Bark and Leaf Lectins of Sophora japonica Are Sequestered in Protein-Storage Vacuoles

The leguminous tree Sophora japonica contains a family of closely related, but distinct, lectins. Different members of this family are independently expressed in seeds, leaves, and bark (CN Hankins, J Kindinger, LM Shannon 1987 Plant Physiol 83: 825-829; 1988 Plant Physiol 86: 67-10). The inter-, and intracellular distribution of the bark and leaf lectins was studied by indirect postembedding immunogold electron microscopy. Aldehyde fixed bark and leaves postifixed with OsO₄ and embedded in LR White resin permitted sensitive and specific immunogold labeling while maintaining cellular ultrastructure. The leaf and bark tissue cells contain protein-filled storage vacuoles which occupy most the cell's interior volume. The leaf and bark vacuoles closely resemble the protein bodies, or protein storage vacuoles, of seed cotyledons. The leaf and bark lectins were found to be exclusively sequestered in the protein-storage vacuoles of these tissues.     

Functional Specialization of Vacuoles in Sugarcane Leaf and Stem

Plant vacuoles are frequently targeted as a storage site for novel products. We have used environment-sensitive fluorescent dyes and the expression of vacuolar marker proteins to characterize the vacuoles in different organs and cell types of sugarcane. The results demonstrated that the lumen of the vacuole in the parenchyma cells of the stem is acidic (<pH 5) and contains active proteases, characteristic of lytic vacuoles. Western blots and tissue labelling with antibodies to vacuolar H⁺-ATPase suggest that this proton pump is involved in acidification of the vacuolar lumen. Quantitative real-time PCR was used to show that the expression of vacuolar proteases and a vacuolar sorting receptor is also coordinately regulated. In contrast to the stem parenchyma cells, the cells of sugarcane leaves contain diverse types of vacuoles. The pH of these vacuoles and their capacity to hydrolyze protease substrates varies according to cell type and developmental stage. Sugarcane suspension-cultures contain cells with vacuoles that resemble those of stem parenchyma cells and are thus a useful model system for investigating the properties of the vacuole. Understanding the growth and development of storage capacity will be useful in designing strategies to maximize the production of sucrose or alternative bioproducts.     

Molecular Markers for Granulovacuolar Degeneration Are Present in Rimmed Vacuoles

Background

Rimmed vacuoles (RVs) are round-oval cytoplasmic inclusions, detected in muscle cells of patients with myopathies, such as inclusion body myositis (IBM) and distal myopathy with RVs (DMRV). Granulovacuolar degeneration (GVD) bodies are spherical vacuoles containing argentophilic and hematoxyphilic granules, and are one of the pathological hallmarks commonly found in hippocampal pyramidal neurons of patients with aging-related neurodegenerative diseases, such as Alzheimer''s disease and Parkinson''s disease. These diseases are common in the elderly and share some pathological features. Therefore, we hypothesized that mechanisms of vacuolar formation in RVs and GVD bodies are common despite their role in two differing pathologies. We explored the components of RVs by immunohistochemistry, using antibodies for GVD markers.

Methods

Subjects included one AD case, eight cases of sporadic IBM, and three cases of DMRV. We compared immunoreactivity and staining patterns for GVD markers. These markers included: (1) tau-modifying proteins (caspase 3, cyclin-dependent kinase 5 [CDK5], casein kinase 1δ [CK1δ], and c-jun N-terminal kinase [JNK]), (2) lipid raft-associated materials (annexin 2, leucine-rich repeat kinase 2 [LRRK2], and flotillin-1), and (3) other markers (charged multi-vesicular body protein 2B [CHMP2B] and phosphorylated transactive response DNA binding protein-43 [pTDP43]) in both GVD bodies and RVs. Furthermore, we performed double staining of each GVD marker with pTDP43 to verify the co-localization.

Results

GVD markers, including lipid raft-associated proteins and tau kinases, were detected in RVs. CHMP2B, pTDP43, caspase 3, LRRK2, annexin 2 and flotillin-1 were detected on the rim and were diffusely distributed in the cytoplasm of RV-positive fibers. CDK5, CK1δ and JNK were detected only on the rim. In double staining experiments, all GVD markers colocalized with pTDP43 in RVs.

Conclusions

These results suggest that RVs of muscle cells and GVD bodies of neurons share a number of molecules, such as raft-related proteins and tau-modifying proteins.     

Small Oligomers of Ribulose-bisphosphate Carboxylase/Oxygenase (Rubisco) Activase Are Required for Biological Activity

Ribulose-bisphosphate carboxylase/oxygenase (Rubisco) activase uses the energy from ATP hydrolysis to remove tight binding inhibitors from Rubisco, thus playing a key role in regulating photosynthesis in plants. Although several structures have recently added much needed structural information for different Rubisco activase enzymes, the arrangement of these subunits in solution remains unclear. In this study, we use a variety of techniques to show that Rubisco activase forms a wide range of structures in solution, ranging from monomers to much higher order species, and that the distribution of these species is highly dependent on protein concentration. The data support a model in which Rubisco activase forms an open spiraling structure rather than a closed hexameric structure. At protein concentrations of 1 μm, corresponding to the maximal activity of the enzyme, Rubisco activase has an oligomeric state of 2–4 subunits. We propose a model in which Rubisco activase requires at least 1 neighboring subunit for hydrolysis of ATP.     

The Role of Tannins in Neutral Red Staining of Pine Leaf Vacuoles

The vital stain, neutral red, was retained by the cell vacuoles of white pine, Pinus strobus L., through the fixing and embedding process. The results obtained were equivalent to living tissue when the material was fixed in 10% formalin or Bouin's solution and embedded in wax. Chromatographic studies indicated that water-soluble tannins were responsible for adsorption of the neutral red, but only after treatment with formaldehyde. Analyses with other mono-, di-, and trihydroxy phenols suggested that ferric ions and phenols with at least 3 hydroxyl groups per molecule were essential to the reaction.     

Immunological Identification of Proteinase Inhibitors I and II in Isolated Tomato Leaf Vacuoles

Proteinase inhibitor I has been identified and quantified in isolated vacuoles from tomato (Lycopersicon esculentum) leaves induced to accumulate inhibitors either by wounding or by supplying excised leaves with the wound hormone, proteinase inhibitor-inducing factor. Proteinase inhibitor II was also identified in the vacuoles but not quantified. Control vacuoles were prepared from unwounded plants that did not contain inhibitors. Vacuole to leaf cell ratios of inhibitors, chlorophyll, and several vacuolar and cytoplasmic enzymes were determined. The inhibitors were found almost entirely in the vacuoles. Acid phosphatase was located in control leaf vacuoles, but was found in both vacuoles and cytoplasm in induced leaves. Carboxypeptidase, induced by wounding, was found distributed between the vacuoles and cytoplasm of induced leaves. Low vacuole to leaf cell ratios of three cytoplasmic markers, triosephosphate isomerase, catalase, and chlorophyll, indicated that the isolated vacuoles were relatively free of intact protoplasts and cell debris.     

PIN1-Independent Leaf Initiation in Arabidopsis

Phyllotaxis, the regular arrangement of leaves and flowers around the stem, is a key feature of plant architecture. Current models propose that the spatiotemporal regulation of organ initiation is controlled by a positive feedback loop between the plant hormone auxin and its efflux carrier PIN-FORMED1 (PIN1). Consequently, pin1 mutants give rise to naked inflorescence stalks with few or no flowers, indicating that PIN1 plays a crucial role in organ initiation. However, pin1 mutants do produce leaves. In order to understand the regulatory mechanisms controlling leaf initiation in Arabidopsis (Arabidopsis thaliana) rosettes, we have characterized the vegetative pin1 phenotype in detail. We show that although the timing of leaf initiation in vegetative pin1 mutants is variable and divergence angles clearly deviate from the canonical 137° value, leaves are not positioned at random during early developmental stages. Our data further indicate that other PIN proteins are unlikely to explain the persistence of leaf initiation and positioning during pin1 vegetative development. Thus, phyllotaxis appears to be more complex than suggested by current mechanistic models.     

Pattern Dynamics in Adaxial-Abaxial Specific Gene Expression Are Modulated by a Plastid Retrograde Signal during Arabidopsis thaliana Leaf Development

The maintenance and reformation of gene expression domains are the basis for the morphogenic processes of multicellular systems. In a leaf primordium of Arabidopsis thaliana, the expression of FILAMENTOUS FLOWER (FIL) and the activity of the microRNA miR165/166 are specific to the abaxial side. This miR165/166 activity restricts the target gene expression to the adaxial side. The adaxial and abaxial specific gene expressions are crucial for the wide expansion of leaf lamina. The FIL-expression and the miR165/166-free domains are almost mutually exclusive, and they have been considered to be maintained during leaf development. However, we found here that the position of the boundary between the two domains gradually shifts from the adaxial side to the abaxial side. The cell lineage analysis revealed that this boundary shifting was associated with a sequential gene expression switch from the FIL-expressing (miR165/166 active) to the miR165/166-free (non-FIL-expressing) states. Our genetic analyses using the enlarged fil expression domain2 (enf2) mutant and chemical treatment experiments revealed that impairment in the plastid (chloroplast) gene expression machinery retards this boundary shifting and inhibits the lamina expansion. Furthermore, these developmental effects caused by the abnormal plastids were not observed in the genomes uncoupled1 (gun1) mutant background. This study characterizes the dynamic nature of the adaxial-abaxial specification process in leaf primordia and reveals that the dynamic process is affected by the GUN1-dependent retrograde signal in response to the failure of plastid gene expression. These findings advance our understanding on the molecular mechanism linking the plastid function to the leaf morphogenic processes.     

生长素对拟南芥叶片发育调控的研究进展

叶片（包括子叶）是茎端分生组织产生的第一类侧生器官,在植物发育中具有重要地位。早期叶片发育包括三个主要过程：叶原基的起始,叶片腹背性的建立和叶片的延展。大量证据表明叶片发育受到体内遗传机制和体外环境因子的双重调节。植物激素,尤其是生长素在协调体内外调节机制中起着不可或缺的作用。生长素的稳态调控、极性运输和信号转导影响叶片发育的全过程。本文着重介绍生长素在叶片生长发育和形态建成中的调控作用,试图了解复杂叶片发育调控网络。     

拟南芥谷氧还蛋白GRXC9负调控叶片大小

谷氧还蛋白(GRX)是一类以CXXC/S基序为活性位点的小分子热稳定蛋白,参与多种谷胱甘肽依赖的氧化还原反应。通过对该家族的GRXC9基因进行克隆、表达、亚细胞定位及功能分析,结果表明,GRXC9基因表达无组织特异性,在拟南芥(Arabidopsis thaliana)的根、茎、叶、花和角果中均能表达,此结果与GUS显色结果基本一致。GRXC9-GFP定位于细胞质和细胞核中,过表达GRXC9的株系叶片明显小于野生型;进一步观察发现,其叶片栅栏细胞明显变小,而细胞总数与野生型差距不大。叶片大小相关基因的表达分析结果表明,过表达株系中AN、LNG1和LNG2的表达量明显下降,说明GRXC9可能通过抑制这些基因的表达从而导致叶片短小。综上所述,GRXC9可能在调控叶片发育方面发挥关键作用。     

生长素对拟南芥叶片发育调控的研究进展

叶片(包括子叶)是茎端分生组织产生的第一类侧生器官, 在植物发育中具有重要地位。早期叶片发育包括三个主要过程: 叶原基的起始, 叶片腹背性的建立和叶片的延展。大量证据表明叶片发育受到体内遗传机制和体外环境因子的双重调节。植物激素, 尤其是生长素在协调体内外调节机制中起着不可或缺的作用。生长素的稳态调控、极性运输和信号转导影响叶片发育的全过程。本文着重介绍生长素在叶片生长发育和形态建成中的调控作用, 试图了解复杂叶片发育调控网络。     

A Small Infrared Thermometer for Measuring Leaf Temperature in Leaf Chambers

A small, inexpensive infrared thermometer is described. Thisinstrument is easily used and is more accurate than thermocouplesfor leaf temperature measurements. Errors are estimated to beless than 0.2 °C when measuring leaf temperatures in a typicalleaf chamber. Key words: Infrared thermometer, Leaf temperature, Leaf chamber     

Stress-Induced Chloroplast Degradation in Arabidopsis Is Regulated via a Process Independent of Autophagy and Senescence-Associated Vacuoles

Two well-known pathways for the degradation of chloroplast proteins are via autophagy and senescence-associated vacuoles. Here, we describe a third pathway that was activated by senescence- and abiotic stress-induced expression of Arabidopsis thaliana CV (for chloroplast vesiculation). After targeting to the chloroplast, CV destabilized the chloroplast, inducing the formation of vesicles. CV-containing vesicles carrying stromal proteins, envelope membrane proteins, and thylakoid membrane proteins were released from the chloroplasts and mobilized to the vacuole for proteolysis. Overexpression of CV caused chloroplast degradation and premature leaf senescence, whereas silencing CV delayed chloroplast turnover and senescence induced by abiotic stress. Transgenic CV-silenced plants displayed enhanced tolerance to drought, salinity, and oxidative stress. Immunoprecipitation and bimolecular fluorescence complementation assays demonstrated that CV interacted with photosystem II subunit PsbO1 in vivo through a C-terminal domain that is highly conserved in the plant kingdom. Collectively, our work indicated that CV plays a crucial role in stress-induced chloroplast disruption and mediates a third pathway for chloroplast degradation. From a biotechnological perspective, silencing of CV offers a suitable strategy for the generation of transgenic crops with increased tolerance to abiotic stress.