Browsing by Author "Lu, Chaofu"

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Creating yellow seed Camelina sativa with enhanced oil accumulation by CRISPR-mediated disruption of Transparent Testa 8
(Wiley, 2024-06) Cai, Yuanheng; Liang, Yuanxue; Shi, Hai; Cui, Jodie; Prakash, Shreyas; Zhang, Jianhui; Anaokar, Sanket; Chai, Jin; Schwender, Jorg; Lu, Chaofu; Yu, Xiao-Hong; Shanklin, John
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.
Enhancing microRNA167A expression in seed decreases the α-linolenic acid content and increases seed size in Camelina sativa
(2019-01) Na, GunNam; Mu, Xiaopeng; Grabowski, Paul; Schmutz, Jeremy A.; Lu, Chaofu
Despite well-established roles of microRNAs in plant development, little has been addressed to understand their effects in seeds especially on lipid metabolism. In this study, we showed that overexpressing microRNA167A (miR167OE) in camelina (Camelina sativa) under a seed-specific promoter changed fatty acid composition and increased seed size. Specifically, the miR167OE seeds had a lower Î±-linolenic acid with a concomitantly higher linoleic acid content than the wild type. This decreased level of fatty acid desaturation corresponded to a decreased transcriptional expression of the camelina fatty acid dsesaturase3 (CsFAD3) in developing seeds. MiR167 targeted the transcription factor auxin response factor (CsARF8) in camelina, as had been reported previously in Arabidopsis. Chromatin immunoprecipitation experiments combined with transcriptome analysis indicated that CsARF8 bound to promoters of camelina bZIP67 and ABI3 genes. These transcription factors directly or through the ABI3-bZIP12 pathway regulate CsFAD3 expression and affect the Î±-linolenic acid accumulation. In addition to decipher the miR167A-CsARF8 mediated transcriptional cascade for CsFAD3 suppression, transcriptome analysis was conducted to implicate mechanisms that regulate seed size in camelina. Expression levels of many genes were altered in miR167OE, including orthologs that have previously been identified to affect seed size in other plants. Most notably, genes for seed coat development such as suberin and lignin biosynthesis were down-regulated.
Generation of a T-DNA Mutagenized Camelina Population
(2013-03) Patel, Jankiben; Lu, Chaofu
A mutagenized Camelina sativa population is being generated with the insertion of T-DNA, which creates random mutations in Camelina plants. Random mutations in Camelina can be detected with a selection marker. Transformation is feasible in Camelina plants due to the infiltration method which easily allows the insertion of T-DNA. The vector used for this experiment has two main advantages: its presence of fluorescent protein marker and it has a plasmid backbone which will be used for plasmid rescuing. Transgenic seeds are obtained with a DsRed fluorescent protein selection marker which makes the Camelina seeds glow red under green light when view through a red filter. Genetic transformation events will be studied by further growth of transgenic seeds. DNA will be extracted and digested from younger leaves and after the DNA extraction, plasmid rescue will be performed for DNA sequencing. Gas chromatography will be used to detect the fatty acid composition of the oil from transgenic seeds. The goal of this project is to identify genes that affect oil quality in camelina seeds.
Genetic dissection of natural variation in oilseed traits of camelina by whole-genome resequencing and QTL mapping
(Wiley, 2021-06) Li, Huang; Hu, Xiao; Lovell, John T.; Grabowski, Paul P.; Mamidi, Sujan; Chen, Cindy; Amirebrahimi, Mojgan; Kahanda, Indika; Mumey, Brendan; Barry, Kerrie; Kudrna, David; Schmutz, Jeremy; Lachowiec, Jennifer; Lu, Chaofu
Camelina [Camelina sativa (L.) Crantz] is an oilseed crop in the Brassicaceae family that is currently being developed as a source of bioenergy and healthy fatty acids. To facilitate modern breeding efforts through marker-assisted selection and biotechnology, we evaluated genetic variation among a worldwide collection of 222 camelina accessions. We performed whole-genome resequencing to obtain single nucleotide polymorphism (SNP) markers and to analyze genomic diversity. We also conducted phenotypic field evaluations in two consecutive seasons for variations in key agronomic traits related to oilseed production such as seed size, oil content (OC), fatty acid composition, and flowering time. We determined the population structure of the camelina accessions using 161,301 SNPs. Further, we identified quantitative trait loci (QTL) and candidate genes controlling the above field-evaluated traits by genome-wide association studies (GWAS) complemented with linkage mapping using a recombinant inbred line (RIL) population. Characterization of the natural variation at the genome and phenotypic levels provides valuable resources to camelina genetic studies and crop improvement. The QTL and candidate genes should assist in breeding of advanced camelina varieties that can be integrated into the cropping systems for the production of high yield of oils of desired fatty acid composition.
Identification of MicroRNAs and Transcript Targets in Camelina sativa by Deep Sequencing and Computational Methods
(2015-03) Poudel, Saroj; Niranjan, Aryal; Lu, Chaofu
Camelina sativa is an annual oilseed crop that is under intensive development for renewable resources of biofuels and industrial oils. MicroRNAs, or miRNAs, are endogenously encoded small RNAs that play key roles in diverse plant biological processes. Here, we conducted deep sequencing on small RNA libraries prepared from camelina leaves, flower buds and two stages of developing seeds corresponding to initial and peak storage products accumulation. Computational analyses identified 207 known miRNAs belonging to 63 families, as well as 5 novel miRNAs. These miRNAs, especially members of the miRNA families, varied greatly in different tissues and developmental stages. The predicted miRNA target genes are involved in a broad range of physiological functions including lipid metabolism. This report is the first step toward elucidating roles of miRNAs in C. sativa and will provide additional tools to improve this oilseed crop for biofuels and biomaterials.
Improved fatty acid profiles in seeds of Camelina sativa by artificial microRNA mediated FATB gene suppression
(2018-09) Ozseyhan, Mehmet E.; Li, Pengcheng; Na, GunNam; Li, Zhenjing; Wang, Changlu; Lu, Chaofu
The fatty acid profile of plant oils determines their quality and uses. Saturated fatty acids are often not desirable from the standpoints of nutrition and some industrial applications. Camelina sativa is a re-emerged oilseed crop, however its oil needs to be improved to meet different application requirements. In this study, saturated fatty acids were greatly reduced by down-regulating genes encoding the fatty acyl-ACP thioesterases (FATB). An artificial microRNA (amiFATB) was created by replacing a microRNA sequence in the camelina Csa-miR159a gene with a FATB gene specific sequence. Seed-specific expression of amiFATB caused a 45% reduction of palmitic acid (16:0) and a 38% reduction of stearic acid (18:0) compared to wildtype seeds. The total saturated fatty acid content was decreased by 35% from 14.6% to 9.4% of total fatty acids. When amiFATB was expressed in a high-oleic acid transgenic line, it caused further increased oleic acid content. This work demonstrates that the FATB genes in camelina can be effectively knocked down by an artificial microRNA targeting gene-specific sequences, thus provides an additional tool to improve seed oils for desired properties.
Mapping quantitative trait loci for seed traits in Camelina sativa
(2019-09-09) King, Kevin; Huang, Li; Kang, Jinling; Lu, Chaofu
Camelina (Camelina sativa L. Crantz) is an oilseed crop that has great potential to provide sustainable feedstock for biofuel production and to improve dryland agriculture. A major breeding objective for camelina is to increase seed size and oil content. Understanding the genetics behind variations of seed size and associated traits such as oil content would help breeders develop varieties of increased oil yield that are more robust, easier to plant and harvest, and better for oil processing. In this study, we developed a recombinant inbred population derived from the two camelina accessions, Suneson and Pryzeth, with contrasting traits, especially seed size and oil content. Using 189 lines, a genetic map was constructed containing 2376 single nucleotide polymorphism markers spanning 2034.6 cM of 20 linkage groups with an average density of 1.5 cM per locus. Field trials were conducted for 2 years (2017 and 2018) in two environments (dryland and irrigated) in Bozeman, Montana. The results revealed important correlations of seed size with other associated traits such as oil content, pod size and seed number per pod. Significant QTLs were also discovered for these traits. The results of this study are the first step to isolate genes controlling seed development and oil accumulation and to develop advanced varieties of camelina better adapted to modern agriculture by marker-assisted breeding.
Mutagenesis of the FAE1 genes significantly changes fatty acid composition in seeds of Camelina sativa
(2018-02) Ozseyhan, Mehmet E.; Kang, Jinling; Mu, Xiaopeng; Lu, Chaofu
Camelina sativa is a re-emerging low-input oilseed crop that has great potentials. It is necessary to ameliorate camelina oils for optimized fatty acid composition that can meet different application requirements. Camelina seed contains significant amounts of C20-C24 very long-chain fatty acids (VLCFAs) that may not be desirable. We demonstrated that these VLCFAs can be effectively reduced by deactivating the Fatty Acid Elongase1 (FAE1) in camelina. The allohexaploid camelina contains three alleles of FAE1 genes. Ethyl methanesulfonate (EMS) induced mutation at the FAE1-B gene caused over 60% reduction of VLCFAs in seed. Homozygous knockout mutants were successfully created in a single generation by simultaneously targeting three FAE1 alleles using the CRISPR technology with an egg cell-specific Cas9 expression. VLCFAs were reduced to less than 2% of total fatty acids compared to over 22% in the wild type, and the C18 unsaturated fatty acids were concomitantly increased. The fae1 mutants were indistinguishable from wild type in seed physiology and plant growth. This study demonstrated that the CRISPR/Cas9 technology can be effectively applied to the polyploid crop camelina to rapidly obtain desired traits such as optimal fatty acid composition in its seed oil. Knocking out FAE1 also provides a means to increase the levels of oleic acid or α-linolenic acid in camelina oils that are desirable for industrial or food/feed uses.
A Phospholipase C-Like Protein From Ricinus communis Increases Hydroxy Fatty Acids Accumulation in Transgenic Seeds of Camelina sativa
(2018-11) Aryal, Niranjan; Lu, Chaofu
There have been strong interests in producing unusual fatty acids in oilseed crops to provide renewable industrial feedstock. Results are so far largely disappointing since much lower amounts of such fatty acids accumulate in genetically engineered seeds than in their original natural sources. It has been suggested that the flux of unusual fatty acids through phosphatidylcholine (PC) represents a major bottleneck for high accumulation of such fatty acids in triacylglycerol (TAG). We show here that a phospholipase C-like protein (RcPLCL1) from castor bean, which accumulates nearly 90% of the hydroxylated ricinoleic acid in its seed TAG, increases the amount of hydroxy fatty acids (HFAs) when co-expresses with the fatty acid hydroxylase (RcFAH12) in transgenic seed of Camelina sativa. RcPLCL1 shows hydrolyzing activities on both PC and phosphatidylinositol substrates in our in vitro assay conditions. The PC-PLC activity of the RcPLCL1 may have increased the efficiency of HFA-PC to diacylglycerol conversion, which explains our observation of increased HFA contents in TAG concomitant with decreased HFA in the membrane lipid PC during seed development. Consequently, this may also alleviate the potential detrimental effect of HFA on germination of the engineered camelina seeds. Our results provide new knowledge that will help design effective strategies to engineer high levels of HFAs in transgenic oilseeds.