Mechanisms Of KdSOC1 And KdN41 Genes Expressed Differentially During Water Deficit Condition In Regulating Plant Flower And Shoot Morphogenesis

The mechanism of gene expression regulation in plant plastic morphogenesis is the key basis of cell and tissue culture.The asexual reproduction of Kalanchoe daigremontiana Hamet&H.Perrier(K.daigremontiana)shares high similarities with plant tissue culture in the process of adventitious shoot formation,adventitious root formation and plantlet development in adapting to environment.Studying the molecular regulation of plantlet formation could do benefit to reveal the regeneration mechanism of agricultural and forestry crops,providing theories and technique foundations to cell engineering assisted breeding and cultivation system.Previous findings showed that under drought stress condition,plantlet could be more easily formed along leaf serration,while K.daigremontiana SUPPRESSOR OF OVEREXPRESSION OF CO 1(KdSOC1)gene was down-regulated and a novel gene with unknown function KdN41 was up-regulated.Thus,these two differentially expressed genes might be involved in the regulation of plantlet morphogenesis.Using techniques like transgenic method and confocal microscopy observation,this research was conducted within three parts to elucidate theses two gene functions and the details were as follows:(1)Delivering KdSOCl gene in socl-2 mutant Arabidopsis,tobacco and K.daigremantiana to check whether KdSOC1 gene possessed two functions,stimulating flowering and regulating shoot regeneration;(2)Transfer KdSOC1 Promoter-GUS,35S-KdSOC1 to both tobacco,K.daigremantiana and DR5-GUS(IAA induced)Arabidopsis to check KdSOC1 gene expression pattern and find out how KdSOC1 gene expression responds to auxin.(3)To identify the function of KdN41 gene expression,this study used KdN41 gene promoter driving GUS protein to obtain its expression patterns during drought stress,employed KdN41-YFP fusion protein to check cellular localization of KdN41 in tobacco leaf epidermal cells,and also constructed KdN41 gene over-expression tobacco to identify whether KdN41 gene could regulate tobacco flowering after drought stress.As a result,KdSOCl gene stimulated flowering in tobacco and Arabidopsis and affected auxin regulated shoot initiation in K.daigremontiana.In response to drought stress signal,KdN41 mainly expressed in leave vein and could participated in regulating plant flowering.The detailed results of this study are listed as follows:1.KdSOC1 gene regulated Arabidopsis,tobacco flowering and K.daigremantiana shoot regenerationThe KdSOC1 gene has the similar function with AtSOC1 gene in promoting plant early flowering and regulating shoot formation along leaf serration.The wild type(WT)Arabidopsis thaliana(with SOC1 gene)was grown in the long day(LD)condition and flowered at 33±2 d;the Arabidopsis socl-2 mutant(loss of SOC1 gene function)flowered at 78±3.1 d,while socl-2 mutant transformed with KdSOC1 gene flowered at 40 ± 2.1 d which was 38 d earlier than socl-2 mutant.The WT tobacco(with NtSOC1)flowered at 179± 3.5 d in the LD condition and 250± 2.6 d in the short day(SD)condition,while the tobacco tranfered with KdSOC1 gene flowered earlier at 128±3.6 d and 192±1.5 d respectively under both LD and SD conditions.Thus,both transgenic results of tobacco and Arabidopsis indicated that KdSOC1 gene positively regulated plant flowering.KdSOC1 gene regulated shoot formation along leaf serration in K.daigremantiana.The K.daigremantiana leaf disk transformed by KdSOC1 gene over-expression(OE)and its RNAi vector could both generated callus and shoot.However,shoot generated from callus which was transformed with KdSOC1 gene RNAi vector,eventually turned yellow and wilted,as a result no positive transgenic plant could be obtained.The transformation of KdSOC1 gene OE vector could initiate the normal regeneration of new plantlet in the callus,and its grown-up plant could just generate 5.3 shoots(p<0.05)which were asymmetrically distributed along the two sides of leaf serration;in WT plant,the average number of shoot were 19 which was symmetrically distributed;The empty vector transgenic lines formed 18 shoots in average which were also symmetrically distributed.2.Four flowering related genes were up-regulated in KdSOCl gene OE tobacco shoot apical meristem(SAM).KdSOCl gene expressed between leaf serration of K.daigremontiana and in the top of tobacco shoot.KdSOCl gene OE resulted in altered auxin distribution in meristem.Four flowering related genes were up-regulated in KdSOC1 gene OE tobacco SAM.Under LD/SD condition,the relative expression level of flowering promotion gene LEAFY was 2.6/2.4 times higher than that of in WT SAM(p<0.05);the relative expression level of GA20 oxidase,the key gene of gibberellin synthesis,was 1.1/2.8 times higher than that of in WT SAM(p<0.05);ABP1(encodes auxin binding protein 1)and PIN1(encodes PIN-FORMED 1 protein)expressed 1.7/1.0 and 3.5/4.1 times higher than those of in WT SAM,respectively(p<0.05).KdSOC1 gene OE tobacco leaf showed increased photosynthetic rate and auxin content under LD condition.For KdSOC1 OE and WT tobaccos,leaf chlorophyll content was 0.47 mg g-1 and 0.35 mg g-1,respectively(p<0.05);the leaf photosynthetic rate at 600?mol m-2 s-1 light intensity was 4.01 ?mol CO2 m-2 s-1 and 2.85 ?mol CO2 m-2 s-1,respectively(p<0.05);IAA content was 407 pg mg' and 287 pg mg,respectively(p<0.05).However,under SD condition,KdSOC1 OE and WT tobaccos showed no significant difference in physiological indexes.Leaf chlorophyll content was 0.35 mg g-1 and 0.36 mg g-1;leaf photosynthetic rate at 0-1400?mol m-2 s--light intensity showed no difference;IAA content was 252 pg mg-1 and 297 pg mg-1,respectively(p<0.05).The KdSOC1 OE K.daigremontiana leaf development is relevant to the ratio of auxin and cytokinin content For KdSOC1 gene OE and WT plants,leaf length/thickness was 3.5 cm/2.59 mm(1.4/1)and 5.1 cm/1.80 mm(2.8/1),respectively[for empty vector transgenic plant,it was 4.5 cm/1.79 mm(2.5/1)](p<0.05);the average width(diameter)of leaf vascular bundle fiber was 18.9 ?M and 9.0 ?M,respectively(p<0.05).KdSOCl gene OE plant,the relative expression of KdSOC1 was 5.6 times than that of in WT(p<0.05);the expression of PIN1 gene showed 2.5 times higher than that of in WT(p<0.05).The auxin(IAA)/cytokinin(Zeatin)content of KdSOC1 gene OE plant and WT plant was 105.3 pg mg-1/260.3 pg mg-1(1/2.5)and 80 pg mg-1/290 pg mg-1(1/3.6)respectively,while for empty vector transgenic plants,it was 83 pg mg-1/287 pg mg-1(1/3.5).These results indicated that the morphologic change in KdSOC1 gene OE plant(shorter and thicker leaves,increasing width of vascular bundle fiber)is related to the enhancement of PIN1 expression and IAA content affected by KdSOC1 OE.KdSOC1 promoter leads tissue specific expression of GUS gene and its activity is related to IAA response.Induced from tobacco seed(T1)transformed with 35S-GUS,both of the callus and shoot could be stained blue,while for KdSOC1 Promoter-GUS transgenic tobacco,globular embryo and shoot apical meristem could be stained blue.When growing on the TIBA(2,3,5-Triiodobenzoic acid)or NPA(1-N-naphthylphtalamic acid)contained medium,for 35S-GUS line all the tissue mentioned above could be stained blue;while for KdSOC1 Promoter-GUS line no blue area could be observed.KdSOC1 gene OE resulted in altered auxin distribution and response during shoot regeneration.For Arabidopsis DR5-GUS reporting lines,the callus,globular embryo,and heart-shaped embryo(induced from its T1 generation seed)were all stained blue,whereas for 35S-KdSOCl-DR5-GUS transgenic plants,areas around globular embryo and areas outside heart-shaped embryo(early and late heart shape stages)could be stained blue.The KdSOCl promoter-GUS transgenic K.daigremontiana could only be stained blue before the formation of shoot primordium and could be no longer stained in later development stages.Thus,KdSOCl gene responded to auxin signaling and regulated primodium initation.3.KdN41 gene was induced to express in tobacco leaf vein by drought stress.Over-expression of KdN41 gene in tobacco resulted in early flowering after drought stress.The GUS gene expression under control of KdN41 gene promoter was detected at tobacco leaf vein under drought stress.The leaf vein of tobacco transformed with ProKdN41GUS construct was stained blue,while under normal watering condition no blue staining area was found in the leaf.The expression of KdN41-YFP fusion protein was found in wild type tobacco leaf epidermis cell nucleus and membrane under confocal laser scanning microscopy(injected by Agrobacterium mediated 35S-KdN41-YFP construct after 48 h).Exogenous plant growth regulators affected KdN41 gene expression profiles in K.daigremontiana leaf.The KdN41 gene relative expression values in K.daigremontiana leaves sprayed with salicylic acid after 4 h and 6 h showed 0.8 and 3.7 times respectively more than those of in leaves sprayed with water.The KdN41 gene relative expression values in K.daigremontiana leaves sprayed with jasmonic acid methyl ester after 4 h and 6 h showed 0.58 and 0.57 times respectively smaller than those of in leaves sprayed with water.Over-expression of KdN41 gene in tobacco resulted in early flowering compared to wild type after drought stress treatment.The wild type tobaccos,KdN41 gene over-expression tobaccos and tobaccos transformed with empty vector were normally watered to grow for 84 d under LD condition.Then after 10 days without watering theses plants(relative soil water content reached to 10%),these plants were normally re-watered again.After 147 d,KdN41 gene over-expression tobaccos showed flowering.However,it took 168 d for wild type tobaccos and tobaccos transformed with empty vector to flower.Thus,KdN41 gene over-expression tobaccos flowered 21 d earlier.