Nina S. Allen

A Sec14p-nodulin domain phosphatidylinositol transfer protein polarizes membrane growth of Arabidopsis thaliana root hairs

Phosphatidylinositol (PtdIns) transfer proteins (PITPs) regulate signaling interfaces between lipid metabolism and membrane trafficking. Herein, we demonstrate that AtSfh1p, a member of a large and uncharacterized Arabidopsis thaliana Sec14p-nodulin domain family, is a PITP that regulates a specific stage in root hair development. AtSfh1p localizes along the root hair plasma membrane and is enriched in discrete plasma membrane domains and in the root hair tip cytoplasm. This localization pattern recapitulates that visualized for PtdIns(4,5)P2 in developing root hairs. Gene ablation experiments show AtSfh1p nullizygosity compromises polarized root hair expansion in a manner that coincides with loss of tip-directed PtdIns(4,5)P2, dispersal of secretory vesicles from the tip cytoplasm, loss of the tip f-actin network, and manifest disorganization of the root hair microtubule cytoskeleton. Derangement of tip-directed Ca2+ gradients is also apparent and results from isotropic influx of Ca2+ from the extracellular milieu. We propose AtSfh1p regulates intracellular and plasma membrane phosphoinositide polarity landmarks that focus membrane trafficking, Ca2+ signaling, and cytoskeleton functions to the growing root hair apex. We further suggest that Sec14p-nodulin domain proteins represent a family of regulators of polarized membrane growth in plants.

Plant nuclei can contain extensive grooves and invaginations

Plant cells can exhibit highly complex nuclear organization. Through dye-labeling experiments in untransformed onion epidermal and tobacco culture cells and through the expression of green fluorescent protein targeted to either the nucleus or the lumen of the endoplasmic reticulum/nuclear envelope in these cells, we have visualized deep grooves and invaginations into the large nuclei of these cells. In onion, these structures, which are similar to invaginations seen in some animal cells, form tubular or planelike infoldings of the nuclear envelope. Both grooves and invaginations are stable structures, and both have cytoplasmic cores containing actin bundles that can support cytoplasmic streaming. In dividing tobacco cells, invaginations seem to form during cell division, possibly from strands of the endoplasmic reticulum trapped in the reforming nucleus. The substantial increase in nuclear surface area resulting from these grooves and invaginations, their apparent preference for association with nucleoli, and the presence in them of actin bundles that support vesicle motility suggest that the structures might function both in mRNA export from the nucleus and in protein import from the cytoplasm to the nucleus.

Increasing plasma membrane phosphatidylinositol(4,5)bisphosphate biosynthesis increases phosphoinositide metabolism in Nicotiana tabacum.

A genetic approach was used to increase phosphatidylinositol(4,5)bisphosphate [PtdIns(4,5)P2] biosynthesis and test the hypothesis that PtdInsP kinase (PIPK) is flux limiting in the plant phosphoinositide (PI) pathway. Expressing human PIPKIα in tobacco (Nicotiana tabacum) cells increased plasma membrane PtdIns(4,5)P2 100-fold. In vivo studies revealed that the rate of 32Pi incorporation into whole-cell PtdIns(4,5)P2 increased >12-fold, and the ratio of [3H]PtdInsP2 to [3H]PtdInsP increased 6-fold, but PtdInsP levels did not decrease, indicating that PtdInsP biosynthesis was not limiting. Both [3H]inositol trisphosphate and [3H]inositol hexakisphosphate increased 3-and 1.5-fold, respectively, in the transgenic lines after 18 h of labeling. The inositol(1,4,5)trisphosphate [Ins(1,4,5)P3] binding assay showed that total cellular Ins(1,4,5)P3/g fresh weight was >40-fold higher in transgenic tobacco lines; however, even with this high steady state level of Ins(1,4,5)P3, the pathway was not saturated. Stimulating transgenic cells with hyperosmotic stress led to another 2-fold increase, suggesting that the transgenic cells were in a constant state of PI stimulation. Furthermore, expressing Hs PIPKIα increased sugar use and oxygen uptake. Our results demonstrate that PIPK is flux limiting and that this high rate of PI metabolism increased the energy demands in these cells.

Demonstration of prominent actin filaments in the root columella

Abstract. The distribution of actin filaments within the gravity-sensing columella cells of plant roots remains poorly understood, with studies over numerous years providing inconsistent descriptions of actin organization in these cells. This uncertainty in actin organization, and thus in actins role in graviperception and gravisignaling, has led us to investigate actin arrangements in the columella cells of Zea mays L., Medicago truncatula Gaertn., Linum usitatissimum L. and Nicotiana benthamiana Domin. Actin organization was examined using a combination of optimized immunofluorescence techniques, and an improved fluorochrome-conjugated phalloidin labeling method reliant on 3-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS) cross-linking combined with glycerol permeabilization. Confocal microscopy of root sections labeled with anti-actin antibodies revealed patterns suggestive of actin throughout the columella region. These patterns included short and fragmented actin bundles, fluorescent rings around amyloplasts and intense fluorescence originating from the nucleus. Additionally, confocal microscopy of MBS-stabilized and Alexa Fluor-phalloidin-labeled root sections revealed a previously undetected state of actin organization in the columella. Discrete actin structures surrounded the amyloplasts and prominent actin cables radiated from the nuclear surface toward the cell periphery. Furthermore, the cortex of the columella cells contained fine actin bundles (or single filaments) that had a predominant transverse orientation. We also used confocal microscopy of plant roots expressing endoplasmic reticulum (ER)-targeted green fluorescent protein to demonstrate rapid ER movements within the columella cells, suggesting that the imaged actin network is functional. The successful identification of discrete actin structures in the root columella cells forms the basis for advancing studies on the role of actin in gravity perception and signaling.

The N-terminal Membrane Occupation and Recognition Nexus Domain of Arabidopsis Phosphatidylinositol Phosphate Kinase 1 Regulates Enzyme Activity

The type I B family of phosphatidylinositol phosphate kinases (PIPKs) contain a characteristic region of Membrane Occupation and Recognition Nexus (MORN) motifs at the N terminus. These MORN motifs are not found in PIPKs from other eukaryotes. To understand the impact of the additional N-terminal domain on protein function and subcellular distribution, we expressed truncated and full-length versions of AtPIPK1, one member of this family of PIPKs, in Escherichia coli and in tobacco cells grown in suspension culture. Deletion of the N-terminal MORN domain (amino acids 1–251) of AtPIPK1 increased the specific activity of the remaining C-terminal peptide (ΔMORN) >4-fold and eliminated activation by phosphatidic acid (PtdOH). PtdOH activation could also be eliminated by mutating Pro396 to Ala (P396A) in the predicted linker region between the MORN and the kinase homology domains. AtPIPK1 is product-activated and the MORN domain binds PtdIns(4,5)P2. Adding back the MORN peptide to ΔMORN or to the PtdOH-activated full-length protein increased activity ∼2-fold. Furthermore, expressing the MORN domain in vivo increased the plasma membrane PtdInsP kinase activity. When cells were exposed to hyperosmotic stress, the MORN peptide redistributed from the plasma membrane to a lower phase or endomembrane fraction. In addition, endogenous PtdInsP kinase activity increased in the endomembrane fraction of hyperosmotically stressed cells. We conclude that the MORN peptide can regulate both the function and distribution of the enzyme in a manner that is sensitive to the lipid environment.

Cortical Actin Interacts with the Plasma Membrane and Microtubules

Cortical actin comprises a dynamic array of single actin microfilaments that interacts with the plasma membrane of most plant cells. These interactions, mediated by as yet unknown actin-binding proteins, may function to anchor the entire actin cytoskeleton, and contribute to cell signaling. In cells elongating by diffuse growth, cortical actin is typically transversely oriented, parallel to microtubules. Drug studies demonstrate that in some cases, cortical actin is necessary for the precise re-arrangements of cortical microtubules that control cell expansion and plant morphogenesis. However, in other cases, cortical actin depends on the cortical microtubules for its organization. This suggests that the cortical cytoskeleton is a closely controlled system involving feedback between actin and microtubules.

Growth dynamics and cytoskeleton organization during stem maturation and gravity-induced stem bending in Zea mays L

Abstract. Characterization of gravitropic bending in the maize stem pulvinus, a tissue that functions specifically in gravity responses, demonstrates that the pulvinus is an ideal system for studying gravitropism. Gravistimulation during the second of three developmental phases of the pulvinus induces a gradient of cell elongation across the non-growing cells of the pulvinus, with the most elongation occurring on the lower side. This cell elongation is spatially and temporally separated from normal internodal cell elongation. The three characterized growth phases in the pulvinus correspond closely to a specialized developmental sequence in which structural features typical of cells not fully matured are retained while cell maturation occurs in surrounding internodal and nodal tissue. For example, the lignification of supporting tissue and rearrangement of transverse microtubules to oblique that occur in the internode when cell elongation ceases are delayed for up to 10 d in the adjacent cells of the pulvinus, and only occurs as a pulvinus loses its capacity to respond to gravistimulation. Gravistimulation does not modify this developmental sequence. Neither wall lignification nor rearrangement of transverse microtubules occurs in the rapidly elongating lower side or non-responsive upper side of the pulvinus until the pulvinus loses the capacity to bend further. Gravistimulation does, however, lead to the formation of putative pit fields within the expanding cells of the pulvinus.

An ER-targeted calcium-binding peptide confers salt and drought tolerance mediated by CIPK6 in Arabidopsis

Different plant organelles have high internal stores of Ca2+ compared to the cytoplasm and could play independent roles in stress responses or signal transduction. We used a GFP fusion with the C-domain of calreticulin, which shows low-affinity, high capacity Ca2+ binding in the ER, as a calcium-binding peptide (CBP) to specifically increase stores in the ER and nucleus. Despite the presence of a signal sequence and KDEL retention sequence, our work and previous studies (Brandizzi et al. Plant Journal 34:269–281, 2003) demonstrated both ER and nuclear localization of GFP-CBP. Under normal conditions, GFP-CBP-expressing lines had ~25% more total Ca2+ and higher levels of chlorophyll and seed yield than wild type and GFP controls. CBP-expressing plants also had better survival under intermittent drought or high salt treatments and increased root growth. One member of the CIPK (calcineurin B-like interacting protein kinase) gene family, CIPK6, was up-regulated in CBP-expressing plants, even under non-stress conditions. A null mutation in cipk6 abolished the increased stress tolerance of CBP-transgenic plants, as well as the CBP-mediated induction of two stress-associated genes, DREB1A and RD29A, under non-stress conditions. Although this suggested that it was the induction of CIPK6, rather than localized changes in Ca2+, that resulted in increased survival under adverse conditions, CIPK6 induction still required Ca2+. This work demonstrates that ER (or nuclear) Ca2+ can directly participate in signal transduction to alter gene expression. The discovery of a method for increasing Ca2+ levels without deleterious effects on plant growth may have practical applications.

Viability and In Vitro Germination of Johnsongrass (Sorghum halepense) Pollen

A high proportion of viable pollen grains must germinate to study the physiology of pollen growth to reduce the confounding effects of environmental influences on pollen germination. The objectives of this study were to evaluate the nuclear state and develop a suitable medium and culture method for in vitro germination of johnsongrass pollen. Johnsongrass pollen was trinucleate, and in vitro tests for pollen viability using Alexanders stain and a fluorochromatic reaction method (FCR) indicated johnsongrass pollen was viable (92.6 to 98.4%). A factorial treatment arrangement of four concentrations of sucrose, two concentrations of boric acid, and two concentrations of calcium nitrate were used to determine the optimum pollen-germination medium composition in suspension culture, agar culture, and cellophane membrane culture. Germination was highest in a suspension culture with a medium containing 0.3 M sucrose, 2.4 mM boric acid, and 3 mM calcium nitrate. Pollen germination using this medium was 78.9% when anthers were harvested just before anthesis. Nomenclature: Johnsongrass, Sorghum halepense (L.) Pers. SORHA

Modified LDLs Induce and Bind to Membrane Ruffles on Macrophages

Macrophage foam cell formation in vitro requires uptake of modified low density lipoproteins (LDL) such as acetylated LDL (AcLDL) and moderately oxidized LDL (OxLDL), or beta‐migrating very low density lipoprotein (βVLDL), a naturally occurring lipoprotein. Incubation of macrophages with AcLDL and OxLDL resulted in stimulation of membrane ruffle formation, while βVLDL primarily resulted in increased numbers of microvilli. Time‐lapse Allen video enhanced contrast differential interference contrast (AVEC‐DIC) light microscopy and correlative whole mount intermediate‐voltage transmission electron microscopy (IVEM) was used to examine the dynamics of AcLDL stimulated membrane ruffling and membrane ruffle ultrastructure. Stereo 3D surface replicas confirmed that AcLDL bound to these AcLDL‐induced membrane ruffles. Quantification of the plasma membrane surface area after incubation with AcLDL, βVLDL or LDL confirmed that AcLDL stimulated membrane ruffling, while βVLDL and LDL stimulated microvilli formation. These studies suggest that modified LDLs induce circular membrane ruffles and modified LDLs bind to these ligand‐induced membrane ruffles. Anat Rec 255:44–56, 1999.