Nitrogen fixation in a landrace of maize is supported by a mucilage-associated diazotrophic microbiota.

Plants are associated with a complex microbiota that contributes to nutrient acquisition, plant growth, and plant defense. Nitrogen-fixing microbial associations are efficient and well characterized in legumes but are limited in cereals, including maize. We studied an indigenous landrace of maize grown in nitrogen-depleted soils in the Sierra Mixe region of Oaxaca, Mexico. This landrace is characterized by the extensive development of aerial roots that secrete a carbohydrate-rich mucilage. Analysis of the mucilage microbiota indicated that it was enriched in taxa for which many known species are diazotrophic, was enriched for homologs of genes encoding nitrogenase subunits, and harbored active nitrogenase activity as assessed by acetylene reduction and 15N2 incorporation assays. Field experiments in Sierra Mixe using 15N natural abundance or 15N-enrichment assessments over 5 years indicated that atmospheric nitrogen fixation contributed 29%-82% of the nitrogen nutrition of Sierra Mixe maize.

Strategy for Structural Elucidation of Polysaccharides: Elucidation of a Maize Mucilage that Harbors Diazotrophic Bacteria.

The recruitment of a bacterial consortium by the host is a strategy not limited to animals but is also used in plants. A maize aerial root mucilage has been found that harbors nitrogen fixing bacteria that are attracted to the carbohydrate rich environment. This synbiotic relationship is facilitated by a polysaccharide, whose complicated structure has been previously unknown. In this report, we present the characterization of the maize polysaccharide by employing new analytical strategies combining chemical depolymerization, oligosaccharide sequencing, and monosaccharide and glycosidic linkage quantitation. The mucilage contains a single heterogeneous polysaccharide composed of a highly fucosylated and xylosylated galactose backbone with arabinan and mannoglucuronan branches. This unique polysaccharide structure may select for the diazotrophic community by containing monosaccharides and linkages that correspond to the glycosyl hydrolases associated with the microbial community. The elucidation of this complicated structure illustrates the power of the analytical methods, which may serve as a general platform for polysaccharide analysis in the future.

An intellectual property sharing initiative in agricultural biotechnology: development of broadly accessible technologies for plant transformation.

The Public Intellectual Property Resource for Agriculture (PIPRA) was founded in 2004 by the Rockefeller Foundation in response to concerns that public investments in agricultural biotechnology benefiting developing countries were facing delays, high transaction costs and lack of access to important technologies due to intellectual property right (IPR) issues. From its inception, PIPRA has worked broadly to support a wide range of research in the public sector, in specialty and minor acreage crops as well as crops important to food security in developing countries. In this paper, we review PIPRA's work, discussing the failures, successes, and lessons learned during its years of operation. To address public sector's limited freedom-to-operate, or legal access to third-party rights, in the area of plant transformation, we describe PIPRA's patent 'pool' approach to develop open-access technologies for plant transformation which consolidate patent and tangible property rights in marker-free vector systems. The plant transformation system has been licensed and deployed for both commercial and humanitarian applications in the United States (US) and Africa, respectively.

Strangers in the matrix: plant cell walls and pathogen susceptibility.

Early in infection, pathogens encounter the outer wall of plant cells. Because pathogen hydrolases targeting the plant cell wall are well-known components of virulence, it has been assumed that wall disassembly by the plant itself also contributes to susceptibility, and now this has been established experimentally. Understanding how plant morphological and developmental remodeling and pathogen cell wall targeted virulence influence infections provides new perspectives about plant-pathogen interactions. The plant cell wall can be an effective physical barrier to pathogens, but also it is a matrix where many proteins involved in pathogen perception are delivered. By breaching the wall, a pathogen potentially reveals itself to the plant and activates responses, setting off events that might halt or limit its advance.

A Model for Nitrogen Fixation in Cereal Crops.

Nitrogen-fixing microbial associations with cereals have been of intense interest for more than a century (Roesch et al., Plant Soil 2008;302:91-104; Triplett, Plant Soil 1996;186:29-38; Mus et al., Appl. Environ. Microbiol. 2016;82:3698-3710; Beatty and Good, Science 2011;333:416-417). A recent report demonstrated that an indigenous Sierra Mixe maize landrace, characterized by an extensive development of aerial roots that secrete large amounts of mucilage, can acquire 28-82% of its nitrogen from atmospheric dinitrogen (Van Deynze et al., PLoS Biol. 2018;16:e2006352). Although the Sierra Mixe maize landrace is unique in the large quantity of mucilage produced, other cereal crops secrete mucilage from underground and aerial roots and we hypothesize that this may represent a general mechanism for cereals to support associations with microbial diazotrophs. We propose a model for the association of nitrogen-fixing microbes with maize mucilage and identify the four main functionalities for such a productive diazotrophic association.

Identification of Nitrogen Fixation Genes in Lactococcus Isolated from Maize Using Population Genomics and Machine Learning.

Sierra Mixe maize is a landrace variety from Oaxaca, Mexico, that utilizes nitrogen derived from the atmosphere via an undefined nitrogen fixation mechanism. The diazotrophic microbiota associated with the plant's mucilaginous aerial root exudate composed of complex carbohydrates was previously identified and characterized by our group where we found 23 lactococci capable of biological nitrogen fixation (BNF) without containing any of the proposed essential genes for this trait (nifHDKENB). To determine the genes in Lactococcus associated with this phenotype, we selected 70 lactococci from the dairy industry that are not known to be diazotrophic to conduct a comparative population genomic analysis. This showed that the diazotrophic lactococcal genomes were distinctly different from the dairy isolates. Examining the pangenome followed by genome-wide association study and machine learning identified genes with the functions needed for BNF in the maize isolates that were absent from the dairy isolates. Many of the putative genes received an 'unknown' annotation, which led to the domain analysis of the 135 homologs. This revealed genes with molecular functions needed for BNF, including mucilage carbohydrate catabolism, glycan-mediated host adhesion, iron/siderophore utilization, and oxidation/reduction control. This is the first report of this pathway in this organism to underpin BNF. Consequently, we proposed a model needed for BNF in lactococci that plausibly accounts for BNF in the absence of the nif operon in this organism.

Diazotrophic bacteria from maize exhibit multifaceted plant growth promotion traits in multiple hosts.

Sierra Mixe maize is a geographically remote landrace variety grown on nitrogen-deficient fields in Oaxaca, Mexico that meets its nutritional requirements without synthetic fertilizer by associating with free-living diazotrophs comprising the microbiota of its aerial root mucilage. We selected nearly 500 diazotrophic (N2-fixing) bacteria isolated from Sierra Mixe maize mucilage and sequenced their genomes. Comparative genomic analysis demonstrated that isolates represented diverse genera and composed three major diazotrophic groups based on nitrogen fixation gene content. In addition to nitrogen fixation, we examined deamination of 1-amino-1-cyclopropanecarboxylic acid, biosynthesis of indole-3-acetic acid, and phosphate solubilization as alternative mechanisms of direct plant growth promotion (PGP). Genome mining showed that isolates of all diazotrophic groups possessed marker genes for multiple mechanisms of direct plant growth promotion (PGP). Implementing in vitro assays corroborated isolate genotypes by measuring each isolate's potential to confer the targeted PGP traits and revealed phenotypic variation among isolates based on diazotrophic group assignment. Investigating the ability of mucilage diazotrophs to confer PGP by direct inoculation of clonally propagated potato plants in planta led to the identification of 16 bio-stimulant candidates. Conducting nitrogen-stress greenhouse experiments demonstrated that potato inoculation with a synthetic community of bio-stimulant candidates, as well as with its individual components, resulted in PGP phenotypes. We further demonstrated that one diazotrophic isolate conferred PGP to a conventional maize variety under nitrogen-stress in the greenhouse. These results indicate that, while many diazotrophic isolates from Sierra Mixe maize possessed genotypes and in vitro phenotypes for targeted PGP traits, a subset of these organisms promoted the growth of potato and conventional maize, potentially through the use of multiple promotion mechanisms.

Genomic characterization of a diazotrophic microbiota associated with maize aerial root mucilage.

A geographically isolated maize landrace cultivated on nitrogen-depleted fields without synthetic fertilizer in the Sierra Mixe region of Oaxaca, Mexico utilizes nitrogen derived from the atmosphere and develops an extensive network of mucilage-secreting aerial roots that harbors a diazotrophic (N2-fixing) microbiota. Targeting these diazotrophs, we selected nearly 600 microbes of a collection obtained from mucilage and confirmed their ability to incorporate heavy nitrogen (15N2) metabolites in vitro. Sequencing their genomes and conducting comparative bioinformatic analyses showed that these genomes had substantial phylogenetic diversity. We examined each diazotroph genome for the presence of nif genes essential to nitrogen fixation (nifHDKENB) and carbohydrate utilization genes relevant to the mucilage polysaccharide digestion. These analyses identified diazotrophs that possessed the canonical nif gene operons, as well as many other operon configurations with concomitant fixation and release of >700 different 15N labeled metabolites. We further demonstrated that many diazotrophs possessed alternative nif gene operons and confirmed their genomic potential to derive chemical energy from mucilage polysaccharide to fuel nitrogen fixation. These results confirm that some diazotrophic bacteria associated with Sierra Mixe maize were capable of incorporating atmospheric nitrogen into their small molecule extracellular metabolites through multiple nif gene configurations while others were able to fix nitrogen without the canonical (nifHDKENB) genes.

Characterization of novel glycosyl hydrolases discovered by cell wall glycan directed monoclonal antibody screening and metagenome analysis of maize aerial root mucilage.

An indigenous maize landrace from the Sierra Mixe region of Oaxaca, Mexico exhibits extensive formation of aerial roots which exude large volumes of a polysaccharide-rich gel matrix or "mucilage" that harbors diazotrophic microbiota. We hypothesize that the mucilage associated microbial community carries out multiple functions, including disassembly of the mucilage polysaccharide. In situ, hydrolytic assay of the mucilage revealed endogenous arabinofuranosidase, galactosidase, fucosidase, mannosidase and xylanase activities. Screening the mucilage against plant cell wall glycan-specific monoclonal antibodies recognized the presence of carbohydrate epitopes of hemicellulosic polysaccharides like xyloglucan (both non-fucosylated and fucosylated), xylan (both substituted and unsubstituted xylan domains) and pectic-arabinogalactans, all of which are potential carbon sources for mucilage microbial residents. Mucilage metagenome annotation using MG-RAST identified the members forming the microbial community, and gene fragments with predicted functions associated with carbohydrate disassembly. Data from the in situ hydrolytic activity and monoclonal antibody screening assays were used to guide the selection of five full length genes with predicted glycosyl hydrolase function from the GenBank database that were similar to gene fragments of high relative abundance in the mucilage metagenomes. These five genes were then synthesized for recombinant production in Escherichia coli. Here we report the characterization of an α-N-arabinofuranosidase (GH51) and an oligosaccharide reducing-end xylanase (GH8) from Flavobacterium johnsoniae; an α-L-fucosidase (GH29) and a xylan ß-1,4 xylosidase (GH39) from Spirosoma linguale, and a ß-mannosidase (GH2) from Agrobacterium fabrum. Biochemical characterization of these enzymes revealed a ß-Mannosidase that also exhibits a secondary activity towards the cleavage of galactosyl residues. We also describe two xylanases (GH8 and GH39) from underexplored glycosyl hydrolase families, one thermostable α-L-Fucosidase (GH29) and a thermostable α-N-Arabinofuranosidase (GH51).

Out of the Amazon: Theobroma cacao enters the genomic era.

Theobroma cacao has long been a crop of near-mystical human importance among indigenous Mesoamerican cultures and its importance in modern culture is growing with new realizations of the potential health benefits of cocoa polyphenolic compounds. However, cultivated T. cacao is vulnerable to emerging disease pressures because it has a narrow genetic base, and systematic genetic improvement of the crop is imperative. A wide range of genomic tools and resources has been developed and are providing the basis for genome-based breeding and gene discovery.

Simultaneous transgenic suppression of LePG and LeExp1 influences fruit texture and juice viscosity in a fresh market tomato variety.

Tomatoes are grown for fresh consumption or for processing of the fruit. Some ripening-associated processes of the fruit can either contribute to or degrade attributes associated with both fresh and processing quality. For example, cell wall disassembly is associated with loss of fresh fruit firmness as well as with loss of processed tomato product viscosity. Several enzymes contribute to cell wall polysaccharide disassembly. Polygalacturonase (PG, poly[1,4-alpha-d-galactouronide] glucanohydrolase, EC 3.2.1.15) is among the most abundant polysaccharide hydrolases in ripening tomato fruit and is the major contributor to pectin depolymerization. Expansin (LeExp1) is also abundant in ripening fruit and is proposed to contribute to cell wall disassembly by nonhydrolytic activity, possibly by increasing substrate accessibility to other enzymes. Suppression of either LePG or LeExp1 expression alone results in altered softening and/or shelf life characteristics. To test whether simultaneous suppression of both LePG and LeExp1 expression influences fruit texture in additive or synergistic ways, transgenic Lycopersicon esculentum var. Ailsa Craig lines with reduced expression of either LePG or LeExp1 were crossed. Fruits from the third generation of progeny, homozygous for both transgenic constructs, were analyzed for firmness and other quality traits during ripening on or off the vine. In field-grown transgenic tomato fruit, suppression of LeExp1 or LePG alone did not significantly increase fruit firmness. However, fruits suppressed for both LePG and LeExp1 expression were significantly firmer throughout ripening and were less susceptible to deterioration during long-term storage. Juice prepared from the transgenic tomato fruit with reduced LePG and LeExp1 expression was more viscous than juice prepared from control fruit.

Transgenic overexpression of expansin influences particle size distribution and improves viscosity of tomato juice and paste.

Suppression of the expression of a ripening-related expansin gene, LeExp1, in tomato enhanced fruit firmness and overexpression of LeExp1 resulted in increased fruit softening. Because of the incompletely understood relationship between fresh fruit texture and the consistency of processed products, we examined the effects of LeExp1 overexpression on the processing characteristics of tomato fruit. As determined by Bostwick consistency and by controlled strain rheometry, juices and pastes prepared from transgenic tomatoes with suppressed LeExp1 expression had a higher viscosity than preparations from control fruits. However, the viscosity of juice and paste prepared from fruit overexpressing LeExp1 was significantly greater than products from controls or lines with reduced LeExp1. Bostwick consistency increased by 9% (juice) and 6% (paste) in lines with suppressed LeExp1 expression but increased by 27.5% (juice) and 19.5% (paste) in lines overexpressing LeExp1, relative to controls. Determined by laser diffraction, the particles in juice and paste prepared from transgenic fruits with reduced LeExp1 expression were smaller, and preparations from fruits overexpressing LeExp1 had a size distribution indicating more large particles. Analysis of cell wall polysaccharides size indicated that LeExp1 overexpression enhanced depolymerization of water soluble pectins as well as tightly bound matrix glycans. LeExp1 overexpression may allow increased cell wall hydration, resulting in expanded particle size and increased viscosity of products. Because either LeExp1 suppression or overexpression leads to improved consistency, the interactions that contribute to optimal product rheological properties are complex.

Taste: unraveling tomato flavor.

New research integrating genetics, chemistry and psychophysics has led to a model for tomato flavor intensity comprising sugars and acids plus six volatile molecules, providing a blueprint for improving the flavor of what has become an iconic symbol of the declining quality of fresh fruits and vegetables.

Transgene mobilization and regulatory uncertainty for non-GE fruit products of transgenic rootstocks.

Genetically engineered (GE) rootstocks may offer some advantages for biotechnology applications especially in woody perennial crops such as grape or walnut. Transgrafting combines horticultural grafting practices with modern GE methods for crop improvement. Here, a non-GE conventional scion (upper stem portion) is grafted onto a transgenic GE rootstock. Thus, the scion does not contain the genetic modification present in the rootstock genome. We examined transgene presence in walnut and tomato GE rootstocks and non-GE fruit-bearing scions. Mobilization of transgene DNA, protein, and mRNA across the graft was not detected. Though transgenic siRNA mobilization was not observed in grafted tomatoes or walnut scions, transgenic siRNA signal was detected in walnut kernels. Prospective benefits from transgrafted plants include minimized risk of GE pollen flow (Lev-Yadun and Sederoff, 2001), possible use of more than one scion per approved GE rootstock which could help curb the estimated US$136 million (CropLife International, 2011) cost to bring a GE crop to international markets, as well as potential for improved consumer and market acceptance since the consumable product is not itself GE. Thus, transgrafting provides an alternative option for agricultural industries wishing to expand their biotechnology portfolio.

Mobility of Transgenic Nucleic Acids and Proteins within Grafted Rootstocks for Agricultural Improvement.

Grafting has been used in agriculture for over 2000 years. Disease resistance and environmental tolerance are highly beneficial traits that can be provided through use of grafting, although the mechanisms, in particular for resistance, have frequently been unknown. As information emerges that describes plant disease resistance mechanisms, the proteins, and nucleic acids that play a critical role in disease management can be expressed in genetically engineered (GE) plant lines. Utilizing transgrafting, the combination of a GE rootstock with a wild-type (WT) scion, or the reverse, has the potential to provide pest and pathogen resistance, impart biotic and abiotic stress tolerance, or increase plant vigor and productivity. Of central importance to these potential benefits is the question of to what extent nucleic acids and proteins are transmitted across a graft junction and whether the movement of these molecules will affect the efficacy of the transgrafting approach. Using a variety of specific examples, this review will report on the movement of organellar DNA, RNAs, and proteins across graft unions. Attention will be specifically drawn to the use of small RNAs and gene silencing within transgrafted plants, with a particular focus on pathogen resistance. The use of GE rootstocks or scions has the potential to extend the horticultural utility of grafting by combining this ancient technique with the molecular strategies of the modern era.

Uniform ripening encodes a Golden 2-like transcription factor regulating tomato fruit chloroplast development.

Modern tomato (Solanum lycopersicum) varieties are bred for uniform ripening (u) light green fruit phenotypes to facilitate harvests of evenly ripened fruit. U encodes a Golden 2-like (GLK) transcription factor, SlGLK2, which determines chlorophyll accumulation and distribution in developing fruit. In tomato, two GLKs--SlGLK1 and SlGLK2--are expressed in leaves, but only SlGLK2 is expressed in fruit. Expressing GLKs increased the chlorophyll content of fruit, whereas SlGLK2 suppression recapitulated the u mutant phenotype. GLK overexpression enhanced fruit photosynthesis gene expression and chloroplast development, leading to elevated carbohydrates and carotenoids in ripe fruit. SlGLK2 influences photosynthesis in developing fruit, contributing to mature fruit characteristics and suggesting that selection of u inadvertently compromised ripe fruit quality in exchange for desirable production traits.

Constitutively expressed DHAR and MDHAR influence fruit, but not foliar ascorbate levels in tomato.

Vitamin C (L-ascorbate, AsA) is an essential nutrient required in key metabolic functions in humans and must be obtained from the diet, mainly from fruits and vegetables. Given its importance in human health and plant physiology we sought to examine the role of the ascorbate recycling enzymes monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) in tomato (Solanum lycopersicum), an economically important fruit crop. Cytosolic-targeted tomato genes Mdhar and Dhar were cloned and over-expressed under a constitutive promoter in tomato var. Micro-Tom. Lines with increased protein levels and enzymatic activity were identified and examined. Mature green and red ripe fruit from DHAR over-expressing lines had a 1.6 fold increase in AsA content in plants grown under relatively low light conditions (150 µmol m(-2) s(-1)). Conversely, MDHAR over-expressers had significantly reduced AsA levels in mature green fruits by 0.7 fold. Neither over-expressing line had altered levels of AsA in foliar tissues. These results underscore a complex regulation of the AsA pool size in tomato.