New insights into natural rubber biosynthesis from rubber-deficient lettuce mutants expressing goldenrod or guayule cis-prenyltransferase.

Lettuce produces natural rubber (NR) with an average Mw of > 1 million Da in laticifers, similar to NR from rubber trees. As lettuce is an annual, self-pollinating, and easily transformable plant, it is an excellent model for molecular genetic studies of NR biosynthesis. CRISPR/Cas9 mutagenesis was optimized using lettuce hairy roots, and NR-deficient lettuce was generated via bi-allelic mutations in cis-prenyltransferase (CPT). This is the first null mutant of NR deficiency in plants. In the CPT mutant, orthologous CPT counterparts from guayule (Parthenium argentatum) and goldenrod (Solidago canadensis) were expressed under a laticifer-specific promoter to examine how the average Mw of NR is affected. No developmental defects were observed in the NR-deficient mutants. The lettuce mutants expressing guayule and goldenrod CPT produced 1.8 and 14.5 times longer NR, respectively, than the plants of their origin. This suggests that, although goldenrod cannot synthesize a sufficiently lengthy NR, goldenrod CPT has the catalytic competence to produce high-quality NR in the cellular context of lettuce laticifers. Thus, CPT alone does not determine the length of NR. Other factors, such as substrate concentration, additional proteins, and/or the nature of protein complexes including CPT-binding proteins, influence CPT activity in determining NR length.

The promoter sequences of lettuce cis-prenyltransferase and its binding protein specify gene expression in laticifers.

MAIN CONCLUSION: Promoters of lettuce cis-prenyltransferase 3 (LsCPT3) and CPT-binding protein 2 (LsCBP2) specify gene expression in laticifers, as supported by in situ ß-glucuronidase stains and microsection analysis. Lettuce (Lactuca sativa) has articulated laticifers alongside vascular bundles. In the cytoplasm of laticifers, natural rubber (cis-1,4-polyisoprene) is synthesized by cis-prenyltransferase (LsCPT3) and CPT-binding protein (LsCBP2), both of which form an enzyme complex. Here we determined the gene structures of LsCPT3 and LsCBP2 and characterized their promoter activities using ß-glucuronidase (GUS) reporter assays in stable transgenic lines of lettuce. LsCPT3 has a single 7.4-kb intron while LsCBP2 has seven introns including a 940-bp intron in the 5'-untranslated region (UTR). Serially truncated LsCPT3 promoters (2.3 kb, 1.6 kb, and 1.1 kb) and the LsCBP2 promoter with (1.7 kb) or without (0.8 kb) the 940-bp introns were fused to GUS to examine their promoter activities. In situ GUS stains of the transgenic plants revealed that the 1.1-kb LsCPT3 and 0.8-kb LsCBP2 promoter without the 5'-UTR intron are sufficient to express GUS exclusively in laticifers. Fluorometric assays showed that the LsCBP2 promoter was several-fold stronger than the CaMV35S promoter and was ~ 400 times stronger than the LsCPT3 promoter in latex. Histochemical analyses confirmed that both promoters express GUS exclusively in laticifers, recognized by characteristic fused multicellular structures. We concluded that both the LsCPT3 and LsCBP2 promoters specify gene expression in laticifers, and the LsCBP2 promoter displays stronger expression than the CaMV35S promoter in laticifers. For the LsCPT3 promoter, it appears that unknown cis-elements outside of the currently examined LsCPT3 promoter are required to enhance LsCPT3 expression.

Creating saponin-free yellow pea seeds by CRISPR/Cas9-enabled mutagenesis on ß-amyrin synthase.

Dry pea (Pisum sativum) seeds are valuable sources of plant protein, dietary fiber, and starch, but their uses in food products are restricted to some extent due to several off-flavor compounds. Saponins are glycosylated triterpenoids and are a major source of bitter, astringent, and metallic off-flavors in pea products. ß-amyrin synthase (BAS) is the entry point enzyme for saponin biosynthesis in pea and therefore is an ideal target for knock-out using CRISPR/Cas9 genome editing to produce saponin deficient pea varieties. Here, in an elite yellow pea cultivar (CDC Inca), LC/MS analysis identified embryo tissue, not seed coat, as the main location of saponin storage in pea seeds. Differential expression analysis determined that PsBAS1 was preferentially expressed in embryo tissue relative to seed coat and was selected for CRISPR/Cas9 genome editing. The efficiency of CRISPR/Cas9 genome editing of PsBAS1 was systematically optimized in pea hairy roots. From these optimization procedures, the AtU6-26 promoter was found to be superior to the CaMV35S promoter for gRNA expression, and the use of 37°C was determined to increase the efficiency of CRISPR/Cas9 genome editing. These promoter and culture conditions were then applied to stable transformations. As a result, a bi-allelic mutation (deletion and inversion mutations) was generated in the PsBAS1 coding sequence in a T1 plant, and the segregated psbas1 plants from the T2 population showed a 99.8% reduction of saponins in their seeds. Interestingly, a small but statistically significant increase (~12%) in protein content with a slight decrease (~5%) in starch content was observed in the psbas1 mutants under phytotron growth conditions. This work demonstrated that flavor-improved traits can be readily introduced in any pea cultivar of interest using CRISPR/Cas9. Further field trials and sensory tests for improved flavor are necessary to assess the practical implications of the saponin-free pea seeds in food applications.

Germacrene A Synthases for Sesquiterpene Lactone Biosynthesis Are Expressed in Vascular Parenchyma Cells Neighboring Laticifers in Lettuce.

Sesquiterpene lactone (STL) and natural rubber (NR) are characteristic isoprenoids in lettuce (Lactuca sativa). Both STL and NR co-accumulate in laticifers, pipe-like structures located along the vasculature. NR-biosynthetic genes are exclusively expressed in laticifers, but cell-type specific expression of STL-biosynthetic genes has not been studied. Here, we examined the expression pattern of germacrene A synthase (LsGAS), which catalyzes the first step in STL biosynthesis in lettuce. Quantitative PCR and Illumina read mapping revealed that the transcripts of two GAS isoforms (LsGAS1/LsGAS2) are expressed two orders of magnitude (~100-200) higher in stems than laticifers. This result implies that the cellular site for LsGAS1/2 expression is not in laticifers. To gain more insights, promoters of LsGAS1/2 were cloned and fused to ß-glucuronidase (GUS), followed by transformations of lettuce with these promoter-GUS constructs. In in situ GUS assays, the GUS expression driven by the LsGAS1/2 promoters was tightly associated with vascular bundles. High-resolution microsections showed that GUS signals are not present in laticifers but are detected in the vascular parenchyma cells neighboring the laticifers. These results suggest that expression of LsGAS1/2 occurs in the parenchyma cells neighboring laticifers, while the resulting STL metabolites accumulate in laticifers. It can be inferred that active metabolite-trafficking occurs from the parenchyma cells to laticifers in lettuce.