Lithospermum erythrorhizon cell cultures have been used to produce plant secondary metabolites, as well as in biosynthetic studies. Shikonin, a representative secondary metabolite of L. erythrorhizon, was first produced industrially by dedifferentiated cell cultures in the 1980s. This culture system has since been used in research on various plant secondary metabolites. Other boraginaceaeous plant species, including Arnebia, Echium, Onosma and Alkanna, have been shown to produce shikonin, and studies have assessed shikonin regulation, including transgene expression, in these plants. This review summarizes current knowledge of shikonin production by L. erythrorhizon cell and hairy root cultures, including the historical aspect of large-scale production, and discusses future biochemical and biological research using this species.
Napier grass (Pennisetum purpureum Schumach.) is a highly productive C4 tropical forage grass that has been targeted as a potential bioenergy crop. To further increase the efficiency of bioethanol production by molecular breeding, a reliable protocol for genetically transforming napier grass is essential. In this study, we report the creation of transgenic napier grass plants derived from embryogenic callus cultures of shoot apices. Embryogenic callus was initiated in three accessions of napier grass and a napier grass×pearl millet hybrid using Murashige and Skoog (MS) medium supplemented with 2.0 mg L－1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 mg L－1 6-benzylaminopurine (BAP) and 50 µM copper sulfate (CuSO4). Of the accessions tested, a dwarf type with late-heading (DL line) had the best response for embryogenic callus formation. Highly regenerative calli that formed dense polyembryogenic clusters were selected as target tissues for transformation. A plasmid vector, pAHC25, containing an herbicide-resistance gene (bar) and the β-glucuronidase (GUS) reporter gene was used in particle bombardment experiments. Target tissues treated with 0.6 M osmoticum were bombarded, and transgenic plants were selected under 5.0 mg L－1 bialaphos selection. Although a total of 1400 target tissues yielded nine GUS-positive bialaphos-resistant calli, only one transgenic line that was derived from target tissue with the shortest culture term produced four transgenic plants. Thus, the length of time that the target tissue is in callus culture was one of the most important factors for acquiring transgenic plants in napier grass. This is the first report of successfully producing transgenic napier grass plants.
Nitrogen limits crop yield, but application of nitrogen fertilizer can cause environmental problems and much fertilizer is lost without being absorbed by plants. Increasing nitrogen use efficiency in plants may help overcome these problems and is, therefore, an important and active subject of agricultural research. Here, we report that the expression of the chimeric repressor for the GATA4 transcription factor (35S:GATA4-SRDX) improved tolerance to nitrogen deficiency in Arabidopsis thaliana. 35S:GATA4-SRDX seedlings were significantly larger than wild type under nitrogen-sufficient and -deficient conditions (10 and 0.5 mM NH4NO3, respectively). 35S:GATA4-SRDX plants exhibited shorter primary roots, fewer lateral roots, and higher root hair density compared with wild type. The expression levels of NITRATE TRANSPORTER 2.1, ASPARAGINE SYNTHETASE and NITRATE REDUCTASE 1 were significantly higher in roots of 35S:GATA4-SRDX plants than in wild type under nitrogen-sufficient conditions. Under nitrogen-deficient conditions, the expression of genes for cytosolic glutamine synthetases was upregulated in shoots of 35S:GATA4-SRDX plants compared with wild type. This upregulation of nitrogen transporter and nitrogen assimilation-related genes might confer tolerance to nitrogen deficiency in 35S:GATA4-SRDX plants.
The unfolded protein response (UPR) mitigates stress caused by accumulation of unfolded proteins in the endoplasmic reticulum (ER). Inositol-requiring enzyme 1 (IRE1) is the most conserved sensor of the UPR with ribonuclease activity that mediates cytoplasmic splicing and decay of mRNA encoding secretory and membrane proteins. In the present study, we demonstrate that the Arabidopsis mutant defective in two IRE1 genes exhibit retarded growth of primary roots under moderate salt stress, although such grow retardation is not observed in wild type plants. Microscopic observation showed decrease in the number of meristematic cells in the mutant under salt stress. This finding suggests that IRE1 plays a role in the maintenance of root meristems under salt stress. Possible connections between the function of IRE1 and the salt sensitivity are discussed.
Eucommia ulmoides, a deciduous dioecious plant species, accumulates trans-1,4-polyisoprene (TPI) in its tissues such as pericarp and leaf. Probable TPI synthase (trans-isoprenyl diphosphate synthase (TIDS)) genes were identified by expressed sequence tags of this species; however, the metabolic pathway of TPI biosynthesis, including the role of TIDSs, is unknown. To understand the mechanism of TPI biosynthesis at the transcriptional level, comprehensive gene expression data from various organs were generated and TPI biosynthesis related genes were extracted by principal component analysis (PCA). The metabolic pathway was assessed by comparing the coexpression network of TPI genes with the isoprenoid gene coexpression network of model plants. By PCA, we dissected 27 genes assumed to be involved in polyisoprene biosynthesis, including TIDS genes, genes encoding enzymes of the mevalonate (MVA) pathway and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, and genes related to rubber synthesis. The coexpression network revealed that 22 of the 27 TPI biosynthesis genes are coordinately expressed. The network was clustered into two modules, and this was also observed in model plants. The first module was mainly comprised of MEP pathway genes and TIDS1 gene, and the second module, of MVA pathway genes and TIDS5 gene. These results indicate that TPI is likely biosynthesized by both the MEP and MVA pathways and that TIDS gene expression is differentially controlled by these pathways.
The unfolded protein response (UPR) or the endoplasmic reticulum (ER) stress response occurs when folding and maturation of secretory and membrane proteins are impaired in the ER. The UPR induces a number of genes that encode ER-localized molecular chaperones and folding enzymes to increase folding capacity in the ER. We have identified Tunicamycin Induced 1 (TIN1), an Arabidopsis gene that is highly induced during the UPR. We have shown that TIN1 protein is localized in the ER but its physiological function remains to be elucidated. In the present study we generated and analyzed transgenic Arabidopsis plants expressing TIN1 under CaMV35S promoter to obtain insights into the physiological role of TIN1. We found that although TIN1-overexpressing plants grew as did wild-type plants under ambient laboratory conditions, their pollen grains exhibited abnormal surface morphology. The result suggests a specific role of TIN1 in secretion of proteins and/or lipids during pollen development.