The Chlamydomonas bZIP transcription factor BLZ8 confers oxidative stress tolerance by inducing the carbon-concentrating mechanism.

Photosynthetic organisms are exposed to various environmental sources of oxidative stress. Land plants have diverse mechanisms to withstand oxidative stress, but how microalgae do so remains unclear. Here, we characterized the Chlamydomonas reinhardtii basic leucine zipper (bZIP) transcription factor BLZ8, which is highly induced by oxidative stress. Oxidative stress tolerance increased with increasing BLZ8 expression levels. BLZ8 regulated the expression of genes likely involved in the carbon-concentrating mechanism (CCM): HIGH-LIGHT ACTIVATED 3 (HLA3), CARBONIC ANHYDRASE 7 (CAH7), and CARBONIC ANHYDRASE 8 (CAH8). BLZ8 expression increased the photosynthetic affinity for inorganic carbon under alkaline stress conditions, suggesting that BLZ8 induces the CCM. BLZ8 expression also increased the photosynthetic linear electron transfer rate, reducing the excitation pressure of the photosynthetic electron transport chain and in turn suppressing reactive oxygen species (ROS) production under oxidative stress conditions. A carbonic anhydrase inhibitor, ethoxzolamide, abolished the enhanced tolerance to alkaline stress conferred by BLZ8 overexpression. BLZ8 directly regulated the expression of the three target genes and required bZIP2 as a dimerization partner in activating CAH8 and HLA3. Our results suggest that a CCM-mediated increase in the CO2 supply for photosynthesis is critical to minimize oxidative damage in microalgae, since slow gas diffusion in aqueous environments limits CO2 availability for photosynthesis, which can trigger ROS formation.

Excretion of triacylglycerol as a matrix lipid facilitating apoplastic accumulation of a lipophilic metabolite shikonin.

Plants produce a large variety of lipophilic metabolites, many of which are secreted by cells and accumulated in apoplasts. These compounds often play a role to protect plants from environmental stresses. However, little is known about how these lipophilic compounds are secreted into apoplastic spaces. In this study, we used shikonin-producing cultured cells of Lithospermum erythrorhizon as an experimental model system to analyze the secretion of lipophilic metabolites, taking advantage of its high production rate and the clear inducibility in culture. Shikonin derivatives are lipophilic red naphthoquinone compounds that accumulate exclusively in apoplastic spaces of these cells and also in the root epidermis of intact plants. Microscopic analysis showed that shikonin is accumulated in the form of numerous particles on the cell wall. Lipidomic analysis showed that L. erythrorhizon cultured cells secrete an appreciable portion of triacylglycerol (24-38% of total triacylglycerol), composed predominantly of saturated fatty acids. Moreover, in vitro reconstitution assay showed that triacylglycerol encapsulates shikonin derivatives with phospholipids to form lipid droplet-like structures. These findings suggest a novel role for triacylglycerol as a matrix lipid, a molecular component involved in the secretion of specialized lipophilic metabolites.

Protein Kinase MpYAK1 Is Involved in Meristematic Cell Proliferation, Reproductive Phase Change and Nutrient Signaling in the Liverwort Marchantia polymorpha.

Plant growth and development are regulated by environmental factors, including nutrient availability and light conditions, via endogenous genetic signaling pathways. Phosphorylation-dependent protein modification plays a major role in the regulation of cell proliferation in stress conditions, and several protein kinases have been shown to function in response to nutritional status, including dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs). Although DYRKs are widely conserved in eukaryotes, the physiological functions of DYRKs in land plants are still to be elucidated. In the liverwort Marchantia polymorpha, a model bryophyte, four putative genes encoding DYRK homologous proteins, each of which belongs to the subfamily yet another kinase 1 (Yak1), plant-specific DYRK, DYRK2, or pre-mRNA processing protein 4 kinase, were identified. MpYAK1-defective male and female mutant lines generated by the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system showed smaller sizes of thalli than did the wild-type plants and repressed cell divisions in the apical notch regions. The Mpyak1 mutants developed rhizoids from gemmae in the gemma cup before release. The Mpyak1 lines developed sexual organs even in non-inductive short-day photoperiod conditions supplemented with far-red light. In nitrogen (N)-deficient conditions, rhizoid elongation was inhibited in the Mpyak1 mutants. In conditions of aeration with 0.08% CO2 (v/v) and N depletion, Mpyak1 mutants accumulated higher levels of sucrose and lower levels of starch compared to the wild type. Transcriptomic analyses revealed that the expression of peroxidase genes was differentially affected by MpYAK1. These results suggest that MpYAK1 is involved in the maintenance of plant growth and developmental responses to light conditions and nutrient signaling.

The bZIP1 Transcription Factor Regulates Lipid Remodeling and Contributes to ER Stress Management in Chlamydomonas reinhardtii.

Endoplasmic reticulum (ER) stress is caused by the stress-induced accumulation of unfolded proteins in the ER. Here, we identified proteins and lipids that function downstream of the ER stress sensor INOSITOL-REQUIRING ENZYME1 (CrIRE1) that contributes to ER stress tolerance in Chlamydomonas (Chlamydomonas reinhardtii). Treatment with the ER stress inducer tunicamycin resulted in the splicing of a 32-nucleotide fragment of a basic leucine zipper 1 (bZIP1) transcription factor (CrbZIP1) mRNA by CrIRE1 that, in turn, resulted in the loss of the transmembrane domain in CrbZIP1, and the translocation of CrbZIP1 from the ER to the nucleus. Mutants deficient in CrbZIP1 failed to induce the expression of the unfolded protein response genes and grew poorly under ER stress. Levels of diacylglyceryltrimethylhomoserine (DGTS) and pinolenic acid (18:3Δ5,9,12) increased in the parental strains but decreased in the crbzip1 mutants under ER stress. A yeast one-hybrid assay revealed that CrbZIP1 activated the expression of enzymes catalyzing the biosynthesis of DGTS and pinolenic acid. Moreover, two lines harboring independent mutant alleles of Chlamydomonas desaturase (CrDES) failed to synthesize pinolenic acid and were more sensitive to ER stress than were their parental lines. Together, these results indicate that CrbZIP1 is a critical component of the ER stress response mediated by CrIRE1 in Chlamydomonas that acts via lipid remodeling.

A plant-specific DYRK kinase DYRKP coordinates cell morphology in Marchantia polymorpha.

Dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs) are activated via the auto-phosphorylation of conserved tyrosine residues in their activation loop during protein translation, and they then phosphorylate serine/threonine residues on substrates. The DYRK family is widely conserved in eukaryotes and is composed of six subgroups. In plant lineages, DYRK homologs are classified into four subgroups, DYRK2s, yet another kinase1s, pre-mRNA processing factor 4 kinases, and DYRKPs. Only the DYRKP subgroup is plant-specific and has been identified in a wide array of plant lineages, including land plants and green algae. It has been suggested that in Arabidopsis thaliana DYRKPs are involved in the regulation of centripetal nuclear positioning induced by dark light conditions. However, the molecular functions, such as kinase activity and the developmental and physiological roles of DYRKPs are poorly understood. Here, we focused on a sole DYRKP ortholog in the model bryophyte, Marchantia polymorpha, MpDYRKP. MpDYRKP has a highly conserved kinase domain located in the C-terminal region and shares common sequence motifs in the N-terminal region with other DYRKP members. To identify the roles of MpDYRKP in M. polymorpha, we generated loss-of-function Mpdyrkp mutants via genome editing. Mpdyrkp mutants exhibited abnormal, shrunken morphologies with less flattening in their vegetative plant bodies, thalli, and male reproductive organs, antheridial receptacles. The surfaces of the thalli in the Mpdyrkp mutants appeared uneven and disordered. Moreover, their epidermal cells were drastically altered to a narrower shape when compared to the wild type. These results suggest that MpDYRKP acts as a morphological regulator, which contributes to orderly tissue morphogenesis via the regulation of cell shape.

Lipid remodeling regulator 1 (LRL1) is differently involved in the phosphorus-depletion response from PSR1 in Chlamydomonas reinhardtii.

The elucidation of lipid metabolism in microalgae has attracted broad interest, as their storage lipid, triacylglycerol (TAG), can be readily converted into biofuel via transesterification. TAG accumulates in the form of oil droplets, especially when cells undergo nutrient deprivation, such as for nitrogen (N), phosphorus (P), or sulfur (S). TAG biosynthesis under N-deprivation has been comprehensively studied in the model microalga Chlamydomonas reinhardtii, during which TAG accumulates dramatically. However, the resulting rapid breakdown of chlorophyll restricts overall oil yield productivity and causes cessation of cell growth. In contrast, P-deprivation results in oil accumulation without disrupting chloroplast integrity. We used a reverse genetics approach based on co-expression analysis to identify a transcription factor (TF) that is upregulated under P-depleted conditions. Transcriptomic analysis revealed that the mutants showed repression of genes typically associated with lipid remodeling under P-depleted conditions, such as sulfoquinovosyl diacylglycerol 2 (SQD2), diacylglycerol acyltransferase (DGTT1), and major lipid droplet protein (MLDP). As accumulation of sulfoquinovosyl diacylglycerol and TAG were suppressed in P-depleted mutants, we designated the protein as lipid remodeling regulator 1 (LRL1). LRL1 mutants showed slower growth under P-depletion. Moreover, cell size in the mutant was significantly reduced, and TAG and starch accumulation per cell were decreased. Transcriptomic analysis also suggested the repression of several genes typically upregulated in adaptation to P-depletion that are associated with the cell cycle and P and lipid metabolism. Thus, our analysis of LRL1 provides insights into P-allocation and lipid remodeling under P-depleted conditions in C. reinhardtii. OPEN RESEARCH BADGES: This article has earned an Open Data Badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. The sequencing data were made publicly available under the BioProject Accession number PRJDB6733 and an accession number LC488724 at the DNA Data Bank of Japan (DDBJ). The data is available at https://trace.ddbj.nig.ac.jp/BPSearch/bioproject?acc=PRJDB6733; http://getentry.ddbj.nig.ac.jp/getentry/na/LC488724. The metabolome data were made publicly available and can be accessed at http://metabolonote.kazusa.or.jp/SE195:/; http://webs2.kazusa.or.jp/data/nur/.

A rice PHD-finger protein OsTITANIA, is a growth regulator that functions through elevating expression of transporter genes for multiple metals.

Essential metal absorption for plant growth is mediated predominantly by metal-specific transporters, with expression that responds to the environmental or cellular conditions of specific metals. Differing from metal-specific regulation, we describe a constitutively expressed transcription factor that regulates the transport of several metals in rice. We characterized the rice mutant LOW CADMIUM 5 (LC5), which exhibited reduced growth and accumulation of essential metals (e.g., copper [Cu], zinc [Zn] and manganese [Mn]) in shoots. LC5 was dwarf and developed less tillers than the wild type, but the structure of vasculature was apparently normal. Molecular genetic analysis revealed that the causal gene of LC5 is an ortholog of the transcriptional regulator Arabidopsis thaliana TITANIA (TTA), known as a transcriptional regulator. Expression analyses demonstrated that the OsTTA gene encodes a nucleus-localized protein containing a plant homeodomain-finger (PHD-finger) domain and is expressed ubiquitously in rice plants. RNA sequencing and quantitative PCR analyses revealed that the mRNA accumulation of transporter genes for essential metals, including iron (Fe), Zn, or Mn, were substantially lower in LC5 roots than in the wild type. Unlike known transcription factors of metal transport regulation, OsTTA transcript accumulation was not affected by metal availability. In addition, the growth defect of LC5 was partially rescued by Fe, Zn, or Mn supplementation, respectively. Taken together, OsTTA is a constitutively expressed regulator of multiple metal transporter genes responsible for essential metals delivery to shoots for their normal growth.

Isolation and Characterization of Chlamydomonas Autophagy-Related Mutants in Nutrient-Deficient Conditions.

Autophagy is a recycling system for amino acids and carbon- and nitrogen (N)-containing compounds. To date, the functional importance of autophagy in microalgae in nutrient-deficient conditions has not been evaluated by using autophagy-defective mutants. Here, we provide evidence which supports the following notions by characterizing an insertional mutant of the autophagy-related gene ATG8, encoding a ubiquitin-like protein necessary for the formation of the autophagosome in the green alga, Chlamydomonas reinhardtii. First, ATG8 is required for maintenance of cell survival and Chl content in N-, sulfur- and phosphate-deficient conditions. Secondly, ATG8 supports the degradation of triacylglycerol and lipid droplets after the resupply of N to cells cultured in N-limiting conditions. Thirdly, ATG8 is also necessary for accumulation of starch in phosphate-deficient conditions. Additionally, autophagy is not essential for maternal inheritance of the organelle genomes in gametogenesis.

Algal Protein Kinase, Triacylglycerol Accumulation Regulator 1, Modulates Cell Viability and Gametogenesis in Carbon/Nitrogen-Imbalanced Conditions.

Nutrient-deprived microalgae accumulate triacylglycerol (TAG) in lipid droplets. A dual-specificity tyrosine phosphorylation-regulated kinase, TAG accumulation regulator 1 (TAR1) has been shown to be required for acetate-dependent TAG accumulation and the degradation of chlorophyll and photosynthesis-related proteins in photomixotrophic nitrogen (N)-deficient conditions (Kajikawa et�al. 2015). However, this previous report only examined particular condition. Here, we report that in photoautotrophic N-deficient conditions, tar1-1 cells, with a mutation in the TAR1 gene, maintained higher levels of cell viability and lower levels of hydrogen peroxide generation and accumulated higher levels of TAG and starch compared with those of wild type (WT) cells with bubbling of air containing 5% carbon dioxide. Transcriptomic analyses suggested that genes involved in the scavenging of reactive oxygen species are not repressed in tar1-1 cells. In contrast, the mating efficiency and mRNA levels of key regulatory genes for gametogenesis, MID, MTD and FUS, were suppressed in tar1-1 cells. Among the TAR1-dependent phosphopeptides deduced by phosphoproteomic analysis, protein kinases and enzymes related to N assimilation and carbon (C) metabolism are of particular interest. Characterization of these putative downstream factors may elucidate the molecular pathway whereby TAR1 mediates cellular propagation and C and N metabolism in C/N-imbalanced stress conditions.

Genomic Insights into the Evolution of the Nicotine Biosynthesis Pathway in Tobacco.

In tobacco (Nicotiana tabacum), nicotine is the predominant alkaloid. It is produced in the roots and accumulated mainly in the leaves. Jasmonates play a central signaling role in damage-induced nicotine formation. The genome sequence of tobacco provides us an almost complete inventory of structural and regulatory genes involved in nicotine pathway. Phylogenetic and expression analyses revealed a series of structural genes of the nicotine pathway, forming a regulon, under the control of jasmonate-responsive ETHYLENE RESPONSE FACTOR (ERF) transcription factors. The duplication of NAD and polyamine metabolic pathways and the subsequent recruitment of duplicated primary metabolic genes into the nicotine biosynthesis regulon were suggested to be the drivers for pyridine and pyrrolidine ring formation steps early in the pathway. Transcriptional regulation by ERF and cooperatively acting MYC2 transcription factors are corroborated by the frequent occurrence of cognate cis-regulatory elements of the factors in the promoter regions of the downstream structural genes. The allotetraploid tobacco has homologous clusters of ERF genes on different chromosomes, which are possibly derived from two ancestral diploids and include either nicotine-controlling ERF189 or ERF199 A large chromosomal deletion was found within one allele of the nicotine-controlling NICOTINE2 locus, which is part of one of the ERF gene clusters, and which has been used to breed tobacco cultivars with a low-nicotine content.

Algal dual-specificity tyrosine phosphorylation-regulated kinase, triacylglycerol accumulation regulator1, regulates accumulation of triacylglycerol in nitrogen or sulfur deficiency.

Although microalgae accumulate triacylglycerol (TAG) and starch in response to nutrient-deficient conditions, the regulatory mechanisms are poorly understood. We report here the identification and characterization of a kinase, triacylglycerol accumulation regulator1 (TAR1), that is a member of the yeast (Saccharomyces cerevisiae) Yet another kinase1 (Yak1) subfamily in the dual-specificity tyrosine phosphorylation-regulated kinase family in a green alga (Chlamydomonas reinhardtii). The kinase domain of TAR1 showed auto- and transphosphorylation activities. A TAR1-defective mutant, tar1-1, accumulated TAG to levels 0.5- and 0.1-fold of those of a wild-type strain in sulfur (S)- and nitrogen (N)-deficient conditions, respectively. In N-deficient conditions, tar1-1 showed more pronounced arrest of cell division than the wild type, had increased cell size and cell dry weight, and maintained chlorophyll and photosynthetic activity, which were not observed in S-deficient conditions. In N-deficient conditions, global changes in expression levels of N deficiency-responsive genes in N assimilation and tetrapyrrole metabolism were noted between tar1-1 and wild-type cells. These results indicated that TAR1 is a regulator of TAG accumulation in S- and N-deficient conditions, and it functions in cell growth and repression of photosynthesis in conditions of N deficiency.

Potential involvement of drought-induced Ran GTPase CLRan1 in root growth enhancement in a xerophyte wild watermelon.

Enhanced root growth is known as the survival strategy of plants under drought. Previous proteome analysis in drought-resistant wild watermelon has shown that Ran GTPase, an essential regulator of cell division and proliferation, was induced in the roots under drought. In this study, two cDNAs were isolated from wild watermelon, CLRan1 and CLRan2, which showed a high degree of structural similarity with those of other plant Ran GTPases. Quantitative RT-PCR and promoter-GUS assays suggested that CLRan1 was expressed mainly in the root apex and lateral root primordia, whereas CLRan2 was more broadly expressed in other part of the roots. Immunoblotting analysis confirmed that the abundance of CLRan proteins was elevated in the root apex region under drought stress. Transgenic Arabidopsis overexpressing CLRan1 showed enhanced primary root growth, and the growth was maintained under osmotic stress, indicating that CLRan1 functions as a positive factor for maintaining root growth under stress conditions.

Isolation and characterization of novel high-CO2-requiring mutants of Chlamydomonas reinhardtii.

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.

The effect of fertilization on cesium concentration of rice grown in a paddy field in Fukushima Prefecture in 2011 and 2012.

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.

Difference in cesium accumulation among rice cultivars grown in the paddy field in Fukushima Prefecture in 2011 and 2012.

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.

Roles of pollen-specific boron efflux transporter, OsBOR4, in the rice fertilization process.

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.

Clustered transcription factor genes regulate nicotine biosynthesis in tobacco.

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.

ANGUSTIFOLIA, a plant homolog of CtBP/BARS, functions outside the nucleus.

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.

Vacuole-localized berberine bridge enzyme-like proteins are required for a late step of nicotine biosynthesis in tobacco.

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.

Dynamic changes in the leaf proteome of a C3 xerophyte, Citrullus lanatus (wild watermelon), in response to water deficit.

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.