Professor John Runions

Plant plasma-membrane (PM) proteins are involved in several vital processes, such as detection of pathogens, solute transport and cellular signalling. For these proteins to function effectively there needs to be structure within the PM allowing,  for example, proteins in the same signalling cascade to be spatially organized. Here we demonstrate that several proteins with divergent functions are located in clusters of differing size in the membrane using sub-diffraction-limited  Airyscan  confocal  microscopy.  Single  particle  tracking  reveals that these  proteins  move  at   different  rates   within  the   membrane.  Actin   and   microtubule cytoskeletons  appear  to  significantly  regulate  the  mobility  of  one  of  these  proteins (the pathogen  receptor  FLS2)  and  we  further demonstrate  that the  cell  wall  is  critical for the regulation of cluster size by quantifying single particle dynamics of proteins with key roles in morphogenesis (PIN3) and pathogen perception (FLS2). We propose a  model in  which the cell wall and cytoskeleton are pivotal for regulation of protein cluster size and dynamics thereby contributing to the formation and functionality of membrane nanodomains.

Studying protein diffusion informs us about how proteins interact with their environment. Work on protein diffusion over the last several decades has illustrated the complex nature of biological lipid bilayers. The plasma membrane contains an array of membrane-spanning proteins or proteins with peripheral membrane associations. Maintenance of plasma membrane microstructure can be via physical features that provide intrinsic ordering such as lipid microdomains, or from membrane-associated structures such as the cytoskeleton. Recent evidence indicates, that in the case of plant cells, the cell wall seems to be a major player in maintaining plasma membrane microstructure. This interconnection / interaction between cell-wall and plasma membrane proteins most likely plays an important role in signal transduction, cell growth, and cell physiological responses to the environment.

SAG21/AtLEA5 belongs to the late embryogenesis-associated (LEA) protein family. Although it has been implicated in growth and redox responses, its precise roles remain obscure. To address this problem, we characterized root and shoot development and response to biotic stress in SAG21/AtLEA5 over-expressor (OEX) and antisense (AS) lines. AS lines exhibited earlier flowering and senescence and reduced shoot biomass. Primary root length was reduced in AS lines, as was the number of laterals relative to the primary root. Root hair number was unchanged but root hair length was proportional to SAG21/AtLEA5 expression level, with longer root hairs in OEX lines and shorter root hairs in AS, relative to wild type. Growth of the fungal nectroph, Botrytis cinerea and of a virulent bacterial pathogen (Pseudomonas syringae pv. tomato) was affected by SAG21/AtLEA5 expression; however, growth of an avirulent P.syringae strain was unaffected. A SAG21/AtLEA5-YFP fusion was localized to mitochondria, raising the intriguing possibility that SAG21 interacts with proteins involved in mitochondrial ROS signalling, which in turn, impacts on root development and pathogen responses.

A cell membrane can be considered a liquid-phase plane in which lipids and proteins theoretically are free to diffuse. Numerous reports, however, describe retarded diffusion of membrane proteins in animal cells. This anomalous diffusion results from a combination of structuring factors including protein–protein interactions, cytoskeleton corralling, and lipid organization into microdomains. In plant cells, plasma-membrane (PM) proteins have been described as relatively immobile, but the control mechanisms that structure the PM have not been studied. Here, we use fluorescence recovery after photobleaching to estimate mobility of a set of minimal PM proteins. These proteins consist only of a PM-anchoring domain fused to a fluorescent protein, but their mobilities remained limited, as is the case for many full-length proteins. Neither the cytoskeleton nor membrane microdomain structure was involved in constraining the diffusion of these proteins. The cell wall, however, was shown to have a crucial role in immobilizing PM proteins. In addition, by single-molecule fluorescence imaging we confirmed that the pattern of cellulose deposition in the cell wall affects the trajectory and speed of PM protein diffusion. Regulation of PM protein dynamics by the plant cell wall can be interpreted as a mechanism for regulating protein interactions in processes such as trafficking and signal transduction.

The constitutive cycling of plant plasma membrane (PM) proteins is an essential component of their function and regulation under resting or stress conditions. Transgenic Arabidopsis plants that express GFP fusions with AtPIP1;2 and AtPIP2;1, two prototypic PM aquaporins, were used to develop a fluorescence recovery after photobleaching (FRAP) approach. This technique was used to discriminate between PM and endosomal pools of the aquaporin constructs, and to estimate their cycling between intracellular compartments and the cell surface. The membrane trafficking inhibitors tyrphostin A23, naphthalene-1-acetic acid and brefeldin A blocked the latter process. By contrast, a salt treatment (100 mm NaCl for 30 min) markedly enhanced the cycling of the aquaporin constructs and modified their pharmacological inhibition profile. Two distinct models for PM aquaporin cycling in resting or salt- stressed root cells are discussed.

Protein sequence analysis of a subfamily of 18 Arabidopsis acyl-activating enzymes (AAE) for organelle targeting signals revealed that eight of them possessed putative peroxisomal targeting signals (PTS1), five of which belonged to Clade VI of the AAE superfamily. Peroxisomal localization was confirmed by confocal microscopy of green fluorescent protein (GFP)-AAE fusion proteins co-localizing with peroxisomal RFP. The sequence analysis also revealed that all enzymes of Clade VI possess N-terminal regions indicative of chloroplast transit peptides (cTP). Among the five Clade VI peroxisomal enzymes tested, masking the PTS1 signal with GFP redirected three to plastids. In addition, three other peroxisomal AAEs appeared to be redirected to plastids in AAE-GFP fusion constructs. Due to the lack of evidence supporting plastid localization, we propose that competition dictates the exclusive localization to peroxisomes. AAE2 of Clade VI was the only enzyme with a putative mitochondrial targeting sequence, and it appeared to be targeted to mitochondria. The remainder of the AAEs appeared to be localized to plastids or cytosol. The AAE9-GFP fusion protein appeared to be located within discreet structures within plastids that may be plastoglobules. AAE15-GFP, but not AAE16-GFP appeared to be located in the chloroplast envelope. The number of examples is increasing whereby proteins located within other compartments contribute to plastid function. We provide an example of this through the light-sensitive phenotype of mutants of AAE2.

G protein-coupled receptor-type G proteins (GTGs) are highly conserved membrane proteins in plants, animals, and fungi that have eight to nine predicted transmembrane domains. They have been classified as G protein-coupled receptor-type G proteins that function as abscisic acid (ABA) receptors in Arabidopsis thaliana. We cloned Arabidopsis GTG1 and GTG2 and isolated new T-DNA insertion alleles of GTG1 and GTG2 in both Wassilewskija and Columbia backgrounds. These gtg1 gtg2 double mutants show defects in fertility, hypocotyl and root growth, and responses to light and sugars. Histological studies of shoot tissue reveal cellular distortions that are particularly evident in the epidermal layer. Stable expression of GTG1(pro):GTG1-GFP (for green fluorescent protein) in Arabidopsis and transient expression in tobacco (Nicotiana tabacum) indicate that GTG1 is localized primarily to Golgi bodies and to the endoplasmic reticulum. Microarray analysis comparing gene expression profiles in the wild type and double mutant revealed differences in expression of genes important for cell wall function, hormone response, and amino acid metabolism. The double mutants isolated here respond normally to ABA in seed germination assays, root growth inhibition, and gene expression analysis. These results are inconsistent with their proposed role as ABA receptors but demonstrate that GTGs are fundamentally important for plant growth and development.

Manipulation of crops to improve their nutritional value (biofortification) and optimisation of plants for removal of toxic metals from contaminated soils (phytoremediation) are major goals. Identification of membrane transporters with roles in zinc and cadmium transport would be useful for both aspects. The P1B-ATPases play important roles in heavy metal allocation and detoxification in Arabidopsis and it is now important to elucidate their roles in monocots. We identified nine P1B-ATPases in barley and this study focuses on the functional characterization of HvHMA2, providing evidence for its role in heavy metal transport. HvHMA2 was cloned using information from EST analysis and 5′ RACE. It possesses the conserved aspartate that is phosphorylated during the reaction cycle of P-type pumps and has motifs and key residues characteristic of P1B-ATPases, falling into the P1B-2 subclass. Homologous sequences occur in three major sub-families of the Poaceae (Gramineae). Heterologous expression inSaccharomyces cerevisiae demonstrates that HvHMA2 functions as a Zn and Cd pump. Mutagenesis studies show that proposed cation coordination sites of the P1B-2 pumps are crucial for the metal responses conferred by HvHMA2 in yeast. HvHMA2 expression suppresses the Zn-deficient phenotype of the Arabidopsis hma2hma4 mutant indicating that HvHMA2 functions as a Zn pump in planta and could play a role in root to shoot Zn transport. When expressed in Arabidopsis, HvHMA2 localises predominantly to the plasma membrane.

Aquaporins of the plasma membrane intrinsic protein (PIP) subfamily are channels which facilitate the diffusion of water across the plant plasma membrane (PM). Although PIPs have been considered as canonical protein markers of this compartment, their endomembrane trafficking is still not well documented. We recently obtained insights into the constitutive cycling of PIPs in Arabidopsis root cells by means of fluorescence recovery after photobleaching (FRAP). This work also uncovered the behavior of the model isoform AtPIP2;1 in response to NaCl. The present addendum connects these findings to another recent work which describes the dynamic properties of AtPIP2;1 in the PM in normal and salt stress conditions by means of single particle tracking (SPT) and fluorescence correlation spectroscopy (FCS). The results suggest that membrane rafts play an important role in the partitioning of AtPIP2;1 in normal conditions and that clathrin-mediated endocytosis is predominant. In salt stress conditions, the rate of AtPIP2;1 cycling was enhanced and endocytosis was cooperated by a membrane raft-associated salt-induced pathway and a clathrin-dependent pathway

A direct interaction of the Arabidopsis thaliana immunophilin ROF1 with phosphatidylinositol-3-phosphate and phosphatidylinositol-3,5-bisphosphate was identified using a phosphatidylinositol-phosphate affinity chromatography of cell suspension extracts, combined with a mass spectrometry (nano LC ESI-MS/MS) analysis. The first FK506 binding domain was shown sufficient to bind to both phosphatidylinositol-phosphate stereoisomers. GFP-tagged ROF1 under the control of a 35S promoter was localised in the cytoplasm and the cell periphery of Nicotiana tabacum leaf explants. Immunofluorescence microscopy of Arabidopsis thaliana root tips verified its cytoplasmic localization and membrane association and showed ROF1 localization in the elongation zone which was expanded to the meristematic zone in plants grown on high salt media. Endogenous ROF1 was shown to accumulate in response to high salt treatment in Arabidopsis thaliana young leaves as well as in seedlings germinated on high salt media (0.15 and 0.2 M NaCl) at both an mRNA and protein level. Plants over-expressing ROF1, (WSROF1OE), exhibited enhanced germination under salinity stress which was significantly reduced in the rof1− knock out mutants and abolished in the double mutants of ROF1 and of its interacting homologue ROF2 (WSrof1−/2−). Our results show that ROF1 plays an important role in the osmotic/salt stress responses of germinating Arabidopsis thaliana seedlings and suggest its involvement in salinity stress responses through a phosphatidylinositol-phosphate related protein quality control pathway.

Actin microfilament (MF) organization and remodelling is critical to cell function. The formin family of actin binding proteins are involved in nucleating MFs in Arabidopsis thaliana. They all contain formin homology domains in the intracellular, C-terminal half of the protein that interacts with MFs. Formins in class I are usually targeted to the plasma membrane and this is true of Formin1 (AtFH1) of A. thaliana. In this study, we have investigated the extracellular domain of AtFH1 and we demonstrate that AtFH1 forms a bridge from the actin cytoskeleton, across the plasma membrane and is anchored within the cell wall. AtFH1 has a large, extracellular domain that is maintained by purifying selection and that contains four conserved regions, one of which is responsible for immobilising the protein. Protein anchoring within the cell wall is reduced in constructs that express truncations of the extracellular domain and in experiments in protoplasts without primary cell walls. The 18 amino acid proline-rich extracellular domain that is responsible for AtFH1 anchoring has homology with cell-wall extensins. We also have shown that anchoring of AtFH1 in the cell wall promotes actin bundling within the cell and that overexpression of AtFH1 has an inhibitory effect on organelle actin-dependant dynamics. Thus, the AtFH1 bridge provides stable anchor points for the actin cytoskeleton and is probably a crucial component of the signalling response and actin-remodelling mechanisms.

Temperature has a direct effect at the cellular level on an organism. For instance, in the case of biomembranes, cooling causes lipids to lose entropy and pack closely together. Reducing temperature should, in the absence of other factors, increase the viscosity of a lipid membrane. We have investigated the effect of temperature variation on plasma membrane (PM) viscosity. We used dispersion tracking of photoactivated green fluorescent protein (GFP) and fluorescence recovery after photobleaching in wild- type and desaturase mutant Arabidopsis thaliana plants along with membrane lipid saturation analysis to monitor the effect of temperature and membrane lipid composition on PM viscosity. Plasma membrane viscosity in A. thaliana is negatively correlated with ambient temperature only under constant-temperature conditions. In the more natural environment of temperature cycles, plants actively manage PM viscosity to counteract the direct effects of temperature. Plasma membrane viscosity is regulated by altering the proportion of desaturated fatty acids. In cold conditions, cell membranes accumulate desaturated fatty acids, which decreases membrane viscosity and vice versa. Moreover, we show that control of fatty acid desaturase 2 (FAD2)-dependent lipid desaturation is essential for this homeostasis of membrane viscosity. Finally, a lack of FAD2 function results in aberrant temperature responses.

A central question in developmental biology concerns the mechanism of generation and maintenance of cell polarity, because these processes are essential for many cellular functions and multicellular development [1]. In plants, cell polarity has an additional role in mediating directional transport of the plant hormone auxin that is crucial for multiple developmental processes [2-4]. In addition, plant cells have a complex extracellular matrix, the cell wall [5, 6], whose role in regulating cellular processes, including cell polarity, is unexplored. We have found that polar distribution of PIN auxin transporters [7] in plant cells is maintained by connections between polar domains at the plasma membrane and the cell wall. Genetic and pharmacological interference with cellulose, the major component of the cell wall, or mechanical interference with the cell wall disrupts these connections and leads to increased lateral diffusion and loss of polar distribution of PIN transporters for the phytohormone auxin. Our results reveal a plant-specific mechanism for cell polarity maintenance and provide a conceptual framework for modulating cell polarity and plant development via endogenous and environmental manipulations of the cellulose-based extracellular matrix.

Cell polarity reflected by asymmetric distribution of proteins at the plasma membrane is a fundamental feature of unicellular and multicellular organisms. It remains conceptually unclear how cell polarity is kept in cell wall-encapsulated plant cells. We have used super-resolution and semi-quantitative live-cell imaging in combination with pharmacological, genetic, and computational approaches to reveal insights into the mechanism of cell polarity maintenance in Arabidopsis thaliana. We show that polar-competent PIN transporters for the phytohormone auxin are delivered to the center of polar domains by super-polar recycling. Within the plasma membrane, PINs are recruited into non-mobile membrane clusters and their lateral diffusion is dramatically reduced, which ensures longer polar retention. At the circumventing edges of the polar domain, spatially defined internalization of escaped cargos occurs by clathrin-dependent endocytosis. Computer simulations confirm that the combination of these processes provides a robust mechanism for polarity maintenance in plant cells. Moreover, our study suggests that the regulation of lateral diffusion and spatially defined endocytosis, but not super-polar exocytosis have primary importance for PIN polarity maintenance. Molecular Systems Biology 7: 540; published online 25 October 2011; doi:10.1038/msb.2011.72

Sad1/UNC-84 (SUN)-domain proteins are inner nuclear membrane (INM) proteins that are part of bridging complexes linking cytoskeletal elements with the nucleoskeleton, and have been shown to be conserved in non-plant systems. In this paper, we report the presence of members of this family in the plant kingdom, and investigate the two Arabidopsis SUN-domain proteins, AtSUN1 and AtSUN2. Our results indicate they contain the highly conserved C-terminal SUN domain, and share similar structural features with animal and fungal SUN-domain proteins including a functional coiled-coil domain and nuclear localization signal. Both are expressed in various tissues with AtSUN2 expression levels relatively low but upregulated in proliferating tissues. Further, we found AtSUN1 and AtSUN2 expressed as fluorescent protein fusions, to localize to and show low mobility in the nuclear envelope (NE), particularly in the INM. Deletion of various functional domains including the N terminus and coiled-coil domain affect the localization and increase the mobility of AtSUN1 and AtSUN2. Finally, we present evidence that AtSUN1 and AtSUN2 are present as homomers and heteromers in vivo, and that the coiled-coil domains are required for this. The study provides evidence suggesting the existence of cytoskeletal-nucleoskeletal bridging complexes at the plant NE.

Controlled movement Of the nucleus is important in a wide variety of plant cellular events Positioning involving intact nuclei occurs in cell division, development, tip growing systems such as the root hair and in response to stimuli, including light, touch and infection. Positioning is also essential in the division and replication of nuclear components, ranging from chromosome attachment to the breakdown and reformation of the nuclear envelope. Although description and understanding of the processes involved have advanced rapidly in recent years, significant gaps remain in our knowledge, especially concerning nuclear proteins involved in anchoring and interacting with cytoskeletal and nucleoskeletal elements involved in movement. In the present review, processes involving the movement and positioning of nuclei and nuclear components are described together with novel proteins implicated in nucleoskeletal and cytoskeletal interactions.

Herein, we report the stepwise transport of multiple plant Golgi membrane markers during disassembly of the Golgi apparatus in tobacco leaf epidermal cells in response to the induced expression of the GTP-locked Sar1p or Brefeldin A (BFA), and reassembly on BFA washout. The distribution of fluorescent Golgi-resident N-glycan processing enzymes and matrix proteins (golgins) with specific cis-trans-Golgi sub-locations was followed by confocal microscopy during disassembly and reassembly. The first event during Golgi disassembly was the loss of trans-Golgi enzymes and golgins from Golgi membranes, followed by a sequential redistribution of medial and cis-Golgi enzymes into the endoplasmic reticulum (ER), whilst golgins were relocated to the ER or cytoplasm. This event was confirmed by fractionation and immuno-blotting. The sequential redistribution of Golgi components in a trans-cis sequence may highlight a novel retrograde trafficking pathway between the trans-Golgi and the ER in plants. Release of Golgi markers from the ER upon BFA washout occurred in the opposite sequence, with cis-matrix proteins labelling Golgi-like structures before cis/medial enzymes. Trans-enzyme location was preceded by trans-matrix proteins being recruited back to Golgi membranes. Our results show that Golgi disassembly and reassembly occur in a highly ordered fashion in plants.

Reticulons are integral endoplasmic reticulum (ER) membrane proteins that have the ability to shape the ER into tubules. It has been hypothesized that their unusually long conserved hydrophobic regions cause reticulons to assume a wedge-like topology that induces membrane curvature. Here we provide proof of this hypothesis. When over-expressed, an Arabidopsis thaliana reticulon (RTNLB13) localized to, and induced constrictions in, cortical ER tubules. Ectopic expression of RTNLB13 was sufficient to induce ER tubulation in an Arabidopsis mutant (pah1 pah2) whose ER membrane is mostly present in a sheet-like form. By sequential shortening of the four transmembrane domains (TMDs) of RTNLB13, we show that the length of the transmembrane regions is directly correlated with the ability of RTNLB13 to induce membrane tubulation and to form low-mobility complexes within the ER membrane. We also show that full-length TMDs are necessary for the ability of RTNLB13 to reside in the ER membrane.

Using a novel analytical tool, this study investigates the relative roles of actin, microtubules, myosin, and Golgi bodies on form and movement of the endoplasmic reticulum (ER) in tobacco (Nicotiana tabacum) leaf epidermal cells. Expression of a subset of truncated class XI myosins, which interfere with the activity of native class XI myosins, and drug-induced actin depolymerization produce a more persistent network of ER tubules and larger persistent cisternae. The treatments differentially affect two persistent size classes of cortical ER cisternae, those >0.3 µm2 and those smaller, called punctae. The punctae are not Golgi, and ER remodeling occurs in the absence of Golgi bodies. The treatments diminish the mobile fraction of ER membrane proteins but not the diffusive flow of mobile membrane proteins. The results support a model whereby ER network remodeling is coupled to the directionality but not the magnitude of membrane surface flow, and the punctae are network nodes that act as foci of actin polymerization, regulating network remodeling through exploratory tubule growth and myosin-mediated shrinkage.

Currently, examination of the cellular structure of plant organs and the gene expression therein largely relies on the production of tissue sections. Here, we present a staining technique that can be used to image entire plant organs using confocal laser scanning microscopy. This technique produces high-resolution images that allow three-dimensional reconstruction of the cellular organization of plant organs. Importantly, three-dimensional domains of gene expression can be analyzed with single-cell precision. We used this technique for a detailed examination of phloem cells in the wild type and mutants. We were also able to recognize phloem sieve elements and their differentiation state in any tissue type and visualize the structure of sieve plates. We show that in the altered phloem development mutant, a hybrid cell type with phloem and xylem characteristics develops from initially normally differentiated protophloem cells. The simplicity of sieve element data collection allows for the statistical analysis of structural parameters of sieve plates, essential for the calculation of phloem conductivity. Taken together, this technique significantly improves the speed and accuracy of the investigation of plant growth and development.

A GFP fusion to the N-terminal 238 amino acids of the mammalian lamin B receptor (LBR) localises to the nuclear envelope (NE) when expressed in Nicotiana tabacum plants, showing properties expected of a native plant NE protein. In this study, we have used this chimaeric construct to explore evidence for common mechanisms of NE targeting and retention between plants and animals, given there is no plant homologue of the mammalian LBR or of one of its binding partners, lamin B. Binding mutants of LBR-GFP were created and fluorescence recovery after photobleaching of mutant and wild type constructs employed to examine their retention in the plant NE. Unmutated LBR-GFP was significantly less mobile in the NE than the lamin binding domain deletion mutant, which was also localised to theER and punctate structures in some cells. Mutation of the chromatin binding domain resulted in localisation of the protein in nuclear inclusions, in which it was immobile. Our findings, that expression of truncated LBR-GFP in plant cells results in altered targeting and retention relative to wt LBR-GFP, suggest that plant cells can recognize the INM-targeting motif of LBR. Altered mobility of the truncated probe indicates that not only do plant cells recognize this signal, but also have nuclear proteins that interact weakly with LBR.

The nuclear envelope (NE) is a double membrane system consisting of the inner nuclear envelope (INE), the outer nuclear envelope (ONE) and nuclear pore complexes (NPCs). Most of our knowledge about the NE proteome comes from studies in animal systems. Recent investigations in plant systems have shown that plants do not have homologues for the majority of animal NE proteins. In a previous study in our laboratory, a construct consisting of the N-terminus of the human lamin B receptor (LBR) fused to GFP was shown to target the plant INE. In mammalian cells, LBR is an intrinsic INE protein, whose targeting to the INE is facilitated by a nuclear localization signal and retention in the INE is achieved by LBR binding mainly to chromatin and lamins. In this study the targeting and retention of LBR–GFP in the plant NE has been investigated by introducing mutations in key domains of LBR and employing fluorescence recovery after photobleaching experiments. Mutation of the chromatin binding domain caused LBR to accumulate in nuclear inclusions in which it was immobile. Deletion of the lamin binding domain resulted in the construct being localized not only to the NE but also ER and to be significantly more mobile then the wild type LBR–GFP in the NE. In the case of both the lamin binding deletion and wild type LBR–GFP, mobility was found to be much greater than previously described in mammalian cells. (Abstracts of the Annual Main Meeting of the Society for Experimental Biology, Glasgow, Scotland, 31st March - 4th April, 2007)

Expression and tracking of fluorescent fusion proteins has revolutionized our understanding of basic concepts in cell biology. The protocol presented here has underpinned much of the in vivo results highlighting the dynamic nature of the plant secretory pathway. Transient transformation of tobacco leaf epidermal cells is a relatively fast technique to assess expression of genes of interest. These cells can be used to generate stable plant lines using a more time-consuming, cell culture technique. Transient expression takes from 2 to 4 days whereas stable lines are generated after approximately 2 to 4 months.

Background information. In a previous study, we showed that GFP (green fluorescent protein) fused to the N-terminal 238 amino acids of the mammalian LBR (lamin B receptor) localized to the NE (nuclear envelope) when expressed in the plant Nicotiana tabacum. The protein was located in the NE during interphase and migrated with nuclear membranes during cell division. Targeting and retention of inner NE proteins requires several mechanisms: signals that direct movement through the nuclear pore complex, presence of a transmembrane domain or domains and retention by interaction with nuclear or nuclear-membrane constituents.

A challenging task in the study of the secretory pathway is the identification and localization of new proteins to increase our understanding of the functions of different organelles. Previous proteomic studies of the endomembrane system have been hindered by contaminating proteins, making it impossible to assign proteins to organelles. Here we have used the localization of organelle proteins by the isotope tagging technique in conjunction with isotope tags for relative and absolute quantitation and 2D liquid chromatography for the simultaneous assignment of proteins to multiple subcellular compartments. With this approach, the density gradient distributions of 689 proteins from Arabidopsis thaliana were determined, enabling confident and simultaneous localization of 527 proteins to the endoplasmic reticulum, Golgi apparatus, vacuolar membrane, plasma membrane, or mitochondria and plastids. This parallel analysis of endomembrane components has enabled protein steady-state distributions to be determined. Consequently, genuine organelle residents have been distinguished from contaminating proteins and proteins in transit through the secretory pathway.

A substantial number of elegant experimental approaches have been developed to image the distribution and dynamics of DNA, mRNA, proteins, organelles, metabolites, and ions in living plant cells. Although the human brain can rapidly assimilate visual information, particularly when presented as animations and movies, it is much more challenging to condense the phenomenal amount of data present in three-, four-, or even five-dimensional images into statistically useful measurements. This review explores a range of in vivo fluorescence imaging applications in plants, with particular emphasis on where quantitative techniques are beginning to emerge.

We have generated a novel genetic system to visualize cell lineages in living tissues at high resolution. Heat shock was used to trigger the excision of a specific transposon and activation of a fluorescent marker gene. A histone-YFP marker was used to allow identification of cell lineages and easy counting of cells. Constitutive expression of a green fluorescent membrane protein was used to provide a precise outline of all surrounding cells. Marked lineages can be induced from specific cells within the organism by targeted laser irradiation, and the fate of the marked cells can be followed non-invasively. We have used the system to map cell lineages originating from the initials of primary and lateral roots in Arabidopsis. The lineage marking technique enabled us to measure the differential contribution of primary root pericycle cell files to developing lateral root primordia. The majority of cells in an emerging lateral root primordium derive from the central file of pericycle founder cells while off-centre founder cells contribute only a minor proliferation of tissue near the base of the root. The system shows great promise for the detailed study of cell division during morphogenesis.

cg12 is an early actinorhizal nodulin gene from Casuarina glauca encoding a subtilisin-like serine protease. Using transgenic Casuarinaceae plants carrying cg12-gus and cg12-gfp fusions, we have studied the expression pattern conferred by the cg12 promoter region after inoculation with Frankia. cg12 was found to be expressed in root hairs and in root and nodule cortical cells containing Frankia infection threads. cg12 expression was also monitored after inoculation with ineffective Frankia strains, during mycorrhizae formation, and after diverse hormonal treatments. None of these treatments was able to induce its expression, therefore suggesting that cg12 expression is linked to plant cell infection by Frankia strains. Possible roles of cg12 in actinorhizal symbiosis are discussed.

Clarkia xantiana has two subspecies that differ in breeding system: ssp. xantiana, which is outcrossing, and ssp. parviflora, which is self-fertilizing. Outcrossing is the ancestral breeding system for the genus Clarkia. Flowers of ssp. parviflora have characteristics commonly associated with selfing taxa: they are smaller and have little temporal and spatial separation between mature anthers and stigma (dichogamy and herkogamy, respectively). Flower morphology and development were studied in four populations of each subspecies to establish the developmental changes that occurred in the evolution of selfing. In particular, we sought to evaluate the hypothesis that the selfing flower may have arisen as a byproduct of selection for rapid maturation in the arid environment occupied by ssp. parviflora. This hypothesis predicts that development time should be reduced in spp. parviflora relative to ssp. xantiana. We also sought to compare the pattern of covariation of flower morphology and development between subspecies to that within subspecies. Similar within vs. between patterns of covariation could be indicative of developmental or functional constraints on the independent evolution of floral parts. In spite of significant variation among populations within subspecies, the subspecies clearly differ in flower morphology and development. All floral organs, except ovaries, are smaller in ssp. parviflora than in ssp. xantiana. The flower plastochron, the duration of flower development from bud initiation to anthesis, and the duration of protandry are all shorter in ssp. parviflora than in ssp. xantiana. Maximum relative growth rates are higher for all organs in ssp. parviflora than in ssp. xantiana. Thus, progenesis (i.e., via a reduction in development time) is combined with growth acceleration in the evolution of the selfing flower. Since reduced development time and growth acceleration both allow selfing flowers to mature earlier than outcrossing ones, selection for early maturation may have contributed to the evolution of the selfing flower form. The pattern of trait covariation differs within spp. parviflora relative to the patterns within spp. xantiana and between the two subspecies, suggesting that floral parts can and have evolved independently of one another.

Characterization of visual opsins can be achieved with the use of specific polyclonal or monoclonal antibodies. In this study, antiserum was raised against a conjugate protein including an N-terminal sequence of the short wavelength sensitive (SWS, commonly referred to as blue-sensitive) visual pigment opsin in goldfish (Carrasius auratus). The antiserum showed immunoreactivity to retinae of phylogenetically distant teleosts, including the goldfish, rainbow trout (Oncorhynchus mykiss), and bluehead wrasse (Thalassoma bisasciatum). Complementary DNA (cDNA) was synthesized from goldfish retinal messenger RNA. An N-terminal sequence of the SWS opsin was PCR-amplified from the cDNA and subsequently cloned into an expression vector. Expressed protein was purified by metal chelate affinity chromatography and used to immunize New Zealand white rabbits. Immune serum was collected and used in immunocytochemical assays, which revealed that the antiserum contains antibodies specific for an opsin that resides in single-cone photoreceptors of the goldfish, rainbow trout, and bluehead wrasse. The results confirm those from earlier studies of goldfish in which short-wavelength sensitivity was localized to single cones, and suggest that the association between short-wavelength sensitivity and single cones may be a feature common to many teleosts. The production of antiserum specific for SWS opsins provides a useful tool in the characterization of opsin expression within teleost retinae.

Sacci of conifer pollen do not function primarily to increase the efficiency of wind pollination as is widely thought. Rather, they are bladders and cause pollen to float upwards in a liquid drop into the ovules. This observation is seemingly unsupported in the case of oriental spruce (Picea orientalis (L.) Link), which has saccate pollen. Ovulate cones are pendant at the time of pollination, which requires that pollen sink into the ovules. Pollen of oriental spruce floats at first but within 1-2 min sinks into the ovule. As sinking does not occur in saccate pollen of other Pinaceae, a variety of techniques was used to determine anatomical differences leading to this uncharacteristic tendency. Light, scanning electron, and confocal microscopy of the pollen surface yielded no significant appearing difference between pollen of oriental spruce and white spruce. However, transmission electron microscopy of freeze-fixed/freeze-substituted hydrated pollen revealed that the ektexine of oriental spruce pollen sacci is porous compared to that of white spruce. Confocal microscopy allowed examination of pollen hydration dynamics. Water enters pollen at the distal pole between sacci, and resulting rapid expansion of the tube cell forces air out of the saccate space. White spruce pollen remains buoyant because of enclosed air pockets in the saccus ektexine. Evolutionary change in pollen wall anatomy with resultant loss of saccus function is correlated with a change in ovulate strobilus orientation at pollination in oriental spruce. A suite of characters interact in the conifer pollination mechanism, and concerted change in these characters may lead to speciation.

Normal sexual reproduction between pollen tube penetration of the archegonium and early embryo formation for interior spruce (Pinaceae) is described. The pollen tube tip penetrates between the neck cells toward the ventral canal cell, and sperm and accompanying cytoplasm are released into the egg. A carbohydrate plug forms around the remains of the pollen tube cell walls and ventral canal cell and seems to plug the archegonium. One sperm fuses with the egg nucleus, and the other stays in the peripheral cytoplasm; modified egg plastids deteriorate, egg mitochondria amass around the zygote nucleus, and male plastids and mitochondria cluster near the zygote nucleus. A 4-nucleate proembryo forms and the nuclei and surrounding neocytoplasm migrate to form a single chalazal tier of proembryo nuclei. Cell divisions and cell wall formations results in a 4-tiered proembryo. The early embryo is forced into the megagametophyte by suspensor tier elongation.

Pollination drops are secreted from the ovules of interior spruce (Picea glauca or Picea engelmannii and their hybrid) as seed cones begin to close at the end of the pollination period. Secreted pollination drops persist within spaces surrounding the micropylar opening in closed seed cones. Saccate pollen floats into the micropyle within the pollination drop. Pollination drops become voluminous enough, within the enclosed spaces, to scavenge pollen adhering to the micropylar arms and other surfaces in proximity with the micropyle. Scavenging of pollen from cone surfaces adjacent to the integuments is sometimes facilitated by rainwater that can float pollen into the opening of the micropyle before cone closure and pollination drop secretion. In practice, periodic, light misting of seed orchard trees during seed cone receptivity might increase pollination efficiency by mimicking rainwater involvement in the pollination mechanism. Rainwater involvement in pollination of some modern conifers may reflect a similar situation in the pollination mechanisms of ancestral conifers. Environments with limited rainfall combined with the requirement for moisture in the pollination mechanism may have provided the selective pressure for evolution of the pollination drop. Keywords: pollination drop, Picea, conifer, sacci.

The pollination mechanism of interior spruce (white spruce, Piceaglauca (Moench) Voss, or Engelmann spruce, Piceaengelmannii Parry, and their hybrid) trees growing in a seed orchard was studied. Seed orchards are established in areas that are warmer and drier than the parent tree natural range. Hot, dry conditions during the pollination period adversely affected secretion of the pollination drop in unbagged seed cones as compared with bagged cones on the same tree. Unbagged cones remained open and apparently receptive for pollination for 5.3 ± 1.6 days (100 ± 31 h at >10°C), but pollination drops did not appear during this period. Pollination drops were secreted as cones began to close. The temporal correlation observed between cone closure and pollination drop secretion occurred as well in the higher relative humidity environment within pollination bags. Pollination drops in unbagged cones were smaller and did not persist as long as those in bagged cones. The micropylar arms, which capture pollen prior to pollination drop secretion, withered completely in unbagged cones before pollination drops were observed. Some withering of micropylar arms during cone receptivity is natural but complete withering means that the arms can not function in pollen capture throughout the period during which cones remain open. For this reason, supplemental mass pollination is best carried out early during the receptive period for trees growing in hot, dry environments. Overhead misting, used periodically during the receptive period to cool orchard trees, might slow seed-cone development and reduce micropylar arm withering, thereby increasing pollination success after supplemental mass pollination.