Bar-expressing peppermint (Mentha × Piperita L. var. Black Mitcham) plants are highly resistant to the glufosinate herbicide Liberty

Weed control is a substantial economic input for production of mint oils, the most commercially important of which are obtained from peppermint. The objective of this research is to obtain peppermint plants resistant to the broad-spectrum herbicide glufosinate, which can be used for development of economically efficacious weed control strategies and, perhaps, serve as a paradigm in perennial crops. The bar gene, which encodes phosphinothricin acetyltransferase (PAT) which inactivates glufosinate-ammonium or phosphinothricin (PPT), was constructed into Agrobacterium tumefaciens binary vectors under the nopaline synthase (NOS) or a chimeric promoter containing a trimer of the OCS-upstream-activating sequence (UAS) to a MAS promoter/activator region[(OCS) ₃ MAS]. A total of 142 independent transgenic peppermint (cv. Black Mitcham) plants were obtained (107 and 35 were obtained with pGPTV (and pCAS1) and pATC940 vectors, respectively) and evaluated for herbicide resistance in the greenhouse after foliar application of glufosinate herbicide Liberty as the commercial product. All transgenic plants exhibited substantially less herbicide symptom development than non-transgenic Black Mitcham or untransformed tissue cultured-derived plants, albeit variation for herbicide resistance occurred amongst the transformed lines. Plants from 35 of the 142 lines were selected at random and all were PCR-positive for the presence of bar. Five lines, that were least affected, exhibited no injury symptoms to Liberty concentrations that are 4 times the standard level for control of weeds in peppermint fields. The most resistant transgenic plants had the greatest steady-state PAT mRNA levels and PAT activities. No experimental difference in herbicide resistance was evident between plant populations obtained with pGPTV (pCAS1)-bar or pATC940-bar vector. However, 4 of 35 lines transformed with (ocs) ₃ MAS-bar exhibited maximal resistance while only 1 of 107 NOS-bar lines has comparable resistance. These herbicide resistant peppermint plants will facilitate development of post-emergent herbicide control strategies that use newer generation herbicides, like glufosinate, which have reduced environmental and product residual because of metabolism by microbes and the transgenic plants.     

Molecular Control of Male Reproductive Development and Pollen Fertility in Rice

Anther development and male fertility are essential biological processes for flowering plants and are important for crop seed production. Genetic manipulation of male fertility/sterility is critical for crop hybrid breeding. Rice (Oryza sativa L.) male sterility phenotypes, including genic male sterility, hybrid male sterility, and cytoplasmic male sterility, are generally caused by mutations of fertility‐related genes, by incompatible interactions between divergent allelic or non‐allelic genes, or by genetic incompatibilities between cytoplasmic and nuclear genomes. Here, we review the recent advances in the molecular basis of anther development and male fertility‐sterility conversion in specific genetic backgrounds, and the interactions with certain environmental factors. The highlighted findings in this review have significant implications in both basic studies and rice genetic improvement. [ Yao‐Guang Liu (Corresponding author)]     

Controlling Transgene Escape in GM Creeping Bentgrass

Trait improvement of turfgrass through genetic engineering is important to the turfgrass industry and the environment. However, the possible transgene escape to wild and non-transformed species raises ecological and commercial concerns. Male sterility provides an effective way for interrupting gene flow. We have designed and synthesized two chimeric gene constructs consisting of a rice tapetum-specific promoter (TAP) fused to either a ribonuclease gene barnase, or the antisense of a rice tapetum-specific gene rts. Both constructs were linked to the bar gene for selection by resistance to the herbicide glufosinate. Agrobacterium-mediated transformation of creeping bentgrass (cv Penn A-4) with both constructs resulted in herbicide-resistant transgenic plants that were also 100% pollen sterile. Mendelian segregation of herbicide resistance and male sterility was observed in T1 progeny derived from crosses with wild-type plants. Controlled self- and cross-pollination studies showed no gene transfer to non-transgenic plants from male-sterile transgenic plants. Thus, male sterility can serve as an important tool to control transgene escape in bentgrass, facilitating the application of genetic engineering in producing environmentally responsible turfgrass with enhanced traits. It also provides a tool to control gene flow in other perennial species using transgenic technology.     

Down‐regulation of OsSPX1 caused semi‐male sterility,resulting in reduction of grain yield in rice

OsSPX1, a rice SPX domain gene, involved in the phosphate (Pi)‐sensing mechanism plays an essential role in the Pi‐signalling network through interaction with OsPHR2. In this study, we focused on the potential function of OsSPX1 during rice reproductive phase. Based on investigation of OsSPX1 antisense and sense transgenic rice lines in the paddy fields, we discovered that the down‐regulation of OsSPX1 caused reduction of seed‐setting rate and filled grain number. Through examination of anthers and pollens of the transgenic and wild‐type plants by microscopy, we found that the antisense of OsSPX1 gene led to semi‐male sterility, with lacking of mature pollen grains and phenotypes with a disordered surface of anthers and pollens. We further conducted rice whole‐genome GeneChip analysis to elucidate the possible molecular mechanism underlying why the down‐regulation of OsSPX1 caused deficiencies in anthers and pollens and lower seed‐setting rate in rice. The down‐regulation of OsSPX1 significantly affected expression of genes involved in carbohydrate metabolism and sugar transport, anther development, cell cycle, etc. These genes may be related to pollen fertility and male gametophyte development. Our study demonstrated that down‐regulation of OsSPX1 disrupted rice normal anther and pollen development by affecting carbohydrate metabolism and sugar transport, leading to semi‐male sterility, and ultimately resulted in low seed‐setting rate and grain yield.     

Cytotoxic Oleanane‐Type Saponins from the Leaves of Albizia anthelmintica Brongn.

Two new oleanane‐type saponins: β‐d ‐xylopyranosyl‐(1 → 4)‐6‐deoxy‐α‐l ‐mannopyranosyl‐(1 → 2)‐1‐O‐{(3β)‐28‐oxo‐3‐[(2‐O‐β‐d ‐xylopyranosyl‐β‐d ‐glucopyranosyl)oxy]olean‐12‐en‐28‐yl}‐β‐d ‐glucopyranose ( 1 ) and 1‐O‐[(3β)‐28‐oxo‐3‐{[β‐d ‐xylopyranosyl‐(1 → 2)‐α‐l ‐arabinopyranosyl‐(1 → 6)‐2‐acetamido‐2‐deoxy‐β‐d ‐glucopyranosyl]oxy}olean‐12‐en‐28‐yl]β‐d ‐glucopyranose ( 2 ), along with two known saponins: (3β)‐3‐[(β‐d ‐Glucopyranosyl‐(1 → 2)‐β‐d ‐glucopyranosyl)oxy]olean‐12‐en‐28‐oic acid ( 3 ) and (3β)‐3‐{[α‐l ‐arabinopyranosyl‐(1 → 6)‐[β‐d ‐glucopyranosyl‐(1 → 2)]‐β‐d ‐glucopyranosyl]oxy}olean‐12‐en‐28‐oic acid ( 4 ) were isolated from the acetone‐insoluble fraction obtained from the 80% aqueous MeOH extract of Albizia anthelmintica Brongn . leaves. Their structures were identified using different NMR experiments including: ¹H‐ and ¹³C‐NMR, HSQC, HMBC and ¹H,¹H‐COSY, together with HR‐ESI‐MS/MS, as well as by acid hydrolysis. The four isolated saponins and the fractions of the extract exhibited cytotoxic activity against HepG‐2 and HCT‐116 cell lines. Compound 2 showed the most potent cytotoxic activity among the other tested compounds against the HepG2 cell line with an IC₅₀ value of 3.60μm . Whereas, compound 1 showed the most potent cytotoxic effect with an IC₅₀ value of 4.75μm on HCT‐116 cells.     

Phenotype Analysis and Fine Mapping of the Male Sterile Mutant <i>ms10</i> in Rice (<i>Oryza sativa</i> L.)

There is a positive correlation between fertility and yield, and the decrease of fertility is bound to a greatly reduced crop yield. Male sterile mutants can be used in hybrid rice. Therefore, rice male sterility has an important value in research and application, and the study of related mutants is also very vital. The mutant ms10 (male sterile 10) reported in this study was induced by ethyl methane sulfonate (EMS) in the indica maintainer line Xinong 1B. There was no significant difference between the ms10 and wild type in the vegetative growth stage. However, in the reproductive growth stage, ms10 showed that the plant became shorter, the anther became smaller and the color became lighter, and finally showed the phenotype of male sterility in comparison to the wild type. I₂-KI staining showed that the pollen was malformed and only a little was active. Scanning electron microscopy observation showed that the exine waxy layer of the ms10 anther decreased, suggesting that the protective effect on pollen was decreased. This may be one of the reasons leading to the phenotype of male sterility. Finally, the pollen showed shrinkage and collapsed, and the structure of germinating pore cover disappeared. This may be the result of sterility. Genetic analysis showed that the male sterility phenotype of the mutant was controlled by a single recessive nuclear gene. MS10 was mapped between the molecular markers IND37 and IND51 on chromosome 4, with a physical distance of 178.6 kb. These results lay the foundation for further studies on MS10.     

Utilization of the herbicide phosphinothricin as a nitrogen source by soil bacteria

Summary Rhodococcus sp., Pseudomonas paucimobilis and five other bacterial soil isolates were found to be capable of utilizing the l-enantiomer of the herbicide phosphinothricin (2-amino-4-(methylphosphinyl)-butanoic acid) as a nitrogen source. This deamination yielded the structural analogue, 2-oxo-4-(methylphosphinyl)-butanoic acid as the main catabolite of phosphinothricin. On prolonged incubation, slow decarboxylation of the main catabolite occurred with the formation of the stable degradation product 3-methylphosphinico-propanoic acid.     

Conformational transformation of ascidiacyclamide analogues induced by incorporating enantiomers of phenylalanine, 1‐naphthylalanine or 2‐naphthylalanine

We designed five ascidiacyclamide analogues [cyclo(‐Xxx¹‐oxazoline²‐d ‐Val³‐thiazole⁴‐l ‐Ile⁵‐oxazoline⁶‐d ‐Val⁷‐thiazole⁸‐)] incorporating l ‐1‐naphthylalanine (l ‐1Nal), l ‐2‐naphthylalanine (l ‐2Nal), d ‐phenylalanine (d ‐Phe), d ‐1‐naphthylalanine (d ‐1Nal) or d ‐2‐naphthylalanine (d ‐2Nal) into the Xxx¹ position of the peptide. The conformations of these analogues were then examined using ¹H NMR, CD spectroscopy, and X‐ray diffraction. These analyses suggested that d ‐enantiomer‐incorporated ASCs [(d ‐Phe), (d ‐1Nal), and (d ‐2Nal)ASC] transformed from the folded to the open structure in solution more easily than l ‐enantiomer‐incorporated ASCs [(l ‐Phe), (l ‐1Nal), and (l ‐2Nal)ASC]. Structural comparison of the two analogues containing isomeric naphthyl groups showed that the 1‐naphthyl isomer induced a more stable open structure than the 2‐naphthyl isomer. In particular, [d ‐1Nal]ASC showed the most significant transformation from the folded to the open structure in solution, and exhibited the strongest cytotoxicity toward HL‐60 cells. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

A novel CCCH‐type zinc finger protein SAW1 activates OsGA20ox3 to regulate gibberellin homeostasis and anther development in rice

Male sterility is a prerequisite for hybrid seed production. The phytohormone gibberellin (GA) is involved in regulating male reproductive development, but the mechanism underlying GA homeostasis in anther development remains less understood. Here, we report the isolation and characterization of a new positive regulator of GA homeostasis, swollen anther wall 1 (SAW1), for anther development in rice (Oryza sativa L.). Rice plants carrying the recessive mutant allele saw1 produces abnormal anthers with swollen anther wall and aborted pollen. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRIPSR‐associated protein 9‐mediated knockout of SAW1 in rice generated similar male sterile plants. SAW1 encodes a novel nucleus‐localizing CCCH‐tandem zinc finger protein, and this protein could directly bind to the promoter region of the GA synthesis gene OsGA20ox3 to induce its anther‐specific expression. In the saw1 anther, the significantly decreased OsGA20ox3 expression resulted in lower bioactive GA content, which in turn caused the lower expression of the GA‐inducible anther‐regulator gene OsGAMYB. Thus, our results disclose the mechanism of the SAW1‐GA20ox3‐GAMYB pathway in controlling rice anther development, and provide a new target gene for the rapid generation of male sterile lines by genome editing for hybrid breeding.     

iTRAQ‐Based Proteomics Reveals that the Tomato ms1035 Gene Causes Male Sterility through Compromising Fat Acid Metabolism

So far, over 50 spontaneous male sterile mutants of tomato have been described and most of them are categorized as genetic male sterility. To date, the mechanism of tomato genetic male sterility remained unclear. In this study, differential proteomic analysis is performed between genetic male sterile line (2‐517), which carries the male sterility (ms10³⁵) gene, and its wild‐type (VF‐11) using isobaric tags for relative and absolute quantification‐based strategy. A total of 8272 proteins are quantified in the 2–517 and VF‐11 lines at the floral bud and florescence stages. These proteins are involved in different cellular and metabolic processes, which express obvious functional tendencies toward the hydroxylation of the ω‐carbon in fatty acids, the tricarboxylic acid cycle, the glycolytic, and pentose phosphate pathways. Based on the results, a protein network explaining the mechanisms of tomato genetic male sterility is proposed, finding the compromising fat acid metabolism may cause the male sterility. These results are confirmed by parallel reaction monitoring, quantitative Real‐time PCR (qRT‐PCR), and physiological assays. Taken together, these results provide new insights into the metabolic pathway of anther abortion induced by ms10³⁵ and offer useful clues to identify the crucial proteins involved in genetic male sterility in tomato.     

Epidermal jasmonate perception is sufficient for all aspects of jasmonate‐mediated male fertility in Arabidopsis

Jasmonate (JA) signaling is essential for several environmental responses and reproductive development in many plant species. In Arabidopsis thaliana, the most obvious phenotype of JA biosynthetic and perception mutants is profound sporophytic male sterility characterized by failure of stamen filament elongation, severe delay of anther dehiscence and pollen inviability. The site of action of JA in the context of reproductive development has been discussed, but the ideas have not been tested experimentally. To this end we used targeted expression of a COI1‐YFP transgene in the coi1‐1 mutant background. As COI1 is an essential component of the JA co‐receptor complex, the null coi1‐1 mutant is male sterile due to lack of JA perception. We show that expression of COI1‐YFP in the epidermis of the stamen filament and anther in coi1 mutant plants is sufficient to rescue filament elongation, anther dehiscence and pollen viability. In contrast, filament expression alone or expression in the tapetum do not restore dehiscence and pollen viability. These results demonstrate that epidermal JA perception is sufficient for anther function and pollen viability, and suggest the presence of a JA‐dependent non‐autonomous signal produced in the anther epidermis to synchronize both anther dehiscence and pollen maturation.     

Expression of <Emphasis Type="Italic">Aprotinin</Emphasis> in Anther Causes Male Sterility in Tobacco var <Emphasis Type="Italic">Petit havana</Emphasis>

Expression of many proteinases has been documented during anther development. Although their roles are not completely understood, their inhibition could possibly result in impairment of anther development leading to male sterility. We proposed that such an impairment of anther development can be engineered in plants resulting in male sterile plants that can be used for hybrid seed production. Here, we report that anther-specific expression of Aprotinin gene (serine proteinase inhibitor) in tobacco has resulted in male sterility. Southern analysis and zymogram analysis confirmed the integration and expression of Aprotinin gene in the anthers of the transgenic plants. Transverse sections of anthers of transgenic male sterile plants showed damaged tapetum. The pollen germination in the transgenic plants ranged between 2% and 65% that confirmed the impairment in pollen production leading to male sterility and low seed yield. Thus, inhibition of serine proteinases that are expressed during anther development has resulted in impaired pollen production and male sterility, though the exact role of these proteinases in anther development still has to be elucidated.     

Natural glufosinate resistance of soil microorganisms and GMO safety

Bacteria and fungi from pristine soil, never exposed to glufosinate herbicide, were isolated and analyzed for glufosinate tolerance. Seven of the 15 tested isolates were sensitive to 1 mM glufosinate (an active ingredient of many nonselective contact herbicides), 5 were resistant to 4 mM glufosinate and 3 even to 8 mM glufosinate in liquid medium. None of the isolated microorganisms carried the gene for glufosinate resistance bar (bialaphos resistance) in its genome and at least in some of glufosinate-resistant isolates the increased glutamine synthetase level was detected as a possible resistance mechanism. The transfer of the bar glufosinate resistance gene from transgenic maize Bt 176 into glufosinate-sensitive soil bacterium Bacillus pumilus S1 was not detected under the laboratory conditions by a classical plate count method and PCR. The ecological risk of potential bar gene transfer from genetically modified plants into soil microcosms under natural circumstances is discussed.     

Allelic relationship of four male sterility genes and nucleo-cytoplasmic interactions in the expression of male sterility in pearl millet,Pennisetum americanum (L.) leeke

Summary Male sterility genes isolated in four inbred lines of pearl millet were found allelic. The differences between male fertile and male sterile phenotypes is mainly due to a single gene. Presence of a dominant gene (Ms) resulted in male fertility and double recessiveness (ms ms) in male sterility. However, genic male sterility (GMS) in Pennisetum is not a simply inherited case of monogenic recessive condition but is influenced by cytoplasmic and several nuclear factors. In a male sterile, the stage at which the male sterility gene is expressed during the development of the male gametophyte resulting in breakdown of the cells is influenced by cytoplasmic and other nuclear factors. Two types of cytoplasm, C-1 and C-2, are recognized. Presence of any two recessive male sterility alleles in C-1 led to breakdown of male development before differentiation of an archesporium in the anther (Arc-type); in C-2 cytoplasm, degeneration started during meiosis with fusion of meiocytes and syncyte formation (Syn-type), or at post-meiotic stages terminating in abortion of microspores before first pollen mitosis (PGM type). The triggering of activity of recessive male sterility genes in C-2 cytoplasm appeared to be regulated by two nuclear factors, R ₁ and R ₂ with duplicate gene action. Recessiveness for both the R factors in C-2 cytoplasm resulted in PGM-type expression. The action of R ₁ and R ₂ is specific to C-2 cytoplasm. Mutation of cytoplasm from C-1 to C-2 and C-2 to C-1 was observed.     

Cotton GhCKI disrupts normal male reproduction by delaying tapetum programmed cell death via inactivating starch synthase

Anther infertility under high temperature (HT) conditions is a critical factor contributing to yield loss in cotton (Gossypium hirsutum). Using large‐scale expression profile sequencing, we studied the effect of HT on cotton anther development. Our analysis revealed that altered carbohydrate metabolism or disrupted tapetal programmed cell death (PCD) underlie anther sterility. Expression of the Gossypium hirsutum casein kinase I (GhCKI) gene, which encodes a homolog of casein kinase I (CKI), was induced in an HT‐sensitive cotton line after exposure to HT. As mammalian homologs of GhCKI are involved in inactivation of glycogen synthase and the regulation of apoptosis, GhCKI may be considered a target gene for improving anther fertility under HT conditions. Our studies suggest that GhCKI exhibits starch synthase kinase activity, increases glucose content in early‐stage buds and activates the accumulation of abscisic acid, thereby disturbing the balance of reactive oxygen species and eventually disrupting tapetal PCD, leading to anther abortion or indehiscence. These results indicate that GhCKI may be a key regulator of tapetal PCD and anther dehiscence, with the potential to facilitate regulation of HT tolerance in crops.     

Tapetum and middle layer control male fertility in Actinidia deliciosa

Background and Aims

Dioecism characterizes many crop species of economic value, including kiwifruit (Actinidia deliciosa). Kiwifruit male sterility occurs at the microspore stage. The cell walls of the microspores and the pollen of the male-sterile and male-fertile flowers, respectively, differ in glucose and galactose levels. In numerous plants, pollen formation involves normal functioning and degeneration timing of the tapetum, with calcium and carbohydrates provided by the tapetum essential for male fertility. The aim of this study was to determine whether the anther wall controls male fertility in kiwifruit, providing calcium and carbohydrates to the microspores.

Methods

The events occurring in the anther wall and microspores of male-fertile and male-sterile anthers were investigated by analyses of light microscopy, epifluorescence, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL assay) and transmission electron microscopy coupled with electron spectroscopy. The possibility that male sterility was related to anther tissue malfunctioning with regard to calcium/glucose/galactose provision to the microspores was also investigated by in vitro anther culture.

Key Results

Both tapetum and the middle layer showed secretory activity and both degenerated by programmed cell death (PCD), but PCD was later in male-sterile than in male-fertile anthers. Calcium accumulated in cell walls of the middle layer and tapetum and in the exine of microspores and pollen, reaching higher levels in anther wall tissues and dead microspores of male-sterile anthers. A specific supply of glucose and calcium induced normal pollen formation in in vitro-cultured anthers of the male-sterile genotype.

Conclusions

The results show that male sterility in kiwifruit is induced by anther wall tissues through prolonged secretory activity caused by a delay in PCD, in the middle layer in particular. In vitro culture results support the sporophytic control of male fertility in kiwifruit and open the way to applications to overcome dioecism and optimize kiwifruit production.