Plant-specific environmental and developmental signals regulate the mismatch repair protein MSH6 in Arabidopsis thaliana.

The DNA mismatch repair (MMR) is a postreplicative system that guarantees genomic stability by correcting mispaired and unpaired nucleotides. In eukaryotic nuclei, MMR is initiated by the binding of heterodimeric MutS homologue (MSH) complexes to the DNA error or lesion. Among these proteins, MSH2-MSH6 is the most abundant heterodimer. Even though the MMR mechanism and proteins are highly conserved throughout evolution, physiological differences between species can lead to different regulatory features. Here, we investigated how light, sugar, and/or hormones modulate Arabidopsis thaliana MSH6 expression pattern. We first characterized the promoter region of MSH6. Phylogenetic shadowing revealed three highly conserved regions. These regions were analyzed by the generation of deletion constructs of the MSH6 full-length promoter fused to the ß-glucuronidase (GUS) gene. Combined, our in silico and genetic analyses revealed that a 121-bp promoter fragment was necessary for MSH6 expression and contained potential cis-acting elements involved in light- and hormone-responsive gene expression. Accordingly, light exposure or sugar treatment of four-day old A. thaliana seedlings triggered an upregulation of MSH6 in shoot and root apical meristems. Appropriately, MSH6 was also induced by the stem cell inducer WUSCHEL. Further, the stimulatory effect of light was dependent on the presence of phyA. In addition, treatment of seedlings with auxin or cytokinin also caused an upregulation of MSH6 under darkness. Consistent with auxin signals, MSH6 expression was suppressed in the GATA23 RNAi line compared with the wild type. Our results provide evidence that endogenous factors and environmental signals controlling plant growth and development regulate the MSH6 protein in A. thaliana.

The multifarious MerR family of transcriptional regulators.

The MerR family of transcriptional regulators includes a variety of bacterial cytoplasmic proteins that respond to a wide range of signals, including toxins, metal ions, and endogenous metabolites. Its best-characterized members share similar structural and functional features with the family founder, the mercury sensor MerR, although most of them do not respond to metal ions. The group of "canonical" MerR homologs displays common molecular mechanisms for controlling the transcriptional activation of their target genes in response to inducer signals. This includes the recognition of distinctive operator sequences located at suboptimal σ70 -dependent promoters. Interestingly, an increasing number of proteins assigned to the MerR family based on their DNA-binding domain do not match in structure, sequence, or mode of action with any of the canonical MerR-like regulators. Here, we analyzed several members of the family, including this last group. Based on a phylogenetic analysis, and similarities in structural/functional features and position of their target operators relative to the promoter elements, we propose to assign these "atypical/divergent" MerR regulators to a phylogenetically separated group. These atypical/divergent homologs represent a new class of transcriptional regulators with novel regulatory mechanisms.

Abscisic Acid Synthesis and Signaling during the Ripening of Raspberry (Rubus idaeus 'Heritage') Fruit.

The raspberry (Rubus idaeus L.) fruit is characterized by its richness in functional molecules and high nutritional value, but the high rate of fruit softening limits its quality during postharvest. Raspberry drupelets have a particular ripening regulation, depending partially on the effect of ethylene produced from the receptacle. However, the possible role of abscisic acid (ABA) in the modulation of quality parameters during the ripening of raspberry is unclear. This study characterized the fruit quality-associated parameters and hormonal contents during fruit development in two seasons. The quality parameters showed typical changes during ripening: a drastic loss of firmness, increase in soluble solids content, loss of acidity, and turning to a red color from the large green stage to fully ripe fruit in both seasons. A significant increase in the ABA content was observed during the ripening of drupelets and receptacles, with the higher content in the receptacle of ripe and overripe stages compared to the large green stage. Moreover, identification of ABA biosynthesis-(9-cis-epoxycarotenoid dioxygenase/NCED) and ABA receptor-related genes (PYRs-like receptors) showed three genes encoding RiNCEDs and nine genes for RiPYLs. The expression level of these genes increased from the large green stage to the full-ripe stage, specifically characterized by a higher expression of RiNCED1 in the receptacle tissue. This study reports a consistent concomitant increase in the ABA content and the expression of RiNCED1, RiPYL1, and RiPYL8 during the ripening of the raspberry fruit, thus supporting the role for ABA signaling in drupelets.

Bolstered plant tolerance to low temperatures by overexpressing NAC transcription factors: identification of critical variables by meta-analysis.

MAIN CONCLUSION: The potential biotechnological application of NAC overexpression has been challenged by meta-analysis, establishing a correlation between the magnitudes of several physiological and biochemical parameters and the enhanced tolerance to cold. Overexpression of various NAC (NAM/ATAF/CUC) transcription factors in different plant systems was shown to confer enhanced tolerance to low temperatures by inducing both common and distinctive stress response pathways. However, lack of consensus on the type of parameters evaluated, their magnitudes, and direction of the responses complicates drawing general conclusions on the effects of NAC expression in plant physiology. We report herein a meta-analysis summarizing the most critical response variables used to study the effect of overexpressing NAC regulators on cold stress tolerance. We found that NAC overexpression affected all of the outcome parameters in stressed plants, and one response in control conditions. Transformed plants displayed an increase of at least 40% in positive responses, while negative outcomes were reduced by at least 30%. The most reported parameters included survival, electrolyte leakage, and malondialdehyde contents, whereas the most sensitive to the treatments were the Fv/Fm parameter, survival, and the activity of catalases. We also explored how different experimental arrangements affected the magnitudes of the responses. NAC-mediated improvements were best observed after severe stress episodes and during brief treatments (ranging from 5 to 24 h), especially in terms of antioxidant activities, accumulation of free proline, and parameters related to membrane integrity. Use of heterologous expression also favored several indicators of plant fitness. Our findings should help both basic and applied research on the influence of NAC expression on enhanced tolerance to cold.

Balance between bacterial extracellular matrix production and intramacrophage proliferation by a Salmonella-specific SPI-2-encoded transcription factor.

Biosynthesis and secretion of a complex extracellular matrix (EM) is a hallmark of Salmonella biofilm formation, impacting on its relationship with both the environment and the host. Cellulose is a major component of Salmonella EM. It is considered an anti-virulence factor because it interferes with Salmonella proliferation inside macrophages and virulence in mice. Its synthesis is stimulated by CsgD, the master regulator of biofilm formation in enterobacteria, which in turn is under the control of MlrA, a MerR-like transcription factor. In this work, we identified a SPI-2-encoded Salmonella-specific transcription factor homolog to MlrA, MlrB, that represses transcription of its downstream gene, orf319, and of csgD inside host cells. MlrB is induced in laboratory media mimicking intracellular conditions and inside macrophages, and it is required for intramacrophage proliferation. An increased csgD expression is observed in the absence of MlrB inside host cells. Interestingly, inactivation of the CsgD-controlled cellulose synthase-coding gene restored intramacrophage proliferation to rates comparable to wild-type bacteria in the absence of MlrB. These data indicate that MlrB represses CsgD expression inside host cells and suggest that this repression lowers the activation of the cellulose synthase. Our findings provide a novel link between biofilm formation and Salmonella virulence.

Photosynthetic characterization of flavodoxin-expressing tobacco plants reveals a high light acclimation-like phenotype.

Flavodoxins are electron carrier flavoproteins present in bacteria and photosynthetic microorganisms which duplicate the functional properties of iron-sulphur containing ferredoxins and replace them under adverse environmental situations that lead to ferredoxin decline. When expressed in plant chloroplasts, flavodoxin complemented ferredoxin deficiency and improved tolerance to multiple sources of biotic, abiotic and xenobiotic stress. Analysis of flavodoxin-expressing plants grown under normal conditions, in which the two carriers are present, revealed phenotypic effects unrelated to ferredoxin replacement. Flavodoxin thus provided a tool to alter the chloroplast redox poise in a customized way and to investigate its consequences on plant physiology and development. We describe herein the effects exerted by the flavoprotein on the function of the photosynthetic machinery. Pigment analysis revealed significant increases in chlorophyll a, carotenoids and chlorophyll a/b ratio in flavodoxin-expressing tobacco lines. Results suggest smaller antenna size in these plants, supported by lower relative contents of light-harvesting complex proteins. Chlorophyll a fluorescence and P700 spectroscopy measurements indicated that transgenic plants displayed higher quantum yields for both photosystems, a more oxidized plastoquinone pool under steady-state conditions and faster plastoquinone dark oxidation after a pulse of saturating light. Many of these effects resemble the phenotypes exhibited by leaves adapted to high irradiation, a most common environmental hardship faced by plants growing in the field. The results suggest that flavodoxin-expressing plants would be better prepared to cope with this adverse situation, and concur with earlier observations reporting that hundreds of stress-responsive genes were induced in the absence of stress in these lines.

Application of a JA-Ile Biosynthesis Inhibitor to Methyl Jasmonate-Treated Strawberry Fruit Induces Upregulation of Specific MBW Complex-Related Genes and Accumulation of Proanthocyanidins.

Fleshy fruits are an important source of anthocyanins and proanthocyanidins (PAs), which protect plants against stress, and their consumption provides beneficial effects for human health. In strawberry fruit, the application of exogenous methyl jasmonate (MeJA) upregulates anthocyanin accumulation, although the relationship between the jasmonate pathway and anthocyanin and PA biosynthesis in fruits remains to be understood. Anthocyanin and PA accumulation is mainly regulated at the transcriptional level through R2R3-MYB and bHLH transcription factors in different plant species and organs. Here, the effect of jarin-1, a specific inhibitor of bioactive JA (jasmonoyl-isoleucine, JA-Ile) biosynthesis, on anthocyanin and PA accumulation was evaluated during strawberry (Fragaria × ananassa) fruit development using an in vitro ripening system for 48 h. Also, we observed the effects of MeJA and the application of jarin-1 to MeJA-treated fruits (MeJA + jarin-1 treatment). We assessed changes of expression levels for the JA-Ile and MeJA biosynthetic (FaJAR1.2 and FaJMT), JA signaling-related (FaMYC2 and FaJAZ1), MYB-bHLH-WD40 (MBW) complex-related (FabHLH3/33, FaMYB9/10/11, and repressor FaMYB1), and anthocyanin and PA biosynthetic (FaANS, FaUFGT, FaANR, and FaLAR) genes. In addition, the promoter region of MBW complex-related MYB genes was isolated and sequenced. We found a higher redness of strawberry fruit skin and anthocyanin content in MeJA-treated fruits with respect to jarin-1-treated ones concomitant with an upregulation of FaANS and FaUFGT genes. Inversely, the PA content was higher in jarin-1- and MeJA + jarin-1-treated than in MeJA-treated fruits. MeJA + jarin-1 treatment resulted in an upregulation of FaANR and associated transcription factors such as FabHLH33 and FaMYB9/11 along with FaJMT and FaJAR1.2. Finally, we found JA-responsive elements in the promoter regions of FaMYB1/9/10/11 genes. It is proposed that PA biosynthesis-related genes can be upregulated by the application of jarin-1 to MeJA-treated fruit, thus increasing PA accumulation in strawberry.

Jasmonate signalling pathway in strawberry: Genome-wide identification, molecular characterization and expression of JAZs and MYCs during fruit development and ripening.

Jasmonates (JAs) are signalling molecules involved in stress responses, development and secondary metabolism biosynthesis, although their roles in fleshy-fruit development and ripening processes are not well known. In strawberry fruit, it has been proposed that JAs could regulate the early development through the activation of the JAs biosynthesis. Moreover, it has been reported that JA treatment increases anthocyanin content in strawberry fruit involving the bioactive jasmonate biosynthesis. Nevertheless, JA signalling pathway, of which main components are the COI1-JAZ co-receptor and the MYC transcription factors (TFs), has not been characterized in strawberry until now. Here we identified and characterized the woodland strawberry (Fragaria vesca) JAZ and MYC genes as well as studied their expression during development and ripening stages in commercial strawberry (Fragaria × ananassa) fruit. We described twelve putative JAZ proteins and two MYC TFs, which showed high conservation with respect to their orthologs in Arabidopsis thaliana and in other fleshy-fruit species such as Malus × domestica, Vitis vinifera and Solanum lycopersicum as revealed by gene synteny and phylogenetic analyses. Noteworthy, their expression levels exhibited a significant decrease from fruit development to ripening stages in F. × ananassa, along with others of the JA signalling-related genes such as FaNINJA and FaJAMs, encoding for negative regulators of JA responses. Moreover, we found that main JA signalling-related genes such as FaMYC2, and FaJAZ1 are promptly induced by JA treatment at early times in F. × ananassa fruit. These results suggest the conservation of the canonical JA signalling pathway in strawberry and a possible role of this pathway in early strawberry fruit development, which also correlates negatively with the beginning of the ripening process.

Optimization of a biotechnological multiproduct batch plant design for the manufacture of four different products: A real case scenario.

In this work a biotechnological multiproduct batch plant that manufactures four different recombinant proteins for human application is described in some detail. This batch plant design is then optimized with regards to the size of equipment using a mixed-integer linear programming (MILP) formulation recently developed by us in order to find a hypothetical new biotechnological batch plant based on the information of real known processes for the production of the four recombinant protein products. The real plant was divided for practical purposes into two sub-processes or facilities: a fermentation facility and a purification facility. Knowing the specific steps conforming the downstream processing of each product, size, and time factors were computed and used as parameters to solve the aforementioned MILP reformulation. New constraints were included to permit the selection of some equipment-such as centrifuges and membrane filters-in a discrete set of sizes. For equipment that can be built according to customer needs-such as reactors-the original formulation was retained. Computational results show the ability of this optimization methodology to deal with real data giving reliable solutions for a multi-product batch plant composed of 44 unit operations in a relatively small amount of time showing that in the case studied it is possible to save up to a 66% of the capital investment in equipment given the cost data used. Biotechnol. Bioeng. 2017;114: 1252-1263. © 2017 Wiley Periodicals, Inc.

Effects of preharvest applications of methyl jasmonate and chitosan on postharvest decay, quality and chemical attributes of Fragaria chiloensis fruit.

Fragaria chiloensis fruit has a short postharvest life mainly due to its rapid softening. In order to improve its postharvest life, preharvest applications of methyl jasmonate (MeJA) and chitosan were evaluated during postharvest storage at room temperature. The quality and chemical parameters, and protection against decay were evaluated at 0, 24, 48 and 72 h of storage from fruits of two subsequent picks (termed as first harvest and second harvest). In general, fruits treated with MeJA and chitosan maintained higher levels of fruit firmness, anthocyanin, and showed significant delays in decay incidence compared to control fruit. MeJA-treated fruits exhibited a greater lignin content and SSC/TA ratio, and delayed decay incidences. Instead, chitosan-treated fruits presented higher antioxidant capacity and total phenol content. In short, both the elicitors were able to increase the shelf life of fruits as evidenced by the increased levels of lignin and anthocyanin, especially of the second harvest.

Methyl jasmonate treatment induces changes in fruit ripening by modifying the expression of several ripening genes in Fragaria chiloensis fruit.

To investigate the role of jasmonates (JAs) in the ripening of Fragaria chiloensis fruit, two concentrations of methyl jasmonate (MeJA, 10 and 100 µM) were evaluated at 2, 5 and 9 d using an in vitro ripening system. Fruit quality parameters; the contents of anthocyanin, lignin and cell wall polymers; and the transcriptional profiles of several ripening-related genes were analyzed. MeJA accelerated fruit ripening by means of a transitory increase in the soluble solid content/titratable acidity ratio, anthocyanin accumulation and an increase in softening at day 5. The expression of several phenylpropanoid-related genes, primarily those associated with anthocyanin biosynthesis, was increased under MeJA treatment, which correlated with an increased accumulation of anthocyanin. MeJA also altered the expression profiles of some cell wall-modifying genes, namely, EG1 and XTH1, and these changes correlated with a transient reduction in the firmness of MeJA-treated fruits. MeJA-responsive elements were observed in the promoter region of the EG1 gene. MeJA also increased the expression of LOX, AOS and OPR3, genes involved in the biosynthesis of JAs, and these changes correlated with the transient activation of fruit ripening observed. Conversely, the expression of ethylene and lignin biosynthesis genes (ACS, ACO, CAD and POD27) increased in MeJA-treated fruits at day 9. The present findings suggest that JAs promote the ripening of non-climacteric fruits through their involvement in anthocyanin accumulation, cell wall modification and the biosynthesis of ethylene and JAs.