Overexpression of SrDXS1 and SrKAH enhances steviol glycosides content in transgenic Stevia plants.

BACKGROUND: Stevia rebaudiana produces sweet-tasting steviol glycosides (SGs) in its leaves which can be used as natural sweeteners. Metabolic engineering of Stevia would offer an alternative approach to conventional breeding for enhanced production of SGs. However, an effective protocol for Stevia transformation is lacking. RESULTS: Here, we present an efficient and reproducible method for Agrobacterium-mediated transformation of Stevia. In our attempts to produce transgenic Stevia plants, we found that prolonged dark incubation is critical for increasing shoot regeneration. Etiolated shoots regenerated in the dark also facilitated subsequent visual selection of transformants by green fluorescent protein during Stevia transformation. Using this newly established transformation method, we overexpressed the Stevia 1-deoxy-d-xylulose-5-phosphate synthase 1 (SrDXS1) and kaurenoic acid hydroxylase (SrKAH), both of which are required for SGs biosynthesis. Compared to control plants, the total SGs content in SrDXS1- and SrKAH-overexpressing transgenic lines were enhanced by up to 42-54% and 67-88%, respectively, showing a positive correlation with the expression levels of SrDXS1 and SrKAH. Furthermore, their overexpression did not stunt the growth and development of the transgenic Stevia plants. CONCLUSION: This study represents a successful case of genetic manipulation of SGs biosynthetic pathway in Stevia and also demonstrates the potential of metabolic engineering towards producing Stevia with improved SGs yield.

Regulation of flowering time by SPL10/MED25 module in Arabidopsis.

Several SQUAMASA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors are involved in plant developmental transition from vegetative to reproductive growth. However, the function of SPL10 in regulating floral transition is largely unknown. It is also not known which Mediator subunit mediates SPL10 transcriptional activity. Here, we used overexpression lines and knockout mutants to examine the role of SPL10 in flowering-time regulation and we investigated possible interactions of SPL10 with several mediator subunits in vitro and in vivo. Plants overexpressing SPL10 showed precocious flowering, whereas the triple loss-of-function mutants of SPL10 and its two homologous genes, SPL2 and SPL11, flowered late compared with wild-type plants. We found that SPL10 interacts with MED25, a subunit of the Mediator complex, which bridges transcription factors and RNA polymerase II to facilitate transcription initiation. Genetic analysis showed that MED25 acts downstream of SPL10 to execute SPL10-regulated floral transition. Furthermore, SPL10 was required for MED25 association with the promoters of two target genes, FUL and LFY. We provide evidence that SPL10 recruits MED25 to the promoters of target genes to regulate flowering time. Our results on the SPL10/MED25 module are relevant to the molecular mechanism of other SPL family members.

A noncoding RNA transcribed from the AGAMOUS (AG) second intron binds to CURLY LEAF and represses AG expression in leaves.

Dispersed H3K27 trimethylation (H3K27me3) of the AGAMOUS (AG) genomic locus is mediated by CURLY LEAF (CLF), a component of the Polycomb Repressive Complex (PRC) 2. Previous reports have shown that the AG second intron, which confers AG tissue-specific expression, harbors sequences targeted by several positive and negative regulators. Using RACE reverse transcription polymerase chain reaction, we found that the AG intron 2 encodes several noncoding RNAs. RNAi experiment showed that incRNA4 is needed for CLF repressive activity. AG-incRNA4RNAi lines showed increased leaf AG mRNA levels associated with a decrease of H3K27me3 levels; these plants displayed AG overexpression phenotypes. Genetic and biochemical analyses demonstrated that the AG-incRNA4 can associate with CLF to repress AG expression in leaf tissues through H3K27me3-mediated repression and to autoregulate its own expression level. The mechanism of AG-incRNA4-mediated repression may be relevant to investigations on tissue-specific expression of Arabidopsis MADS-box genes.

Spearmint R2R3-MYB transcription factor MsMYB negatively regulates monoterpene production and suppresses the expression of geranyl diphosphate synthase large subunit (MsGPPS.LSU).

Many aromatic plants, such as spearmint, produce valuable essential oils in specialized structures called peltate glandular trichomes (PGTs). Understanding the regulatory mechanisms behind the production of these important secondary metabolites will help design new approaches to engineer them. Here, we identified a PGT-specific R2R3-MYB gene, MsMYB, from comparative RNA-Seq data of spearmint and functionally characterized it. Analysis of MsMYB-RNAi transgenic lines showed increased levels of monoterpenes, and MsMYB-overexpressing lines exhibited decreased levels of monoterpenes. These results suggest that MsMYB is a novel negative regulator of monoterpene biosynthesis. Ectopic expression of MsMYB, in sweet basil and tobacco, perturbed sesquiterpene- and diterpene-derived metabolite production. In addition, we found that MsMYB binds to cis-elements of MsGPPS.LSU and suppresses its expression. Phylogenetic analysis placed MsMYB in subgroup 7 of R2R3-MYBs whose members govern phenylpropanoid pathway and are regulated by miR858. Analysis of transgenic lines showed that MsMYB is more specific to terpene biosynthesis as it did not affect metabolites derived from phenylpropanoid pathway. Further, our results indicate that MsMYB is probably not regulated by miR858, like other members of subgroup 7.

Metabolic engineering of terpene biosynthesis in plants using a trichome-specific transcription factor MsYABBY5 from spearmint (Mentha spicata).

In many aromatic plants including spearmint (Mentha spicata), the sites of secondary metabolite production are tiny specialized structures called peltate glandular trichomes (PGT). Having high commercial values, these secondary metabolites are exploited largely as flavours, fragrances and pharmaceuticals. But, knowledge about transcription factors (TFs) that regulate secondary metabolism in PGT remains elusive. Understanding the role of TFs in secondary metabolism pathway will aid in metabolic engineering for increased yield of secondary metabolites and also the development of new production techniques for valuable metabolites. Here, we isolated and functionally characterized a novel MsYABBY5 gene that is preferentially expressed in PGT of spearmint. We generated transgenic plants in which MsYABBY5 was either overexpressed or silenced using RNA interference (RNAi). Analysis of the transgenic lines showed that the reduced expression of MsYABBY5 led to increased levels of terpenes and that overexpression decreased terpene levels. Additionally, ectopic expression of MsYABBY5 in Ocimum basilicum and Nicotiana sylvestris decreased secondary metabolite production in them, suggesting that the encoded transcription factor is probably a repressor of secondary metabolism.

Rapid Detection and Quantification of Plant Innate Immunity Response Using Raman Spectroscopy.

We have developed a rapid Raman spectroscopy-based method for the detection and quantification of early innate immunity responses in Arabidopsis and Choy Sum plants. Arabidopsis plants challenged with flg22 and elf18 elicitors could be differentiated from mock-treated plants by their Raman spectral fingerprints. From the difference Raman spectrum and the value of p at each Raman shift, we derived the Elicitor Response Index (ERI) as a quantitative measure of the response whereby a higher ERI value indicates a more significant elicitor-induced immune response. Among various Raman spectral bands contributing toward the ERI value, the most significant changes were observed in those associated with carotenoids and proteins. To validate these results, we investigated several characterized Arabidopsis pattern-triggered immunity (PTI) mutants. Compared to wild type (WT), positive regulatory mutants had ERI values close to zero, whereas negative regulatory mutants at early time points had higher ERI values. Similar to elicitor treatments, we derived an analogous Infection Response Index (IRI) as a quantitative measure to detect the early PTI response in Arabidopsis and Choy Sum plants infected with bacterial pathogens. The Raman spectral bands contributing toward a high IRI value were largely identical to the ERI Raman spectral bands. Raman spectroscopy is a convenient tool for rapid screening for Arabidopsis PTI mutants and may be suitable for the noninvasive and early diagnosis of pathogen-infected crop plants.

Gene silencing of Sugar-dependent 1 (JcSDP1), encoding a patatin-domain triacylglycerol lipase, enhances seed oil accumulation in Jatropha curcas.

BACKGROUND: Triacylglycerols (TAGs) are the most abundant form of storage oil in plants. They consist of three fatty acid chains (usually C16 or C18) covalently linked to glycerol. SDP1 is a specific lipase for the first step of TAG catabolism in Arabidopsis seeds. Arabidopsis mutants deficient in SDP1 accumulate high levels of oils, probably due to blockage in TAG degradation. We applied this knowledge from the model plant, Arabidopsis thaliana, to engineer increased seed oil content in the biodiesel plant Jatropha curcas using RNA interference (RNAi) technology. RESULTS: As Jatropha is a biodiesel crop, any significant increase in its seed oil content would be an important agronomic trait. Using A. thaliana as a model plant, we found that a deficiency of SDP1 led to higher TAG accumulation and a larger number of oil bodies in seeds compared with wild type (Columbia-0; Col-0). We cloned Jatropha JcSDP1, and verified its function by complementation of the Arabidopsis sdp1-5 mutant. Taking advantage of the observation with Arabidopsis, we used RNAi technology to generate JcSDP1 deficiency in transgenic Jatropha. We found that Jatropha JcSDP1-RNAi plants accumulated 13 to 30% higher total seed storage lipid, along with a 7% compensatory decrease in protein content, compared with control (CK; 35S:GFP) plants. Free fatty acid (FFA) content in seeds was reduced from 27% in control plants to 8.5% in JcSDP1-RNAi plants. CONCLUSION: Here, we showed that SDP1 deficiency enhances seed oil accumulation in Arabidopsis. Based on this result, we generated SDP1-deficient transgenic Jatropha plants using by RNAi technology with a native JcSDP1 promoter to silence endogenous JcSDP1 expression. Seeds of Jatropha JcSDP1-RNAi plants accumulated up to 30% higher total lipid and had reduced FFA content compared with control (CK; 35S:GFP) plants. Our strategy of improving an important agronomic trait of Jatropha can be extended to other oil crops to yield higher seed oil.

Engineering geminivirus resistance in Jatropha curcus.

BACKGROUND: Jatropha curcus is a good candidate plant for biodiesel production in tropical and subtropical regions. However, J. curcus is susceptible to the geminivirus Indian cassava mosaic virus (ICMV), and frequent viral disease outbreaks severely limit productivity. Therefore the development of J. curcus to carry on durable virus resistance remains crucial and poses a major biotechnological challenge. RESULTS: We generated transgenic J. curcus plants expressing a hairpin, double-stranded (ds) RNA with sequences homologous to five key genes of ICMV-Dha strain DNA-A, which silences sequence-related viral genes thereby conferring ICMV resistance. Two rounds of virus inoculation were conducted via vacuum infiltration of ICMV-Dha. The durability and heritability of resistance conferred by the dsRNA was further tested to ascertain that T1 progeny transgenic plants were resistant to the ICMV-SG strain, which shared 94.5% nucleotides identity with the ICMV-Dha strain. Quantitative PCR analysis showed that resistant transgenic lines had no detectable virus. CONCLUSIONS: In this study we developed transgenic J. curcus plants to include a resistance to prevailing geminiviruses in Asia. These virus-resistant transgenic J. curcus plants can be used in various Jatropha breeding programs.

Development of marker-free transgenic Jatropha plants with increased levels of seed oleic acid.

BACKGROUND: Jatropha curcas is recognized as a new energy crop due to the presence of the high amount of oil in its seeds that can be converted into biodiesel. The quality and performance of the biodiesel depends on the chemical composition of the fatty acids present in the oil. The fatty acids profile of the oil has a direct impact on ignition quality, heat of combustion and oxidative stability. An ideal biodiesel composition should have more monounsaturated fatty acids and less polyunsaturated acids. Jatropha seed oil contains 30% to 50% polyunsaturated fatty acids (mainly linoleic acid) which negatively impacts the oxidative stability and causes high rate of nitrogen oxides emission. RESULTS: The enzyme 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine delta 12-desaturase (FAD2) is the key enzyme responsible for the production of linoleic acid in plants. We identified three putative delta 12 fatty acid desaturase genes in Jatropha (JcFAD2s) through genome-wide analysis and downregulated the expression of one of these genes, JcFAD2-1, in a seed-specific manner by RNA interference technology. The resulting JcFAD2-1 RNA interference transgenic plants showed a dramatic increase of oleic acid (> 78%) and a corresponding reduction in polyunsaturated fatty acids (< 3%) in its seed oil. The control Jatropha had around 37% oleic acid and 41% polyunsaturated fatty acids. This indicates that FAD2-1 is the major enzyme responsible for converting oleic acid to linoleic acid in Jatropha. Due to the changes in the fatty acids profile, the oil of the JcFAD2-1 RNA interference seed was estimated to yield a cetane number as high as 60.2, which is similar to the required cetane number for conventional premium diesel fuels (60) in Europe. The presence of high seed oleic acid did not have a negative impact on other Jatropha agronomic traits based on our preliminary data of the original plants under greenhouse conditions. Further, we developed a marker-free system to generate the transgenic Jatropha that will help reduce public concerns for environmental issues surrounding genetically modified plants. CONCLUSION: In this study we produced seed-specific JcFAD2-1 RNA interference transgenic Jatropha without a selectable marker. We successfully increased the proportion of oleic acid versus linoleic in Jatropha through genetic engineering, enhancing the quality of its oil.

Tapetum determinant1 is required for cell specialization in the Arabidopsis anther.

In flowering plants, pollen formation depends on the differentiation and interaction of two cell types in the anther: the reproductive cells, called microsporocytes, and somatic cells that form the tapetum. The microsporocytes generate microspores, whereas the tapetal cells support the development of microspores into mature pollen grains. Despite their importance to plant reproduction, little is known about the underlying genetic mechanisms that regulate the differentiation and interaction of these highly specialized cells in the anther. Here, we report the identification and characterization of a novel tapetum determinant1 (TPD1) gene that is required for the specialization of tapetal cells in the Arabidopsis anther. Analysis of the male-sterile mutant, tpd1, showed that functional interruption of TPD1 caused the precursors of tapetal cells to differentiate and develop into microsporocytes instead of tapetum. As a results, extra microsporocytes were formed and tapetum was absent in developing tpd1 anthers. Molecular cloning of TPD1 revealed that it encodes a small protein of 176 amino acids. In addition, tpd1 was phenotypically similar to excess microsporocytes1/extra sporogenous cells (ems1/exs) single and tpd1 ems1/exs double mutants. These data suggest that the TPD1 product plays an important role in the differentiation of tapetal cells, possibly in coordination with the EMS1/EXS gene product, a Leu-rich repeat receptor protein kinase.