaves five times

higher as compared to the fully irrigated control (Dry et al., 2000a,b; Stoll et al., 2000).

However, water stress induces other root-to-shoot signal, that can significantly change

concentration of both cytokinins (Kudoyarova et al., 2007) and ethylene (Sobeih et al., 2004).

In this regards, Dry et al. (2001) noted a correlation between reduction of shoot growth in PRD

grapevines and a evident reduction in the concentration of cytokinins in shoots and roots.

Comparing PRD and SDI techniques, no significant differences on grapevine physiology and

WUE were observed in low vigour vineyards in Mediterranean conditions (Portugal) with the

cultivar “Tempranilho” (Lopes et al., 2011) and the same also applies to other cultivars in other

areas (Pudney and McCarthy, 2004; Bravdo et al., 2004; Gu et al., 2004; Baeza and

Lissarrague, 2005). On the other hand, other authors have shown positive effects using PRD

compared to other techniques namely that it tends to reduce vigour due to smaller canopy leaf

area and less pruning weight (Souza et al., 2003; Santos et al., 2005). Experiments conducted

in Australia have observed a significant reduction in vegetative growth, with only a small

22

reduction of photosynthesis (Stoll et al., 2000) but still maintaining high standards of yield and

quality, saving a large quantity of water (Dry et al., 2001). Results obtained can be justified by

different root response to drought, which is deeply influenced by soil properties and genotypic

differences in stomatal sensitivity to ABA signalling and hydraulic signals (Antolín et al.,

2006; De la Hera et al., 2007; Lovisolo et al., 2016). Although the soil effect seem to be

predominant over vine genetic (Tramontini et al., 2013), but it remains to be determined how

it affects yield and composition of the berries.

However, in order to adopt the PRD technique in the field, compared to other DI techniques,

more costs and time are required for the installation of adequate tubes. In fact, PRD can

demand the double amount of tubes as well as more accurate control of the irrigation system

as well as of the irrigation scheduling, wich demands more labour. In addition, some authors

speculate that the PRD technique function in the field cannot be comparable to the results

obtained during pot trials. In fact, in field conditions, where the roots are not clearly separated

into two different sides, there could be a redistribution of water from deeper roots to more

superficial roots (Bravdo, 2005). This led to the intuition that it could be the amount of water

used rather than the PRD irrigation strategy that explained the effects obtained (Gu et al.,

2004).

4.1.4 Some additional considerations

These modern water saving irrigation strategies allow: (i) a maintenance of high grape quality

characters; (ii) reasonable yield losses as compared to full irrigation; (iii) to maximize the WUE.

However, and very importantly, in Mediterranenan area and, in general in warm climate,

application of DI strategies in vineyards must be carefully done because when drought and

heat waves are combined this results in extreme stomatal closure what is a risk for leaf

condition. The grapevines, even if only mildly stressed, if exposed to these conditions become

much more vulnerable to leaf burn by sun, as leaf temperature can drastically increase (up to

7◦C above air temperature) (Blum, 2015). In these moments, deficit irrigation should be

substituted by full irrigation in order to increase evaporative cooling (Lopes et al., 2014), as if

this situation stands for long periods it could give rise to leaf photo-damage and/or xylem

embolism, causing dramatic problems to the plants. In fact, it is well accepted that the proper

agronomic strategy to minimize heat wave effects is abudant irrigation (Webb et al., 2009).

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4.2 Sensors for precise soil and plant water status

There are several methods to assess plant water status and soil water content. They can be

classified according to their characteristics (e.g. destructive/non destructive, low cost/high cost,

direct/indirect plant-based method). Below they are divided into indirect methods, soil-based

or atmospheric based methods and direct plant-based methods.

4.2.1 Indirect methods

4.2.1.1 Soil-based methods

Soil-based methods allow to define soil moisture by volumetric methods or by tensiometric

methods. Volumetric methods express the amount of water in a volume of soil (can be

expressed in % or mm/m depth). The instruments are inserted into the soil at various depths

to measure the dielectric constant that is a linear function of the soil moisture itself. The most

used tools in a production context are neutron moisture probes and capacitance sensors

(Townend et al., 2001). Instead, the tensiometric methods quantify voltage expressed in -MPa

with which water is retained by solid particles. There are two types of sensors that can be used

for this purpose:

(i) based on the measurement of soil electrical resistance

(ii) tensiometers.

The main advantage of soil-based methods is to allow remote monitoring of soil moisture.

Measurements are carried out continuously, allowing the soil water content to be monitored

even during the winter to asses the refilling of soil water capacity.

However, the use of soil-based methods to develop and manage the irrigation strategy has

several drawbacks. A common drawback in vineyards is the soil/plots heterogeneity, often

even adjacent ones. Therefore, before installation of sensors it would be appropriate to perform

a soil mapping and then adapt the results obtained with the different soil characteristics of the

parcels. This implies the need of installing many sensors in the vineyard resulting in higher

costs. However, through soil-based methods, it remains complex to accurately assess the

plant water status (Lavoie-Lamoureux et al., 2017) considering the water redistribution from

high soil moisture regions to drier regions (Smart et al., 2006) and the roots capacity to get

water in deeper soil layers where sensors do not have access.

24

4.2.1.2 Atmosphere-based methods

A basic tool to support the monitorization of plant needs in water is the installation of a meteo

station. This is the basic procedure to have an idea on the water needs of the vines. More

complex approaches to estimate grapevine water consumption can be deduced from the total

vineyard evapotranspiration (ET) utilizing two atmosphere-based methods:

(i) eddy covariance method

(ii) Bowen ratio energy balance method

These methods are mainly used for research purposes since they are complex and expensive

(Drexler et al., 2004). In addition, these methods present some critical issues. For the

estimation of vineyard actual evapotranspiration (ETa) several factors must be considered

such as row spacing, trellis system and interrow cover crops. In turn, this last parameter

depends on the climate, water availability, soil properties and it may be present throughout the

entire vineyard or in alternating rows, in permanent way or not.

The estimation of each factor influencing ETa is very complex, especially since it varies

throughout the season. Atmosphere-based methods are a promising approach to monitor

irrigated strategy although it requires even more research, in particular on partitioning of ETa

between grapevine transpiration and other component.

4.2.2 Direct or plant-based methods

4.2.2.1 Visual observation

It is a simple and inexpensive method to assess the vine water status based on visual

observation carried out in the field. Slowing the growth of apical meristem or the vine apex is

the first symptom of limited water availability (De Toda et al., 2010). Instead, a light to moderate

state of stress is indicated by leaves that tend to take a vertical orientation and by wider angles

between the direction of shoot apex and the apical tendril. Severe water stress leads to

premature drying and yellowing of the basal leaves followed by a premature fall. An additional

indicator of water stress is the tendrils development. In moderately stressed vine their withering

is visible until complete abscission when the deficit becomes severe (Keller, 2010). This is only

a qualitative and approximate method, related to detector subjectivity.

25

4.2.2.2 Pressure chamber

The pressure chamber (Fig. 4.2) is the tool most used in viticulture to monitoring plant water

status (Van Leeuwen et al., 2009), thanks to its manageability and being relatively cheap.

Procedure of use is also quite simple: the leaf, or a petiole, is inserted into the pressure

chamber with the cut end coming out of the sample carrier device. Increasing pressure is slowly

applied, until the internal tension is exceeded, and xylem sap begins to come out of the cut

section. The water potential is equal and opposite to the pressure value used by the chamber.

The major constraint is the time required for the numerous measurements to be carried out

considering the plots heterogeneity and the short validity, for example after a rainfall (Yuste et

al., 2004).

Figure 4.2. Pressure chamber used to determine water potential in plants (Chavarria and Santos,

2012).

It is possible to make several measurements based on time, protocol and plant organ

evaluated (i.e. leaves, stem):

▪ Leaf water potential (Ѱleaf) is the simplest measure, usually taken at midday on a well

exposed adult leaf. The main disadvantages of this quick measurement which make

the results unsatisfactory are (i) rapid fluctuations in water potential throughout the day

depending on environmental conditions; (ii) the different microclimatic conditions of the

leaves (Jones, 2004); (iii) the an-or isohydric behaviour influencing stomatal control

and therefore also variations in the leaf water potential (Rienth and Scholasch, 2019).

26

▪ Stem water potential (Ѱstem) is an accurate and stable measure with respect to the leaf

water potential (Rienth and Scholasch, 2019). The stem water potential is generally

determined by inserting a leaf in a plastic bag surrounded by aluminium foil for 45–120

minutes, to balance the leaf water potential with that of the stem. The measurement is

usually carried out between 13.00 h and 16.30 h, when values reach a minimum.

▪ Predawn leaf water potential (ѰPD) The measurement of the pre-dawn leaf water

potential is conducted one or two hours before sunrise (Rienth and Scholasch, 2019),

that is, when the transpiration is almost zero and the leaf is in hydraulic balance with

the root system. ѰPD measurements present the advantages of not being affected by

climatic conditions. Moreover, the predawn water potential has also the big advantage

to indicate soil water availability, as the water status of the plant is in equilibrium with

the soil. However, it presents the disadvantage that ѰPD will be in homeostasis with

the wettest layer of soil, without considering other portions of soil. Therefore, the

measure of water available in the soil could be overestimated (Améglio et al., 1999).

These last two types of measurement (i.e. pre-dawn and stem water potential) remain reliable

indicators of plant-water status and can be useful for irrigation scheduling (García-Tejero et

al., 2011).

Threshold values have been proposed by Carbonneau (1998), Lovisolo et al. (2010, 2016) and

Van Leeuwen et al. (2009) (Tab. 1).

4.2.2.3 Carbon isotope δ13C

In the atmosphere there are two stable carbon isotopes of CO2: 12C and 13C. The former is

usually present in larger quantities and is preferred by enzymes involved in photosynthesis.

However, the sugars produced during periods of water stress contain more 13C compared to

those produced when there is high water availability. Therefore, the carbon isotope index,

based on the 13C/12C ratio in grape sugar, allows to understand the water deficit level

experienced by the grapevine (Rienth and Scholasch, 2019). Different values correspond to

different water stress conditions, as shown in the Tab. 1. The results obtained with the carbon

isotope indicator showed an excellent correlation with those obtained with pressure chamber

(Spangenberg and Zufferey, 2018). However, in this case, measurements can only be made

at the end of the growing season.

27

Table 1. Threshold values of water potential (Ѱ), stomatal conductance (gs) and carbon isotope (δ13C) (adapted from Rienth and Scholasch, 2019).

4.2.2.4 Sap flow sensors

Sap flow measurements are used to monitor the vine water status through the examination of

fluid movement inside the xylem to get information about plant hydraulic function or dysfunction

(Steppe et al., 2015). There are two techniques for calculating sap flow:

▪ Thermal dissipation probes method. The method of thermal dissipation probe is not

suitable for commercial use because it does not provide reliable results.

▪ The stem heat balance method (Fig. 4.3). It overcomes the limitations of the previous

method. In this case it is non-intrusive and is reliable for calculating grapevine

transpiration, separating it from other ET components. Therefore, this is a technique

successfully adopted to guide irrigation strategies (Scholasch, 2018).

Figure 4.3. A sap flow sensor installed in the vineyard (Rienth and Scholasch, 2019).

28

4.2.2.5 Leaf gas exchange

Stomatal conductance changes are particularly sensitive to grapevine water deficits and that

is why it has been identified as a good indicator to monitor the water status (Chaves et al.,

2010; Urban et al., 2017). In fact, gs variation may occur before any measurable change in

plant water status (Jones, 2004). Stomatal conductance can be measured by porometer or by

measuring leaf gas exchange with an infrared gas analyzer (IRGA). In the first case, gs is

measured only considering the diffusion of water vapour from the leaf while in the second case,

the diffusion of CO2 from the leaf is also considered, according to their infrared absorption

wavelength.

Since results obtained through porometers are not always reliable, in recent years the

measurements are carried out through the IRGA which offers better results in terms of

sensitivity and precision (Ciccarese et al., 2011). However, this tool has the drawback of being

expensive and complex to use. In addition, many replicates are necessary to have a real

evaluation, making these measurements more complicated to apply in the open field (Costa et

al., 2010, 2013).

4.2.2.6 Thermal data

Plant or leaf temperature can be measured with several methods to monitor vine’s water status

(Grant et al., 2007, Möller et al., 2007). Thermography is based on IR radiation emitted by an

object, and the warmer the object is the more it will emit IR radiation, in accordance with the

Stefan-Boltzmann law that establishes that the emittance of a black object is proportional to

the fourth power of its absolute temperature.

The fact that leaf temperature can work as an indicator of water stress in crops, has given the

opportunity to use thermography as a fast, non-invasive and robust technique to assess plant

water status and to support irrigation strategies in vineyards. These positive features should

encourage the use of thermography in field conditions (Costa et al., 2012; Grant, 2012;

Fuentes et al., 2014). However, efficient irrigation management requires the use of the most

appropriate thermal index taking into account the combined effects of the climatic conditions

and the genotype, as well as the best time of day to perform measurement. In detail, the

combined effects of the genotype and climate become more significant in situations of severe

water stress (García-Tejero et al., 2016).

29

Different thermal indices were developed when using IR thermal measurements. These indices

normalize absolute values of Tleaf or Tc (Maes and Steppe, 2012). The most widely used in

different crops, such as in grapevine are the crop-water stress index (CWSI), the index of

relative stomatal conductance (IG), and the different between canopy and air temperature

(ΔTcanopy-air) (Jones et al., 2002; Costa et al., 2012; Bellvert et al., 2014) and are obtained as

follows:

(i) ΔTcanopy-air = Tc - Tair

(ii) CWSI = (Tc - Twet) / (Tdry – Twet)

(iii) IG = (Tdry - Tc) / (Tc – Twet)

Tdry and Twet refer to temperature values (maximum and minimum) for a leaf with fully closed

stomata under severe water stress and for a fully transpiring leaf under optimal watering

condition, respectively; Tc is the canopy temperature and Tair the temperature of the

surrounding air.

The non-normalized thermal indicator Tc, and the thermal indices have shown a high

correlation with leaf gas exchange parameters (e.g. gs and An) (García-Tejero et al., 2016). Tc

is the easiest index to support thermal remote sensing of water stress in grapevine. However,

Tc is extremely affected by environmental conditions, especially when measurements occur

under highly variable climate conditions (e.g. windy, slightly cloudy, Tair). Instead, CWSI and Ig

thermal indices, can weigh up the problem of the variability of climatic conditions during the

day. They can also provide stronger results in case there are limitations in obtaining a precise

Tair near vine canopies (García-Tejero et al. 2016).

The timing of measurements is also relevant. Some authors show that the best time to perform

robust thermal measurements was at midday (Fuentes et al., 2012; Pou et al., 2014; Bellvert

et al., 2015) that partially agrees with García-Tejero et al. (2016), who suggest assessing vine

water status through thermal data between 11:00 h and 14:00 h. Other authors also propose

afternoon measurements (Deery et al., 2019) at the most stressful moments of the day (Costa

et al., 2013). Other relevant aspects of thermal measurements related to the phenological

stage (Deery et al. 2019) as well as with the sun exposition (sunlit vs shadow site) which can

also interfere with stomatal behaviour (Grant et al., 2007).

30

V. THE ROLE OF IRRIGATION FREQUENCY

Irrigation is an increasingly important agronomic practice in modern viticulture in dry areas with

the aim of reducing the vine water deficit level as well as to minimize risks and guarantee

yields. Planning site-specific irrigation, to reduce water stress level to which the vine is

subjected, should take into account several fators such as rootstock-scion combination, soil

texture, climatic conditions, vine phenological stages, water salinity, trellis system and canopy

architecture (Scholasch and Rienth, 2019). Considering the large number of factors and the

combination effects between them that are still not entirely clear, it makes an optimal irrigation

hard to schedule.

An appropiate irrigation strategy should also consider the effects induced by different irrigation

frequency on the vine water status. In fact, this technique can affect WUE and the vine

ecophysiological perfomances (Scholasch, 2018), although, recent scientific research

regarding irrigation intervals has shown conflicting results due to various abiotic and biotic

factors.

Myburgh (2012), when working with grapevines grown in sandy soils, reported that the more

frequent irrigation strategies resulted in an increase in yield, while decreasing the frequency it

resulted in lower yields. This effect can be explained because a lower irrigation frequency leads

to greater water constraint.

In turn, Bowen et al. (2012a,b) in a 4-year study evaluated the effect of irrigation frequency of

1 and 3 days in a loamy sandy soil for cvs Cabernet Sauvignon, Merlot and Syrah, observing

higher yields while preserving quality for the less frequent irrigation treatments.

Also, Selles et al. (2004) observed similar effect, but dealing with table-grapes growing in a

clay loamy soil. They found that lower frequency application implied better plant water status

that resulted in an increased yield and pruning weight, concluding that less frequent irrigation

resulted in a better distribution of water in this particular soil profile and larger root

development.

This type of results emphasizes the role of soil characteristics in vines response to irrigation

strategies. Different soil textures induce differences in soil moisture and wetting pattern,

affecting vine water use regulation under water deficit, even when the same quantity of water

is applied but under different irrigation frequencies (Wang et al., 2006; Sebastian et al., 2015).

For example, Tramontini et al. (2013; 2014) showed that a clay-rich soil can induce a non-

31

hydraulic root-to-shoot signal causing stomatal closure that may overlap with the putatively

variety-induced anisohydric response to water stress, reducing the extent of embolism

formation. Therefore, under mild water stress conditions, in clay soil, the grapevines seem to

express a “more isohydric” behaviour, maintaining high levels of water potential, without

significant metabolic alterations.

Sebastian et al. (2015) noted that applying a small irrigation dose with a high frequency (i.e.

every two days or less) in a heavy clay soil may lead to an efficiency loss under low water

availability conditions, as the irrigation bulbs created is small and close to the soil surface

(Amin and Ekhmaj, 2006), favouring soil water evaporation and resulting in a relatively fast soil

water depletion (Montoro et al., 2016).

Hence, if low irrigation frequency does not reduce yield and/or wine quality significantly, it could

be assumed that less frequent irrigation permits to use water more efficiently (Scholasch,

2018). In fact, less frequent irrigation could reduce evaporation losses from the soil surface

and reduce vineyard evapotranspiration (Sebastian et al., 2015; Montoro et al., 2016) in

particular in semi-arid areas.

In this way, evaporation related to irrigation can be reduced by irrigating at low frequency and

a greater intensity, consequently, high irrigation frequency should be questioned in semi-arid

areas (Montoro et al., 2016). In addition, Montoro et al. (2016) conclude that the results

obtained can be summarized in a sentence: "the lower the volume of irrigation applied, the

greater the fraction of evaporated water", although the results may have been influenced by

other factors not considered in this study. In this research where several irrigation strategies

were applied to establish the effect of the amount of irrigation water on evaporation, the most

promising results in increasing WUE were obtained when a higher amount of irrigation was

applied with low irrigation frequency. Supporting the findings and conclusions of Montoro et al.

(2016), Gardner and Gardner (1969) have shown that applying 96 mm or four 23 mm water

irrigation the total water loss due to evaporation from a bare soil surface represents a 38% for

the single irrigation and 62% for the multiple irrigations, in a total experiment period of 24 days.

Hence, it can be assumed that adopting a high irrigation frequency, which leads to having

smaller irrigation bulbs and bulbs closer to the soil surface, inhibits translocation to the leaf of

the hypothetical ABA synthetized in deep root, because there is suficient water in the

superficial soil (Fig. 5.1A). Instead, low irrigation frequency with greater water supplement may

result in a better distribution of water in the soil profile. In fact, when water goes down in the

soil, the shallow roots can synthetized ABA that can be transported to leaves (Fig. 5.1B). This

32

phenomenon allows to have an effect similar to PRD strategy. In fact, in the vine coexist ABA

production and water availability, without the additional costs of PRD. To confirm this

hypotheis, drying of the superficial roots has been observed under field condition in several

grapevines subjected to dry soil conditions or PRD treatment (Smart et al., 2005; Bauerle et

al., 2008). However, the water redistribution process from wet to dry roots (the so-called

‘hydraulic lift’) in response to water potential gradients could contribute to decrease of ABA

biosynthesis (De la Hera et al., 2007) compared to ABA levels observed in pot experiments.

Figure 5.1. Representative scheme of soil water distribution and ABA metabolism. (A) On the left side of the figure it show how ABA signal synthetized in the roots does not reach the leaves, (B) while on the right side, where the production of ABA signal takes place in the shallow roots, translocation of

ABA signal to the leaves is possible.

5.1 Root development and distribution

Root growth is extremely plastic. Under mild water stress conditions, there is a change in the

allocation of plant resources, mainly carbohydrates, towards root growth at the expense of the

shoots because root development is less sensitive to mild water stress than shoot growth and

because plants concentrate resources mainly on organs that obtain the limiting resource

(Poorter et al., 2012; Ledo et al., 2018). This allows the plant to increase soil exploration for

water uptake, while reducing transpiration (Cramer et al., 2013; Simonneau et al., 2017).

In general, all conditions that lead to depletion of soil water-nutrient resources stimulate radical

branching, inducing a better response to abiotic stresses (Palliotti et al., 2018). However, the

maintenance of grapevine root growth capacity and plasticity in root development during water

33

scarcity depend also on the rootstocks (Bauerle et al., 2008; Tramontini et al., 2013) and on

water stress level. In fact, when grapevine is subject to a severe water deficit, the limited water

available in the soil will cause complete cessation of root growth (Robbins and Dinneny, 2018).

Development and distribution of root absorption sites along the soil profile can be altered

according to different irrigation strategies (Bou Nader et al., 2019), among these also the

frequency and the volume of irrigation can affect the roots architecture. Edwards and

Clingeleffer (2013) have shown that reducing prolonged periods of drought allows vines to

develop a greater root system. Indeed, the study of total root dry mass in a vertical soil portion

75 cm deep, showed that it increased in irrigated vines with high irrigation frequency. However,

the main inconvenience is that the root absorption sites were concentrated more in the surface

soil (0-25 cm) around the dripper lines, making the vine water state much more dependent on

changes in soil moisture content because the topsoil layer loses moisture easily due to

evaporation.

In turn, Myburgh (2011a,b) observed that a different irrigation frequency (less frequent

irrigation, i.e. at pea size, véraison and post-harvest; more frequent irrigations, i.e. at pea size,

midway between pea size and véraison, at véraison, midway between véraison and harvest

and post-harvest) caused no significant differences on root density along the grapevine row as

compared to the non-irrigated control.

Developing an adequate irrigation strategy may be important to relieve the water stress of adult

plants but it is also crucial for newly planted grapevines. In fact, after planting, if a small amount

of irrigation water is frequently applied, the roots tend to develop only in the wettest part of the

soil, that is, in the surface part, where less energy is required to absorb water. Already a year

after deep soil preparation carried out before planting, many vineyard soils tend to re-compact

naturally after an irrigated event or rainfall (Myburgh, 2018). For this reason, a high irrigation

frequency in the early years of the plant limits the roots development in the subsoil in the

following years, although subsequently more irrigation water is applied to wet the subsoil, root

development will be limited by soil compactness.

5.2 Irrigation volumes and irrigation frequency

The study carried out by Scholasch et al. (2009) in Napa Valley highlights the effects induced

by a low and high irrigation frequency. In the same season, the same volume of water is applied

by single irrigation event (18 mm) or by three smaller irrigation events (6 mm each) in the same

vineyard within a eleven day period (Fig. 5.2). We note that when irrigation volumes and

34

intervals are shorter, transpiration levels are also lower and tend to decrease more rapidly in

between. In fact, stomatal opening is reduced already after a few days, making grapevine more

susceptible to drought stress (Sebastian et al., 2016). Conversely, when applying a single

irrigation event, average transpiration is greater than 50% (1.2 mm per day vs 0.6 mm per day)

and transpiration rates tend to gradually decrease over more than 10 days, even if the same

total amount of water is applied over the period. Under these experimental conditions, a low

irrigation frequency resulted in higher WUE and the irrigation effect can last longer (Scholasch,

2018).

Based on the results obtained (Fig. 5.2) the heavy variation in transpiration levels induced by

high irrigation frequency (6mm every 3/4 days), is due to a hydraulic response implemented

by the vine. In fact, stomatal regulation is controlled by both hydraulic (water potential-

mediated) and chemical (ABA-mediated) mechanisms (Pantin et al., 2013; Tombesi et al.,

2015; Bonada et al., 2018). It has been suggested that in V. vinifera passive hydraulic control

of stomatal closure seems to precede ABA signalling during early onset of drought conditions

(Tombesi et al., 2015; Lovisolo et al., 2016) and that ABA accumulation in the leaf is an

additional signal involved in maintaining stomatal closure in case of prolonged water stress

and/or under post-drought recovery of grapevine water status (Tombesi et al., 2015).

The hypothesis is that in V. vinifera under high irrigation frequency the stomatal closure is

primarily triggered by water potential decrease and coordinated with xylem embolism

formation. In fact, the short interval between irrigations does not trigger the ABA root-to-shoot

signal, which is probably activated in the case of low irrigation frequency, represented in the

figure 5.2. A large irrigation water (18 mm) induces, after the peak maximum of transpiration,

a gradual stomatal closure and probably a related increase in ABA biosynthesis.

35

Figure 5.2. Grapevine transpiration variation in response to different irrigation frequency during a short period of eleven days (Scholasch, 2018).

In addition to analyzing the effects induced by the irrigation frequency in the short term (about

eleven days) Scholasch (2018) also considered the effects over a three-year consecutive

period in the same vineyard with the aim of changing the irrigation frequency from high to low.

The first year of the experiment (year 1), the irrigation event took place every three to seven

days, while the second year (year 2) every ten to fifteen. In Fig. 5.3, Scholasch (2018)

compares the effects caused by a different irrigation frequency, and shows that in the first year,

high irrigation frequency resulted in a total of fifteen events while in the second year with a less

frequent irrigation, nine. The second year the amount of water per irrigation event is slightly

greater as compared to the first year (8mm versus 5mm). However, this allows to achieve

higher water deficit index (WDI) values and have a lower seasonal deficit than in the first year

of the experiment, even if the period between irrigations is greater in the second year. In

practice, WDI is a percentage that goes from 100 to 0 indicating the grapevine transpiration

level: 100 percent means that a vine is transpiring at its maximal level whereas 0 percent the

vine is no longer transpiring. The results obtained throughout the vegetative cycle are

correlated with those observed previously considering a short period. Low irrigation frequency

allows greater vine transpiration, and less total water use compared to frequent irrigation

(72mm versus 75mm).

36

Figure 5.3. Grapevine transpiration variation (expressed as WDI) in response to decreasing irrigation frequency between year 1 and year 2. Each number on the graph corresponds to one irrigation event

(Scholasch, 2018).

The results obtained in the third year of the experiment validate the previous results (Fig. 5.4).

The figure compares the results obtained during the first and the third years of the experiment.

The total amount of irrigation water is the same (75-76 mm) but the irrigation events in the third

year are about half compared to the first year (7 against 15 events). In the third year of

research, it has been shown that by further reducing the irrigation frequency compared to even

the second year (data shown in the previous figure), and by increasing the water volume per

irrigation event, the water deficit level is further reduced. Consequently, applying a larger water

volume in combination with low irrigation frequency for two consecutive years (year 2 and year

3) seems to improve vine water use regulation and the severity of seasonal water deficit

compared to a higher irrigation frequency (year 1). These results disagree with previous

studies (De Souza et al., 2005; Romero et al., 2010; Sebastian et al., 2016), which instead

noted that the effects of irrigation frequency on plant water status and gas exchange

parameters were found to be relatively small compared to the effects of different water volumes

returned to the plant.

37

Figure 5.4. Grapevine transpiration variation (expressed as WDI) in response to decreasing irrigation frequency between year 1 and year 3. Each number on the graph corresponds to one irrigation event

(Scholasch, 2018).

5.3 Grape ripening

It is known that too severe water deficit post-véraison can lead to a loss of berry volume (Delrot

et al., 2014) and a reduction in net photosynthesis activity with negative effects on grape

maturation processes (Martínez-Lüscher et al., 2015). For this reason, low irrigation frequency

that positively affect the grapevine water status throughout their vegetative cycle can induce

positive effects even on berry ripening. In fact, Fig. 5.5 shows that adopting an irrigation

strategy that involves a high irrigation frequency (year 1) can lead to a sharp decrease in the

berry weight by comparing the results with a low irrigation frequency (year 2 and year 3). In

the first year, under high irrigation frequency, berry weight accumulation is interrupted and

delayed. This is caused by a major water deficit and a consequent manifestation of shriveling

symptoms, shortly before harvest. On the other hand, during year 2 and year 3, when the

irrigation frequency was reduced, the berry volume accumulation profile is smooth, reaches a

peak and maintains its value until harvest.

38

Figure 5.5. Effect of different irrigation frequency on berry weight (Scholasch, 2018).

Instead, Fig. 5.6 shows that during the first year, under high irrigation frequency, berry sugar

accumulation is discontinuous, probably due to the low-optimal grapevine water status. While

decreasing the irrigation frequency, sugar accumulation is smooth until it reaches its maximum

value (Year 2 and 3) due probably to the best vine water status.

Figure 5.6. Effect of different irrigation frequency on berry sugar accumulation (Scholasch, 2018).

39

These results show that a low irrigation frequency allows to maximize the WUE. In fact, WUE

is optimized when: (i) you get more yield using the same amount of irrigation water without

compromising the grape quality, or (ii) you can maintain grape quality and yield using less

water (Myburgh, 2018). In this experiment it was observed that, increasing the interval between

irrigations, not only the grape weight increased (and consequently the yield) but there were

also no disorders occurred during berry ripening, probably also improving the berry quality. On

the contrary, a high irrigation frequency combined with low amounts of irrigation water induces

significant variation in vine water deficit that negatively reflects on grape yield and quality.

In addition, reducing the irrigation frequency by imposing prolonged drought periods seems to

improve the berry skin resistance to mechanical deformation and it seems that these changes

in berry cell wall composition led to an easier color extraction (Cooley et al., 2017), which is

desirable for red wine.

40

VI. CONCLUDING REMARKS AND FUTURE PROSPECTS

The effects of climate change are visible and are striking Mediterranean regions, with a

negative impact for viticulture and demanding alternative management solutions in the

vineyard. Some of these solutions involve the use of irrigation water.

Mediterranean vineyards are increasingly prone to stress, soil is being increasingly exploited

and exposed to erosion and water for irrigation is an increasingly scarce resource in both

quantity and quality. These negative trends and climatic projection had however, resulted in

some positive aspects. For example, they have made vinegrowers aware of the importance of

their agronomic choices for soil and water management, forcing them to adopt more

sustainable strategies (Clothier et al., 2010; Stefanelli et al., 2010). This is for example, the

OIV resolution focused on sustainability.

In areas most affected by climate change, grapevine yield and quality are greatly limited by

water deficit, temperature, and evaporative demand during summer (Santesteban and Royo,

2006; Chaves et al., 2007; Fraga et al., 2019). Irrigation therefore plays a key role in alleviating

plant stresses, but water use needs to be optimised to improve WUE and save water and

maintain or even improve berry quality.

A further drawback in view of global warming and the use of irrigation is recent discoveries

concerning the potassium channels involved in potassium transport to the berry apoplast. In

fact, the results suggest that higher irrigation regimes could promote an accumulation of

potassium in the berry (Nieves-Cordones et al., 2019) which favours potassium tartrate

precipitation. In addition, the acceleration of malic acid degradation (Rienth et al., 2016) could

lead to a reduction in the total acidity of wines (Mira de Orduna, 2010).

The fact that grapevine perceives water stress as a hazard and that responds to it by

accumulating secondary metabolites in berry pulps, seeds and skins is a strategy to prevent

cell damages (Cramer et al., 2011) that leads to increase the final quality of the berry (Tenore

et al., 2011; De Nisco et al., 2013). With careful viticultural practices, such as emergency

irrigation, regulated deficit irrigation, partial root-zone drying, the use of rootstocks tolerant to

water scarcity, the use of controlled cover crops and other techniques (Keller, 2005; Dai et al.,

2010, 2011) and considering different response to water stress for different varieties (Palliotti

et al., 2014; Hochberg et al., 2015; Theodorou et al., 2019) and the different site-specific

characteristics, controlling the thermal regulation of the vine organs, monitoring the water

status of the plant and soil also using different sensors together to have a wider view, it is

41

possible to stimulate the mechanisms of response of the vine to environmental stress and

obtain grapes rich in secondary metabolites and improve berry composition.

Therefore, the threshold and frequency of irrigation should be defined according to the

exclusive varietal specific and site-specific response induced by water stress to the

ecophysiological performance of the vine and the ripening and composition of the berry

(Scholasch and Rienth, 2019). Site-specific features also include soil characteristics, which

affect ABA metabolism, fundamental in stomatal response, especially but not only during water

stress conditions (Lovisolo et al., 2016). For this reason, it is of primary importance to gain

deeper knowledge about the direct/indirect influence (ABA-mediated) of soil properties on both

vegetative growth and berry composition.

Based on the results obtained by Scholasch (2018) a promising irrigation strategy that aims to

promote water and energy saving and wants to maintain high quality standards should avoid

high irrigation frequency which induces a heavy variation in plant water status but should

induce more gradual water deficit variations as observed as a result of larger irrigation (Cooley

et al., 2017; Linares et al., 2018; Scholasch and Rienth, 2019).

In addition, winegrowers can choose the duration of drought periods between the two irrigation

events causing water deficits of varying intensity according to production objectives. However,

large intervals between irrigated events can stimulate ABA biosynthesis and following

rehydration, high concentration of ABA is found in the leaves, even higher that in not-irrigated

plants, due to fast transport of ABA by the transpiration stream from the roots to the recovering

leaves (Lovisolo et al., 2002; Perrone et al., 2012b) and/or due to an in loco biosynthesis,

which promotes genes expression involved in biosynthetic pathway of phenylpropanoid and

flavonoid, contributing to increase the quality and antioxidant power of grapes and must and

the wine longevity (Ferrandino and Lovisolo, 2014). Due to the dependence of the response to soil type, water availability, and many other factors,

it is necessary to increase the existing knowledge on how different irrigation frequency affect

vineyard performance and how the combined effect of irrigation frequency and the genotypes

can influence frequency strategies. It will be also relevant to quantify to which extent irrigation

frequency can help to save water with minor or positive impacts on berry composition.

What is more, it becomes more and more important expanding our knowledge on the

physiological and molecular adaptations that the vine implements to counter water stress

(Hochberg et al., 2017; Gambetta et al., 2020). Studies on the long-term effects of DI strategy

42

and irrigation frequency on plant performance are important for crops with long commercial life

like fruit trees or grapevines because a choice can have repercussions even in the following

years.

43

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