A122S, A205V, D376E, W574L and S653N substitutions in acetolactate synthase (ALS) from Amaranthus palmeri show different functional impacts on herbicide resistance.

BACKGROUND: Amaranthus palmeri S. Watson, a problematic weed infesting summer crops in Argentina, has developed multiple herbicide resistance. Resistance to acetolactate synthase (ALS)-inhibiting herbicides is particularly common, with high-level resistance mostly caused by different mutations in the ALS enzyme. Six versions of the enzyme were identified from a resistant A. palmeri population, carrying substitutions D376E, A205V, A122S, A282D, W574L and S653N. This work aims to provide a comparative analysis of these mutants and the wild-type (WT) enzyme to fully understand the herbicide resistance. Thus, all the versions of the ALS gene from A. palmeri were heterologously expressed and purified to evaluate their kinetics and inhibitory response against imazethapyr, diclosulam, chlorimuron-ethyl, flucarbazone-sodium and bispyribac-sodium. RESULTS: A decrease in catalytic efficiency was detected in the A205V, A122S-A282D, W574L and S653N ApALS enzymes, whereas only A205V and W574L substitutions also produced a decrease in the substrate affinity. In vitro ALS inhibition assays confirmed cross-resistance to almost all the herbicides tested, with the exception of A282D ApALS, which was as susceptible as WT ApALS. Moreover, the results confirmed that the novel substitution A122S provides cross-resistance to at least one herbicide within each of the five families of ALS inhibitors, and this property could be explained by a lower number of hydrophobic interactions between the herbicides and the mutant enzyme. CONCLUSION: This is the first report to compare various mutations in vitro from A. palmeri ALS. Our data contribute to understanding the impacts of herbicide resistance in this species. © 2021 Society of Chemical Industry.

Secretome characterization of the lignocellulose-degrading fungi Pycnoporus sanguineus and Ganoderma resinaceum growing on Panicum prionitis biomass.

C4 grasses are common species in rangelands around the world and represent an attractive option for second-generation biofuel production. Although they display high polysaccharide content and reach great levels of biomass accumulation, there is a major technical issue to be addressed before they can be used for bioethanol industrial production: lignin removal. Concerning this, Pycnoporus and Ganoderma fungal genera have been highlighted due to their ability to hydrolyze lignocellulose in biological pretreatments. Our goals here were to evaluate the pretreatment efficiency using the secretome of species from Pycnoporus and Ganoderma spp. harvested from a glucose-free inductive medium (using a C4 grass) and to identify the fungal enzymatic activities responsible for the lignin degradation and glucose release. Our results show that P. sanguineus secretome exhibits a higher activity of lignocellulolytic enzymes such as cellulases, xylanases, laccases, and manganese peroxidases compared with that from G. resinaceum. Interestingly, zymograms in the presence of 2 M glucose suggest that a ß-glucosidase isoform from P. sanguineus could be glucose tolerant. The proteomic approach carried out allowed the identification of 73 and 180 different proteins in G. resinaceum and P. sanguineus secretomes, respectively, which were functionally classified in five main categories and a miscellaneous group. These results open new avenues for future experimental work that lead to a deeper comprehension and a greater application of the mechanisms underlying lignocellulosic biomass degradation.

Genetic Transformation of Apomictic Grasses: Progress and Constraints.

The available methods for plant transformation and expansion beyond its limits remain especially critical for crop improvement. For grass species, this is even more critical, mainly due to drawbacks in in vitro regeneration. Despite the existence of many protocols in grasses to achieve genetic transformation through Agrobacterium or biolistic gene delivery, their efficiencies are genotype-dependent and still very low due to the recalcitrance of these species to in vitro regeneration. Many plant transformation facilities for cereals and other important crops may be found around the world in universities and enterprises, but this is not the case for apomictic species, many of which are C4 grasses. Moreover, apomixis (asexual reproduction by seeds) represents an additional constraint for breeding. However, the transformation of an apomictic clone is an attractive strategy, as the transgene is immediately fixed in a highly adapted genetic background, capable of large-scale clonal propagation. With the exception of some species like Brachiaria brizantha which is planted in approximately 100 M ha in Brazil, apomixis is almost non-present in economically important crops. However, as it is sometimes present in their wild relatives, the main goal is to transfer this trait to crops to fix heterosis. Until now this has been a difficult task, mainly because many aspects of apomixis are unknown. Over the last few years, many candidate genes have been identified and attempts have been made to characterize them functionally in Arabidopsis and rice. However, functional analysis in true apomictic species lags far behind, mainly due to the complexity of its genomes, of the trait itself, and the lack of efficient genetic transformation protocols. In this study, we review the current status of the in vitro culture and genetic transformation methods focusing on apomictic grasses, and the prospects for the application of new tools assayed in other related species, with two aims: to pave the way for discovering the molecular pathways involved in apomixis and to develop new capacities for breeding purposes because many of these grasses are important forage or biofuel resources.

Herbicide resistant weeds: A call to integrate conventional agricultural practices, molecular biology knowledge and new technologies.

Herbicide resistant (HR) weeds are of major concern in modern agriculture. This situation is exacerbated by the massive adoption of herbicide-based technologies along with the overuse of a few active ingredients to control weeds over vast areas year after year. Also, many other anthropological, biological, and environmental factors have defined a higher rate of herbicide resistance evolution in numerous weed species around the world. This review focuses on two central points: 1) how these factors have affected the resistance evolution process; and 2) which cultural practices and new approaches would help to achieve an effective integrated weed management. We claim that global climate change is an unnoticed factor that may be acting on the selection of HR weeds, especially those evolving into non-target-site resistance mechanisms. And we present several new tools -such as Gene Drive and RNAi technologies- that may be adopted to cope with herbicide resistance spread, as well as discuss their potential application at field level. This is the first review that integrates agronomic and molecular knowledge of herbicide resistance. It covers not only the genetic basis of the most relevant resistance mechanisms but also the strengths and weaknesses of traditional and forthcoming agricultural practices.

A novel triple amino acid substitution in the EPSPS found in a high-level glyphosate-resistant Amaranthus hybridus population from Argentina.

BACKGROUND: The evolution of herbicide-resistant weeds is one of the most important concerns of global agriculture. Amaranthus hybridus L. is a competitive weed for summer crops in South America. In this article, we intend to unravel the molecular mechanisms by which an A. hybridus population from Argentina has become resistant to extraordinarily high levels of glyphosate. RESULTS: The glyphosate-resistant population (A) exhibited particularly high parameters of resistance (GR50 = 20 900 g ai ha-1 , Rf = 314), with all plants completing a normal life cycle even after 32X dose application. No shikimic acid accumulation was detected in the resistant plants at any of the glyphosate concentrations tested. Molecular and genetic analyses revealed a novel triple substitution (TAP-IVS: T102I, A103V, and P106S) in the 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) enzyme of population A and an incipient increase on the epsps relative copy number but without effects on the epsps transcription levels. The novel mechanism was prevalent, with 48% and 52% of the individuals being homozygous and heterozygous for the triple substitution, respectively. In silico conformational studies revealed that TAP-IVS triple substitution would generate an EPSPS with a functional active site but with an increased restriction to glyphosate binding. CONCLUSION: The prevalence of the TAP-IVS triple substitution as the sole mechanism detected in the highly glyphosate resistant population suggests the evolution of a new glyphosate resistance mechanism arising in A. hybridus. This is the first report of a naturally occurring EPSPS triple substitution and the first glyphosate target-site resistance mechanism described in A. hybridus. © 2018 Society of Chemical Industry.

A Plant-Specific TGS1 Homolog Influences Gametophyte Development in Sexual Tetraploid Paspalum notatum Ovules.

Aposporous apomictic plants form clonal maternal seeds by inducing the emergence of non-reduced (2n) embryo sacs in the ovule nucellus and the development of embryos by parthenogenesis. In previous work, we reported a plant-specific TRIMETHYLGUANOSINE SYNTHASE 1 (TGS1) gene (PN_TGS1-like) showing expression levels positively correlated with sexuality rates in facultative apomictic Paspalum notatum. PN_ TGS1-like displayed contrasting in situ hybridization patterns in apomictic and sexual plant ovules from premeiosis to anthesis. Here we transformed sexual P. notatum with a TGS1-like antisense construction under a constitutive promoter, in order to produce lines with reduced transcript representation. Antisense plants developed prominent trichomes on the adaxial leaf surface, a trait absent from control genotypes. Reproductive development analysis revealed occasional formation of twin ovules. While control individuals typically displayed a single meiotic embryo sac per ovule, antisense lines showed 12.93-15.79% of ovules bearing extra nuclei, which can be assigned to aposporous-like embryo sacs (AES-like) or, alternatively, to gametophytes with a misguided cell fate development. Moreover, around 8.42-9.52% of ovules showed what looked like a combination of meiotic and aposporous-like sacs. Besides, 32.5% of ovules at early developmental stages displayed nucellar cells with prominent nuclei resembling apospory initials (AIs), which surrounded the megaspore mother cell (MMC) or the MMC-derived meiotic products. Two or more concurrent meiosis events were never detected, which suggest a non-reduced nature for the extra nuclei observed in the mature ovules, unless they were generated by proliferation and misguided differentiation of the legitimate meiotic products. The antisense lines produced a similar amount of viable even-sized pollen with respect to control genotypes, and formed an equivalent full seed set (∼9% of total seeds) after self-pollination. Flow cytometry analyses of caryopses derived from antisense lines revealed that all full seeds had originated from meiotic embryo sacs (i.e. by sexuality). A reduction of 25.55% in the germination percentage was detected when comparing antisense lines with controls. Our results indicate that PN_ TGS1-like influences ovule, gametophyte and possibly embryo development.

The MAP3K-Coding QUI-GON JINN (QGJ) Gene Is Essential to the Formation of Unreduced Embryo Sacs in Paspalum.

Apomixis is a clonal mode of reproduction via seeds, which results from the failure of meiosis and fertilization in the sexual female reproductive pathway. In previous transcriptomic surveys, we identified a mitogen-activated protein kinase kinase kinase (N46) displaying differential representation in florets of sexual and apomictic Paspalum notatum genotypes. Here, we retrieved and characterized the N46 full cDNA sequence from sexual and apomictic floral transcriptomes. Phylogenetic analyses showed that N46 was a member of the YODA family, which was re-named QUI-GON JINN (QGJ). Differential expression in florets of sexual and apomictic plants was confirmed by qPCR. In situ hybridization experiments revealed expression in the nucellus of aposporous plants' ovules, which was absent in sexual plants. RNAi inhibition of QGJ expression in two apomictic genotypes resulted in significantly reduced rates of aposporous embryo sac formation, with respect to the level detected in wild type aposporous plants and transformation controls. The QGJ locus segregated independently of apospory. However, a probe derived from a related long non-coding RNA sequence (PN_LNC_QGJ) revealed RFLP bands cosegregating with the Paspalum apospory-controlling region (ACR). PN_LNC_QGJ is expressed in florets of apomictic plants only. Our results indicate that the activity of QGJ in the nucellus of apomictic plants is necessary to form non-reduced embryo sacs and that a long non-coding sequence with regulatory potential is similar to sequences located within the ACR.

Target-site resistance to acetolactate synthase (ALS)-inhibiting herbicides in Amaranthus palmeri from Argentina.

BACKGROUND: Herbicide-resistant weeds are a serious problem worldwide. Recently, two populations of Amaranthus palmeri with suspected cross-resistance to acetolactate synthase (ALS)-inhibiting herbicides (R1 and R2) were found by farmers in two locations in Argentina (Vicuña Mackenna and Totoras, respectively). We conducted studies to confirm and elucidate the mechanism of resistance. RESULTS: We performed in vivo dose-response assays, and confirmed that both populations had strong resistance to chlorimuron-ethyl, diclosulam and imazethapyr when compared with a susceptible population (S). In vitro ALS activity inhibition tests only indicated considerable resistance to imazethapyr and chlorimuron-ethyl, indicating that other non-target mechanisms could be involved in diclosulam resistance. Subsequently, molecular analysis of als nucleotide sequences revealed three single base-pair mutations producing substitutions in amino acids previously associated with resistance to ALS inhibitors, A122, W574, and S653. CONCLUSION: This is the first report of als resistance alleles in A. palmeri in Argentina. The data support the involvement of a target-site mechanism of resistance to ALS-inhibiting herbicides. © 2017 Society of Chemical Industry.

Streptomyces thermoautotrophicus does not fix nitrogen.

Streptomyces thermoautotrophicus UBT1 has been described as a moderately thermophilic chemolithoautotroph with a novel nitrogenase enzyme that is oxygen-insensitive. We have cultured the UBT1 strain, and have isolated two new strains (H1 and P1-2) of very similar phenotypic and genetic characters. These strains show minimal growth on ammonium-free media, and fail to incorporate isotopically labeled N2 gas into biomass in multiple independent assays. The sdn genes previously published as the putative nitrogenase of S. thermoautotrophicus have little similarity to anything found in draft genome sequences, published here, for strains H1 and UBT1, but share >99% nucleotide identity with genes from Hydrogenibacillus schlegelii, a draft genome for which is also presented here. H. schlegelii similarly lacks nitrogenase genes and is a non-diazotroph. We propose reclassification of the species containing strains UBT1, H1, and P1-2 as a non-Streptomycete, non-diazotrophic, facultative chemolithoautotroph and conclude that the existence of the previously proposed oxygen-tolerant nitrogenase is extremely unlikely.

Evaluation of biological pretreatments to increase the efficiency of the saccharification process using Spartina argentinensis as a biomass resource.

Second generation bioethanol obtained from native perennial grasses offers a promising alternative for biofuel production, avoiding land use competition for crops production. Spartina argentinensis is a native perennial C4 grass with high photosynthetic rates, well adapted to halo-hydromorphic soils, though its forage quality (palatability and digestibility) for livestock is quite low due to its high lignin content. Hence, cattle raisers burn these grasslands frequently in order to stimulate the emergence of new leaves with higher digestibility for cattle feeding. Lignin is the main barrier to overcome in order to efficiently hydrolyze the cellulose for bioethanol production. In this work, we evaluate different pretreatments (phosphoric acid, ligninolytic enzymes and fungal supernatants) aimed to remove lignin and improving cellulose hydrolysis efficiency. Results show that pretreatment with Pycnoporus sanguineus supernatant improves fermentable carbohydrates availability, compared with a conventional chemical pretreatment, and that 56.84% of cellulose can be hydrolyzed using this pretreatment.

Detection and quantification of transgenes in grains by multiplex and real-time PCR.

Multiplex PCR reactions were developed for detecting simultaneously the CryIA(b) and pat genes from events 176, MON810, BT11, and T25 of transgenic maize, using only two pairs of primers, one for the CryIA(b) gene and the other for the pat gene. The Roundup Ready soybean can be precisely detected by a multiplex PCR reaction using known primers, amplifying fragments of the NOS and the epsps sequences simultaneously. Transgenic events such as Roundup Ready soybean and GA21 maize, among others, can be quantified by real-time PCR using a pair of primers and a probe specifically designed for annealing to the NOS ending region. As an alternative to amplifying an endogenous gene, the addition of a foreign gene in a percentage equal to the required level of detection, in a parallel reaction, is proposed. The use of hexane to homogenize large flour samples is suggested.

Stable wheat transformation obtained without selectable markers.

Transgenic wheat plants without the selectable marker gene were obtained either in the presence or in the absence of selective pressure during the transformation protocol. When using hygromycin as selective agent in a co-transformation experiment involving a mixture of plasmids pGL2, containing the hpt gene, and pAI1Gus, containing the uidA gene, 3 out of 19 transgenic wheat plants had the uidA gene alone as shown by Southern blots. The gene was transmitted to the progeny following Mendelian rules. Segregation and loss of the selectable marker gene was also found in three out of six events from other experiments where high-molecular-weight glutenin genes were expressed or over-expressed. On the other hand, in 7 experiments where no selective pressure was applied and that involved 1016 bombarded explants, 23 transgenic wheat plants were obtained. The uidA gene was stably integrated as suggested by its transmission to the progeny.

Bt protein rhizosecreted from transgenic maize does not accumulate in soil

The persistence of CryIAb protein rhizosecreted in soil is important in the assessment of its environmental risk. Here we report that CryIAb protein from transgenic maize does not accumulate at high levels in soils. Levels of CryIAb protein rhizosecreted by three maize transgenic events (BT11, MON810 and 176) were studied in hydroponic cultures and found only in the MON810 and BT11 events but not in event 176 or control plants. Under field conditions, the cryIAb gene and a basal level of CryIAb protein was detected in soils from plots cultivated with transgenic and non-transgenic maize, possibly from Bacillus thuringiensis present in the soils.