RNA Isolation Method in Marginal Crops with High Agronomic Potential.

Ribonucleic Acid (RNA) isolation is a basic technique in the field of molecular biology. The purpose of RNA isolation is to acquire pure and complete RNA that can be used to evaluate gene expression. Many methods can be used to perform RNA isolation, all of them based on the chemical properties of nucleic acids. However, some of them do not achieve high RNA yields and purity levels when used in a number of marginally studied crops of agronomic importance, such as grain and vegetable amaranth plants. In the method described here, the use of guanidinium thiocyanate and two additional precipitation steps with different reagents designed to obtain high yields and RNA purity levels from diverse plant species employed for plant functional genomics studies is described.

Physiological, Ecological, and Biochemical Implications in Tomato Plants of Two Plant Biostimulants: Arbuscular Mycorrhizal Fungi and Seaweed Extract.

The worldwide use of plant biostimulants (PBs) represents an environmentally friendly tool to increase crop yield and productivity. PBs include different substances, compounds, and growth-promoting microorganism formulations, such as those derived from arbuscular mycorrhizal fungi (AMF) or seaweed extracts (SEs), which are used to regulate or enhance physiological processes in plants. This study analyzed the physiological, ecological, and biochemical implications of the addition of two PBs, AMF or SE (both alone and in combination), on tomato plants (Solanum lycopersicum L. cv. "Rio Fuego"). The physiological responses evaluated were related to plant growth and photosynthetic performance. The ecological benefits were assessed based on the success of AMF colonization, flowering, resistance capacity, nonphotochemical quenching (NPQ), and polyphenol content. Biochemical effects were evaluated via protein, lipid, carbohydrate, nitrogen, and phosphorous content. Each PB was found to benefit tomato plants in a different but complementary manner. AMF resulted in an energetically expensive (high ETRMAX but low growth) but protective (high NPQ and polyphenol content) response. AMF + nutritive solution (NS) induced early floration but resulted in low protein, carbohydrate, and lipid content. Both AMF and AMF + NS favored foliar instead of root development. In contrast, SE and SE + NS favored protein content and root development and did not promote flowering. However, the combination of both PBs (AMF + SE) resulted in an additive effect, reflected in an increase in both foliar and root growth as well as protein and carbohydrate content. Moreover, a synergistic effect was also found, which was expressed in accelerated flowering and AMF colonization. We present evidence of benefits to plant performance (additive and synergistic) due to the interactive effects between microbial (AMF) and nonmicrobial (SEs) PBs and propose that the complementary modes of action of both PBs may be responsible for the observed positive effects due to the new and emerging properties of their components instead of exclusively being the result of known constituents. These results will be an important contribution to biostimulant research and to the development of a second generation of PBs in which combined and complementary mechanisms may be functionally designed.

Distinct gene expression and secondary metabolite profiles in suppressor of prosystemin-mediated responses2 (spr2) tomato mutants having impaired mycorrhizal colonization.

Arbuscular mycorrhizal fungi (AMF) colonization, sampled at 32-50 days post-inoculation (dpi), was significantly reduced in suppressor of prosystemin-mediated responses2 (spr2) mutant tomato plants impaired in the ω-3 FATTY ACID DESATURASE7 (FAD7) gene that limits the generation of linolenic acid and, consequently, the wound-responsive jasmonic acid (JA) burst. Contrary to wild-type (WT) plants, JA levels in root and leaves of spr2 mutants remained unchanged in response to AMF colonization, further supporting its regulatory role in the AM symbiosis. Decreased AMF colonization in spr2 plants was also linked to alterations associated with a disrupted FAD7 function, such as enhanced salicylic acid (SA) levels and SA-related defense gene expression and a reduction in fatty acid content in both mycorrhizal spr2 roots and leaves. Transcriptomic data revealed that lower mycorrhizal colonization efficiency in spr2 mutants coincided with the modified expression of key genes controlling gibberellin and ethylene signaling, brassinosteroid, ethylene, apocarotenoid and phenylpropanoid synthesis, and the wound response. Targeted metabolomic analysis, performed at 45 dpi, revealed augmented contents of L-threonic acid and DL-malic acid in colonized spr2 roots which suggested unfavorable conditions for AMF colonization. Additionally, time- and genotype-dependent changes in root steroid glycoalkaloid levels, including tomatine, suggested that these metabolites might positively regulate the AM symbiosis in tomato. Untargeted metabolomic analysis demonstrated that the tomato root metabolomes were distinctly affected by genotype, mycorrhizal colonization and colonization time. In conclusion, reduced AMF colonization efficiency in spr2 mutants is probably caused by multiple and interconnected JA-dependent and independent gene expression and metabolomic alterations.

Network Analysis of the Papaya Orchard Virome from Two Agroecological Regions of Chiapas, Mexico.

The study of complex ecological interactions, such as those among host, pathogen, and vector communities, can help to explain host ranges and the emergence of novel pathogens. We evaluated the viromes of papaya orchards, including weed and insect viromes, to identify common viruses in intensive production of papaya in the Pacific Coastal Plain and the Central Depression of Chiapas, Mexico. Samples of papaya cultivar Maradol, susceptible to papaya ringspot virus (PRSV), were categorized by symptoms by local farmers (papaya ringspot symptoms, non-PRSV symptoms, or asymptomatic). These analyses revealed the presence of 61 viruses, where only 4 species were shared among both regions, 16 showed homology to known viruses, and 36 were homologous with genera including Potyvirus, Comovirus, and Tombusvirus (RNA viruses) and Begomovirus and Mastrevirus (DNA viruses). We analyzed the network of associations between viruses and host-location combinations, revealing ecological properties of the network, such as an asymmetric nested pattern, and compared the observed network to null models of network association. Understanding the network structure informs management strategies, for example, revealing the potential role of PRSV in asymptomatic papaya and that weeds may be an important pathogen reservoir. We identify three key management implications: (i) each region may need a customized management strategy; (ii) visual assessment of papaya may be insufficient for PRSV, requiring diagnostic assays; and (iii) weed control within orchards may reduce the risk of virus spread to papaya. Network analysis advances understanding of host-pathogen interactions in the agroecological landscape.IMPORTANCE Virus-virus interactions in plants can modify host symptoms. As a result, disease management strategies may be unsuccessful if they are based solely on visual assessment and diagnostic assays for known individual viruses. Papaya ringspot virus is an important limiting factor for papaya production and likely has interactions with other viruses that are not yet known. Using high-throughput sequencing, we recovered known and novel RNA and DNA viruses from papaya orchards in Chiapas, Mexico, and categorized them by host and, in the case of papaya, symptom type: asymptomatic papaya, papaya with ringspot virus symptoms, papaya with nonringspot symptoms, weeds, and insects. Using network analysis, we demonstrated virus associations within and among host types and described the ecological community patterns. Recovery of viruses from weeds and asymptomatic papaya suggests the need for additional management attention. These analyses contribute to the understanding of the community structure of viruses in the agroecological landscape.

Fruits of wild and semi-domesticated Diospyros tree species have contrasting phenological, metabolic, and antioxidant activity profiles.

BACKGROUND: In contrast to commercial Diospyros species, Mesoamerican fruit-producing species are scarcely known, particularly wild species that might harbor desirable traits suitable for breeding. Thus, metabolomic, chemical, and antioxidant profiles of fruits harvested from cultivated Diospyros digyna and wild Diospyros rekoi trees during consecutive winter seasons were obtained. Fruits were harvested in habitats having marked differences in soil quality, climate, and luminosity. RESULTS: D. digyna fruits were larger and less acid than D. rekoi fruits, whereas antioxidant activity tended to be higher in D. rekoi fruits. Phenolic, flavonoid, and sugar contents also varied significantly between species. Metabolomic analysis allowed the pre-identification of 519 and 1665 metabolites in negative and positive electrospray ionization (ESI) modes, respectively. Principal component analysis of the positive ESI data explained 51.8% of the variance and indicated clear metabolomic differences between D. rekoi and D. digyna fruits that were confirmed by direct-injection ESI mass spectrometry profiles. Twenty-one discriminating metabolites were detected in fruits of both species; D. digyna fruits differentially accumulated lysophospholipids, whereas discriminating metabolites in D. rekoi fruits were chemically more diverse than those in D. digyna fruits. CONCLUSION: Domesticated D. digyna fruits have improved physicochemical fruit traits compared with wild D. rekoi fruits, including larger size and lower acidity. The metabolomic and chemical composition of their respective fruits were also significantly different, which in D. rekoi was manifested as a notable season-dependent increase in antioxidant capacity. Therefore, wild D. rekoi can be considered as an important genetic resource for the improvement of commercial Diospyros fruit quality. © 2019 Society of Chemical Industry.

Modulation of steroidal glycoalkaloid biosynthesis in tomato (Solanum lycopersicum) by jasmonic acid.

Jasmonic acid (JA) is a phytohormone involved in plant development and defense. A major role of JA is the enhancement of secondary metabolite production, such as response to herbivory. Systemin is a bioactive plant peptide of 18 amino acids that contributes to the induction of local and systemic defense responses in tomato (Solanum lycopersicum) through JA biosynthesis. The overexpression of systemin (PS-OE) results in constitutive JA accumulation and enhances pest resistance in plants. Conversely, mutant plants affected in linolenic acid synthesis (spr2) are negatively compromised in the production of JA which favors damage and oviposition by insect herbivores. With undirected mass fingerprinting analyses, we found global metabolic differences between genotypes with modified jasmonic acid production. The spr2 mutants were enriched in di-unsaturated fatty acids and generally showed more changes. The PS-OE genotype produced an unidentified compound with a mass-to-charge ratio of 695 (MZ695). Most strikingly, the steroidal glycoalkaloid biosynthesis was negatively affected in the spr2 genotype. Complementation with jasmonic acid could restore the tomatine pathway, which strongly suggests the control of steroidal glycoalkaloid biosynthesis by jasmonic acid. spr2 plants were more susceptible to fungal infection with Fusarium oxysporum f.sp. ciceris, but not to bacterial infection with Clavibacter michiganensis subsp. michiganensis which supports the involvement of steroidal glycoalkaloids in the plant response against fungi.

Treatment of Amaranthus cruentus with chemical and biological inducers of resistance has contrasting effects on fitness and protection against compatible Gram positive and Gram negative bacterial pathogens.

Amaranthus cruentus (Ac) plants were treated with the synthetic systemic acquired resistance (SAR) inducer benzothiadiazole (BTH), methyl jasmonate (MeJA) and the incompatible pathogen, Pseudomonas syringae pv. syringae (Pss), under greenhouse conditions. The treatments induced a set of marker genes in the absence of pathogen infection: BTH and Pss similarly induced genes coding for pathogenesis-related and antioxidant proteins, whereas MeJA induced the arginase, LOX2 and amarandin 1 genes. BTH and Pss were effective when tested against the Gram negative pathogen Ps pv. tabaci (Pst), which was found to have a compatible interaction with grain amaranth. The resistance response appeared to be salicylic acid-independent. However, resistance against Clavibacter michiganensis subsp. michiganensis (Cmm), a Gram positive tomato pathogen also found to infect Ac, was only conferred by Pss, while BTH increased susceptibility. Conversely, MeJA was ineffective against both pathogens. Induced resistance against Pst correlated with the rapid and sustained stimulation of the above genes, including the AhPAL2 gene, which were expressed both locally and distally. The lack of protection against Cmm provided by BTH, coincided with a generalized down-regulation of defense gene expression and chitinase activity. On the other hand, Pss-treated Ac plants showed augmented expression levels of an anti-microbial peptide gene and, surprisingly, of AhACCO, an ethylene biosynthetic gene associated with susceptibility to Cmm in tomato, its main host. Pss treatment had no effect on productivity, but compromised growth, whereas MeJA reduced yield and harvest index. Conversely, BTH treatments led to smaller plants, but produced significantly increased yields. These results suggest essential differences in the mechanisms employed by biological and chemical agents to induce SAR in Ac against bacterial pathogens having different infection strategies. This may determine the outcome of a particular plant-pathogen interaction, leading to resistance or susceptibility, as in Cmm-challenged Ac plants previously induced with Pss or BTH, respectively.

Transcriptomic analysis of grain amaranth (Amaranthus hypochondriacus) using 454 pyrosequencing: comparison with A. tuberculatus, expression profiling in stems and in response to biotic and abiotic stress.

BACKGROUND: Amaranthus hypochondriacus, a grain amaranth, is a C4 plant noted by its ability to tolerate stressful conditions and produce highly nutritious seeds. These possess an optimal amino acid balance and constitute a rich source of health-promoting peptides. Although several recent studies, mostly involving subtractive hybridization strategies, have contributed to increase the relatively low number of grain amaranth expressed sequence tags (ESTs), transcriptomic information of this species remains limited, particularly regarding tissue-specific and biotic stress-related genes. Thus, a large scale transcriptome analysis was performed to generate stem- and (a)biotic stress-responsive gene expression profiles in grain amaranth. RESULTS: A total of 2,700,168 raw reads were obtained from six 454 pyrosequencing runs, which were assembled into 21,207 high quality sequences (20,408 isotigs + 799 contigs). The average sequence length was 1,064 bp and 930 bp for isotigs and contigs, respectively. Only 5,113 singletons were recovered after quality control. Contigs/isotigs were further incorporated into 15,667 isogroups. All unique sequences were queried against the nr, TAIR, UniRef100, UniRef50 and Amaranthaceae EST databases for annotation. Functional GO annotation was performed with all contigs/isotigs that produced significant hits with the TAIR database. Only 8,260 sequences were found to be homologous when the transcriptomes of A. tuberculatus and A. hypochondriacus were compared, most of which were associated with basic house-keeping processes. Digital expression analysis identified 1,971 differentially expressed genes in response to at least one of four stress treatments tested. These included several multiple-stress-inducible genes that could represent potential candidates for use in the engineering of stress-resistant plants. The transcriptomic data generated from pigmented stems shared similarity with findings reported in developing stems of Arabidopsis and black cottonwood (Populus trichocarpa). CONCLUSIONS: This study represents the first large-scale transcriptomic analysis of A. hypochondriacus, considered to be a highly nutritious and stress-tolerant crop. Numerous genes were found to be induced in response to (a)biotic stress, many of which could further the understanding of the mechanisms that contribute to multiple stress-resistance in plants, a trait that has potential biotechnological applications in agriculture.