Patent Application: US-12845602-A

Abstract:
the present invention concerns a transgenic plant with enhanced tolerance to reduced water availability . the plant includes an expression cassette including an abscisic acid responsive element binding factor , which is capable of expressing the recombinant abf . a method to improve plant &# 39 ; s tolerance to reduced water availability is also provided .

Description:
in order to investigate the in vivo functions of abf3 and abf4 , we employed an overexpression approach . the coding region of abf3 or abf4 was fused to the cauliflower mosaic virus 35s promoter and each construct was used to transform arabidopsis ( ecotype ler ) plants . thirtyeight and twelve t3 homozygous lines were recovered from the 35s - abf3 and the 35s - abf4 constructs , respectively , and , after preliminary analysis , transgenic lines with higher abf expression levels were selected for more detailed analysis . compared with wild type plants , 35s - abf3 transgenic plants exhibited mild growth retardation in the aerial parts ; petioles were slightly shorter and leaves were rounder in shape ( fig1 a ). the degree of retardation was not severe , however , and overall growth patterns were similar to wild type plants except that siliques were somewhat shorter and thicker ( fig1 a , inset ). in contrast , 35s - abf4 transgenic plants exhibited severe growth retardation ( fig1 a ), which was dependent on the abf4 expression level ( fig1 b ). petioles were shorter , leaves were smaller , flowering was delayed , and plants were shorter . also , germination of 35s - abf3 plants was delayed several hours compared with that of wild type plants in the absence of aba ( fig1 c ). 35s - abf4 plants , on the other hand , germinated normally ( data not shown ), and , thus , the growth retardation observed with 35s - abf4 plants was a post - germination process . to test whether abf3 or abf4 overexpression affected aba sensitivity , 35s - abf3 and 35s - abf4 transgenic plants were germinated and grown on media containing various concentrations of aba . when aba concentration was 0 . 5 μm or above , the growth of 35s - abf plants was completely arrested after radicles emerged , i . e ., cotyledon greening / expansion and root growth were severely inhibited and none of the transgenic seedlings developed to have true leaves ( fig2 a ). at the same conditions , wild type plants continued to grow and develop although at slower rates than on aba - free medium . the aba hypersensitivity of 35s - abf transgenic plants was also observed at 0 . 25 μm aba ( fig2 b ), although the transgenic seedlings eventually grew to have true leaves ( data not shown ). to see the stage - specificity of aba response , aba dose - response was examined during and after germination . as shown in fig2 c , 35s - abf transgenic plants were hypersensitive to aba at the germination stage , 0 . 5 μm aba being sufficient to inhibit their germination efficiencies to 8 ( abf3 ) or 28 % ( abf4 ) while wild type plants retained 80 % germination at the same condition . likewise , root growth of the 35s - abf transgenic lines was hypersensitive to aba ( fig2 d ). at 0 . 5 μm aba , wild type root growth was 84 % of its control rate , whereas those of 35s - abf4 and 35s - abf3 plants were 35 % and 51 % of their control rates , respectively . at 1 μm aba , root growth of the 35s - abf4 plants reduced to 6 % and the 35s - abf3 plants to 37 %. in addition , lateral root and aerial part growth of 35s - abf transgenic plants was significantly inhibited at this concentration ( data not shown ). wild type plants grew at 62 % of the control rate at the same aba concentration . transgenic root growth was almost completely arrested when aba concentration was above 5 μm , whereas wild type plants still continued to grow . the results indicate that both germination and post germination growth of 35s - abf transgenic plants are hypersensitive to aba . high concentrations of salts inhibit the germination of arabidopsis ( werner and finkelstein , 1995 ; leon - kloosterziel et al ., 1996 ; quesada et al ., 2000 ; zhu , 2000 ). several studies show that aba plays a role in the inhibition process . although not all salt - insensitive mutants are aba - insensitive ( warner and finkelstein , 1995 ), all aba deficient ( aba ) and aba insensitive ( abi ) mutants exhibit salt - insensitivity during germination ( leon - kloosterziel et al ., 1996 ). this is probably because aba , whose level increases by high salt , promotes the inhibition process . since the expression of both abf3 and abf4 is salt - inducible ( choi et al ., 2000 ), they may participate in salt response , i . e ., salt - induced germination inhibition in this case . to test this , 35s - abf transgenic plants were germinated on media containing various concentrations of nacl . fig3 a shows that the germination of wild type and aba1 , abi1 and abi2 mutant plants was not affected by nacl below 100 mm . in contrast , germination and growth of the 35s - abf plants were significantly affected by 100 mm nacl ( fig3 a and b ); radicle emergence , root growth and cotyledon opening / expansion were severely inhibited . in a parallel experiment , the transgenic plants responded to kcl in a similar way to nacl ( fig3 b ). on the other hand , their response to the same concentration or twice the concentration ( data not shown ) of mannitol , which gives the same osmotic pressure , was normal . thus , in contrast to the salt - insensitive phenotype of aba deficient or aba insensitive mutants , abf3 and abf4 overexpression both resulted in salt hypersensitivity at the germination / young seedling stage , and the hypersensitivity appeared to be ionic rather than osmotic in nature . at higher concentrations , sugars inhibit the development of young seedlings , i . e ., they inhibit cotyledon greening / expansion and shoot growth ( jang et al ., 1997 ). according to works done by others , aba plays an essential role in the glucose or sucrose signal transduction ( arenas - huertero et al ., 2000 ; huijser et al ., 2000 ; laby et al ., 2000 ). for example , the aba - deficient , aba2 mutation is allelic to the sugar insensitive , sis4 mutation , and the glucose or sugar insensitive mutations , gin6 , sis5 and sun6 are allelic to the aba insensitive mutation abi4 . also , the studies show that other aba mutants and , to some degree , abi5 mutants are insensitive to glucose . thus , abf overexpression might have affected sugar sensitivity as well , if indeed abf3 and abf4 mediate aba signaling . we addressed this by examining their response to glucose , which exerts more severe growth inhibition than other sugars ( jang et al ., 1997 ). under our experimental conditions , wild type seedlings showed growth defects such as the inhibition of cotyledon greening and true leaf development when glucose concentration was above 4 % ( data not shown ). the aerial part growth of 35s - abf transgenic lines , on the other hand , was arrested completely at 3 % glucose , at which wild type plants developed fully ( fig3 c ). thus , 35s - abf transgenic plants were hypersensitive to glucose . this enhanced response of the transgenic plants was not observed with the same concentration of mannitol , which inhibited the growth of both wild type and 35s - abf transgenic plants significantly but similarly . thus , the hypersensitivity was glucose - specific rather than osmotic . recent genetic studies show that aba signaling pathways interact with those of ethylene . according to the studies , aba - mediated inhibition of germination is negatively regulated by ethylene , whereas ethylene signaling components are required for the aba inhibition of root growth ( beaudoin et al ., 2000 ; ghassemian et al ., 2000 ). the involvement of auxin in aba - dependent stress response has also been demonstrated ( vartanian et al ., 1994 ). when exposed to progressive drought , new lateral roots take a short and tuberized form . the process , known as ‘ drought rhizogenesis ’, is impaired not only in the aba deficient aba - 1 and aba - insensitive abi1 - 1 mutants but also in the auxin resistant axr1 - 3 mutant . to test whether the overexpression of abf3 or abf4 affected ethylene or auxin sensitivity , we compared the effects of the ethylene precursor 1 - aminocyclopropane - 1 - carboxylic acid ( acc ) and indole - 3 - acetic acid ( iaa ) on the growth of 35s - abf and wild type plants . we did not see any differences in their responses . however , leaves of 35s - abf4 plants have a tendency to have epinastic curvature when grown on plates ( fig4 a ), which is generally attributed to ethylene action under stress conditions ( jackson , 1997 ). also , roots of 35s - abf4 transgenic plants exhibited abnormality in the obstacle - touching response ( okada and shimura , 1990 ; simmons et al ., 1995 ). as shown in fig4 b , roots of wild type plants grow in a wavy pattern when grown on a hard agar surface at an inclined position . the wavy pattern of root growth , however , was significantly diminished in 35s - abf4 plants . the obstacle - touching response is impaired in several auxin resistant mutants , indicating that auxin signalling components are involved in the process ( okada and shimura , 1992 ). thus , the result implies that auxin signaling pathway ( s ) might have been perturbed in 35s - abf4 plants . taken together , our results suggest that abf4 overexpression might have affected some aspects of ethylene and auxin responses . one of the key aba - controlled processes is the stomatal closure under water stress conditions , which minimizes water loss through transpiration ( leung and giraudat , 1998 ). aba biosynthesis mutants and some of the aba response mutants ( i . e ., abi1 and abi2 ), therefore , are very susceptible to drought because of the impaired stomatal aperture regulation ( leung and giraudat , 1998 ; schroeder et al ., 2001 ). thus , abf3 and abf4 overexpression lines are expected to exhibit altered response to water deficit conditions if the factors are involved in aba / stress signaling . to address this , we examined the drought tolerance of 35s - abf plants . as shown in fig5 a , wild type plants withered completely when withdrawn from water for 11 days and only 16 % of them survived to maturity when re - watered afterwards . 35s - abf3 plants , however , were not affected noticeably and all survived the treatment to set seeds . in a similar experiment , 35s - abf4 plants also exhibited higher survival rates under water deficit condition ; all of them survived a 12 day drought treatment , whereas 33 % of the wild type plants survived to set seeds ( fig5 b ). thus , both 35s - abf3 and 35s - abf4 plants survived the drought conditions better than wild type plants . the enhanced drought tolerance of the transgenic plants could be attributed , at least in part , to their lower transpiration rates . when measured by the fresh weight loss of detached rosette leaves , the water loss rates of 35s - abf3 and 35s - abf4 transgenic lines were less than half and approximately 70 % of the wild type plants &# 39 ;, respectively ( fig5 c and d ). consistent with the result , the stomata of the transgenic plants had smaller openings than wild type plants ( fig5 e ). under normal growth condition , ca . 85 % ( 89 / 104 ) of the wild type stomata were open , whereas approximately 20 % of them were open in 35s - abf3 ( 15 / 71 ) and 35s - abf4 ( 17 / 71 ) plants when observed in the middle of watering period . thus , constitutive overexpression of abf3 or abf4 resulted in partial stomatal closure , reduced transpiration and enhanced drought tolerance . to investigate the transcriptional regulatory roles of abf3 and abf4 in planta , expression of various aba / stress responsive genes in 35s - abf plants was determined . as shown in fig6 the transcript levels of a number of aba - regulated genes ( group i ) were enhanced in 35s - abf3 and 35s - abf4 transgenic lines . these include lea class genes rd29b ( yamaguchi - shinozaki and shinozaki , 1994 ) and rab18 ( lang and palva , 1992 ), whose expression is induced by aba and abiotic stresses . expression of the aba - inducible , cell cycle regulator gene ick1 ( cyclin - dependent kinase inhibitor ) ( wang et al ., 1998 ) was also elevated in the 35s - abf transgenic lines . the ick1 gene has been suggested to mediate cell division arrest by aba . in the 35s - abf3 lines , strong enhancement of abi1 ( leung et al , 1994 ; meyer et al ., 1994 ) rna level was observed , and abi2 ( leung et al ., 1997 ) transcript level was elevated albeit the degree of increase was lower . increase in the abi1 rna level was also observed in the 35s - abf4 line with higher abf4 expression ( a405 ). expression of abi1 and abi2 , which encode homologous protein phosphatase 2cs and whose mutations result in defective stomatal closing and wilty phenotype ( schroeder et al ., 2001 ), is enhanced by aba and water stress ( leung et al ., 1997 ). meanwhile , the rna level of the aba - repressible gene skor ( gaymard et al ., 1998 ) was significantly reduced or undetectable in 35s - abf4 transgenic lines ( group ii ). the gene encodes a root - specific k + outward rectifying channel , and aba repression of its expression has been suggested to be a part of adaptive water stress response . similarly , guard cell ion channel genes kat1 and kat2 ( anderson et al ., 1992 ; pilot et al ., 2001 ) were negatively regulated in the 35s - abf3 line with higher abf3 level ( a319 ). the two ion channels normally mediate k + influx , enabling stomatal opening , but their activity is inhibited by aba . also , stress responsive biosynthetic genes ( group iii ), chalcone synthase gene chs ( feinbaum and ausubel , 1988 ) and alcohol dehydrogenase gene adh1 ( de bruxelles et al ., 1996 ), were down - regulated . the transcript levels of the two genes were , however , higher in the 35s - abf transgenic lines when plants were treated with high salt , suggesting that stress - induced post - translational modification of abf3 and abf4 may be required for the regulation of these genes . in summary , overexpression of abf3 or abf4 resulted in the modulation of aba / stress responsive gene expression and the two factors played positive or negative roles depending on specific genes and environmental conditions . the phenotypes described so far indicated that overexpression of abf3 or abf4 enhanced various aspects of aba response . to assess the physiological relevance of the results and to obtain further clues about their functions , we investigated their temporal and spatial expression patterns . we first investigated the tissue - specificity of their uninduced , basal expression . since the basal expression levels of abf3 and abf4 are very low ( choi et al ., 2000 ), we employed the coupled reverse transcription and polymerase chain reaction ( rt - pcr ) for the analysis . as shown in fig7 a , relatively higher expression of both abf3 and abf4 was detected in roots . also , lower abf3 expression was observed in flowers but not in leaves and siliques at the same condition . on the other hand , weak abf4 expression was detectable in leaves , flowers and siliques . more detailed temporal and spatial expression patterns of abf3 and abf4 were determined by histochemical gus staining of transgenic plants that harboured an abf promoter - gus reporter construct . with the abf3 promoter ( 2 . 1 kb ) construct , gus activity was undetectable in embryos , but it was observed in the emerging radicles at the germination stage ( fig7 b , a ) and in most of the vegetative tissues at later stages ( fig7 b , b and c ). roots were stained most strongly except the tip area , and petioles , leaf vascular tissues and guard cells exhibited relatively strong gus activity ( fig7 b , b - f ). emerging shoots and younger leaves ( fig7 b , b and c ), on the other hand , exhibited gus staining only after aba , salt or mannitol treatment ( fig7 b , d ). in mature plants , gus staining was detected also in anthers , stigma , and siliques ( abscission zone , replum , and funiculi ) ( fig7 b , g and h ). the abf4 promoter was active in embryos from green siliques ( fig7 c , a ), but its activity decreased as embryos became mature and was not detected in newly germinated seedlings except in some limited regions ( radicle tip , shoot meristem region , and cotyledon tips ) ( fig7 c , a ). at later stages , starting from the stage of fully - expanded cotyledons ( fig7 c , b ), abf4 promoter activity was observed in all vegetative tissues ( fig7 c , c - f ), and also in floral organs and siliques ( abscission zone , replum , and funiculi ) ( fig7 c , g and h ). the abf4 promoter was most active in roots , especially in the growing regions ( meristem , elongation zone and lateral root primordia ) ( fig7 c , e ), suggesting its role in growth regulation . also , it exhibited strong activity in petioles and guard cells ( fig7 c , c and f ). salt treatment of seedlings enhanced the abf4 promoter activity somewhat ( fig7 c , d ). aba - regulated gene expression plays a central role in aba signaling , and numerous aba / stress responsive genes are regulated by the ( c / t ) acgtggc - or cgcgtg - containing . abres . thus , identifying relevant transcription factors is critical for the delineation of aba signal transduction cascades . many studies show that aba signaling pathways are tissue - specific ( leung and giraudat , 1998 ; giraudat et al ., 1994 ), and several seed - specific aba signaling components ( abi3 , abi4 , and abi5 ) have been identified by genetic screens . abi3 and abi4 encode transcription factors ( giraudat et al ., 1992 ; finkelstein et al ., 1998 ), whose binding sites and immediate target genes are unknown . recently , abi5 has been shown to encode a bzip factor that belongs to a seed - specific subfamily of abf - related factors ( finkelstein and lynch , 2000 ; lopez - molina and chua , 2000 ) and its role in post germination developmental arrest has also been demonstrated ( lopez - molina et al ., 2001 ). however , abre binding factors whose major function is to mediate the aba signaling during vegetative growth have not been reported , although numerous bzip factors are known to interact with the abres in vitro ( foster et al ., 1994 ). abfs are unique among the abre binding bzip factors in that , unlike most of the other plant bzip factors , they can interact with both the g - box type and the cgcgtg - containing abres ( choi et al ., 2000 ). the broad binding specificity , together with their transactivation capability of an abre - containing reporter gene and the stress - inducibility of their expression , suggested that abfs have a potential to regulate a large number of aba / stress responsive genes and , thus , are likely to participate in stress responsive aba signaling . in order to address this question , we employed an overexpression approach . considering the potential functional redundancy of abfs and numerous other bzip factors interacting with abres ( foster et al ., 1994 ), the approach would be a better way than loss of function approaches such as antisense , knockout and rna interference . the overexpression of abfs ( we estimate that abf3 and abf4 levels in the 35s - abf transgenic lines used in our study range from approximately 2 to 10 folds of their aba - induced levels ), however , might have caused non - natural conditions . for example , genes that are not normally regulated by abfs might have been turned on or off . also , it may have affected the functions of other members of abfs or potentially other bzip factors , by titrating them out via non - natural heterdimerization . thus , the overexpression phenotypes need to be interpreted with caution , and their roles can be further confirmed by other experimental means . nevertheless , our results show that abf3 or abf4 overexpression conferred several aba - associated phenotypes such as aba hypersensitivity , sugar hypersensitivity and enhanced drought tolerance , with altered expression of aba / stress responsive genes . thus , our data provide a strong in vivo case for the involvement of abf3 and abf4 in stress responsive aba signaling . whereas the aba hypersensitivity conferred by abf3 or abf4 overexpression was very distinct and observed both at the germination and at later growth stages ( fig2 ), the overexpression effects on growth in the absence of exogenous aba were either moderate ( abf3 ) or developmental stage - dependent ( abf4 ) ( fig1 ). abf3 exerted inhibitory effect on both germination and seedling growth . however , the low degree of inhibition compared with that in the presence of exogenous aba suggests that abf3 alone is not sufficient for the inhibitory function . on the other hand , abf4 overexpression had little effect on germination but had a severe effect on seedling growth , suggesting that abf4 activity is developmentally modulated . alternatively , the result may indicate that aba inhibition of seedling growth is mediated by a mechanism that differs from the germination inhibition mechanism . also , the developmental stage - dependency of the abf4 effects implies that the growth retardation of 35s - abf4 plants result probably from the constitutive operation of a part of aba signal transduction cascades rather than from the pleiotropic effects of abf4 overexpression . except the varying degrees of growth retardation , neither 35s - abf4 nor 35s - abf3 plants did show abnormality in general development . thus , their overexpression affected growth rate , but not developmental processes . other aba - or stress - associated phenotypes of 35s - abf3 and 35s - abf4 transgenic plants include their hypersensitivities to salt and glucose , and 35s - abf4 plants exhibited additional phenotypes ( i . e ., epinasty of leaves and abnormal obstacle - touching response ) that can be related to altered ethylene or auxin response . the salt and glucose hypersensitivities may reflect the increased sensitivity to high osmolarity , since both high salt and high sugar accompany increase in osmolarity . however , 35s - abf plants responded normally to mannitol , indicating that osmotic sensitivity was not affected . thus , it appears that abf3 and abf4 are involved only in the non - osmotic branches of salt and glucose signaling pathways . the sugar - mediated developmental arrest is confined to a narrow window of developmental stages , approximately 2 days post germination , and is mediated by increased aba level via an abi4 - dependent signaling cascade ( gazzarrini and mccourt , 2001 ). also , it has been reported that aba mediates developmental arrest at the similar stage and that the inhibition process requires abi5 ( lopez - molina et al ., 2001 ), whose mutations result in weak sugar insensitivity . thus , our results suggest that abf3 and abf4 have overlapping functions with abi4 and abi5 in mediating the sugar - and aba - induced developmental arrest . this is particularly so in the case of abf3 , since the onset of its expression coincides with the early developmental stage ( fig7 b , a ). stomatal closure is a key aba - controlled process in coping with water deficit conditions . our data indicate that abf3 and abf4 are involved in this process . their overexpression resulted in lower transpiration and enhanced drought tolerance ( fig5 ), which are reminiscent of the phenotypes of the aba hypersensitive mutant era1 ( pei et al ., 1998 ). furthermore , the stomatal openings of 35s - abf transgenic plants were smaller than those of wild type plants , and altered expression of several genes involved in stomatal aperture regulation has been observed in the transgenic plants ( fig6 ). among the genes we investigated , abi1 was the most strongly affected , especially in abf3 overexpression lines . abi1 is known to be a negative regulator of aba signaling ( gosti et al ., 1999 ), although its expression is enhanced by aba and high osmolarity while reduced in the aba1 and abi1 mutants ( leung et al ., 1997 ). it is not clear , thus , whether the increased abi1 level played a positive or a negative role in the stomatal closing . whatever the abi1 &# 39 ; s role might be , our results indicate that abi1 expression is subject to abf3 regulation and that abf3 and abf4 overexpression affected the expression of genes involved in stomatal movement and / or guard cell aba signaling ( schroeder et al ., 2001 ), the net result being enhanced stomatal closure . the transcript level changes of aba responsive genes in 35s - abf transgenic lines demonstrate that abf3 and abf4 function as transcriptional regulators in planta . as shown in fig6 both positive and negative changes were observed depending on specific genes . the result suggests that different subsets of abf3 and abf4 target genes are regulated via different mechanisms . also , the negative regulation of some genes under normal growth condition but positive regulation of the same genes after salt treatment ( fig6 group iii ) suggest that stress - induced modification of abfs activities is required for the regulation of these genes . the modifying activity may be limiting under normal condition . thus , the negative regulation of some genes can be explained , for example , by the binding of transcriptionally inactive , unmodified abfs , whose proportion increases with higher abf levels . the modification may involve phosphorylation of abfs . involvement of kinase / phosphatases in aba / stress signalling has been well known ( leung and giraudat , 1998 ), and several phosphorylation sites are highly conserved among abfs ( choi et al ., 2000 ). more recently , uno et al . ( uno et al ., 2000 ) reported an aba - activated kinase activity in cultured cells that phosphorylates areb1 ( abf2 ) and areb2 ( abf4 ). alternatively , it may involve interaction with other regulatory proteins . the expression patterns of abf3 and abf4 were consistent with their functions suggested by their overexpression phenotypes . spatially , both promoters were most active in roots and guard cells , consistent with their roles during water stress response . also , abf3 , which exerted only minor growth inhibition , was weakly expressed in the growing tissues ( root tips , new shoots , and new leaves ), whereas abf4 was strongly expressed in the growing regions of roots ( meristem , elongation zone and lateral root primordia ). temporally , strong abf3 promoter activity was observed in the newly germinated seedlings , consistent with its more pronounced effect on the germination ( fig1 c and 2c ). on the other hand , the abf4 promoter exhibited major activity at the onset of seedling growth , in agreement with its severe effect on seedling growth . our results also show that both abf3 and abf4 might function during reproductive stages and seed abscission . both promoters exhibited strong activity in the abscission zone , replum and funiculi of siliques , and relatively strong activity was also detected in stigma and anthers . the isolation of multiple factors with similar binding activities but with different expression patterns suggested that the aba / stress signaling involving the abres is likely to be mediated by multiple factors ( choi et al ., 2000 ). our current results further support the observation . although their overexpression phenotypes and expression patterns were similar , abf3 and abf4 were different from each other in several respects . the details of the temporal and spatial expression patterns differed ( fig7 ). growth retardation , root growth inhibition and impaired stimulus - touching response were more prominent in abf4 transgenic lines . also , minor differences were observed at the molecular level . abi1 and abi2 expression levels were higher in 35s - abf3 transgenic lines , while down - regulation of skor expression was observed only in 35s - abf4 plants . the function of other abfs , abf1 and abf2 , remains to be determined , but their roles appear to be quite different from each other and also from abf3 and abf4 according to our preliminary data . thus , abre - dependent aba / stress signaling in vegetative tissues appears to be mediated by multiple factors with overlapping , but distinct functions . our data presented and discussed above indicate that abf3 and abf4 are involved in stress - responsive aba signaling . in particular , overexpression of abf3 and abf4 clearly improved the survival rate of arabidopsis plants under drought conditions . in our experiments , we used a constitutive promoter ( i . e , 35s promoter ). however , other promoters , which are tissue - specific or inducible , also can be employed to drive the expression of abf genes in transgenic plants . inducible promoters that are active only under stress conditions will be particularly effective in the case of abf4 , whose constitutive overexpression results in growth retardation . also , it will be possible to alter stress tolerance of plants by decreasing the expression levels of abfs or its homologs employing established technologies such as antisense , rna interference and knockdown . aba controls not only the drought response but also responses to other environmental stresses such as high salt ( shinozaki and yamaguchi - shinozaki , 1997 ), cold / freezing ( llorente et al , 2000 ), heat ( larkindale j , knight m r ., 2002 ) and wounding ( giraudat et al , 1994 ). thus , abfs can be used to develop transgenic plants that are more tolerant than untransformed plants to these multiple stresses . furthermore , the cis - regulatory elements which control stress - responsive gene expression and to which abfs bind are highly conserved among various plants , in both monocot and dicot plants . thus , overexpression of abfs in plants other than arabidopsis will also enhance their drought and other stress tolerance , and it will be possible to develop transgenic vegetable or cereal crop plants with enhanced stress tolerance by expressing abf genes in them . [ 0050 ] arabidopsis thaliana ecotype landsberg erecta ( ler ) was used in this study . aba1 - 1 , abi1 - 1 , and abi2 - 1 seeds ( koornneef et al ., 1982 , 1984 ) were obtained from the arabidopsis biological resource center at the ohio state university , and their phenotypes were confirmed before use . plants were grown at 22 ° c . under long day condition ( 16 hr light / 8 hr dark cycle ) aseptically or on soil . for soil growth , seeds were sown on 1 : 1 : 1 mixture of vermiculite , perlite and peat moss irrigated with 0 . 1 % hyponex , placed at 4 ° c . for 4 days in the dark to break residual dormancy , and transferred to normal growth conditions . unless otherwise stated , the plants were watered once a week . for aseptic growth , seeds were treated with 70 % ethanol for 5 min and then with 30 % household bleach for 5 min , washed 5 times with sterile water , and plated on ms medium ( murashige and skoog , 1962 ) solidified with 0 . 8 % phytoagar . the ms medium was supplemented with 1 % sucrose and , as described in the text , was also supplemented with aba , salts , glucose , or mannitol as needed . for germination test , seeds collected at the same or similar times were used . for root growth measurement , plants were germinated and grown at vertical position . the 35s promoter - abf coding region constructs ( 35s - abf3 and 35s - abf4 ) were prepared by replacing the β - glucuronidase ( gus ) coding region of pbi121 ( jefferson et al ., 1987 ) with the coding region of abf3 or abf4 . the gus sequence was removed after bamhi - saci digestion , and , after t4 dna polymerase treatment to remove the 3 ′ overhang , the remaining portion of pbi121 was ligated with the abf3 or the abf4 coding region , which was prepared by polymerase chain reaction ( pcr ) followed by bamhi digestion . the abf coding regions included their entire coding regions with the stop codons and a bamhi linker sequence was attached in front of the initiation codons for the cloning purpose . the abf promoter - gus reporter fusions were prepared by inserting 2 . 1 kb ( abf3 ) or 1 . 2 kb ( abf4 ) of their 5 ′ flanking sequences from the initiation codons in front of the gus reporter gene of pbi101 . 2 ( jefferson et al ., 1987 ). the promoter fragments were prepared by pcr using arabidopsis ( ecotype , columbia ) genomic dna as a template and primer sets , 5 ′- caaacttaccctgttgttgcaact - 3 ′ ( seq id no : 1 ) and 5 ′- ctagtctagaaggatcaagcttctggatatttac - 3 ′ ( seq id no : 2 ) for abf3 , and 5 ′- gatcaatttgaatttttgatatacatc - 3 ′ ( seq id no : 3 ) and 5 ′- ctagtctagattcaatgaaaacaaagcatccaag - 3 ′ ( seq id no : 4 ) for abf4 . the pcr fragments were digested with xbai and ligated with the pbi101 . 2 , which was prepared by hindiii digestion followed by klenow treatment and xbai digestion . dna manipulation was according to the standard procedures ( ausubel et al ., 1994 ; sambrook et al ., 1989 ), and the intactness of the abf coding regions and the junction sequences was confirmed by dna sequencing . transformation of arabidopsis was according to the vacuum infiltration method ( bechtold and pelletier , 1998 ), using a . tumefaciens strain gv3101 . for the phenotypic investigation , t3 or t4 homozygous lines were used . gus staining patterns were confirmed by observing at least 5 different transgenic lines and t3 homozygous lines were used for detailed analysis . rna was isolated by the method of chomczynski and mackey ( 1995 ), with a minor modification ( choi et al ., 2000 ). rna gel blot analysis and rt - pcr were performed as described ( choi et al ., 2000 ) with following modifications . for rna gel blot analysis , hybridization was performed at 65 ° c . in the rapid - hyb buffer from amersham pharmacia biotech . exposure time was 6 ( abf3 ) or 20 hr ( abf4 ). rt - pcr was performed using the access rt - pcr system from promega or the superscript ™ one - step rt - pcr system from gibco brl . each rt - pcr result was confirmed by several independent reactions , and free of dna contamination in rna preparations was confirmed by using primer sets spanning introns whenever possible . primers used in the rt - pcr reactions are presented in table 1 . in situ assay of gus activity was performed as described by jefferson et al . ( 1987 ). whole plants were immersed in 1 mm x - gluc ( 5 - bromo - 4 - chloro - 3 - indolyl - β - glucuronic acid ) solution in 100 mm sodium phosphate , ph 7 . 0 , 0 . 1 mm edta , 0 . 5 mm ferricyanide , 0 . 5 mm ferrocyanide , and 0 . 1 % triton x - 100 , and after applying vacuum for 5 min , incubated at 37 ° c . for indicated times . chlorophyll was cleared from the plant tissues by immersing them in 70 % ethanol . for drought treatment , 3 week - old , soil - grown plants were withheld completely from water for the specified times . to minimize experimental variations , same numbers of plants were grown on the same tray . with 35s - abf4 plants , which show growth retardation , two batches of plants , one of the same age and the other of similar developmental stages ( i . e ., wild type seeds were sown 7 days later so that they were at the similar developmental stages at the end of the treatment ) were tested , and similar results were obtained . the whole test was repeated at least four times , sometimes using different arrangements of plants ( i . e ., test plants on different containers etc . ), and the results were consistent . transpiration rate of detached leaves was measured by weighing freshly harvested leaves placed abaxial side up on open petri dishes on the laboratory bench . leaves of similar developmental stages ( third to fifth true rosette leaves ) from three week - old , soil - grown plants were used . to examine guard cells , leaves were excised from 3 week - old , soil - grown plants in the middle of watering ( 3 days after watering ) and light periods . leaves of similar developmental stages ( third to sixth true rosette leaves ) from 20 different plants of wild type and transgenic lines , respectively , were placed on slides abaxial side up immediately after excision and pictures were taken . the number of guard cells was then counted in the randomly chosen fields , usually 6 - 7 . anderson , j . a ., huprikar , s . s ., kochian , l . v ., lucas , w . j ., and gaber , r . f . 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