Depolarization of alfalfa root hair membrane potential by Rhizobium meliloti Nod factors.

Although much is known about the bacterial genetics of early nodulation, little is known about the plant cell response. Alfalfa root hair cells were impaled with intracellular microelectrodes to measure a membrane potential depolarizing activity in Rhizobium meliloti cell-free filtrates, a plant response dependent on the bacterial nodulation genes. The depolarization was desensitized by repeated exposure to factors and was not observed in a representative nonlegume. A purified extracellular Nod factor, NodRm-IV(S), caused membrane potential depolarization at nanomolar concentrations. This rapid single-cell assay provides a tool for dissecting the mechanisms of host cell response in early nodulation.

Visualization of cellulose synthases in Arabidopsis secondary cell walls.

Cellulose biosynthesis in plant secondary cell walls forms the basis of vascular development in land plants, with xylem tissues constituting the vast majority of terrestrial biomass. We used plant lines that contained an inducible master transcription factor controlling xylem cell fate to quantitatively image fluorescently tagged cellulose synthase enzymes during cellulose deposition in living protoxylem cells. The formation of secondary cell wall thickenings was associated with a redistribution and enrichment of CESA7-containing cellulose synthase complexes (CSCs) into narrow membrane domains. The velocities of secondary cell wall-specific CSCs were faster than those of primary cell wall CSCs during abundant cellulose production. Dynamic intracellular of endomembranes, in combination with increased velocity and high density of CSCs, enables cellulose to be synthesized rapidly in secondary cell walls.

Density-dependent flowering phenology, outcrossing, and reproduction in Impatiens capensis.

We investigated the effects of plant density on cleistogamous (CL) and chasmogamous (CH) flowering phenology and seed production in a natural Impatiens capensis population, by censusing individually marked plants at experimentally reduced and natural densities. CL flowering was earlier at natural density. This plastic density response may have resulted from a stress-related threshold for CL flowering; slower growing plants at natural density flowered earlier. Although apparently triggered by slow early growth, early CL flowering also involved an additional cost for later growth rate. In contrast, CH flowering was unrelated to relative growth rate, but apparently required a size threshold. Experimental density reduction resulted in earlier CH flowering and a dramatic increase in the percentage of plants producing CH flowers. Individual CL and CH flowering duration and flower production were greater at reduced density. These density-dependent effects caused differences between treatments in the shape and location of population flowering phenology curves. Moreover, the percentage of CH seeds produced per individual was much higher at reduced density. At natural density total seed production per plant was lower and more hierarchical than at lower density, suggesting that dominance and suppression shape jewelweed fitness distributions.

Colocalization of cell division proteins FtsZ and FtsA to cytoskeletal structures in living Escherichia coli cells by using green fluorescent protein.

In the current model for bacterial cell division, FtsZ protein forms a ring that marks the division plane, creating a cytoskeletal framework for the subsequent action of other proteins such as FtsA. This putative protein complex ultimately generates the division septum. Herein we report that FtsZ and FtsA proteins tagged with green fluorescent protein (GEP) colocalize to division-site ring-like structures in living bacterial cells in a visible space between the segregated nucleoids. Cells with higher levels of FtsZ-GFP or with FtsA-GFP plus excess wild-type FtsZ were inhibited for cell division and often exhibited bright fluorescent spiral tubules that spanned the length of the filamentous cells. This suggests that FtsZ may switch from a septation-competent localized ring to an unlocalized spiral under some conditions and that FtsA can bind to FtsZ in both conformations. FtsZ-GFP also formed nonproductive but localized aggregates at a higher concentration that could represent FtsZ nucleation sites. The general domain structure of FtsZ-GFP resembles that of tubulin, since the C terminus of FtsZ is not required for polymerization but may regulate polymerization state. The N-terminal portion of Rhizobium FtsZ polymerized in Escherichia coli and appeared to copolymerize with E. coli FtsZ, suggesting a degree of interspecies functional conservation. Analysis of several deletions of FtsA-GFP suggests that multiple segments of FtsA are important for its localization to the FtsZ ring.

Calcium spiking in plant root hairs responding to Rhizobium nodulation signals.

SUMMARY: Rhizobium lipochitooligosaccharide signal molecules stimulate multiple responses in legume host plants, including changes in host gene expression, cell growth, and mitoses leading to root nodule development. The basis for signal transduction in the plant is not known. We examined cytoplasmic free calcium in host root hairs using calcium-sensitive reporter dyes. Image analysis of injected dyes revealed localized periodic spikes in cytoplasmic calcium levels that ensued after a characteristic lag following signal application. Structural features of the signal molecules required to cause nodulation responses in alfalfa are also essential for stimulating calcium spiking. A nonnodulating alfalfa mutant is defective in calcium spiking, consistent with the possibility that this mutant is blocked in an early stage of nodulation signal perception.

Random GFP::cDNA fusions enable visualization of subcellular structures in cells of Arabidopsis at a high frequency.

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 approximately 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 low 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.

In vitro sulfotransferase activity of NodH, a nodulation protein of Rhizobium meliloti required for host-specific nodulation.

Early stages of nodulation involve the exchange of signals between the bacterium and the host plant. Bacterial nodulation (nod) genes are required for Rhizobium spp. to synthesize lipooligosaccharide morphogens, termed Nod factors. The common nod genes encode enzymes that synthesize the factor core structure, which is modified by host-specific gene products. Here we show direct in vitro evidence that Rhizobium meliloti NodH, a host-specific nodulation gene, catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to the terminal 6-O position of Nod factors, and we show substrate requirements for the reaction. Our results indicate that polymerization of the chitooligosaccharide backbone likely precedes sulfation and that sulfation is not absolutely dependent on the presence or the particular structure of the N-acyl modification. NodH sulfation provides a tool for the enzymatic in vitro synthesis of novel Nod factors, or putative Nod factors intermediates, with high specific activity.