Patent Application: US-201013148655-A

Abstract:
the present invention relates to the use of trehalase to obtain drought and / or salt resistance in plants . more specifically , it relates to genetically modified constructs , transformed into plants and resulting in overexpression of trehalase , whereby the transformed plants show a significantly better drought resistance during the drought period , and a better recovery when water is supplied after the drought period .

Description:
table i plant lines name ecotype source sa tre ox ( salk_151791 ) colombia abrc * gt tre ko ( gt_16843 ) landsberg erecta cshl * * from the ordered lines , homozygous plants were obtained in this study table iv primers to check insertion in arabidopsis mutants seq id primer sequence mutant line no : lba1 tggttcacgtagtgggccatcg salk 13 ds3 ccgtcccgcaagttaaatatg gt 14 attrelp1 gcctttttcaaactcaagttgcac sa tre ox 15 attrerp1 gagtttattatgtgtgtgcgtgga sa tre ox 16 attrelp2 actcatctccacgcacacac gt tre ko 17 attrerp2 aatctcgaaccgggaatgat gt tre ko 18 abbreviations : sa ( salk lines ); gt ( genetrap line ), lp ( left primer ), and rp ( right primer ). trehalase cds was cloned ( source material : siliques and flowers of wild - type arabidopsis thaliana ecotype colombia ) in plant vector pgfp ( kovtun et al ., 1998 ; 2000 ; hwang and sheen , 2001 ). pgfp = puc18 modified for gfp fusion . cds sequence was blasted against tair arabidopsis genome initiative coding sequence datasets ( www . arabidopsis . org ) and mutations , leading to amino acid changes , were corrected by site - directed mutagenesis ( sdm ). transgenic and wild - type arabidopsis , ecotype col and ecotype ler seeds were vapor - phase sterilized according to a protocol adapted from ye et al . ( 1999 ). seeds were placed in open micro - centrifuge tubes and put inside a desiccator jar , where bleach - chlorine fumes sterilize the seeds for a period of 3 hours - 9 hours ( bleach + concentrated hcl ). sterile water is added afterwards working in laminar flow , followed by a 48 - hour cold treatment under constant light for imbibition and stratification . vapor - sterilized seeds were sown , after a 48 - hour stratification period , on wet soil and germinated for two days in high humidity . in general , they were grown in 12 hours - 12 hours , 16 hours - 8 hours day / night cycle or continuous light ( 21 ° c ., 60 % humidity , 100 μmol s − 1 m − 2 ). vapor - sterilized seeds were sown after a 48 - hour stratification period in vertically or horizontally oriented petri dishes on a full strength ( or mentioned otherwise ) murashige and skoog salt mixture including vitamins ( ms ) medium ( 4 . 3 g / l ) with mes ( 0 . 5 g / l ) and solidified with phytagar ( 8 g / l ) ( duchefa , haarlem , the netherlands ). the media was enriched with sucrose / glucose ( 0 - 6 %) or trehalose , depending on the tested conditions , which are specified . val a was added in a concentration of 10 μm . for homozygous plant selection , salk lines and gt lines ( with the ntpii marker gene as selection marker ) were screened on 1 × ms + kanamycin ( 50 μg / ml , sigma - aldrich ). for growth on ms media with selective media , seeds were germinated for seven days on ms and antibiotics , positives were transferred to soil to flower and to set seeds . plant samples ( seedlings ) were harvested and immediately frozen in liquid nitrogen . rna extraction of plant material was performed with trizol reagent ( invitrogen ), according to the manufacturer &# 39 ; s instructions . for each sample , reverse transcription ( rt ) was performed using 1 μg of total rna ( reverse transcription system , promega ). after grinding a medium size leaf with screwdriver - pestle in 100 μl tps buffer ( 100 mm tris - hcl , ph 9 . 5 , 1 m kcl , 10 mm edta ), the samples were incubated at 70 ° c . for 20 - 30 minutes . the debris was pelleted by centrifugation at top speed for 10 minutes at rt and supernatans transferred to a new eppendorf tube . for further pcr reactions , 0 . 5 to 1 μl supernatans was used in a 20 - 50 μl reaction . pcr reactions were performed using different polymerase kits , according to manufacturer &# 39 ; s recommendations ; high - fidelity taq polymerases ( roche ), phusion high - fidelity dna polymerase ( finnzymes ) and ultra pfu hf dna polymerase ( stratagene ) for cloning and extaq ( takara ) for diagnostic pcr . equal amount of pci was added to the pcr product ( 45 μl - 45 μl ), vortex , and centrifuged at maximum speed , for 5 minutes . to the transferred supernatans , naac 3m ( 3 . 6 μl ) and ice - cold pure ethanol ( 109 μl ) was added , vortex , and spinned down for 1 minute at maximum speed . after additional wash with ice - cold 80 % ethanol , the pellet was dried out and dissolved in 12 . 5 μl h 2 o . pcr products could also be purified with using the wizard sv gel and pcr clean - up system ( promega , usa ), according to manufacturer &# 39 ; s guidelines . pcr was performed on cdna samples equally diluted to 250 ng / μl and a control gene was used as control . the pcr product was run on 1 % agarose gel and visualized with sybr safe dna gel stain ( invitrogen ). 25 μl real - time pcr reaction was composed of 12 . 5 μl platinum sybr green qpcr supermix - udg ( invitrogen ), 0 . 5 μl of rox reference dye , 1 . 25 μl of each primer of 500 nm stock solution and 5 μl of cdna mix ( 100 ng / μl ). the pcr program comprised an initial denaturation of 2 minutes at 95 ° c ., amplification by 50 cycles of 15 seconds at 95 ° c ., 1 minute at 58 ° c . the expression levels were analyzed with abi prism and were all normalized to actin2 expression . site - directed mutagenesis ( sdm ) was performed according to the stratagene &# 39 ; s quickchange site - directed mutagenesis kit . as such , point mutations , insertions and deletions were created in a plasmid with ultra pfu hf dna polymerase ( stratagene ). three to four week - old arabidopsis plants ( in soil , 12 hours - 12 hours day / night cycle , under low light , 50 - 75 μmol m − 2 s − 1 ) with healthy , unstressed , and well - expanded true leaves were used . this technique is detailed described by yoo et al . ( 2007 ) ( genetics . mgh . harvard . edu / sheenweb / protocols_reg . html ). briefly , after the removal of cell walls , protoplasts are released , washed and collected . dna - peg4000 ( platelets , fluka )- calcium transfection is performed using 2 × 10 4 protoplasts per 10 - 20 μg of endotoxin - free ( maxiprep ) plasmid dna . the transfected protoplasts are incubated for 6 hours in the light before harvesting . subcellular localization in mesophyll protoplast cells of the protein of interest can be determined by fluorescence microscopic analysis , using the gfp - tagged vectors . crude leaf material ( 300 mg , mix of plant material / different plants ) was homogenized and powdered on dry ice , suspended in 2 ml ice - cold extraction buffer ( 0 . 1 m mes / k + , ph 6 ; 1 mm pmsf 2 mm edta , 10 mg / gfw insoluble pvp , polyvinylpyrrolidone ). the suspension was vortexed and centrifuged ( 13000 rpm , cold , 10 minutes ). the supernatant was dialyzed ( brl microdialysis system ) on at 4 ° c . against mes buffer ph 7 with 50 μl cacl 2 . trehalase activity was determined by a method described by pernambuco et al . ( 1996 ) adapted to microtiter plates . 10 μl dialyzed sample was added to 50 μl trehalose buffer ( 250 mm trehalose , 25 mm mes , ph 7 , 50 μl cacl 2 ). the microtiter plate was incubated for 30 minutes at 30 ° c . and reaction was stopped by boiling 5 minutes at 95 ° c . warm waterbath . glucose was determined using glucose oxidase - peroxidase method by adding 200 μl god - pap ( dialab ) ( blancs : residual glucose content in each sample determined by boiling samples before trehalose addition ). a505 was measured after 15 minutes of incubation at 30 ° c . the amount of protein was determined using lowry method with bsa as standard ( lowry et al ., 1951 ). the transposon insertion genetrap ( gt ) line gt 16843 ( a . thaliana , ecotype ler ), was originally produced in the martienssen &# 39 ; s cold spring harbor laboratory ( sundaresan et al ., 1995 ; martienssen , 1998 ). based on the flanking sequences of the insertion site obtained by tail - pcr ( liu et al ., 1995 ), line gt — 16843 was predicted to carry a unique insertion of a gt transposable ds element , 395 by downstream of the atg start codon , in the middle of the first exon of the attre1 gene ( fig1 , panel a ). t3 descendants showed 3 : 1 ( resistant - susceptible ) segregation on kan selection media , consistent with a single insertion . homozygous lines were obtained and confirmed by pcr ( ds3 , attrelp2 - rp2 primer set ) and the cshl predicted insertion site was confirmed . semi - quantitative rt - pcr ( primer set attrep1fw - rv ) and real - time pcr ( primer set attrep2fw - rv , fig1 , panel b ) revealed strong down - regulation of attre1 in the homozygous gt — 16843 plants , hereinafter designed as “ gt tre ko ” plants . the salk — 151791 line ( a . thaliana , ecotype col ) ( fig1 , panel a ), originated from the salk institute ( alonso et al ., 2003 ), was available from the arabidopsis biological resource center ( abrc ), and ordered via nasc . from the ordered seeds , we selected plants , homozygous for a single t - dna insertion in the attre1 gdna . after kan selection , plants were checked for homozygousity with primers lbal and attrelp1 - rp1 primer set ( designed with signal ). flanking t - dna insertion sequences were obtained 304 bp upstream of the start codon ( tair predicted 211 bp in front of the atg start codon ). surprisingly , semi - quantitative rt - pcr ( attrep1fw - rv ) revealed higher attre1 expression in the mutants , suggesting ox instead of ko . this was confirmed by real time - pcr ( fig1 , panel c , homozygous salk — 151791 plants are hereinafter designed as sa tre ox ). closer inspection of the left border of the t - dna reveals a 35s camv promoter , which might be responsible for this overexpression . also , insertion of the t - dna in an important region for transcription regulation ( e . g ., a repressor binding site ) is possible . measurements of trehalase activity in different plant tissues demonstrated that sa tre ox plants exhibited much higher trehalase activity than control wild - type plants . for example , up to 25 times more trehalase activity was observed in adult leaves of tre ox , compared to wild - type col leaves ( fig2 , panel a ). in contrast , very low to no detectable trehalase activity was measured in the leaves and siliques ( fig2 , panel b ) of the gt tre ko plants compared to the control plants . trehalose causes dramatic effects on plant metabolism , growth and development . validamycin a ( val a ) specifically inhibits trehalase activity and prohibits the use of trehalose as an extra source of glucose . it is worth noting that , while val a treatment alters adult plant stress and reproduction responses and even causes phytotoxicity , no phenotypic irregularities are observed in seedlings ( wingler et al ., 2000 ; müller et al ., 2001 ; ishikawa et al ., 2005 ; 2007 ; ramon et al ., 2007 ). in our growth assay , no extra sugar was added to the media . as known from literature , gradually increased levels of trehalose in the presence of val a , cause increased inhibition of root growth and development of wild - type seedlings ( fig3 , panels a , b ). the sensitivity to trehalose differed between the ecotypes ler and col , with a rather gradual inhibitory effect in ler wild - type plants , and a more dramatic decrease in root length of col wild - type plants at 10 mm trehalose . gt tre ko lines exhibited similar growth inhibition on trehalose as wild - type ler seedlings in the presence of val a ( fig3 , panel a ), consistent with lack of trehalase activity in both plants . in contrast , sa tre ox plants were significantly more resistant to higher trehalose levels than wild - type controls in the presence of val a ( fig3 , panel b ), suggesting that the amount of val a ( 10 μm ) is not sufficient to inhibit increased trehalase activity in the ox lines . in the absence of val a , external supplied trehalose can be used as an extra glucose source for growth . on high trehalose concentrations , wild - type seedlings arrest with development shortly after germination , show altered cotyledons extension , and develop only short primary roots ( wingler et al ., 2000 ). interestingly , at low trehalose contents , gt tre ko seedlings do not seem to be able to use the extra glucose source for growth ( fig3 , panel c ), because of total lack of trehalase activity . moreover , with increasing trehalose concentrations , ko mutants are significantly more affected in development , compared to wild - type ler controls ( fig3 , panel c , fig4 ). in contrast , sa tre ox plants seem to be insensitive to high trehalose concentrations compared to wild - type seedlings ( fig3 , panel d , fig4 ). mutants in trehalose metabolism have been reported to exhibit sensitivity to supplemented sugars , and t6p has been highlighted as an important regulator for carbohydrate utilization ( schluepmann et al ., 2003 ; avonce et al ., 2004 ). when grown on gradually increasing sugar concentrations , sa tre ox seedlings exhibit similar leaf and hypocotyl development as control seedlings ( horizontally orientated plates , 16 hours - 8 hours day / night cycle ( fig5 , panel a ). however , root growth of sa tre ox seedlings was significantly affected , with reduced length and increased root branching . this aberrant phenotype was also observed on vertically oriented plates in continuous light with 1 % sucrose ( fig5 , panel c ). however , the mutants displayed a somewhat accelerated root growth on media without sugars . all together , it seems that overexpressing trehalase allows seedlings to efficiently use carbon supplies in low energy conditions , but makes them unable to deal with high energy conditions . in contrast , a slightly longer root growth of gt tre ko could be observed when more energy was available ( fig5 , panels b , d ), and interestingly , gt tre ko plants seemed to germinate slower in media without sucrose . plants engineered in trehalose metabolism typically exhibit dramatic phenotypic irregularities , with altered physiology and morphology . in addition , val a treatment can alter plant reproduction and can even cause phytotoxicity ( müller et al ., 1995 ; müller et al ., 2001 ; ishikawa et al ., 2005 ; 2007 ). however , to our surprise , altering endogenous trehalase activities did not seem to cause severe phenotypic irregularities in arabidopsis . we did notice a slower germination rate of gt tre ko seeds in soil in a 12 hours - 12 hours day / night cycle , and this phenotype was abrogated when increasing the light period to 16 hours . moreover , in a 12 hours - 12 hours day / night cycle , leaves of the gt tre ko plants were smaller ( fig6 , panel a ), and when increasing the day length , the growth rate of these ko plants increased ( fig6 , panel b , growth stages according to boyes et al . ( 2001 ), 1 . 10 for wt and 1 . 11 for ko ). this observation again suggests that growth of plants , altered in trehalase activity , strongly depends on the available energy . gt tre ko plants seem to have problems to cope with low energy conditions , whereas higher growth rates are achieved when more energy is available , e . g ., through longer photosynthesis or supplied sugars . in contrast , sa tre ox plants grow slightly better than controls in low carbohydrate conditions ( fig6 , panel c , e . g ., rosette leaf nr 9 ), while at a 16 - hour - 8 - hour day / night cycle , no obvious growth phenotype was observed ( fig6 , panel d ). interestingly , gt tre ko plants started flowering earlier than wild - types ( 16 hours - 8 hours day / night cycle ) ( fig7 , panel a ), displayed longer inflorescence stems , and altered silique orientation ( fig7 , panels b and d ). stems of sa tre ox plants ( fig7 , panel c ) appeared somewhat smaller , but no obvious other phenotypes could be observed . given trehalose - modified plants performed better in multiple stress treatments , we tested trehalase ox and ko plants under variable stress conditions . when performing heat - stress during germinating ( 33 ° c ., continuous low light , 1 × ms , 1 % suc ), we noticed a better early seedling development of sa tre ox seeds , compared to wild - type seeds , in contrast to gt tre ko lines ( fig8 ). these results , together with ample circumstantial evidence of pivotal roles of trehalose metabolism in abiotic stress responses , give us incentives to look closer to more environmental stresses such as drought and dehydration stresses in the vegetative stage . four - week - old ko and ox plants grown in a 16 - hour / 8 - hour day / night cycle ( in soil with controlled watering regime ), were subjected to drought stress for two weeks and then rewatered . in contrast to the performances of gt tre ko lines , the sa tre ox plants withstand and recovered much better the dehydration stress ( fig9 ). given trehalase controls extracellular trehalose levels , but seems also to regulate plant trehalose ( sugar ) metabolism , we analyzed the fluorescence of attre1 - gfp constructs in arabidopsis mesophyll protoplasts , to gain more insight in the exact location of trehalase . attre1 seemed to be mainly localized in the plasma - membrane ( fig1 ). this result was recently confirmed and published by frison and coworkers ( 2007 ). the expression was further analyzed by fusing the trehalase promoter to a gus / gfp construct and analyzing the plants after gus staining . in leafs and leaf primordia , a clear specificity of the expression is seen in the stomatal guard cells ( fig1 a and 11b ); expression could also be detected in the flowers ( in stomata in sepals of inflorescence , stomata in pedicel of inflorescence , petal veins and chalaza in siliques ) and in the root tips . to analyze further the effect of modulation of trehalase expression on stomata , mutants of attre1 were grown in soil under normal moist conditions in a growth room . 21 days after sowing , leaf 1 or leaf 2 were cut out of the plant and submerged for the abaxial side in aperture buffer ( 10 mm mes , 10 mm cacl2 , 10 mm kcl , ph 6 . 1 ) under light conditions , or closure buffer ( 10 mm mes , 10 mm cacl2 , ph 6 . 1 ) under dark conditions . after two hours , leaves were submerged in aperture buffer containing 20 um aba under light conditions for 2 hours , the abaxial epidermis was mounted on a slide with double - sided tape and stomata aperture measurements ( width ) were recorded under a microscope , n = 50 . trehalase overexpression plants showed a decrease in opening of stomata in aperture buffer , whereas in closure buffer , no significant difference between the wild - types , overexpression of ko plant could be detected ( fig1 ). to analyze the effect of trehalase expression on number of stomata , mutants of attre1 were grown on ½ ms , 1 % sucrose horizontal plates , under 16 hours light / 8 hours dark regime for 21 days . leaf 1 or leaf 2 were cut off and cleared in lactic acid for 2 days under constant shaking . leaves were put on a slide and pictures ( 0 . 142 mm 2 ) of 2 areas ( tip and base ) of each leaf were taken to count stomata and pavement cells , n = 10 and to calculate the stomatal index . the stomatal index corresponds to the ratio of stomata per total number of cells in the epidermis (# stomata /( 2 ×# stomata +# pavement cells ). compared with their respective wild - types , there is a slight decrease in the number of stomata ( fig1 a ) and in stomatal index ( fig1 ). whereas , for the overexpressing plant no influence on the number of pavement cells was seen , the ko showed a small increase in pavement cell number ( fig1 b ). the results indicate that the trehalase modulates drought resistance by modulating the opening of the stomatal guard cells , and / or the number of stomatal guard cells per surface unit and / or the stomatal index . alonso j . m ., a . n . stepanova , t . j . leisse , c . j . kim , h . chen , p . shinn , d . k . stevenson , j . zimmerman , p . barajas , r . cheuk , c . gadrinab , c . heller , a . jeske , e . koesema , c . c . meyers , h . parker , l . prednis , y . ansari , n . choy , h . deen , m . geralt , n . hazari , e . horn , m . karnes , c . mulholland , r . ndubaku , i . schmidt , p . guzman , l . aguilar - 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