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At least 19 records

Data for Expression of a Bacterial Trehalose 6-Phosphate Synthase Gene otsA in Camelina sativa Seeds Promotes the Channelling of Carbon Towards Oil Accumulation

Improving seed oil yield is essential for developing Camelina sativa as a sustainable biofuel crop. Fatty acid synthesis depends on the production of acetyl-CoA from photosynthetically derived sugars. Trehalose 6-phosphate (T6P), a proxy for sucrose availability, can link sugar status to plant growth and development. Synthesised by trehalose 6-phosphate synthase (TPS) from UDP-glucose and glucose-6-phosphate, T6P plays a regulatory role in metabolism. Our previous studies on Arabidopsis transgenic lines constitutively expressing the E. coli otsA (encoding TPS) showed increased T6P levels and seed triacylglycerol, along with stunted growth. In the present study we express otsA in camelina under the control of a seed-specific Phaseolin promoter. Seeds of the resulting transgenic lines accumulated high levels of T6P, and a 15%–20% increase in total fatty acids and triacylglycerol compared to wild-type. Molecular analysis showed the transgenic seeds had reduced SnRK1 activity, elevated WRI1 protein levels, and increased the levels of WRI1 and its target genes, along with enhanced rates of fatty acid synthesis that increased seed weights relative to wild type. Notably, the increase in oil did not affect seed protein levels but did reduce the soluble metabolite fraction. Crucially, seed-specific expression of otsA mitigated the growth defects associated with constitutive otsA expression, and the transgenic lines showed normal seed development and germination. These findings demonstrate that targeted T6P modulation via seed-specific otsA expression is an effective metabolic engineering strategy to boost oil production in camelina and potentially in other oilseed crops and bioenergy crops such as energycane, sorghum and miscanthus.

Lipids

Rapid quantification of whole seed fatty acid amount, composition, and shape phenotypes from diverse oilseed species with large differences in seed size

Seed oils are widely used in the food, biofuel, and industrial feedstock industries, with their utility and value determined by total oil content and fatty acid composition. Current high throughput seed oil analysis methods either lack accuracy in total fatty acid profiling or require extensive labor for lipid extraction prior to derivatization to fatty acid methyl esters (FAME) and quantification by gas chromatography (GC). Alternatively, direct whole seed FAME production methods have been developed for the very small seeds in the model species Arabidopsis thaliana but these have generally not been adapted to larger seeds of most oilseed crops. High-throughput direct whole seed FAME production methods were optimized for seeds up to 5 mg each utilizing acid-catalyzed esterification. For the oilseed species Camelina sativa, Thlaspi avernse (pennycress), Cuphea viscosissima, and Brassica napus (var. Canola), the total seed fatty acid content and composition from direct seed esterification to FAME matched that of lipid extract derivatization demonstrating the accuracy of the methods. In combination with seed phenotyping using GridFree, this approach enabled the development of a rapid pipeline for simultaneous seed weight, count, size/shape phenotyping, and oil analysis. For the larger and tougher seeds produced by Limnanthes alba (Meadowfoam) and Cannabis sativa L. (hemp) the whole seed acid-based method proved insufficient, and prior laborious homogenization of seeds was required. Therefore, a rapid one-tube bead homogenization and base catalyzed-esterification method was developed. Base-derived fatty acid esterification cannot derivatize free fatty acids leading to slightly lower total seed fatty acid than acid-catalyzed methods, however the seed oil content and fatty acid composition that is valuable for screening large numbers of samples in research populations was accurately measured. New rapid whole seed fatty acid esterification and phenotyping protocols were developed to accurately assess oilseed lipid content. These methods are particularly valuable in oilseed research, breeding, and engineering applications where efficient analysis of large numbers of samples and accurate oil fatty acid profiling is essential. While having been developed for current and emerging oilseed crops, these methods also provide a foundation from which protocols might be established for new and emerging crop species.

59 BASIC BIOLOGICAL SCIENCES

Data for Creating Yellow Seed Camelina sativa with Enhanced Oil Accumulation by CRISPR-Mediated Disruption of Transparent Testa 8

Camelina ( Camelina sativa L.), a hexaploid member of the Brassicaceae family, is an emerging oilseed crop being developed to meet the increasing demand for plant oils as biofuel feedstocks. In other Brassicas, high oil content can be associated with a yellow seed phenotype, which is unknown for camelina. We sought to create yellow seed camelina using CRISPR/Cas9 technology to disrupt its Transparent Testa 8 (TT8) transcription factor genes and to evaluate the resulting seed phenotype. We identified three TT8 genes, one in each of the three camelina subgenomes, and obtained independent CsTT8 lines containing frameshift edits. Disruption of TT8 caused seed coat colour to change from brown to yellow reflecting their reduced flavonoid accumulation of up to 44%, and the loss of a well-organized seed coat mucilage layer. Transcriptomic analysis of CsTT8-edited seeds revealed significantly increased expression of the lipid-related transcription factors LEC1, LEC2, FUS3, and WRI1 and their downstream fatty acid synthesis-related targets. These changes caused metabolic remodelling with increased fatty acid synthesis rates and corresponding increases in total fatty acid (TFA) accumulation from 32.4% to as high as 38.0% of seed weight, and TAG yield by more than 21% without significant changes in starch or protein levels compared to parental line. These data highlight the effectiveness of CRISPR in creating novel enhanced-oil germplasm in camelina. The resulting lines may directly contribute to future net-zero carbon energy production or be combined with other traits to produce desired lipid-derived bioproducts at high yields.

Biofuels

Seed coat transcriptomic profiling of 5-593, a genotype important for genetic studies of seed coat color and patterning in common bean ( Phaseolus vulgaris L.)

Common bean (Phaseolus vulgaris L.) market classes have distinct seed coat colors, which are directly related to the diverse flavonoids found in the mature seed coat. To understand and elucidate the molecular mechanisms underlying the regulation of seed coat color, RNA-Seq data was collected from the black bean 5-593 and used for a differential gene expression and enrichment analysis from four different seed coat color development stages. 5-593 carries dominant alleles for 10 of the 11 major genes that control seed coat color and expression and has historically been used to develop introgression lines used for seed coat genetic analysis. Pairwise comparison among the four stages identified 6,294 differentially expressed genes (DEGs) varying from 508 to 5,780 DEGs depending on the compared stages. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that phenylpropanoid biosynthesis, flavonoid biosynthesis, and plant hormone signal transduction comprised the principal pathways expressed during bean seed coat pigment development. Transcriptome analysis suggested that most structural genes for flavonoid biosynthesis and some potential regulatory genes were significantly differentially expressed. Further studies detected 29 DEGs as important candidate genes governing the key enzymatic flavonoid biosynthetic pathways for common bean seed coat color development. Additionally, four gene models, Pv5-593.02G016100, 593.02G078700, Pv5-593.02G090900, and Pv5-593.06G121300, encode MYB-like transcription factor family protein were identified as strong candidate regulatory genes in anthocyanin biosynthesis which could regulate the expression levels of some important structural genes in flavonoid biosynthesis pathway. These findings provide a framework to draw new insights into the molecular networks underlying common bean seed coat pigment development.

60 APPLIED LIFE SCIENCES

LIPID DROPLET PROTEIN OF SEEDS is involved in the control of lipid droplet size in Arabidopsis seeds and seedlings

Abstract In oilseeds, energy-rich carbon is stored as triacylglycerols in organelles called lipid droplets (LDs). While several of the major biogenetic proteins involved in LD formation have been identified, the full repertoire of LD proteins and their functional roles remains incomplete. Here, we show that the low-abundance, seed-specific LD protein LIPID DROPLET PROTEIN OF SEEDS (LDPS) contains an amphipathic α-helix and proline hairpin motif that serves as an LD-targeting signal and a separate region that binds to the LD protein OLEOSIN 1 (OLEO1). Loss of LDPS function results in smaller LDs and less seed oil in comparison with wild type, while overexpression of LDPS results in an increase in LD size and seed oil content. Loss of LDPS function also results in an inability of LDs to undergo fusion during postgerminative seedling growth. Analysis of oleo1 and ldps single- and double-mutant seeds and freeze–thaw treatment of seeds revealed that OLEO1 suppresses the ability of LDPS to promote larger LDs. Collectively, our results identify LDPS as an important player in LD biology that functions together with OLEO1 to determine LD size in Arabidopsis (Arabidopsis thaliana) seeds and seedlings through a process that involves LD–LD fusion.

Biochemistry & Molecular Biology

Gravity independence of seed-to-seed cycling in Brassica rapa

Growth of higher plants in the microgravity environment of orbital platforms has been problematic. Plants typically developed more slowly in space and often failed at the reproductive phase. Short-duration experiments on the Space Shuttle showed that early stages in the reproductive process could occur normally in microgravity, so we sought a long-duration opportunity to test gravity's role throughout the complete life cycle. During a 122-d opportunity on the Mir space station, full life cycles were completed in microgravity with Brassica rapa L. in a series of three experiments in the Svet greenhouse. Plant material was preserved in space by chemical fixation, freezing, and drying, and then compared to material preserved in the same way during a high-fidelity ground control. At sampling times 13 d after planting, plants on Mir were the same size and had the same number of flower buds as ground control plants. Following hand-pollination of the flowers by the astronaut, siliques formed. In microgravity, siliques ripened basipetally and contained smaller seeds with less than 20% of the cotyledon cells found in the seeds harvested from the ground control. Cytochemical localization of storage reserves in the mature embryos showed that starch was retained in the spaceflight material, whereas protein and lipid were the primary storage reserves in the ground control seeds. While these successful seed-to-seed cycles show that gravity is not absolutely required for any step in the plant life cycle, seed quality in Brassica is compromised by development in microgravity.

Flight Experiment

Effect of cultural conditions on the seed-to-seed growth of Arabidopsis and Cardamine - A study of growth rates and reproductive development as affected by test tube seals

The effects of test tube seals on the growth, flowering, and seed pod formation of Arabidopsis thaliana (L.) Heynh., mouse ear cress, and Cardamine oligosperma Nutt, bitter cress, are studied in order to assess the conditions used in weightlessness experiments. Among other results, it is found that the growth (height) and flowering (date of bud appearance) were suppressed in mouse ear cress in tubes sealed with Saran. Seed pod formation which occurred by day 45 in open-to-air controls, was still lacking in the sealed plants even up to day 124. The growth and flowering of bitter cress were also suppressed by the Saran seal, although up to day 55 the Saran-sealed plants were taller. It is suggested that atmospheric composition was the cause of the suppression of growth, flowering, and seed pod development in these plants, since the mouse ear cress renewed their growth and then set seed pods after the Saran seal was ruptured.

Hoshizaki, T.

Seed-to-seed growth of superdwarf wheat and arabidopsis using red light-emitting diodes (LED's): A report on baseline tests conducted for NASA's proposed Plant Research Unit (PRU)

To determine the influence of narrow-spectrum red light-emitting diodes (LED's) on plant growth and seed production, wheat (Triticum aestivum L.cv Superdwarf) and Arabidopsis (Arabidopsis thaliana (L.) Heynh, race Columbia) plants were grown under red LED's (peak emission 660 nm) and compared to plants grown under daylight fluorescent (white) light and red LED's supplemented with either 1 percent or 10 percent blue fluorescent (BF) light. Wheat growth under red LED's alone appeared normal, whereas Arabidopsis under red LED's alone developed curled leaf margins and a spiraling growth pattern. Both wheat and Arabidopsis under red LED's alone or red LED's + 1 percent BF light had significantly lower seed yield than plants grown under white light. However, the addition of 10 percent BF light to red LED's partially alleviated the adverse effect of red LED's on yield. Irrespective of the light treatment, viable seeds were produced by wheat(75-92 percent germination rate) and Arabidopsis (85-100 percent germination rate). These results indicate that wheat, and to a lesser extent Arabidopsis, can be successfully grown under red LED's alone, but supplemental blue light is required with red LED's to sufficiently match the growth characteristics and seed yield associated with plants grown under white light.

Goins, G. D.

Seed-to-seed growth of Arabidopsis thaliana on the International Space Station

The assembly of the International Space Station (ISS) as a permanent experimental outpost has provided the opportunity for quality plant research in space. To take advantage of this orbital laboratory, engineers and scientists at the Wisconsin Center for Space Automation and Robotics (WCSAR), University of Wisconsin-Madison, developed a plant growth facility capable of supporting plant growth in the microgravity environment. Utilizing this Advanced Astroculture (ADVASC) plant growth facility, an experiment was conducted with the objective to grow Arabidopsis thaliana plants from seed-to-seed on the ISS. Dry Arabidopsis seeds were anchored in the root tray of the ADVASC growth chamber. These seeds were successfully germinated from May 10 until the end of June 2001. Arabidopsis plants grew and completed a full life cycle in microgravity. This experiment demonstrated that ADVASC is capable of providing environment conditions suitable for plant growth and development in microgravity. The normal progression through the life cycle, as well as the postflight morphometric analyses, demonstrate that Arabidopsis thaliana does not require the presence of gravity for growth and development. c2003 COSPAR. Published by Elsevier Ltd. All rights reserved.

ISS Project

Greenhouse (III): Gas-Exchange and Seed-to-Seed Experiments on the Russian Space Station MIR and Earth-grown, Ethylene-Treated Wheat Plants

The Mir Space Station provided an outstanding opportunity to study long-term plant responses when exposed to a microgravity environment. Furthermore, if plants can be grown to maturity in a microgravity environment, they might be used in future bioregenerative life-support systems (BLSS). The primary objective of the Greenhouse experiment onboard Mir was to grow Super Dwarf and Apogee wheat through complete life cycles in microgravity; i.e., from seed-to-seed-to-seed. Additional objectives were to study chemical, biochemical, and structural changes in plant tissues as well as photosynthesis, respiration, and transpiration (evaporation of water from plants). Another major objective was to evaluate the suitability clothe facilities on Mir for advanced research with plants. The Greenhouse experiment was conducted in the Russian/Bulgarian plant growth chamber, the Svet, to which the United States added instrumentation systems to monitor changes in CO2 and water vapor caused by the plants (with four infrared gas analyzers monitoring air entering and leaving two small plastic chambers). In addition, the US instrumentation also monitored O2; air, leaf (IR), cabin pressure; photon flux; and substrate temperature and substrate moisture (16 probes in the root module). Facility modifications were first performed during the summer of 1995 during Mir 19, which began after STS-72 left Mir. Plant development was monitored by daily observations and some photographs.

Campbell, William F.

[Growth of wheat from seed-to-seed in space flight]

In our earlier space experiment with super dwarf wheat we found the spikes developed in space to be barren. The cause of the full crop sterility was sensitivity of this wheat species to the ethylene concentration of 0.3-0.8 mg/m3 during the experiment. The follow-up ground experiments were made to identify species of dwarf wheat that could be raised in space greenhouse Svet and are distinguished by partial tolerance of their reproductive organs to elevated ethylene in air. The choice fell on the USU-Apogee cultivar specially developed for planting in growth chambers as an integral part of various bioregenerative life support systems, including the space ones. An experiment with wheat Apogee was performed in greenhouse Svet on board MIR. The period of the full crop vegetation cycle was not significantly altered under the spaceflight conditions. The experiment yielded 508 seeds from 12 plants, i.e. by 38% less than in laboratory experiments and by 69% more as compared with results of growing crops in ethylene-contaminated atmosphere (1 mg/m3). Mass of the space seeds was low if compared with the laboratory crops. This was the first time when the feasibility of gathering seeds from wheat that had passed the whole vegetation cycle in space flight was demonstrated. The experiment will give mightly impetus to the advancement of research on space biological LSS and gravitational biology.

long duration

In vitro seed to seed growth on clinostats

The effect of a long term micro-gravity environment on the life cycle of plants is unknown. Whether higher plants have evolved to a stage where removal or reduction of gravity is detrimental to plant life cycle and thus fatal to the plant species, is an unanswered question in space plants which were successfully grown through the various stages of their life cycle. Attempts to grow plants as a continuous integral process from seed to seed through one generation were successful until recently. Culture of plants through multiple generations was not accomplished in space nor in ground based studies. The effect of long term simulated weightlessness by growing consecutive generations of plants continuously on clinostats using the cruciferous plants, Arabidopsis thaliana (L.) Heyn. and Cardamine oligosperma Nutt. is being investigated.

Hoshizaki, T.

X-Ray Characterization of Structural Defects in Seeded and Self-Seeded ZnSe Crystal Grown by PVT in Horizontal and Vertical Configurations

As part of a pre-flight ground based investigation of crystal growth of II-VI compound semiconductors, a number of ZnSe boules have been grown by physical vapor transport (PVT) at Marshall Space Flight Center. Boules were grown in both horizontal and vertical configurations and seeded and self-seeded growth techniques were employed. As-grown and/or cleaved boules were examined by a combination of synchrotron white beam x-ray topography (SWBXT) and high resolution triple axis diffraction (HRTXD) to characterized the structural defects and correlate them with the growth conditions. Horizontal grown boules tend to grow away from the ampoule wall (contactless growth) and generally exhibit large (110) facets parallel to the gravity vector. Vertical grown boules grew to the full diameter of the ampoule and exhibited no faceting. X-ray topography combined with back reflection x-ray diffraction revealed the presence of lamellar twins (180 deg type about the [111] axis) in horizontal grown boules while vertically grown boules contain a few large grains, some of which are twinned. X-ray topographs and reciprocal space maps recorded from the boules show the better crystal quality of horizontal grown boules. The relationship between crystal quality and gravity vector is investigated. Further, an attempt is made to extend the Hurle theory of twin nucleation in Czochralski grown crystals to explain the twinning mechanisms in horizontal grown boules.

Raghothamachar, B.

Automated seed manipulation and planting

The Mechanical Division fabricated three seed separators utilizing pressure gradients to move and separate wheat seeds. These separators are called minnow buckets and use air, water, or a combination of both to generate the pressure gradient. Electrostatic fields were employed in the seed separator constructed by the Electrical Division. This separator operates by forcing a temporary electric dipole on the wheat seeds and using charged electrodes to attract and move the seeds. Seed delivery to the hydroponic growth tray is accomplished by the seed cassette. The cassette is compatible with all the seed separators, and it consists of a plastic tube threaded with millipore filter paper. During planting operations, the seeds are placed in an empty cassette. The loaded cassette is then placed in the growth tray and nutrient solution provided. The solution wets the filter paper and capillary action draws the nutrients up to feed the seeds. These seeding systems were tested and showed encouraging results. Seeds were effectively separated and the cassette can support the growth of wheat plants. Problems remaining to be investigated include improving the success of delivering the seeds to the cassette and providing adequate spacing between seeds for the electric separator.

Garcia, Ray

Continued results of the seeds in space experiment

Two million seeds of 120 different varieties representing 106 species, 97 genera, and 55 plant families were flown aboard the Long Duration Exposure Facility (LDEF). The seed were housed on the Space Exposed Experiment Developed for Students (SEEDS) tray in the sealed canister number 6 and in two small vented canisters. The tray was in the F-2 position. The seed were germinated and the germination rates and the development of the resulting plants were compared to the performance of the control seed that stayed in Park Seed's seed storage facility. The initial results were presented in a paper at the First LDEF Post-Retrieval Symposium. There was a better survival rate of the seed in the sealed canister in space than in the storage facility at Park Seed. At least some of the seed in each of the vented canisters survived the exposure to vacuum for almost six years. The number of observed apparent mutations was very low. In the initial testing, the small seeded crops were not grown to maturity to check for mutations and obtain second generation seed. These small seeded crops have now been grown for evaluation and second generation seed collected.

Alston, Jim A.

Germination, growth rates, and electron microscope analysis of tomato seeds flown on the LDEF

The tomato seeds were flown in orbit aboard the Long Duration Exposure Facility (LDEF) for nearly six years. During this time, the tomato seeds received an abundant exposure to cosmic radiation and solar wind. Upon the return of the LDEF to earth, the seeds were distributed throughout the United States and 30 foreign countries for analysis. The purpose of the experiment was to determine the long term effect of cosmic rays on living tissue. Our university analysis included germination and growth rates as well as Scanning Electron Microscopy and X-ray analysis of the control as well as Space-exposed tomato seeds. In analyzing the seeds under the Electron Microscope, usual observations were performed on the nutritional and epidermis layer of the seed. These layers appeared to be more porous in the Space-exposed seeds than on the Earth-based control seeds. This unusual characteristic may explain the increases in the space seeds growth pattern. (Several test results show that the Space-exposed seeds germinate sooner than the Earth-Based seeds. Also, the Space-exposed seeds grew at a faster rate). The porous nutritional region may allow the seeds to receive necessary nutrients and liquids more readily, thus enabling the plant to grow at a faster rate. Roots, leaves and stems were cut into small sections and mounted. After sputter coating the specimens with Argon/Gold Palladium Plasma, they were ready to be viewed under the Electron Microscope. Many micrographs were taken. The X-ray analysis displayed possible identifications of calcium, potassium, chlorine, copper, aluminum, silicon, phosphate, carbon, and sometimes sulfur and iron. The highest concentrations were shown in potassium and calcium. The Space-exposed specimens displayed a high concentration of copper and calcium in the two specimens. There was a significantly high concentration of copper in the Earth-based specimens, whereas there was no copper in the Space-exposed specimens.

Hammond, Ernest C., Jr.

Dynamics of storage reserve deposition during Brassica rapa L. pollen and seed development in microgravity

Pollen and seeds share a developmental sequence characterized by intense metabolic activity during reserve deposition before drying to a cryptobiotic form. Neither pollen nor seed development has been well studied in the absence of gravity, despite the importance of these structures in supporting future long-duration manned habitation away from Earth. Using immature seeds (3-15 d postpollination) of Brassica rapa L. cv. Astroplants produced on the STS-87 flight of the space shuttle Columbia, we compared the progress of storage reserve deposition in cotyledon cells during early stages of seed development. Brassica pollen development was studied in flowers produced on plants grown entirely in microgravity on the Mir space station and fixed while on orbit. Cytochemical localization of storage reserves showed differences in starch accumulation between spaceflight and ground control plants in interior layers of the developing seed coat as early as 9 d after pollination. At this age, the embryo is in the cotyledon elongation stage, and there are numerous starch grains in the cotyledon cells in both flight and ground control seeds. In the spaceflight seeds, starch was retained after this stage, while starch grains decreased in size in the ground control seeds. Large and well-developed protein bodies were observed in cotyledon cells of ground control seeds at 15 d postpollination, but their development was delayed in the seeds produced during spaceflight. Like the developing cotyledonary tissues, cells of the anther wall and filaments from the spaceflight plants contained numerous large starch grains, while these were rarely seen in the ground controls. The tapetum remained swollen and persisted to a later developmental stage in the spaceflight plants than in the ground controls, even though most pollen grains appeared normal. These developmental markers indicate that Brassica seeds and pollen produced in microgravity were physiologically younger than those produced in 1 g. We hypothesize that microgravity limits mixing of the gaseous microenvironments inside the closed tissues and that the resulting gas composition surrounding the seeds and pollen retards their development.

short duration