A tobacco homolog of DCN1 is involved in pollen development and embryogenesis.

KEY MESSAGE: We show that DCN1 binds ubiquitin and RUB/NEDD8, associates with cullin, and is functionally conserved. DCN1 activity is required for pollen development transitions and embryogenesis, and for pollen tube growth. Plant proteomes show remarkable plasticity in reaction to environmental challenges and during developmental transitions. Some of this adaptability comes from ubiquitin-mediated protein degradation regulated by cullin-RING E3 ubiquitin ligases (CRLs). CRLs are activated through modification of the cullin subunit with the ubiquitin-like protein RUB/NEDD8 by an E3 ligase called defective in cullin neddylation 1 (DCN1). Here we show that tobacco DCN1 binds ubiquitin and RUB/NEDD8 and associates with cullin. When knocked down by RNAi, tobacco pollen formation was affected and zygotic embryogenesis was blocked around the globular stage. Additionally, we found that RNAi of DCN1 inhibited the stress-triggered reprogramming of cultured microspores from their intrinsic gametophytic mode of development to an embryogenic state. This stress-induced developmental switch is a known feature in many important crops and leads ultimately to the formation of haploid embryos and plants. Compensating the RNAi effect by re-transformation with a promoter-silencing construct restored pollen development and zygotic embryogenesis, as well as the ability for stress-induced formation of embryogenic microspores. Overexpression of DCN1 accelerated pollen tube growth and increased the potential for microspore reprogramming. These results demonstrate that the biochemical function of DCN1 is conserved in plants and that its activity is involved in transitions during pollen development and embryogenesis, and for pollen tube growth.

Two tobacco proline dehydrogenases are differentially regulated and play a role in early plant development.

Proline dehydrogenase is the rate-limiting enzyme in proline degradation and serves important functions in the stress responses and development of plants. We isolated two tobacco proline dehydrogenases, NtPDH1 and NtPDH2, in the course of screening for genes upregulated in stressed tobacco (Nicotiana tabacum) microspores. Expression analysis revealed that the two genes are differentially regulated. Under unstressed conditions, their steady-state transcript levels were similar in mature pollen and apical meristems, whereas NtPDH2 was expressed predominantly in vegetative organs, styles, and ovules. The expression of NtPDH1 was maintained at a constant low level during 24 h of dehydration, whereas NtPDH2 was upregulated within 1 h after the onset of stress and subsequently downregulated to undetectable levels. Differential and sustained expression was also found for the two enzymatic isoforms of Arabidopsis thaliana AtPDH. Silencing of the NtPDH genes by RNA interference using the CaMV 35S promoter led to increased proline contents, decreased seed set, delayed seed germination and retarded seedling development pointing towards an important function of at least one of the two NtPDH genes during plant reproductive development.

Transcriptional and metabolic profiles of stress-induced, embryogenic tobacco microspores.

Higher plant microspores, when subjected to various stress treatments in vitro, are able to reprogram their regular gametophytic development towards the sporophytic pathway to form haploid embryos and plants. Suppression subtractive hybridization (SSH) and metabolic profiling were used to characterize this developmental switch. Following differential reverse Northern hybridizations 90 distinct up-regulated sequences were identified in stressed, embryogenic microspores (accessible at www.univie.ac.at/ntsm). Sequence analyses allowed the classification of these genes into functional clusters such as metabolism, chromosome remodeling, signaling, transcription and translation, while the putative functions of half of the sequences remained unknown. A comparison of metabolic profiles of non-stressed and stressed microspores using gas chromatography/mass spectrometry (GC/MS) identified 70 compounds, partly displaying significant changes in metabolite levels, e.g., highly elevated levels of isocitrate and isomaltose in stressed microspores compared to non-stressed microspores. The formation of embryogenic microspores is discussed on the basis of the identified transcriptional and metabolic profiles.

A model system to study the environment-dependent expression of the Bet v 1a gene encoding the major birch pollen allergen.

BACKGROUND: The major birch pollen allergen Bet v 1 (or Bet v 1a) is one of the main causes of seasonal type I allergies. Various environmental factors such as light, temperature and air pollution may influence the activity of the Bet v 1a gene. The creation of a model system to evaluate the role of environmental factors affecting the Bet v 1a gene expression would be highly desirable. We suggest the use of transgenic tobacco plants carrying a Bet v 1a promoter-reporter gene fusion as such a system. METHODS: The promoter of the Bet v 1a gene was isolated with the use of the Universal Genome Walker kit (BD Biosciences Clontech, USA). Web Software was used to search for putative cis-regulatory elements within the promoter. Transgenic tobacco plants harboring the promoter-beta-glucuronidase (GUS) reporter gene fusion were obtained via Agrobacterium tumefaciens-mediated transformation. Promoter activity was examined with histochemical and quantitative assays. RESULTS: Structural analysis predicted elements responsible for pollen-specific, light-, stress- and hormone-mediated induction within the Bet v 1a promoter. The evaluation of GUS activity in transgenic tobacco plants showed that the Bet v 1a promoter is pollen-specific. Moreover, the Bet v 1a promoter is considered to be the strongest isolated pollen-specific promoter reported to date. It was shown that temperature and abscisic acid positively regulate the activity of the Bet v 1a promoter during pollen development, providing evidence for environment-dependent regulation of the Bet v 1a gene. CONCLUSIONS: A model system to study the effect of environmental factors on the expression of the Bet v 1a gene encoding the major birch allergen in pollen was generated. Additionally, we suggest that this system could be used to search for factors that inhibit the activity of the gene in pollen in order to reduce the potential allergenicity of birch trees.