The Arabidopsis ROCK-N-ROLLERS gene encodes a homolog of the yeast ATP-dependent DNA helicase MER3 and is required for normal meiotic crossover formation

Recent studies in Saccharomyces cerevisiae have unveiled that meiotic recombination crossovers are formed by two genetically distinct pathways: a major interference-sensitive pathway and a minor interference-insensitive pathway. Several proteins, including the MSH4/MSH5 heterodimer and the MER3 DNA helicase, are indispensable for the interference-sensitive pathway. MSH4 homologs have been identified in mice and Arabidopsis and shown to be required for normal levels of crossovers, suggesting that the function of MSH4 may be conserved among major eukaryotic kingdoms. However, it is not known whether an MER3-like function is also required for meiosis in animals and plants. We have identified an Arabidopsis gene that encodes a putative MER3 homolog and is preferentially expressed in meiocytes. T-DNA insertional mutants of this gene exhibit defects in fertility and meiosis. Detailed cytological studies indicate that the mutants are defective in homolog synapsis and crossover formation, resulting in a reduction of bivalents and in the formation of univalents at late prophase I. We have named this gene ROCK-N-ROLLERS (RCK) to reflect the mutant phenotype of chromosomes undergoing the meiotic 'dance' either in pairs or individually. Our results demonstrate that an MER3-like function is required for meiotic crossover in plants and provide further support for the idea that Arabidopsis, like the budding yeast, possesses both interference-sensitive and insensitive pathways for crossover formation.     

Jekyll encodes a novel protein involved in the sexual reproduction of barley

Cereal seed development depends on the intimate interaction of filial and maternal tissues, ensuring nourishment of the new generation. The gene jekyll, which was identified in barley (Hordeum vulgare), is preferentially expressed in the nurse tissues. JEKYLL shares partial similarity with the scorpion Cn4 toxin and is toxic when ectopically expressed in Escherichia coli and tobacco (Nicotiana tabacum). In barley, jekyll is upregulated in cells destined for autolysis. The gene generates a gradient of expression in the nucellar projection, which mediates the maternal-filial interaction during seed filling. Downregulation of jekyll by the RNA interference technique in barley decelerates autolysis and cell differentiation within the nurse tissues. Flower development and seed filling are thereby extended, and the nucellar projection no longer functions as the main transport route for assimilates. A slowing down in the proliferation of endosperm nuclei and a severely impaired ability to accumulate starch in the endosperm leads to the formation of irregular and small-sized seeds at maturity. Overall, JEKYLL plays a decisive role in the differentiation of the nucellar projection and drives the programmed cell death necessary for its proper function. We further suggest that cell autolysis during the differentiation of the nucellar projection allows the optimal provision of basic nutrients for biosynthesis in endosperm and embryo.     

ASK1, a SKP1 homolog, is required for nuclear reorganization, presynaptic homolog juxtaposition and the proper distribution of cohesin during meiosis in Arabidopsis

Nuclear reorganization and juxtaposition of homologous chromosomes at late leptotene/early zygotene are essential steps before chromosome synapsis at pachytene. We report the results of detailed studies, which demonstrate that nuclear reorganization and homolog juxtapositioning processes are defective in a null mutant, ask1-1. Our results from 4, 6-diamino-2-phenylindole (DAPI)-stained spreads showed that the “synizetic knot”, which is typically found in wild type (WT) meiosis during late leptotene and zygotene, was missing in the ask1-1 mutant. Furthermore, ask1-1 meiocytes exhibited only limited homolog juxtaposition at centromere regions at early zygotene. Immunodetection of the cohesin protein SYN1 identified ask1 defects in cohesin distribution from zygotene to anaphase I. Analysis of meiotic chromosomes in ask1-1 and syn1 single mutants, as well as an ask1-1 syn1 double mutant indicate that ASK1 is required for normal SYN1 distribution during meiotic prophase I and suggest that ask1 associated defects may be primarily related to SYN1 mislocalization.     

Subtissue-specific evaluation of promoter efficiency by quantitative fluorometric assay in laser microdissected tissues of rapeseed

β-glucuronidase (GUS) is a useful reporter for the evaluation of promoter characteristics in transgenic plants. Here, we introduce an original technique to quantify the strength of promoters at subtissue resolution of cell clusters. The method combines cryotomy, laser microdissection, and improved fluorometric analysis of GUS activity using 6-chloro-4-methylumbelliferyl-β-D-glucuronide as an efficient fluorogenic substrate for kinetic studies in plants. The laser microdissection/6-chloro-4-methylumbelliferyl-β-D-glucuronide method is robust and reliable in a wide range of GUS expression levels and requires extremely low (few cells) tissue amounts. Suitability of the assay was demonstrated on rapeseed (Brassica napus) plants transformed with a P35S2::GUS construct. GUS expression patterns were visualized and quantified in approximately 30 tissues of vegetative and generative organs. Considerable differences in promoter activity within the tissues are discussed in relation to the cell type and developmental state.     

Transient expression of storage-protein genes during early embryogenesis ofVicia faba: synthesis and metabolization of vicilin and legumin in the embryo,suspensor and endosperm

Seed storage proteins are thought to be accumulated exclusively in the cell-expansion phase of embryogenesis and metabolized during germination and seedling growth. Here we show by a sensitive immunohistological technique that the two Vicia faba L. storage proteins vicilin and legumin are accumulated in substantial amounts in the suspensor and coenocytic endosperm and to a lesser extent in the mid-globular embryo. Both proteins appear and disappear at precise stages specific for each tissue. In the endosperm the accumulation starts around 12 d after pollination (DAP). After a maximum attained at 14–15 DAP, storage proteins are degraded within about 4 d. Accumulation is restricted to that part of the endosperm which covers the embryo and displays the highest levels of endoploidy (maximum 96n). In all other parts of the endosperm, storage proteins do not appear to accumulate, although storage-protein-specific mRNA synthesis takes place. In the suspensor, storage proteins are already observed at 6 DAP and disappear very quickly at approximately 10 DAP. Low amounts of legumin and vicilin are also detectable in the mid-globular embryo, but disappear completely as the embryo enters the heart stage. We conclude that storage proteins of Vicia faba accumulated transiently during early seed development are used as nutritive reserves for the growing embryo.Abbreviation DAP days after pollination Dedicated to Prof. Rigomar Rieger in the occasion of his 65th birthdayThis research was supported by the Ministry of Science and Research, Land Sachsen-Anhalt, Germany. U.W. acknowledges additional support by the Fonds der Chemischen Industrie.     

Controlling seed development and seed size in Vicia faba: a role for seed coat-associated invertases and carbohydrate state

We previously provided evidence that seed coat-associated invertase is involved in controlling the carbohydrate state of developing seeds and, by this way, triggering developmental processes (Weber et al . (1995) Plant Cell , 7, 1835–1846). To verify our postulate, we compared seed development of two genotypes of Vicia faba differing in seed weight. The seed coat of the large-seeded genotype formed a higher number of parenchymatous cell layers and matured later. VfCWINV1 encoding a cell wall-bound invertase is expressed in the unloading zone of the seed coat. mRNA levels peaked later in 'large' coats and mRNA was present in more cell layers over a longer time period. Cell wall-bound invertase activity revealed a similar accumulation pattern, obviously generating the high hexose conditions present in the endospermal cavity bathing the premature cotyledons and thus controlling their carbohydrate state. High hexose conditions were correlated with an extended mitotic activity of the 'large' cotyledons. In 'large' and 'small' cotyledons, sucrose levels rose when hexoses decreased apparently terminating cell divisions and initiating differentiation and storage activities. This developmental switch was delayed in 'large' embryos. To prove the outlined relationship, sucrose was added in vitro to mitotically active cotyledons. This treatment favoured nuclear expansion and starch accumulation over cell division. In contrast, a hexose-based medium maintained cell divisions. We conclude that development of the embryo is coordinately regulated with that of the maternal seed coat which controls, by metabolic signals, the phase of cell division of the embryo and consequently also seed size.     

The role of oxygen-binding properties of hemoglobin in the development of oxygen deficiency in acute exogenous hyperthermia

Acid-base balance and oxygen-binding hemoglobin properties in mixed venous blood have been studied in 25 mongrel rabbits with acute environmental hyperthermia. As oxygen-hemoglobin affinity at standard pH, pCO2 and temperature increases, the effect of heat on oxygen-hemoglobin interaction is considerably attenuated. The Bohr effect increases. The mechanisms of changes in oxygen-binding properties of hemoglobin and their role in development of oxygen deficiency are discussed.     

Energy status and its control on embryogenesis of legumes: ATP distribution within Vicia faba embryos is developmentally regulated and correlated with photosynthetic capacity

To analyse the energy status of Vicia faba embryos in relation to differentiation processes, we measured ATP concentrations directly in cryosections using a quantitative bioluminescence-based imaging technique. This method provides a quantitative picture of the ATP distribution close to the in vivo situation. ATP concentrations were always highest within the axis. In pre-storage cotyledons, the level was low, but it increased strongly in the course of further development, starting from the abaxial region of cotyledons and moving towards the interior. Greening pattern, chlorophyll distribution and photosynthetic O2 production within embryos temporally and spatially corresponded to the ATP distribution, implicating that the overall increase of the energy state is associated to the greening process. ATP patterns were associated to the photosynthetic capacity of the embryo. The general distribution pattern as well as the steady state levels of ATP were developmentally regulated and did not change upon dark/light conditions. The major storage protein legumin started to accumulate in abaxial regions with high ATP, whereas starch localized in regions with relatively lower ATP levels. This suggests a role of the energy state in the partitioning of assimilates into the different storage-product classes. Highest biosynthetic rates occurred when cotyledons became fully green and contained high ATP levels, implicating that a photoheterotrophic state was required to ensure high fluxes. Based on these data, we propose a model for the role of embryonic photosynthesis to improve the energy status of the embryo.