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Increased acidification of the external medium by an activated H super( + ) -ATPase results in cell expansion, in the absence of upstream activating signaling. The plasma membrane H super(+)-ATPase couples ATP hydrolysis with proton transport outside the cell, and thus creates an electrochemical gradient, which energizes secondary transporters. According to the acid growth theory, this enzyme is also proposed to play a major role in cell expansion, by acidifying the external medium and so activating enzymes that are involved in cell wall-loosening. However, this theory is still debated. To challenge it, we made use of a plasma membrane H super(+)-ATPase isoform from Nicotiana plumbaginifolia truncated from its C-terminal auto-inhibitory domain ( Delta CPMA4), and thus constitutively activated. This protein was expressed in Nicotiana tabacum BY-2 suspension cells using a heat shock inducible promoter. The characterization of several independent transgenic lines showed that the expression of activated Delta CPMA4 resulted in a reduced external pH by 0.3-1.2 units, as well as in an increased H super(+)-ATPase activity by 77-155 % (ATP hydrolysis), or 70-306 % (proton pumping) of isolated plasma membranes. In addition, Delta CPMA4-expressing cells were 17-57 % larger than the wild-type cells and displayed abnormal shapes. A proteomic comparison of plasma membranes isolated from Delta CPMA4-expressing and wild-type cells revealed the altered abundance of several proteins involved in cell wall synthesis, transport, and signal transduction. In conclusion, the data obtained in this work showed that H super(+)-ATPase activation is sufficient to induce cell expansion and identified possible actors which intervene in this process.

Black shank, a devastating disease affecting tobacco production worldwide, is caused by Phytophthora nicotianae. However, few genes related to Phytophthora resistance have been reported in tobacco. Here, we identified NpPP2-B10, a gene strongly induced by P. nicotianae race 0, with a conserved F-box motif and Nictaba (tobacco lectin) domain, in the highly resistant tobacco species Nicotiana plumbaginifolia. NpPP2-B10 is a typical F-box-Nictaba gene. When it was transferred into the black shank-susceptible tobacco cultivar ‘Honghua Dajinyuan’, it was found to promote resistance to black shank disease. NpPP2-B10 was induced by salicylic acid, and some resistance-related genes (NtPR1, NtPR2, NtCHN50, and NtPAL) and resistance-related enzymes (catalase and peroxidase) were significantly upregulated in the overexpression lines after infection with P. nicotianae. Furthermore, we showed that NpPP2-B10 actively regulated the tobacco seed germination rate, growth rate, and plant height. The erythrocyte coagulation test of purified NpPP2-B10 protein showed that NpPP2-B10 had plant lectin activity, and the lectin content in the overexpression lines was significantly higher than that in the WT, which could lead to accelerated growth and improved resistance of tobacco. SKP1 is an adaptor protein of the E3 ubiquitin ligase SKP1, Cullin, F-box (SCF) complex. We demonstrated that NpPP2-B10 could interact with the NpSKP1-1A gene in vivo and in vitro through yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC), indicating that NpPP2-B10 likely participates in the plant immune response by mediating the ubiquitin protease pathway. In conclusion, our study provides some important insights concerning NpPP2-B10-mediated regulation of tobacco growth and resistance.

Fructans play an important role in assimilate partitioning and possibly in stress tolerance in many plant families. Sucrose:fructan 6-fructosyltransferase (6-SFT), an enzyme catalyzing the formation and extension of beta-2,6-linked fructans typical of grasses, was purified from barley (Hordeum vulgare L.). It occurred in two closely similar isoforms with indistinguishable catalytic properties, both consisting of two subunits with apparent masses of 49 and 23 kDa. Oligonucleotides, designed according to the sequences of tryptic peptides from the large subunit, were used to amplify corresponding sequences from barley cDNA. The main fragment generated was cloned and used to screen a barley cDNA expression library. The longest cDNA obtained was transiently expressed in Nicotiana plumbaginifolia protoplasts and shown to encode a functional 6-SFT. The deduced amino acid sequence of the cDNA comprises both subunits of 6-SFT. It has high similarity to plant invertases and other beta-fructosyl hydrolases but only little to bacterial fructosyltransferases catalyzing the same type of reaction as 6-SFT

The stress induced by allelochemicals present in stem aqueous extract (SAE) of Nicotiana plumbaginifolia on alterations in growth, ultrastructure on Cassia tora L., and mitotic changes on Allium cepa L. were inspected. Application of SAE at different concentrations (0.5, 1, 2, and 4%) expressively reduced the growth of C. tora in terms of seedling length and dry biomass. Moreover, the ultrastructural variations induced in the epidermis of Cassia leaf (adaxial and abaxial surface) of 15-day-old saplings were analyzed through scanning electron microscopy (SEM). The variations noticed are rupturing and shrinking of cells along epidermis; damaged margins, extensively curled leaf apex along with the appearance of puff-like structures, grooves, and thread-like structures on the leaf surface. The epidermal cells of samples exposed to treatment no longer appear smooth relative to control, besides showing necrosis as well. Upon exposure to different concentrations of extract, A. cepa root tip cells showed aberrations in chromosome arrangement and disparity in the shape of the interphase and prophase nuclei along various phases of mitotic cycle as compared to control. The mitotic index (MI) showed a concentration-dependent decline in onion root tips exposed to SAE. The aberrations appearing frequently were formation of multinucleated cells, sticky metaphase and anaphase with bridges, sticky telophase, disturbed polarity, etc. The results also show the induction of elongated cells, giant cells, and cells with membrane damage by extract treatment. To our knowledge, this is the first gas chromatography-mass spectrometry (GC-MS) analysis of the methanolic extract of N. plumbaginifolia stem. Overall, 62 compounds were reported, covering 99.61% of the entire constituents, which can be considered responsible for the allelopathic suppression of C. tora. The chief component was 4-tert-butylcalix[4]arene with the highest composition of 19.89%, followed by palmitic acid (12.25%), palmitoleic acid (8.23%), precocene 2 (7.53%), isophytyl acetate (4.01%), and betastigmasterol (3.95%).

Roots of weeds that are left behind in the soil after removal of their aerial parts can reduce the growth of crops. Here we conducted an assessment to evaluate the allelopathic interference of Nicotiana plumbaginifolia roots and its rhizosphere soil on the growth of Pisum sativum L., identification of chemicals involved, the role of charcoal, the role of N and other macronutrients. Growth responses of P. sativum were analysed for (a) rhizosphere soil with and without N supplementation and (b) soil amendment with Nicotiana roots. Scanning electron microscopy-energy dispersive spectrometry (SEM–EDS) analysis of the rhizosphere soil was conducted to look for concentration of essential nutrients. Rhizosphere soil showed an inhibitory effect even with N supplementation. SEM–EDS showed a healthy concentration of essential nutrients. Root residues of Nicotiana integrated with soil do cause a reduction in seedling length (root length, shoot length) and dry biomass of recipient plant. However, a partial alleviation in growth inhibition occurred upon the addition of activated charcoal. Soils amended with root residues were rich in phenolics as compared to control. Overall, 34 compounds were reported upon GC–MS analysis which can be considered responsible for the allelopathic suppression of P. sativum. The chief component was guanosine (26.21%) followed by n-hexadecanoic acid (18.61%), oleic acid (18.29%), palmitoleic acid (4.80%), -(-)nicotine (5.09%) and solasodine (2.54%). These results show a definite role of putative allelochemicals that exerted allelopathic effects on P. sativum.

The identification of a family of NAR2-type genes in higher plants showed that there was a homolog in Arabidopsis ( Arabidopsis thaliana), AtNAR2.1. These genes encode part of a two-component nitrate high-affinity transport system ( HATS). As the Arabidopsis NRT2 gene family of nitrate transporters has been characterized, we tested the idea that AtNAR2.1 and AtNRT2.1 are partners in a two-component HATS. Results using the yeast split-ubiquitin system and Xenopus oocyte expression showed that the two proteins interacted to give a functional HATS. The growth and nitrogen (N) physiology of two Arabidopsis gene knockout mutants, atnrt2.1-1 and atnar2.1-1, one for each partner protein, were compared. Both types of plants had lost HATS activity at 0.2 mM nitrate, but the effect was more severe in atnar2.1-1 plants. The relationship between plant N status and nitrate transporter expression revealed a pattern that was characteristic of N deficiency that was again stronger in atnar2.1-1. Plants resulting from a cross between both mutants (atnrt2.1-1 x atnar2.1- 1) showed a phenotype like that of the atnar2.1- 1 mutant when grown in 0.5 mM nitrate. Lateral root assays also revealed growth differences between the two mutants, confirming that atnar2.1- 1 had a stronger phenotype. To show that the impaired HATS did not result from the decreased expression of AtNRT2.1, we tested if constitutive root expression of a tobacco ( Nicotiana plumbaginifolia) gene, NpNRT2.1, previously been shown to complement atnrt2.1-1, can restore HATS to the atnar2.1- 1 mutant. These plants did not recover wild-type nitrate HATS. Taken together, these results show that AtNAR2.1 is essential for HATS of nitrate in Arabidopsis.

We developed transgenic Nicotiana plumbaginifolia hairy roots with sucrose-inducible minimal promoter (Spomin)-β-glucuronidase (GUS) gene–fused constructs with signal sequences for sorting to cytosol, apoplast, and ER, and we analyzed the GUS activities of hairy roots after sucrose treatment. Induced GUS activities by Spomin were about 10 times higher than those by the CaMV 35S promoter. GUS activities in hairy roots induced with a Spomin-UTR-GUS construct after 6% sucrose treatment were higher than those with either Spomin-UTR-GUS construct after 10% sucrose treatment, Spomin-apoplast or ER-GUS constructs after sucrose treatment. High GUS activities in hairy roots with Spomin constructs were induced during 20 wk by culturing hairy roots in LS liquid medium containing 6% sucrose in 300 ml conical flasks. Hairy roots in Linsmaier and Skoog (LS) liquid medium showed the increase of fresh weight (FW) for 16 wk. Total yield (µg) of GUS by the hairy roots with Spomin-UTR-GUS construct using 5 l culture container was calculated to about 6742 µg after 8-wk culture. Productive efficiencies of GUS were over 3.0% of total soluble protein after 1–2 wk 6% sucrose treatment. Productive efficiencies of GUS in transgenic N. plumbaginifolia hairy roots were higher than that in transgenic N. plumbaginifolia leaves, stems, and roots. And growth speeds of transgenic N. plumbaginifolia hairy roots were faster than transgenic N. plumbaginifolia plants. These results showed that N. plumbaginifolia hairy roots with Spomin-heterologous construct systems and sucrose treatment would be useful tools to develop protein production system.

The plasma membrane H⁺-ATPase couples ATP hydrolysis with proton transport outside the cell, and thus creates an electrochemical gradient, which energizes secondary transporters. According to the acid growth theory, this enzyme is also proposed to play a major role in cell expansion, by acidifying the external medium and so activating enzymes that are involved in cell wall-loosening. However, this theory is still debated. To challenge it, we made use of a plasma membrane H?⁺-ATPase isoform from Nicotiana plumbaginifolia truncated from its C-terminal auto-inhibitory domain (ΔCPMA4), and thus constitutively activated. This protein was expressed in Nicotiana tabacum BY-2 suspension cells using a heat shock inducible promoter. The characterization of several independent transgenic lines showed that the expression of activated ΔCPMA4 resulted in a reduced external pH by 0.3–1.2 units, as well as in an increased H⁺-ATPase activity by 77–155 % (ATP hydrolysis), or 70–306 % (proton pumping) of isolated plasma membranes. In addition, ΔCPMA4-expressing cells were 17–57 % larger than the wild-type cells and displayed abnormal shapes. A proteomic comparison of plasma membranes isolated from ΔCPMA4-expressing and wild-type cells revealed the altered abundance of several proteins involved in cell wall synthesis, transport, and signal transduction. In conclusion, the data obtained in this work showed that H⁺-ATPase activation is sufficient to induce cell expansion and identified possible actors which intervene in this process.

Mitochondria fulfil essential functions in respiration and metabolism as well as regulating stress responses and apoptosis. Most native mitochondrial proteins are encoded by nuclear genes and are imported into mitochondria via one of several receptors that recognize N-terminal signal peptides. The targeting of recombinant proteins to mitochondria therefore requires the presence of an appropriate N-terminal peptide, but little is known about mitochondrial import in monocotyledonous plants such as rice (Oryza sativa). To gain insight into this phenomenon, we targeted nuclear-encoded enhanced green fluorescent protein (eGFP) to rice mitochondria using six mitochondrial pre-sequences with diverse phylogenetic origins, and investigated their effectiveness by immunoblot analysis as well as confocal and electron microscopy. We found that the ATPA and COX4 (Saccharomyces cerevisiae), SU9 (Neurospora crassa), pFA (Arabidopsis thaliana) and OsSCSb (Oryza sativa) peptides successfully directed most of the eGFP to the mitochondria, whereas the MTS2 peptide (Nicotiana plumbaginifolia) showed little or no evidence of targeting ability even though it is a native plant sequence. Our data therefore indicate that the presence of particular recognition motifs may be required for mitochondrial targeting, whereas the phylogenetic origin of the pre-sequences probably does not play a key role in the success of mitochondrial targeting in dedifferentiated rice callus and plants.

Secondary walls in vessels and fibers of dicotyledonous plants are mainly composed of cellulose, xylan, and lignin. Although genes involved in biosynthesis of cellulose and lignin have been intensively studied, little is known about genes participating in xylan synthesis. We found that Arabidopsis thaliana fragile fiber8 (fra8) is defective in xylan synthesis. The fra8 mutation caused a dramatic reduction in fiber wall thickness and a decrease in stem strength. FRA8 was found to encode a member of glycosyltransferase family 47 and exhibits high sequence similarity to tobacco (Nicotiana plumbaginifolia) pectin glucuronyltransferase. FRA8 is expressed specifically in developing vessels and fiber cells, and FRA8 is targeted to Golgi. Comparative analyses of cell wall polysaccharide fractions from fra8 and wild-type stems showed that the xylan and cellulose contents are drastically reduced in fra8, whereas xyloglucan and pectin are elevated. Further structural analysis of cell walls revealed that although wild-type xylans contain both glucuronic acid and 4-O-methylglucuronic acid residues, xylans from fra8 retain only 4-O-methylglucuronic acid, indicating that the fra8 mutation results in a specific defect in the addition of glucuronic acid residues onto xylans. These findings suggest that FRA8 is a glucuronyltransferase involved in the biosynthesis of glucuronoxylan during secondary wall formation.