Hanna Levanony

Purification, Characterization, and Intracellular Localization of Glycosylated Protein Disulfide Isomerase from Wheat Grains

Wheat (Triticum aestivum) storage proteins fold and assemble into complexes that are linked by intra- and intermolecular disulfide bonds, but it is not yet clear whether these processes are spontaneous or require the assistance of endoplasmic reticulum (ER)-resident enzymes and molecular chaperones. Aiming to unravel these processes, we have purified and characterized the enzyme protein disulfide isomerase (PDI) from wheat endosperm, as well as studied its developmental expression and intracellular localization. This ER-resident enzyme was previously shown to be involved in the formation of disulfide bonds in secretory proteins. Wheat PDI appears as a 60-kD glycoprotein and is among the most abundant proteins within the ER of developing grains. PDI is notably up-regulated in developing endosperm in comparison to embryos, leaves, and roots. In addition, the increase in PDI expression in grains appears at relatively early stages of development, preceding the onset of storage protein accumulation by several days. Subcellular localization analysis and immunogold labeling of electron micrographs showed that PDI is not only present in the lumen of the ER but is also co-localized with the storage proteins in the dense protein bodies. These observations are consistent with the hypothesis that PDI is involved in the assembly of wheat storage proteins within the ER.

Root surface colonization of non-cereal crop plants by pleomorphic Azospirillum brasilense Cd

Summary: Root surface colonization by Azospirillum brasilense Cd of tomato, pepper and cotton plants under normal growth conditions and soybean plants under normal and water-stress conditions was monitored by scanning electron microscopy and bacterial counts. A. brasilense Cd was capable of efficiently colonizing the elongation and root-hair zones of all four plant species tested. In these zones, the bacteria mainly colonized the root surface (tomato, soybean), root-hairs (pepper), or both (cotton), by single cells (tomato, soybean), micro-aggregates (pepper), or a combination of the two (cotton). All inoculated plants demonstrated (i) larger amounts of mucigel-like substance on the root surface than non-inoculated plants and (ii) fibrillar material which anchored the bacterial cells to the root surface and established connections between cells within bacterial aggregates. On non-water-stressed soybean plants, most A. brasilense Cd cells in the rhizosphere occurred as vibroid forms whereas those on water-stressed plants (wilting) were cyst-like. A lower rhizosphere bacterial population was observed on water-stressed plants. When water-stress conditions were eliminated, the bacterial cells reverted to the vibroid form and a concomitant increase in the bacterial population was observed. It is suggested that cyst-like formation is a natural response for A. brasilense Cd in the rhizosphere of water-stressed plants.

An autophagy-associated Atg8 protein is involved in the responses of Arabidopsis seedlings to hormonal controls and abiotic stresses

Eukaryotes contain a ubiquitous family of autophagy-associated Atg8 proteins. In animal cells, these proteins have multiple functions associated with growth, cancer, and degenerative diseases, but their functions in plants are still largely unknown. To search for novel functions of Atg8 in plants, the present report tested the effect of expression of a recombinant AtAtg8 protein, fused at its N-terminus to green fluorescent protein (GFP) and at its C-terminus to the haemagglutinin epitope tag, on the response of Arabidopsis thaliana plants to the hormones cytokinin and auxin as well as to salt and osmotic stresses. Expression of this AtAtg8 fusion protein modulates the effect of cytokinin on root architecture. Moreover, expression of this fusion protein also reduces shoot anthocyanin accumulation in response to cytokinin feeding to the roots, implying the participation of AtAtg8 in cytokinin-regulated root–shoot communication. External application of cytokinin leads to the formation of novel GFP–AtAtg8-containing structures in cells located in the vicinity of the root vascular system, which are clearly distinct in size and dynamic movement from the GFP–AtAtg8-containing autophagosome-resembling structures that were observed in root epidermis cells. Expression of the AtAtg8 fusion construct also renders the plants more sensitive to a mild salt stress and to a lesser extent to a mild osmotic stress. This sensitivity is also associated with various changes in the root architecture, which are morphologically distinct from those observed in response to cytokinin. The results imply multiple functions for AtAtg8 in different root tissues that may also be regulated by different mechanisms.

Assembly and transport of seed storage proteins.

Plant seeds store nitrogen by accumulating storage proteins in protein bodies within various compartments of the endomembrane system. The prolamin storage proteins of some cereal species are normally retained and assembled into protein bodies within the ER. Yet, these proteins lack a C-terminal KDEL/HDEL signal, suggesting that their retention is regulated by novel mechanisms. Furthermore, in other cereal species, such protein bodies formed within the ER may be subsequently internalized into vacuoles by a special route that does not utilize the Golgi complex. Thus, studies of the routing of seed storage proteins are revealing novel mechanisms of protein assembly and transport in the endomembrane system.

Horizontal and Vertical Movement of Azospirillum brasilense Cd in the Soil and along the Rhizosphere of Wheat and Weeds in Controlled and Field Environments

SUMMARY: Horizontal movement of Azospirillum brasilense Cd in soil and its vertical movement in the plant rhizosphere were studied. No movement was detected in the absence of living plants. In a controlled environment the bacteria moved horizontally at least 30 cm from the inoculation point to the first growing plant. Once the first root system was colonized, all the neighbouring plants became inhabited. Horizontal movement under field conditions was at least 160 cm, and depended on the presence of live plant roots. Several weeds that grew in the passes between plots acted as efficient vectors. The numbers of A. brasilense Cd decreased with increasing distance from the inoculated plot. Vertical movement in soil columns in a controlled environment was up to 40 cm. Under field conditions, bacteria were detected as deep as 50 cm in the root systems of wheat plants in various types of soil. During the growing season bacteria were mostly found on and in young roots at a depth of 20–50 cm and near the soil surface. A map of depth distribution of A. brasilense Cd showed an uneven colonization pattern within the same root system or between adjacent plants. It was concluded that A. brasilense Cd moved horizontally and vertically in various soil types and that this movement was mainly dependent on the presence of plants.

Ultrastructural localization and identification ofAzospirillum brasilense Cd on and within wheat root by immuno-gold labeling

Azospirillum brasilense Cd localization in wheat roots was studied by light microscopy, by scanning, and by transmission electron microscopy.A. brasilense Cd cells were specifically identified immunocytochemically around and within root tissues.A. brasilense Cd cells found both outside and inside inoculated roots were intensively labeled with colloidal gold. In non-axenic cultures other bacterial strains or plant tissue were not labeled, thereby providing a non-interfering background. The roots of axenic grown wheat plants were colonized both externally and internally byA. brasilense Cd after inoculation, whereas non-axenic cultures were colonized by other bacterial strains as well.A. brasilense Cd cells were located on the root surface along the following zones: the root tip, the elongation, and the root-hair zone. However, bacteria were located within the cortex only in the latter two zones. In a number of observations, an electron dense material mediated the binding of bacterial cells to outer surfaces of epidermal cells, or between adjacent bacterial cells.A. brasilense Cd were found in root cortical intercellular spaces, but were not detected in either the endodermal layer or in the vascular system. This study proposes that in addition to root surface colonization,A. brasilense Cd forms intercellular associations within wheat roots.

Arabidopsis ATG8-INTERACTING PROTEIN1 Is Involved in Autophagy-Dependent Vesicular Trafficking of Plastid Proteins to the Vacuole

A previously unrecognized plastid-to-vacuole protein trafficking pathway that is stress induced and autophagy dependent appears to involve ATI1 interaction with plastid proteins and ATG8 of the core autophagy machinery. Selective autophagy has been extensively studied in various organisms, but knowledge regarding its functions in plants, particularly in organelle turnover, is limited. We have recently discovered ATG8-INTERACTING PROTEIN1 (ATI1) from Arabidopsis thaliana and showed that following carbon starvation it is localized on endoplasmic reticulum (ER)-associated bodies that are subsequently transported to the vacuole. Here, we show that following carbon starvation ATI1 is also located on bodies associating with plastids, which are distinct from the ER ATI bodies and are detected mainly in senescing cells that exhibit plastid degradation. Additionally, these plastid-localized bodies contain a stroma protein marker as cargo and were observed budding and detaching from plastids. ATI1 interacts with plastid-localized proteins and was further shown to be required for the turnover of one of them, as a representative. ATI1 on the plastid bodies also interacts with ATG8f, which apparently leads to the targeting of the plastid bodies to the vacuole by a process that requires functional autophagy. Finally, we show that ATI1 is involved in Arabidopsis salt stress tolerance. Taken together, our results implicate ATI1 in autophagic plastid-to-vacuole trafficking through its ability to interact with both plastid proteins and ATG8 of the core autophagy machinery.

Active Attachment of Azospirillum brasilense Cd to Quartz Sand and to a Light-textured Soil by Protein Bridging

SUMMARY: Inoculation and incubation of Azospirillurn brasilense Cd in pure quartz sand resulted in their attachment to sand particles by fibrillar material. Addition of low concentrations of nutrients, such as fructose or malate together with NH4C1 or root extract, enhanced bacterial multiplication and attachment. This attachment was relatively weak, grid depended on the presence of living bacterial cells and on growth conditions. Sequential washing after bacterial attachment decreased the number of bacterial cells in the sand. The killing of A. brasilense Cd, either before or after attachment, resulted in elimination of most of the applied cells from the sand. By comparison, light-textured soil adsorbed both dead and live bacteria. Addition of protease or EDTA to attached bacteria in sand significantly reduced the number of bacteria. The addition of various inhibitors or exposure to high temperatures yielded a reduced attachment of bacteria, proportionate to their relative inhibitory effect on growth of A. brasilense Cd. Agitating A. brasilense Cd cells immediately after addition to sand reduced bacterial attachment, but increased bacterial multiplication proportionate to the increase in agitation. Attachment of A. brasilense Cd under microaerophilic conditions was lower than under aerobic conditions, and depended on the amount, quality and composition of the available nutrients. The richer the mixture, the greater the attachment and multiplication of bacteria. Similar trends, but at a lower magnitude, were observed in light-textured soil.

Evidence for a Weak Active External Adsorption of Azospirillum brasilense Cd to Wheat Roots

SUMMARY: Azospirillum brasilense Cd, when inoculated onto wheat roots, multiplied and formed aggregates on the root surfaces and established an internal root population. Washing the roots removed most of the external but not the internal bacterial population. Killing the bacteria before or after their interaction with the roots eliminated the adsorbed bacteria from the root surface. The external adsorption of A. brasilense to wheat roots can be categorized as a weak active process.

Adsorption of the Rhizosphere Bacterium Azospirillum brasilense Cd to Soil, Sand and Peat Particles

Summary: The rhizosphere bacterium Azospirillum brasilense Cd adsorbed strongly to light-texture and heavy-texture soils, but only slightly to quartz sand. Increase in clay and organic matter content, decrease in soil pH, or flooding the soil enhanced adsorption, whereas the presence of a bacterial attractant, increase in soil pH or drying of the soil decreased adsorption. The cells adsorbed to the upper fraction of the soil profile, but were able to infiltrate deeper if very dry soil was wetted. Washing the soil did not desorb the bacteria from soil particles, but it did from the sand particles. Overwashing soil recovered relatively few cells, whereas overwashing sand detached most of the bacteria. Survival time of A. brasilense Cd was short in soil but long in peat inoculant.