Genetic engineering for drought tolerance - DistaGenomics

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Dec 11, 2012 (4 years and 7 months ago)

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Genetic engineering for
drought tolerance
silvio.salvi@unibo.it
WUEMED training course
June 5-10, 2006
Transcriptionunit
Codingregion
Promoter
Exon
Intron
Translationend
Translationstart
Gene structure (eucaryote)
DNA
mRNA
Enhancers, silencers,
MARS, etc
Control of eucaryoticgene expression
•Chromatin
•Transcription
factor
Mod. from Albertset al., Molecular Biology of the Cell, 4th edition
Methodsof transformation
1.Agrobacterium

c/o tissueculture

c/o floraldip
2.Particlebombardment(gun)
3.PEG-mediated
4.Electroporation
5.Siliconcarbidefibers(WhiskersTM)
Detailof Ti plasmid
Agrobacteriumtumefaciens
Mod from: Griffith et al., Modern Genetic Analysis, Freeman, andBrown, Genomes, Synauer.
Exampleof binaryvectorsystem
Helper
plasmid
+
ParticleGun
Schemeof particlegun
Selectionand regeneration
Constructsforgeneticengineering: design
Plantcells
Gene of
interest
Promoter
and/or
enhancer
•constitutive
35-S, Ubi, actin
•inducible
Byartificial
stimula
Environmentally
Endogenously
Terminator
e.g.: nos
Selectionmarker
forplantcells
Provide
resistance
to:
•Antibiotic
Kanamycin
Igromycin
•Herbicide
Bialaphos
Bromoxynil
Glyphosate
Selectionmarker
forbacterialcells
Provide
resistance
to:
•Antibiotic
Kanamycin
Igromycin
Reporter gene
Toverifyefficacy/rate of
transformationor promoter
functionality
E.g.: gus, gfp(green fluorescentprotein),
luciferase
PrTerm
PrTerm
MARS
PrTerm
Strategiesforthe genetic
engineeringof droughttolerance
Synthesis of compatible solutes
•Almost all organisms, ranging from microbes to animals and plants,
synthesize compatible solutes in response to osmotic stress
•Compatible solutes are nontoxic molecules such as amino acids, glycine
betaine, sugars, or sugar alcohols which can accumulate at high
concentration without interfering with normal metabolism
•They may have a role in osmotic adjustment, stabilizing proteinsand cell
structures, scavenging reactive oxygen species
•Strategies for manipulation:
require knowledge of biosynthetic pathways
include up and down regulation of key regulatory enzymes involved
in synthesis and degradation, use of feedback inhibition-insensitive
forms, ecc.
Synthesis of compatible solutes
•Mannitolis normally synthesized in numerous plant species, but not in
wheat.
•MtlD(from E.coli) encodes for mannitol-1-phosphate dehydrogenasethat
catalyzes the reversible conversion of Fru-6-phosphate to mannitol-1-
phosphate. In transgenic plants, mannitol-1-phosphate is converted to
mannitolvia nonspecific phosphatases
•Transgenic wheat accumulated mannitolfrom 0.6 to 2.0 mol g-1
fresh
weight in the mature fifth leaf. This amount was inadequate to
account for osmotic effects
•Plants that accumulated higher mannitolhad severe abnormalities
use of stress-inducible expression systems, which should lack potential
detrimental effects on growth
•The beneficial effect of mannitolmight result from protective
mechanisms like scavenging of hydroxyl radicals (OH•) and
stabilization of macromolecular structures
•Unlike osmotic adjustment, OH• scavenging and other protective
functions require only small amounts of mannitol
•The mechanism by which mannitolinteracts with OH• remains to be
explained
•Prolineis the most widely distributed osmolyte; it occurs in plant and in
many other organisms. Its accumulation correlates with tolerance to
drought and salt stress
•Roles: osmotic adjustment, membranes protection, sink of energy and
reducing power, C and N source, OH• scavenger
•Synthesis can occurs via two biosynteticpathways: the ornithine-
dependent and the glutamate dependent (predominant under stress
conditions)
•Constitutive overexpressionrequires extra energy and building blocks
which may hamper the plant growth. Thus, it is desirable to use a stress-
inducible promoter to drive the expression of such new functions.
Zhu et al., (1998) Plant Science 139, 41–48
Overexpressionof a Δ
ΔΔ
Δ1-pyrroline-5-carboxylate synthetasegene and
analysis of tolerance to water-and salt-stress in transgenic rice
Zhu et al., (1998) Plant Science 139, 41–48
Overexpressionof a Δ
ΔΔ
Δ1-pyrroline-5-carboxylate synthetasegene and
analysis of tolerance to water-and salt-stress in transgenic rice
49-bp ABA-responsive element
from the barley Hva22 gene
180-bp minimum rice
actin1promoter
Hva22 intron
Inducible promoter
Selection marker gene
PC5CSgene from
Vignaaconitifolia
•Stress-inducible expression of
P5CStransgeneresulted in the
overproduction of the P5CS
enzyme in transgenic rice plants
as well as an increase in proline
accumulation
•Increased levels of prolinemay
contribute, at least in part, to an
enhanced biomass production,
as reflected as higher level of
fresh shoot and root weight, of
transgenic rice plants under
water-or salt-stress conditions
Zhu et al., (1998) Plant Science 139, 41–48
Overexpressionof a Δ
ΔΔ
Δ1-pyrroline-5-carboxylate synthetasegene and
analysis of tolerance to water-and salt-stress in transgenic rice
Increasingprotectiveproteins
Lateembriogenesisabundant
(LEA)
•Lea genesencodea diverse groupof stress-protectionproteins
expressedduringembryomaturationin allangiosperms
•Accumulationof LEA proteinsduringembryogenesiscorrelates
withincreasedlevelsof ABA and withacquisitionof desiccation
tolerance
•LEA proteinsare notnormallyexpressedin vegetative tissues
buttheyare inducedbyosmoticstress or exogenous
applicationof ABA
•Evidencederivedfromexpressionprofilesstronglysupportsa
roleforLEA proteinsasprotectivemolecules, whichenablethe
cellstosurviveprotoplasmicwater depletion
•FromLEA group1 togroup5. LEA-2 are dehydrins
Hva1 (LEA3 protein) frombarleytorice
ROS-scavenging
AlteringABA concentration
DroughttolerancebymodulatingABA
content: ABA synthesis(usingAtNCED3)
Key enzyme in ABA synthesis. Overexpressionof AtNCED3increase ABA and drought
tolerance. Antisensesuppression lead to low ABA and drought sensitivity.
DroughttolerancebymodulatingABA
content: ABA catabolism(usingCYP707A3)
•The major ABA catabolicpathwayistriggeredbycytochromeP450 CYP707A family.
•The expressionof CYP707A3 wasmosthighlyinducedin responsetoboth
dehydrationand subsequentrehydration.
•Theyproposedthatthe pairof AtNCED3and CYP707A3hasa pivotalroleforABA
biosynthesisand catabolismduringdehydrationor rehydration.
(Shinozakiand coll, PlantJ. 2006)
Knockouts
AVP1 (H+PPase) in Arabidopsisand tomato
•AVP1 isa vacuolarH+pumpabletogenerate pHgradientsinvolvedin auxin
distribution
•Modificationof Avp1 expressionimpactsauxin-mediatedorganogenesis(especially
roots!)
Engineeringdroughttolerance
usingtranscriptionfactors
•Conventional and transcriptome-based analyses have revealed that
dozens of transcription factors (TFs) are involved in plant response to
drought stress
•Most TFsfall into large gene families like AP2/ERF, bZIP, NAC, MYB,
MYC, Cys2His2zinc-finger and WRKY
•TFsregulates downstream genes which more directly act on drought
response
•Two important regulation pathways exist, one ABA-indipendent
(CBF/DREB TFsacting on genes carrying the CRT/DRE –C-
repeat/dehydration responsive -elements) and one ABA-dependent
(ABF/AREB TFs, acting on genes carrying the ABRE element).
•Genes with both (and other) cis-elements exist!
Transcriptional regulatory networks (cis-acting elements and transcription
factors) involved in osmotic and cold-stress responsiveness in Arabidopsis
Mod. from: Yamaguchi-Shinozaki and Shinozaki, 2006, Ann Rev Plant Biol
= TF
= cis-element
(Drought and salinity)
Transcriptional regulatory networks (cis-acting elements and transcription
factors) involved in osmotic and cold-stress responsiveness in Arabidopsis
Yamaguchi-Shinozaki and Shinozaki, 2006, Ann Rev Plant Biol
Engineeringdroughttolerance
usingtranscriptionfactors(Table1)
Engineeringdroughttolerance
usingtranscriptionfactors(Table2)
•CBF3/DREB1Aand ABF3are two ArabidopsisTFsrelated to the ABA-
independent and ABA-dependent pathways, respectively
•CBF3enhances tolerance to cold and other abioticstresses and the
expression of target genes (corgenes, rd29a, etc) involved in cold
responses
•ABF3enhances tolerance to drought and expression of target genes
(LEA genes rd29band rab18; protein phosphatase, ecc) involved in
drought responses
Aim
To test the expression of ArabidopsisCBF3and ABF3under constitutive
promoter in rice
Matrix attachment regions
from chicken lysozymegene
Maize ubiquitin1promoter and
its first 5’ UTR (leader) intron
CBF3(or ABF3)coding
sequences from Arabidopsis
3’ UTR from potato
proteinaseinhibitor II
Selection marker gene
•Agrobacterium-mediatedtransformation
+5 day+7 day
RNA from Ubi1:CBF3, Ubi1:ABF3
and control, 14-days seedling
58,417 known
or predicted
ORFs
2 slides
70-mer oligos
•Ubi1:CBF3activated 12 genes
•Ubi1:ABF3activated 7 genes
•3 genes (Hsp70, PP2Caand a receptor kinasegene) were activated by
both
•The 12 CBF3-activate genes had one ore more DRE elements while the 7
ABF3-activatedgenes had one ore more ABRE element in their promoters
•CBF3over-expression improved tolerance to drought and salinity and only
marginally to cold
The low effect on cold correlates with the low number of regulated
genes in rice (overexpressionof CBF3: 38 genes activated in
Arabidopsis vs12 genes in rice)
This could be due to i) lack of appropriated target genes and/orii)
Arabidopsis CBF3does not efficiently recognize rice promoters
•ABF3over-expression improved tolerance to drought only
•Lack of negative or pleiotropiceffects is probably due to low number of
genes which are altered by Ubi:CBF3 and Ubi:ABF3
Genetic engineering of maize using the rice transcription factorOsMyb4 for
adaptation to abioticstresses
Natoli, Tuberosa, Coraggioet al., in preparation
•OsMyb4is expressed in rice following cold stress and is able to activate the
expression of downstream genes enhancing cold stress tolerance in Arabidopsis
•Constitutive expression of OsMyb4in Arabidopsisresulted in improved cold
tolerance and variable dwarf phenotypes
•Aim:
to test the effect on cold and drought stresses of regulated andectopicexpression
of OsMyb4in maize
DRE
ABRE
MYB4NOS-t
UBI1-P
MYB4NOS-t
UBI1-P
BARNOS-t
UBI1-P
BARNOS-t
•T4 plants homozygous for transgenic events were identified usingPCR.
•Drought treatment:
OsMyb4 +/+ and the isogenic-/-were tested. At 3-leaf stage, water was
withheld until 60% RWC (-/-) was reached (+/+ reached ca. 80% RWC).
+/+-/-A188
+/+
-/-
A188
Genetic engineering of maize using the rice transcription factorOsMyb4 for
adaptation to abioticstresses
Natoli, Tuberosa, Coraggioet al., in preparation
•No regenerantswere obtained when OsMyb4was driven
by a constitutive (Ubi) promoter
•OsMyb4expression under the control of an inducible (cold
and ABA responsive element) promoter conferred drought
and cold tolerance in maize
•Absence of negative pleiotropiceffects on phenotype when
plants were grown in well-watered and mild drought-stress
conditions
Genetic engineering of maize using the rice transcription factorOsMyb4 for
adaptation to abioticstresses
Natoli, Tuberosa, Coraggioet al., in preparation
Engineeringdroughttolerance
usingsignalingfactors
Wang et al., 2005, Plant J 43, 416-424
•The ERA1mutation in Arabidopsis increases sensitivity to ABA (tight
closure of guard cells with reduced wilting under drought stress) but has
severe pleiotropicphenotypes
•ERA1encodes farnesyltransferaseb-subunity(Farnesilationis a type of
regulative protein modification where a lipid chain -15 C-is attached to
the aachain)
•A negative regulator of guard cell sensitivity to ABA signallingmust be
farnesylatedto modulate ABA response
•Aim: to place under control such regulative function in order toenhance
a positive response to drought in Arabidopsisand Brassicanapus
Wang et al., 2005, Plant J 43, 416-424
Open stomataClose stomata
ABA signal
Farnesyltransferase
(AtFTB)
negative regulator (unknown)
Rd29:anti-AtFTB
Wang et al., 2005, Plant J 43, 416-424
Engineeringof stomata
Itwouldbebeneficialtoagricultureforcropplantstoshow widestomatalopeningfor
CO2 intakewhenwater isavailable, buttoclosestomataduringdroughtperiods,
therebyslowingdesiccationand damage. However, conventionalhigh-yield
breedingapproachesmayhavecontributedtoselectionof cropplantswithreduced
stomatalABA responsiveness, becausegenescontrollingguardcellsignallingare
alsoexpressedin othertissuesand control otheryieldparameters.
(Schroederetal, Guard cell abscisicacid signallingand engineering drought hardiness in plants, Nature, 2001)
Aim:
tooptimizethe balancebetweenstomatalCO2 influxand water effluxin orderto
providethe best droughttolerance
Strategy:
Geneticengineeringtocontrol guardcellmovements
Possibleavenues:
•Toimproveguardcellsensitivitytobothincreaseand decreaseof water availability
•Tomodifyguardcellsensitivityin ordertomeetcrops’and environments’
specificities
•AtMYB60isa R2R3-MYB gene specificiallyexpressedin guard
celland down regulatedupondroughtstress
•A nullmutation(atmyb60-1) showeda constitutivereductionof
stomataopeningand decreasedwiltingunder droughtstress
•the atmyb60-1 mutationresultsin guard-cell-specificdefects
withoutaffectingotherdevelopmentaland physiological
processes
Laporteetal.J ExpBot(2002) 53, 699
•High concentrationof ionsincreasesturgor
pressureof guardcellsand increasedstomatal
poresize
•Ionsaccumulatedin guardcellsduringstomatal
openinginclude K, Cl and malate
•In transgenicplantsexpressinga NADP malic
enzyme, malate concentrationwasdecreased
•Transgenicplantshaddecreasedstomatal
conductanceand gainedmore freshmass per unit
water consumedwhiletheyweresimilartowild
typein theirgrowthrate and development
Relevant references
Reviews
•Bartels D, SunkarR: Drought and salt tolerance in plants. CritRev Plant Sci2005, 24:23-58.
•Schroeder JI, KwakJM, Allen GJ: Guard cell abscisicacid signalling and engineering drought hardiness in plants. Nature 2001,
410:327-330.
•UmezawaT, Fujita M, Fujita Y, Yamaguchi-Shinozaki K, Shinozaki K. Engineering drought tolerance in plants: discovering and
tailoring genes to unlock the future. CurrOpinBiotechnol. 2006 Apr;17(2):113-22.
•Yamaguchi-Shinozaki K, Shinozaki K. Transcriptional regulatory networks incellular responses and tolerance to dehydration
and cold stresses. AnnuRev Plant Biol. 2006 Jun 2;57:781-803.
Research papers
•Abede, T., Guenzi, A. C., Martin, B., and Cushman, J. C. 2003. Tolerance of mannitol-accumulating transgenic wheat to water stress and
salinity. Plant Physiol. 131: 1748–1755.
•Chandra BabuR, JingxianZ, BlumcA, David HodT-H, WueR, NguyenfHT: HVA1, a LEA gene from barley confers dehydration tolerance in
transgenic rice (OryzasativaL.) via cell membrane protection. Plant Sci2004, 166:855-862.
•Clough SJ, Bent A (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thali-ana. Plant J 16:735-
743
•CominelliE, GalbiatiM, VavasseurA, Conti L, SalaT, VuylstekeM, LeonhardtN, DellaportaSL, TonelliC: A guard-cell-specific MYB
transcription factor regulates stomatalmovements and plant drought tolerance. CurrBiol2005, 15:1196-1200.
•IuchiS, Kobayashi M, TajiT, NaramotoM, Seki M, Kato T, TabataS, KakubariY, Yamaguchi-Shinozaki K, Shinozaki K: Regulation of
drought tolerance by gene manipulation of 9-cis-epoxycarotenoid dioxygenase, a key enzyme in abscisicacid biosynthesis in Arabidopsis.
Plant J 2001, 27:325-333.
•LaporteMM, ShenB, TarczynskiMC: Engineering for drought avoidance: expression of maize NADP-malicenzyme in tobacco results in
altered stomatalfunction. J Exp Bot2002, 53:699-705.
•Oh SJ, Song SI, Kim YS, Jang HJ, Kim SY, Kim M, Kim YK, NahmBH, Kim JK: Arabidopsis CBF3/DREB1A and ABF3 in transgenic rice
increased tolerance to abioticstress without stunting growth. Plant Physiol2005, 138:341-351.
•Park S, Li J, Pittman JK, Berkowitz GA, Yang H, UndurragaS, Morris J, HirschiKD, GaxiolaRA. Up-regulation of a H+-pyrophosphatase
(H+-PPase) as a strategy to engineer drought-resistant crop plants. Proc NatlAcadSciU S A. 2005 Dec 27;102(52):18830-5.
•UmezawaT, Okamoto M, Kushiro T, NambaraE, OonoY, Seki M, Kobayashi M, KoshibaT, KamiyaY, Shinozaki K: CYP707A3, a major
ABA 80-hydroxylase involved in dehydration and rehydrationresponse in Arabidopsis thaliana. Plant J 2006:46,171-182
•Wang Y, Ying J, KuzmaM, ChalifouxM, Sample A, McArthur C, UchaczT, SarvasC, Wan J, Dennis DT et al.: Molecular tailoring of
farnesylationfor plant drought tolerance and yield protection. Plant J 2005,43:413-424.
•Zhu, B. C., Su, J., Chan, M. C., Verma, D. P. S., Fan, Y. L., and Wu, R. 1998. Over-expression of a -pyrroline-5-carboxylate synthetasegene
and analysis of tolerance to water-stress and salt-stress in transgenic rice. Plant Sci. 139:41–48.