An efficient method for flow cytometric analysis of pollen and detection of 2n nuclei in Brassica napus pollen

A simple and reliable method was developed for isolating pollen nuclei from Brassica napus and Triticum aestivum for DNA analysis using flow cytometry. The nuclei were released from pollen by ultrasonic treatment. The isolated nuclei following filtration through nylon mesh and a purification procedure were suitable for flow cytometric analysis as well as for isolating genomic DNA. Ultrasonic treatment time was optimized for B. napus pollen at different developmental stages. The method is effective and suitable for the preparation of many samples. We analyzed the nuclear DNA levels in pollen of B. napus at three major developmental stages as well as in mature wheat pollen. Only a single 1C peak representing the haploid DNA level was detected in the nuclei isolated from Brassica uninucleate microspores as well as in mature Triticum pollen. Interestingly, diploid nuclei were detected in both binucleate and mature pollen of B. napus. The possible origins of the diploid nuclei are discussed.Abbreviations DAPI 4,6-Diamidino-2-phenylindole - NIB Nuclear isolation buffer     

Presence of plastid and absence of mitochondrial DNA in male reproductive cells as evidence for cytoplasmic inheritance in Turnera ulmifolia and Zantedeschia aethiopica

Summary. Epifluorescence microscopy of mature pollen grains of Turnera ulmifolia and Zantedeschia aethiopica stained with 4,6-diamidino-2-phenylindole demonstrated the presence of fluorescent cytoplasmic DNA aggregates in the male reproductive cells of both species. Double staining of the cells with 4,6-diamidino-2-phenylindole and 3,3-dihexyloxacarbocyanine iodide in Technovit resin sections showed that the mitochondria of these cells did not correspond to the fluorescent cytoplasmic DNA aggregates. Electron microscopy studies revealed both plastids and mitochondria in the cells of these species. In addition, immunoelectron microscopy using an anti-DNA monoclonal antibody showed clear labeling of plastids but not mitochondria. These data provide cytological evidence for biparental plastid inheritance and maternal mitochondrial inheritance in these species.Correspondence and reprints: College of Life Sciences, Peking University, Beijing 100871, Peoples Republic of China.     

Behavior of organelle nuclei (nucleoids) in generative and vegetative cells during maturation of pollen inLilium longiflorum andPelargonium zonale

Summary The behavior of organelle nuclei during maturation of the male gametes ofLilium longiflorum andPelargonium zonale was examined by fluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI) and Southern hybridization. The organelle nuclei in both generative and vegetative cells inL. longiflorum were preferentially degraded during the maturation of the male gametes. In the mature pollen grains ofL. longiflorum, there were absolutely no organelle nuclei visible in the cytoplasm of the generative cells. In the vegetative cells, almost all the organelle nuclei were degraded. However, in contrast to the situation in generative cells, the last vestiges of organelle nuclei in vegetative cells did not disappear completely. They remained in evidence in the vegetative cells during germination of the pollen tubes. InP. zonale, however, no evidence of degradation of organelle nuclei was ever observed. As a result, a very large number of organelle nuclei remained in the sperm cells during maturation of the pollen grains. When the total DNA isolated from the pollen or pollen tubes was analyzed by Southern hybridization with a probe that contained therbc L gene, for detection of the plastid DNA and a probe that contained thecox I gene, for detection of the mitochondrial DNA, the same results were obtained. Therefore, the maternal inheritance of the organelle genes inL. longiflorum is caused by the degradation of the organelle DNA in the generative cells while the biparental inheritance of the organelle genes inP. zonale is the result of the preservation of the organelle DNA in the generative and sperm cells. To characterize the degradation of the organelle nuclei, nucleolytic activities in mature pollen were analyzed by an in situ assay on an SDS-DNA-gel after electrophoresis. The results revealed that a 40kDa Ca²⁺-dependent nuclease and a 23 kDa Zn²⁺ -dependent nuclease were present specifically among the pollen proteins ofL. longiflorum. By contrast, no nucleolytic activity was detected in a similar analysis of pollen proteins ofP. zonale.     

DNA repair in pollen: Range of mutagens inducing repair, effect of replication inhibitors and changes in thymidine nucleotide metabolism during repair

Summary Pollen of Petunia hybrida carry out DNA repair during the first two hours of germination when certain mutagens are included in the germination medium. This repair, detected readily as unscheduled DNA synthesis, since there is no replicative DNA synthesis in Petunia pollen, can be induced by the chemical mutagens N-methyl-N-nitro-N-nitrosoguanidine, 4-nitroquinoline-1-oxide, azaserine and methyl methanesulphonate. These compounds are all considered to be capable of direct covalent interaction with DNA. Mutagens requiring metabolic activation before interaction with DNA did not induce DNA repair synthesis in pollen. The practice of solubilizing water-insoluble chemical mutagens with dimethyl sulphoxide did not prove practical, due to the extremely harmful effects of dimethyl sulphoxide on pollen. Pretreatment of pollen before germination with pure ether, however, had no harmful effect on either repair or pollen germination. Therefore water-insoluble, ether-soluble mutagens were tested by pretreatment of the pollen with mutagens in ether solution. By this means it was shown that the direct-acting mutagen, diethyl sulphate, would also bring about unscheduled DNA synthesis in pollen, while 2-acetylaminofluorence and dimethyl-p-aminobenzene, both requiring metabolic activation, did not do so. Inhibitors of DNA replicative synthesis, hydroxyurea, azaserine, azauridine and fluorodeoxyuridine did not inhibit unscheduled DNA synthesis brought about by N-methyl-N-nitro-N-nitrosoguanidine. On the contrary, these compounds stimulated repair synthesis to varying degrees, hydroxyurea having the greatest effect. Pollen uptake of ³H-thymidine and the amount of radioactive label subsequently appearing in dTMP and dTDP+dTTP was increased by 4-nitroquinoline-1-oxide. Partial inhibition of these increases and of 4-nitroquinoline-1-oxide induced repair synthesis by 3,5-cyclic AMP suggested that thymidine:AMP phosphotransferase rather than thymidine kinase was responsible for thymidine phosphorylation in pollen. Enzyme assays on pollen extracts confirmed this.     

Preferential degradation of plastid DNA with preservation of mitochondrial DNA in the sperm cells ofPelargonium zonale during pollen development

Summary In the present study, we studied changes in organellar DNA in the sperm cells of maturing pollen ofPelargonium zonale, a plant typical to exhibit biparental inheritance, by fluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI) and by immunogold electron microscopy using anti-DNA antibody. Fluorescence intensities of DAPI-stained plastid nuclei in generative and sperm cells at various developmental stages were quantified with a video-intensified microscope photon counting system (VIMPCS). Results indicated that the amount of DNA per plastid in generative cells increased gradually during pollen development and reached a maximum value (about 70 T per plastid; 1 T represents the amount of DNA in a particle of T4 phage) in young sperm cells at 5 days before flowering. However, the DNA content of plastids was subsequently reduced to about 20% of the maximum value on the day of flowering. Moreover, the DNA content of the plastid further decreased to 4% of the maximum value when pollen grains were cultured for 6 h in germination medium. In contrast, the amount of DNA per mitochondrion did not decrease significantly around the flowering day. Similar results were also obtained by immunogold electron microscopy using anti-DNA antibody. The density of gold particles on plastids decreased during pollen maturation whereas labelling density on mitochondria remained relatively constant. The number of plastids and mitochondria per generative cell or per pair of sperm cells did not change significantly, indicating that the segregation of DNA by plastid division was not responsible for the decrease in the amount of DNA per plastid. These results indicate that the plastid DNA is preferentially degraded, but the mitochondrial DNA is preserved, in the sperm cells ofP. zonale. While the plastid DNA of the sperm cells decreased before fertilization, it was also suggested that the low DNA contents that remain in the plastids of the sperm cells are enough to account for the biparental inheritance of plastids inP. zonale.Abbreviations DAPI 4,6-diamidino-2-phenylindole - VIMPCS video-intensified microscope photon counting system     

Ca2+-induced fragmentation of actin filaments in pollen tubes

Summary To control the intracellular free Ca²⁺ concentration from the cell exterior, pollen tubes ofLilium longiflorum were treated with a Ca²⁺ ionophore, A23187. Cytoplasmic streaming was inhibited when the free Ca²⁺ concentration of the external medium ([Ca²⁺]) was raised to 5×10^–6 M or higher. At [Ca²⁺] below 1×10^–6 M, the rhodamine-phalloidin stained actin filaments appeared straight and thin. However, at [Ca²⁺] which inhibited cytoplasmic streaming, the actin filaments appeared fragmented. In pollen tubes, Ca²⁺ regulation of cytoplasmic streaming may be linked not only to myosin (Shimmen 1987) but also to actin.Abbreviations ATP adenosine-5-triphosphoric acid - [Ca²⁺] concentration of free Ca²⁺ - EGTA ethyleneglycol-bis-(-aminoethylether)N,N,N,N-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) - Rh-ph rhodamine-conjugated phalloidin     

Unique positioning of mitochondria in developing microspores and pollen grains inPharbitis nil: mitochondria cover the nuclear surface at specific developmental stages

Summary Changes in the number and distribution of mitochondria in microspores and pollen grains during male gametogenesis inPharbitis nil were examined with Technovit sections stained with 3,3-dihexyloxacarbocyanine iodide. The number of mitochondria per microspore or pollen grain ofP. nil increased constantly and dramatically during male gametogenesis. During this process, mitochondria exhibited characteristic localizations: subpopulations of mitochondria covered the surface of the microspore and vegetative nuclei before and again just after postmeiotic mitosis I (9 and 5 days before flowering, respectively). The mitochondria also surrounded the generative nucleus 2 days after postmeiotic mitosis I (5 days before flowering), although the density of mitochondria on the nuclear surface was lower. Electron microscopy showed that the mitochondria were about 30 nm from the nuclear envelope and that each mitochondrion was located near a nuclear pore. The characteristic localization of mitochondria inP. nil pollen may serve as a model to analyze the mechanisms that control mitochondrial positioning within a cell and interactions between mitochondria and nuclei.Abbreviations DAPI 4,6-diamidino-2-phenylindole - DiOC₆ 3,3-dihexyloxacarbocyanine iodide - PM I postmeiotic mitosis I     

Characterization of the translocator associated with pollen tube organelles

Summary In pollen tubes, the motive force of cytoplasmic streaming is assumed to be generated by the sliding of the translocator associated with cell organelles along actin filaments. In the present study, the characteristics of the translocator were studied by reconstituting the movement of pollen tube organelles along characean actin bundles. Movement of pollen tube organelles proceeded from the pointed end to the barbed end of the actin filaments of the characean cells. The reconstituted movement was not inhibited by vanadate. KCL at higher concentrations inhibited the movement. Furthermore, heavy meromyosin (HMM) prepared from rabbit skeletal muscle myosin partially inhibited the reconstituted movement and pCMB-modified HMM inhibited it completely. The present results strongly support our previous conclusion that the translocator which generates the motive force of cytoplasmic streaming in pollen tube is myosin.Abbreviations AMP-PNP adenylyl-imidodiphosphate - ATP adenosine-5-triphosphate - ATP--S adenosine-5-0-(3-thiotriphosphate) - BSA bovine serum albumin - CCCP carbonylcyanide m-chlorophenylhydrazone - DTT dithiothreitol - EDTA ethylenediamine tetraacetic acid - EGTA ethyleneglycol-bis-(-aminoethyl ether)N,N,N,N-tetraacetic acid - HB homogenization buffer - HMM heavy meromyosin - NEM N-ethylmaleimide - pCMB p-chloromercuribenzoic acid - PIPES piperazine-N,N-bis-(2-ethanesulfonic acid) - PPi pyrophosphate     

Behavior of organelles and their nucleoids in the shoot apical meristem during leaf development in Arabidopsis thaliana L.

The behavior of organelle nucleoids and cell nuclei was studied in the shoot apical meristem and developing first foliage leaves of Arabidopsis thaliana. Samples were embedded in Technovit 7100 resin, cut into thin sections and stained with 4-6-diamidino-2-phenylindole to observe DNA. Fluorimetry was performed using a video-intensified microscope photon-counting system. The DNA content of individual mitochondria was more than 1 Mbp in the shoot apical meristem and the young leaf primordium, and decreased to approximately 170 kbp in the mature foliage leaf. In contrast, the DNA content of individual plastids was low in the shoot apical meristem and increased until day 7 after sowing. Application of 5-bromo-2-deoxyuridine, an analogue of thymidine, was usesd to investigate DNA synthesis in situ. The activities of DNA synthesis in the mitochondria and plastids changed according to the stage of development. Mitochondrial DNA was actively synthesized in the shoot apical meristem and young leaf primordia. This strongly suggests that the amount of mitochondrial DNA per mitochondrion, which has been synthesized in the shoot apical meristem and young leaf primordium, is gradually reduced due to continual divisions of the mitochondria during low levels of mitochondrial DNA synthesis. Synthesis of DNA in the plastid became active in the leaf primordia following DNA synthesis in the mitochondria, and the small plastids were filled with large plastid nucleotids. This enlargement of the plastid nucleoids occurred before the synthesis of ribulose-1,5-bisphosphate carboxylase/oxygenase and the development of thylakoids.Abbreviations BrdU 5-bromo-2-deoxyuridine - DAPI 4-6-diamidino-2-phenylindole - DiOC₆a 3,3-dihexyloxacarbocyanine - mtDNA mitochondrial DNA - mt-nucleoid mitochondrial nucleoid - ptDNA plastid DNA - pt-nucleoid plastid nucleoid - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase This work was supported by grant No. 2553 to M.F. and Nos. 04454019, 03304005 and 06262204 to T.K. from the Ministry of Education, Science and Culture of Japan, and by a grant for a pioneering research project in biotechnology from the Ministry of Agriculture, Forestry and Fisheries of Japan.     

Five transfer RNA genes lacking CCA termini are clustered in the chromosome of Streptomyces rimosus

Summary The nucleotide sequence of a 1105 by Streptomyces rimosus DNA fragment containing five transfer RNA genes was determined. Two tRNA^Gln (CUG) genes, differing by 1 by in the aminoacyl stem, and three identical tRNA^Glu (CUC) genes were identified. The five tRNA genes, arranged in the order: Gln1-Glul-Glu2-Gln2-Glu3, were separated by short, nonhomologous intergenic regions. Surprisingly, none of these tRNA genes encoded the CCA 3 terminus of mature tRNAs. All five encoded tRNAs for the translation of GC rich codons, which are preferentially used in Streptomyces genes (CAG and GAG, respectively). We recently reported nucleotide sequences of two initiator tRNA genes from S. rimosus, which also do not encode the CCA end of mature tRNAs. It is therefore very likely that S. rimosus represents an example of those eubacteria in which the majority of tRNA genes do not encode the 3 terminal CCA end of mature tRNAs. Evolutionary implications of this finding remain to be elucidated.     

Successful expression in pollen of various plant species of in vitro synthesized mRNA introduced by particle bombardment

Gold particles coated with -glucuronidase (GUS) mRNA with a 5 cap structure that had been synthesized in vitro were introduced, by use of a pneumatic particle gun, into pollen grains of lily (Lilium longiflorum), freesia (Freesia refracta) and tulip (Tulipa gesneriana). A fluorometric assay for the GUS activity indicated that in vitro synthesized GUS mRNA introduced into these pollen cells by particle bombardment was successfully expressed. GUS activity in extracts of the bombarded lily pollen became detectable fluorometrically within 30 min after bombardment, peaked at 6 h, then gradually decreased. This activity changed as a function of the developmental stage of the pollen cell of lily.     

Molecular characterization of rice genes specifically expressed in the anther tapetum

In situ localization of mRNA was carried out on two cDNAs (Osc4 and Osc6) that had been isolated from rice anthers at the microspore stage. The mRNA corresponding to each cDNA was shown to be localized only in the tapetal cells of the rice immature anthers, but not in the microspores or the mature pollen. The corresponding genomic clone, Osg6B, was isolated, and its 5-upstream region was found to regulate -glucuronidase expression in the tapetum of transgenic tobacco. A set of 5 deletions was also generated and a 1095 bp 5 region was revealed to be necessary for activation of the Osg6B promoter in transgenic tobacco.     

Disappearance of plastid and mitochondrial nucleoids during the formation of generative cells of higher plants revealed by fluorescence microscopy

Summary The fate of plastid and mitochondrial nucleoids (pt and mt nucleoids) ofTriticum aestivum was followed during the reproductive organ formation using fluorescence microscopy after staining with 4'6-diamidino-2-phenylindole (DAPI). This investigation showed a drastic morphological change of pt nucleoids during the differentiation of reproductive organs from the shoot apex. Dot-shaped pt nucleoids grew into ring-shaped ones, which divided into small pieces in the monocellular pollen grain, as observed in this plant's earlier stage of leaf development. During the development of mature pollen grain from monocellular pollen grain, pt and/or mt nucleoids disappeared through the division of the male generative cell ofT. aestivum. Cytologically, this observation is direct evidence of the maternal inheritance of higher plants. Thus far, cytological evidence of this phenomenon has been found mostly by morphological criteria using electron microscopy, which admits some ambiguity. In the plants exemplified byLilium longiflorum, pt and/or mt nucleoids disappeared after the first pollen grain mitosis, which precededT. aestivum. In the plants exemplified byTrifolium repens, pt and/or mt nucleoids existed in the generative cells of the mature pollen grain.The significance of these observations was discussed in relation to the interaction between nuclear and organelle genomes during plant development.Abbreviations DAPI 4'6 diamidino-2-phenylindole - Mt DNA Mitochondrial DNA - Mt nucleoid Mitochondrial nucleoid - Pt DNA Plastid DNA - Pt nucleoid Plastid nucleoid On leave from Department of Biology, Nagoya University, Furocho, Chikusaku, Nagoya 464, Japan.     

Partial purification of myosin from lily pollen tubes by monitoring with in vitro motility assay

Summary Myosin in pollen tubes ofLilium longiflorum was partially purified, using an in vitro motility assay as a monitor. The main components in the partially purified preparation had molecular masses of 110, 120, and 140 kDa in SDS-PAGE. They became bound to actin filaments in an ATP-dependent manner. Among the components, only that of 120 kDa became bound to ATP and was concluded to be the heavy chain of pollen tube myosin.Abbreviations ATP adenosine-5-triphosphate - DTT dithiothreitol - EB extraction buffer - EGTA ethyleneglycol-bis-(-aminoethylether) N, N, N, N-tetraacetic acid - PAGE polyacrylamide gel electrophoresis - PIPES piperazine-N,N-bis-(2-ethanesulfonic acid) - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecylsulfate - TBS Tris buffered saline - TEB Tris-EGTA buffer     

Multicopy plasmid suppression of stationary phase chaperone toxicity in Escherichia coli by phosphogluconate dehydratase and the N-terminus of DnaK

Overproduction of DnaK in Escherichia coli results in a bacteriocidal effect. This effect is most acute in stationary phase cells. A selection scheme was developed to isolate multicopy suppressors from an E. coli plasmid expression library, which overcome the stationary phase toxicity of excess DnaK. Two suppressor plasmids were recovered which contained inserts of 1.85 kb and 2.69 kb, respectively. Rearranged and deleted plasmid derivatives were constructed and used to further localize the suppressors. DNA sequence analysis demonstrated that one suppressor encoded phosphogluconate dehydratase (Edd) while the other suppressor encoded the N-terminal 237 amino acids of DnaK itself (DnaK). Strains bearing the suppressor plasmids constitutively overproduced proteins with apparent masses of 66 kDa (Edd) and 37 kDa (DnaK) as determined by gel electrophoresis. Western blot analysis using polyclonal antisera specific for either Edd or DnaK confirmed the identity of these overproduced proteins. Suppression of DnaK toxicity was eliminated by the introduction of a + 1 frameshift mutation early in the respective coding regions of either of the two suppressors. These results suggest that suppressor gene translation plays a role in the mechanism of DnaK suppression.     

PO149, a new member of pollen pectate lyase-like gene family from alfalfa

PO149 is a low-copy-number gene expressed in the late stages of pollen development. The promoter region contains no similarities in DNA sequence to those of other pollen-specific genes, except for a tobacco sequence (AAATGA), which occurs four times in this alfalfa gene and much further upstream than in tobacco. Four distinct TATA boxes were detected in the promoter with the distal and proximal TATA boxes being separated by a spacer of 269 nucleotides. Hairpin loop structures were found in the 5-and 3-untranslated regions of PO149 mRNA. The coding region of PO149 is interrupted by two introns and encodes a putative prepeptide of 450 amino acids with homology to pollen pectate lyase-like proteins and pollen allergens. The coding region also contains sequences characteristic of both a signal peptide and a nuclear localization signal.     

Development of a pollen-mediated transformation method forNicotiana glutinosa

The development is described of a new procedure to genetically transform plant species using the male gametophyte as a natural transformation vector. Our system avoids the need for complicated regeneration procedures thus making it broadly applicable. Naked plasmid DNA encoding kanamycin resistance and GUS activity was introduced by particle gun bombardment into mature pollen grains ofNicotiana glutinosa. Bombarded pollen was used for pollinations and the resulting seeds were selected for kanamycin resistance. Two different kanamycin-resistant plants, designated VIP A and VIP B, were obtained in two independent experiments. In VIP A, TR2-driven GUS activity was observed in vascular bundles, trichomes and in a small number of pollen grains. DNA gel blot analysis indicated that the introduced DNA was integrated independently into the genome of VIP A and VIP B. It was shown that male and female gametophyte development and seed set were highly aberrant in both VIP A and VIP B and that the offspring of self- and cross-pollinations did not contain the transgenes. This might be caused by a recombination event during the integration of the naked DNA resulting in a deletion of part of the target chromosome. After meiosis such a deletion is lethal for the gametes. Our observation that the transgenes were detected in DNA isolated from sporophytic tissues but not in DNA from VIP A and VIP B pollen grains is in line with this explanation. Future experiments designed to increase the frequency of transformation and to transfer the transgenes to the offspring are discussed.     

Dissection of a pollen-specific promoter from maize by transient transformation assays

We have previously reported the isolation and characterization of a gene (Zm 13) from Zea mays which shows a pollen-specific pattern of expression. Stably transformed tobacco plants containing a reporter gene linked to portions of the Zm 13 5 flanking region show correct temporal and spatial expression of the gene. Here we present a more detailed analysis of the 5 regions responsible for expression in pollen by utilizing a transient expression system. Constructs containing the -glucuronidase (GUS) gene under the control of various sized fragments of the Zm13 5 flanking region were introduced into Tradescantia and Zea mays pollen via high-velocity microprojectile bombardment, and monitored both visually and with a fluorescence assay. The results suggest that sequences necessary for expression in pollen are present in a region from –100 to –54, while other sequences which amplify that expression reside between –260 and –100. The replacement of the normal terminator with a portion of the Zm13 3 region containing the putative polyadenylation signal and site also increased GUS expression. While the –260 to –100 region contains sequences similar to other protein-binding domains reported for plants, the –100 to –54 region appears to contain no significant homology to other known promoter fragments which direct pollen-specific expression. The microprojectile bombardment of Tradescantia pollen appears to be a good test system for assaying maize and possibly other monocot promoter constructs for pollen expression.     

Involvement of phenylalanine ammonia-lyase in the development of pollen in broccoli (Brassica oleracea L.)

Summary To determine whether phenylalanine ammonia-lyase (EC 4.3.1.5) is involved in the maturation of microspores to fertile pollen, anthers of a fertile strain of broccoli (Brassica oleracea L.) were studied in a comparison with anthers of a cytoplasmic male sterile strain. In the normal fertile strain, immature anthers of about 2 mm in length exhibited higher phenylalanine ammonia-lyase activity than mature anthers or those shorter than 2 mm. The 2-mm-long anthers corresponded to the mononucleate stage, just after release of the microspores during pollen development. Immunohistochemical localization of phenylalanine ammonia-lyase in the anthers indicated that the protein was present predominantly in the tapetal cells. The immature anthers of cytoplasmic male sterile broccoli had a lower phenylalanine ammonia-lyase activity than those of the normal fertile strain. The level of phenylalanine ammonia-lyase activity in the immature anthers was positively correlated with the number of fertile pollen grains at the flowering stage in both strains. It seems possible, therefore, that phenylpropanoid metabolism, which involves phenylalanine ammonia-lyase, may play an important role in the maturation of microspores in flowering plants.Abbreviations CHS chalcone synthase - CMS cytoplasmic male sterility - DAPI 4, 6-diamidmo-2-phenylindole dihydrochloride - PAL L-phenylalanine ammonia-lyase