Bjørn K. Drøbak

Maize Profilin Isoforms Are Functionally Distinct

Profilin is an actin monomer binding protein that, depending on the conditions, causes either polymerization or depolymerization of actin filaments. In plants, profilins are encoded by multigene families. In this study, an analysis of native and recombinant proteins from maize demonstrates the existence of two classes of functionally distinct profilin isoforms. Class II profilins, including native endosperm profilin and a new recombinant protein, ZmPRO5, have biochemical properties that differ from those of class I profilins. Class II profilins had higher affinity for poly-L-proline and sequestered more monomeric actin than did class I profilins. Conversely, a class I profilin inhibited hydrolysis of membrane phosphatidylinositol-4,5-bisphosphate by phospholipase C more strongly than did a class II profilin. These biochemical properties correlated with the ability of class II profilins to disrupt actin cytoplasmic architecture in live cells more rapidly than did class I profilins. The actin-sequestering activity of both maize profilin classes was found to be dependent on the concentration of free calcium. We propose a model in which profilin alters cellular concentrations of actin polymers in response to fluctuations in cytosolic calcium concentration. These results provide strong evidence that the maize profilin gene family consists of at least two classes, with distinct biochemical and live-cell properties, implying that the maize profilin isoforms perform distinct functions in the plant.

Inositol(1,4,5)trisphosphate production in plant cells: an early response to salinity and hyperosmotic stress.

Salinity and hyperosmotic stress are environmental factors that severely affect the growth and development of plants. Adaptation to these stresses is known to be a complex multistep process, but a rise in cytoplasmic Ca2+ and increased polyphosphoinositide turnover have now been identified as being amongst the early events leading to the development of tolerance. To determine whether a causal link exists between these two events we have investigated the effects of several salts and osmotic agents on levels of inositol(1,4,5)trisphosphate (Ins(1,4,5)P3) in plant cells. Our data show that salts as well as osmotic agents induce a rapid and up to 15‐fold increase in cellular Ins(1,4,5)P3 levels. The increase in Ins(1,4,5)P3 occurs in a dose‐dependent manner and levels remain elevated for at least 10 min. These data indicate that increased Ins(1,4,5)P3 production is a common response to salt and hyperosmotic stresses in plants and that it may play an important role in the processes leading to stress tolerance.

Association of Phosphatidylinositol 3-Kinase with Nuclear Transcription Sites in Higher Plants

The kinases responsible for phosphorylation of inositol-containing lipids are essential for many aspects of normal eukaryotic cell function. Genetic and biochemical studies have established that the phosphatidylinositol (PtdIns) 3-kinase encoded by the yeast VPS34 gene is essential for the efficient sorting and delivery of proteins to the vacuole; the kinase encoded by the human VPS34 homolog has been equally implicated in the control of intracellular vesicle traffic. The plant VPS34 homolog also is required for normal growth and development, and although a role for PtdIns 3-kinase in vesicle trafficking is likely, it has not been established. In this study, we have shown that considerable PtdIns 3-kinase activity is associated with the internal matrix of nuclei isolated from carrot suspension cells. Immunocytochemical and confocal laser scanning microscopy studies using the monoclonal antibody JIM135 (John Innes Monoclonal 135), raised against a truncated version of the soybean PtdIns 3-kinase, SPI3K-5p, revealed that this kinase appears to have a distinct and punctate distribution within the plant nucleus and nucleolus. Dual probing of root sections with JIM135 and anti–bromo-UTP antibodies, after in vitro transcription had been allowed to proceed in the presence of bromo-UTP, showed that SPI3K-5p associates with active nuclear and nucleolar transcription sites. These findings suggest a possible link between PtdIns 3-kinase activity and nuclear transcription in plants.

Association of Phosphatidylinositol 4-Kinase with the Plant Cytoskeleton.

In eukaryotic cells, phosphatidylinositol 4-hydroxy kinase and phosphatidylinositol-4-phosphate 5-hydroxy kinase are responsible for the formation of the two second messenger precursors phosphatidylinositol-4-phosphate (Ptdlns(4)P) and phosphatidylinositol-4,5-bisphosphate (Ptdlns(4,5)P2). In plant cells, these kinases have been considered to be exclusively membrane associated, with the majority of activity residing in the inner leaflet of the plasmalemma. By sequentially extracting carrot protoplasts with the detergent Nonidet P-40 then more rigorously with Triton X-100, we were able to remove the activity of three separate plasma membrane marker enzymes and to demonstrate that a significant proportion of cellular Ptdlns 4-kinase is associated with the cytoskeleton. When only endogenous substrates were present, Nonidet P-40-permeabilized protoplasts and Nonidet P-40-extracted cytoskeletons displayed a pattern of lipid phosphorylation similar to that obtained with isolated plant membranes or permeabilized cells, whereas the Triton X-100-extracted cytoskeletons showed little or no activity. In contrast, when exogenous substrates were added, a major proportion of PtdlnsP formed was due to kinase activity associated with the cytoskeleton as well as nuclei. However, by subtracting the activity of isolated nuclei, it could be demonstrated that a significant proportion of the detergent-resistant Ptdlns kinase activity resides with the cytoskeletal fraction. These findings suggest that the pathways of polyphosphoinositide biosynthesis in plant cells should be reevaluated to take account of the cytoskeleton and that Ptdlns(4)P itself may play a unique role in modulation of plant cytoskeletal integrity and cellular signal transduction.

Phosphoinositide kinases and the synthesis of polyphosphoinositides in higher plant cells

Phosphoinositides are a family of inositol-containing phospholipids which are present in all eukaryotic cells. Although in most cells these lipids, with the exception of phosphatidylinositol, constitute only a very minor proportion of total cellular lipids, they have received immense attention by researchers in the past 15-20 years. This is due to the discovery that these lipids, rather than just having structural functions, play key roles in a wide range of important cellular processes. Much less is known about the plant phosphoinositides than about their mammalian counterparts. However, it has been established that a functional phosphoinositide system exists in plant cells and it is becoming increasingly clear that inositol-containing lipids are likely to play many important roles throughout the life of a plant. It is not our intention to give an exhaustive overview of all aspects of the field, but rather we focus on the phosphoinositide kinases responsible for the synthesis of all phosphorylated forms of phosphatidylinositol. Also, we mention some of the aspects of current phosphoinositide research which, in our opinion, are most likely to provide a suitable starting point for further research into the role of phosphoinositides in plants.

Nuclear phosphoinositides could bring FYVE alive

Phosphoinositide signalling systems exist in all eukaryotes. A high degree of evolutionary conservation is found at the functional level, but distinct phylogenetic differences are also becoming evident. Although the nuclear phosphoinositide system is likely to be a primordial forerunner of the plasma membrane system, relatively little is known about it. However, nuclear phosphoinositides might have far more diverse roles than hitherto envisaged and interact specifically with regulatory proteins containing phosphoinositide-binding domains. A novel family of proteins, so far only identified in plants, display domain structures that might link phosphoinositide metabolism to nuclear function in an unexpected way.

ATP‐dependent regulation of nuclear Ca2+ levels in plant cells

Localised alterations in cytoplasmic Ca2+ levels are an integral part of the response of eukaryotic cells to a plethora of external stimuli. Due to the large size of nuclear pores, it has generally been assumed that intranuclear Ca2+ levels reflect the prevailing cytoplasmic Ca2+ levels. Using nuclei prepared from carrot (Daucus carota L.) cells, we now show that Ca2+ can be transported across nuclear membranes in an ATP‐dependent manner and that over 95% of Ca2+ is accumulated into a pool releasable by the Ca2+ ionophore A.23187. ATP‐dependent nuclear Ca2+ uptake did not occur in the presence of ADP or ADPγS and was abolished by orthovanadate. Confocal microscopy of nuclei loaded with dextran‐linked Indo‐1 showed that the initial ATP‐induced rise in [Ca2+] occurs in the nuclear periphery. The occurrence of ATP‐dependent Ca2+ uptake in plant nuclei suggests that alterations of intranuclear Ca2+ levels may occur independently of cytoplasmic [Ca2+] changes.

Alarms and diversions: the biochemistry of development

In considering biochemical aspects of development over a range of different organisms--plants, animals, fungi and bacteria--some ubiquitous themes emerge. Many of the regulatory mechanisms being discovered in higher organisms have already been found in yeast, and there are examples of similar mechanisms in bacteria, notably, analogies and even homologies in multistep cascades involving phosphorylation and negatively acting steps; interplay between development and the cell cycle; and emerging evidence that metabolic pathways can be important developmental agents. On the other hand, those topics that remain resolutely organism-specific may serve as a warning to those who tend to overgeneralize, or as the nucleus for the next generation of general insights.

Second-messenger-induced signalling events in pollen tubes of Papaver rhoeas

A role for cytosolic free Ca2+ (Ca i 2+ ) in the regulation of growth of Papaver rhoeas pollen tubes during the self-incompatibility response has recently been demonstrated [Franklin-Tong et al. Plant J. 4:163–177 (1993); Franklin-Tong et al. Plant J. 8:299–307 (1995); Franklin-Tong et al. submitted to Plant J.]. We have investigated the possibility that Ca i 2+ is more generally involved in the regulation of pollen tube growth using confocal laser scanning microscopy (CLSM). Data obtained using Ca2+ imaging, in conjunction with photolytic release of caged inositol 1,4,5-trisphosphate [Ins(1,4,5)P 3], point to a central role of the phosphoinositide signal transduction pathway in the control of Ca“ fluxes and control of pollen tube growth. These experiments further revealed that increases in cytosolic levels of Ins(1,4,5)P 3 resulted in the formation of distinct Ca2+ waves. Experiments using the pharmacological agents heparin, neomycin and mastoparan further indicated that Ca2+ waves are propagated, at least in part, by Ins(1,4,5)P 3-induced Ca2+ release rather than by simple diffusion or by “classic” Ca2+-induced Ca2+ release mechanisms. We also have data which suggest that Ca2+ waves and oscillations may be induced by photolytic release of caged Ca2+. Ratio-imaging has enabled us to identify an apical oscillating Ca2+ gradient in growing pollen tubes, which may regulate normal pollen tube growth. We also present evidence for the involvement of Ca2+ waves in mediating the self-incompatibility response. Our data suggest that changes in Ca i 2+ and alterations in growth rate/patterns are likely to be closely correlated and may be causally linked to events such as Ca2+-induced, or Ins(1,4,5)P 3-induced wave formation and apical Ca2+ oscillations.