Review

New Biotechnology �Volume 00, Number 00 �November 2012 REVIEW

Challenges and perspectives to improvecrop drought and salinity tolerance

Eleonora Cominelli1, Lucio Conti2,3, Chiara Tonelli2,3, and Massimo Galbiati2,3

1 Istituto di Biologia e Biotecnologia Agraria, CNR, Via E. Bassini 15, 20133 Milano, Italy2Dipartimento di Bioscienze, Universita degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy3 Fondazione Filarete, Viale Ortles 22/4, 20139 Milano, Italy

Drought and high salinity are two major abiotic stresses affecting crop productivity. Therefore, the

development of crops better adapted to cope with these stresses represents a key goal to ensure global

food security to an increasing world population.

Although many genes involved in the response to these abiotic stresses have been extensively

characterised and some stress tolerant plants developed, the success rate in producing stress-tolerant

crops for field conditions has been thus far limited. In this review we discuss different factors

hampering the successful transfer of beneficial genes from model species to crops, emphasizing some

limitations in the phenotypic characterisation and definition of the stress tolerant plants developed so

far. We also highlight some technological advances and different approaches that may help in

developing cultivated stress tolerant plants.

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

Conventional breeding and biotechnological approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

The importance of phenotyping: field conditions versus laboratory conditions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

Integration of different strategies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

The key role of regulatory sequences to express transgenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

New strategies from epigenetic and post-transcriptional control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

IntroductionGlobal population is expected to rise above 9 billion in less than 40

years. Average living standards are also increasing, impacting on

food consumption, demand for grain for livestock sustenance and

ultimately on agricultural land use. To meet the target of 70%

more food by 2050 defined under the Declaration of the World

Summit on Food Security [1], an average annual increase in cereal

Please cite this article in press as: Cominelli, E. et al., Challenges and perspectives to improvj.nbt.2012.11.001

Corresponding author: Tonelli, C. (chiara.tonelli@unimi.it)

1871-6784/$ - see front matter � 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.nbt.2012.1

production of 44 million metric tons per year is required. This food

supply will need to originate from current arable lands, with very

little potential for future expansion [2]. Moreover, in the face of

the projected scenario of global warming [3] how can we reconcile

the need to produce more food and the increasingly environmen-

tally challenging conditions to obtain it? To meet these demands,

there is an obvious and urgent need to further increase crop

productivity. In the sixties the so-called ‘green revolution’ mark-

edly increased crop yield through genetic improvements of major

e crop drought and salinity tolerance, New Biotechnol. (2012), http://dx.doi.org/10.1016/

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food crops, increased mechanisation, improved pest control and

improved soil fertility [4]. Today scientists envisage a crucial need

for a ‘second green revolution’ to enhance crop yield and yield

stability under abiotic stresses that affect and are predict to increas-

ingly affect future plant productivity. Many plant scientists believe

that the use of modern biotechnology, molecular breeding tech-

niques, and genetic engineering of crop species can contribute

significantly to achieve these goals [5].

Soil water deficits and salinisation are the two most crucial

abiotic stresses that limit the production of the world’s food crops

[6]. To meet the growing demand for food and to contrast the

detrimental effects of climate change on crop yields, it is impera-

tive to develop new crops that have improved resistance to

drought and salinity stress and crops that have improved water

use efficiency (WUE), that is, able to reach great yields using less

water. Not only is the development of plants with higher WUE

important to overcome problems caused by drought and salinity,

but also to achieve a sustainable use of water worldwide.

Water will increasingly become scarcer in the scenario of cli-

mate change. On our planet there is approximately 1400 million

km3 of water, but after subtracting the salt water of the oceans and

the freshwater locked up in ice caps, only around 9000–

14,000 km3 of freshwater is potentially available for human use

[7]. Food production is a water-intensive process, as agriculture is

responsible for the consumption of over 70% of the entire avail-

able freshwater [5,8]. Producing a kilogram of corn requires 900 l of

water, for the same amount of wheat and rice, 1350 and 3000 l of

water respectively are required [9]. However, while only 16% of

cropland is irrigated, it produces about 40% of the world’s food

[10]. This data is worrying, considering that, even in the most

productive cropping environments, short periods of water defi-

ciency are responsible for considerable reductions in seed and

biomass yields each year [5].

Drought affects plant performance and productivity by causing

cellular dehydration that reduces the cytosolic and vacuolar

volumes and stimulates the production of reactive oxygen species

that negatively affect cellular structures and metabolism [11]. High

salinity, typically accompanying water scarcity, causes both ionic

and osmotic stresses [12,13], modifying plant cell plasma mem-

brane, lipid and protein composition, ultimately impairing opti-

mal growth and development [14,15].

Different strategies have been employed to improve plants WUE

and drought and salinity tolerance. We will provide an overview of

the current state of the art in the field and what we consider to be

highly promising strategies for the future.

Conventional breeding and biotechnologicalapproachesThe classical strategy to obtain crops more tolerant to drought and

salinity is through breeding. In this approach the genetic varia-

bility underlying stress resilience is identified by screening germ-

plasm collections. Beneficial traits are subsequently introduced

into cultivars/lines through different mating designs. Although

some success in improving stress tolerance has been obtained so

far using conventional breeding, as reviewed by [16,17], this

procedure is time-consuming, cost- and labour-intensive. It also

suffers from a poor selectivity as, along with desirable traits,

unwanted linked traits can be also transferred.

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2 www.elsevier.com/locate/nbt

Biotechnological approaches such as molecular breeding and

genetic engineering offer the possibility to obtain better results in a

shorter time. Genetic engineering may also allow to overcome the

reproductive barrier among different plant species.

Molecular breeding combines the process of genetic improve-

ment of conventional breeding with DNA markers. It increases the

genetic gain by enhancing the selection efficiency and reducing

the length of breeding cycles. With such strategy some important

results have been obtained in the field of drought and high salinity

tolerance, reviewed by [16,17]. Genome sequences are now avail-

able for different crops, including species with large and complex

genomes. Moreover the advent of so-called ‘next-generation

sequencing’ technologies offers the possibility to sequence, rela-

tively quickly and cheaply, new crops and different varieties of the

same plant to develop new markers to be used in molecular

breeding, reviewed by [18].

In the past years many efforts have been directed to the devel-

opment of plants tolerant to water or high salinity stress through

genetic engineering, as recently reviewed by [19,20]. Different

approaches, based on genetic and molecular studies have shown

that many genes, proteins and metabolites modulate in a very

complex network plant adaptation to environmental stresses [12].

Based on their biological function, genes involved in these

responses can be grouped in two main classes: single function

genes and regulatory genes [21]. Genes belonging to the first group

encode enzymes associated with the accumulation of osmolytes,

proteins and enzymes scavenging oxygen radicals (ROS), molecu-

lar chaperones, ion transporters, channels, proteins involved in

lipid biosynthesis and examples of the successful use of this

strategy are reviewed by [19,20]. Genes belonging to the second

group are involved in transcriptional or post-transcriptional reg-

ulation of gene expression such as transcription factors, protein

kinases, protein phosphatases and proteinases [19,20]. Conse-

quently two strategies have been mainly used to develop trans-

genic plants tolerant to abiotic stresses: (1) the overexpression or

down-regulation of single action genes, and (2) the manipulation

of regulatory genes, expected to modulate the expression of

numerous downstream genes. The first approach has not been

always successful in conferring tolerance, because multiple and

complex pathways are involved in controlling plant abiotic stress

responses [19,20]. Conversely the modification of the expression

of a regulatory gene has been more efficacious and is probable to be

widely used in the next generation of genetically modified crops

[19,20]. Many transcriptional regulators are known to be involved

in plant responses to drought or high salinity stress, most belong-

ing to one of the large transcription factor families (AP2/ERF, bZIP,

NAC, MYB, MYC, Cys2His2 zinc-finger, NFY and WRKY). Different

recently published reviews summarise results obtained in improv-

ing abiotic stress tolerance by the overexpression or down-regula-

tion of a single transcription factor [22–25]. Particularly DREB

(Dehydration Responsive Element Binding) proteins, belonging

to AP2/ERF family, have been extensively characterised for their

role in response to abiotic stresses, as reviewed by [22,25]. The

practical and application value of DREBs in crop improvement,

such as stress tolerance engineering as well as marker-assisted

selection (MAS), has been demonstrated in different crops [25].

Although numerous genes conferring tolerance to different

abiotic stresses have been identified (particularly in model species

e crop drought and salinity tolerance, New Biotechnol. (2012), http://dx.doi.org/10.1016/

New Biotechnology �Volume 00, Number 00 �November 2012 REVIEW

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such as Arabidopsis thaliana), the successful transfer to crops has

been limited. Moreover, those plant varieties where abiotic stress

tolerance has indeed been achieved through genetic engineering

are yet to be released for the benefit of the farmers [20,26].

Different explanations for the low translatability for crop improve-

ment can be given and perspectives in this field are discussed

below.

The importance of phenotyping: field conditions versuslaboratory conditionsA key obstacle hindering the generation and commercialisation of

stress tolerant crops is the direct transfer of studies performed

under laboratory conditions to the field. While survival (or recov-

ery) is the major trait from a purely physiological perspective, it is

crop yield the likely determinant of successful stress tolerant crops

from an agronomical point of view. The majority of studies

reporting abiotic stress resistance in model plants, reviewed by

[27–29], tested tolerance by measuring plant recovery after a

drastic and agronomically unrealistic stress episode (i.e. very high

salinity, severe dehydration, osmotic shock, among others). This is

in contrast with field conditions in temperate climates, where

plants experience drought gradually and limited water availability

rarely causes plant death, while normally restricts biomass and

seed yield. Improved survival rate under lethal conditions does not

predict superior growth performance and, thus, biomass yield

gain, under moderate drought often encountered in the field

[30]. This was shown by comparing several Arabidopsis transgenic

lines, previously characterised for their enhanced survival under

severe drought. Under relatively mild drought stress conditions all

plants showed a comparable growth reduction caused by drought.

Thus, enhanced survival under severe stress conditions is largely a

function of water-saving mechanisms rather than a net improve-

ment in plant production [30].

A strictly connected aspect is the developmental stage at which

abiotic stress event is applied. In fact, although the majority of

crops are highly sensitive to abiotic stresses during flowering, with

devastating effects on yield [31–33], most laboratory studies,

especially those performed with Arabidopsis, do not address the

effects of abiotic stress on seed productivity, the most important

parameter from an agronomical point of view. Only a few genes

have been characterised that enhance plant stress tolerance and

result in increased seed yields in the field [34–42].

In nature plants are often simultaneously subjected to multiple

rather than single environmental perturbations. Only few studies

addressed plant responses to environmental stresses applied in

combination. In some cases the combination of different stresses

may cause more than additive negative effects on plant perfor-

mance, as summarised by the so called ‘stress matrix’, in which the

impact of different combinations of stresses is classified as poten-

tially negative, potentially positive or with no effect [43]. For

example, the interaction between drought or salinity with heat

or nutrient starvation causes more deleterious effects on crop

productivity than the single stresses applied individually, as

reviewed by [44].

The prospect of transferring genes to confer broader plant

protection to a combination of different abiotic plant stresses is

extremely attractive [12]. Some genes implicated in response to

multiple stress responses have been identified, the majority of

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which encode transcription factors. For example the Arabidopsis

DREB2a transcription factor has been shown to be implicated in

response to drought, salinity and high temperature responses [45],

while the barley HvCBF4, belonging to the same DREB/CBF family,

when overexpressed, confers increased tolerance to drought, high

salinity and cold [46]. Also the overexpression of AP37 and AP59 in

rice increased tolerance to drought and salinity [36]. Members of

the WRKY family, such as the soybean GmWRKY13, GmWRKY21,

GmWRKY54 [47], of the bZIP family, like the rice OsABF1 [48] and

the wheat WLIP19 [49] and of the MYB family, as the rice OsMYB4

[50,51] have a role in general abiotic stress responses. These

transcription factors provide potentially valuable starting points

to develop crop protection against multiple stresses, strengthening

future stress tolerance in crops.

Integration of different strategiesThe majority of researchers interested in improving plant response

to abiotic stresses focused on either molecular breeding or genetic

engineering approaches [17], while an integration of these differ-

ent strategies is desirable. An interesting example of this combined

approach is the Water Efficient Maize in Africa (WEMA; http://

www.aatf-africa.org/wema/en/) project, strictly connected to the

Drought Tolerant Maize for Africa (DTMA; http://dtma.cimmyt.

org/) project. WEMA, involving public research centres such as

CIMMYT (International Maize and Wheat Improvement Center)

and AATF (African Agricultural Technology Foundation) together

with multinational private companies, is integrating conventional

and molecular breeding with genetic engineering approaches to

develop and disseminate new drought-tolerant African maize

varieties. The DTMA project has already developed, through con-

ventional breeding, different maize varieties adapted to tropical

mid-altitude agro-ecologies found in Sub-Saharan Africa with 20–

30% higher yields. WEMA is incorporating a drought tolerance

trait developed by Monsanto and BASF into DTMA-developed

varieties, using transgenic breeding. The transgene used in these

plants is the CspB gene from B. subtilus, encoding a cold shock

protein with RNA chaperone activity [38]. The WEMA project

started in 2008 and access to maize hybrids developed through

biotechnology is expected to be ready in about eight years (http://

www.aatf-africa.org/wema/en/).

New technologies such as ‘next-generation sequencing’ (NGS)

and systems biology offer other opportunities that should be

integrated to the already discussed approaches to develop plants

more tolerant to drought and high salinity stress. The advent of

NGS technologies makes it feasible to explore the natural variation

to mine for genes or alleles variants conferring stress tolerance

[52]. The increasing number of Single Nucleotide Polymorphysm

(SNPs), obtained through sequencing and re-sequencing, allow

statistically robust associations between phenotypes and geno-

types through Genome Wide Association Studies (GWAS)

[53,54]. To this extent, several sequencing projects are currently

being developed for Arabidopsis (http://www.1001genomes.org/).

As observed previously, responses to drought and salinity are

complex and controlled by many genes. Moreover plants in the

field are mainly simultaneously subjected to multiple stresses.

Systems biology approaches could help in this field through the

generation of models defining the contribution of different signal-

ling pathways and of different stresses, defining plant ‘-omic’

e crop drought and salinity tolerance, New Biotechnol. (2012), http://dx.doi.org/10.1016/

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architectural responses in relation to stresses and to the changing

environmental conditions [12].

The key role of regulatory sequences to expresstransgenesLittle efforts have been made in the study of regulatory sequences

controlling gene expression. The majority of transgenic plants

tolerant to abiotic stresses have been obtained through the use

of strong constitutive promoters such as Cauliflower mosaic virus

35S (CaMV35S) [55] or plant promoters like ubiquitin [56], actin

[57], and cytochrome c [58]. Unfortunately constitutive expres-

sion results in a yield penalty even under non-stress conditions,

illustrating the pitfalls of constitutively activating metabolically

costly stress response pathways [59–61]. A refinement of the

temporal and spatial pattern of gene expression is therefore impor-

tant in avoiding pleiotropic effects. This objective can be reached

through the use of inducible or organ-, tissue-, cell-specific pro-

moters (or combinations of these) to allow expression of a trans-

gene only in defined conditions [59–61]. For example, the

expression in Arabidopsis of DREB1/CBF3 confers tolerance to

stress, but causes severe growth retardation under normal growth

conditions [59]. However, when expressed under the control of an

osmotic stress-inducible promoter like rd29A promoter, growth

occurs normally [59,61,62]. Inducible down-regulation is an

equivalently effective strategy to improve tolerance. The a- and

b-subunits of the Arabidopsis farnesyltransferase ERA1 are

involved in the regulation of ABA signalling (ABA, Abscisic acid,

a phytohormone that plays a fundamental role in drought

response). Inducible down-regulation of ERA1 enhances the

responses to the hormone and results in increased stress tolerance

[39].

A similar strategy consists in expressing a gene of interest in an

organ-, tissue- or cell-specific manner. Two main target tissues for

such gene mis-expression approach for stress tolerance can be

envisaged: roots and guard cells. Roots were traditionally difficult

to study, but recent progresses have made the manipulation of

root architecture and physiology a feasible strategy to produce

crops with better yields [63]. Water is absorbed from the soil

through the root system and deep rooting can overcome soil

drying. The manipulation of root system architecture is therefore

an important target to enhance tolerance to soil water deficit stress

[64] and the availability of root-specific promoter is important to

achieve this result. Transcriptomic analysis of the multiple root

cell types and tissues in Arabidopsis allowed the identification of

candidate promoters controlling gene expression in specific root

cell types and at a particular developmental stage of primary and

lateral root formation [65,66]. Also, numerous microarray studies

of root gene expression responses to abiotic stresses, such as high

salinity in Arabidopsis [67] or tomato [68,69] or water-stress in

maize plants [70], constitute a source of information to identify

new root-specific promoters to allow root gene expression modu-

lated by abiotic stresses.

Interesting applications of root-specific gene expression has

been developed to combat salinity and drought. For example,

the Na+ transporter HKT1;1, when specifically expressed in the

mature root stele of Arabidopsis thaliana, resulted in an increased

salinity tolerance by causing an increased influx of Na+ into stellar

root cells and decreased Na+ accumulation in the shoot by

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4 www.elsevier.com/locate/nbt

37–64%. By contrast, the overexpression of the same gene under

the control of the CaMV35S promoter resulted in high shoot Na+

concentration and poor plant growth [71]. The expression of two

NAC transcription factors under the control of root-specific pro-

moter demonstrated the general efficacy of root-specific

approaches in response to drought in rice [40,41].

A promising approach to reduce the water requirement of crops

and to enhance tolerance to stresses is the manipulation of guard

cells [72]. However, targeting gene expression specifically in these

cells is an important preliminary requirement. Guard cells sur-

round stomatal pores through which land plants uptake carbon

dioxide for photosynthesis and lose water vapour by transpiration.

Only few guard cell-specific promoters have been isolated so far

[73–77]. Several sources and tools for guard cells specific promoters

derive from functional characterisation of single genes [78–83],

large scale gene- or enhancer-trap screens [73,84,85], transcrip-

tomic and proteomic studies [75,86–88]. However, no example of

the use of guard cell-specific promoters to improve abiotic stress

tolerance has been reported to our knowledge so far, despite their

great potential.

Also shoot-specific promoters are important to improve stress

tolerance, as shown by the use of the promoter of Arabidopsis

hydroxypyruvate reductase (AtHPR1) to down regulate the

expression of the alpha-subunit of farnesyltransferase (AtFTA)

in canola [42]. This promoter contains the core motif of the

well-characterised dehydration-responsive cis-acting element

and is inducible by drought stress. Conditional and specific

down-regulation of FTA in canola, using the AtHPR1 promoter

driving an RNAi construct, resulted in yield protection against

drought stress in the field [42].

The functionality of different cis-elements conferring organ-,

tissue- or cell-specificity appears to be conserved among different

plant species, as demonstrated in some cases [89,90]. For example

promoters of root hair-specific AtEXPA7 orthologous and paralo-

gous genes from different angiosperm species contained conserved

root hair-specific cis-elements either in EXPAs genes and also in

other root hair-specific promoters [89], although these species

have different root hair distribution pattern. Also in the case of

the Arabidopsis AtMYB60 gene [76,91] and of its grape ortholog

VvMYB60 [90] strong conservation was reported among the cis-

elements controlling guard cell-specific expression. In fact cis-

elements recognised by DOF transcription factors, necessary for

AtMYB60 promoter activity in guard cells are also present in

VvMYB60 promoter and maintain a similar cluster organisation

in the two promoter sequences [76,90]. Moreover the expression

of the two genes is down-regulated by drought and ABA, suggest-

ing the presence in the two promoters of cis-elements important

for the responses to the stress and the hormone.

The conservation of cis-regulatory motifs offers the opportunity

to use a well-characterised promoter to manipulate expression in

distantly related species and to develop synthetic promoter with the

desired characteristics. In fact the ideal combination to regulate the

expression of a transgene is to achieve both temporal and spatial

regulation. Synthetic promoters, containing a functional combina-

tion of different cis-elements represent an attractive tool to reach

this goal [92]. The manipulation of the architecture of the promoter

sequence through ‘cis re-arrangement’ may allow to optimise the

relative strength and tissue specificity. There is currently a paucity of

e crop drought and salinity tolerance, New Biotechnol. (2012), http://dx.doi.org/10.1016/

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engineered promoters designed for plant biotechnology applica-

tions, although this strategy can offer many advantages [93]. Several

hybrid or chimeric recombinant plant promoters have been devel-

oped mainly to improve pathogen and wounding responses [94,95]

and to obtain strong ‘superpromoters’ [96–98], while in the field of

abiotic stress response very little progress has been achieved. A

synthetic promoter, combining a sequence that confers guard

cell-specificificity (gcPEPC) and a sequence ethanol-inducible

(AlcR/alcA) was developed [99]. Preliminary results suggest that

the minimal AtMYB60 promoter sequence, required for guard

cell-specificity, can be combined with other cis-regulatory modules

stress-responsive to produce functional guard cell-specific chimeric

promoters (our unpublished results).

New strategies from epigenetic and post-transcriptional controlRecent data indicate epigenetic processes (DNA methylation,

histone modifications, generation of small RNAs molecules and

transposable element activity) as a novel important layer of

regulation of gene activity in response to abiotic stresses

[20,100–103].

The importance of DNA methylation has been suggested in the

halophyte Mesembryanthemum crystallinum L. where salt stress

induces specific CpHpG-hypermethylation activating the switch

in photosynthesis mode from C3 to CAM for a better adaptation to

stress [104]. Also, the use of the methylation inhibitor 5-azacyti-

dine resulted in the increased tolerance to salt stress at the seedling

stage in wheat [105].

Histone modifications such as acetylation and methylation may

contribute to gene expression regulation in response to stress. In

fact decreased levels of histone acetylation through antisense

strategy in tomato resulted in higher photosynthetic rates under

water-stress [106]. Moreover tobacco and Arabidopsis cells respond

to high salinity by transient up-regulation of H3 phosphoacetyla-

tion and histone H4 acetylation [107]. Acetylation of H3K23 and

H3K27 was also observed in response to drought stress on RD29B,

RD20 and At2g20880 genes [108]. Histone H3K4 methylation

patterns change in response to dehydration stress in Arabidopsis

[109]. Also ABA and salt stress modulate the histone acetylation

and methylation of abiotic stress responsive genes [110].

miRNAs (micro RNAs) are emerging as important players in

stress response as well as proving a valuable tool for the manip-

ulation of gene expression to improve stress tolerance. Although

Please cite this article in press as: Cominelli, E. et al., Challenges and perspectives to improvj.nbt.2012.11.001

in plants miRNAs only target a low percentage of mRNAs (less than

1% of protein coding genes versus 60% in animals), their direct

target are mainly transcription factors, thus amplifying enor-

mously the number of genes controlled by miRNAs activity

[111]. miRNAs expression is altered in response to drought and

salinity as demonstrated in different species and their activity is

seemingly important to attenuate plant growth and development

in response to stress [111]. The overexpression of miR169 causes

drought-sensitivity in Arabidopsis, while the overexpression of

NFYA5, a transcription factor targeted by miR169, improves

drought tolerance [112]. Importantly, a correlation exists between

miRNAs expression levels and sensitivity to stress among different

genotypes of soybean: some miRNAs were up-regulated in

drought-sensitive soybean genotypes while they were down-regu-

lated in drought-tolerant genotypes [113]. A similar correlation

could be made in maize where some miRNAs were differentially

expressed in lines showing different sensitivity to high salt con-

centration [114]. These data indicate that miRNAs engineering to

enhance plant stress tolerance is highly promising.

ConclusionsBiotechnological approaches allowed the generation of different

plants tolerant to drought and high salinity stresses, but the

transfer of these results to agriculture is still lagging behind. Under

field conditions, plants are often subjected to multiple stresses,

varying in intensity and duration and differentially affecting plant

performance depending on the plant developmental stage. There-

fore, stress tolerance traits should be evaluated in more realistic

conditions, mimicking what really happens in the field. Trans-

genic approaches can be integrated with conventional and mole-

cular breeding but also with more innovative strategies, taking

advantage of the most recent technologies in plant research.

Finally, in our opinion the different opportunities offered by

targeted engineering of regulatory sequences for precise expres-

sion of transgenes have not been sufficiently exploited in biotech-

nological applications in agriculture.

AcknowledgementsThis work was partially supported by: Progetto AGER, bando

Viticoltura da Vino (SERRES, 2010-2105); Progetto Agrisost,

Fondazione Umberto Veronesi per il Progresso delle Scienze,

Milano, Italy; Progetto BIOGESTECA 15083/RCC and funded by

Regione Lombardia.

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