Xingxiang Li

Segregation of storage protein mRNAs on the rough endoplasmic reticulum membranes of rice endosperm cells

Developing rice endosperm cells display two distinct rough endoplasmic reticula (ER), cisternal ER (C-ER) and protein body ER (PB-ER), the latter delimiting the prolamine protein bodies. These ER membranes are utilized for the simultaneous synthesis of glutelin and prolamine storage proteins, which are subsequently routed into separate protein bodies. We demonstrate by blot hybridization, and by visualization of the spatial distributions and densities of these transcripts in endosperm cells via high resolution in situ hybridization analysis, that prolamine transcripts are associated primarily with the PB-ER, while glutelin mRNAs are enriched on the C-ER. The results suggest that the initial targeting process of these storage proteins into distinct protein bodies is the segregation of their transcripts on the ER membranes.

Isolation of a crystal matrix protein associated with calcium oxalate precipitation in vacuoles of specialized cells

The formation of calcium (Ca) oxalate crystals is considered to be a high-capacity mechanism for regulating Ca in many plants. Ca oxalate precipitation is not a stochastic process, suggesting the involvement of specific biochemical and cellular mechanisms. Microautoradiography of water lettuce (Pistia stratiotes) tissue exposed to 3H-glutamate showed incorporation into developing crystals, indicating potential acidic proteins associated with the crystals. Dissolution of crystals leaves behind a crystal-shaped matrix “ghost” that is capable of precipitation of Ca oxalate in the original crystal morphology. To assess whether this matrix has a protein component, purified crystals were isolated and analyzed for internal protein. Polyacrylamide gel electrophoresis revealed the presence of one major polypeptide of about 55 kD and two minor species of 60 and 63 kD. Amino acid analysis indicates the matrix protein is relatively high in acidic amino acids, a feature consistent with its solubility in formic acid but not at neutral pH. 45Ca-binding assays demonstrated the matrix protein has a strong affinity for Ca. Immunocytochemical localization using antibody raised to the isolated protein showed that the matrix protein is specific to crystal-forming cells. Within the vacuole, the surface and internal structures of two morphologically distinct Ca oxalate crystals, raphide and druse, were labeled by the antimatrix protein serum, as were the surfaces of isolated crystals. These results demonstrate that a specific Ca-binding protein exists as an integral component of Ca oxalate crystals, which holds important implications with respect to regulation of crystal formation.

Targeting of mRNAs to domains of the endoplasmic reticulum.

The targeting of proteins to specific regions of the cell by signal elements within the polypeptide sequence has received much attention, but proteins can also be directed to their appropriate cellular locations by localization of their mRNAs. This mechanism is seen clearly in polar cells like germ and embryonic cells, neurons and epithelia. Recent evidence indicates that mRNAs may also be localized to morphologically and functionally distinct endoplasmic reticulum membranes, thereby facilitating sorting of the proteins they encode to subdomains of the reticulum or to polarized plasma membranes.

Localization of RNA by high resolution in situ hybridization.

Publisher Summary Histological localization of RNA involves in situ hybridization techniques. Light microscopy (LM) in situ hybridization techniques have allowed localization of messenger RNAs (mRNAs) to specific regions of the cell. The mechanism of intracellular RNA localization involves a two-step process: transport from the perinuclear region and then anchoring of the RNA to a specific subcellular location. Transport processes involve assembly of the RNA with cellular factors and movement of the complex along microfilaments and/or microtubules. In contrast little is known about the anchoring process. To identify the cellular structures that provide RNA anchoring sites, high resolution in situ hybridization techniques at the electron microscopy (EM) level are required. The success of EM in situ hybridization relies on experimental conditions for production of high specific activity nonisotopic probes, saturated hybridization kinetics, and efficient detection of the hybridization signal. This chapter describes a protocol for detecting the nonrandom distribution of rice storage protein mRNAs on the endoplasmic reticulum (ER) using nonradioactive RNA probes.