Biomineralization Mechanisms Of Calcium Oxalate Raphides In Banana (Musa Spp.)

Calcium oxalate biomineralization formed in specialized cells (idioblast) is a basic and important process in many plant families. They often possess diverse and species-specific morphologies and structures. During their formation there exist the synergetic interactions between cell expansion/growth and crystal nucleation/growth, showing that the mineralization process is precisely controlled by biomacromolecules (protein, polysaccharide, glycoprotein and so on) through the exquisite bio-regulation mechanisms rather than a simple random physical-chemical precipitation reaction. The mineralized phases and the final mature products formed inside idioblasts are shaped and thermodynamically stabilized by various biomacromolecules expressed and synthesized in corresponding bio-pathways. Biological organisms possess an unparalleled ability to control crystallization of biominerals with convoluted molecular and biochemical mechanisms. For example, an occluded organic matrix can interact with the mineral during its formation to control its morphology and structure. Although related matrix proteins that preferentially nucleate minerals in plants have been identified, the mechanisms elucidating the structural and chemical complexity of calcium oxalate biominerals in Musa spp. remain unclear. In this paper the morphology and structure of raphide idioblast (RI) and isolated raphides (R) from pseudostem of banana(Musa spp.), and the formation mechanism of raphide regulated by Ca will be discussed. In addition, the main research will be focused on dynamics of calcium oxalate crystallization in vitro at the near-molecular level. The main results are summarized as following:(1) Morphology and distribution of raphide idioblasts forming calcium oxalate monohydrate in MusaRaphide crystals in Musa form within crystal idioblasts that develop in the pseudostem of the plant. RI developed in large air spaces and had a distinct elongated shape in contrast to the isodiametric parenchyma cells. An isolated raphide idioblast contained 200 needle-shaped (acicular) raphide crystals. The individual raphides with high-resolution field emission scanning electron microscopy (FSEM) showed that they were 4 μm width and 150μm length and exhibited hexagonal or octahedral facets along their length and gradually taper at both ends of the crystal to form needle-like points. Raphides extracted from the pseudostem were identified by X-ray diffraction (XRD), Fourier transformation infrared spectroscopy (FTIR), thermo gravimetry/differential thermal analysis (TG/DTA) and Raman spectroscopy as calcium oxalate monohydrate (COM). Brazil and Thailand banana with the same genotype and different sources were similar in crystal type, distribution and size, indicating the genetic stability in different species.(2) Ca2+ -mediated formation of raphide idioblasts in MusaThe result of non-invasive micro-test technique (NMT) showed that the Ca2+ efflux of RI was reduced 8 times compared to the control when the concentration of Ca2+ increased to 10 mmol/L, but the length and width of raphide crystals and idioblasts were increased, indicating the Ca2+ absorbed by RI was increased and the remarkable coordination of crystal growth and cell expansion.(3) Matrix associated with needle-shaped calcium oxalate crystalSections of individual raphide crystals were subjected to in situ elemental microanalysis using secondary ion mass spectrometry (NanoSIMS). The result showed that an organic film (Aceton-Soluble Matrix, MA) was present in the outer layers of individual raphides, detected by NanoSIMS as 16O- and 12C14N- secondary ions. Raphides treated first with acetone and then etched in EDTA (Ethylene Diamine Tetraacetic Acid) solution were examined with in situ atomic force microscopy (AFM), which revealed a filamentous component that forms a double-stranded structure associated with the raphides. AFM height images of partly demineralized needle-like crytals show obvious fibrous structures with a diameter of about 9 nm embedded in the individual crystalline COM needles. The EDTA-insoluble matrix could dissolve in formic acid, defined as formic acid-soluble matrix, MF. Protein molecular mass (14 kDa) and sequence were obtained by LTQ-ESI-MS/MS (Linear Ion Trap Quadrupole-Electrospray Ionization-Tandem Mass Spectrometry/Mass Spectrometry) analysis. BLAST analysis showed that the 14 kD protein had a high degree of similarity with to chaperone protein DnaJ of a bacteria (Thermoplasma acidophilum DSM 1728). Derived from 101 to 111 of the carboxy terminus from 14 kD protein was high conserved and rich in proline (11-mer proline-rich peptide, PRP).(4) Templated elongate needle-shaped calcium oxalate crystal biomineralization on self-assembled protein nanofibers11-mer PRP peptide molecules synthesized in vitro can spontaneously form fiber structures under different solution conditions. The formation of crystals along the fiber axis was COM identified by XRD. The results of non-seeded nucleation experiments showed significantly shorter induction time in the presence of the PRP peptide segments than in control supersaturated solution. COM crystallites nucleated in solutions with PRP exhibited considerably elongated morphology with length-to-width aspect ratio. Interestingly, quantitative determination of crystal growth rates at the near-molecular level were obtained by using in situ AFM to measure the step velocity on (-101) and (010) faces, respectively. No roughening of the steps was observed. These results indicated that PRP peptide segments influence nucleation rather than growth of COM crystals.(5) Self-assembled flexible needle-shaped calcium oxalate crystalDemineralization experiments of individual needle-shaped crystals by in situ AFM showed that the (100)-like surface of the hexagonal needle consists of regular bands (about 130 nm in width) aligned parallel to one another, each consisting of calcium oxalate nanoparticles (about 65 nm in diameter). Cross-sectional analyses of a fractured needle at the micron scale reveal that the needles have a lamellar structure, close to the band width (130 nm). Based on evidence observed in this study, we propose a model in which the protein fiber assemblies embedded in the core of needle-like crystals control the arrangement of calcium oxalate nanoparticles into organized and elongated structures. In this model mineral nanoparticles assemble around the template of protein fibers into parallel arrays, and the nanoparticles fuse to form regular lamellar structures, leading to the final product of elongated and tapered crystallites.(6) Inhibition of calcium oxalate monohydrate crystallization through selective binding to atomic stepsGenerally, proteins associated calcium oxalate biomineralization are present phosphate sites. In this study, calsequestrin (CS) reported related to the formation of calcium oxalate was selected, and the related peptides CS-1 and (p)CS-1 were synthetized in vitro. The results of non-seeded nucleation experiments showed significantly longer induction timesin the presence of CS-1 or (p)CS-1 peptide segments. COM crystallites nucleated in solutions with CS-1 or (p)CS-1 produced diamond-shaped crystals through decreases the aspect ratio. Quantitative determination of crystal growth rates at the near-molecular level were obtained by using in situ AFM to measure the step velocity on (-101) and (010) faces, respectively. The step velocity and morphology were changed (step pinning) in the presenceCS-1 or (p)CS-1 segment, roughening of the steps on (-101) were observed, too. This effect on (010) face was reversible because the steps once again become linear after addition of pure calcium oxalate supersaturated solution, but irreversible on (-101) face, indicating that CS-1 or (p)CS-1 bound to the (-101) face (positive) of CaOx crystal surface was selectively, but to the (010) face (negative) was limit. The inhibiting effect was increased by phosphorylated threonine in CS-1, which indicates that the electrostatic interaction between negatively charged phosphate and COM crystal faces is enhanced, which makes bonding strength improved, and significantly inhibits the crystal nucleation and growth.In conclusion, we anticipate that the present investigation of the structural and morphological complexity of plant calcium oxalate crystals and the underlying mechanisms of their formation will contribute to our understanding how plants evolved these sophisticated structures and morphologies for survival and adaptation, and ultimately provide useful clues about how to maximally sequester calcium ions and/or oxalate in a confined compartment.