Over-accumulation of chloroplast-nucleus located WHIRLY1 in barley leads to a decrease in growth and an enhanced stress resistance.

WHIRLY1 is a chloroplast-nucleus located DNA/RNA-binding protein with functions in development and stress tolerance. By overexpression of HvWHIRLY1 in barley, one line with a 10-fold and two lines with a 50-fold accumulation of the protein were obtained. In these lines, the relative abundance of the nuclear form exceeded that of the chloroplast form. Growth of the plants was shown to be compromised in a WHIRLY1 abundance-dependent manner. Over-accumulation of WHIRLY1 in chloroplasts had neither an evident impact on nucleoid morphology nor on the composition of the photosynthetic apparatus. Nevertheless, oeW1 plants were found to be compromised in the light reactions of photosynthesis as well as in carbon fixation. The reduction in growth and photosynthesis was shown to be accompanied by a decrease in the levels of cytokinins and an increase in the level of jasmonic acid. Gene expression analyses revealed that in nonstress conditions the oeW1 plants had enhanced levels of pathogen response (PR) gene expression indicating activation of constitutive defense. During growth in continuous light of high irradiance PR gene expression increased indicating that under stress conditions oeW1 are capable to further enhance defense.

Barley cysteine protease PAP14 plays a role in degradation of chloroplast proteins.

Chloroplast protein degradation is known to occur both inside chloroplasts and in the vacuole. Genes encoding cysteine proteases have been found to be highly expressed during leaf senescence. However, it remains unclear where they participate in chloroplast protein degradation. In this study HvPAP14, which belongs to the C1A family of cysteine proteases, was identified in senescing barley (Hordeum vulgare L.) leaves by affinity enrichment using the mechanism-based probe DCG-04 targeting cysteine proteases and subsequent mass spectrometry. Biochemical analyses and expression of a HvPAP14:RFP fusion construct in barley protoplasts was used to identify the subcellular localization and putative substrates of HvPAP14. The HvPAP14:RFP fusion protein was detected in the endoplasmic reticulum and in vesicular bodies. Immunological studies showed that HvPAP14 was mainly located in chloroplasts, where it was found in tight association with thylakoid membranes. The recombinant enzyme was activated by low pH, in accordance with the detection of HvPAP14 in the thylakoid lumen. Overexpression of HvPAP14 in barley revealed that the protease can cleave LHCB proteins and PSBO as well as the large subunit of Rubisco. HvPAP14 is involved in the normal turnover of chloroplast proteins and may have a function in bulk protein degradation during leaf senescence.

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.

CAM-related changes in chloroplastic metabolism of Mesembryanthemum crystallinum L.

Crassulacean acid metabolism (CAM) is an intriguing metabolic strategy to maintain photosynthesis under conditions of closed stomata. A shift from C(3) photosynthesis to CAM in Mesembryanthemum crystallinum plants was induced by high salinity (0.4 M NaCl). In CAM-performing plants, the quantum efficiencies of photosystem II and I were observed to undergo distinct diurnal fluctuations that were characterized by a strong decline at the onset of the day, midday recovery, and an evening drop. The temporal recovery of both photosystems' efficiency at midday was associated with a more rapid induction of the electron transport rate at PSII. This recovery of the photosynthetic apparatus at midday was observed to be accompanied by extreme swelling of thylakoids. Despite these fluctuations, a persistent effect of CAM was the acceptor side limitation of PSI during the day, which was accompanied by a strongly decreased level of Rubisco protein. Diurnal changes in the efficiency of photosystems were parallel to corresponding changes in the levels of mRNAs for proteins of PSII and PSI reaction centers and for rbcL, reaching a maximum in CAM plants at midday. This might reflect a high demand for new protein synthesis at this time of the day. Hybridization of run-on transcripts with specific probes for plastid genes of M. crystallinum revealed that the changes in plastidic mRNA levels were regulated at the level of transcription.

The Enigma of Interspecific Plasmodesmata: Insight From Parasitic Plants.

Parasitic plants live in intimate physical connection with other plants serving as their hosts. These host plants provide the inorganic and organic compounds that the parasites need for their propagation. The uptake of the macromolecular compounds happens through symplasmic connections in the form of plasmodesmata. In contrast to regular plasmodesmata, which connect genetically identical cells of an individual plant, the plasmodesmata that connect the cells of host and parasite join separate individuals belonging to different species and are therefore termed "interspecific". The existence of such interspecific plasmodesmata was deduced either indirectly using molecular approaches or observed directly by ultrastructural analyses. Most of this evidence concerns shoot parasitic Cuscuta species and root parasitic Orobanchaceae, which can both infect a large range of phylogenetically distant hosts. The existence of an interspecific chimeric symplast is both striking and unique and, with exceptions being observed in closely related grafted plants, exist only in these parasitic relationships. Considering the recent technical advances and upcoming tools for analyzing parasitic plants, interspecific plasmodesmata in parasite/host connections are a promising system for studying secondary plasmodesmata. For open questions like how their formation is induced, how their positioning is controlled and if they are initiated by one or both bordering cells simultaneously, the parasite/host interface with two adjacent distinguishable genetic systems provides valuable advantages. We summarize here what is known about interspecific plasmodesmata between parasitic plants and their hosts and discuss the potential of the intriguing parasite/host system for deepening our insight into plasmodesmatal structure, function, and development.

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.

WHIRLY2 plays a key role in mitochondria morphology, dynamics, and functionality in Arabidopsis thaliana.

WHIRLY2 is a single-stranded DNA binding protein associated with mitochondrial nucleoids. In the why 2-1 mutant of Arabidopsis thaliana, a major proportion of leaf mitochondria has an aberrant structure characterized by disorganized nucleoids, reduced abundance of cristae, and a low matrix density despite the fact that the macroscopic phenotype during vegetative growth is not different from wild type. These features coincide with an impairment of the functionality and dynamics of mitochondria that have been characterized in detail in wild-type and why 2-1 mutant cell cultures. In contrast to the development of the vegetative parts, seed germination is compromised in the why 2-1 mutant. In line with that, the expression level of why 2 in seeds of wild-type plants is higher than that of why 3, whereas in adult plant no difference is found. Intriguingly, in early stages of shoots development of the why 2-1 mutant, although not in seeds, the expression level of why 3 is enhanced. These results suggest that WHIRLY3 is a potential candidate to compensate for the lack of WHIRLY2 in the why 2-1 mutant. Such compensation is possible only if the two proteins are localized in the same organelle. Indeed, in organello protein transport experiments using intact mitochondria and chloroplasts revealed that WHIRLY3 can be dually targeted into both, chloroplasts and mitochondria. Together, these data indicate that the alterations of mitochondria nucleoids are tightly linked to alterations of mitochondria morphology and functionality. This is even more evident in those phases of plant life when mitochondrial activity is particularly high, such as seed germination. Moreover, our results indicate that the differential expression of why 2 and why 3 predetermines the functional replacement of WHIRLY2 by WHIRLY3, which is restricted though to the vegetative parts of the plant.

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.

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.

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.

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.