During sulfur-limited growth, the cyanobacterium Synechococcus sp. strain PCC 7942 loses most of its photosynthetic pigments and develops an increased capacity to acquire sulfate. Sulfur deprivation also triggers the synthesis of several soluble polypeptides. We have isolated a prominent polypeptide of 33 kDa that accumulates specifically under sulfur-limiting conditions. This polypeptide was localized to the periplasmic space. The gene for this protein (designated rhdA) was isolated and discovered to lie within a region of the Synechococcus sp. strain PCC 7942 genome that encodes components of the sulfate permease system. The mRNA for the 33-kDa protein accumulates to high levels within an hour after the cells are deprived of sulfur and drops rapidly when sulfur is added back to the cultures. The amino acid sequence of the protein has similarity to bovine liver rhodanese, an enzyme that transfers the thiol group of thiosulfate to a thiophilic acceptor molecule, and a rhodaneselike protein of Saccharopolyspora erythraea. A strain in which rhdA was interrupted by a drug resistance marker exhibited marginally lower levels of rhodanese activity but was still capable of efficiently utilizing a variety of inorganic sulfur sources. The possible role of this protein in the transport of specific sulfur compounds is discussed.

We describe a general approach for identifying components of subcellular structures in a multicellular organism by exploiting the ability to generate thousands of independent transformants in Arabidopsis thaliana. A library of Arabidopsis cDNAs was constructed so that the cDNAs were inserted at the 3' end of the green fluorescent protein (GFP) coding sequence. The library was introduced en masse into Arabidopsis by Agrobacterium-mediated transformation. Fluorescence imaging of 5,700 transgenic plants indicated that approximate to 2% of lines expressed a fusion protein with a different subcellular distribution than that of soluble GFP, About half of the markers identified were targeted to peroxisomes or other subcellular destinations by non-native coding sequence (i.e., out-of-frame cDNAs). This observation suggests that some targeting signals are of sufficiently law information content that they can be generated frequently by chance, The potential of the approach for identifying markers with unique dynamic processes is demonstrated by the identification of a GFP fusion protein that displays a cell-cycle regulated change in subcellular distribution. Our results indicate that screening GFP-fusion protein libraries is a useful approach for identifying and visualizing components of subcellular structures and their associated dynamics in higher plant cells.

Live-cell imaging has yielded surprising pictures of subcellular structures and dynamics in living plant cells. Recent studies illustrate the power of live-cell observation for revealing new biological phenomena and for generating new questions about plant cell structure and function.

Most microalgae are obligate photoautotrophs and their growth is strictly dependent on the generation of photosynthetically derived energy. We show that the microalga Phaeodactylum tricornutum can be genetically engineered to thrive on exogenous glucose in the absence of light through the introduction of a gene encoding a glucose transporter (glut1 or hup1). This demonstrates that a fundamental change in the metabolism of an organism can be accomplished through the introduction of a single gene. This also represents progress toward the use of fermentation technology for large-scale commercial exploitation of algae by reducing limitations associated with light-dependent growth.

The view of plant-cell cytokinesis commonly depicted in textbooks is of a symmetrical process, with the phragmoplast initiating in the center of the cell and growing outward to the parental cell membrane. In contrast to this picture, we observe that cell-plate development in Arabidopsis shoot cells is highly polarized along the plane of division. Three-dimensional live-cell imaging reveals that the mitotic spindle and phragmoplast are laterally displaced, and that the growing cell plate anchors on one side of the cell at an early stage of cytokinesis. Growth of phragmoplast across the cell creates a new partition in its wake, giving the visual effect of a curtain being pulled across the cell. Throughout this process, the advancing front of the phragmoplast is in intimate contact with the parental wall, suggesting that short-range interactions between the phragmoplast and plasma membrane may play important roles in guiding the cell plate throughout much of its development. Polarized cytokinesis was observed in a wide variety of vacuolate shoot cells and in some small root cells, implying that it is not solely a function of cell size. This mode of cytokinesis may provide a mechanically robust mechanism for cell-plate formation in large cells and suggests a simple explanation for the occurrence of cell wall stubs observed upon drug treatment or in cytokinetic mutants.

Plant cells create highly structured microtubule arrays at the cell cortex without a central organizing center to anchor the microtubule ends. In vivo imaging of individual microtubules in Arabidopsis plants revealed that new microtubules are initiated at the cell cortex and exhibit dynamics at both ends. Polymerization-biased dynamic instability at one end and slow depolymerization at the other end result in sustained microtubule migration across the cell cortex by a hybrid treadmilling mechanism. This motility causes widespread microtubule repositioning and contributes to changes in array organization through microtubule reorientation and bundling.

Fluorescent proteins are generating fresh insight into plant cell function by providing new opportunities to visualize structure and dynamic events in live cells. Novel and transient structures, such as discreet locations in the nucleus where activated photoreceptor proteins accumulate, have recently been identified with fluorescent protein tags. Fluorescent proteins have also enabled the discovery of new dynamic molecular behaviors, such as the repositioning of cortical microtubules by polymer treadmilling. The early potential of fluorescent proteins to reveal protein interactions in living cells is being realized, as demonstrated in recent studies of transcription factors and signal transduction proteins. A promising new approach to the creation of fluorescent-protein-based biosensors has produced an exciting family of tools for visualizing small sugars, and perhaps will produce a wide variety of other small molecules in the future.

We developed a high-throughput methodology, termed fluorescent tagging of full-length proteins (FTFLP), to analyze expression patterns and subcellular localization of Arabidopsis gene products in planta. Determination of these parameters is a logical first step in functional characterization of the approximately one-third of all known Arabidopsis genes that encode novel proteins of unknown function. Our FTFLP-based approach offers two significant advantages: first, it produces internally-tagged full-length proteins that are likely to exhibit native intracellular localization, and second, it yields information about the tissue specificity of gene expression by the use of native promoters. To demonstrate how FTFLP may be used for characterization of the Arabidopsis proteome, we tagged a series of known proteins with diverse subcellular targeting patterns as well as several proteins with unknown function and unassigned subcellular localization.

The sku6-1 mutant of Arabidopsis thaliana exhibits altered patterns of root and organ growth. sku6 roots, etiolated hypocotyls, and leaf petioles exhibit right-handed axial twisting, and root growth on inclined agar media is strongly right skewed. The touch-dependent sku6 root skewing phenotype is suppressed by the antimicrotubule drugs propyzamide and oryzalin, and right skewing is exacerbated by cold treatment. Cloning revealed that sku6-1 is allelic to spiral1-1 (spr1-1). However, modifiers in the Columbia (Col) and Landsberg erecta (Ler) ecotype backgrounds mask noncomplementation in sku6-1 (Col)/spr1-1 (Ler) F1 plants. The SPR1 gene encodes a plant-specific 12-kD protein that is ubiquitously expressed and belongs to a six-member gene family in Arabidopsis. An SPR1:green fluorescent protein (GFP) fusion expressed in transgenic seedlings localized to microtubules within the cortical array, preprophase band, phragmoplast, and mitotic spindle. SPR1:GFP was concentrated at the growing ends of cortical microtubules and was dependent on polymer growth state; the microtubule-related fluorescence dissipated upon polymer shortening. The protein has a repeated motif at both ends, separated by a predicted rod-like domain, suggesting that it may act as an intermolecular linker. These observations suggest that SPR1 is involved in microtubule polymerization dynamics and/or guidance, which in turn influences touch-induced directional cell expansion and axial twisting.