Ventilation-induced massive lethal air embolism and subcutaneous emphysema in a patient with a lung cavern.

The simultaneous occurrence of subcutaneous emphysema and intravascular air due to an air embolism is a rare condition. Here, we report a patient with COPD who developed a severe episode of hemoptysis due to rupture of a previously undiagnosed lung cavern. Intubation and ventilation led to the development of both massive subcutaneous emphysema and a massive air embolism, resulting from aspiration of air through a torn pulmonary vessel in the cavern. The dramatic amount of intravenous air and subsequent conduction along the venous system to the right heart and pulmonary trunk caused major hemodynamic compromise and ultimately death. The degree of subcutaneous emphysema, especially the massive venous air embolism, was unprecedented.

Molecular and physiological analysis of growth-limiting drought stress in Brachypodium distachyon leaves.

The drought-tolerant grass Brachypodium distachyon is an emerging model species for temperate grasses and cereal crops. To explore the usefulness of this species for drought studies, a reproducible in vivo drought assay was developed. Spontaneous soil drying led to a 45% reduction in leaf size, and this was mostly due to a decrease in cell expansion, whereas cell division remained largely unaffected by drought. To investigate the molecular basis of the observed leaf growth reduction, the third Brachypodium leaf was dissected in three zones, namely proliferation, expansion, and mature zones, and subjected to transcriptome analysis, based on a whole-genome tiling array. This approach allowed us to highlight that transcriptome profiles of different developmental leaf zones respond differently to drought. Several genes and functional processes involved in drought tolerance were identified. The transcriptome data suggest an increased energy availability in the proliferation zones, along with an up-regulation of sterol synthesis that may influence membrane fluidity. This information may be used to improve the tolerance of temperate cereals to drought, which is undoubtedly one of the major environmental challenges faced by agriculture today and in the near future.

Addressing the role of microRNAs in reprogramming leaf growth during drought stress in Brachypodium distachyon.

Plant responses to drought are regulated by complex genetic and epigenetic networks leading to rapid reprogramming of plant growth. miRNAs have been widely indicated as key players in the regulation of growth and development. The role of miRNAs in drought response was investigated in young leaves of Brachypodium distachyon, a drought-tolerant monocot model species. Adopting an in vivo drought assay, shown to cause a dramatic reduction in leaf size, mostly due to reduced cell expansion, small RNA libraries were produced from proliferating and expanding leaf cells. Next-generation sequencing data were analyzed using an in-house bioinformatics pipeline allowing the identification of 66 annotated miRNA genes and 122 new high confidence predictions greatly expanding the number of known Brachypodium miRNAs. In addition, we identified four TAS3 loci and a large number of siRNA-producing loci that show characteristics suggesting that they may represent young miRNA genes. Most miRNAs showed a high expression level, consistent with their involvement in early leaf development and cell identity. Proliferating and expanding leaf cells respond differently to drought treatment and differential expression analyses suggest novel evidence for an miRNA regulatory network controlling cell division in both normal and stressed conditions and demonstrate that drought triggers a genetic reprogramming of leaf growth in which miRNAs are deeply involved.

Brachypodium distachyon promoters as efficient building blocks for transgenic research in maize.

The biotechnological approach to improve performance or yield of crops or for engineering metabolic pathways requires the expression of a number of transgenes, each with a specific promoter to avoid induction of silencing mechanisms. In maize (Zea mays), used as a model for cereals, an efficient Agrobacterium tumefaciens-mediated transformation system has been established that is applied for translational research. In the current transformation vectors, the promoters of the 35S gene of the cauliflower mosaic virus and of the ubiquitin gene of maize are often used to drive the bialaphos-selectable marker and the transgene, respectively. To expand the number of promoters, genes with either constitutive or seed-specific expression were selected in Brachypodium distachyon, a model grass distantly related to maize. After the corresponding Brachypodium promoters had been fused to the ß-glucuronidase reporter gene, their activity was followed throughout maize development and quantified in a fluorimetric assay with the 4-methylumbelliferyl ß-D-glucuronide substrate. The promoters pBdEF1α and pBdUBI10 were constitutively and highly active in maize, whereas pBdGLU1 was clearly endosperm-specific, hence, expanding the toolbox for transgene analysis in maize. The data indicate that Brachypodium is an excellent resource for promoters for transgenic research in heterologous cereal species.

How MIKC* MADS-box genes originated and evidence for their conserved function throughout the evolution of vascular plant gametophytes.

Land plants have a remarkable life cycle that alternates between a diploid sporophytic and a haploid gametophytic generation, both of which are multicellular and changed drastically during evolution. Classical MIKC MADS-domain (MIKCC) transcription factors are famous for their role in sporophytic development and are considered crucial for its evolution. About the regulation of gametophyte development, in contrast, little is known. Recent evidence indicated that the closely related MIKC* MADS-domain proteins are important for the functioning of the Arabidopsis thaliana male gametophyte (pollen). Furthermore, also in bryophytes, several MIKC* genes are expressed in the haploid generation. Therefore, that MIKC* genes have a similar role in the evolution of the gametophytic phase as MIKCC genes have in the sporophyte is a tempting hypothesis. To get a comprehensive view of the involvement of MIKC* genes in gametophyte evolution, we isolated them from a broad variety of vascular plants, including the lycophyte Selaginella moellendorffii, the fern Ceratopteris richardii, and representatives of several flowering plant lineages. Phylogenetic analysis revealed an extraordinary conservation not found in MIKCC genes. Moreover, expression and interaction studies suggest that a conserved and characteristic network operates in the gametophytes of all tested model organisms. Additionally, we found that MIKC* genes probably evolved from an ancestral MIKCC-like gene by a duplication in the Keratin-like region. We propose that this event facilitated the independent evolution of MIKC* and MIKCC protein networks and argue that whereas MIKCC genes diversified and attained new functions, MIKC* genes retained a conserved role in the gametophyte during land plant evolution.

Abscisic acid, ethylene and gibberellic acid act at different developmental stages to instruct the adaptation of young leaves to stress.

Drought stress represents a particularly great environmental challenge for plants. A decreased water availability can severely limit growth, and this jeopardizes the organism's primary goal-to survive and sustain growth long enough to ensure the plentiful production of viable seeds within the favorable growth season. It is therefore vital for a growing plant to sense oncoming drought as early as possible, and to respond to it rapidly and appropriately in all organs. A typical, fast energy-saving response is the arrest of growth in young organs, which is likely mediated by root-derived signals. A recent publication indicates that three plant hormones (abscisic acid, ethylene and gibberellic acid) mediate the adaptation of leaf growth in response to drought, and that they act at different developmental stages. Abscisic acid mainly acts in mature cells, while ethylene and gibberellic acid function in expanding and dividing leaf cells. This provides the plant with a means to differentially control the developmental zones of a growing leaf, and to integrate environmental signals differently in sink and source tissues. Here we discuss the biological implications of this discovery in the context of long-distance xylem and phloem transport.

The class E floral homeotic protein SEPALLATA3 is sufficient to loop DNA in 'floral quartet'-like complexes in vitro.

The organs of a eudicot flower are specified by four functional classes, termed class A, B, C and E, of MADS domain transcription factors. The combinatorial formation of tetrameric complexes, so called 'floral quartets', between these classes is widely believed to represent the molecular basis of floral organ identity specification. As constituents of all complexes, the class E floral homeotic proteins are thought to be of critical relevance for the formation of floral quartets. However, experimental support for tetrameric complex formation remains scarce. Here we provide physico-chemical evidence that in vitro homotetramers of the class E floral homeotic protein SEPALLATA3 from Arabidopsis thaliana bind cooperatively to two sequence elements termed 'CArG boxes' in a phase-dependent manner involving DNA looping. We further show that the N-terminal part of SEPALLATA3 lacking K3, a subdomain of the protein-protein interactions mediating K domain, and the C-terminal domain, is sufficient for protein dimerization, but not for tetramer formation and cooperative DNA binding. We hypothesize that the capacity of class E MADS domain proteins to form tetrameric complexes contributes significantly to the formation of floral quartets. Our findings further suggest that the spacing and phasing of CArG boxes are important parameters in the molecular mechanism by which floral homeotic proteins achieve target gene specificity.

MADS-complexes regulate transcriptome dynamics during pollen maturation.

BACKGROUND: Differentiation processes are responsible for the diversity and functional specialization of the cell types that compose an organism. The outcome of these processes can be studied at molecular, physiologic, and biochemical levels by comparing different cell types, but the complexity and dynamics of the regulatory processes that specify the differentiation are largely unexplored. RESULTS: Here we identified the pollen-specific MIKC* class of MADS-domain transcription factors as major regulators of transcriptome dynamics during male reproductive cell development in Arabidopsis thaliana. Pollen transcript profiling of mutants deficient in different MIKC* protein complexes revealed that they control a transcriptional switch that directs pollen maturation and that is essential for pollen competitive ability. We resolved the functional redundancy among the MIKC* proteins and uncovered part of the underlying network by identifying the non-MIKC* MADS-box genes AGL18 and AGL29 as downstream regulators of a subset of the MIKC* MADS-controlled genes. CONCLUSION: Our results provide a first, unique, and compelling insight into the complexity of a transcription factor network that directs cellular differentiation during pollen maturation, a process that is essential for male reproductive fitness in flowering plants.

MIKC* MADS-protein complexes bind motifs enriched in the proximal region of late pollen-specific Arabidopsis promoters.

The genome of Arabidopsis (Arabidopsis thaliana) encodes over 100 MADS-domain transcription factors, categorized into five phylogenetic subgroups. Most research efforts have focused on just one of these subgroups (MIKC(c)), whereas the other four remain largely unexplored. Here, we report on five members of the so-called Mdelta or Arabidopsis MIKC* (AtMIKC*) subgroup, which are predominantly expressed during the late stages of pollen development. Very few MADS-box genes function in mature pollen, and from this perspective, the AtMIKC* genes are therefore highly exceptional. We found that the AtMIKC* proteins are able to form multiple heterodimeric complexes in planta, and that these protein complexes exhibit a for the MADS-family unusual and high DNA binding specificity in vitro. Compared to their occurrence in promoters genome wide, AtMIKC* binding sites are strongly overrepresented in the proximal region of late pollen-specific promoters. By combining our experimental data with in silico genomics and pollen transcriptomics approaches, we identified a considerable number of putative direct target genes of the AtMIKC* transcription factor complexes in pollen, many of which have known or proposed functions in pollen tube growth. The expression of several of these predicted targets is altered in mutant pollen in which all AtMIKC* complexes are affected, and in vitro germination of this mutant pollen is severely impaired. Our data therefore suggest that the AtMIKC* protein complexes play an essential role in transcriptional regulation during late pollen development.

Analysis of an Arabidopsis thaliana protein family, structurally related to cytochromes b561 and potentially involved in catecholamine biochemistry in plants.

Cytochromes b561 (cyts b561) constitute a family of eukaryotic membrane proteins, catalysing ascorbate (Asc)-mediated trans-membrane electron transport, and hence likely involved in Asc regeneration. A class of proteins (DoH-CB) has been identified in plants and animals, containing the cyt b561 electron-transport domain (CB), combined with the catecholamine-binding regulatory domain of dopamine-beta-hydroxylase (DoH). A mammalian DoH-CB protein was previously reported to function as a cell-derived growth factor receptor (SDR2). We have performed an in silico analysis on DoH-CB proteins from Arabidopsis thaliana and demonstrate that structural features of both CB and DoH domains are well conserved. The combination of both domains may have evolved from a functional interaction between a cyt b561 and a DoH-containing protein, illustrating the so-called "Rosetta Stone" evolutionary principle, and this hypothesis is supported by sequence comparisons. DoH-CB proteins form a newly identified group of proteins, likely to play a key role in catecholamine action in plants. It is suggested that these proteins may function as trans-membrane electron shuttles, possibly regulated by catecholamines. The role and action of catecholamines in plants is poorly documented, but it is clear that they are involved in many aspects of growth and development. Whether the DoH-CB proteins functionally interact with Asc, as is the case for cyts b561, remains to be determined.

Tissue-specific expression and developmental regulation of cytochrome b561 genes in Arabidopsis thaliana and Raphanus sativus.

Ascorbate (Asc) is an essential molecule in many aspects of development and stress responses in plants and animals. Cytochromes b561 (cyts b561) are tightly coupled to Asc homeostasis. These proteins are found in mammalian tissues, where they are involved in the regeneration of Asc, serving the synthesis of catecholamine neurotransmitters, and in intestinal iron reduction. Plant genomes encode homologous membrane-associated, Asc-reducible cyts b561. The expression of these proteins in plants, however, has so far not been studied. We have now examined the expression of two Arabidopsis thaliana cyt b561-encoding genes-Artb561-1 and Artb561-2-using relative-quantitative RT-PCR and in situ hybridization (ISH) techniques. The genes show overlapping and distinct tissue- and organ-specific expression patterns. Transcripts of both genes are found in leaf epidermal cells, and expression seems to correlate with leaf maturation and cessation of cell elongation. Both genes are also expressed in the epidermal cell layer of stems and roots in the L1 layer of the shoot apex, in the vascular system of leaves, stems and roots, and in the root pericycle. In addition, Artb561-1 is expressed in the root cap, whereas Artb561-2 mRNA is found in the epidermis of lateral roots, in the root meristem, and in unfertilized ovules. These observations provide important information for the elucidation of the physiological function of cyts b561 in plants.

A phylogenetic study of cytochrome b561 proteins.

BACKGROUND: As an antioxidant and cofactor to numerous metabolic enzymes, ascorbate has an essential role in plants and animals. Cytochromes b561 constitute a class of intrinsic membrane proteins involved in ascorbate regeneration. Despite their importance in ascorbate metabolism, no evolutionary analysis has been presented so far on this newly described protein family. RESULTS: Cytochromes b561 have been identified in a large number of phylogenetically distant species, but are absent in fungi and prokaryotes. Most species contain three or four cytochrome b561 paralogous proteins, and the encoding genes usually have four or five exons. At the protein level, sequence similarities are rather low between cytochromes b561 within a single species (34-45% identity), and among phylogenetically distant species (around 30% identity). However, particular structural features characterizing this protein family are well conserved in members from all species investigated. These features comprise six transmembrane helices, four strictly conserved histidine residues, probably coordinating the two heme molecules, and putative ascorbate and monodehydro-ascorbate (MDHA) substrate-binding sites. Analysis of plant cytochromes b561 shows a separation between those from monocotyledonous and dicotyledonous species in a phylogenetic tree. CONCLUSIONS: All cytochromes b561 have probably evolved from a common ancestral protein before the separation of plants and animals. Their phyletic distribution mirrors the use of ascorbate as primary antioxidant, indicating their role in ascorbate homeostasis and antioxidative defense. In plants, the differentiation into four cytochrome b561 isoforms probably occurred before the separation between monocots and dicots.