Eight-carbon (C8) volatiles, such as 1-octen-3-ol, octan-3-one, and octan-3-ol, are ubiquitously found among fungi and bryophytes. In this study, it was found that the thalli of the common liverwort Marchantia polymorpha, a model plant species, emitted high amounts of C8 volatiles mainly consisting of (R)-1-octen-3-ol and octan-3-one upon mechanical wounding. The induction of emission took place within 40min. In intact thalli, 1-octen-3-yl acetate was the predominant C8 volatile while tissue disruption resulted in conversion of the acetate to 1-octen-3-ol. This conversion was carried out by an esterase showing stereospecificity to (R)-1-octen-3-yl acetate. From the transgenic line of M. polymorpha (des6(KO)) lacking arachidonic acid and eicosapentaenoic acid, formation of C8 volatiles was only minimally observed, which indicated that arachidonic and/or eicosapentaenoic acids were essential to form C8 volatiles in M. polymorpha. When des6(KO) thalli were exposed to the vapor of 1-octen-3-ol, they absorbed the alcohol and converted it into 1-octen-3-yl acetate and octan-3-one. Therefore, this implied that 1-octen-3-ol was the primary C8 product formed from arachidonic acid, and further metabolism involving acetylation and oxidoreduction occurred to diversify the C8 products. Octan-3-one was only minimally formed from completely disrupted thalli, while it was formed as the most abundant product in partially disrupted thalli. Therefore, it is assumed that the remaining intact tissues were involved in the conversion of 1-octen-3-ol to octan-3-one in the partially disrupted thalli. The conversion was partly promoted by addition of NAD(P)H into the completely disrupted tissues, suggesting an NAD(P)H-dependent oxidoreductase was involved in the conversion.
Aquatic microalgae induce a carbon-concentrating mechanism (CCM) to maintain photosynthetic activity in low-CO2 (LC) conditions. Although the molecular mechanism of the CCM has been investigated using the single-cell green alga Chlamydomonas reinhardtii, and several CCM-related genes have been identified by analyzing high-CO2 (HC)-requiring mutants, many aspects of the CO2-signal transduction pathways remain to be elucidated. In this study, we report the isolation of novel HC-requiring mutants defective in the induction of CCM by DNA tagging. Growth rates of 20,000 transformants grown under HC and LC conditions were compared, and three HC-requiring mutants (H24, H82, and P103) were isolated. The photosynthetic CO2-exchange activities of these mutants were significantly decreased compared with that of wild-type cells, and accumulation of HLA3 and both LCIA and HLA3 were absent in mutants H24 and H82, respectively. Although the insertion of the marker gene and the HC-requiring phenotype were linked in the tetrad progeny of H82, and a calcium-sensing receptor CAS was disrupted by the insertion, exogenous expression of CAS alone could not complement the HC-requiring phenotype.
Arabidopsis thaliana BOR1 was the first boron (B) transporter identified in living systems. There are four AtBOR1-like genes, OsBOR1, 2, 3 and 4, present in the rice genome. We characterized the activity, expression and physiological function of OsBOR4. OsBOR4 is an active efflux transporter of B. Quantitative PCR analysis and OsBOR4 promoter-green fluorescent protein (GFP) fusion revealed that OsBOR4 was both highly and specifically expressed in pollen. We obtained five Tos17 insertion mutants of osbor4. The pollen grains were viable and development of floral organs was normal in the homozygous osbor4 mutants. We observed that in all Tos17 insertion lines tested, the frequency of osbor4 homozygous plants was lower than expected in the progeny of self-fertilized heterozygous plants. These results establish that OsBOR4 is essential for normal reproductive processes. Pollen from osbor4 homozygous plants elongated fewer tubes on wild-type stigmas, and tube elongation of mutant pollen was less efficient compared with the wild-type pollen, suggesting reduced competence of osbor4 mutant pollen. The reduced competence of mutant pollen was further supported by the crosses of independent Tos17-inserted alleles of OsBOR4. Our results suggest that OsBOR4, a boron efflux transporter, is required for normal pollen germination and/or tube elongation.
After the accident of the Fukushima 1 nuclear power plant in March 2011, radioactive cesium was released and paddy field in a wide area of Fukushima Prefecture was contaminated. To reduce radioactive Cs uptake by rice, it is important to understand factors that affect Cs uptake in rice. Here we describe our study in 2011 and 2012 to investigate Cs concentration in two rice cultivars, Koshihikari and Hitomebore, the top two cultivars in Fukushima prefecture, grown under different fertilizer conditions in the contaminated paddy field. Our study demonstrated that high nitrogen and low potassium conditions increase Cs concentrations both in straw and brown rice.
After the accident of the Fukushima 1 Nuclear Power Plant in March 2011, radioactive cesium was released and paddy fields in a wide area including Fukushima Prefecture were contaminated. To estimate the levels of radioactive Cs accumulation in rice produced in Fukushima, it is crucial to obtain the actual data of Cs accumulation levels in rice plants grown in the actual paddy field in Fukushima City. We herein conducted a two-year survey in 2011 and 2012 of radioactive and non-radioactive Cs accumulation in rice using a number of rice cultivars grown in the paddy field in Fukushima City. Our study demonstrated a substantial variation in Cs accumulation levels among the cultivars of rice.
Boron is an essential nutrient for plants, but it is toxic in excess. Transgenic rice plants expressing an Arabidopsis thaliana borate efflux transporter gene, AtBOR4, at a low level exhibited increased tolerance to excess boron. Those lines with high levels of expression exhibited reduced growth. These findings suggest a potential of the borate transporter BOR4 for the generation of high-boron tolerant rice.
CtBP/BARS is a unique protein family in having quite diversified cellular functions, intercellular localizations, and developmental roles. ANGUSTIFOLIA (AN) is the sole homolog of CtBP/BARS from Arabidopsis thaliana, although it has plant AN-specific motifs and a long C-terminus. Previous studies suggested that AN would function in the nucleus as a transcriptional co-repressor, as CtBPs function in animals; however, precise verification has been lacking. In this paper, we isolated a homologous gene (MAN) of AN from liverwort, Marchantia polymorpha. Transformation of the Arabidopsis an-1 mutant with 35S-driven MAN completely complemented the an-1 phenotype, although it lacks the putative nuclear localization signal (NLS) that exists in AN proteins isolated from other plant species. We constructed several plasmids for expressing modified ANs with amino acid substitutions in known motifs. The results clearly indicated that modified AN with mutations in the putative NLS-like domain could complement the an-1 phenotype. Therefore, we re-examined localization of AN using several techniques. Our results demonstrated that AN localizes on punctuate structures around the Golgi, partially overlapping with a trans-Golgi network resident, which highlighted an unexpected link between leaf development and membrane trafficking. We should reconsider the roles and evolutionary traits of AN based on these findings.
Tobacco (Nicotiana tabacum) plants synthesize nicotine and related pyridine-type alkaloids, such as anatabine, in their roots and accumulate them in their aerial parts as chemical defenses against herbivores. Herbivory-induced jasmonate signaling activates structural genes for nicotine biosynthesis and transport by way of the NICOTINE (NIC) regulatory loci. The biosynthesis of tobacco alkaloids involves the condensation of an unidentified nicotinic acid-derived metabolite with the N-methylpyrrolinium cation or with itself, but the exact enzymatic reactions and enzymes involved remain unclear. Here, we report that jasmonate-inducible tobacco genes encoding flavin-containing oxidases of the berberine bridge enzyme family (BBLs) are expressed in the roots and regulated by the NIC loci. When expression of the BBL genes was suppressed in tobacco hairy roots or in tobacco plants, nicotine production was highly reduced, with a gradual accumulation of a novel nicotine metabolite, dihydromethanicotine. In the jasmonate-elicited cultured tobacco cells, suppression of BBL expression efficiently inhibited the formation of anatabine and other pyridine alkaloids. Subcellular fractionation and localization of green fluorescent protein-tagged BBLs showed that BBLs are localized in the vacuoles. These results indicate that BBLs are involved in a late oxidation step subsequent to the pyridine ring condensation reaction in the biosynthesis of tobacco alkaloids.
Wild watermelon (Citrullus lanatus) is a xerophyte native to the Kalahari Desert, Africa. To better understand the molecular mechanisms of drought resistance in this plant, we examined changes in the proteome in response to water deficit. Wild watermelon leaves showed decreased transpiration and a concomitant increase in leaf temperature under water deficit conditions. Comparison of the proteome of stressed plants with that of unstressed plants by two-dimensional gel electrophoresis revealed that the intensity of 40 spots increased in response to the stress, and the intensity of 11 spots decreased. We positively identified 23 stress-induced and 6 stress-repressed proteins by mass spectrometry and database analyses. Interestingly, 15 out of the 23 up-regulated proteins (65% of annotated up-regulated proteins) were heat shock proteins (HSPs). Especially, 10 out of the 15 up-regulated HSPs belonged to the small heat shock protein (sHSP) family. Other stress-induced proteins included those related to antioxidative defense and carbohydrate metabolism. Fifteen distinct cDNA sequences encoding the sHSP were characterized from wild watermelon. Quantitative real-time PCR analysis of the representative sHSP genes revealed strong transcriptional up-regulation in the leaves under water deficit. Moreover, immunoblot analysis confirmed that protein abundance of sHSPs was massively increased under water deficit. Overall, these observations suggest that the defense response of wild watermelon may involve orchestrated regulation of a diverse array of functional proteins related to cellular defense and metabolism, of which HSPs may play a pivotal role on the protection of the plant under water deficit in the presence of strong light.
Tobacco (Nicotiana tabacum) synthesizes nicotine and related pyridine alkaloids in the root, and their synthesis increases upon herbivory on the leaf via a jasmonate-mediated signaling cascade. Regulatory NIC loci that positively regulate nicotine biosynthesis have been genetically identified, and their mutant alleles have been used to breed low-nicotine tobacco varieties. Here, we report that the NIC2 locus, originally called locus B, comprises clustered transcription factor genes of an ethylene response factor (ERF) subfamily; in the nic2 mutant, at least seven ERF genes are deleted altogether. Overexpression, suppression, and dominant repression experiments using transgenic tobacco roots showed both functional redundancy and divergence among the NIC2-locus ERF genes. These transcription factors recognized a GCC-box element in the promoter of a nicotine pathway gene and specifically activated all known structural genes in the pathway. The NIC2-locus ERF genes are expressed in the root and upregulated by jasmonate with kinetics that are distinct among the members. Thus, gene duplication events generated a cluster of highly homologous transcription factor genes with transcriptional and functional diversity. The NIC2-locus ERFs are close homologs of ORCA3, a jasmonate-responsive transcriptional activator of indole alkaloid biosynthesis in Catharanthus roseus, indicating that the NIC2/ORCA3 ERF subfamily was recruited independently to regulate jasmonate-inducible secondary metabolism in distinct plant lineages.
Root vigor is an important trait for the growth of terrestrial plants, especially in water-deficit environments. Although deserts plants are known for their highly developed root architecture, the molecular mechanism responsible for this trait has not been determined. Here we established an efficient protocol for the genetic manipulation of two varieties of watermelon plants: a desert-grown wild watermelon that shows vigorous root growth under drought, and a domesticated cultivar showing retardation of root growth under drought stress. Agrobacterium rhizogenes-mediated transgenic hairy roots were efficiently induced and selected from the hypocotyls of these plants. Transgenic GUS expression was detected in the roots by RT-PCR and histochemical GUS staining. Moreover, a liquid culture system for evaluating their root growth was also established. Interestingly, growth of the hairy roots derived from domesticated variety of watermelon strongly inhibited under high osmotic condition, whereas the hairy roots derived from wild variety of watermelon retained substantial growth rates under the stress condition. The new protocol presented here offers a powerful tool for the comparative study of the molecular mechanism underlying drought-induced root growth in desert plants.
Plants in the Nicotiana genus produce nicotine and related pyridine alkaloids as a part of their chemical defense against insect herbivores. These alkaloids are formed by condensation of a derivative of nicotinic acid, but the enzyme(s) involved in the final condensation step remains elusive. In Nicotiana tabacum, an orphan reductase A622 and its close homolog A622L are coordinately expressed in the root, upregulated by methyl jasmonate treatment, and controlled by the NIC regulatory loci specific to the biosynthesis of tobacco alkaloids. Conditional suppression of A622 and A622L by RNA interference inhibited cell growth, severely decreased the formation of all tobacco alkaloids, and concomitantly induced an accumulation of nicotinic acid beta-N-glucoside, a probable detoxification metabolite of nicotinic acid, in both hairy roots and methyl jasmonate-elicited cultured cells of tobacco. N-methylpyrrolinium cation, a precursor of the pyrrolidine moiety of nicotine, also accumulated in the A622(L)-knockdown hairy roots. We propose that the tobacco A622-like reductases of the PIP family are involved in either the formation of a nicotinic acid-derived precursor or the final condensation reaction of tobacco alkaloids.
Euphorbia tirucalli L., which is also known as a petroleum plant, produces a large amount of phytosterols and triterpenes. During their biosynthesis, squalene synthase converts two molecules of the hydrophilic substrate farnesyl diphosphate into a hydrophobic product, squalene. An E. tirucalli cDNA clone of a putative squalene synthase gene (EtSS) was isolated by RT-PCR followed by 5- and 3-RACE. The restriction fragment polymorphisms revealed by Southern blot analysis suggest that EtSS is a single copy gene. The glycine at the 287th residue from the N-terminal end of domain C has replaced alanine, which is conserved among all the other SS sequences deposited in the Genbank database. The N-terminal 380 residues of the hydrophilic sequence was expressed as a peptide-tagged protein in E. coli, and the resultant bacterial crude extract was incubated with farnesyl diphosphate and NADPH. GC-MS analysis showed that squalene was detected in the in vitro reaction mixture. E. tirucalli transgenic callus lines, in which EtSS was overexpressed, accumulated increased amounts of phytosterols as compared with that of wild type callus. RT-PCR analysis of wild type E. tirucalli plants revealed that the EtSS transcript accumulated in almost equal amounts in the stems and the leaves with a stalk, while a lower amount was detected in the roots. In situ hybridization analysis revealed that prominent antisense-probe signal was detected in the cambia within bundle sheathes. These results indicate that EtSS functions prominently in cambia, which are located adjacent to conductive tubes, and that this gene plays important roles in phytosterol accumulation in petroleum plants.
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