In situ molecular identification to correlate molecular targets and associated subcellular changes involved in microspore embryogenesis icon

In situ molecular identification to correlate molecular targets and associated subcellular changes involved in microspore embryogenesis


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ABSTRACTS

Working Group 1 Workshop on: "Technology advancement in gametic embryogenesis of recalcitrant genotypes"

Palermo, 11-13 November, 2004.


ORAL PRESENTATIONS


IN SITU MOLECULAR IDENTIFICATION TO CORRELATE MOLECULAR TARGETS AND ASSOCIATED SUBCELLULAR CHANGES INVOLVED IN MICROSPORE EMBRYOGENESIS

María C. Risueño and Pilar S. Testillano

Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.

E-mail: risueno@cib.csic.es


In situ molecular identification methods constitute powerful approaches to characterise molecular and cellular targets involved in the stress response and the progression of microspore embryogenesis, as well as the associated changes in different cellular compartments. Fluorescence in situ hybridisation, FISH, technology and confocal analysis have been used to study spatial and temporal expression patterns of embryogenesis specific genes during early microspore embryogenesis in maize. Results showed that various genes expressed during early zygotic embryogenesis were also transcribed in young microspore-derived proembryos, endosperm and embryo-specific genes being expressed at early and late developmental stages respectively. Electron microscopy ISH permitted to identify the ultrastructural component housing the signal.

The search of molecular and cellular markers during the early stages of microspore embryogenesis constitutes an important goal to monitor the physiological processes involved in the induction and to identify cells committed to the new developmental programme. The identification of signals and transduction elements in defined target cells would inform on how a microspore becomes competent. In situ molecular identification methods constitute a convenient technology to approach these questions, especially in recalcitrant species in which very scarce knowledge of the cellular basis has been obtained. Various subcellular and cell wall components, and signalling molecules (MAPKs) have been investigated during early microspore embryogenesis in woody and herbaceous species: Quercus suber, Citrus clementina, Prunus persica and Capsicum annuum.

Ultrastructural and immunocytochemical analysis revealed changes in cell organisation after induction at different developmental stages. Cellular features displayed were in relation to proliferation activity and the beginning of differentiation in young and late pro-embryos. Different levels of pectin esterification in walls were found to be associated with either proliferation or differentiation events. Expression ERK1/2 MAPK homologues increased and entered the nucleus during early microspore embryogenesis, suggesting MAPK modules would operate in the reprogramming of the microspore and would confer information for the cell fate.

The reported findings provide new information which could direct future strategies for microspore embryogenesis induction, holding great promise for its advancement in recalcitrant species.


Ramírez C. et al. 2004. Eur. J. Cell Biol. 83, 213-225. Ramírez C. et al. 2003. Acta Biol. Cracov. Bot. 45, 53-58. Barany et al. 2001. Int. J Dev Bio.l 45 Supp.1, 39-40. Préstamo et al 1999, J Cell Sci 106, 1333-1346

Supported by Projects of Spanish MCyT BOS2002-03572, CM 07G/0026/2003, Spanish-Italian HI2002-99.


Immature pollen-derived doubled haploid plant formation from the barley cultivar 'Golden Promise' as a tool for efficient recombination of transgenes

Maria-José Coronado and Jochen Kumlehn

^ Institute of Plant Genetics and Crop Plant Research, Plant Reproductive Biology

Corrensstrasse 3, D-06466 Gatersleben, Germany.

Routine genetic transformation of barley by use of Agrobacteria is performed in a number of laboratories world-wide. Based upon recent substantial improvement of transformation efficiency, we increasingly focus our interest on quality features of the transgenic plants produced. In particular, there is a demand for true-breeding, selectable marker-free transgenic plants with single transgene copy number. As an elegant solution to tackle all these requirements at once, we suggest a concept that includes: (1) the generation of primary transgenic plants via Agrobacterium-mediated transformation of immature embryos, followed by (2) androgenetic formation of a comprehensively segregating T1-generation consisting of entirely true-breeding plants as a basis for, (3) the identification of selectable marker-free and/or single copy individuals which will stably transmit and express the gene of interest to the following generations. Unfortunately, Agrobacterium-mediated transformation based upon barley immature embryos is highly genotype-dependent, i.e. efficient transformation is so far confined to the spring type cv. 'Golden Promise'. In turn, this particular genotype turned out to be fairly recalcitrant in pollen embryogenesis. Therefore, we have carried out experiments to establish a protocol for efficient formation of doubled haploids from 'Golden Promise'. As a result, we present a useful method that combines conventional cold-treatment of spikes for two to three weeks, subsequent exposure of the isolated microspores to starvation for one or two days and appropriate feeding of the culture medium by immature pistils to support embryonic development. Experiments on the rapid and efficient generation of selectable marker-free, true-breeding, single-copy transgenic plants are in progress.


^ Haploid production in maize, barley, wheat, flax and potato

Pretova Anna¹, Obert Bohus¹, Zofajova Alzbeta², Bartosova Zuzana¹

¹ Instituite of Plant Genetics and Biotechnology, SAS, Akademicka 2, P.O.Box 39A, 950 07 Nitra, Slovak Republic. E-mail: anna.pretova@savba.sk

² Institute of Plant Production, Bratislavska cesta 64, 921 01 Piestany, Slovak Republic.


The value of haploids in genetic analysis and modern plant breeding has been known for a long time. Haploids in nature occur only sporadically. Induced androgenesis and gynogenesis are promising sources for haploid production in a large number of crops, but some plants are recalcitrant.

Isolated microspores or microspores in anthers were cultivated in our laboratory under specific conditions necessary for gradual recession of the pre-programmed gametophytic development. The cultivation conditions of the donor plants, the developmental stage of microspores, genotypes and some induction factors are responsible for microspore reprogramming. In maize possible pathways for androgenesis were described along with the participation of the microspore nucleus in the process. Cold treatment and starvation could promote the androgenic response. The dynamics of actin filaments around microspore nuclei has also been followed. The late uni-nucleate microspore was the most responsive stage for maize androgenesis. The size of microspores above 75μ, the spinul density and the “star-like” organisation of the microspore cytoplasm can be considered as structural markers for androgenic induction. The androgenic response is a heritable trait, so we tried to incorporate this trait into commercial maize lines by crossing with exotic lines. In barley anther cultures the first division occurred on the third day in culture and after 10 days embryo-like structures appeared. The most responsive genotype was Vladan (15.38 structures per 100 anthers). In microspore cultures (genotype Dissa) the process of embryo-like structure formation was delayed by 2-3 days compared to anther cultures. In both cultures we obtained about 7% of albino regenerants. We also followed the conditions influencing the occurrence of albino plants. In wheat the best response was obtained if the donor plants were cultivated under field conditions compared to those from glasshouse. The androgenic response in 4 genotypes and 10 crossing combinations was tested and the conditions for better yield of green plants were followed. Derived DH lines had very good performance in field conditions (yield and resistance against fungi diseases) compared to classical wheat lines. In flax the effect of culture conditions and genotype were tested as well as the cultivation conditions of donor plants.

Only anther cultures gave callus formation and subsequent plant regeneration. The most promising genotypes were PR FGL 77 and Red Wing. We reported also for the first time a gynogenic response in flax. In potato differences in androgenic response were found between tetraploid and dihaploid genotypes. In tetraploid cultivar Albina the first division of the microspore was asymmetric and as a consequence of such division for the first time a suspensor-like structure was reported.


^ Characterisation of proteins secreted during maize microspore culture: Arabinogalactan-proteins (AGPs) stimulate embryo development

Elisabeth Matthys-Rochon

Laboratoire de Reproduction et développement des Plantes, UMR 5667 CNRS/INRA/ENS/UNIV/LYON1 Ecole Normale Supérieure, 6,Allée d'Italie 69364 LYON Cédex O7 France.


In order to study molecules secreted from cultured plant cells that promote embryo development, maize microspores were transferred into cultures and the conditioned media were collected over time and analysed. Electrophoresis indicated that both non-glycosylated and glycosylated proteins including arabinogalactan-proteins (AGPs) appeared in the media and their concentration increased during the time of culture. The development of embryos was correlated with the presence of specific extra-cellular proteins, using an experimental system based on a tunicamycin inhibition test. In addition, a precise protein analysis was conducted using MALDI-TOF and ESI-MS-MS techniques. These approaches have allowed the identification of 5 other types of proteins: a cell wall invertase, two thaumatin isoforms, one 1-3 beta-glucanase and two chitinase isoforms. These experiments and results open ways for research aimed at understanding which molecules stimulate embryo formation. Moreover, AGPs may be used to stimulate the development of microspores (pollen embryogenesis) prepared from non-responsive genotypes. The possible role of the secreted molecules is discussed and an analagy is made between the development of an embryo in liquid medium and in planta.


EFFECT OF THE D GENOME ON Anther culture RESPONSE IN wheat:

^ PRELIMINARY RESULTS

M. Tersi1, I. N. Xynias2, E. Gouli-Vavdinoudi1 and D. G. Roupakias1.

1, Lab of Genetics and Plant Breeding, School of Agriculture, Aristotle University of Thessaloniki, Greece.

2, Department of Plant Production, School of Agricultural Technology, Technological and Educational Foundation of Kalamata, Antikalamos, 24 100 Kalamata, Greece.


Durum wheat is an important crop for the Southern part of Europe. The weather conditions in this area result in the production of semolina and pasta of excellent quality. However durum wheat has a disadvantage in shortening the time needed to release a new variety. The doubled haploid technique, although effective in bread wheat, fails to produce green and doubled haploid plants when applied in durum wheat. It has been proposed that the genes responsible for anther culture response are located on B genome chromosomes of bread wheat. Thus, one could presume that if these genes could be transferred to the corresponding B genome chromosomes of durum wheat the latter could have a better response to anther culture. For this to be accomplished five durum wheat cultivars (three modern, one older and a local population) were crossed to three bread wheat cultivars, two of which were found in our previous research to respond well and the third to be very recalcitrant to anther culture. The resultant F1 plants along with their parental cultivars were grown on the field and anthers containing microspores at the proper stage were cultured on potato 2 and W14 media, with and without low temperature treatment. No green plants were produced from the parental durum wheat cultivars. In contrast, green plants were produced from the F1 pentaploid plants. Root-tips of the green plants were examined cytologically and their chromosome number counted. It was observed that almost all of the green plants produced carried D-genome chromosomes. Spontaneous

chromosome doubling was effective only in plants carrying a number of D-genome chromosomes. These results indicate that D-genome chromosomes are necessary for anther culture response and spontaneous chromosome doubling. Further work, however, is needed for this to be verified.


^ Effects of different osmotica on androgenesis and albinism in barley

Geneviève Wojnarowiez1, Stéphane Caredda1, Pierre Devaux2, Rajbir Sangwan3, Christophe Clément and Rajbir S. Sangwan3

1 Université de Reims Champagne Ardenne; UFR Sciences; Biologie et Physiologie Végétales; BP 1039; 51687 Reims Cédex 2; France2 Florimond Desprez Ind.; Section Biotechnologies; BP 41; 59242 Cappelle en Pévèle; France.

3 Université de Picardie Jules Verne; Androgenèse et Biotechnologies; 33, rue Saint-Leu; 80039 Amiens; France.


Sugars and polyols were tested at different steps of anther culture in barley (^ Hordeum vulgare L.) to elucidate their influence on the yield of androgenesis and albinism. During the pre-treatment period, the osmotic shock was beneficial for androgenesis whatever the type and concentration of the tested osmoticum. The use of mannitol (300 mosm/kg), sorbitol (180 mosm/kg), PEG (240 mosm/kg) and sucrose (180 mosm/kg) gave the best results in terms of green plant production although the influence of each substance was different according to the studied parameter. During anther culture, we found that the regulation of the osmotic pressure in the culture medium had various effects, according to the osmoticum used. The best results were obtained using mannitol providing a total osmotic pressure of 364 mosm/kg in the medium whereas other combinations dramatically decreased the yield of androgenesis.

Plastids were examined by electron microscopy following both pre-treatment and culture. In the presence of mannitol and PEG, plastids did not accumulate starch at any step of the protocol but they started to differentiate into chloroplasts in the microspore derived embryos. Using sorbitol and sucrose, plastids poorly differentiated, but accumulated large amounts of starch, suggesting that these sugars are metabolised by microspores and microspore derived structures. However, the accumulation of starch was not correlated with the occurrence of albinism. These results indicated that, in barley, the osmotic shock was favourable to switch the microspore gametophytic programme towards a sporophytic programme whatever the nature of the osmoticum. During the pre-treatment period, mannitol was found to be the most suitable osmoticum for subsequent embryo development.



^ Isolated microspore culture in Durum Wheat: effect of pretreatments

De Buyser Jacques, Labbani Zelikha, Richard Nathalie and Picard Emmanuel

Université Paris-Sud, Laboratoire MVEH, bât 360, Orsay 91405-France.


Isolated microspore culture in vitro is a haplodiploidisation method recently used. However the high level in regenerating albinos plantlets limited this technology for crop improvement especially in durum wheat which remains recalcitrant because of the low frequency of embryo production and low yield of regenerated plants, which are mostly albinos.

Two durum wheat varieties (Triticum turgidum subs. durum), tetraploid (2n = 4x = 28, AABB) Cham1 and Jennah Khetifa (JK) and Pavon 76 (Triticum aestivum subsp. Aestivum) hexaploid (2n = 6x = 42, AABBDD) were tested. Microspores extraction followed protocol de Buyser et al., 2002. We compared pretreatments during isolated microspore culture: cold pretreatment for 3 days and 5 weeks at 4°C, pretreatment using a mannitol 0,1M solution at 4°C for 3 days, and an inverted combination of cold (4°C) before mannitol (0,1M) in order to improve green regeneration plants. This work is concerned with the main phases of androgenesis: induction, embryos formation and regeneration of embryos in green plantlets. Some algerian cultivars were also tested. The cultivar Cham1 has a poor response in induction and embryo formation for all

pretreatments. However the cv.JK possesses good induction properties for all pretreatments, but principally when used mannitol 0,1M for 3 days at 4°C. Under this condition, we obtained 0,71 percent (0,71%) of embryo formation, which exceeded the level unregistered with Pavon 76 in this experiment.

As far as green plants regeneration is concerned, a cold pretreatment was found to be best for durum wheat. It favoured a net improved yield in microspore culture responses, 3% and 7 % green plantlets were obtained respectively in JK. and Cham1. Results show that it possible to obtain, with a few durum wheat lines, a high level of induction of embryos. Yet, in most of lines, microspores begin to develop many proembryos during the first ten days of culture but they stay inside the exine and cease to develop further into true embryos. The same observations were found with accessions of T. monococcum, T. dicoccoides and T. dicoccum.

For lines that give embryos, we observe a high level of regeneration but most of the plants are albinos. Some pretreatments improve green regeneration but not enough to use this method in durum wheat breeding.



^ Microspore culture of small grain cereals (wheat, triticale, rice)

Csaba Lantos, Mihály Jancsó, Robert Mihály and Janos Pauk

Cereal Research Non-profit Company, Szeged, Alsó kikötő sor 9. H-6726 Hungary.


Isolated microspore culture is a special intensively developed field of haploid technology. We are doing research on isolated microspore culture of wheat, triticale and rice. We use the isolated microspore culture to in vitro selection of cells, genetic transformation and to study androgenesis and in vitro embryogenesis.

Wheat experiments were carried out on the spring genotype CY-45. The effect of cold pre-treatment on the induction of androgenesis was studied and a two-week cold pre-treatment on the donor heads was found to have a positive effect. We have tested four media (W14, AA2, P4 and CHB3) in microspore culture of wheat (Triticum aestivum L.). We have found the best mediums W14 and AA2, so we recently use W14 medium for wheat microspore culture.

Successful isolated microspore culture of triticale have been developed too. In the latest experiments five winter triticale genotypes (Bobcat, Bobcat×Bogo, Bogo, 7F95×Bogo, 7F95) were involved. Induction of androgenesis, production of embryos and regenerate plants were made from every genotype. We also observed good effects on ovaries in this system. We were not able to produce embryos without ovaries in this winter triticale genotype group.

We studied rice microspore culture in a part of our haploid research programme. Meaningful differences in androgenesis reaction was observed among the genotype series. We could regenerate plants by indirect embryogenesis in rice microspore culture, while we produced green plants by direct embryogenesis in wheat and triticale isolated microspore culture. We tested three genotypes (Dáma, Janka and Sandora) in microspore culture, and we regenerated green plants from everyone, but the best was Dáma. It is useful to develop the system of isolated microspore culture in rice.

We can see differences and similarites between androgenesis of cereals which are very important from scientific respect. We are improving this method in three cereals. Recently, we can produce lots of embryos or calli, but we have to increase efficient of green plant regeneration to exploit the microspore system in breeding.


^ Isolated microspore culture in winter wheat

Anni Jensen

Pajbergfonden, Grindsnabevej 25, DK-8300, Denmark.


Open discussion: are we interested in joining our forces in improving the method and is it practical and political possible? Status of the art at Pajbjerg.


^ Microspore embryogenesis: news from the basic science front

Erwin Heberle-Bors

Max F. Perutz Laboratories, University Departments at the Vienna Biocenter, Institute of Microbiology and Genetics, Vienna Univerisity, A-1030, Dr. Bohrgasse 9/4, Vienna, Austria.

Tel: +43 1 4277 54603; Fax: +43 1 4277 9546; E-mail: Erwin.Heberle-Bors@univie.ac.at


Microspore embryogenesis is not only a process of biotechnological importance but also a model system to study plant cell totipotency. Isolated and in vitro cultured higher plant microspores can be reprogrammed by various stresses from their normal gametophytic mode of development to form haploid embryos via the sporophytic pathway, or in other words, a cell with a restricted developmental potential can be converted into a totipotent cell. Suppression subtractive hybridization (SSH) method was used to isolate genes involved in this developmental switch. Analysis of selected clones by multiple tissue Northern and in situ hybridisation confirmed their stronger expression in stressed microspores and microspore-derived embryos. Sequence analysis revealed several genes involved in metabolism, chromosome remodelling, transcription and translation, while about 35% of sequences did not significantly match any known gene or published ESTs.

Several of the isolated genes are being analyzed at present by reverse genetics, both in tobacco and Arabidopsis. Knock-out, knock-down, and over-expressing transgenic plants showed interesting phenotypes, particularly also with respect to microspore culture.


^ Mutation assisted breeding - AN IAEA project with A doubled haploid component

S. Mohan Jain, FAO/IAEA Joint Division, International Atomic Energy Agency, Box 100, Wagramerstreaase 5, A-1400, Vienna, Austria.

Email: s.m.jain@iaea.org


The International Atomic Energy Agency (IAEA) has a mandate to apply nuclear techniques for peaceful purposes in food and agriculture, especially for genetic improvement of both seed and vegetatively propagated plants. Since 1966, IAEA started maintaining a mutant variety database of officially released mutant varieties world-wide, website http://www-mvd.iaea.org/ and so far over 2,300 mutant varieties are registered in this database. Co-ordinated Research Programmes (CRP) and Technical Co-operation

Projects (TCP) are two major activities at IAEA to serve Member States at the national, regional, and interregional levels. Most of the projects involve nuclear techniques, biotechnology, and breeding or vegetative propagation, and have recently include molecular marker analysis. Doubled haploid production has become an important tool in our breeding programmes, especially in cereal and Brassica breeding. The most common method used for haploid production is by anther /microspore culture. Guha and Maheswari (1964, 1966), University of Delhi, India developed this method; the developmental stage of the pollen is crucial for androgenesis, and pollen at the uninucleate stage yields androgenic cultures. Microspore cultures of cereals are quite successful in haploid production and an ideal system for mutation induction by radiation treatment. Low dosage of gamma irradiation enhances androgenic capacity in few crop plants. Recently, we have started a new CRP and TCP on genetic improvement for enhancing salinity and drought tolerance in cereals and other crops. Our Tunisian counterpart has tested 20 durum wheat varieties for DH production by anther culture. The local varieties are better for DH production, albinism is a major problem by anther culture. Gynogenesis has resulted in more haploid green plants. Similarly, our Chinese counterpart irradiated young spikes from F1 crosses with 100-150Gy gamma radiation, and callus was induced in the presence of 0.4-0.6% NaCl. Callus was differentiated into shoots and plants were regenerated under saline conditions. Seed propagation was done up to M2 generation in the absence of any salt selection pressure. The final selection of saline mutants was done in saline field conditions. They have released several salt tolerant wheat mutant varieties including H-112 and H 78-2, which can grow comfortably in soil containing 0.4-0.5% NaCl salt tolerant wheat mutant varieties. Trait specific molecular markers for salt tolerance will be identified to develop marker-assisted breeding programme for developing salinity and drought tolerant mutant varieties.


^ NEW STRESSES AND THEIR APPLICATIONS TO INDUCE MICROSPORE EMBRYOGENESIS

Alisher Touraev

Max F. Perutz Laboratories, University Departments at the Vienna Bioenter, Institute of Microbiology and Genetics, Vienna University, A-1030, Dr. Bohrgasse 9/4, Vienna, Austria. Email: Alishertouraev@univie.ac.at


Various stresses used widely to induce microspore embryogenesis and their characteristics are presented. The data summarized show that stress applied to microspores affects different processes (e.g. cell cycle regulation, cytoskeleton organization, carbohydrate metabolism, protein biosynthesis and phosphorylation) and structures (membrane, plastid, and mitochondrion structure) simultaneously resulting in de-differentiation of the microspore with features typical of actively proliferating cells. Moreover, cross-talk between signal transduction pathways for different stresses may take place. In some cases, the signal transduction pathways triggered by different stresses are common to more than one type of stress.

Stress in general changes the morphology of microspores: a vacuolated and the polarized microspore is transformed into a depolarized and dedifferentiated cell, which has a centrally-positioned and de-condensed nucleus, typical for cells preparing for a change in development. After transfer to a suitable rich medium, the “microspore” becomes enriched in cytoplasm and the cell cycle is reactivated. The mechanisms integrating stress-responses and cell-cycle checkpoint pathways in microspores are still unknown.
An important conclusion is also that we are still far from knowing exactly what stress means in the context of microspore reprogramming. While heat and starvation treatment shared certain features in their effect on microspore reprogramming in a number of different species, could cannot really be considered a stress, rather an anti-stress, while for colchicine we lack evidence for its general relevance in microspore embryogenesis induction and for its stress effect. However, high pH can now be added to the list of stresses effectively reprogramming microspores. A better understanding of stress in general, and of the events underlying cellular reprogramming will surely help to identify new stresses which alone, or in combination, may lead to more efficient induction of

microspore embryogenesis for doubled haploid production or allow to induce it in still recalcitrant species.

 


^ Understanding plant morphogenesis to overcome constraints in regenerating recalcitrant genotypes

Ana M. Fortes and M. Salomé Pais

Unit of Plant Molecular Biology and Biotechnology – ICAT, ed. ICAT, Campo Grande, 1749-016, Lisboa, Portugal.


The totipotent character of plant cells and tissues can be expressed by their ability to regenerate into plants via embryogenesis or organogenesis. Both processes lead to in vitro regeneration and are a major prerequisite for high yield mass propagation and for genetic transformation leading to crop improvement. Morphogensis is defined as the development of shape but also as a change of metabolic pattern. Plant or animal developmental processes are gene-switching processes. These running changes in gene products lead to a new stable homeostatic set and involves changes in inorganic elements (Fraústo da Silva & Williams, 1991). A given free element content of a cell or organism is a “metalome” which reflects its speciation in a given state as DNA (genome) does when it is expressed. There must have a link between the two with protein production (proteome) and from all of them with metabolites production (metabolome). Although morphogenic processes such as somatic embryogenesis and organogenic nodule formation play pivotal roles in plant biotechnology, little is known on signalling and signal transduction processes involved in their induction. The widespread application of mass plant propagation and gene transfer techniques in crop improvement cannot be successfully achieved if the processes leading to morphogenesis are not well understood.

In this presentation we report on some of the available data that appear determinant for a controlled induction of plant morphogenesis.


^ Alterations in the chloroplast genome of microspore-derived albino plants

E. Ankele1, BJ Hofinger2, E. Heberle-Bors1 and M. Pfosser3

1Institute of Microbiology and Genetics, University of Vienna, Dr. Bohrgasse 9, 1030 Vienna, Austria.

2Institute of Botany, University of Agriculture, Muthgasse 18, 1190 Vienna, Austria.

3Institute of Botany, Rennweg 14, 1030 Vienna, Austria.


In cereals, but largely unknown in dicots, chlorophyll-deficient (albino) plants are frequently found among regenerants derived from in vitro cultures. A variety of factors influences the expression of the albino phenotype. Molecular studies suggest that the albino phenotype may be the result of large-scale deletions in the chloroplast genome. Small deletions have been found already in freshly isolated microspores. Occasionally, microspore-derived albino plants are reported which contain plastid DNA molecules indistinguishable from that of green plants. However, these lines displayed altered transcription and translation patterns when compared to green regenerants. Other studies hint towards genetic and epigenetic factors.

Although the chloroplast gene content is highly conserved within land plants, differences in their ORFs were shown to exist. The most striking difference between dicots and monocots concerns the two largest ORFs designated ycf1 and ycf2. Bioinformatic data revealed similarities between Ycf1 and the E. coli SSB-protein that functions in DNA repair, replication and recombination. In contrast, in monocot plants like cereals, only truncated versions of the ycf1-gene (ψycf1) are present. To analyse whether the lack of a possible cpDNA stabilizing protein or the truncated ψycf1 sequence as a target for illegitimate recombination are responsible for the albino formation, the tobacco ycf1 locus had been modified. Tobacco ycf1 knockout lines stayed heteroplasmic, indicating that the ycf1 locus is essential for the plants. The replacement of the tobacco ycf1 with ψycf1 of rice resulted in a chlorophyll-deficient phenotype displaying yellowish-white leaves with little green spots. Further analysis showed that rearrangements between part of the insertion site and the psbA gene occurred, resulting in the loss of one inverted repeat and in an altered psbA gene. A knock out of the psbA gene had been shown to result in a chlorophyll-deficient phenotype. However, other sequences of the insertion site did not result in rearranged plastid genomes, which indicate that only particular sequences (“hot spot regions”) contribute or initiate rearrangements and deletions in the chloroplast genome.

In chloroplasts, at least two different DNA-dependent RNA polymerases exist – the NEP (nuclear-encoded polymerase) and the PEP (plastid-encoded polymerase). The NEP enzyme transcribes genes with a novel type promoter that is preferentially found in genes with housekeeping functions whereas genes transcribed by the PEP enzyme have bacteria-like promoters that are mainly found in promoters of photosynthesis genes. Since we obtained albino plants without any detectable deletions in their plastid genomes, we had to find an alternative explanation for albino formation. We studied the expression pattern of plastid- encoded genes known to be expressed by either NEP or PEP. Indeed, we found typical alterations in RNA levels of these albino lines. The RNA pattern of these albino plants indicates the absence of the PEP polymerase. Control experiments of plastid gene translation revealed strongly reduced levels of plastid encoded proteins, one of them an essential component of plastid ribosomes that suggest a complete deficiency of plastid protein synthesis in microspore-derived albino plants.

It seems that several factors lead to the formation of albino phenotype in cereals. The alteration of plastid-encoded gene expression results in the absence of the PEP enzyme necessary for transcription of photosynthesis genes. On the other hand, small deletions occur already in developing microspores, which might be followed by further rearrangements and large-scale deletions via hot spot regions encoded in the plastid genome.





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