Wetting of phase-separated droplets on plant vacuole membranes leads to a competition between tonoplast budding and nanotube formation.

Seeds of dicotyledonous plants store proteins in dedicated membrane-bounded organelles called protein storage vacuoles (PSVs). Formed during seed development through morphological and functional reconfiguration of lytic vacuoles in embryos [M. Feeney et al., Plant Physiol. 177, 241-254 (2018)], PSVs undergo division during the later stages of seed maturation. Here, we study the biophysical mechanism of PSV morphogenesis in vivo, discovering that micrometer-sized liquid droplets containing storage proteins form within the vacuolar lumen through phase separation and wet the tonoplast (vacuolar membrane). We identify distinct tonoplast shapes that arise in response to membrane wetting by droplets and derive a simple theoretical model that conceptualizes these geometries. Conditions of low membrane spontaneous curvature and moderate contact angle (i.e., wettability) favor droplet-induced membrane budding, thereby likely serving to generate multiple, physically separated PSVs in seeds. In contrast, high membrane spontaneous curvature and strong wettability promote an intricate and previously unreported membrane nanotube network that forms at the droplet interface, allowing molecule exchange between droplets and the vacuolar interior. Furthermore, our model predicts that with decreasing wettability, this nanotube structure transitions to a regime with bud and nanotube coexistence, which we confirmed in vitro. As such, we identify intracellular wetting [J. Agudo-Canalejo et al., Nature 591, 142-146 (2021)] as the mechanism underlying PSV morphogenesis and provide evidence suggesting that interconvertible membrane wetting morphologies play a role in the organization of liquid phases in cells.

ABCC Transporters Mediate the Vacuolar Accumulation of Crocins in Saffron Stigmas.

Compartmentation is a key strategy enacted by plants for the storage of specialized metabolites. The saffron spice owes its red color to crocins, a complex mixture of apocarotenoid glycosides that accumulate in intracellular vacuoles and reach up to 10% of the spice dry weight. We developed a general approach, based on coexpression analysis, heterologous expression in yeast (Saccharomyces cerevisiae), and in vitro transportomic assays using yeast microsomes and total plant metabolite extracts, for the identification of putative vacuolar metabolite transporters, and we used it to identify Crocus sativus transporters mediating vacuolar crocin accumulation in stigmas. Three transporters, belonging to both the multidrug and toxic compound extrusion and ATP binding cassette C (ABCC) families, were coexpressed with crocins and/or with the gene encoding the first dedicated enzyme in the crocin biosynthetic pathway, CsCCD2. Two of these, belonging to the ABCC family, were able to mediate transport of several crocins when expressed in yeast microsomes. CsABCC4a was selectively expressed in C. sativus stigmas, was predominantly tonoplast localized, transported crocins in vitro in a stereospecific and cooperative way, and was able to enhance crocin accumulation when expressed in Nicotiana benthamiana leaves.plantcell;31/11/2789/FX1F1fx1.

Aquaporins influence seed dormancy and germination in response to stress.

Aquaporins influence water flow in plants, yet little is known of their involvement in the water-driven process of seed germination. We therefore investigated their role in seeds in the laboratory and under field and global warming conditions. We mapped the expression of tonoplast intrinsic proteins (TIPs) during dormancy cycling and during germination under normal and water stress conditions. We found that the two key tonoplast aquaporins, TIP3;1 and TIP3;2, which have previously been implicated in water or solute transport, respectively, act antagonistically to modulate the response to abscisic acid, with TIP3;1 being a positive and TIP3;2 a negative regulator. A third isoform, TIP4;1, which is normally expressed upon completion of germination, was found to play an earlier role during water stress. Seed TIPs also contribute to the regulation of depth of primary dormancy and differences in the induction of secondary dormancy during dormancy cycling. Protein and gene expression during annual cycling under field conditions and a global warming scenario further illustrate this role. We propose that the different responses of the seed TIP contribute to mechanisms that influence dormancy status and the timing of germination under variable soil conditions.

Enhancing the Secretion of a Glyco-Engineered Anti-CD20 scFv-Fc Antibody in Hairy Root Cultures.

Hairy root (HR) cultures represent an attractive platform for the production of heterologous proteins, due to the possibility of secreting the molecule of interest in the culture medium. The main limitation is the low accumulation yields of heterologous proteins. The aim of this study is to enhance the accumulation of a tumor-targeting antibody with a human-compatible glycosylation profile in HR culture medium. To this aim, the authors produce Nicotiana benthamiana HR cultures expressing the red fluorescent protein (RFP) to easily screen for different auxins able to induce heterologous protein secretion in the medium. The hormone 2,4-dichlorophenoxyacetic acid (2,4-D) is found to induce high accumulation levels (334 mg L-1 ) of RFP in the culture medium. The same protocol is used to improve the secretion of the tumor-targeting, CD20-specific 2B8-FcΔXF recombinant antibody from glyco-engineered ΔXTFT N. benthamiana HR cultures. The addition of 2,4-D determine a 28-fold increase of the accumulation of fully functional 2B8-FcΔXF in the culture medium, at levels of ≈16 mg L-1 . Antibody N-glycosylation profiling reveal the prominent occurrence of GnGn structures and low levels of xylose- and fucose-containing counterparts. This result is the first example of the expression of an engineered anti-CD20 antibody with a scFv-Fc format at high levels in HR.

Protein Storage Vacuoles Originate from Remodeled Preexisting Vacuoles in Arabidopsis thaliana.

Protein storage vacuoles (PSV) are the main repository of protein in dicotyledonous seeds, but little is known about the origins of these transient organelles. PSV are hypothesized to either arise de novo or originate from the preexisting embryonic vacuole (EV) during seed maturation. Here, we tested these hypotheses by studying PSV formation in Arabidopsis (Arabidopsis thaliana) embryos at different stages of seed maturation and recapitulated this process in Arabidopsis leaves reprogrammed to an embryogenic fate by inducing expression of the LEAFY COTYLEDON2 transcription factor. Confocal and immunoelectron microscopy indicated that both storage proteins and tonoplast proteins typical of PSV were delivered to the preexisting EV in embryos or to the lytic vacuole in reprogrammed leaf cells. In addition, sectioning through embryos at several developmental stages using serial block face scanning electron microscopy revealed the 3D architecture of forming PSV. Our results indicate that the preexisting EV is reprogrammed to become a PSV in Arabidopsis.

Subcellular localization and interactions among rubber particle proteins from Hevea brasiliensis.

Natural rubber (polyisoprene) from the rubber tree Hevea brasiliensis is synthesized by specialized cells called laticifers. It is not clear how rubber particles arise, although one hypothesis is that they derive from the endoplasmic reticulum (ER) membrane. Here we cloned the genes encoding four key proteins found in association with rubber particles and studied their intracellular localization by transient expression in Nicotiana benthamiana leaves. We show that, while the cis-prenyltransferase (CPT), responsible for the synthesis of long polyisoprene chains, is a soluble, cytosolic protein, other rubber particle proteins such as rubber elongation factor (REF), small rubber particle protein (SRPP) and Hevea rubber transferase 1-REF bridging protein (HRBP) are associated with the endoplasmic reticulum (ER). We also show that SRPP can recruit CPT to the ER and that interaction of CPT with HRBP leads to both proteins relocating to the plasma membrane. We discuss these results in the context of the biogenesis of rubber particles.

Hemp (Cannabis sativa L.).

Hemp (Cannabis sativa L.) suspension culture cells were transformed with Agrobacterium tumefaciens strain EHA101 carrying the binary plasmid pNOV3635. The plasmid contains a phosphomannose isomerase (PMI) selectable marker gene. Cells transformed with PMI are capable of metabolizing the selective agent mannose, whereas cells not expressing the gene are incapable of using the carbon source and will stop growing. Callus masses proliferating on selection medium were screened for PMI expression using a chlorophenol red assay. Genomic DNA was extracted from putatively transformed callus lines, and the presence of the PMI gene was confirmed using PCR and Southern hybridization. Using this method, an average transformation frequency of 31.23% ± 0.14 was obtained for all transformation experiments, with a range of 15.1-55.3%.

Reprogramming cells to study vacuolar development.

During vegetative and embryonic developmental transitions, plant cells are massively reorganized to support the activities that will take place during the subsequent developmental phase. Studying cellular and subcellular changes that occur during these short transitional periods can sometimes present challenges, especially when dealing with Arabidopsis thaliana embryo and seed tissues. As a complementary approach, cellular reprogramming can be used as a tool to study these cellular changes in another, more easily accessible, tissue type. To reprogram cells, genetic manipulation of particular regulatory factors that play critical roles in establishing or repressing the seed developmental program can be used to bring about a change of cell fate. During different developmental phases, vacuoles assume different functions and morphologies to respond to the changing needs of the cell. Lytic vacuoles (LVs) and protein storage vacuoles (PSVs) are the two main vacuole types found in flowering plants such as Arabidopsis. Although both are morphologically distinct and carry out unique functions, they also share some similar activities. As the co-existence of the two vacuole types is short-lived in plant cells, how they replace each other has been a long-standing curiosity. To study the LV to PSV transition, LEAFY COTYLEDON2, a key transcriptional regulator of seed development, was overexpressed in vegetative cells to activate the seed developmental program. At the cellular level, Arabidopsis leaf LVs were observed to convert to PSV-like organelles. This presents the opportunity for further research to elucidate the mechanism of LV to PSV transitions. Overall, this example demonstrates the potential usefulness of cellular reprogramming as a method to study cellular processes that occur during developmental transitions.

Following vegetative to embryonic cellular changes in leaves of Arabidopsis overexpressing LEAFY COTYLEDON2.

Embryogenesis in flowering plants is controlled by a complex interplay of genetic, biochemical, and physiological regulators. LEAFY COTYLEDON2 (LEC2) is among a small number of key transcriptional regulators that are known to play important roles in controlling major events during the maturation stage of embryogenesis, notably, the synthesis and accumulation of storage reserves. LEC2 overexpression causes vegetative tissues to change their developmental fate to an embryonic state; however, little information exists about the cellular changes that take place. We show that LEC2 alters leaf morphology and anatomy and causes embryogenic structures to form subcellularly in leaves of Arabidopsis (Arabidopsis thaliana). Chloroplasts accumulate more starch, the cytoplasm fills with oil bodies, and lytic vacuoles (LVs) appear smaller in size and accumulate protein deposits. Because LEC2 is responsible for activating the synthesis of seed storage proteins (SSPs) during seed development, SSP accumulation was investigated in leaves. The major Arabidopsis SSP families were shown to accumulate within small leaf vacuoles. By exploiting the developmental and tissue-specific localization of two tonoplast intrinsic protein isoforms, the small leaf vacuoles were identified as protein storage vacuoles (PSVs). Confocal analyses of leaf vacuoles expressing fluorescently labeled tonoplast intrinsic protein isoforms reveal an altered tonoplast morphology resembling an amalgamation of a LV and PSV. Results suggest that as the LV transitions to a PSV, the tonoplast remodels before the large vacuole lumen is replaced by smaller PSVs. Finally, using vegetative and seed markers to monitor the transition, we show that LEC2 induces a reprogramming of leaf development.

Hemp (Cannabis sativa L.).

Hemp (Cannabis sativa L.) suspension culture cells were transformed with Agrobacterium tumefaciens strain EHA101 carrying the binary plasmid pNOV3635. The plasmid contains a phosphomannose isomerase (PMI) selectable marker gene. Cells transformed with PMI are capable of metabolizing the selective agent, mannose, whereas cells not expressing the gene are incapable of using the carbon source and will stop growing. Callus masses proliferating on selection were screened for PMI expression using a chlorophenol red assay. Genomic DNA was extracted from putatively transformed callus lines and the presence of the PMI gene was confirmed using polymerase chain reaction and Southern hybridization. Using this method, an average transformation frequency of 31.23% +/- 0.14 was obtained for all transformation experiments, with a range of 15.1 to 55.3%.