Recipient Organization
PURDUE UNIVERSITY
(N/A)
WEST LAFAYETTE,IN 47907
Performing Department
Botany & Plant Pathology
Non Technical Summary
Most groups of higher plants accumulate crystalline calcium oxalate inside cell vacuoles (Zindler-Frank 1976). These include important crop plants such as soybean and other legumes (Ilarslan et al. 1997), tomato and other members of the Solanaceae (de Kreij et al. 1992), sugar beet (Mostafa and Ulrich 1976), grape (Webb et al. 1995), banana (Li et al, 2014) and many other fruits, vegetables, and horticultural crops. Calcium oxalate crystals in plants exhibit distinct patterns of morphology and distribution, indicating that plants control the sites and modes of calcium oxalate crystallization (reviewed in Webb 1999; Franceschi and Nakata 2005).Calcium oxalate provides several important functions in plants:Calcium oxalate crystallization removes excess calcium from active plant metabolism. Many plants respond to increases in external calcium supply by increased accumulation of calcium oxalate crystals (reviewed in Webb 1999; Franceschi and Nakata 2005). Thus calcium oxalate accumulation provides an essential mechanism for sequestering excess calcium in plants.Calcium oxalate crystals protect plants against animal herbivory (Salz and Ward 2000; Molano-Flores 2001; Korth et al. 2006). In some plants raphides are associated with toxic or acrid proteins (Perera et al. 1990), which confer a bitter taste and/or cause an inflammatory reaction when the plant is eaten (Bradbury and Nixon 1998). Raphides are particularly common in monocots, but also occur in dicots (www.mobot.org).Calcium oxalate in deciduous leaves favorably impacts retention and recycling of calcium in soils of forest ecosystems (Bailey et al. 2003). The oxalate component of calcium oxalate also provides a store for CO2, relevant to amelioration of climate change.Recent studies have revealed that calcium oxalate crystals and calcium carbonate cystoliths in some plants function to scatter light within leaves to promote photosynthesis (Gal et al. 2012).Calcium oxalate can also be harmful to humans and other animals that consume plants. High levels of oxalates in plant foods can be toxic, and in some house or garden plants poisonous substances are associated with calcium oxalate crystals. Calcium transport and partitioning contribute to disorders in fruits and vegetables, such as bitter pit, blossom end rot, and fruit softening, resulting in localized calcium deficiency, even in plants that contain large amounts of calcium oxalate (Mostafa and Ulrich 1976; Millaway and Wiersholm 1979). Some plants accumulate levels of calcium oxalate up to 80% of plant dry weight (Zindler-Frank 1976) or 80-90% of total calcium (Fink 1991). Research that clarifies factors mediating calcium oxalate pools and crystallization of calcium oxalate in cells will contribute to understanding harmful diseases or disorders caused by excessive calcium oxalate.Calcium bioavailability is reduced in foods containing calcium oxalate (Heaney et al 1988; McConn and Nakata 2004). Foods with high oxalate content contribute to elevated urinary oxalate and can lead to kidney disease (Holmes et al. 2001) and diets for patients with susceptibility to kidney stones recommend eliminating high-oxalate foods. Normal human urine contains a variety of proteins, some of which inhibit crystal growth (Ryall et al. 2000). Identification of additional inhibitors of calcium oxalate crystallization in plants could contribute to prevention and treatment for this serious disease.
Animal Health Component
20%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
(N/A)
Goals / Objectives
In previous research we extracted a complex mix of proteins associated with raphides isolated from grape (Vitis labrusca) (Webb et al., 1995) and banana (Musa acuminata) (Li et al., 2014). Separation of raphide-associated proteins by SDS-PAGE yielded dissimilar profiles of protein bands in the two species that may relate to differences in raphide morphology between the two species. Although we previously identified raphide-associated proteins in both grape and banana, improvements in the sensitivity of mass spectrometry make it worthwhile to repeat these analyses. In continuing research we propose to compare the proteomes associated with developing raphides in grape and banana, including constituents of the membranes that enclose the raphides and any proteins present within the calcium oxalate matrix.1. To extract, separate, and identify proteins from developing raphides in grape and banana leaves, including surface and intracrystalline proteins2. a) To compare the set of proteins identified in the grape and banana extracts to distinguish proteins that are common to both, as well as proteins unique to either grape or banana b) To identify selected proteins associated with crystal growth in vitro3. To gain information about the function of raphide-associated proteins in grape and banana by suppressing expression of genes encoding the proteins via RNA-interference (RNAi) silencing
Project Methods
Objective 1: To extract, separate, and identify proteins extracted from raphides in grape and banana leaves, including surface and intracrystalline proteinsIsolation of raphide bundles. Fresh plant leaves will be macerated in absolute EtOH in a blender. Frozen leaves will be ground in a mortar and pestle in liquid nitrogen and then transferred to EtOH. Coherent bundles of raphides, each representing the raphide content of a single cell vacuole, will be isolated from the crude extract in absolute EtOH. When crude extracts are swirled in the spot plate wells, raphide bundles collect at the bottom of the wells and can be retrieved with minimal impurities. The process can be repeated until the raphide bundles are separated from other cell debris.Extraction and separation of raphide-associated proteins. To dissolve the carbohydrate-rich matrix surrounding the bundles, they will be transferred from EtOH into dH20 and washed multiple times. Raphides will be separated from the solubilized matrix by centrifugation and the supernatant discarded. SDS-PAGE sample buffer will be added to the raphide pellet and boiled to release proteins from the surface of the raphides (Webb et al, 1995). This procedure yields a complex mix of proteins (Webb et al. 1995). Following the previous extractions, EDTA and formic acid will be used to dissolve the crystal matrix, allowing collection of any intracrystalline proteins (Li et al., 2003; Li et al., 2014). All protein extracts obtained from raphides will be separated by SDS-PAGE.Identification of raphide-associated proteins. Protein bands will be submitted for sequencing by MALDI-TOF mass spectrometry, and identified via BLAST analysis of sequences obtained.Objective 2: a) To compare the set of proteins identified in grape and banana extracts to distinguish proteins that are common to both, as well as proteins unique to either grape or bananab) To identify selected proteins associated with crystal growth in vitroProteins identified in banana and grape via MALDI-TOF spectrometry will be compared to identify proteins common to raphides in these two species, as well as proteins distinct to either banana or grape. We expect that among the proteins common to each will be some that influence generic features of raphides. Proteins that differ between banana and grape may include those involved in structural or developmental features unique to grape or banana raphides.Proteins associated with raphide development may be involved in crystal initiation, promoting or inhibiting crystal growth, or stabilization of the crystal structure. To specifically target such proteins, we will use a "crystal blot" previously developed in my lab. In this procedure proteins extracted from raphides will be separated by SDS-PAGE in duplicate gels. One of the duplicate gels will be stained with Coomassie Blue to reveal protein bands, and the second gel will be blotted onto nitrocellulose and incubated in a calcium oxalate solution. We previously observed blots incubated in the calcium oxalate solution formed discrete bands of crystals on the blot. This method will allow us to identify proteins directly interacting with calcium oxalate.Objective 3: To gain information about the function of raphide-associated proteins by suppressing expression of genes encoding the proteins via RNA-interference (RNAi) silencingTo gain knowledge about the functions of proteins associated with raphides, we will use RNAi silencing to suppress gene expression. Selected raphide-associated proteins identified via MALDI-TOF spectrometry and/or crystal blots (see Objective 2) will be targeted for RNAi studies. Grape roots are a particularly good system for these studies because raphide-forming cells develop in files that initiate near the root meristem and differentiate away from the root tip as it continues to grow. We previously found that this developmental pattern persists in cultured roots. We plan to use Agrobacterium rhizogenes (Giri and Narasu, 1994) to introduce into grape roots RNAi constructs that will express double-stranded RNA corresponding to selected genes encoding raphide-associated proteins. We will utilize constructs designed for RNAi mediated silencing, as well as silencing rescue of the targeted gene, available in the Golden Gate system (Binder et al., 2014). An alternative approach that has used effectively in grape would be virus-induced gene silencing via RNAi silencing (Kurth et al., 2012). Our initial studies with grape will inform further research with banana. In banana A. tumefaciens will be used to introduce RNAi constructs, corresponding to genes encoding raphide-associated proteins, into plants (Dang et al., 2014). Raphide-forming cells in banana protrude into large intercellular channels in the leaf petiole, so they are readily identified and easily accessible. Vectors without RNAi constructs will be used as controls.RT-PCR will be used to confirm the presence of RNAi constructs in plants. Effects of RNAi silencing will be assessed initially in cultured grape roots using light and electron microscopy to observe raphide structure and development of raphide-forming cells in comparison to plants not subjected to RNAi. This will help us to separate outcomes that are particularly interesting in response to RNAi silencing before transforming plants with the RNAi construct. Using these methods we expect to gain knowledge about the functions of specific proteins in raphide structure and development. These studies will be performed in collaboration with Dr. Sue Loesch-Fries.