Karin Krupinska

Pheophytin pheophorbide hydrolase (pheophytinase) is involved in chlorophyll breakdown during leaf senescence in Arabidopsis.

During leaf senescence, chlorophyll is removed from thylakoid membranes and converted in a multistep pathway to colorless breakdown products that are stored in vacuoles. Dephytylation, an early step of this pathway, increases water solubility of the breakdown products. It is widely accepted that chlorophyll is converted into pheophorbide via chlorophyllide. However, chlorophyllase, which converts chlorophyll to chlorophyllide, was found not to be essential for dephytylation in Arabidopsis thaliana. Here, we identify pheophytinase (PPH), a chloroplast-located and senescence-induced hydrolase widely distributed in algae and land plants. In vitro, Arabidopsis PPH specifically dephytylates the Mg-free chlorophyll pigment, pheophytin (phein), yielding pheophorbide. An Arabidopsis mutant deficient in PPH (pph-1) is unable to degrade chlorophyll during senescence and therefore exhibits a stay-green phenotype. Furthermore, pph-1 accumulates phein during senescence. Therefore, PPH is an important component of the chlorophyll breakdown machinery of senescent leaves, and we propose that the sequence of early chlorophyll catabolic reactions be revised. Removal of Mg most likely precedes dephytylation, resulting in the following order of early breakdown intermediates: chlorophyll → pheophytin → pheophorbide. Chlorophyllide, the last precursor of chlorophyll biosynthesis, is most likely not an intermediate of breakdown. Thus, chlorophyll anabolic and catabolic reactions are metabolically separated.

Leaf senescence and nutrient remobilisation in barley and wheat

Extensive studies have been undertaken on senescence processes in barley and wheat and their importance for the nitrogen use efficiency of these crop plants. During the senescence processes, proteins are degraded and nutrients are re-mobilised from senescing leaves to other organs, especially the developing grain. Most of the proteins degraded reside in the chloroplasts, with Rubisco constituting the most dominant protein fraction. Despite intensive studies, the proteases responsible for Rubisco degradation have not yet been identified. Evidence for degradation of stromal proteins outside of chloroplasts is summarised. Rubisco is thought to be released from chloroplasts into vesicles containing stroma material (RCB = Rubisco-containing bodies). These vesicles may then take different routes for their degradation. Transcriptome analyses on barley and wheat senescence have identified genes involved in degradative, metabolic and regulatory processes that could be used in future strategies aimed at modifying the senescence process. The breeding of crops for characters related to senescence processes, e.g. higher yields and better nutrient use efficiency, is complex. Such breeding has to cope with the dilemma that delayed senescence, which could lead to higher yields, is correlated with a decrease in nutrient use efficiency. Pinpointing regulatory genes involved in senescence might lead to tools that could effectively overcome this dilemma.

Plant senescence and crop productivity

Senescence is a developmental process which in annual crop plants overlaps with the reproductive phase. Senescence might reduce crop yield when it is induced prematurely under adverse environmental conditions. This review covers the role of senescence for the productivity of crop plants. With the aim to enhance productivity, a number of functional stay-green cultivars have been selected by conventional breeding, in particular of sorghum and maize. In many cases, a positive correlation between leaf area duration and yield has been observed, although in a number of other cases, stay-green cultivars do not display significant effects with regards to productivity. In several crops, the stay-green phenotype is observed to be associated with a higher drought resistance and a better performance under low nitrogen conditions. Among the approaches used to achieve stay-green phenotypes in transgenic plants, the expression of the IPT gene under control of senescence-associated promoters has been the most successful. The promoters employed for senescence-regulated expression contain cis-elements for binding of WRKY transcription factors and factors controlled by abscisic acid. In most crops transformed with such constructs the stay-green character has led to increased biomass, but only in few cases to increased seed yield. A coincidence of drought stress resistance and stay-green trait is observed in many transgenic plants.

Complete DNA sequences of the plastid genomes of two parasitic flowering plant species, Cuscuta reflexa and Cuscuta gronovii

BackgroundThe holoparasitic plant genus Cuscuta comprises species with photosynthetic capacity and functional chloroplasts as well as achlorophyllous and intermediate forms with restricted photosynthetic activity and degenerated chloroplasts. Previous data indicated significant differences with respect to the plastid genome coding capacity in different Cuscuta species that could correlate with their photosynthetic activity. In order to shed light on the molecular changes accompanying the parasitic lifestyle, we sequenced the plastid chromosomes of the two species Cuscuta reflexa and Cuscuta gronovii. Both species are capable of performing photosynthesis, albeit with varying efficiencies. Together with the plastid genome of Epifagus virginiana, an achlorophyllous parasitic plant whose plastid genome has been sequenced, these species represent a series of progression towards total dependency on the host plant, ranging from reduced levels of photosynthesis in C. reflexa to a restricted photosynthetic activity and degenerated chloroplasts in C. gronovii to an achlorophyllous state in E. virginiana.ResultsThe newly sequenced plastid genomes of C. reflexa and C. gronovii reveal that the chromosome structures are generally very similar to that of non-parasitic plants, although a number of species-specific insertions, deletions (indels) and sequence inversions were identified. However, we observed a gradual adaptation of the plastid genome to the different degrees of parasitism. The changes are particularly evident in C. gronovii and include (a) the parallel losses of genes for the subunits of the plastid-encoded RNA polymerase and the corresponding promoters from the plastid genome, (b) the first documented loss of the gene for a putative splicing factor, MatK, from the plastid genome and (c) a significant reduction of RNA editing.ConclusionOverall, the comparative genomic analysis of plastid DNA from parasitic plants indicates a bias towards a simplification of the plastid gene expression machinery as a consequence of an increasing dependency on the host plant. A tentative assignment of the successive events in the adaptation of the plastid genomes to parasitism can be inferred from the current data set. This includes (1) a loss of non-coding regions in photosynthetic Cuscuta species that has resulted in a condensation of the plastid genome, (2) the simplification of plastid gene expression in species with largely impaired photosynthetic capacity and (3) the deletion of a significant part of the genetic information, including the information for the photosynthetic apparatus, in non-photosynthetic parasitic plants.

Constitutive overexpression of barley 4-hydroxyphenylpyruvate dioxygenase in tobacco results in elevation of the vitamin E content in seeds but not in leaves1

With the aim to enhance the plant vitamin E content, the barley gene encoding 4‐hydroxyphenylpyruvate dioxygenase was overexpressed in tobacco plants under control of the 35S promoter. Transgenic lines have a higher capacity for homogentisate biosynthesis as evident by a more than 10‐fold higher resistance towards the bleaching herbicide sulcotrione. Seeds from transgenic lines have an up to two‐fold enhanced level of vitamin E without a change in the ratio of γ‐tocopherol and γ‐tocotrienol. While the vitamin E content is not affected in leaves, the level of plastoquinone is enhanced in leaves of transgenic lines during leaf senescence.

Disruption of plastid-encoded RNA polymerase genes in tobacco : expression of only a distinct set of genes is not based on selective transcription of the plastid chromosome

Abstract Plastids of higher plants operate with at least two distinct DNA-dependent RNA polymerases, which are encoded in the organelle (PEP) and in the nucleus (NEP), respectively. Plastid run-on assays and Northern analyses were employed to analyse gene expression in tobacco mutant plastids lacking the PEP genes rpoA, rpoB or rpoC1. Hybridisation of run-on transcripts to restriction fragments representing the entire tobacco plastid chromosome, as well as to selected plastid gene-specific probes, shows that all parts of the plastid DNA are transcribed in rpo-deficient plastids. In comparison to wild-type chloroplasts, which are characterized by preferential transcription of photosynthesis-related genes in the light, mutant plastids exhibit a different transcription pattern with less pronounced differences in the hybridisation intensities between the individual genes. The analysis of steady-state transcript patterns and transcription rates of selected genes in both types of plastids demonstrates that differences in transcription rates are not necessarily paralleled by corresponding changes in transcript levels. The accumulation of large transcripts in the mutant plastids indicates that processing of primary transcripts may be impaired in the absence of PEP. These data suggest that, contrary to the prevailing view, much of the regulation of NEP-driven plastid gene expression in the rpo-deficient mutants is not based on differential promoter usage but is exerted at post-transcriptional levels.

A Plant Locus Essential for Phylloquinone (Vitamin K1) Biosynthesis Originated from a Fusion of Four Eubacterial Genes

Phylloquinone is a compound present in all photosynthetic plants serving as cofactor for Photosystem I-mediated electron transport. Newly identified seedling-lethal Arabidopsis thaliana mutants impaired in the biosynthesis of phylloquinone possess reduced Photosystem I activity. The affected gene, called PHYLLO, consists of a fusion of four previously individual eubacterial genes, menF, menD, menC, and menH, required for the biosynthesis of phylloquinone in photosynthetic cyanobacteria and the respiratory menaquinone in eubacteria. The fact that homologous men genes reside as polycistronic units in eubacterial chromosomes and in plastomes of red algae strongly suggests that PHYLLO derived from a plastid operon during endosymbiosis. The principle architecture of the fused PHYLLO locus is conserved in the nuclear genomes of plants, green algae, and the diatom alga Thalassiosira pseudonana. The latter arose from secondary endosymbiosis of a red algae and a eukaryotic host indicating selective driving forces for maintenance and/or independent generation of the composite gene cluster within the nuclear genomes. Besides, individual menF genes, encoding active isochorismate synthases (ICS), have been established followed by splitting of the essential 3′ region of the menF module of PHYLLO only in genomes of higher plants. This resulted in inactivation of the ICS activity encoded by PHYLLO and enabled a metabolic branch from the phylloquinone biosynthetic route to independently regulate the synthesis of salicylic acid required for plant defense. Therefore, gene fusion, duplication, and fission events adapted a eubacterial multienzymatic system to the metabolic requirements of plants.

Comparative survey of plastid and mitochondrial targeting properties of transcription factors in Arabidopsis and rice

A group of nuclear transcription factors, the Whirly proteins, were recently shown to be targeted also to chloroplasts and mitochondria. In order to find out whether other proteins might share this feature, an in silico-based screening of transcription factors from Arabidopsis and rice was carried out with the aim of identifying putative N-terminal chloroplast and mitochondrial targeting sequences. For this, the individual predictions of several independent programs were combined to a consensus prediction using a naïve Bayes method. This consensus prediction shows a higher specificity at a given sensitivity value than each of the single programs. In both species, transcription factors from a variety of protein families that possess putative N-terminal plastid or mitochondrial target peptides as well as nuclear localization sequences, were found. A search for homologues within members of the AP2/EREBP protein family revealed that target peptide-containing proteins are conserved among monocotyledonous and dicotyledonous species. Fusion of one of these proteins to GFP revealed, indeed, a dual targeting activity of this protein. We propose that dually targeted transcription factors might be involved in the communication between the nucleus and the organelles in plant cells. We further discuss how recent results on the physical interaction between the organelles and the nucleus could have significance for the regulation of the localization of these proteins.

The hydroxyphenylpyruvate dioxygenase from Synechocystis sp. PCC 6803 is not required for plastoquinone biosynthesis

The disruption of the Synechocystis open reading frame Δslr0090 encoding a gene with high homology to plant genes encoding 4‐hydroxyphenylpyruvate dioxygenase results in an impairment of tocopherol biosynthesis without affecting levels of plastoquinone, carotenoids and chlorophyll as well as cell growth and photosynthesis. Our results indicate that in Synechocystis in contrast to the situation in higher plants the 4‐hydroxyphenylpyruvate dioxygenase is not required for the synthesis of plastoquinone.

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.