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This work describes the genetic transformation of a strain of Aspergillus niger with five different constructs containing 16 different heterologous genes, coding for four oxidoreductases, two cellobiohydrolases, one endoglucanase, one β-glucosidase, six enzymes involved in xylose metabolism, and two enzymes involved in fermentation. The aim was to try and engineer a consolidated bioprocessing in A. niger. The fungus already contains most of these enzymes and we only enhanced endogenous activities. We recovered nine transformants containing all genes, as indicated by polymerase chain reaction (PCR). To confirm that the products of the genes were functional, we measured the activity of five different enzymes in all the strains, and they all showed enhanced activity over the wild-type (wt) strain. The strains were grown on carboxymethyl cellulose (CMC) and xylan as substrates, and they produced considerably more ethanol than the wt. The levels of ethanol production were comparable to those reported in the literature., (© 2022 International Union of Biochemistry and Molecular Biology, Inc.)

Background: Metabolic reconfiguration in plants is a hallmark response to insect herbivory that occurs in the attack site and systemically in undamaged tissues. Metabolomic systemic responses can occur rapidly while the herbivore is still present and may persist in newly developed tissue to counterattack future herbivore attacks. This study analyzed the metabolic profile of local and newly developed distal (systemic) leaves of husk tomato (Physalis philadelphica) plants after whitefly Trialeurodes vaporariorum infestation. In addition, the effect of these metabolomic adjustments on whitefly oviposition and development was evaluated., Results: Our results indicate that T. vaporariorum infestation induced significant changes in husk tomato metabolic profiles, not only locally in infested leaves, but also systemically in distal leaves that developed after infestation. The distinctive metabolic profile produced in newly developed leaves affected whitefly nymphal development but did not affect female oviposition, suggesting that changes driven by whitefly herbivory persist in the young leaves that developed after the infestation event to avoid future herbivore attacks., Conclusions: This report contributes to further understanding the plant responses to sucking insects by describing the metabolic reconfiguration in newly developed, undamaged systemic leaf tissues of husk tomato plants after whitefly infestation. © 2022 Society of Chemical Industry., (© 2022 Society of Chemical Industry.)

The Physalis genus includes species of commercial importance due to their ornamental, edible and medicinal properties. These qualities stem from their variety of biologically active compounds. We performed a metabolomic analysis of three Physalis species, i.e., P. angulata, P. grisea , and P. philadelphica , differing in domestication stage and cultivation practices, to determine the degree of inter-species metabolite variation and to test the hypothesis that these related species mount a common metabolomic response to foliar damage caused by Trichoplusia ni larvae. The results indicated that the metabolomic differences detected in the leaves of these species were species-specific and remained even after T. ni herbivory. They also show that each Physalis species displayed a unique response to insect herbivory. This study highlighted the metabolite variation present in Physalis spp. and the persistence of this variability when faced with biotic stressors. Furthermore, it sets an experimental precedent from which highly species-specific metabolites could be identified and subsequently used for plant breeding programs designed to increase insect resistance in Physalis and related plant species.

Aspergillus niger has been employed to produce heterologous proteins due to its high capacity for expression and secretion; nevertheless, expression levels of human proteins have been modest. We were interested in investigating whether A. niger can express and secret human erythropoietin (HuEPO) at high yields. Our strategy was to combine the presence of introns with CRISPR-Cas9 to increase the yield of the recombinant protein. The epo gene was codon-optimized and its expression driven by the PmbfA promoter. Another version of epo contained introns from the fructose-1,6-bisphosphatase (fbp) gene. Two recombinant clones, uME12 (no introns) and uME23 (with introns), were selected based on the resistance to the antibiotic and because they showed a protein profile different from that of the parental strain, as shown by SDS-PAGE. Expression of epo was confirmed by RT-PCR in both colonies but the recombinant EPO protein (rHUEPO) was detected by Western blot only in uME23. The rHuEPO yield from uME23 was estimated at about 1.8 mg L -1  by ELISA, demonstrating that the presence of introns resulted in higher yield, possibly by conferring more stability to mRNA. On the other hand, as part of our strategy we decided to inactivate in the strain uME23 the following genes vps, prtT, algC and och1 which are involved in protein secretion, regulating of protease expression and protein glycosylation in A. niger, with CRISPR-Cas9, yielding the muPS20 transformant. muPS20 is a protease-free strain and its rHuEPO production level was increased 41.1-fold. Moreover, its molecular weight was ≈27 kDa showing that mutations in the above mentioned genes improved secretion, prevented proteolytic degradation and hyperglycosylation of heterologous protein., Competing Interests: Declaration of competing interest The authors have no competing interests to declare., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Domesticated tomato ( Solanum lycopersicum L.) crops have presented an increased susceptibility to pests under field and greenhouse conditions. Among these pests is tomato/potato psyllid, Bactericera cockerelli Sulc (Hemiptera: Triozidae), a major pest in solanaceous crops. In this study, we evaluated volatile organic compound (VOC) emissions from the headspace in three healthy varieties of tomato plants (Floradade, Micro-Tom and wild) under greenhouse conditions using solid-phase microextraction and gas chromatography-mass spectrometry (SPME/GC-MS). Later, independent bioassays were performed to evaluate VOC emissions with three varieties infested with nymphs of B. cockerelli . The results in healthy plants showed markedly different VOC profiles in each variety (14 compounds for wild, 17 for Floradade and 4 for Micro-Tom). Plants infested with nymphs showed changes in VOC emissions distinctly in Floradade and wild varieties. We suggest that these qualitative differences in VOC profiles by the degree of domestication could explain the preferences of B. cockerelli .

A novel bipartite begomovirus, Blechum interveinal chlorosis virus (BleICV), was characterized at the genome level. Comparative analyses revealed that BleICV coat protein (CP) gene promoter is highly divergent from the equivalent region of other begomoviruses (BGVs), with the single exception of Tomato chino La Paz virus (ToChLPV) with which it shares a 23-bp phylogenetic footprint exhibiting dyad symmetry. Systematic examination of the homologous CP promoter segment of 132 New World BGVs revealed the existence of a quasi-palindromic DNA segment displaying a strongly conserved ACTT-(N7)-AAGT core. The spacer sequence between the palindromic motifs is constant in length, but its sequence is highly variable among viral species, presenting a relaxed consensus (TT)GGKCCCY, which is similar to the Conserved Late Element or CLE (GTGGTCCC), a putative TrAP-responsive element. The homologous CP promoter region of Old World BGVs exhibited a distinct organization, with the putative TATA-box overlapping the left half of the ACTT-N7 composite element. Similar CP promoter sequences, dubbed "TATA-associated composite element" or TACE, were found in viruses belonging to different Geminiviridae genera, hence hinting unsuspected evolutionary relationships among those lineages. To get cues about the TACE function, the regulatory function of the CLE was explored in distinct experimental systems. Transgenic tobacco plants harboring a GUS reporter gene driven by a promoter composed by CLE multimers expressed high beta-glucuronidase activity in absence of viral factors, and that expression was increased by begomovirus infection. On the other hand, the TrAP-responsiveness of a truncated CP promoter of Tomato golden mosaic virus (TGMV) was abolished by site-directed mutation of the only CLE present in it, whereas the artificial addition of one CLE to the -125 truncated promoter strongly enhanced the transactivation level in tobacco protoplasts. These results indicate that the CLE is a TrAP-responsive element, hence providing valuable clues to interpret the recurrent association of the CLE with the TACE. On the basis of the aforesaid direct evidences and the insights afforded by the extensive comparative analysis of BleICV CP promoter, we propose that the TACE might be involved in the TrAP-mediated derepression of CP gene in vascular tissues., Competing Interests: The authors have declared that no competing interests exist.

Capsaicinoids are responsible for the hot taste of chili peppers. They are restricted to the genus Capsicum and are synthesized by the acylation of the aromatic compound vanillylamine (derived from the phenylpropanoid pathway) with a branched-chain fatty acid by the catalysis of the putative enzyme capsaicinoid synthase. R2R3-MYB transcription factors have been reported in different species of plants as regulators of structural genes of the phenylpropanoid pathway; therefore, we hypothesized that MYB genes might be involved in the regulation of the biosynthesis of pungent compounds. In this study, an R2R3-MYB transcription factor gene, designated CaMYB31 , was isolated and characterized in Capsicum annuum 'Tampiqueño 74'. Bioinformatic analysis suggested that CaMYB31 could be involved in secondary metabolism, stress and plant hormone responses, and development. CaMYB31 expression analysis from placental tissue of pungent and nonpungent chili pepper fruits showed a positive correlation with the structural genes Ca4H , Comt , Kas , pAmt , and AT3 expression and also with the content of capsaicin and dihydrocapsacin during fruit development. However, CaMYB31 also was expressed in vegetative tissues (leaves, roots, and stems). Moreover, CaMYB31 silencing significantly reduced the expression of capsaicinoid biosynthetic genes and the capsaicinoid content. Additionally, CaMYB31 expression was affected by the plant hormones indoleacetic acid, jasmonic acid, salicylic acid, and gibberellic acid or by wounding, temperature, and light, factors known to affect the production of capsaicinoids. These findings indicate that CaMYB31 is indeed involved in the regulation of structural genes of the capsaicinoid biosynthetic pathway., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

Small RNA (sRNA)-mediated gene silencing represents a conserved regulatory mechanism controlling a wide diversity of developmental processes through interactions of sRNAs with proteins of the ARGONAUTE (AGO) family. On the basis of a large phylogenetic analysis that includes 206 AGO genes belonging to 23 plant species, AGO genes group into four clades corresponding to the phylogenetic distribution proposed for the ten family members of Arabidopsis thaliana. A primary analysis of the corresponding protein sequences resulted in 50 sequences of amino acids (blocks) conserved across their linear length. Protein members of the AGO4/6/8/9 and AGO1/10 clades are more conserved than members of the AGO5 and AGO2/3/7 clades. In addition to blocks containing components of the PIWI, PAZ, and DUF1785 domains, members of the AGO2/3/7 and AGO4/6/8/9 clades possess other consensus block sequences that are exclusive of members within these clades, suggesting unforeseen functional specialization revealed by their primary sequence. We also show that AGO proteins of animal and plant kingdoms share linear sequences of blocks that include motifs involved in posttranslational modifications such as those regulating AGO2 in humans and the PIWI protein AUBERGINE in Drosophila. Our results open possibilities for exploring new structural and functional aspects related to the evolution of AGO proteins within the plant kingdom, and their convergence with analogous proteins in mammals and invertebrates.

Background: Fructans obtained from agave, called agavins, have recently shown significant benefits for human health including obesity. Therefore, we evaluated the potential of agavins as neuroprotectors and antioxidants by determining their effect on brain-derived neurotrophic factor (BDNF) and glial-derived neurotrophic factor (GDNF) as well as oxidative brain damage in of obese mice., Methods: Male C57BL/6J mice were fed a high-fat diet (HFD) and treated daily with 5% (HFD/A5) or 10% (HFD/A10) of agavins or a standard diet (SD) for 10 weeks. The levels of BDNF and GDNF were evaluated by ELISA. The oxidative stress was evaluated by lipid peroxidation (TBARS) and carbonyls. SCFAs were also measured with GC-FID. Differences between groups were assessed using ANOVA and by Tukey's test considering p < 0.05., Results: The body weight gain and food intake of mice HFD/A10 group were significantly lower than those in the HFD group. Agavins restored BDNF levels in HFD/A5 group and GDNF levels of HFD/A5 and HFD/A10 groups in cerebellum. Interestingly, agavins decreased TBARS levels in HFD/A5 and HFD/A10 groups in the hippocampus, frontal cortex and cerebellum. Carbonyl levels were also lower in HFD/A5 and HFD/A10 for only the hippocampus and cerebellum. It was also found that agavins enhanced SCFAs production in feces., Conclusion: Agavins may act as bioactive ingredients with antioxidant and protective roles in the brain.

Pseudomonas syringae pv. maculicola is a natural pathogen of members of the Brassicaceae plant family. Using a transposon-based mutagenesis strategy in Pseudomonas syringaepv. maculicola M2 (PsmM2), we conducted a genetic screen to identify mutants that were capable of growing in M9 medium supplemented with a crude extract from the leaves of Arabidopsis thaliana. A mutant containing a transposon insertion in the hrpZ gene (PsmMut8) was unable to infect adult plants from Arabidopsis thaliana or Brassica oleracea, suggesting a loss of pathogenicity. The promotorless cat reporter present in the gene trap was expressed if PsmMut8 was grown in minimal medium (M9) supplemented with the leaf extract but not if grown in normal rich medium (KB). We conducted phylogenetic analysis using hrpAZB genes, showing the classical 5-clade distribution, and nucleotide diversity analysis, showing the putative position for selective pressure in this operon. Our results indicate that the hrpAZB operon from Pseudomonas syringaepv. maculicola M2 is necessary for its pathogenicity and that its diversity would be under host-mediated diversifying selection.

The biochemical processes underlying variations of tolerance are often accompanied by source-sink transitions affecting carbon (C) metabolism. We investigated the tolerance of Amaranthus cruentus L. to total mechanical defoliation through development and in different growing seasons. Defoliated A. cruentus recovered ∼80% of their above-ground biomass and ∼100% of grain yield compared to intact plants if defoliation occurred early during ontogeny, but could not compensate when defoliation occurred during flowering. Tolerance index was higher in the summer season (-0.3) than in the winter season (-0.7). Overall, defoliation tolerance was closely related to phosphoenolpyruvate carboxylase (PEPC) activity in leaves and the subsequent accumulation of starch (∼500 μmol/gDW) and sucrose (∼140 μmol/gDW) in stems and roots. Thus, A. cruentus accumulated sufficient C in roots and stem to allow branching and shoot re-growth after defoliation, but it only possessed sufficient C reserves to maintain <19% seed yield in the absence of new vegetative tissue. Seed size was larger during the warm season but it was not affected by foliar damage. Seed chemical composition was altered by defoliation at flowering. We conclude that A. cruentus defoliation tolerance depends on both, the re-allocation of starch from stem and roots, and the activation of dormant meristems before flowering to generate new photosynthetic capacity to sustain seed filling., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

In angiosperms, the transition to the female gametophytic phase relies on the specification of premeiotic gamete precursors from sporophytic cells in the ovule. In Arabidopsis thaliana, a single diploid cell is specified as the premeiotic female gamete precursor. Here, we show that ecotypes of Arabidopsis exhibit differences in megasporogenesis leading to phenotypes reminiscent of defects in dominant mutations that epigenetically affect the specification of female gamete precursors. Intraspecific hybridization and polyploidy exacerbate these defects, which segregate quantitatively in F2 populations derived from ecotypic hybrids, suggesting that multiple loci control cell specification at the onset of female meiosis. This variation in cell differentiation is influenced by the activity of ARGONAUTE9 (AGO9) and RNA-DEPENDENT RNA POLYMERASE6 (RDR6), two genes involved in epigenetic silencing that control the specification of female gamete precursors. The pattern of transcriptional regulation and localization of AGO9 varies among ecotypes, and abnormal gamete precursors in ovules defective for RDR6 share identity with ectopic gamete precursors found in selected ecotypes. Our results indicate that differences in the epigenetic control of cell specification lead to natural phenotypic variation during megasporogenesis. We propose that this mechanism could be implicated in the emergence and evolution of the reproductive alternatives that prevail in flowering plants., (© 2015 American Society of Plant Biologists. All rights reserved.)

In this study we investigate the effect that Agave fructans as new prebiotics have on mineral absorption improvement. Forty-eight 12-week-old C57BL/6J mice were used in this study. Forty mice were ovariectomized and eight were sham-operated controls. Mice were fed standard diets or diets supplemented with 10% Agave fructans or 10% inulin fructans. Calcium and magnesium were evaluated as well as their excretion in feces. Osteocalcin levels were also measured; femur structure was studied by scanning electron microscopy. Other parameters, such as food intake, body weight, glucose, and short-chain fatty acid content, were recorded. Calcium in plasma and bone increased in Agave fructan groups (from 53.1 to 56 and 85 mg/L and from 0.402 to 0.474 and 0.478 g/g, respectively) and osteocalcin increased in all fructan groups (>50%). Scanning electron microscopy showed that fructans were able to mitigate bone loss. In conclusion, we demonstrated that supplementation with Agave fructans prevents bone loss and improves bone formation.

Filamentous fungi are used in several industries and in academia to produce antibiotics, metabolites, proteins and pharmaceutical compounds. The development of valuable strains usually requires the insertion of recombinant deoxyribonucleic acid; however, the protocols to transfer DNA to fungal cells are highly inefficient. Recently, underwater shock waves were successfully used to genetically transform filamentous fungi. The purpose of this research was to demonstrate that the efficiency of transformation can be improved significantly by enhancing acoustic cavitation using tandem (dual-pulse) shock waves. Results revealed that tandem pressure pulses, generated at a delay of 300 μs, increased the transformation efficiency of Aspergillus niger up to 84% in comparison with conventional (single-pulse) shock waves. This methodology may also be useful to obtain new strains required in basic research and biotechnology., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

The production of transgenic fungi is a routine process. Currently, it is possible to insert genes from other fungi, viruses, bacteria and even animals, albeit with low efficiency, into the genomes of a number of fungal species. Genetic transformation requires the penetration of the transgene through the fungal cell wall, a process that can be facilitated by biological or physical methods. Novel methodologies for the efficient introduction of specific genes and stronger promoters are needed to increase production levels. A possible solution to this problem is the recently discovered shock-wave-mediated transformation. The objective of this article is to review the state of the art of the physical methods used for genetic fungi transformation and to describe some of the basic physics and molecular biology behind them., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The expression of genes coding for sucrose:sucrose 1-fructosyltransferase (1-SST; EC 2.4.1.99) and fructan:fructan 1-fructosyltransferase (1-FFT; EC 2.4.1.100), both fructan biosynthesizing enzymes, characterization by TLC and HPAEC-PAD, as well as the quantification of the fructo-oligosaccharides (FOS) accumulating in response to the exogenous application of sucrose, kinetin (cytokinin) or other plant hormones associated with (a)biotic stress responses were determined in two Agave species grown in vitro, domesticated Agave tequilana var. azul and wild A. inaequidens. It was found that elicitors such as salicylic acid (SA), and jasmonic acid methyl ester (MeJA) had the strongest effect on fructo-oligosaccharide (FOS) accumulation. The exogenous application of 1mM SA induced a 36-fold accumulation of FOS of various degrees of polymerization (DP) in stems of A. tequilana. Other treatments, such as 50mM abscisic acid (ABA), 8% Sucrose (Suc), and 1.0 mg L(-1) kinetin (KIN) also led to a significant accumulation of low and high DP FOS in this species. Conversely, treatment with 200 μM MeJA, which was toxic to A. tequilana, induced an 85-fold accumulation of FOS in the stems of A. inaequidens. Significant FOS accumulation in this species also occurred in response to treatments with 1mM SA, 8% Suc, and 10% polyethylene glycol (PEG). Maximum yields of 13.6 and 8.9 mg FOS per g FW were obtained in stems of A. tequilana and A. inaequidens, respectively. FOS accumulation in the above treatments was tightly associated with increased expression levels of either the 1-FFT or the 1-SST gene in tissues of both Agave species., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

To investigate the genetic and molecular regulation that the female gametophyte could exert over neighboring sporophytic regions of the ovule, we performed a quantitative comparison of global expression in wild-type and nozzle/sporocyteless (spl) ovules of Arabidopsis thaliana (Arabidopsis), using Massively Parallel Signature Sequencing (MPSS). This comparison resulted in 1517 genes showing at least 3-fold increased expression in ovules lacking a female gametophyte, including those encoding 89 transcription factors, 50 kinases, 25 proteins containing a RNA-recognition motif (RRM), and 20 WD40 repeat proteins. We confirmed that eleven of these genes are either preferentially expressed or exclusive of spl ovules lacking a female gametophyte as compared to wild-type, and showed that six are also upregulated in determinant infertile1 (dif1), a meiotic mutant affected in a REC8-like cohesin that is also devoided of female gametophytes. The sporophytic misexpression of IOREMPTE, a WD40/transducin repeat gene that is preferentially expressed in the L1 layer of spl ovules, caused the arrest of female gametogenesis after differentiation of a functional megaspore. Our results show that in Arabidopsis, the sporophytic-gametophytic cross talk includes a negative regulation of the female gametophyte over specific genes that are detrimental for its growth and development, demonstrating its potential to exert a repressive control over neighboring regions in the ovule.

RING finger E3 ligases are components of the ubiquitin proteasome system (UPS) that mediate the transfer of ubiquitin to substrates. Single-subunit RING finger E3s binds the E2 ubiquitin-conjugating enzyme and contains recognition sequences for the substrate within the same polypeptide. Here we describe the characterization of a class of RING finger E3 ligases that is conserved among eukaryotes. This class encodes a RING-H2 domain related in sequence to the ATL RING-H2 domain, another class of E3 ligases, and a C2/C2 zing finger at the amino-terminus, formerly described as BZF. In viridiplantae (green algae and land plants), we designed this family as BTL for BZF ATLs. BTLs are putative orthologs of the mammalian Rabring7/BCA2 RING-H2 E3s that have expanded in angiosperms. They are found in numbers ranging from three to thirty-one, which is in contrast to the one to three members normally found in animals, fungi, and protists. Furthermore, the number of sequence LOGOs generated in angiosperms is four times greater than that in other eukaryotes. In contrast to ATLs, which show expansion by tandem duplication, tandemly duplicated BTLs are scarce. The mode of action of Rabring7/BCA2 and BTLs may be similar since both the Rabring7/BCA2 BZF and the ath|BTL4 BZF are likely to mediate the binding of ubiquitin. This study introduces valuable information on the evolution and domain structure of the Rabring7/BCA2/BTL class of E3 ligases which may be important for core eukaryotic genes.

Tolerance to defoliation can be defined as the degree to which productivity is affected by photosynthetic area reduction. This trait was studied in grain amaranth (Amaranthus cruentus and A. hypochondriacus), which are considered to be a highly defoliation-tolerant species. The physiological and biochemical responses to increasing levels of mechanical leaf removal up to total defoliation were quantified. Tolerance appeared to be dependent on various factors: ( i) amount of lost tissue; (ii) mechanics of leaf tissue removal; (iii) environment, and (iv) species tested. Thus, grain amaranth was found to be a highly tolerant species under green-house conditions when leaf tissue loss was performed by gradual perforation. However, tolerance was compromised under similar conditions when defoliation was done by gradual cutting of the leaf. Also tolerance in completely defoliated plants tended to decrease under field conditions, where differences between A. cruentus and A. hypochondriacus were observed. All non-structural carbohydrate (NSC) levels were reduced in stems and roots of totally defoliated amaranths one day after treatment. Such depletion probably provided the carbon (C) resources needed to sustain the early recovery process in the absence of photosynthetic capacity. This was corroborated by shading of intact plants, which produced the same rapid and drastic reduction of NSC levels in these tissues. These results emphasize the role of stored NSCs, particularly starch, in buffering the impact of severe defoliation in amaranth. The fall in sucrose synthase and cell wall invertase activity observed in stems and roots soon after defoliation was consistent with their predicted shift from sink to source tissues. It is concluded that mobilization of C stores in stems and roots, is a physiologically important trait underlying tolerance to defoliation in grain amaranth.

Genetic transformation of filamentous fungi is an essential tool in many areas such as biotechnology, medicine, phytopathology and genetics. However, available protocols to transform fungi are inefficient, laborious and have low reproducibility. We report the use of underwater shock waves as a novel method to transform filamentous fungi. An experimental piezoelectric shock wave generator was designed to expose fungal conidia to heterologous DNA. The device was successfully tested in Aspergillus niger, Fusarium oxysporum, Trichoderma reesei and Phanerochaete chrysosporium. The transformation frequency per number of conidia was between two and four orders of magnitude higher in comparison to previously published methods. For example, the frequency of transformation in A. niger was improved up to 5400-fold as compared with Agrobacterium protocols. Transformation was verified by expression of the green fluorescent protein, PCR and Southern blot. Our method offers new possibilities for fast, easy and efficient genetic manipulation of diverse fungal species., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Female gametogenesis in most flowering plants depends on the predetermined selection of a single meiotically derived cell, as the three other megaspores die without further division or differentiation. Although in Arabidopsis thaliana the formation of the functional megaspore (FM) is crucial for the establishment of the gametophytic generation, the mechanisms that determine the specification and fate of haploid cells remain unknown. Here, we show that the classical arabinogalactan protein 18 (AGP18) exerts an active regulation over the selection and survival of megaspores in Arabidopsis. During meiosis, AGP18 is expressed in integumentary cells located in the abaxial region of the ovule. Overexpression of AGP18 results in the abnormal maintenance of surviving megaspores that can acquire a FM identity but is not sufficient to induce FM differentiation before meiosis, indicating that AGP18 positively promotes the selection of viable megaspores. We also show that all four meiotically derived cells in the ovule of Arabidopsis are competent to differentiate into a gametic precursor and that the function of AGP18 is important for their selection and viability. Our results suggest an evolutionary role for arabinogalactan proteins in the acquisition of monospory and the developmental plasticity that is intrinsic to sexual reproduction in flowering plants.

In higher plants, the phloem plays a central role in the delivery of nutrients and signals from source to sink tissues. These signals likely coordinate different aspects of plant development, as well as its response to environmental cues. Although some phloem-transported proteins and RNAs may function as signaling molecules in plants, their mode of action remains poorly understood. Previous analysis of transcripts from CMV-infected pumpkin (Cucurbita maxima cv Big Max) identified a Translationally-Controlled Tumor Protein (TCTP) mRNA homolog, designated CmTCTP. In the present work this transcript was analyzed in terms of its expression pattern. This RNA accumulates, both in healthy and CMV-infected plants, in developing and mature phloem in petiole and roots, as well as in apices at high levels. The protein was present at lower levels in most cell types, and almost no signal was detected in apices, suggesting translational regulation of this RNA. Additionally, CmTCTP harbored by Agrobacterium rhizogenes is capable of inducing whole plant regeneration. These data suggest a role for CmTCTP in growth regulation, possibly through long-distance signaling.

Volatile organic compounds (VOCs) emitted from plants in response to insect infestation can function as signals for the attraction of predatory/parasitic insects and/or repulsion of herbivores. VOCs also may play a role in intra- and inter-plant communication. In this work, the kinetics and composition of VOC emissions produced by tomato (Solanum lycopersicum) plants infested with the greenhouse whitefly Trialeurodes vaporariorum was determined within a 14 days period. The VOC emission profiles varied concomitantly with the duration of whitefly infestation. A total of 36 different VOCs were detected during the experiment, 26 of which could be identified: 23 terpenoids, plus decanal, decane, and methyl salicylate (MeSA). Many VOCs were emitted exclusively by infested plants, including MeSA and 10 terpenoids. In general, individual VOC emissions increased as the infestation progressed, particularly at 7 days post-infestation (dpi). Additional tunnel experiments showed that a 3 days exposure to VOC emissions from whitefly-infested plants significantly reduced infection by a biotrophic bacterial pathogen. Infection of VOC-exposed plants induced the expression of a likely tomato homolog of a methyl salicylate esterase gene, which preceded the expression of pathogenesis-related protein genes. This expression pattern correlated with reduced susceptibility in VOC-exposed plants. The observed cross-kingdom effect of plant-plant signaling via VOCs probably represents a generalized defensive response that contributes to increased plant fitness, considering that resistance responses to whiteflies and biotrophic bacterial pathogens in tomato share many common elements.

Ustilago maydis (DC) Cda., a phytopathogenic Basidiomycota, is the causal agent of corn smut. During its life cycle U. maydis alternates between a yeast-like, haploid nonpathogenic stage, and a filamentous, dikaryotic pathogenic form that invades the plant and induces tumor formation. As all the members of the Subphylum Ustilaginomycotina, U. maydis is unable to form basidiocarps, instead it produces teliospores within the tumors that germinate forming a septate basidium (phragmobasidium). We have now established conditions allowing a completely different developmental program of U. maydis when grown on solid medium containing auxins in dual cultures with maize embryogenic calli. Under these conditions U. maydis forms large hemi-spheroidal structures with all the morphological and structural characteristics of gastroid-type basidiocarps. These basidiocarps are made of three distinct hyphal layers, the most internal of which (hymenium) contains non-septate basidia (holobasidia) from which four basidiospores develop. In basidiocarps meiosis and genetic recombination occur, and meiotic products (basidiospores) segregate in a Mendelian fashion. These results are evidence of sexual cycle completion of an Ustilaginomycotina in vitro, and the demonstration that, besides its quasi-obligate biotrophic pathogenic mode of life, U. maydis possesses the genetic program to form basidiocarps as occurs in saprophytic Basidiomycota species., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Amaranthus cruentus and A. hypochondriacus are crop plants grown for grain production in subtropical countries. Recently, the generation of large-scale transcriptomic data opened the possibility to study representative genes of primary metabolism to gain a better understanding of the biochemical mechanisms underlying tolerance to defoliation in these species. A multi-level approach was followed involving gene expression analysis, enzyme activity and metabolite measurements., Results: Defoliation by insect herbivory (HD) or mechanical damage (MD) led to a rapid and transient reduction of non-structural carbohydrates (NSC) in all tissues examined. This correlated with a short-term induction of foliar sucrolytic activity, differential gene expression of a vacuolar invertase and its inhibitor, and induction of a sucrose transporter gene. Leaf starch in defoliated plants correlated negatively with amylolytic activity and expression of a β-amylase-1 gene and positively with a soluble starch synthase gene. Fatty-acid accumulation in roots coincided with a high expression of a phosphoenolpyruvate/phosphate transporter gene. In all tissues there was a long-term replenishment of most metabolite pools, which allowed damaged plants to maintain unaltered growth and grain yield. Promoter analysis of ADP-glucose pyrophosphorylase and vacuolar invertase genes indicated the presence of cis-regulatory elements that supported their responsiveness to defoliation. HD and MD had differential effects on transcripts, enzyme activities and metabolites. However, the correlation between transcript abundance and enzymatic activities was very limited. A better correlation was found between enzymes, metabolite levels and growth and reproductive parameters., Conclusions: It is concluded that a rapid reduction of NSC reserves in leaves, stems and roots followed by their long-term recovery underlies tolerance to defoliation in grain amaranth. This requires the coordinate action of genes/enzymes that are differentially affected by the way leaf damage is performed. Defoliation tolerance in grain is a complex process that can't be fully explained at the transcriptomic level only.

An abundant class of E3 ubiquitin ligases encodes the RING-finger domain. The RING finger binds to the E2 ubiquitin-conjugating enzyme and brings together both the E2 and substrate. It is predicted that 477 RING finger E3 ligases exist in Arabidopsis thaliana. A particular family among them, named Arabidopsis Tóxicos en Levadura (ATL), consists of 91 members that contain the RING-H2 variation and a hydrophobic domain located at the N-terminal end. Transmembrane E3 ligases are important in several biological processes. For instance, some transmembrane RING finger E3 ligases are main participants in the endoplasmic reticulum-associated degradation pathway that targets misfolded proteins. Functional analysis of a number of ATLs has shown that some of them regulate distinct pathways in plants. Several ATLs have been shown to participate in defense responses, while others play a role in the regulation of the carbon/nitrogen response during post-germinative seedling growth transition, in the regulation of cell death during root development, in endosperm development, or in the transition to flowering under short day conditions. The ATL family has also been instrumental in evolution studies for showing how gene families are expanded in plant genomes.

The life cycle of flowering plants alternates between a predominant sporophytic (diploid) and an ephemeral gametophytic (haploid) generation that only occurs in reproductive organs. In Arabidopsis thaliana, the female gametophyte is deeply embedded within the ovule, complicating the study of the genetic and molecular interactions involved in the sporophytic to gametophytic transition. Massively parallel signature sequencing (MPSS) was used to conduct a quantitative large-scale transcriptional analysis of the fully differentiated Arabidopsis ovule prior to fertilization. The expression of 9775 genes was quantified in wild-type ovules, additionally detecting >2200 new transcripts mapping to antisense or intergenic regions. A quantitative comparison of global expression in wild-type and sporocyteless (spl) individuals resulted in 1301 genes showing 25-fold reduced or null activity in ovules lacking a female gametophyte, including those encoding 92 signalling proteins, 75 transcription factors, and 72 RNA-binding proteins not reported in previous studies based on microarray profiling. A combination of independent genetic and molecular strategies confirmed the differential expression of 28 of them, showing that they are either preferentially active in the female gametophyte, or dependent on the presence of a female gametophyte to be expressed in sporophytic cells of the ovule. Among 18 genes encoding pentatricopeptide-repeat proteins (PPRs) that show transcriptional activity in wild-type but not spl ovules, CIHUATEOTL (At4g38150) is specifically expressed in the female gametophyte and necessary for female gametogenesis. These results expand the nature of the transcriptional universe present in the ovule of Arabidopsis, and offer a large-scale quantitative reference of global expression for future genomic and developmental studies.

To explore the molecular mechanisms that prevail during the establishment of the arbuscular mycorrhiza symbiosis involving the genus Glomus, we transcriptionally analysed spores of Glomus intraradices BE3 during early hyphal growth. Among 458 transcripts initially identified as being expressed at presymbiotic stages, 20% of sequences had homology to previously characterized eukaryotic genes, 30% were homologous to fungal coding sequences, and 9% showed homology to previously characterized bacterial genes. Among them, GintPbr1a encodes a homolog to Phenazine Biosynthesis Regulator (Pbr) of Burkholderia cenocepacia, an pleiotropic regulatory protein that activates phenazine production through transcriptional activation of the protein D isochorismatase biosynthetic enzyme phzD (Ramos et al., 2010). Whereas GintPbr1a is expressed during the presymbiotic phase, the G. intraradices BE3 homolog of phzD (BGintphzD) is transcriptionally active at the time of the establishment of the arbuscular mycorrhizal symbiosis. DNA from isolated bacterial cultures found in spores of G. intraradices BE3 confirmed that both BGintPbr1a and BGintphzD are present in the genome of its potential endosymbionts. Taken together, our results indicate that spores of G. intraradices BE3 express bacterial phenazine biosynthetic genes at the onset of the fungal-plant symbiotic interaction.

Chili pepper (Capsicum spp.) is a very important horticultural crop around the world and is especially important for Mexicans because of its impact in the culture and the cuisine. Biotechnological tools such as tissue culture techniques and specifically anther culture may be applied successfully for plant breeding and genetic improvement in order to generate isogenic lines (100% homozygous) in a shorter time in comparison with the classic breeding methods. In this chapter, a protocol for efficient recovery of chili pepper haploid plants from in vitro cultured anthers is described.

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.

In flowering plants, the formation of gametes depends on the differentiation of cellular precursors that divide meiotically before giving rise to a multicellular gametophyte. The establishment of this gametophytic phase presents an opportunity for natural selection to act on the haploid plant genome by means of epigenetic mechanisms that ensure a tight regulation of plant reproductive development. Despite this early acting selective pressure, there are numerous examples of naturally occurring developmental alternatives that suggest a flexible regulatory control of cell specification and subsequent gamete formation in flowering plants. In this review, we discuss recent findings indicating that epigenetic mechanisms related to the activity of small RNA pathways prevailing during ovule formation play an essential role in cell specification and genome integrity. We also compare these findings to small RNA pathways acting during gametogenesis in animals and discuss their implications for the understanding of the mechanisms that control the establishment of the female gametophytic lineage during both sexual reproduction and apomixis., (© Springer-Verlag 2011)

Capsicum species produce fruits that synthesize and accumulate unique hot compounds known as capsaicinoids in placental tissues. The capsaicinoid biosynthetic pathway has been established, but the enzymes and genes participating in this process have not been extensively studied or characterized. Capsaicinoids are synthesized through the convergence of two biosynthetic pathways: the phenylpropanoid and the branched-chain fatty acid pathways, which provide the precursors phenylalanine, and valine or leucine, respectively. Capsaicinoid biosynthesis and accumulation is a genetically determined trait in chili pepper fruits as different cultivars or genotypes exhibit differences in pungency; furthermore, this characteristic is also developmentally and environmentally regulated. The establishment of cDNA libraries and comparative gene expression studies in pungent and non-pungent chili pepper fruits has identified candidate genes possibly involved in capsaicinoid biosynthesis. Genetic and molecular approaches have also contributed to the knowledge of this biosynthetic pathway; however, more studies are necessary for a better understanding of the regulatory process that accounts for different accumulation levels of capsaicinoids in chili pepper fruits.

DEAD-box proteins are found in all domains of life and participate in almost all cellular processes that involve RNA. The presence of DEAD and Helicase&#95;C conserved domains distinguish these proteins. DEAD-box proteins exhibit RNA-dependent ATPase activity in vitro, and several also show RNA helicase activity. In this study, we analyzed the distribution and architecture of DEAD-box proteins among bacterial genomes to gain insight into the evolutionary pathways that have shaped their history. We identified 1,848 unique DEAD-box proteins from 563 bacterial genomes. Bacterial genomes can possess a single copy DEAD-box gene, or up to 12 copies of the gene, such as in Shewanella. The alignment of 1,208 sequences allowed us to perform a robust analysis of the hallmark motifs of DEAD-box proteins and determine the residues that occur at high frequency, some of which were previously overlooked. Bacterial DEAD-box proteins do not generally contain a conserved C-terminal domain, with the exception of some members that possess a DbpA RNA-binding domain (RBD). Phylogenetic analysis showed a separation of DbpA-RBD-containing and DbpA-RBD-lacking sequences and revealed a group of DEAD-box protein genes that expanded mainly in the Proteobacteria. Analysis of DEAD-box proteins from Firmicutes and γ-Proteobacteria, was used to deduce orthologous relationships of the well-studied DEAD-box proteins from Escherichia coli and Bacillus subtilis. These analyses suggest that DbpA-RBD is an ancestral domain that most likely emerged as a specialized domain of the RNA-dependent ATPases. Moreover, these data revealed numerous events of gene family expansion and reduction following speciation.

Plants are exposed to several biotic and abiotic stresses. A common environmental stress that plants have to face both in natural and agricultural ecosystems that impacts both its growth and development is low phosphate (Pi) availability. There has been an important progress in the knowledge of the molecular mechanisms by which plants cope with Pi deficiency. However, the mechanisms that mediate alterations in the architecture of the Arabidopsis root system responses to Pi starvation are still largely unknown. One of the most conspicuous developmental effects of low Pi on the Arabidopsis root system is the inhibition of primary root growth that is accompanied by loss of root meristematic activity. To identify signalling pathways potentially involved in the Arabidpsis root meristem response to Pi-deprivation, here we report the global gene expression analysis of the root tip of wild type and low phosphorus insensitive4 (lpi4) mutant grown under Pi limiting conditions. Differential gene expression analysis and physiological experiments show that changes in the redox status, probably mediated by jasmonic acid and ethylene, play an important role in the primary root meristem exhaustion process triggered by Pi-starvation.

Transgenic tobacco plants capable of over-expressing Xenopus PPARα (xPPARα), a transcription factor known to be required for peroxisome proliferation in animals, were recently generated. These plants (herewith referred to as PPAR-OE) were found to have increased peroxisome abundance, higher peroxisomal acyl-CoA oxidase and catalase activity and modified fatty acid metabolism. Further characterization of PPAR-OE plants revealed a higher susceptibility to virulent and a partial loss of resistance to avirulent Pseudomonas syringae pathogens, whereas the basal resistance response remained unaffected. Biochemical- and defense-related gene expression analyses showed that increased susceptibility to bacterial invasion coincided with the generalized reduction in H(2)O(2) and salicylic acid (SA) levels observed within the first 24 h of bacterial contact. Decreased H(2)O(2) levels were correlated with modified activity levels of catalase and other antioxidant enzymes. A correspondence between a rapid (within 1-24 hpi; ACCO and AOC) and sustained increase (up to 6 days pi; ACCO) in the expression levels of ethylene (ACCO) and jasmonic acid (AOC) biosynthetic genes and a higher susceptibility to virulent bacterial invasion was also observed in PPAR-OE plants. Conversely, no apparent differences in the short- and/or long-term expression levels of markers for the hypersensitive-response, oxidative burst and systemic-acquired resistance were observed between wild type and PPAR-OE plants. The results suggest that peroxisome proliferation could lead to increased susceptibility to bacterial pathogens in tobacco by altering the redox balance of the plant and the expression pattern of key defense signaling pathway genes.

Among the major cucurbit vegetables, melon (Cucumis melo) has one of the greatest polymorphic fruit types and botanical varieties. Some melon fruits have excellent aroma, variety of flesh colors, deeper flavor, and more juice compared to other cucurbits. Despite numerous available melon cultivars, some of them are exceedingly susceptible to several diseases. The genetic background carrying the genes for tolerance and/or resistance for those diseases is found in wild melon landraces. Unfortunately, the commercial melon varieties are not able to produce viable hybrids when crossed with their wild melon counterparts. Plant tissue culture techniques are needed to surpass those genetic barriers. In vitro melon embryo rescue has played a main role to obtain viable hybrids originated from commercial versus wild melon crosses. In this chapter, an efficient and simple embryo rescue melon protocol is thoroughly described.

Ubiquitin-ligases or E3s are components of the ubiquitin proteasome system (UPS) that coordinate the transfer of ubiquitin to the target protein. A major class of ubiquitin-ligases consists of RING-finger domain proteins that include the substrate recognition sequences in the same polypeptide; these are known as single-subunit RING finger E3s. We are studying a particular family of RING finger E3s, named ATL, that contain a transmembrane domain and the RING-H2 finger domain; none of the member of the family contains any other previously described domain. Although the study of a few members in A. thaliana and O. sativa has been reported, the role of this family in the life cycle of a plant is still vague. To provide tools to advance on the functional analysis of this family we have undertaken a phylogenetic analysis of ATLs in twenty-four plant genomes. ATLs were found in all the 24 plant species analyzed, in numbers ranging from 20-28 in two basal species to 162 in soybean. Analysis of ATLs arrayed in tandem indicates that sets of genes are expanding in a species-specific manner. To get insights into the domain architecture of ATLs we generated 75 pHMM LOGOs from 1815 ATLs, and unraveled potential protein-protein interaction regions by means of yeast two-hybrid assays. Several ATLs were found to interact with DSK2a/ubiquilin through a region at the amino-terminal end, suggesting that this is a widespread interaction that may assist in the mode of action of ATLs; the region was traced to a distinct sequence LOGO. Our analysis provides significant observations on the evolution and expansion of the ATL family in addition to information on the domain structure of this class of ubiquitin-ligases that may be involved in plant adaptation to environmental stress.

Recent evidence indicates that the establishment of the haploid phase of the plant life cycle requires epigenetic mechanisms that control reproductive cell fate. We previously showed that in Arabidopsis thaliana (Arabidopsis) mutations in ARGONAUTE9 (AGO9) result in defective cell specification during megasporogenesis. AGO9 preferentially interacts with 24 nucleotide (nt) small RNAs (sRNAs) derived from transposable elements (TEs), and its sporophytic activity is required to silence TEs in the female gametophyte. Here we show that AGO9 can bind in vitro to 24 nt sRNAs corresponding to Athila retrotransposons expressed in the ovule prior to pollination. We also show that AGO9 is necessary to inactivate a significant proportion of long terminal repeat retrotransposons (LTRs) in the ovule, and that its predominant TE targets are located in the pericentromeric regions of all 5 chromosomes, suggesting a link between the AGO9-dependent sRNA pathway and heterochromatin formation. Our extended results point towards the existence of a tissue-specific mechanism of sRNA-dependent TE silencing in the ovule.

We generated a conditional CCase mutant of Bacillus subtilis to explore the participation in vivo of the tRNA nucleotidyltransferase (CCA transferase or CCase) in the maturation of the single-copy tRNA(Cys), which lacks an encoded CCA 3' end. We observed that shorter tRNA(Cys) species, presumably lacking CCA, only accumulated when the inducible Pspac : cca was introduced into an rnr mutant strain, but not in combination with pnp. We sequenced the tRNA 3' ends produced in the various mutant tRNA(Cys) species to detect maturation and decay intermediates and observed that decay of the tRNA(Cys) occurs through the addition of poly(A) or heteropolymeric tails. A few clones corresponding to full-size tRNAs contained either CCA or other C and/or A sequences, suggesting that these are substrates for repair and/or decay. We also observed editing of tRNA(Cys) at position 21, which seems to occur preferentially in mature tRNAs. Altogether, our results provide in vivo evidence for the participation of the B. subtilis cca gene product in the maturation of tRNAs lacking CCA. We also suggest that RNase R exoRNase in B. subtilis participates in the quality control of tRNA.