The Major Storage Protein in Potato Tuber Is Mobilized by a Mechanism Dependent on Its Phosphorylation Status

The role of the protein phosphorylation mechanism in the mobilization of vegetative storage proteins (VSPs) is totally unknown. Patatin is the major VSP of the potato (Solanum tuberosum L.) tuber that encompasses multiple differentially phosphorylated isoforms. In this study, temporal changes in the phosphorylation status of patatin isoforms and their involvement in patatin mobilization are investigated using phosphoproteomic methods based on targeted two-dimensional electrophoresis (2-DE). High-resolution 2-DE profiles of patatin isoforms were obtained in four sequential tuber life cycle stages of Kennebec cultivar: endodormancy, bud break, sprouting and plant growth. In-gel multiplex identification of phosphorylated isoforms with Pro-Q Diamond phosphoprotein-specific stain revealed an increase in the number of phosphorylated isoforms after the tuber endodormancy stage. In addition, we found that the phosphorylation status of patatin isoforms significantly changed throughout the tuber life cycle (P < 0.05) using the chemical method of protein dephosphorylation with hydrogen fluoride-pyridine (HF-P) coupled to 2-DE. More specifically, patatin phosphorylation increased by 32% from endodormancy to the tuber sprouting stage and subsequently decreased together with patatin degradation. Patatin isoforms were not randomly mobilized because highly phosphorylated Kuras-isoforms were preferably degraded in comparison to less phosphorylated non-Kuras isoforms. These results lead us to conclude that patatin is mobilized by a mechanism dependent on the phosphorylation status of specific isoforms.

Soybean vegetative lipoxygenases are not vacuolar storage proteins

The paraveinal mesophyll (PVM) of soybean is a distinctive uniseriate layer of branched cells situated between the spongy and palisade chlorenchyma of leaves that contains an abundance of putative vegetative storage proteins, Vspα and Vspβ, in its vacuoles. Soybean vegetative lipoxygenases (five isozymes designated as Vlx(A–E)) have been reported to co-localise with Vsp in PVM vacuoles; however, conflicting results regarding the tissue-level and subcellular localisations of specific Vlx isozymes have been reported. We employed immuno-cytochemistry with affinity-purified, isozyme-specific antibodies to reinvestigate the subcellular locations of soybean Vlx isozymes during a sink limitation experiment. VlxB and VlxC were localised to the cytoplasm and nucleoplasm of PVM cells, whereas VlxD was present in the cytoplasm and nucleoplasm of mesophyll chlorenchyma (MC) cells. Label was not associated with storage vacuoles or any evident protein bodies, so our results cast doubt on the hypothesis that Vlx isozymes function as vegetative storage proteins.

The functional status of paraveinal mesophyll vacuoles changes in response to altered metabolic conditions in soybean leaves

Antibodies raised against tonoplast intrinsic proteins (TIPs) were used to probe the functional status of the soybean [Glycine max (L.) Merr.] paraveinal mesophyll (PVM) vacuole during changes in nitrogen metabolism within the leaf. Young plants grown under standard conditions had PVM vacuoles characterised by the presence of γ-TIP, which is indicative of a lytic function. When plants were then subjected to shoot tip removal for a period of 15 d, forcing a sink-limited physiological condition, the γ-TIP marker diminished while the δ-TIP marker became present in the PVM vacuole, indicating the conversion of the PVM vacuole to a storage function. When the shoot tips were allowed to regrow, the γ-TIP marker again became dominant demonstrating the reversion of these PVM vacuoles back to a lytic compartment. The changes in TIP markers correlated with the accumulation of vegetative storage proteins and vegetative lipoxygenases, proteins implicated in nitrogen storage and assimilate partitioning. This research suggests that the PVM vacuole is able to undergo dynamic conversion between lytic and storage functions and further implicates this cell layer in assimilate storage and mobilisation in soybeans.

DISTRIBUTION AND ULTRASTRUCTURE OF VEGETATIVE STORAGE PROTEINS IN LEGUMINOSAE

The distribution and ultrastructure of vegetative storage proteins in 44 species and one variety of 31 genera of Leguminosae were investigated by light- and electron microscopy and SDS-PAGE. Leguminosae are as a whole a vegetative storage protein-rich family, abundant with vacuolar protein inclusions in deciduous trees while much less so in evergreen trees. Several prominent proteins with molecular weights ranging from 15 to 45 kDa were isolated and identified to be vegetative storage proteins on the basis of their association with vacuolar protein inclusions and seasonal fluctuation. Vacuolar protein inclusions were present in protein body-like organelles in temperate species while localized in large central vacuoles in tropical ones during leafless periods. The inclusions varied in forms among species or in the same species, but the different forms were present in different cells, suggesting that vegetative storage proteins may be cell-type specific to some extent.

Vegetative storage proteins in overwintering storage organs of forage legumes: roles and regulation

In perennial forage legumes such as alfalfa (Medicago sativa L.) and white clover (Trifolium repens L.), vegetative storage proteins are extensively mobilized to meet the nitrogen requirements of new shoot growth in spring or after cutting in summer. The 32-kDa alfalfa storage protein possesses high homology with class III chitinases, belonging to a group of pathogenesis-related proteins that possess antifreeze protein properties in some species and exhibit chitinolytic activity in vitro. This protein and the corresponding mRNA accumulate in taproots of cold-hardy culti vars during acclimation for winter, and in response to short-day conditions in controlled environments. The 17.3-kDa storage protein of white clover possesses high homology with pathogenesis-related proteins and abscisic- acid-responsive proteins from several legume species and has characteristics common to stress-responsive proteins. Low temperature enhances accumulation of this 17.3-kDa protein and its corresponding transcript. Exogenous abscisic acid stimulates the accumulation of vegetative storage proteins and their transcripts in both legume species. These observations suggest that vegetative storage proteins do not exclusively serve as nitrogen reserves during specific phases of legume development, but may play important adaptive roles in plant protection against abiotic (low temperature) and biotic (pathogen attack) stresses.Key words: nitrogen reserves, vegetative storage proteins, regulation, cold tolerance, chitinase, pathogenesis-related proteins.

Vegetative storage proteins in the tropical tree Swietenia macrophylla: seasonal fluctuation in relation to a fundamental role in the regulation of tree growth

The protein-storing cells in Swietenia macrophylla King were investigated. They were found to be of the Populus type, i.e., ordinary parenchyma cells containing both vacuole protein inclusion and starch grains. Vegetative storage proteins with molecular masses of 18 and 21 kDa were separated by SDS–PAGE (sodium dodecyl sulfate – polyacrylamide gel electrophoresis). Immunoblotting with the 21-kDa protein antiserum showed that the 18- and 21-kDa proteins shared common epitopes. The 21-kDa protein and presumably the 18-kDa protein were demonstrated by immunogold labeling to be the main components of the vacuole protein inclusion of the protein-storing cells. At the late stage of an annual growth cycle, vegetative storage proteins were found in the branchlets, trunk, large roots, and small roots. They were stored in large amounts in the secondary phloem of these organs and also in the secondary xylem of the terminal branchlets and small roots. In a new growth cycle, the consumption of the previously accumulated vegetative storage proteins began in the terminal branchlets of the last growth cycle. The vegetative storage proteins in the branchlets were exhausted completely when the new shoot leaves matured, while the storage proteins in the trunk and large roots had no detectable changes in abundance. On the other hand, the tree started to accumulate the two proteins in the stem of the new shoots as early as 1 week after the new shoot leaves matured. These results suggested that the previously accumulated vegetative storage proteins were used for new shoot growth and cambial activity in preference to the newly assimilated nitrogen and that vegetative storage proteins existed in considerable amounts in the stems throughout an annual growth cycle. This seasonal fluctuating pattern of vegetative storage proteins in the whole tree may be an important mechanism by which the tree regulates its growth.Key words: vegetative storage proteins, nitrogen metabolism, Populus-type of protein-storing cells, tropical hardwoods, Swietenia macrophylla King.