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

Protein storage vacuoles originate by remodelling of pre-existing vacuoles in Arabidopsis thaliana

Protein storage vacuoles (PSV) are the main repository of protein in dicotyledonous seeds. Little is known about the origins of these transient organelles. PSV are hypothesised to either arise de novo or to originate from the pre-existing embryonic vacuole (EV) during seed maturation. We have tested these hypotheses by studying PSV formation in Arabidopsis embryos at different stages of seed maturation and have recapitulated this process in Arabidopsis leaves reprogrammed to an embryogenic fate by inducing expression of the LEAFY COTYLEDON2 transcription factor. Confocal and immunoelectron microscopy indicate that both storage proteins and tonoplast proteins typical of PSV are delivered to the pre-existing 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 reveals the 3D architecture of forming PSV. Our results indicate that in Arabidopsis the pre-existing vacuole is reprogrammed to become a PSV

Create the Scene and Watch the Show Unfold: Following Vegetative to Embryonic Developmental Transitions by Over-Expressing LEC2 in Leaves of Arabidopsis thaliana

During seed development, the lytic vacuole (LV) is replaced by a protein storage vacuole (PSV) which specializes in accumulating seed storage proteins (SSPs). As seed protein reserves are mobilized upon germination, the PSV is once again replaced by the LV which takes on different roles in vegetative tissues. Cellular events occurring during these developmental transitions are not well understood, particularly, the transition between vacuole types. This research investigates whether PSVs can exist in leaves. To study vacuole transitions in leaves, an Arabidopsis thaliana line over-expressing the LEAFY COTYLEDON2 (LEC2) transcription factor was used. LEC2 is a master regulator of embryogenesis responsible for creating a cellular environment that promotes embryogenic development. Over-expression of LEC2 causes vegetative tissues to change their developmental fate to an embryonic state. LEC2 alters the leaf phenotype at the subcellular level; chloroplasts de-differentiated and contained more starch. The cytoplasm becomes filled with oil bodies, which are typically seed organelles. The large LV was replaced by small-sized vacuoles that accumulated protein deposits. Since LEC2 is responsible for activating the synthesis of SSPs during seed development, SSP accumulation was investigated in leaves. The major Arabidopsis SSP families were shown to accumulate within small sized vacuoles in leaf cells. By exploiting the developmental and tissue specific localization of two tonoplast intrinsic protein (TIP) isoforms, the small vacuoles were identified as PSVs. A time course following the cellular alterations and accumulation of seed proteins in leaves after induction of LEC2 activity with dexamethasone (DEX) revealed the appearance of embryonic characteristics as early as 4 days on DEX and became more prominent over time. Additionally, a dynamic view of the transition between vacuole types was observed using TIP isoforms fused to fluorescent markers. The morphology of leaf vacuoles was altered to resemble an amalgamation of a LV and PSV. Results suggest that as the LV transitions to a PSV, the tonoplast remodels before the large vacuole is replaced by smaller PSVs. The formation of PSVs in leaves in response to LEC2 over-expression is a novel approach to study vacuoles and will lead to a better understanding of their basic biology

Progress in vegetable crop transformation and future prospects and challenges

Genetically Modified Crops: Their Development, Uses, and Risks examines cutting-edge methods for integrating foreign DNA into plant cells. This volume details the tools and techniques for gene transfer, and identifies the benefits, risks, and limitations of these methods in horticulture and agriculture. Complete with references, figures, and photographs, this book is a valuable manual for agronomists, plant and molecular geneticists, and students of agronomy, genetics, entomology, horticulture, and plant pathology

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

Hemp (Cannabis sativa L.)

A gold standard collection of Agrobacterium-mediated transformation techniques for state-of-the-art plant genetic engineering, functional genomic analysis, and crop improvement. Volume 1 details the most updated techniques available for twenty-six plant species drawn from cereal crops, industrial plants, legume plants, and vegetable plants, and presents various methods for introducing DNA into three major model plant species, Arabidopsis thaliana, Medicago truncatula, and Nicotiana. The authors also outline the basic methods in Agrobacterium manipulation and strategies for vector construction. Volume 2 contains another thirty-three proven techniques for root plants, turf grasses, woody species, tropic plants, nuts and fruits, ornamental plants, and medicinal plants. Additional chapters provide methods for introducing DNA into non-plant species, such as bacteria, fungi, algae, and mammalian cells. The protocols follow the successful Methods in Molecular Biology series format, each offering step-by-step laboratory instructions, an introduction outlining the principles behind the technique, lists of the necessary equipment and reagents, and tips on troubleshooting and avoiding known pitfalls

Tissue culture and Agrobacterium-mediated transformation of hemp (Cannabis sativa L.)

Hemp (Cannabis sativa L.) is cultivated in many parts of the world for ils fiber, oil, and seed. The development of new hemp cultivars with improved traits could be facilitated through the application of biotechnological strategies. The purpose of this study was to investigate the propagation of hemp in tissue culture and to establish a protocol for Agrobacterium-mediated transformation for foreign gene introduction. Stem and leaf segments from seedlings of four hemp varieties were placed on Murashige and Skoog medium with Gamborg B5 vitamins (MB) supplemented with 5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 μM kinetin, 3% sucrose, and 8 gl−1 agar. Large masses of callus were produced within 4 wk for all cultivars. Suspension cultures were established in MB medium containing 2.5 μM 2,4-D. To promote embryogenesis or organogenesis, explants, callus, and suspension cultures derived from a range of explant sources and seedling ages were exposed to variations in the culture medium and changes to the culture environment. None of the treatments tested were successful in promoting plantlet regeneration. Suspension cells were transformed with Agrobacterium tumefaciens strain EHA101 carrying the binary vector pNOV3635 with a gene encoding phosphomannose isomerase (PMI). Transformed callus was selected on medium containing 1–2% mannose. A chlorophenol red assay was used to confirm that the PMI gene was expressed. Polymerase chain reaction and Southern hybridization detected the presence of the PMI gene. Copy number in different lines ranged from one to four

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 %

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%