R. Howard Berg

Ultrastructure of the membrane systems in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803

Summary.Among prokaryotes, cyanobacteria are unique in having highly differentiated internal membrane systems. Like other Gram-negative bacteria, cyanobacteria such as Synechocystis sp. strain PCC 6803 have a cell envelope consisting of a plasma membrane, peptidoglycan layer, and outer membrane. In addition, these organisms have an internal system of thylakoid membranes where the electron transfer reactions of photosynthesis and respiration occur. A long-standing controversy concerning the cellular ultrastructures of these organisms has been whether the thylakoid membranes exist inside the cell as separate compartments, or if they have physical continuity with the plasma membrane. Advances in cellular preservation protocols as well as in image acquisition and manipulation techniques have facilitated a new examination of this topic. We have used a combination of electron microscopy techniques, including freeze-etched as well as freeze-substituted preparations, in conjunction with computer-aided image processing to generate highly detailed images of the membrane systems in Synechocystis cells. We show that the thylakoid membranes are in fact physically discontinuous from the plasma membrane in this cyanobacterium. Thylakoid membranes in Synechocystis sp. strain PCC 6803 thus represent bona fide intracellular organelles, the first example of such compartments in prokaryotic cells.

Loss‐of‐function mutations and inducible RNAi suppression of Arabidopsis LCB2 genes reveal the critical role of sphingolipids in gametophytic and sporophytic cell viability

Serine palmitoyltransferase (SPT) catalyzes the first step in sphingolipid biosynthesis, and downregulation of this enzyme provides a means for exploring sphingolipid function in cells. We have previously demonstrated that Arabidopsis SPT requires LCB1 and LCB2 subunits for activity, as is the case in other eukaryotes. In this study, we show that Arabidopsis has two genes (AtLCB2a and AtLCB2b) that encode functional isoforms of the LCB2 subunit. No alterations in sphingolipid content or growth were observed in T-DNA mutants for either gene, but homozygous double mutants were not recoverable, suggesting that these genes are functionally redundant. Reciprocal crosses conducted with Atlcb2a and Atlcb2b mutants indicated that lethality is associated primarily with the inability to transmit the lcb2 null genotype through the haploid pollen. Consistent with this, approximately 50% of the pollen obtained from plants homozygous for a mutation in one gene and heterozygous for a mutation in the second gene arrested during transition from uni-nucleate microspore to bicellular pollen. Ultrastructural analyses revealed that these pollen grains contained aberrant endomembranes and lacked an intine layer. To examine sphingolipid function in sporophytic cells, Arabidopsis lines were generated that allowed inducible RNAi silencing of AtLCB2b in an Atlcb2a mutant background. Studies conducted with these lines demonstrated that sphingolipids are essential throughout plant development, and that lethality resulting from LCB2 silencing in seedlings could be partially rescued by supplying exogenous long-chain bases. Overall, these studies provide insights into the genetic and biochemical properties of SPT and sphingolipid function in Arabidopsis.

GS52 Ecto-Apyrase Plays a Critical Role during Soybean Nodulation

Apyrases are non-energy-coupled nucleotide phosphohydrolases that hydrolyze nucleoside triphosphates and nucleoside diphosphates to nucleoside monophosphates and orthophosphates. GS52, a soybean (Glycine soja) ecto-apyrase, was previously shown to be induced very early in response to inoculation with the symbiotic bacterium Bradyrhizobium japonicum. Overexpression of the GS52 ecto-apyrase in Lotus japonicus increased the level of rhizobial infection and enhanced nodulation. These data suggest a critical role for the GS52 ecto-apyrase during nodulation. To further investigate the role of GS52 during nodulation, we used RNA interference to silence GS52 expression in soybean (Glycine max) roots using Agrobacterium rhizogenes-mediated root transformation. Transcript levels of GS52 were significantly reduced in GS52 silenced roots and these roots exhibited reduced numbers of mature nodules. Development of the nodule primordium and subsequent nodule maturation was significantly suppressed in GS52 silenced roots. Transmission electron micrographs of GS52 silenced root nodules showed that early senescence and infected cortical cells were devoid of symbiosome-containing bacteroids. Application of exogenous adenosine diphosphate to silenced GS52 roots restored nodule development. Restored nodules contained bacteroids, thus indicating that extracellular adenosine diphosphate is important during nodulation. These results clearly suggest that GS52 ecto-apyrase catalytic activity is critical for the early B. japonicum infection process, initiation of nodule primordium development, and subsequent nodule organogenesis in soybean.

A member of the highly conserved FWL (tomato FW2.2-like) gene family is essential for soybean nodule organogenesis

A soybean homolog of the tomato FW2.2 gene, here named GmFWL1 (Glycine max FW2.2-like 1), was found to respond strongly to inoculation with the nitrogen-fixing symbiotic bacterium Bradyrhizobium japonicum. In tomato, the FW2.2 gene is hypothesized to control 30% of the variance in fruit weight by negatively regulating cell division. In the present study, the induction of GmFWL1 expression in root hair cells and nodules in response to B. japonicum inoculation was documented using quantitative RT-PCR and transcriptional fusions to both beta-glucuronidase (GUS) and green fluorescent protein (GFP). RNAi-mediated silencing of GmFWL1 expression resulted in a significant reduction in nodule number, with a concomitant reduction in nuclear size and changes in chromatin structure. The reduction in nuclear size is probably due to a change in DNA heterochromatinization, as the ploidy level of wild-type and RNAi-silenced nodule cells was similar. GmFWL1 was localized to the plasma membrane. The data suggest that GmFWL1 probably acts indirectly, perhaps through a cellular cascade, to affect chromatin structure/nuclei architecture. As previously proposed in tomato, this function may be a result of effects on plant cell division.

Fluorescent protein applications in plants.

Study of plant cell biology has benefited tremendously from the use of fluorescent proteins (FPs). Development of well-established techniques in genetics, by transient expression or by Agrobacterium-mediated plant cell transformation, makes it possible to readily create material for imaging molecules tagged with FPs. Confocal microscopy of FPs is routine and, in highly scattering tissues, multiphoton microscopy improves deep imaging. The abundance of autofluorescent compounds in plants in some cases potentially interferes with FP signals, but spectral imaging is an effective tool in unmixing overlapping signals. This approach allows separate detection of DsRed and chlorophyll, DsRed and GFP, and green fluorescent protein (GFP) and yellow fluorescent protein (YFP). FPs have been targeted to most plant organelles. Free (untargeted) FPs in plant cells are not only cytoplasmic, but also go into the nucleus due to their small size. FP fluorescence is potentially unstable in acidic vacuoles. FPs have been targeted to novel compartments, including protein storage vacuoles in seeds. Endoplasmic reticulum (ER)-targeted GFP has identified novel inclusion bodies that are surprisingly dynamic. FP-tagged Rab GTPases have allowed documentation of the dynamics of membrane trafficking. Investigation of virus infections has progressed significantly with the aid of FP-tagged virus proteins. Advanced techniques are giving plant scientists the ability to quantitatively analyze the behavior of FP-tagged proteins. Fluorescence lifetime microscopy is becoming the method of choice for fluorescence resonance energy transfer (FRET) analysis of FP-tagged proteins. Fluorescence correlation spectroscopy (FCS) of FPs provides information on molecular diffusion and intermolecular interactions. Use of FPs in elucidating the behavior of plant cells has a bright future.

Unique Thylakoid Membrane Architecture of a Unicellular N2-Fixing Cyanobacterium Revealed by Electron Tomography

Cyanobacteria, descendants of the endosymbiont that gave rise to modern-day chloroplasts, are vital contributors to global biological energy conversion processes. A thorough understanding of the physiology of cyanobacteria requires detailed knowledge of these organisms at the level of cellular architecture and organization. In these prokaryotes, the large membrane protein complexes of the photosynthetic and respiratory electron transport chains function in the intracellular thylakoid membranes. Like plants, the architecture of the thylakoid membranes in cyanobacteria has direct impact on cellular bioenergetics, protein transport, and molecular trafficking. However, whole-cell thylakoid organization in cyanobacteria is not well understood. Here we present, by using electron tomography, an in-depth analysis of the architecture of the thylakoid membranes in a unicellular cyanobacterium, Cyanothece sp. ATCC 51142. Based on the results of three-dimensional tomographic reconstructions of near-entire cells, we determined that the thylakoids in Cyanothece 51142 form a dense and complex network that extends throughout the entire cell. This thylakoid membrane network is formed from the branching and splitting of membranes and encloses a single lumenal space. The entire thylakoid network spirals as a peripheral ring of membranes around the cell, an organization that has not previously been described in a cyanobacterium. Within the thylakoid membrane network are areas of quasi-helical arrangement with similarities to the thylakoid membrane system in chloroplasts. This cyanobacterial thylakoid arrangement is an efficient means of packing a large volume of membranes in the cell while optimizing intracellular transport and trafficking.

Characterization of mutant tobacco mosaic virus coat protein that interferes with virus cell-to-cell movement

Expression of tobacco mosaic virus (TMV) coat protein (CP) in plants confers resistance to infection by TMV and related tobamoviruses. Certain mutants of the CP (CPT42W) provide much greater levels of resistance than wild-type (wt) CP. In the present work, infection induced by RNA transcripts of TMV clones that contain wt CP or mutant CPT42W fused to the green fluorescent protein (GFP) (TMV-CP:GFP, TMV-CPT42W:GFP) and clones harboring TMV movement protein (MP):GFP were followed in nontransgenic and transgenic tobacco BY-2 protoplasts and Nicotiana tabaccum Xanthi-nn plants that express wt CP or CPT42W. On nontransgenic and wt CP transgenic plants, TMV-CP:GFP produced expanding, highly fluorescent disk-shaped areas. On plants expressing CPT42W, infection by TMV-CP:GFP or TMV-MP:GFP-CP produced infection sites of smaller size that were characterized by low fluorescence, reflecting reduced levels of virus spread and reduced accumulation of both CP:GFP and MP:GFP. TMV-CPT42W:GFP failed to produce visible infection sites on nontransgenic plants, yet produced normal infection sites on MP-transgenic plants that produce MP. TMV infection of transgenic BY-CPT42W protoplasts resulted in very low levels of MP accumulation, whereas on BY-CP protoplasts (containing wt CP), infection produced higher levels of MP than in nontransgenic BY-2 cells. The results suggest that wt CP has a positive effect on the production of MP, whereas the CPT42W has a negative effect on MP accumulation and/or function. This effect results in very high levels of resistance to TMV infection in plants containing CPT42W. This report shows that the CP of a plant virus regulates production of the MP, and that a mutant CP interferes with MP accumulation and cell-to-cell movement of infection.

Cell Biology of Plant Nematode Parasitism

Plant Infection by Root-Knot Nematode.- Parasitism Genes: What They Reveal about Parasitism.- Molecular Insights in the Susceptible Plant Response to Nematode Infection.- Resistant Plant Responses.- Development of the Root-Knot Nematode Feeding Cell.- Structure of Cyst Nematode Feeding Sites.- Transcriptomic Analysis of Nematode Infestation.- Genomic Analysis of the Root-Knot Nematode Genome.- Molecular Approaches Toward Resistance to Plant-Parasitic Nematodes.

14-3-3 proteins SGF14c and SGF14l play critical roles during soybean nodulation.

The soybean (Glycine max) genome contains 18 members of the 14-3-3 protein family, but little is known about their association with specific phenotypes. Here, we report that the Glyma0529080 Soybean G-box Factor 14-3-3c (SGF14c) and Glyma08g12220 (SGF14l) genes, encoding 14-3-3 proteins, appear to play essential roles in soybean nodulation. Quantitative reverse transcription-polymerase chain reaction and western-immunoblot analyses showed that SGF14c mRNA and protein levels were specifically increased in abundance in nodulated soybean roots 10, 12, 16, and 20 d after inoculation with Bradyrhizobium japonicum. To investigate the role of SGF14c during soybean nodulation, RNA interference was employed to silence SGF14c expression in soybean roots using Agrobacterium rhizogenes-mediated root transformation. Due to the paleopolyploid nature of soybean, designing a specific RNA interference sequence that exclusively targeted SGF14c was not possible. Therefore, two highly similar paralogs (SGF14c and SGF14l) that have been shown to function as dimers were silenced. Transcriptomic and proteomic analyses showed that mRNA and protein levels were significantly reduced in the SGF14c/SGF14l-silenced roots, and these roots exhibited reduced numbers of mature nodules. In addition, SGF14c/SGF14l-silenced roots contained large numbers of arrested nodule primordia following B. japonicum inoculation. Transmission electron microscopy further revealed that the host cytoplasm and membranes, except the symbiosome membrane, were severely degraded in the failed nodules. Altogether, transcriptomic, proteomic, and cytological data suggest a critical role of one or both of these 14-3-3 proteins in early development stages of soybean nodules.

Alfalfa mosaic virus replicase proteins, P1 and P2, localize to the tonoplast in the presence of virus RNA.

To identify the virus components important for assembly of the Alfalfa mosaic virus replicase complex, we used live cell imaging of Arabidopsis thaliana protoplasts that expressed various virus cDNAs encoding native and GFP-fusion proteins of P1 and P2 replicase proteins and full-length virus RNAs. Expression of P1-GFP alone resulted in fluorescent vesicle-like bodies in the cytoplasm that colocalized with FM4-64, an endocytic marker, and RFP-AtVSR2, RabF2a/Rha1-mCherry, and RabF2b/Ara7-mCherry, all of which localize to multivesicular bodies (MVBs), which are also called prevacuolar compartments, that mediate traffic to the lytic vacuole. GFP-P2 was driven from the cytosol to MVBs when expressed with P1 indicating that P1 recruited GFP-P2. P1-GFP localized on the tonoplast, which surrounds the vacuole, in the presence of infectious virus RNA, replication competent RNA2, or P2 and replication competent RNA1 or RNA3. This suggests that a functional replication complex containing P1, P2, and a full-length AMV RNA assembles on MVBs to traffic to the tonoplast.