An official website of the United States government
Abstract Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) and phospholipid:diacylglycerol acyltransferase 1 (PDAT1) share responsibility for triacylglycerol (TAG) biosynthesis, and their selectivities control TAG fatty acid (FA) compositions. For rational metabolic engineering of seed oils, replacing endogenous TAG biosynthesis with exogenous enzymes containing different substrate FA selectivities is desirable; however, the dgat1-1/pdat1-2 double mutant is pollen lethal. Here, we evaluated the ability of 3 DGAT1s, from phylogenetically diverse plants with distinct TAG assembly processes, to completely replace endogenous TAG biosynthesis in Arabidopsis (Arabidopsis thaliana). We transformed dgat1-1 mutant plants with expression constructs for DGAT1s from Camelina sativa, Physaria fendleri, and castor (Ricinus communis). Transgene expression was properly “contextualized” by using a previously determined minimum necessary expression unit containing the promoter/5′ UTR and first intron of native AtDGAT1; both of these DNA elements are essential for pollen expression. Next, we crossed homozygous lines with a DGAT1/DGAT1/PDAT1/pdat1-2 parent. C. sativa and P. fendleri DGAT1s restored the FA compositions and transcriptional differences of dgat1-1 to near wild-type and rescued the dgat1-1/pdat1-2 pollen lethality. R. communis DGAT1 was active in dgat1-1 seeds but produced unique oil profiles and alterations in the expression of lipid metabolic genes; it also failed to rescue dgat1-1/pdat1-2 lethality. This study confirms that the promoter and first intron of AtDGAT1 can modulate the expression of foreign DGAT1 genes to fit the correct spatiotemporal profile necessary for completely replacing endogenous TAG biosynthesis. Furthermore, it demonstrates an additional layer of unexpected enzyme incompatibility between oilseed lineages, which may complicate bioengineering approaches that seek to replace essential genes with orthologs.
more »
« less
The first intron and promoter of Arabidopsis DIACYLGLYCEROL ACYLTRANSFERASE 1 exert synergistic effects on pollen and embryo lipid accumulation
Summary Accumulation of triacylglycerols (TAGs) is crucial during various stages of plant development. InArabidopsis, two enzymes share overlapping functions to produce TAGs, namely acyl‐CoA:diacylglycerol acyltransferase 1 (DGAT1) and phospholipid:diacylglycerol acyltransferase 1 (PDAT1). Loss of function of both genes in adgat1‐1/pdat1‐2double mutant is gametophyte lethal. However, the key regulatory elements controlling tissue‐specific expression of either gene has not yet been identified.We transformed adgat1‐1/dgat1‐1//PDAT1/pdat1‐2parent with transgenic constructs containing theArabidopsis DGAT1promoter fused to theAtDGAT1open reading frame either with or without the first intron.Triple homozygous plants were obtained, however, in the absence of theDGAT1first intron anthers fail to fill with pollen, seed yield isc. 10% of wild‐type, seed oil content remains reduced (similar todgat1‐1/dgat1‐1), and non‐Mendelian segregation of thePDAT1/pdat1‐2locus occurs. Whereas plants expressing theAtDGAT1pro:AtDGAT1transgene containing the first intron mostly recover phenotypes to wild‐type.This study establishes that a combination of the promoter and first intron ofAtDGAT1provides the proper context for temporal and tissue‐specific expression ofAtDGAT1in pollen. Furthermore, we discuss possible mechanisms of intron mediated regulation and how regulatory elements can be used as genetic tools to functionally replace TAG biosynthetic enzymes inArabidopsis.
more »
« less
- Award ID(s):
- 2242822
- PAR ID:
- 10574967
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- New Phytologist
- Volume:
- 245
- Issue:
- 1
- ISSN:
- 0028-646X
- Format(s):
- Medium: X Size: p. 263-281
- Size(s):
- p. 263-281
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Summary In seed plants, 1‐aminocyclopropane‐1‐carboxylic acid (ACC) is the precursor of the plant hormone ethylene but also has ethylene‐independent signaling roles. Nonseed plants produce ACC but do not efficiently convert it to ethylene. InArabidopsis thaliana, ACC is transported by amino acid transporters, LYSINE HISTIDINE TRANSPORTER 1 (LHT1) and LHT2. In nonseed plants,LHThomologs have been uncharacterized.Here, we isolated an ACC‐insensitive mutant (Mpain) that is defective in ACC uptake in the liverwortMarchantia polymorpha. Mpaincontained a frameshift mutation (1 bp deletion) in the MpLHT1coding sequence, and was complemented by expression of a wild‐type MpLHT1transgene. Additionally, ACC insensitivity was re‐created in CRISPR/Cas9‐Mplht1knockout mutants. We found that MpLHT1 can also transportl‐hydroxyproline andl‐histidine.We examined the physiological functions of MpLHT1in vegetative growth and reproduction based on mutant phenotypes. Mpainand Mplht1plants were smaller and developed fewer gemmae cups compared to wild‐type plants. Mplht1mutants also had reduced fertility, and archegoniophores displayed early senescence.These findings reveal that MpLHT1 serves as an ACC and amino acid transporter inM. polymorphaand has diverse physiological functions. We propose that MpLHT1 contributes to homeostasis of ACC and other amino acids inM. polymorphagrowth and reproduction.more » « less
-
Summary Integration ofAgrobacterium tumefacienstransferred DNA (T‐DNA) into the plant genome is the last step required for stable plant genetic transformation. The mechanism of T‐DNA integration remains controversial, although scientists have proposed the participation of various nonhomologous end‐joining (NHEJ) pathways. Recent evidence suggests that inArabidopsis, DNA polymerase θ (PolQ) may be a crucial enzyme involved in T‐DNA integration.We conducted quantitative transformation assays of wild‐type andpolQmutantArabidopsisand rice, analyzed T‐DNA/plant DNA junction sequences, and (forArabidopsis) measured the amount of integrated T‐DNA in mutant and wild‐type tissue.Unexpectedly, we were able to generate stable transformants of all tested lines, although the transformation frequency ofpolQmutants was c.20% that of wild‐type plants. T‐DNA/plant DNA junctions from these transformed rice andArabidopsis polQmutants closely resembled those from wild‐type plants, indicating that loss of PolQ activity does not alter the characteristics of T‐DNA integration events.polQmutant plants show growth and developmental defects, perhaps explaining previous unsuccessful attempts at their stable transformation.We suggest that either multiple redundant pathways function in T‐DNA integration, and/or that integration requires some yet unknown pathway.more » « less
-
Summary Plasmodesmata (PD) allow direct communication across the cellulosic plant cell wall, facilitating the intercellular movement of metabolites and signaling molecules within the symplast. InArabidopsis thalianaembryos with reduced levels of the chloroplast RNA helicase ISE2, intercellular trafficking and the number of branched PD were increased. We therefore investigated the relationship between alteredISE2expression and intercellular trafficking.Gene expression analyses in Arabidopsis tissues whereISE2expression was increased or decreased identified genes associated with the metabolism of glucosinolates (GLSs) as highly affected.Concomitant with changes in the expression of GLS‐related genes, plants with abnormalISE2expression contained altered GLS metabolic profiles compared with wild‐type (WT) counterparts. Indeed, changes in the expression of GLS‐associated genes led to altered intercellular trafficking in Arabidopsis leaves. Exogenous application of GLSs but not their breakdown products also resulted in altered intercellular trafficking.These changes in trafficking may be mediated by callose levels at PD as exogenous GLS treatment was sufficient to modulate plasmodesmal callose in WT plants. Furthermore, auxin metabolism was perturbed in plants with increased indole‐type GLS levels. These findings suggest that GLSs, which are themselves transported between cells via PD, can act on PD to regulate plasmodesmal trafficking capacity.more » « less
-
Summary IRE1, BI‐1, and bZIP60 monitor compatible plant–potexvirus interactions though recognition of the viral TGB3 protein. This study was undertaken to elucidate the roles of threeIRE1isoforms, thebZIP60UandbZIP60S, andBI‐1roles in genetic reprogramming of cells during potexvirus infection.Experiments were performed usingArabidopsis thalianaknockout lines andPlantago asiatica mosaic virusinfectious clone tagged with the green fluorescent protein gene (PlAMV‐GFP).There were more PlAMV‐GFP infection foci inire1a/b,ire1c,bzip60, andbi‐1knockout than wild‐type (WT) plants. Cell‐to‐cell movement and systemic RNA levels were greaterbzip60andbi‐1than in WT plants. Overall, these data indicate an increased susceptibility to virus infection. Transgenic overexpression ofAtIRE1borStbZIP60inire1a/borbzip60mutant background reduced virus infection foci, whileStbZIP60expression influences virus movement. Transgenic overexpression ofStbZIP60also confers endoplasmic reticulum (ER) stress resistance following tunicamycin treatment. We also show bZIP60U and TGB3 interact at the ER.This is the first demonstration of a potatobZIPtranscription factor complementing genetic defects in Arabidopsis. Evidence indicates that the three IRE1 isoforms regulate the initial stages of virus replication and gene expression, while bZIP60 and BI‐1 contribute separately to virus cell‐to‐cell and systemic movement.more » « less
