Nam-Hai Chua, PhD - US grants

Most, if not all, multimeric enzymes in the chloroplast(ct) are made up of subunits whose structural genes are dispersed in two different cellular compartments (nucleus and ct). Our long term objective is to understand how the expression of these genes are coordinated and regulated. As a first step toward this goal, we wish to gather more data on the fine structures of ct genes themselves. Although ct DNA has the potential to code for more than 100 polypeptides, only two of them have been identified so far. This proposal deals with the identification and characterization of cucumber ct genes encoding polypeptide subunits of the photosystem (PS) II complex. Polypeptide subunits that are made inside the ct will be determined by pulse-labeling in the absence or presence of specific inhibitors. Antibodies will be raised against the ct-synthesized subunits which are likely to be ct gene products. Restriction fragments of cucumber ct DNA will be used to select for specific mRNA from a total ct RNA mixture. The selected mRNA will be translated in a reticulocyte lysate system and the products identified by immunoprecipitation with antibodies to PS II polypeptides. These experiments will identify ct genes encoding PS II polypeptides and localize them to specific restriction fragments of known map positions. The 5' initiation site of each mRNA encoding PS II polypeptide will be mapped by a modification of the S1 technique. Each gene will be characterized with respect to its direction of transcription and whether it contains any introns. The 3' and 5' regions of the gene will be sequenced by the Maxam-Gilbert technique to see if there are consensus sequences that may be important in gene regulation. To study ct gene expression and regulation in vitro we will establish a DNA-dependent soluble transcription system using plastid lysates. Truncated fragments of the carboxylase large subunit (LS) gene and the 32 kd polypeptide gene will be used as templates. We predict transcription of both genes in the ct lysate but only the LS gene in the etioplast lysate. We hope to exploit this differential transcriptional response of lysates from the two plastid types of isolate a factor that is specifically required for the transcription of the 32 kd polypeptide gene in ct.

Plants are dependent upon light as a signal for regulating both developmental and metabolic processes, and phytochrome is held to be the principal plant photoreceptor that regulates these responses. The long- term objective of our laboratory is to identify the components of phytochrome signaling pathways and to understand how these pathways are regulated. By developing a microinjection-based experimental system, we have demonstrated the existence of three signal transduction pathways that are downstream of one of the phytochromes (PHYA), and which regulate the expression of genes encoding chloroplast components and anthocyanin biosynthetic enzymes. One pathway is dependent upon calcium, the second upon cGMP, and the third upon both of these effectors. We have established CAB, CHS, and FNR, as specific reporter genes for the activity of each of the three PHYA signaling pathways respectively. We now propose to investigate these pathways further using four approaches: A) To identify individual cis-elements within the CAB, CHS and FNR promoters that can respond to calcium and/or cGMP. This will be carried out by microinjection experiments using various promoter deletions and synthetic promoters containing one or more previously identified cis-elements. Our attention will be focused on binding sites for the transcription factor GT-1 in the CAB promoter, and on G-boxes and Myb-factor binding sites within the CHS promoter. B) In addition to the PHYA signaling intermediates already elucidated, we hope to use our microinjection techniques to characterize others. In particular, we will examine the possible participation of calcium/calmodulin-dependent protein kinase II and guanylyl cyclase. C) Using a mutant isolation screen in Arabidopsis based upon the aberrant expression of a photoregulated reporter gene (CAB- LUC) in response to light, we have isolated a number of mutants which may be altered in PHYA signal transduction. The photobiology and PHYA dependency of mutant phenotypes will be characterized and those clearly affected in PHYA signal transduction will be analyzed to determine the sites of lesions within the signaling pathways. D) Based upon recent experiments e now have some understanding of how individual PHYA signaling pathways are regulated, and how cross-talk mechanisms between each pathway operate. We have devised new screening strategies to isolate mutants in such regulatory mechanisms, based upon the aberrant expression of the CAB- LUC reporter gene or a CHS-LUC reporter gene which has recently been inserted into the Arabidopsis genome in our laboratory. Using these approaches we hope to expand our knowledge not only of the processes by which plants perceive light via phytochrome, but also the regulatory processes which operate to coordinate the expression of genes regulated by phytochrome.

Pattern formation in plants depends upon the precise spatial and temporal control of cell growth. To dissect the molecular mechanisms controlling pattern formation, these investigators have isolated Arabidopsis mutants with altered body organizations. One of these mutants, emb30, is unable to form a true root, a true hypocotyl, stems, or flowers. Instead, most emb30 seedlings contain only abnormal cotyledon tissue and sometimes an abnormal shoot apical meristem. As a result, emb30 mutants are seedling lethals. Microscopy analysis has shown that EMB30 is essential for normal cell division, cell expansion, and cell adhesion in plants and molecular studies have revealed that a region in the predicted EMB30 protein is similar to a region in the yeast Sec7 protein, which is involved in the secretory pathway. The PIs have termed this region the Sec7 domain. Two other open reading frames, one from humans and one from C. elegans, also contain Sec7 domains. The goal of this proposal is to define the specific role of EMB30 in cell growth and organ development. Cell biology and molecular techniques will be employed to examine the effect emb30 mutations have on undifferentiated tissue and adult organ development. Experiments in which the EMB30 gene is placed under the control of an inducible promoter will also allow the examination of the role of EMB30 in the initiation and maintenance of organs, such as the root. To elucidate the specific function of EMB30 in plant cell growth, the components of processes that have been shown or suggested to be affected in emb30 mutants, such as glycoproteins and the cell wall, will be analyzed. The subcellular localization of the EMB30 protein will also be determined. Experiments to determine if EMB30 can complement a yeast sec7 mutant will also be performed. In addition, an Arabidopsis EMB30 homolog will be cloned and characterized. These experiments should elucidate the specific function of EMB30 in particular, and provide i nsight into the processes regulating cell growth and pattern formation, in general.

DESCRIPTION (provided by applicant): Light is an important signal for regulation of plant development and metabolic processes. The photoreceptors for perception of different wavelengths of light have been characterized in Arabidopsis. Amongst these, phytochrome A (phyA), which is activated by far-red light, has emerged as the photoreceptor that plays a major role in seedling de-etiolation. Our long-term objective is to identify and characterize the signaling intermediates and mechanisms involved in transduction of the phyA signal to enable downstream developmental responses. To this end, we have identified mutants pat and lafdefective in phyA signaling. We will focus on identification of the biochemical mechanisms of action of PAT and LAF proteins and how they act with other intermediates in the phyA signaling network. In particular, we are interested in how phyA signals modify these proteins and regulate their abundance. A major focus is to study the regulation of the myb-type transcription activator, LAF1, its downstream targets and interacting proteins. We have shown that COP1, a repressor of photomorphogenesis, is an E3 ligase and can ubiquitinate LAF1 in vitro. The regulation of LAF1 by COP1-mediated ubiquitination in phyA signaling will be investigated in vitro and in transgenic plants. Conjugation of proteins by SUMO (small ubiquitin-like modifier) is emerging as an important regulatory mechanism of signaling. We have indirect evidence that LAF1 could be sumoylated as it is co-localized with COP1 in nuclear speckles, a subnuclear localization pattern associated with SUMO conjugation. We will investigate the role of sumoylation in LAF1 regulation and transmission of phyA responses. The other mutant we will analyze is pat1, which belongs to the plant-specific GRAS family of signal regulators. The possible redundant functional relationship between PAT1 and its close homolog SCL21 in phyA signaling will be examined. A third gene PAT3 and a close homolog PAT3h seem to participate only in a subset of phyA signaling responses. We will characterize the role of these genes in detail in the phyA signal transduction. Characterization of the above proteins will uncover previously unknown mechanisms of phyA signal transduction.

Filamentous fungi grow as a cellular syncytium where individual cells are
interconnected by perforate crosswalls. Cell lysis in these systems triggers a rapid
mechanism of septal pore sealing that is mediated by a dense-core vesicle known as the
Woronin body. The Woronin body has been observed in over 50 species but its origin in
the peroxisome and function associated with the cellular response to cell lysis have only
recently been defined at the molecular level (Jedd and Chua, 2000). In the model system
Neurospora crassa, the Woronin-body core is comprised of Hex1, a protein that is both
necessary and sufficient for Woronin-body core formation. Deletion of the HEX1 gene
eliminates the Woronin body from the cytoplasm and produces hyphae that bleed
cytoplasm following cell lysis. Thus, Hex1 defines a novel peroxisomal vesicle that
functions in the cellular response to cell lysis.
The goal of this research is to understand basic mechanisms of Woronin body
biogenesis and function. As part of this effort, the following specific aims will be
addressed. i) Woronin-body associated proteins will be identified. The Woronin body
will be biochemically purified and associated proteins identified by amino acid
sequencing. Corresponding genes will then be cloned and analyzed to assess their role in
the biogenesis and function of the Woronin body using genetic, biochemical and cell
biological techniques. ii) The conservation of Woronin body structure and function will
be determined by analyzing HEX1 and recently identified HEX1 alternative-splicing
variants in several model ascomycetes using genetic and cell biological techniques. iii) A
mechanism of Woronin body formation will be determined by analyzing the phenotype of
Woronin-body-associated protein deletion strains and by reconstituting Woronin body
formation in yeast cells using heterologous expression of Hex1 and protein interactants. In
addition, the crystal structure of Hex1 will be determined and used to identify amino acid
residues that mediate Hex1 self-assembly. Mutations in these residues will then be used to
determine how Hex1 self-assembly influences Woronin body biogenesis. iv) The
importance of the Woronin body for the pathogenicity of disease-causing fungi will be
determined by deleting HEX1 from the genome of the rice blast fungus Magnaporthe
grisea. The deletion strain will then be examined with respect to infection-related
morphogenesis and host colonization.
Results from this study will enhance our understanding of basic mechanisms
governing vesicle formation and define a previously unrecognized pathway for protein
sorting in the peroxisome. In addition, the possibility that the Woronin body functions as
a pathogenicity factor in disease-causing fungi may define new strategies for combating
fungal pathogens in both plants and humans.

molecular biology; transcription factor; biomedical resource; macromolecule; clinical research; mass spectrometry;

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