Sterile Tobacco Research Articles

The role of exogenous glycinebetaine on some antioxidant activity of non-T and T tobacco (Nicotiana tabacum L.) under in vitro salt stress

Glycine betaine is an osmoprotectant compound which enhances cell tolerance in plant species in response to environmental stresses. This study aimed to investigate the effect of exogenous application of glycine betaine on some antioxidant activities of tobacco plants overexpressing P5CS gene. Sterile tobacco seedlings with four to six leaves were transferred to MS medium containing 0, 100, and 200 mM NaCl, after which glycine betaine (20 and 40 mg l-1) were foliar sprayed on the surface of the plants. After four weeks, glycine betaine treatment enhanced the antioxidant capacity of the plant through activation of catalase (CAT), superoxide dismutase (SOD), and ascorbate peroxidase (APX). In contrast, H2O2 content and MDA level were reduced by glycine betaine under similar conditions. Therefore, application of exogenous glycine betaine under salt stress improved stress tolerance in T and non-T plants. Meanwhile, our results indicated the positive effect of glycine betaine in T plants was greater than in non-T plants. On the other hand, this result suggested that the synergistic effects of glycine betaine and proline in plants enhanced the antioxidant defense system in T plants overexpressing P5CS gene.

Role of MS1 homolog Ntms1 gene of tobacco infertility.

The sterile line is the basis of crop heterosis utilization. To broaden the sources of male sterility in tobacco, the Ntms1 (Nicotiana tabacum L. ms1) gene was cloned from the tobacco variety K326 by homologous cloning based on the Cams1 (Capsicum annuum L. ms1) gene sequence of male-sterility genes in pepper. The protein structure and physicochemical properties of the two genes were determined by bioinformatics analysis, and the function of the Ntms1 gene was verified by the CRISPR/Cas9 system. The results showed that the sequences of Ntms1 and Cams1 were 85.25% similar, and plant homeodomains were found in both genes; the physical and chemical properties were also very similar. It is speculated that the Ntms1 gene had the same function as the Cams1 gene in controlling male sterility. Compared to the wild-type plants, the filaments of the Ntms1 knockout mutant plants were shorter, and the stamen was shorter than the pistil. The anthers did not develop fully and had few viable pollen grains; the tapetum and the anther wall had developed abnormally, and the anther chamber was severely squeezed. The malondialdehyde content in the mutant plants was significantly higher than that in the wild-type plants, while self-fertility was significantly lower in the mutant plants. The results showed that the Ntms1 gene plays an important role in regulating fertility in tobacco.

Comparative analysis of mitochondrial genome and expression variation between UG93A and UG93B reveals a candidate gene related to cytoplasmic male sterility in kenaf

Plant cytoplasmic male sterility (CMS) is a maternal genetic trait that results from incompatibilities between the organellar and nuclear genomes which causes pollen abortion. Cytoplasmic male sterility plays an important role in hybrid seed production and heterosis utilization. In kenaf, cytoplasmic male sterility has been exploited in hybrid seed production for several years, but its molecular mechanism has not yet been disclosed. In the present work, the mitochondrial genome of the isonuclear cytoplasmic male sterile line UG93A compared with maintainer line UG93B was described and a total of 398 single nucleotide polymorphisms (SNPs) and 230 insertions/deletions (InDels) were identified in the UG93A mitochondrial DNA. A total of 26 single nucleotide polymorphisms and insertions/deletions were identified in the coding region of atp6, indicating active mitochondrial genome rearrangement. An expression cassette containing atp6 of UG93A without a fused mitochondrial transit peptide induced male sterility in tobacco and Arabidopsis. This indicated abnormal pollen grains and lack of pollen activity. Conclusively, coding sequence of atp6, which contains single nucleotide polymorphism and indel variations may play a crucial role in regulating male sterility in kenaf. These data suggested that atp6 of UG93A may be a candidate gene for kenaf cytoplasmic male sterility and provided basic information for further study of the molecular mechanisms of cytoplasmic male sterility in kenaf.

Induction of Male Sterility in Tobacco by Anther-Specific Expression of the Gene for Ricin Enzymatic Subunit A Chain RTA.

Targeted gene expression in plants allows us to further study biological traits of interest, such as reproductive and developmental processes. Here, the tobacco TA29 anther-specific promoter was used to direct the expression of the ricin enzymatic subunit A (RTA) in transgenic tobacco plants, phenotypic analysis of the resulting positive transgenic tobacco (Nicotiana tabacum L.) plants demonstrated that RTA expression led to a reduction in pistil length and shriveling of anthers, as well as the grayish-brown color of anthers, the reduced pollen viability and male sterility. For the first time, a plant-derived ricin gene enzymatic subunit A (RTA) expression system under the tissue-specific promoter was demonstrated to be sensitive and efficient in controlling plant sterility and creating male-sterile materials. Consequently, it could be used to control other agronomic traits and produce hybrid seeds in plants in the future.

A proteomic study of cysteine protease induced cell death in anthers of male sterile tobacco transgenic plants.

Manifestation of male sterility in plants is an important requirement for hybrid seed production. Tapetum cell layer of anther is a primary target for genetic manipulation for male sterility. In our previous report, the targeted expression of Arachis cysteine protease in tapetum led to premature degeneration of tapetal layer that resulted in complete male sterility in transgenic tobacco plants. To correlate cysteine protease mediated cell death of tapetum, transmission electron microscopy (TEM) and proteomic pattern of anthers of cysteine protease induced male sterile plant were compared with the untransformed control plant. TEM study revealed the abnormal growth of tapetal cells exhibiting excessive vacuolization that synchronized with irregular exine wall formation of the microspores. In anther proteome, a total 250 protein spots were detected that were reproducible and exhibited similar distribution pattern. Further, anther proteome of male sterile plant showed the significant upregulation (≥ 1.5) of 56 protein spots. Using Mass spectroscopy (MALDI TOF/TOF), we have identified 14 protein spots that were involved in several processes such as energy metabolism, protein synthesis, plastid protein, lipid metabolism, and cell wall assembly. Upregulation of patatin-like protein-2 homolog, carboxylesterase 17 and dicer like protein-4 in male sterile anthers that have been demonstrated to induce cell death, suggesting that cysteine protease mediated premature tapetal cell death might involve the lipid peroxidation pathway in coordination with gene silencing mechanism.

Targeted expression of cystatin restores fertility in cysteine protease induced male sterile tobacco plants

Fertility restoration in male sterile plants is an essential requirement for their utilization in hybrid seed production. In an earlier investigation, we have demonstrated that the targeted expression of a cysteine protease in tapetal cell layer resulted in complete male sterility in tobacco transgenic plants. In the present investigation, we have used a cystatin gene, which encodes for a cysteine protease inhibitor, from a wild peanut, Arachis diogoi and developed a plant gene based restoration system for cysteine protease induced male sterile transgenic tobacco plants. We confirmed the interaction between the cysteine protease and a cystatin of the wild peanut, A. diogoi through in silico modeling and yeast two-hybrid assay. Pollen from primary transgenic tobacco plants expressing cystatin gene under the tapetum specific promoter- TA29 restored fertility on cysteine protease induced male sterile tobacco plants developed earlier. This has confirmed the in vivo interaction of cysteine protease and cystatin in the tapetal cells, and the inactivation of cysteine protease and modulation of its negative effects on pollen fertility. Both the cysteine protease and cystatin genes are of plant origin in contrast to the analogous barnase–barstar system that deploys genes of prokaryotic origin. Because of the deployment of genes of plant origin, this system might not face biosafety problems in developing hybrids in food crops.

Enzymatic conversion from pyridoxal to pyridoxine caused by microorganisms within tobacco phyllosphere

Vitamin B6 (VB6) comprises six interconvertible pyridine compounds (vitamers), among which pyridoxal 5′-phosphate (PLP) is a coenzyme involved in a high diversity of biochemical reactions. In plants, PLP is de novo synthesized, and pyridoxine (PN) is usually maintained as the predominant B6 vitamer. Although the conversion from pyridoxal (PL) to PN catalyzed by PL reductase in plants has been confirmed, the enzyme itself remains largely unknown. We previously found pre-incubation at 35 °C dramatically enhanced PL reductase activity in tobacco leaf homogenate. In this study, we demonstrated that the increase in the reductase activity was a consequence of phyllosphere microbial proliferation. VB6 was detected from tobacco phyllosphere, and PL level was the highest among three non-phosphorylated B6 vitamers. When the sterile tobacco rich in PL were kept in an open, warm and humid environment to promote microorganism proliferation, a significant change from PL to PN was observed. Our results suggest that there may be a plant–microbe interaction in the conversion from PL to PN within tobacco phyllosphere.

Tapetum-specific expression of harpin<sub>Pss</sub> causes male sterility in transgenic tobacco

Harpin, an elicitor molecule of bacterial origin induces hypersensitive response (HR) in non-host plants. In an attempt to induce male sterility, harpin was tagged with a signal peptide and expressed downstream to tapetum-specific TA29 promoter resulting in extracellular secretion, subsequent degeneration of tapetum and development of male sterility in tobacco. Putative transgenics were analyzed by PCR amplification of transgene, semiquantitative RT-PCR analysis from total RNA extracts from anther tissue with transgene specific probe, Western blotting using polyclonal antibody raised against harpin, by transmission and scanning electron microscopy, and by confocal microscopy of anthers and pollen at various stages of development. Varying degrees of male sterility (30-100 %) was observed with plants showing complete and partial male sterility as well as several morphological variations were seen especially in leaves and flowers. Further, some of the transgenics showed un-induced of HR-like local lesions in the vegetative tissues. HarpinPss got deposited on the pollen grains upon tapetal degeneration resulting in significant alterations in the morphology of pollen cell wall. However, megagametogenesis was not affected in complete and partial male sterile plants and female gametes were completely fertile. The complete male sterility was attributed to premature tapetal cell death due to sufficient extracellular harpinPss accumulation whereas insufficient protein content might be the reason for partial male sterility. These findings indicate the possible use of cytotoxic harpinPss for the development of male sterile plants.

Cover Caption: Man‐made Male Sterile Technology

Male sterility is a highly valuable technology in modern agriculture, as it provides economic means to produce hybrid seeds and may also reduce the risk of ‘gene‐flow’ from genetically‐modified crops to sounding species. However, this technology is useful only in few crops, with cytoplasmic male sterility and nuclear controlled restoration traits available. The paper of Shaya et al. (pp 115–129) reports a novel technology for generating male sterility in tobacco based on the expression of a truncated mitochondrial gene fragments in the nuclei.

Intron hairpin and transitive RNAi mediated silencing of orfH522 transcripts restores male fertility in transgenic male sterile tobacco plants expressing orfH522

The present work was aimed at developing vector construct(s) suitable for restoring fertility in transgenic male sterile tobacco plants expressing male-sterility-inducing ORFH522 in tapetal cell layer (Nizampatnam et al. Planta 229:987-1001, 2009). PTGS vectors that could produce either intron spliced hairpin RNA against the orfH522 or induce silencing of orfH522 by heterologous 3'UTR region were developed using the selected 316bp (orf316) fragment of orfH522. The constructs were independently mobilized into Agrobacterium and used for transforming tobacco. The T(1) generation plants carrying the restorer gene cassettes in homozygous condition were identified and crossed with the male sterile transgenic tobacco plants to obtain the hybrid seeds. PCR analysis of hybrid plants indicated segregation for the sterility inducing cassette while all the plants carried the restorer cassette. Hybrid plants produced fertile pollen grains and formed normal capsules upon selfing. Further molecular analyses of these hybrid plants with RT-PCR, Northern blotting and siRNA detection, revealed that intron interrupted hairpin RNA (ihp-RNA) mediated gene silencing was more effective compared to silencing by heterologous 3'UTR (SHUTR) as indicated by the complete degradation of orfH522 transcripts and formation of higher levels of orf316 specific siRNA molecules in plants carrying ihp-RNA restorer construct. Segregation analyses of F(2) (selfed hybrid) plants confirmed the co-segregation of gene cassettes and the traits in Mendelian di-hybrid ratio (9:3:3:1). Taken together, the results established that intron hairpin and transitive RNAi mediated silencing of orfH522 transcripts restored fertility in transgenic male sterile tobacco plants expressing orfH522 and ihp-RNA was more efficient in silencing orfH522 transcripts.

Modification of tobacco plant development by sense and antisense expression of the tomato viroid-induced AGC VIIIa protein kinase PKV suggests involvement in gibberellin signaling

BackgroundThe serine-threonine protein kinase gene, designated pkv (protein kinase- viroid induced) was previously found to be transcriptionally activated in tomato plants infected with the plant pathogen Potato spindle tuber viroid (PSTVd). These plants exhibited symptoms of stunting, and abnormal development of leaf, root, and vascular tissues. The encoded protein, PKV, is a novel member of the AGC VIIIa group of signal-transducing protein kinases; however, the role of PKV in plant development is unknown. In this communication, we report the phenotypic results of over expression and silencing of pkv in transgenic tobacco.ResultsOver expression of pkv in Nicotiana tabacum cv. Xanthi (tobacco) resulted in stunting, reduced root formation, and delay in flowering, phenotypes similar to symptoms of PSTVd infection of tomato. In addition, homozygous T2 tobacco plants over expressing PKV were male sterile. Antisense expression of pkv, on the other hand, resulted in plants that were taller than non-transformed plants, produced an increased number of flowers, and were fertile. Exogenous application of GA3 stimulated stem elongation in the stunted, sense-expressing plants. PKV sense and antisense expression altered transcript levels of GA biosynthetic genes and genes involved in developmental and signaling pathways, but not genes involved in salicylic acid- or jasmonic acid-dependent pathways. Our data provide evidence suggesting that PKV plays an important role in a GA signaling pathway that controls plant height and fertility.ConclusionWe have found that the over expression of the tomato protein kinase PKV resulted in stunting, modified vascular tissue development, reduced root formation, and male sterility in tobacco, and we propose that PKV regulates plant development by functioning in critical signaling pathways involved in gibberellic acid metabolism.

Restoring full pollen fertility in transgenic male-sterile tobacco (Nicotiana tabacum L.) by Cre-mediated site-specific recombination

Producing hybrid seed requires an efficient pollination control system to prevent unwanted self-pollination. For further breeding, it would be advantageous to restore pollen fertility in the hybrids. In this work we demonstrate the use of tapetum-specific expression of a stilbene synthase (sts) transgene to induce pollen sterility in tobacco as has been shown previously. The sts-coding region was flanked by loxP recognition sites for Cre-recombinase. From 10 T0-plants obtained, five proved to be male-sterile. They had smaller flowers with shorter stamina, but the vegetative phenotype was just as in the wild-type. Crossing male-sterile sts-plants with tobacco lines expressing the cre recombinase transgene resulted in site-specific recombination in the hybrids. GUS activity caused by fusion of the tap1-promoter with a promoterless gusA coding region indicated recombination events already in early stages of flower bud development. In all plants which had contained single or double sts-copies before crossing, these were excised, and pollen fertility was fully restored. The phenotype of these restored plants was as in wild-type controls. Contrary, from male sterile plants containing multiple copies of the sts-gene, not all copies were removed, and pollen sterility was maintained.

Overexpression of human erythropoietin (EPO) affects plant morphologies: retarded vegetative growth in tobacco and male sterility in tobacco and Arabidopsis

Erythropoietin (EPO) is a glycoprotein used for curing human anemia by regulating the differentiation of erythroid progenitors and the production of red blood cells. To examine the expression of recombinant EPO in plants, pPEV-EP21, in which human epo cDNA under the control of the CaMV 35S promoter, was introduced into tobacco and Arabidopsis via Agrobacterium tumefaciens-mediated transformation. The RNA expression level of epo in the transgenic lines was initially estimated by Northern blot analysis. Two transgenic lines, which exhibited a high expression level of epo mRNA determined by Northern analysis, were chosen for Western blot analysis to examine the production of EPO proteins. Those two lines, EP21-12 and EP21-14, revealed detectable bands on the immunoblot. Interestingly, constitutive expression of the human epo gene affected the morphologies in transgenic plants such that vegetative growth of transgenic tobacco was retarded, and male sterility was induced in transgenic tobacco and Arabidopsis.

Production of Transgenic Male Sterile Tobacco Plants with the cDNA Encoding a Ribosome Inactivating Protein in Dianthus sinensis L.

Production of Transgenic Male Sterile Tobacco Plants with the cDNA Encoding a Ribosome Inactivating Protein in Dianthus sinensis L.

Prevention of flower formation in dicotyledons

Prevention of flower formation is important, for example for preventing the spread of transgenes from genetically modified plants or the spread of non-native species, for increasing vegetative growth or preventing the formation of allergenic pollen. The aim of this study was to determine whether flowering of dicotyledonous plants can be prevented by genetic manipulation without harmful effects on vegetative growth. Here we describe isolation of the BpMADS1 gene (similar to SEP3, formerly AGL9) from birch and show that it is expressed only in the inflorescences. In tobacco and Arabidopsis, the expression of BpMADS1::GUS was also virtually inflorescence-specific. Transgenic tobacco and Arabidopsis containing a BpMADS1::BARNASE construct grew well. In one tobacco line the formation of the inflorescence was completely prevented; in several other lines the flowers lacked stamens and carpels and therefore were sterile. The final dry weights of the shoots of the sterile tobacco lines were 140–200% of those of controls. In Arabidopsis, some of the transgenic lines containing the BpMADS1::BARNASE construct formed inflorescences. Some of these lines formed never flowers and some others formed occasionally single fertile flowers. Some other lines did not form inflorescences, but formed up to about one hundred leaves, even in long-day conditions. These results suggest that formation of flowers or inflorescences in widely different dicotyledonous plants could be prevented using the BpMADS1::BARNASE construct and that prevention of flowering may lead to increased vegetative mass.

The restoration of fertility in male sterile tobacco demonstrates that transgene silencing can be mediated by T-DNA that has no DNA homology to the silenced transgene.

Male sterile tobacco plants expressing a pathogenesis-related (PR) beta-1,3-glucanase gene driven by the Arabidopsis thaliana A3 or A9 tapetum-specific promoter, were partially restored to fertility by retransformation with a range of pA9-driven sense and antisense PR glucanase fragments. The restored plants exhibited improved seed set. PR glucanase protein was undetectable in the anthers of these plants and there was an associated increase in microsporocyte callose, the structural target of the A3 and A9-driven PR glucanase. This phenotype was not solely dependent on interactions between sense and antisense PR glucanase transcripts since a pA9-driven restorer was also capable of down regulating a pA3-GUS construct in the absence of extensive promoter, coding region, or terminator sequence homology. Since the A3 and A9 promoters have similar temporal and spatial expression patterns, it is possible that trans-acting factors common to both promoters become limiting in the PR glucanase double transformants resulting in improved levels of fertility. An alternative hypothesis is that additional sequences present in both the silencing and target T-DNAs can mediate the silencing of adjacent non-homologous transgenes.

Stilbene synthase gene expression causes changes in flower colour and male sterility in tobacco

SummaryStilbene synthase genes (STS) have previously been used to enhance disease resistance in plants. In order to study the effects of modified STS expression patterns in plants, heterologous promoters were fused to an STS gene and the chimeric genes were transferred to tobacco. Very high constitutive expression of STS mediated by a duplicated upstream region of the 35S RNA promoter from CaMV affected secondary biosynthetic pathways. STS over‐expression caused altered flower pigmentation and male sterility, probably due to competition between the introduced STS and the endogenous chalcone synthase for the substrates 4‐coumaroyl CoA and malonyl CoA. Furthermore, tobacco plants with tapetum‐specific STS expression were male‐sterile. Thus, STS genes are promising tools in strategies for engineering altered flower colours and the development of a novel hybrid seed system.

Cytoplasmic male sterility in Nicotiana tabacum L. obtained through interspecific hybridization

Three new sources of cytoplasmic male sterility in tobacco with interspecific origin are reported. The wild species Nicotiana excelcior Black (n = 19), N. amplexicaulis Burbidge (n = 18) and N. rustica L. (n = 24) are used as donor of cytoplasm. Nuclear donor is N. tabacum L. (n = 24). The genetic effects of cytoplasmic-nuclear interactions of the studied species are discussed. Our experiments prove that N. excelcior, N. amplexicaulis and N. rustica are carriers of cytoplasmic genes for male sterility that express in combination with nuclear genes of N. tabacum.

Novel ‘homeotic’ CMS patterns generated inNicotianavia cybridization withHyoscyamusandScopolia

Cytoplasmic hybrids (cybrids) that contain nuclear genetic material from Nicotiana tabacum and cytoplasms from Hyoscyamus niger or Scopolia carniolica were constructed by protoplast fusions. Both types of hybrids exhibited cytoplasmic male sterility (CMS). Furthermore, unusual floral morphogenesis marked by 'green flowers' deprived of corolla and stamens occurred in the majority of the lines. Backcrosses of these plants with wild-type tobacco demonstrated a maternal inheritance of the 'green flower' trait. After repeated transfer of cytoplasm ('donor-recipient fusion') from cytoplasmic hybrid N. tabacum (+H. niger) to albino plastome mutant N. tabacum DSR A15, male sterile tobacco plants with two types of flowers were recovered ('green flowers' and corolla-containing flowers with transformed stamens). RFLP analysis confirmed that N. tabacum (+ H. niger) and N. tabacum (+S. carniolica) as well as their sexual progenies contained plastids from H. niger and S. carniolica, respectively. Mitochondrial DNA within the hybrids N. tabacum (+H. niger) originated from H. niger, but was obviously altered. Repeated parasexual transmission, cybrids in the combination of N. tabacum+N. tabacum (+H. niger), reflected similar characteristics. Cybrids, N. tabacum (+S. carniolica) and their sexual progeny, which resulted after pollination with wild-type tobacco, contained a modified mtDNA generally originating from tobacco. Further histological analysis established the dramatic difference in the composition of 'green flowers' and flowers of wild-type tobacco. Therefore, the construction of tobacco cybrids with foreign cytoplasms provides a functional method for the de novo generation of alternative CMS types.

RNA editing in wheat mitochondria

C to U transitions in plant mitochondrial mRNA (RNA editing) lead to amino acid changes as well as to the creation of new initiation or termination codons. We established an in vitro system to assay and to dissect the process of wheat mitochondrial mRNA editing. A deamination mechanism explains most easily the observed C to U transitions. Several fractions of organellar protein participate in the editing machinery. Some of these proteins presumably carry the catalytic activity while others are typical RNA binding proteins and may confer specificity to the ‘editosome’ complex. To investigate the functional properties of protein products synthesized from unedited mRNAs, we constructed transgenic tobacco plants carrying an unedited gene coding for subunit 9 (ATP9) of the ATP synthase complex. The nuclear encoded ‘unedited’ protein product is targeted to the mitochondria with a heterologous presequence. A significant number of male sterile tobacco plants were obtained suggesting that at least the functional ATP9 protein requires RNA editing. This result suggests a novel approach to obtain artificial male sterile plants by using a physiological effect resulting in CMS which mimics the situation found in many natural populations.