Ellen M. Reardon

A guide to naming sequenced plant genes

A scientist preparing an article on bacterial genes knows that there are canonical designations for genes ofE. coli: araA, dnaJ, lacZ .... (CGSC Files and Database). Similarly, another scientist writing about genes of chloroplasts or mitochondria knows that these also have standard designations: atpE, cox3, rbcL, ... [3, 4, 6]. The advantages to scientific communication in having a common genetic language are obvious, but, until recently, names for genes of nuclear genomes of plants have resembled a Tower of Babel. The purpose of this article is to identify sources for recommended designations for sequenced plant genes and the avenues for researchers to propose designations for newly discovered plant genes.

[16] Isolation of plastids in density gradients of percoll and other silica sols

Publisher Summary This chapter discusses methods for the isolation of plastids in density gradients of Percoll and other silica sols. The most commonly used silica sols are Percoll, which is quite expensive, and Ludox HS and Ludox AM, which are very cheap. Silica sol gradients are generally useful for the isolation of different kinds of plastids from a wide variety of higher plants, including mesophyll and bundle sheath chloroplasts and heat-bleached chloroplasts, and from at least three kinds of algae. Separation in silica sol gradients has become the method of choice for the study of plastid composition, for the isolation of plastids other than chloroplasts, and to reisolate plastids from a complex incubation mixture where it is necessary. Plastids recovered from silica sol gradients have been used for the isolation of plastid DNA from spinach, tobacco, pepper, and Cyanophora paradoxas. Gradient-purified plastids have also been used for the studies on photosynthesis and on salt and calmodulin contents of chloroplasts.

Plastid genomes of three non-green algae are sequenced

T he total sequences of the chloroplast genomes of Marchantia poh/nlorpha (Ohyama et al., 1986) and Nicotiana tabacum (Shinozaki et al., 1986) were reported just nine years ago (see box borrowed from Stirewalt et al., 1995). Apart from the excitement caused by having complete sequences of the largest genomes to that time, these historic discoveries shifted our focus from the question, What genes are present ? to How is their expression regulated? The sequencing of plastid genomes of liverwort and tobacco were followed by those of rice (Hiratsuka et al., 1989), black pine (Wakasugi et al., 1994), Epifagus virginiana (Wolfe et al., 1992), Euglena gracilis (Hallick et al., 1993), Zea mays (Maier et al., 1995), and now Chlorella ellipsoidea (M. Sugiura, personal communication). The accumulated data permit us to make detailed comparisons among plastid genomes: The similarities between vascular and non-vascular plants and between dicots and monocots are far more abundant than are the differences.

Mendel, a database of nomenclature for sequenced plant genes.

The Mendel database contains names for plant-wide families of sequenced plant genes. The names have either been approved by the Commission on Plant Gene Nomenclature (CPGN), an organization of the International Society for Plant Molecular Biology (ISPMB), or are identified as provisional or temporary names. Mendel also identifies the corresponding genes in individual species of plants. Mendel can be searched through the mirror sites at Cornell (http://genome. cornell.edu/cgi-bin/WebAce/webace?db=mendel) and Stanford (http://genome-www.stanford.edu/Mendel/). In addition, parts of Mendel can be downloaded from the CPGN Web site (http://mbclserver. rutgers.edu/CPGN/).

Isolation of chloroplasts and chloroplast DNA

That chloroplasts are green and therefore recognizable by eye should have been an advantage in developing methods for their isolation, but historically it has been a disadvantage. It was relatively easy to grind up cells, subject the brei to differential centrifugation, and obtain a green pellet. It was also easy to infer that the pellet contained chloroplasts when it actually consisted of the swollen relics of thylakoid membranes. Much was learned about the light reactions of photosynthesis from studying such suspensions of thylakoids, but they were useless for the study of CO2 fixation or other processes that we now know occur in chloroplasts. A fundamental principle of biochemistry is (or should be) that we can only claim that a process has been reconstructed in vitro when the isolated system is shown to mimic the process in vitro both qualitatively and quantitatively. Failure to do so should not be explained away by ad-hoc hypotheses.

Chapter 14 Chromoplasts

Publisher Summary Plastids have many differentiation states, including proplastids, chloroplasts, amyloplasts, etioplasts, gerontoplasts, and chromoplasts. These various forms of organelles play very different roles and are mostly interconvertible. An exception is gerontoplasts, the terminal state of chloroplasts in senescent leaves. Chromoplasts are plastids in which exceptional amounts of carotenoids accumulate. As a consequence, chromoplasts confer bright colors to plant tissues: shades of orange, yellow, and red. Chromoplasts, therefore, are the quintessential plastids of flowers and fruits, attracting insects for pollination and animals for seed dispersal. Chromoplasts may originate from chloroplasts, proplastids, or leukoplasts. The accumulation of carotenoids is independent of the presence or absence of chlorophyll. The function of chromoplasts is esthetic, making plant organs attractive to animals, such as humans. Therefore, they are not essential to the life of the plant. Chromoplast carotenoids have provided a rich substrate for organic chemistry with many chemical species serving as botanical species markers.

Cytoplasmic regulation of chloroplast translation in Euglena gracilis

A regulatory role for cytoplasmically derived proteins in chloroplast translation in organello was examined by analyzing protein synthesis in plastids isolated from cells of Euglena gracilis which had been treated with cycloheximide (CHI). Incorporation of [35S]methionine by chloroplasts from CHI-inhibited Euglena was reduced approximately 40 and 90% by exposure of the cells to the antibiotic for 2 and 4 h, respectively. The chloroplast translation products were then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. The synthesis of polypeptides in the soluble compartment of the plastid was substantially diminished by as little as 15 min of CHI pretreatment. No qualitative alterations of the polypeptide pattern were detected. Qualitative changes were seen in the thylakoid fraction, however. Comparison of the stainable polypeptides and fluorographs of thylakoid membranes from CHI-treated cells with those of controls showed several instances in which the more slowly migrating member of a doublet accumulated with a concomitant depletion of a more rapidly migrating component. A pair of polypeptides at 28 and 30 kDa, which we believe are the Euglena homologs of the photogene product and its precursor, respectively, are representative of this phenomenon. Additionally, thylakoids from cells pretreated with CHI sometimes synthesized novel polypeptides larger than 65 kDa. Finally, when intact chloroplasts from CHI-inhibited Euglena were incubated with a postchloroplast supernatant from normal cells, there was a partial reversion of the anomalies seen in the fluorographs. These data are interpreted to indicate the cytoplasmic origin of one or more proteins whose function is to process chloroplast translation products.

Release of 7 of Mendel, the CPGN's Database of Sequenced Plant Genes

Mendel 7is more than twice as large as its predecessor and is contemporary with EMBL/GenBank sequence databases to 5 February 1999; it will be updated monthly. In addition, all protein sequences from Swiss-Prot , release 37, are included with monthly updates expected. Mendel 7includes the complete accessions of non-green algae and cyanobacteria in addition to the continuing coverage of higher plants and green algae. Mendel 7lists many new gene family names, including those related to alcohol dehydrogenase, light-harvesting proteins, methyl transferases, transporters, and chloroplast YCFs. Many gene families that had been omitted or obscured in Me del 6are restored; these include families encoding RNAs, catalase, sucrose synthase, and subunits of RNA polymerase subunits. A new feature on the CPGN Web site entitled ‘Conversations’ is available to facilitate interactive discussions of nomenclature for proposed or provisional gene families. The CPGN is restoring a number of other features to Mendel 7, including the fields defining alleles, subgenomes and links to other databases. It retains, however, the model of sorting proteins by sequence similarity, which was introduced by the Norich group under the direction of David Lonsdale. This feature was the key to making Mendelcontemporary with the sequence

Tenth Annual Penn State Symposium in Plant Physiology: Phytochemicals and Health

The Tenth Annual Penn State Symposium in Plant Physiology was organized by Hector Flores and David Gustine and held in State College, Pennsylvania, USA, from 18 to 20 May 1995. The topic this year was Phytochemicals and Health and if anything, exceeded the reputation for excellence of previous symposia. The casual browser of the Reporter is probably asking how a meeting on phytochemicals relates to molecular biology; the answer is that the time for molecular exploitation of phytochemicals is now. Acknowledging that 80 percent of the pharmaceuticals now marketed are derived from plants, and the explosion of research in the chemistry and biochemistry of secondary metabolites, it follows that phytochemicals will represent a vitally important area of research well into the next millennium. Six sessions categorized the topics introduced: biochemistry and regulation of phytochemicals, their use in plant, their importance to human health, nitrogenous phytochemicals, steroid phytochemicals, and the future of phytochemical diversity. The latter topic, biodiversity, reflects a nagging theme [cf. PMBR 12(3):285-290] that must continue to be addressed. The f o u n d a t i o n for u n d e r s t a n d i n g the b road d iv is ions of phytochemicals, their synthesis and relationships was laid by Eric Conn (Davis). The early products of photosynthesis provide the primary sources of carbon atoms for thousands of natural products that occur in plants. Most phytochemicals derive from three metabolic pathways and a few amino acids: 9 The shikimate pathway: phenylalanine, tyrosine and tryptophan, end products of the pathway, are precursors of many alkaloids; phenylalanine is also the precursor of many phenylpropanoids that include phenolic acids, coumarins, flavonoids and lignin. 9 The isoprenoid pathway: utilizes acetyl CoA to form mevalonic acid, the precursor of numerous classes of terpenoid compounds including sterols and carotenoids.