Results Directory UMM :Data Elmu:jurnal:P:PlantScience:PlantScience_Elsevier:Vol148.Issue1.2000:

on MSAR supplemented with kanamycin to select for the presence of the transgene. The F4 and F5 generations of the 35S::AP 3 -F N , 35S::AP 3 -F N u6h 1 , C19 transgenic control line and C19 u6h 1 lines were grown on soil in the greenhouse or growth cabinets without prior selection on kanamycin. Visual inspection of flowers was per- formed to identify the F4 mutants.

3. Results

3 . 1 . Strategy for isolating mutants of genes directly regulated by AP 3 The type IIS FokI restriction enzyme is com- posed of two distinct and separable domains, one for the specific recognition of DNA and the other, called F N , for the endonuclease activity that cleaves the DNA nearby the recognition site 9 bp for one strand and 13 bp for the other, regardless of the DNA sequence. The F N domain has been fused to various heterologous DNA-binding do- mains or proteins [23 – 25]. However, only the Sp1- F N hybrid protein has been used in vivo in transient expression experiments in animal cells. In these cells, Sp1-F N induces single-strand breaks SSBs and double-strand breaks DSBs of a target vector [26]. No hybrid protein with the F N domain has been expressed in a whole organism. Our approach to isolate mutants of AP3 direct target genes is based on conferring a nuclease activity to AP3 by translationally fusing the F N domain to the entire AP3 protein. Fig. 1 illustrates the design of the experiment. By expressing the AP3-F N hybrid protein in plants, DNA cleavages in AP3 target genes could occur in cells of whorls 2 and 3 where PI is present and could induce misrepair by homologous or non-homologous re- pair pathways [27 – 29]. Since AP 3 expression is detected early at floral stage 3 defined in [30], before stamen primordium specification, precur- sors of pollen-mother cells will carry a mutation. This mutation will therefore be transmitted through pollen to offspring, thereby creating mu- tants after two generations in the case of a reces- sive mutation. The efficiency of the AP3- F N protein in vivo should be increased in a mutant deficient in DNA repair mechanisms see below. 3 . 2 . Fusion of AP 3 to F N and acti6ity of AP 3 -F N in 6itro AP 3 and F N were PCR-amplified so that a first XhoI site replaced the AP 3 stop codon and a second XhoI site was placed at the 5 end and in frame of the F N domain see Section 2. The AP 3 -F N fusion gene was cloned in the eukaryotic pSPUTK expression plasmid in order to express the hybrid protein in reticulocyte lysates as de- scribed for AP3 and PI [15]. In vitro transcription and translation in the presence of 35 S-methionine demonstrated that the expected AP3-F N hybrid protein was produced Fig. 2a. The ability of the AP3-F N PI complex to bind specific target se- quences was investigated in vitro. AP3-F N or AP3 were synthesised by cotranslation with PI and analysed using a fragment of the AP 3 promoter probe A by electrophoretic mobility shift assays EMSA. As shown in Fig. 2b, probe A was shifted when AP3PI heterodimers were present in the reaction. The binding of AP3PI was competed away by increasing concentrations of unlabelled probe A but not by a probe containing a mutated CArG element oligo B [20]. When AP3-F N PI heterodimers were present in the binding reaction, probe A was also shifted. The intensity of the shift was lower compared to the shift obtained with AP3PI heterodimers. Nevertheless, the binding of AP3-F N PI to probe A was competed away by Fig. 1. The rationale of the AP3-F N strategy. When expressed constituvely in vivo, the AP3- F N fusion protein should form dimers with PI in second petal and third stamen whorl precursor cells. Upon binding of the AP3-F N PI heterodimer, DNA cleavage of target genes by F N occur which can induce mutations in vivo by misrepair via homologous or non-ho- mologous recombination pathways [27 – 29]. Later on in de- velopment, pollen-mother cells will appear in stamen primordia and will give rise to pollen grains carrying muta- tions in target genes of AP3. These mutations should be transmitted to the next generations. SP, sepal primordia. non-labelled probe A, but not by oligo B. We conclude that AP3-F N PI heterodimers have re- tained the ability to bind specifically in vitro to CArG elements. The nuclease activity assay was performed with reticulocyte lysates containing AP3PI, AP3-F N PI heterodimers or AP3-F N in the presence of Mg 2 + which is required for F N activity. The sin- gular need for AP3 and PI to be co-translated in a eucaryotic expression system to form an active heterodimer [15,16] renders the cleavage test chal- lenging since it is performed with crude extracts containing endogenous nuclease activities. We have optimised the incubation time, lysate vol- ume range and Mg 2 + concentrations that min- imised these endogenous activities although they could not be totally abolished as shown in Fig. 2c where the intensity of the probe P is lower after incubation with AP3PI lysate for instance. Nevertheless, the intensity of a probe A-contain- ing fragment decreased significantly when incu- bated with increasing amounts of AP3-F N PI lysate but remained constant with AP3PI or AP3-F N lysates. This result indicates that AP3-F N shows a specific nuclease activity only when PI is present in vitro and is consistent with the specific binding shown in Fig. 2b. In the case of a DSB activity, a 200 bp fragment was expected to ap- pear see Section 2 but was not visible under our conditions presumably because the F N domain is present as a single copy in the heterodimer see Section 4 and induces SSBs in the target DNA which becomes more sensitive to endogenous nu- cleases. Fig. 2. Fig. 2. DNA-binding and nuclease activity of the AP3-F N PI heterodimer. a Expression of AP3, AP3-F N and PI in reticu- locyte lysates. Proteins were synthesised using the TNT reticu- locyte lysate system Promega. Labelled 35 S-methionine in vitro translation reactions demonstrated that the expected polypeptides were produced. The number of methionines in these proteins is AP3, 4; AP3-F N , 10; PI, 10. Molecular weight predicted from the sequence of the proteins are indi- cated. b DNA-binding activity of the AP3-F N PI het- erodimer. AP3 or AP3-F N were cosynthesized with PI in vitro. Reticulocyte lysates were incubated with the radiola- belled CArG-box-containing fragment referred as probe A in [15]. AP3-F N PI heterodimers bind to probe A although with a lower affinity than AP3PI heterodimers. Competition was done with two concentrations × 50 and × 200 of non-ra- dioactive probe A indicated as ‘A’ in the figure filled trian- gles or a fragment carrying a mutated binding site of the MADS-domain Serum Response Factor referred as oligo B in [20] and indicated as ‘B’ in the figure open triangles. When synthesised separately and mixed afterward, AP3PI het- erodimers displayed a drastically reduced binding activity data not shown. A control with a lysate containing a pSPUTK without insert is included L. The probe P shown on the left lane was not incubated with the lysate. c Nucle- ase activity of the AP3-F N PI heterodimer. A radiolabelled probe A-carrying fragment was incubated for 4 h in the presence of 2 mM MgCl 2 with increasing concentrations 1, 2 and 3 ml of reticulocytes lysates containing AP3PI, AP3-F N PI or AP3-F N as indicated. After a phenolchloroforme treat- ment, the DNA was precipitated and loaded on an agarose gel. A decrease in probe intensity is detected specifically with AP3-F N PI but not with AP3PI nor AP3-F N lysates. The probe P shown on the left lane was not incubated with the lysate. A decrease of the intensity with AP3PI, AP3-F N -con- taining lysates or the lysate alone data not shown compared with the probe alone indicates the presence of endogenous nuclease activities in the lysate. Fig. 3. RT-PCR amplification of AP 3 and AP 3 -F N cDNAs. Total RNA from inflorescences was used for reverse tran- scription using an oligodT primer. PCR amplifications was performed with primers AP3-1 and F N -2 to amplify the entire AP 3 -F N fusion gene and with primers AP3-1 and AP3-2 see Section 2 to amplify the entire AP 3 cDNA. The expression of adenine phosphoribosyltransferase APT was used as a control. AP 3 expression in all three lines is similar to the wildtype expression. While no expression was detected in the wild-type, an AP 3 -F N amplification product was observed at the expected size 1.3 kb in lines 67-2 and 67-3. A weakly detectable, higher molecular weight product, whose specificity is unclear, is detected in line 67-16. second-whorl organs are found instead of petals and third whorl organs are abnormal stamens producing no pollen [31,32]. The AP 3 -F N trans- genic line 67-2 that segregates for one T-DNA insertion was crossed with ap 3 -1 homozygous plants. F2 plants that were resistant to kanamycin were observed. If AP 3 -F N fully complemented the ap 3 -1 mutation, all F2 kanamycin-resistant lines would be expected to form fertile flowers with four fully developed petals while a segregation of 3 wild-type to 1 ap 3 -1 plant phenotypes would indicate a failure of the hybrid protein to comple- ment the mutant. All F2 plants were fertile at 22°C. However, one quarter of the plants was distinguishable from the rest of the population. Firstly, although these plants made flowers with white petals instead of green second whorl organs in ap 3 -1 flowers Fig. 4a, the petals were short compared to wild-type Fig. 4b and c, respec- tively. Secondly, stamens did not release high amounts of pollen relative to wild-type stamens compare Fig. 4b and c which led to the forma- tion of siliques containing fewer seeds than wild- type siliques. We conclude from these results that AP3-F N can suppress the ap 3 -1 phenotype and therefore is able, at least partially, to fulfil AP 3 functions in vivo. 3 . 5 . Analysis of 35 S :: AP 3 -F N progeny Several thousands of 35S::AP 3 -F N plants were observed in T2, T3 and T4 generations. No defects were observed in flowers or in the overall develop- ment of these plants. This lack of abnormal phe- notype could indicate that the F N domain was not functional in vivo or alternatively that plant cells were able to repair efficiently DNA breaks gener- ated by F N . To test the latter alternative, line 67-2 was crossed with the DNA-break repair deficient mutant u6h 1 [33,34]. Upon crossing, two 35S::AP 3 -F N u6h 1 plants were identified in the F2 population. The misrepaired breaks of AP3-F N would occur for the first time in these F2 plants. Therefore, it was necessary to propagate 35S::AP 3 -F N u6h 1 plants for two additional gener- ations i.e. F4 generation to detect potential reces- sive mutants in the offspring. About 3000 F4 plants deriving from each of the following genotypes: 35S::AP 3 -F N , 35S::AP 3 -F N u6h 1 , C19 transgenic control and C19 u6h 1 were 3 . 3 . 35 S :: AP 3 -F N transgenic lines The AP 3 -F N fusion gene was cloned in a pCGN18-derived plasmid [11] downstream of the constitutive cauliflower mosaic virus 35S promoter and transgenic lines were obtained. The presence of the fusion gene was assessed by PCR for three lines: 67-2, 67-3 and 67-16. All lines had integrated the full length gene fusion data not shown. The expression of the endogenous AP 3 and of the transgene were assessed by RT-PCR using total RNA isolated from inflorescences Fig. 3. The endogenous AP 3 mRNA level in these lines was comparable to the wild-type level. The AP 3 -F N mRNA was detected in two of the lines and its abundance was similar than the endogenous AP 3 mRNA level. This results indicate that the AP 3 -F N transgene is expressed in transgenic lines 67-2 and 67-3. 3 . 4 . Complementation of the ap 3 - 1 mutant by 35 S :: AP 3 -F N To determine whether the AP3-F N protein was functional in vivo, we attempted to genetically complement the ap 3 -1 mutant with the 35S::AP 3 - F N construct. ap 3 -1 is a weak, temperature-sensi- tive allele and is sterile at 22°C: green, sepaloid Fig. 4. Complementation of ap 3 -1. a ap 3 -1 flower. Second and third whorl organs are short green sepaloid organs and short abnormal green staminoid organs respectively. b ap 3 -1 35S::AP 3 -F N . Short white petals are visible. Stamens are nearly as tall as wild-type stamens with yellow anthers releasing some pollen grains. c Wild-type flower ecotype Ler. Plants were grown on soil at 22°C under long-day photoperiod. Fig. 5. Phenotype of mutants isolated in the progeny of 35S::AP 3 -F N u6h 1 plants. Shown are inflorescences and flowers of F5 plants. a Wild-type immature flower, ecotype WS. Petals are small and narrow. Stamens are small and yellow with no visible released pollen grains. The stigmatic tissue on top of pistil is underdevelopped. b Wild-type mature flower, ecotype WS. Petals are large and bent backwards one petal was manually removed. Stamens are tall and pollen is being released. Stigmatic tissue is fully developed. c – d Inflorescence and individual flower respectively from mutant SS1. c The inflorescence shows almost wild-type flowers. Siliques seen on the picture did not contain seeds. d Petals are slightly shorter than wild-type. Stamens produce pollen but flowers are sterile. Pistil is underdevelopped. e – f Inflorescence and individual flower respectively from mutant SS2. e The inflorescence shows normal-looking flowers. f Stamens have anthers with little pollen. Pistil is normal. g – h Inflorescence and individual flower respectively from mutant SS4. g Inflorescence shows flowers with variable shorter-than-wild- type petal lengths. h Petals are narrow. Stamens are short producing some pollen. i – j Inflorescence and individual flower respectively from mutant AFB9. i Flowers of the inflorescence do not open. j Petals and stamens are small and pale yellow anthers are often seen with a heart-like shape producing no pollen. k – l Inflorescence and individual flower respectively from mutant AFB10. k Flowers do not open. The missing flower at the bottom right corner is shown in l. l Stamen do not elongate and anthers have a narrow shape producing no pollen. Pistil is underdevelopped. All individual flowers were picked below the main inflorescence, a position that normally shows a mature flower such as the flower in b. visually inspected. Plants were sown and grown under the same conditions. The C19 control plants showed no abnormal phenotype neither in their general growth or floral development. C19 u6h 1 plants behaved similarly indicating that, from the initial cross to the F4 plants, the u6h 1 background had no indirect effect on genotype or phenotype of the plants during their growth on soil and selections on kanamycin when cultured in Petri dishes. Conversely, while 35S::AP 3 -F N lines in a wild-type background were normal as described above, 19 plants in the F4 progeny of the 35S::AP 3 -F N u6h 1 plants showed, to various degrees, a lack of fertility. These 19 plants were isolated from the progeny of six individual F3 families out of 17 tested. Four phenotypic classes were distinguished: completely sterile CS, seven plants, severely sterile SS, seven plants, aborted flower bud AFB, four plants and unbent petals UP, one plant. When seeds were produced classes SS, AFB and UP, plants in the F5 gen- eration were observed and showed the same phe- notype as F4 mutants, therefore indicating that the mutations are transmitted. 3 . 6 . Analysis of the mutants Mutants of class CS produced no progeny which prevented their further analysis. Mutants of class SS show severe sterility with normal flow- ers. Fig. 5 illustrates representative phenotypes of mutants SS1, SS2 and SS4 Fig. 5 c – d, e – f and g – h, respectively. In these plants, flowers have normal pollen-releasing stamens. The sterility phenotype seems therefore to be the consequence of a non-functional pollen. Petals are slightly shorter than wild-type. Mutants of class AFB develop unopened flowers. The sterility is often total until very late in plant development when the last four to five flowers make short siliques containing few seeds. Mutants AFB9 and AFB10 illustrate this class Fig. 5 i – j and k – l, respec- tively. For both mutants, the sterility comes ob- viously from the fact that stamen development is impaired. Mutant UP17 was the only member of class UP with partial sterility homogenous along the plant. Most of the mutants were isolated in a green- house under shortday SD conditions. We looked at the phenotype of mutant classes SS, AFB and UP under longday LD conditions. For most mutants, especially mutants AFB, the penetrance of the phenotypes was incomplete un- der LD conditions. The phenotype of SS mutants was less sensitive to day length. To ensure that the sterility phenotype was not related to the presence of the AP3-F N hybrid protein in the u6h 1 background, mutants SS1 and SS2 were crossed with wild-type plants and off- spring were observed without selection on kanamycine. F1 plants showed a partial sterility suggesting that the mutations are semi-dominant. In both F2 populations, sterile plants were ob- served in a 3:1 to 2:1 ratio depending on the penetrance of the phenotype. Therefore, the sterility phenotype is not dependent on the pres- ence of the transgene in the u6h 1 background. Furthermore, offsprings of F5 plants showing a SS phenotype were sown on MSAR medium con- taining kanamycine. Several families showed a kanamycine-sensitive phenotype indicating that the SS phenotype was not dependent on the pres- ence of the transgene.

4. Discussion