No abstract

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.

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 () embryos at different stages of seed maturation and recapitulated this process in Arabidopsis leaves reprogrammed to an embryogenic fate by inducing expression of the 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.

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.

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.

The major proteins of rubber particles from Hevea brasiliensis associate with the endoplasmic reticulum membrane and can interact with the key cytosolic biosynthetic enzyme cis-prenyltransferase.

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.

A complete method to regenerate adventitious shoots and to produce field-ready trees from three commercial cultivars of sweet cherry (Prunus avium L.) is described. The effects of explant types, pre-treatments, basal media, and phloroglucinol on cultivars Bing, Sweetheart, and Lapins were investigated. Callus developed on four explant types: apical shoot tips isolated from orchard trees; and punctured shoot tips, stem sections, and shoot bases of in vitro shoot cultures. Callus formed on Bing (5%), Sweetheart (8%), and Lapins (20%) shoot tips from orchard trees after 4 months on Murashige and Skoog medium (MS) at half-strength with 3 mM benzylaminopurine (BA). In vitro-derived explants formed callus after 3 months on Woody Plant Medium with 3 mM BA (W3B): punctured shoot tips (Sweetheart and Lapins 67%), stem sections (Sweetheart 31%, Lapins 27%), and shoot bases (Sweetheart 10%, Lapins 17%). Pre-treatment of shoot cultures on MS with 3 mM BA and 1 mM phloroglucinol increased callus formation three-fold on shoot base explants. Callus was separated from parental explants and maintained on MS with 3 mM BA. Shooting was induced by transferring callus to W3B. At 2 weeks, shoot development approached 100%. By 4 weeks, 7-17 shoots had formed on each explant. Callus was maintained for 1.5 years with no decrease in shoot production. Shoots were grafted onto Mazzard (P. avium) rootstocks with 54% (Sweetheart), 57% (Lapins), and 21% (Bing) success after 5 weeks.