Maria Mulisch

DNA-binding proteins of the Whirly family in Arabidopsis thaliana are targeted to the organelles

Arabidopsis thaliana contains three genes with high homology to potato p24 which was described as a member of the Whirly family of nuclear transcriptional activators. Computer‐based analysis revealed that all Arabidopsis Whirly (Why) proteins contain targeting sequences for either plastids or mitochondria. The functionality of these sequences was demonstrated by in vitro import assays into isolated organelles. Transient expression of GFP fusion proteins in protoplasts and onion epidermal cells confirmed the localisation of these proteins in plastids or mitochondria, respectively. The possession of organellar targeting sequences seems to be conserved among Why proteins of higher plant species, including potato p24.

Recombinant Whirly1 translocates from transplastomic chloroplasts to the nucleus

Whirly1 was shown to be dually located in chloroplasts and nucleus of the same cell. To investigate whether the protein translocates from chloroplasts to the nucleus, we inserted a construct encoding an HA‐tagged Whirly1 into the plastid genome of tobacco. Although the tagged protein was synthesized in plastids, it was detected in nuclei. Dual location of the protein was confirmed by immunocytological analyses. These results indicate that the plastidial Whirly1 is translocated from the plastid to the nucleus where it affects expression of target genes such as PR1. Our results support a role of Whirly1 in plastid‐nucleus communication.

Single-Stranded DNA-Binding Protein Whirly1 in Barley Leaves Is Located in Plastids and the Nucleus of the Same Cell

This article concerns the intriguing protein Whirly1 (Why1) that belongs to a small family of single-stranded DNA-binding proteins and has been described to have functions in the nucleus ([Desveaux et al., 2002][1]; [Yoo et al., 2007][2]). In contrast, in vitro import assays with isolated organelles

Whirly1 in chloroplasts associates with intron containing RNAs and rarely co-localizes with nucleoids

The nucleic acid binding protein Whirly1 of barley has been located to both chloroplasts and the nucleus of the same cell. Immunogold labelling furthermore showed that in vivo Whirly1 does not strictly co-localize with DNA in chloroplasts, while it is closely associated with DNA in the nucleus. High-resolution imaging of Whirly1-GFP and PEND-RFP fusion proteins revealed that only a minor part of Whirly1 co-localizes with nucleoids. The co-localization with nucleoids is in accordance with the detection of Whirly1 in a conventionally prepared fraction of the transcriptionally active chromosome (TAC). By further purification and enrichment of transcriptional activity Whirly1, however, was lost from the TAC fraction. Knockdown of Whirly1 in transgenic barley plants had neither impact on transcription of selected protein coding genes nor on genes coding for ribosomal RNAs or tRNAs. The results of RIP-chip experiments showed that barley Whirly1 as its maize orthologue associates with a set of intron containing plastid RNAs. Taken together, the results suggest that plastid-located Whirly1 functions primarily in RNA metabolism rather than as a DNA binding protein.

An alternative strategy of dismantling of the chloroplasts during leaf senescence observed in a high-yield variety of barley

Changes in function and composition of the photosynthetic apparatus as well as the ultrastructure of chloroplasts in mesophyll cells were analyzed in flag leaves of the high-yield barley (Hordeum vulgare) variety cv. Lomerit during senescence under field conditions in two successive years. In contrast to previous results obtained with the elder variety cv. Carina photosystem II efficiency measured by F(v)/F(m) was found to be rather stable until a very late stage of senescence. Chlorophyll a fluorescence and P700 absorbance measurements revealed that the activities of the two photosystems declined in parallel. An increase in the chlorophyll a/b ratio at a late stage of senescence was observed to coincide with a decline in the level of the Lhcb1 apoprotein of the light harvesting complex (LHC) and the level of the corresponding transcript. Ultrastructural investigations revealed the presence of gerontoplasts that have long, single or pairwise thylakoids and lack large grana stacks. It is hypothesized that the early degradation of grana thylakoids harboring the major LHC reduced the risk of photoinhibition and might be causally related to the high yield of the barley variety cv. Lomerit.

WHIRLY1 is a major organizer of chloroplast nucleoids

WHIRLY1 is an abundant protein of chloroplast nucleoids, which has also been named pTAC-1 with regard to its detection in the proteome of transcriptionally active chromosomes (TAC). In barley primary foliage leaves, expression of the WHIRLY1 gene is highest at the base whereas protein accumulation is highest in the middle of the leaf where young developing chloroplasts are found. In order to elucidate the function of WHIRLY1 in chloroplast nucleoids, transgenic barley plants with an RNAi-mediated knock-down of the HvWHIRLY1 gene (RNAi-W1) were generated. The homozygous RNAi-W1-7 plants, barely containing traces of the WHIRLY1 protein, were chosen for detailed analyses of nucleoids. Nucleic acid specific-staining with YO-PRO®-1 revealed that in comparison to wild type chloroplasts, which have multiple small nucleoids attached to thylakoids, chloroplasts of the transgenic plants contain large irregularly formed patches of DNA besides nucleoids that are similar in size and shape to those of wild type chloroplasts. In large electron lucent areas, filamentous structures were detected by conventional transmission electron microscopy. Analyses of ptDNA levels by both DNA dot-blot hybridization and quantitative PCR showed that leaves of the transgenic plants have a two- to three-fold higher level of ptDNA than the wild type. The higher ptDNA level in RNAi-W1 plants coincided with an enhanced expression of the gene encoding a putative organelle targeted DNA polymerase in the mid part of primary foliage leaves. Furthermore, overexpression of the barley WHIRLY1 gene in E. coli cells revealed a higher compaction of bacterial nucleoids. These results suggest that WHIRLY1 belongs to the group of plastid nucleoid associated proteins (ptNAP) having a function in compacting a subpopulation of chloroplast nucleoids thereby affecting DNA replication.

The Tr-cp 14 cysteine protease in white clover (Trifolium repens) is localized to the endoplasmic reticulum and is associated with programmed cell death during development of tracheary elements.

Cysteine proteases are known to be associated with programmed cell death, developmental senescence and some types of pathogen and stress-induced responses. In the present study, we have characterized the cysteine protease Tr-cp 14 in white clover (Trifolium repens). Tr-cp 14 belongs to the C1A family of cysteine proteases with homology to XCP1 and XCP2 from Arabidopsis thaliana and p48h-17 from Zinnia elegans, which previously have been reported to be associated with tracheary element differentiation. The proform as well as the processed form of the protein was detected in petioles, flowers and leaves, but the processed form was more abundant in leaves and petioles than in flowers. The Tr-cp 14 protein was localized to differentiating tracheary elements within the xylem, indicating that the cysteine protease is involved in protein re-mobilization during tracheary element differentiation. Immunogold studies suggest that the protease prior to the burst of the vacuole was associated to the ER cisternae. After disruption of the tonoplast, it was found in the cytoplasm, and, in later stages, associated with disintegrating material dispersed throughout the cell.

Ultrastructural Analyses of Senescence Associated Dismantling of Chloroplasts Revisited

During leaf senescence, chloroplasts are transformed into gerontoplasts involving typical structural changes that have been revealed by electron microscopy for more than 30 years. The structural changes involved in chloroplast-to-gerontoplast transition affect the organization of the thylakoid membrane system which is progressively degraded. In parallel, the number and size of plastoglobules was observed to increase. The internal changes in the structure of chloroplasts occurring during leaf senescence are accompanied by a change from an ellipsoid to a round shape and by a reduction in volume. Recent results on Rubisco degradation involving modern cell biology approaches suggest that plastids during senescence release material including Rubisco and other stromal proteins for degradation outside the organelle. In order to get further insight into the structural changes associated with chloroplast dismantling, we have revisited the pertinent literature and furthermore analyzed the ultrastructure of chloroplasts at different stages of barley leaf senescence and under different conditions leading to yellowing of the leaves. Specific changes at the periphery of chloroplasts at certain stages during aging might be related to an exchange of material between chloroplasts and the endoplasmic reticulum. Electron microscopy cannot, however, discriminate between anterograde and retrograde vesicle movements. Electron lucent areas in the matrix of chloroplasts indicate that protein degradation occurs not only outside but also inside the organelle.

Lack of tocopherols influences the PSII antenna and the functioning of photosystems under low light.

As tocopherols are expected to protect PSII against toxic singlet oxygen it is surprising that the null tocopherol mutant vte1 has been reported to show only a weak enhancement of photosystem II photoinhibition under high irradiance. Based on the view that singlet oxygen is formed also in unstressed conditions, such as low light (LL), we hypothesized that some defense strategies are activated in vte1 in these light conditions. In support for that we noted several symptoms of stress at PSII in the mutant under LL, by means of parameters of fast and slow kinetics of chlorophyll fluorescence and of changes in the relative contribution of PSII antenna in comparison to those of PSI. This was associated with a lower extent of phosphorylation of PSII core proteins (D1 and CP43). PSII RCs do not totally recover from stress in vte1 even after the nocturnal phase. As a clear compensation for the impeded performance of PSII in the vte1 we noted an increased quantum efficiency of PSI. A pronounced changes between WT and the vte1 mutant were also related to conformation of LHCII at the beginning of photoperiod, suggesting the absence of LHCII trimers in the mutant. The thylakoids thickness was similar in WT and vte1 under LL, but a pronounced unstacking of thylakoids was evoked by HL only in vte1. In conclusion, we postulate that action of 1O2 on PSII in vte1 leads to some permanent damage at PSII core and at LHCII already under LL.

Fixierungen für Licht- und Elektronenmikroskopie

Viele mikroskopische Verfahren (z. B. REM, TEM), histologische Farbungen und Nachweise eignen sich nicht fur Lebendmaterial. Daher steht am Anfang vieler Praparationsgange das Abtoten der Zellfunktionen bei gleichzeitiger Erhaltung der Zellstrukturen, die sogenannte Fixierung. Fixierte Proben konnen, je nach Eignung und Fragestellung, geschnitten, gefarbt und mikroskopiert werden. Sie konnen fur das REM getrocknet und beschichtet werden (7 Kap. 8). Oder Sie werden in Paraffin bzw. Kunststoff eingebettet und fur licht- oder elektronenmikroskopische Untersuchungen geschnitten (7 Kap. 6, 7).