Cellular and subcellular localization of phototropin 1

Phototropin 1 (phot1) is a Ser/Thr photoreceptor kinase that binds two molecules of flavin mononucleotide as its chromophores and undergoes autophosphorylation in response to blue light. Phot1 is plasma membrane associated and, as with phot2, has been shown to function as a photoreceptor for phototropism, blue light-induced chloroplast movement, and blue light-induced stomatal opening. Phot1 likely also plays a redundant role with phot2 in regulating the rate of leaf expansion. Understanding the mechanism(s) by which phot1 initiates these four different responses requires, at minimum, knowledge of where the photoreceptor is located. Therefore, we transformed a phot1 null mutant of Arabidopsis with a construct encoding translationally fused phot1-green fluorescent protein (GFP) under the control of the endogenous PHOT1 promoter and investigated its cellular and subcellular distribution. This PHOT1-GFP construct complements the mutant phenotype, restoring second positive curvature. Phot1 is expressed strongly in dividing and elongating cortical cells in the apical hook and in the root elongation zone in etiolated seedlings. It is localized evenly to the plasma membrane region in epidermal cells but is confined largely to the plasma membrane region of the transverse cell walls in the cortical cells of both root and hypocotyl. It is found at both apical and basal ends of these cortical cells. In light-grown plants, phot1-GFP is localized largely in the plasma membrane regions adjacent to apical and basal cell end walls in the elongating inflorescence stem, where the photoreceptor is expressed strongly in the vascular parenchyma and leaf vein parenchyma. Phot1 also is localized to the plasma membrane region of leaf epidermal cells, mesophyll cells, and guard cells, where its distribution is uniform. Although phot1 is localized consistently to the plasma membrane region in etiolated seedlings, a fraction becomes released to the cytoplasm in response to blue light. Possible relationships between observed phot1 distribution and the various physiological responses activated by blue light are discussed.     

The C-terminal kinase fragment of Arabidopsis phototropin 2 triggers constitutive phototropin responses

Phototropins mediate various blue-light responses such as phototropism, chloroplast relocation, stomatal opening and leaf flattening in plants. Phototropins are hydrophilic chromoproteins that are mainly bound to the plasma membrane. One of two phototropins in Arabidopsis thaliana, phot2, associates with the Golgi apparatus in a light-dependent manner. In this study, we analyzed the biological activities of the N-terminal photosensory and C-terminal kinase domains of phot2. For this purpose, these domains were fused to green fluorescent protein (GFP) and ectopically expressed in the wild-type and a phot1 phot2 double mutant of Arabidopsis. The kinase domain fused to GFP (P2CG) was localized to the plasma membrane and the Golgi apparatus, whereas the photosensory domain fused to GFP (P2NG) was uniformly localized in the cytosol. Hence, the kinase domain rather than the photosensory domain is responsible for the membrane association. Interestingly, the P2CG plants exhibited constitutive blue-light responses even in dark conditions, i.e. stomata were open and chloroplasts were in the avoidance position. By contrast, P2CG with a mutation that abolishes the kinase activity (P2C[D720/N]G) failed to exhibit these responses. phot2 kinase is therefore suggested to be correctly localized to functional sites in the cell and to trigger light signal transduction through its kinase activity. In contrast to P2CG, P2NG did not affect the phot2 responses, except for partial inhibition of the phototropic response caused by the endogenous phototropins.     

Blue light-induced association of phototropin 2 with the Golgi apparatus

Phototropins 1 and 2 (phot1 and phot2) function as blue light (BL) photoreceptors for phototropism, chloroplast relocation, stomatal opening and leaf flattening in Arabidopsis thaliana. Phototropin consists of two functional domains, the N-terminal photosensory domain and the C-terminal Ser/Thr kinase domain. However, little is known about the signal transduction pathway that links the photoreceptors and the physiological responses downstream of BL perception. To understand the mechanisms by which phot2 initiates these responses, we transformed the phot1phot2 double mutant of Arabidopsis with constructs encoding translationally fused phot2:green fluorescent protein (P2G). P2G was fully functional for the phot2-specific physiological responses in these transgenic plants. It localized strongly to the plasma membrane and weakly to the cytoplasm in the dark. Upon illumination with BL, punctate P2G staining was formed within a few minutes in addition to the constitutive plasma membrane staining. This punctate distribution pattern matched well with that of the Golgi-localized KAM1DeltaC:mRFP. Brefeldin A (BFA), an inhibitor of vesicle trafficking, induced accumulation of P2G around the perinuclear region even in darkness, but the punctate pattern was not observed. After treatment of these cells with BL, P2G exhibited the punctate distribution pattern that matched with that of the Golgi marker. Hence, the light-dependent association of P2G with the Golgi apparatus was BFA-insensitive. A structure/function analysis indicated that the kinase domain was essential for the Golgi localization of phot2. The BL-induced Golgi localization of phot2 may be one of important signaling steps in the phot2 signal transduction pathway.     

Functional analysis and intracellular localization of rice cryptochromes

Blue-light-receptor cryptochrome (CRY), which mediates cotyledon expansion, increased accumulation of anthocyanin, and inhibition of hypocotyl elongation, was first identified in Arabidopsis. Two Arabidopsis cryptochromes (AtCRY1 and AtCRY2) have been reported to be localized to the nucleus. However, there is no information on the cryptochromes in monocotyledons. In this study, we isolated two cryptochrome cDNAs, OsCRY1 and OsCRY2, from rice (Oryza sativa) plants. The deduced amino acid sequences of OsCRY1 and OsCRY2 have a photolyase-like domain in their N termini and are homologous to AtCRY1. To investigate the function of OsCRY1, we overexpressed a green fluorescence protein-OsCRY1 fusion gene in Arabidopsis and assessed the phenotypes of the resulting transgenic plants. When the seedlings were germinated in the dark, no discernible effect was observed. However, light-germinated seedlings showed pronounced inhibition of hypocotyl elongation and increased accumulation of anthocyanin. These phenotypes were induced in a blue-light-dependent manner, indicating that OsCRY1 functions as a blue-light-receptor cryptochrome. We also examined the intracellular localization of green fluorescence protein-OsCRY1 in the transgenic plants. It was localized to both the nucleus and the cytoplasm. We identified two nuclear localization domains in the primary structure of OsCRY1. We discuss the relationship between the function and intracellular localization of rice cryptochromes by using additional data obtained with OsCRY2.     

Subcellular localization and turnover of Arabidopsis phototropin 1

We reported recently that internalization of the plant blue light receptor phototropin 1 (phot1) from the plasma membrane in response to irradiation is reliant on receptor autophosphorylation. Pharmacological interference and co-immunoprecipitation analyses also indicated that light-induced internalization of phot1 involves clathrin-dependent processes. Here, we describe additional pharmacological studies that impact the subcellular localization and trafficking of Arabidopsis phot1. Alterations in the microtububle cytoskeleton led to dramatic differences in phot1 localization and function. Likewise, inhibition of phosphatidic acid (PA) signaling was found to impair phot1 localization and function. However, action of PA inhibition on phot1 function may be attributed to pleiotropic effects on cell growth. While phot1 kinase activation is necessary to stimulate its internalization, autophosphorylation is not required for phot1 turnover in response to prolonged blue light irradiation. The implications of these findings in regard to phot1 localization and function are discussed.Key words: phototropin 1 (phot1), phototropism, subcellular trafficking, autophosphorylation, protein turnover     

Detection of intracellular phosphatidylserine in living cells

To demonstrate the intracellular phosphatidylserine (PS) distribution in neuronal cells, neuroblastoma cells and hippocampal neurons expressing green fluorescence protein (GFP)-AnnexinV were stimulated with a calcium ionophore and localization of GFP-AnnexinV was monitored by fluorescence microscopy. Initially, GFP-AnnexinV distributed evenly in the cytosol and nucleus. Raising the intracellular calcium level with ionomycin-induced translocation of cytoplasmic GFP-AnnexinV to the plasma membrane but not to the nuclear membrane, indicating that PS distributes in the cytoplasmic side of the plasma membrane. Nuclear GFP-AnnexinV subsequently translocated to the nuclear membrane, indicating PS localization in the nuclear envelope. GFP-AnnexinV also localized in a juxtanuclear organelle that was identified as the recycling endosome. However, minimal fluorescence was detected in any other subcellular organelles including mitochondria, endoplasmic reticulum, Golgi complex, and lysosomes, strongly suggesting that PS distribution in the cytoplasmic face in these organelles is negligible. Similarly, in hippocampal primary neurons PS distributed in the inner leaflet of plasma membranes of cell body and dendrites, and in the nuclear envelope. To our knowledge, this is the first demonstration of intracellular PS localization in living cells, providing an insight for specific sites of PS interaction with soluble proteins involved in signaling processes.     

Determinants of AQP6 trafficking to intracellular sites versus the plasma membrane in transfected mammalian cells

BACKGROUND INFORMATION: Most AQPs (aquaporins) function at the plasma membrane, however AQP6 is exclusively localized to membranes of intracellular vesicles in acid-secreting type-A intercalated cells of renal collecting ducts. The intracellular distribution indicates that AQP6 has a function distinct from trans-epithelial water movement. RESULTS: We show by mutational analyses and immunofluorescence that the N-terminus of AQP6 is a determinant for its intracellular localization. Presence or absence at the plasma membrane of AQP6 constructs was confirmed by electrophysiological methods. Addition of a GFP (green fluorescent protein) or a HA (haemagglutinin) epitope tag (GFP-AQP6 or HA-AQP6) to the N-terminus of AQP6, directed AQP6 to the plasma membranes of transfected Madin-Darby canine kidney cells. In contrast, addition of a GFP tag to the C-terminus (AQP6-GFP) caused the protein to remain intracellular, similar to untagged wild-type AQP6. Replacement of the N-terminus of AQP6 by that of AQP1 also directed AQP6 to the plasma membranes, whereas the N-terminus of AQP6 retained AQP1 in cytosolic sites. CONCLUSION: Our results suggest that the N-terminus of AQP6 is critical for trafficking of the protein to the intracellular sites. Moreover, our studies provide an approach for future identification of proteins involved in vesicle sorting in the acid-secreting type-A intercalated cells.     

Mapping of the phosphorylation sites on the phototropic signal transducer, NPH3

The phototropic response in Arabidopsis thaliana is initiated by the blue-light photoreceptors, phototropin (phot)1 and phot2. A recent study has shown that one of the phototropic signal transducers, NPH3, is phosphorylated under dark conditions and dephosphorylated by blue-light irradiation. To further understand the function of phosphorylation and dephosphorylation of NPH3 during this phototropic response, we have mapped the phosphorylation sites of NPH3 in our current study. The NPH3 protein has at least three phosphorylation sites at serine residues, Ser212, Ser222, and Ser236, and these sites are dephosphorylated by blue-light irradiation. By immunoblotting analysis, these phosphorylation sites in phot1 mutants are not dephosphorylated following blue-light irradiation at both low and high fluence rates, even though such irradiations induce the phot2-dependent phototropic response in phot1. These results suggest that the dephosphorylated NPH3 is involved in the phot1-dependent phototropic response and is not essential for the phot2-dependent phototropic response. We generated two types of transgenic nph3 plants expressing a NPH3^{S212A/S222A/S232A/S236A} protein and a NPH3^Δ212–238 protein in which the phosphorylation region is deleted, and assessed the phototropic phenotype of these. Based upon our present findings, we discuss the role of dephosphorylated and phosphorylated NPH3 in mediating the phototropic response.     

FLOWERING LOCUS T regulates stomatal opening

Stomatal pores surrounded by a pair of guard cells in the plant epidermis control gas exchange for photosynthesis in response to light, CO(2), and phytohormone abscisic acid. Phototropins (phot1 and phot2) are plant blue-light receptor kinases and mediate stomatal opening via activation of the plasma membrane H(+)-ATPase. However, the signaling mechanism from phototropins to the H(+)-ATPase has yet to be determined. Here, we show that FLOWERING LOCUS T (FT) is expressed in guard cells and regulates stomatal opening. We isolated an scs (suppressor of closed-stomata phenotype in phot1 phot2) 1-1 mutant of Arabidopsis thaliana and showed that scs1-1 carries a novel null early flowering 3 (elf3) allele in a phot1 phot2 background. scs1-1 (elf3 phot1 phot2 triple mutant) had an open-stomata phenotype with high H(+)-ATPase activity and showed increased levels of FT mRNA in guard cells. Transgenic plants overexpressing FT in guard cells showed open stomata, whereas a loss-of-function FT allele, ft-1, exhibited closed stomata and failed to activate the H(+)-ATPase in response to blue light. Our results define a new cell-autonomous role for FT and demonstrate that the flowering time genes ELF3 and FT are involved in the regulation of H(+)-ATPase by blue light in guard cells.     

The signal transducer NPH3 integrates the phototropin1 photosensor with PIN2-based polar auxin transport in Arabidopsis root phototropism

Under blue light (BL) illumination, Arabidopsis thaliana roots grow away from the light source, showing a negative phototropic response. However, the mechanism of root phototropism is still unclear. Using a noninvasive microelectrode system, we showed that the BL sensor phototropin1 (phot1), the signal transducer NONPHOTOTROPIC HYPOCOTYL3 (NPH3), and the auxin efflux transporter PIN2 were essential for BL-induced auxin flux in the root apex transition zone. We also found that PIN2-green fluorescent protein (GFP) localized to vacuole-like compartments (VLCs) in dark-grown root epidermal and cortical cells, and phot1/NPH3 mediated a BL-initiated pathway that caused PIN2 redistribution to the plasma membrane. When dark-grown roots were exposed to brefeldin A (BFA), PIN2-GFP remained in VLCs in darkness, and BL caused PIN2-GFP disappearance from VLCs and induced PIN2-GFP-FM4-64 colocalization within enlarged compartments. In the nph3 mutant, both dark and BL BFA treatments caused the disappearance of PIN2-GFP from VLCs. However, in the phot1 mutant, PIN2-GFP remained within VLCs under both dark and BL BFA treatments, suggesting that phot1 and NPH3 play different roles in PIN2 localization. In conclusion, BL-induced root phototropism is based on the phot1/NPH3 signaling pathway, which stimulates the shootward auxin flux by modifying the subcellular targeting of PIN2 in the root apex transition zone.     

Primary inhibition of hypocotyl growth and phototropism depend differently on phototropin-mediated increases in cytoplasmic calcium induced by blue light

The phototropin photoreceptors transduce blue-light signals into several physiological and developmental responses in plants. A transient rise in cytoplasmic calcium (Ca2+) that begins within seconds of phototropin 1 (phot1) excitation is believed to be an important element in the transduction pathways leading to one or more of the phot1-dependent responses. The goal of the present work was to determine whether the Ca2+ response was necessary for (a). the inhibition of hypocotyl elongation that develops within minutes of the irradiation, and (b). hypocotyl phototropism (curved growth of the stem in response to asymmetric illumination). After determining that pulses of light delivering photon fluences of between 1 and 1000 micromol m-2 induced growth inhibition mediated by phot1 without significant interference from other photosensory pathways, the effect of blocking the Ca2+ rise was assessed. Treatment of seedlings with a Ca2+ chelator prevented the rise in cytoplasmic Ca2+ and prevented phot1-mediated growth inhibition. However, the same chelator treatment did not impair phot1-mediated phototropism. Thus, it appears that the early, transient rise in cytoplasmic Ca2+ is an important intermediary process in at least one but not all phot1-signaling pathways.     

Physiological roles of the light, oxygen, or voltage domains of phototropin 1 and phototropin 2 in Arabidopsis

Phototropins (phot1 and phot2) are plant blue-light receptors that mediate phototropism, chloroplast movement, stomatal opening, rapid inhibition of growth of etiolated seedlings, and leaf expansion in Arabidopsis (Arabidopsis thaliana). Their N-terminal region contains two light, oxygen, or voltage (LOV) domains, which bind flavin mononucleotide and form a covalent adduct between a conserved cysteine and the flavin mononucleotide chromophore upon photoexcitation. The C-terminal region contains a serine/threonine kinase domain that catalyzes blue-light-activated autophosphorylation. Here, we have transformed the phot1 phot2 (phot1-5 phot2-1) double mutant with PHOT expression constructs driven by the cauliflower mosaic virus 35S promoter. These constructs encode either wild-type phototropin or phototropin with one or both LOV-domain cysteines mutated to block their photochemistry. We selected multiple lines in each of the eight resulting categories of transformants for further physiological analyses. Specifically, we investigated whether LOV1 and LOV2 serve the same or different functions for phototropism and leaf expansion. Our results show that the LOV2 domain of phot1 plays a major role in phototropism and leaf expansion, as does the LOV2 domain of phot2. No complementation of phototropism or leaf expansion was observed for the LOV1 domain of phot1. However, phot2 LOV1 was unexpectedly found to complement phototropism to a considerable level. Similarly, transformants carrying a PHOT transgene with both LOV domains inactivated developed strong curvatures toward high fluence rate blue light. However, we found that the phot2-1 mutant is leaky and produces a small level of full-length phot2 protein. In vitro experiments indicate that cross phosphorylation can occur between functional phot2 and inactivated phot1 molecules. Such a mechanism may occur in vivo and therefore account for the functional activities observed in the PHOT transgenics with both lov domains inactivated. The implications of this mechanism with respect to phototropin function are discussed.     

Functional analyses of the activation loop of phototropin2 in Arabidopsis

Phototropins (phot1 and phot2) are autophosphorylating blue-light receptor kinases that mediate blue-light responses such as phototropism, chloroplast accumulation, and stomatal opening in Arabidopsis (Arabidopsis thaliana). Only phot2 induces the chloroplast avoidance response under strong blue light. The serine (Ser) residues of the kinase activation loop in phot1 are autophosphorylated by blue light, and autophosphorylation is essential for the phot1-mediated responses. However, the role of autophosphorylation in phot2 remains to be determined. In this study, we substituted the conserved residues of Ser-761 and Ser-763 with alanine (S761A S763A) in the phot2 activation loop and analyzed their function by investigating the phot2-mediated responses after the transformation of phot1 phot2 double mutant with this mutant phot2 gene. Transgenic plants expressing the mutant phot2 protein exhibited impaired responses in chloroplast movement, stomatal opening, phototropic bending, leaf flattening, and plant growth; and those expressing phot2 with S761D S763D mutations showed the normal responses. Substitution of both Ser-761 and Ser-763 with alanine in phot2 did not significantly affect the kinase activity in planta. From these results, we conclude that phosphorylation of Ser-761 and Ser-763 in the activation loop may be a common primary step for phot2-mediated responses.     

SCARFACE encodes an ARF-GAP that is required for normal auxin efflux and vein patterning in Arabidopsis

To identify molecular mechanisms controlling vein patterns, we analyzed scarface (sfc) mutants. sfc cotyledon and leaf veins are largely fragmented, unlike the interconnected networks in wild-type plants. SFC encodes an ADP ribosylation factor GTPase activating protein (ARF-GAP), a class with well-established roles in vesicle trafficking regulation. Quadruple mutants of SCF and three homologs (ARF-GAP DOMAIN1, 2, and 4) showed a modestly enhanced vascular phenotype. Genetic interactions between sfc and pinoid and between sfc and gnom suggest a possible function for SFC in trafficking of auxin efflux regulators. Genetic analyses also revealed interaction with cotyledon vascular pattern2, suggesting that lipid-based signals may underlie some SFC ARF-GAP functions. To assess possible roles for SFC in auxin transport, we analyzed sfc roots, which showed exaggerated responses to exogenous auxin and higher auxin transport capacity. To determine whether PIN1 intracellular trafficking was affected, we analyzed PIN1:green fluorescent protein (GFP) dynamics using confocal microscopy in sfc roots. We found normal PIN1:GFP localization at the apical membrane of root cells, but treatment with brefeldin A resulted in PIN1 accumulating in smaller and more numerous compartments than in the wild type. These data suggest that SFC is required for normal intracellular transport of PIN1 from the plasma membrane to the endosome.     

Functional expression and cellular localization of the Na+/H+ exchanger Sod2 of the fission yeast Schizosaccharomyces pombe

In the fission yeast Schizosaccharomyces pombe, the Na+/H+ exchanger, Sod2, plays a major role in the removal of excess intracellular sodium, and its disruption results in a sodium-sensitive phenotype. We examined the subcellular distribution and dynamics of Sod2 expression in S. pombe using a sod2-GFP fusion protein under the control of an attenuated version of the inducible nmt promoter. Sod2 was localized throughout the plasma membrane, the nuclear envelope, and some internal membrane systems. In exponentially growing cells, in which sod2-GFP was expressed and then the promoter turned-off, previously synthesized sod2-GFP was stable for long periods and found localized to the plasma membrane in the medial regions of the cell. It was not present at the actively growing cell ends. This suggests that these regions of the cell contain old plasma membrane protein vs. newly synthesized plasma membrane without Sod2 at the growing ends. Sod2 localization was not affected by salt stress. The results suggest that Sod2 is both a plasma membrane protein and is present in intracellular membranes. It is likely tethered within discrete regions of the plasma membrane and is not free to diffuse throughout the bilayer.     

Tomato LeAGP-1 is a plasma membrane-bound, glycosylphosphatidylinositol-anchored arabinogalactan-protein

Arabinogalactan-proteins (AGPs) are a class of highly glycosylated, hydroxyproline-rich glycoproteins that function in plant growth and development. Tomato LeAGP-1 represents a major AGP expressed in cultured cells and plants. Based on cDNA and amino acid sequence analyses along with carbohydrate and other biochemical analyses, tomato LeAGP-1 is hypothesized to be a classical AGP localized to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. Here, this hypothesis was tested and supported with the following experiments. First, tomato ( Lycopersicon esculentum , cv. UC82B) cotyledon protoplasts were isolated following cell wall digestion with cellulase and pectinase, and LeAGP-1 was immunolocalized to the plasma membrane with a LeAGP-1 antibody. Second, LeAGP-1 was shown to be a major AGP component in plasma membrane vesicles from tomato cv. Bonnie Best suspension-cultured cells by Western blot analysis with the LeAGP-1 antibody. Third, fluorescence microscopy of plasmolysed, transgenic tobacco ( Nicotiana tabacum BY-2) suspension-cultured cells expressing a green fluorescent protein (GFP)-LeAGP-1 fusion product demonstrated localization to the plasma membrane and Hechtian threads. Fourth, the GFP-LeAGP-1 fusion protein was present in plasma membrane preparations from these transgenic tobacco cells by Western blot analysis with a GFP antibody. Fifth, GFP-LeAGP-1 secreted into the culture media contained ethanolamine, presumably attached to the C-terminal amino acid residue, consistent with its processing and release from the plasma membrane. Thus, these data support the hypothesis that LeAGP-1 is localized to the plasma membrane via a GPI anchor and suggest possible roles for LeAGP-1 in cellular signalling and matrix remodelling.     

H-Ras dynamically interacts with recycling endosomes in CHO-K1 cells: involvement of Rab5 and Rab11 in the trafficking of H-Ras to this pericentriolar endocytic compartment

H-, N-, and K-Ras are isoforms of Ras proteins, which undergo different lipid modifications at the C terminus. These post-translational events make possible the association of Ras proteins both with the inner plasma membrane and to the cytosolic surface of endoplasmic reticulum and Golgi complex, which is also required for the proper function of these proteins. To better characterize the intracellular distribution and sorting of Ras proteins, constructs were engineered to express the C-terminal domain of H- and K-Ras fused to variants of green fluorescent protein. Using confocal microscopy, we found in CHO-K1 cells that H-Ras, which is palmitoylated and farnesylated, localized at the recycling endosome in addition to the inner leaflet of the plasma membrane. In contrast, K-Ras, which is farnesylated and nonpalmitoylated, mainly localized at the plasma membrane. Moreover, we demonstrate that sorting signals of H- and K-Ras are contained within the C-terminal domain of these proteins and that palmitoylation on this region of H-Ras might operate as a dominant sorting signal for proper subcellular localization of this protein in CHO-K1 cells. Using selective photobleaching techniques, we demonstrate the dynamic nature of H-Ras trafficking to the recycling endosome from plasma membrane. We also provide evidence that Rab5 and Rab11 activities are required for proper delivery of H-Ras to the endocytic recycling compartment. Using a chimera containing the Ras binding domain of c-Raf-1 fused to a fluorescent protein, we found that a pool of GTP-bound H-Ras localized on membranes from Rab11-positive recycling endosome after serum stimulation. These results suggest that H-Ras present in membranes of the recycling endosome might be activating signal cascades essential for the dynamic and function of the organelle.     

Immunolocalization of alkaloids and X-ray microanalysis of elements in lupin seeds

Summary Immunolocalization of alkaloids in lupin seeds (Lupinus spp.) has been performed by cryofixation and conventional methods. Alkaloids were localized in the protein bodies of the cotyledon cells. Some immunogold particles in the walls of these cells were also observed. There were no differences in the sites of localization between the two mentioned methods. X-ray microanalysis of elements showed the presence of P, Mg, S, and K in the protein bodies of cotyledon cells in lupin seeds. The role of K⁺ in alkaloids transport is discussed.