Role In Ripening Research Articles

New Genetic Determinants for qPCR Identification and the Enumeration of Selected Lactic Acid Bacteria in Raw-Milk Cheese.

Lactic acid bacteria (LAB) play an important role in the ripening of cheeses and contribute to the development of the desired profile of aroma and flavor compounds. Therefore, it is very important to monitor the dynamics of bacterial proliferation in order to obtain an accurate and reliable number of their cells at each stage of cheese ripening. This work aimed to identify and conduct a quantitative assessment of the selected species of autochthonous lactic acid bacteria from raw cow's milk cheese by the development of primers and probe pairs based on the uniqueness of the genetic determinants with which the target microorganisms can be identified. For that purpose, we applied real-time quantitative PCR (qPCR) protocols to quantify Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, and Lactococcus lactis subsp. cremoris cells in cheese directly after production and over three-month and six-month ripening periods. While L. lactis subsp. cremoris shows good acidification ability and the ability to produce antimicrobial compounds, L. delbrueckii subsp. bulgaricus has good proteolytic ability and produces exo-polysaccharides, and S. thermophilus takes part in the formation of the diacetyl flavor compound by metabolizing citrate to develop aroma, they all play an important role in the cheese ripening. The proposed qPCR protocols are very sensitive and reliable methods for a precise enumeration of L. delbrueckii subsp. bulgaricus, S. thermophilus, and L. lactis subsp. cremoris in cheese samples.

Insights into the Genes Involved in ABA Biosynthesis and Perception during Development and Ripening of the Chilean Strawberry Fruit.

Hormones act as master ripening regulators. In non-climacteric fruit, ABA plays a key role in ripening. Recently, we confirmed in Fragaria chiloensis fruit that in response to ABA treatment the fruit induces ripening-associated changes such as softening and color development. In consequence of these phenotypic changes, transcriptional variations associated with cell wall disassembly and anthocyanins biosynthesis were reported. As ABA stimulates the ripening of F. chiloensis fruit, the molecular network involved in ABA metabolism was analyzed. Therefore, the expression level of genes involved in ABA biosynthesis and ABA perception was quantified during the development of the fruit. Four NCED/CCDs and six PYR/PYLs family members were identified in F. chiloensis. Bioinformatics analyses confirmed the existence of key domains related to functional properties. Through RT-qPCR analyses, the level of transcripts was quantified. FcNCED1 codifies a protein that displays crucial functional domains, and the level of transcripts increases as the fruit develops and ripens, in parallel with the increment in ABA. In addition, FcPYL4 codifies for a functional ABA receptor, and its expression follows an incremental pattern during ripening. The study concludes that FcNCED1 is involved in ABA biosynthesis; meanwhile, FcPYL4 participates in ABA perception during the ripening of F. chiloensis fruit.

Ethylene-induced potassium transporter AcKUP2 gene is involved in kiwifruit postharvest ripening

BackgroundPotassium (K) is important in the regulation of plant growth and development. It is the most abundant mineral element in kiwifruit, and its content increases during fruit ripening. However, how K+ transporter works in kiwifruit postharvest maturation is not yet clear.ResultsHere, 12 K+ transporter KT/HAK/KUP genes, AcKUP1 ~ AcKUP12, were isolated from kiwifruit, and their phylogeny, genomic structure, chromosomal location, protein properties, conserved motifs and cis-acting elements were analysed. Transcription analysis revealed that AcKUP2 expression increased rapidly and was maintained at a high level during postharvest maturation, consistent with the trend of K content; AcKUP2 expression was induced by ethylene, suggesting that AcKUP2 might play a role in ripening. Fluorescence microscopy showed that AcKUP2 is localised in the plasma membrane. Cis-elements, including DER or ethylene response element (ERE) responsive to ethylene, were found in the AcKUP2 promoter sequence, and ethylene significantly enhanced the AcKUP2 promoter activity. Furthermore, we verified that AcERF15, an ethylene response factor, directly binds to the AcKUP2 promoter to promote its expression. Thus, AcKUP2 may be an important potassium transporter gene which involved in ethylene-regulated kiwifruit postharvest ripening.ConclusionsTherefore, our study establishes the first genome-wide analysis of the kiwifruit KT/HAK/KUP gene family and provides valuable information for understanding the function of the KT/HAK/KUP genes in kiwifruit postharvest ripening.

Ethylene application at the immature stage of Fragaria chiloensis fruit represses the anthocyanin biosynthesis with a concomitant accumulation of lignin

Ethylene seems to play a secondary role in non-climacteric strawberry ripening compared to abscisic acid. However, this does not exclude that ethylene can regulate some specific events related to the ripening process. Preliminary experiments of applications of ethylene or its inhibitor 1-MCP to strawberry fruits have reinforced this hypothesis. Here, we reveal some previously non-covered physiological effects of ethylene using an in vitro strawberry ripening system. Fruits of Fragaria chiloensis treated with ethephon at the large green developmental stage showed inhibition of anthocyanin biosynthesis and downregulation of essential anthocyanin biosynthesis genes during the ripening. At the same time, ethylene stimulated lignin biosynthesis and remarkably upregulated the expression of FcPOD27. Since contrasting results have been reported when ethylene was applied at late ripening developmental stages, our findings support the hypothesis of a temporal-specific ethylene role in the ripening of strawberry fruits.

Interactions Between Strawberry ABA Receptor PYR/PYLs and Protein Phosphatase PP2Cs on Basis of Transcriptome and Yeast Two-Hybrid Analyses

Strawberry (Fragaria × ananassa) is an ideal model plant for studies on non-climacteric fruit ripening. Although much progress has been made towards understanding the molecular mechanism of strawberry fruit ripening regulated by abscisic acid (ABA), little is known about the interactions between members of the FaPYR/PYL and FaPP2C families. Here, using RNA-seq, qPCR, and yeast two-hybrid analyses, we found that the FaPYR/PYL and FaPP2C gene families both have nine members, respectively, among which FaPYL2/4/8/9/11/12 and FaABI1/FaPP2C16/51/16L2/16L1/37 showed relatively higher expression at the mRNA level during ripening. The transcript abundance of candidate members of the FaPYR/PYLs and FaPP2Cs families tended to increase and decrease, respectively, at the onset of fruit reddening. In phylogenetic analyses, FaPYL2, FaPYL4, FaPYL11/FaPYL12, and FaPYL8/FaPYL9 were classified into four subgroups, and FaABI1/FaPP2C16/FaPP2C16L/FaPP2C6, FaPP2C24/FaPP2C37, and FaPP2C8/FaPP2C51 were classified into three subgroups. In yeast two-hybrid analyses, FaPYL4/9/11 and FaABI1/FaPP2C16 were able to interact with each other. ABA could not promote interactions between un-interacted pairs of FaPYL2/8/12 and FaPP2C16/51/37. FaABI1 and FaPYL2 interaction may play a major role in ripening. This is the first report on the analysis of interactions between members of the FaPYR/PYLs and FaPP2Cs families in strawberry. The results provide insights into ABA signal transduction during non-climacteric fruit ripening.

Molecular Insights into FaEG1, a Strawberry Endoglucanase Enzyme Expressed during Strawberry Fruit Ripening.

The endo-β-1,4-glucanases (EGs) that belong to the glycosyl hydrolase family 9 (GH9) have roles in cell wall synthesis, remodeling and degradation. Previous studies have suggested that EGs may play a key role in the ripening of different fruits including strawberries. In this study, we used reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assays to determine the transcript accumulation of an endo-β-1,4-glucanase (FaEG1) during fruit development in two different strawberry ‘Camarosa’ and ‘Monterey’ with contrasting softening ratios. Phylogenetic analyses suggest that FaEG1 belongs to the α group of the GH9 family with other proteins previously described with roles in elongation, abscission and ripening. Comparative modeling was used to obtain the FaEG1 structure. The model displays a α-barrel–type structure that is typical of the GH9 enzyme family, and comprises 12 α-helices, 2 310 helices and 6 β-sheets. The catalytic residues were oriented to the solvent in the middle of an open groove. Protein–ligand interactions were explored with cellulose and two xyloglucans as ligands; the results suggest that the FaEG1-cellulose and FaEG1-XXXGXXXG (the most abundant xyloglucan in strawberries) complexes were more stable complexes than XXFGXXFG. The cell wall degradation was observed by scanning electron microscopy (SEM). The data are congruent with the probable role of the FaEG1 protein in the dissembly of the cellulose-hemicellulose fraction during the ripening of strawberry fruit.

Polyamines Regulate Strawberry Fruit Ripening by Abscisic Acid, Auxin, and Ethylene.

Polyamines (PAs) participate in many plant growth and developmental processes, including fruit ripening. However, it is not clear whether PAs play a role in the ripening of strawberry (Fragaria ananassa), a model nonclimacteric plant. Here, we found that the content of the PA spermine (Spm) increased more sharply after the onset of fruit coloration than did that of the PAs putrescine (Put) or spermidine (Spd). Spm dominance in ripe fruit resulted from abundant transcripts of a strawberry S-adenosyl-l-Met decarboxylase gene (FaSAMDC), which encodes an enzyme that generates a residue needed for PA biosynthesis. Exogenous Spm and Spd promoted fruit coloration, while exogenous Put and a SAMDC inhibitor inhibited coloration. Based on transcriptome data, up- and down-regulation of FaSAMDC expression promoted and inhibited ripening, respectively, which coincided with changes in several physiological parameters and their corresponding gene transcripts, including firmness, anthocyanin content, sugar content, polyamine content, auxin (indole-3-acetic acid [IAA]) content, abscisic acid (ABA) content, and ethylene emission. Using isothermal titration calorimetry, we found that FaSAMDC also had a high enzymatic activity with a Kd of 1.7 × 10-3 m In conclusion, PAs, especially Spm, regulate strawberry fruit ripening in an ABA-dominated, IAA-participating, and ethylene-coordinated manner, and FaSAMDC plays an important role in ripening.

Characterization of an ABA-Induced and K+ Channel Gene FaKAT1 that Regulates Strawberry Fruit Ripening

Potassium (K+) plays an important role in the regulation of plant growth and development. However, how potassium works in K+ channels in strawberry fruit development is yet unknown. Here, a 2205 bp potassium channel cDNA encoding 734 amino acids, named FaKAT1, was cloned from strawberry fruits. Bioinformatics analysis showed that the cis-element ABRE responsive to ABA and conserved domains characteristic to the KAT1 superfamily were found in the promoter and encoding protein of FaKAT1. Transcription analysis revealed that FaKAT1 was expressed higher in stems and leaves, followed by fruits, but was less in roots and flowers, and that coupled with the fruit red-coloring, its transcripts increased rapidly, suggesting that FaKAT1 might play a role in ripening. Using tobacco rattle virus-induced gene silencing, downregulation of the FaKAT1 transcripts resulted in significant unripening of fruits, which is consistent with changes of the ripening-related events and genes, such as fruit firmness, contents of titratable acid, sugar, anthocyanin, and abscisic acid (ABA), as well as transcripts of PYR1, ABI1, SnRK2, GAL6, PE5, EXP2, XYL2, CA4H, CHS, CHI, DFR, and 4CL. Furthermore, ABA and the ABA inhibitor, fluridone, promoted and inhibited the mRNA expression levels of FaKAT1, respectively. Importantly, a K+ channel blocker, tetraethylammonium, also inhibited FaKAT1 expression and fruit red coloring. In conclusion, FaKAT1 is an ABA-induced K+ channel, which regulates strawberry fruit ripening.

Identification, cloning and characterization of the tomato TCP transcription factor family.

BackgroundTCP proteins are plant-specific transcription factors, which are known to have a wide range of functions in different plant species such as in leaf development, flower symmetry, shoot branching, and senescence. Only a small number of TCP genes has been characterised from tomato (Solanum lycopersicum). Here we report several functional features of the members of the entire family present in the tomato genome.ResultsWe have identified 30 Solanum lycopersicum SlTCP genes, most of which have not been described before. Phylogenetic analysis clearly distinguishes two homology classes of the SlTCP transcription factor family - class I and class II. Class II differentiates in two subclasses, the CIN-TCP subclass and the CYC/TB1 subclass, involved in leaf development and axillary shoots formation, respectively. The expression patterns of all members were determined by quantitative PCR. Several SlTCP genes, like SlTCP12, SlTCP15 and SlTCP18 are preferentially expressed in the tomato fruit, suggesting a role during fruit development or ripening. These genes are regulated by RIN (RIPENING INHIBITOR), CNR (COLORLESS NON-RIPENING) and SlAP2a (APETALA2a) proteins, which are transcription factors with key roles in ripening. With a yeast one-hybrid assay we demonstrated that RIN binds the promoter fragments of SlTCP12, SlTCP15 and SlTCP18, and that CNR binds the SlTCP18 promoter. This data strongly suggests that these class I SlTCP proteins are involved in ripening. Furthermore, we demonstrate that SlTCPs bind the promoter fragments of members of their own family, indicating that they regulate each other. Additional yeast one-hybrid studies performed with Arabidopsis transcription factors revealed binding of the promoter fragments by proteins involved in the ethylene signal transduction pathway, contributing to the idea that these SlTCP genes are involved in the ripening process. Yeast two-hybrid data shows that SlTCP proteins can form homo and heterodimers, suggesting that they act together in order to form functional protein complexes and together regulate developmental processes in tomato.ConclusionsThe comprehensive analysis we performed, like phylogenetic analysis, expression studies, identification of the upstream regulators and the dimerization specificity of the tomato TCP transcription factor family provides the basis for functional studies to reveal the role of this family in tomato development.

Network Inference Analysis Identifies an APRR2-Like Gene Linked to Pigment Accumulation in Tomato and Pepper Fruits

Carotenoids represent some of the most important secondary metabolites in the human diet, and tomato (Solanum lycopersicum) is a rich source of these health-promoting compounds. In this work, a novel and fruit-related regulator of pigment accumulation in tomato has been identified by artificial neural network inference analysis and its function validated in transgenic plants. A tomato fruit gene regulatory network was generated using artificial neural network inference analysis and transcription factor gene expression profiles derived from fruits sampled at various points during development and ripening. One of the transcription factor gene expression profiles with a sequence related to an Arabidopsis (Arabidopsis thaliana) ARABIDOPSIS PSEUDO RESPONSE REGULATOR2-LIKE gene (APRR2-Like) was up-regulated at the breaker stage in wild-type tomato fruits and, when overexpressed in transgenic lines, increased plastid number, area, and pigment content, enhancing the levels of chlorophyll in immature unripe fruits and carotenoids in red ripe fruits. Analysis of the transcriptome of transgenic lines overexpressing the tomato APPR2-Like gene revealed up-regulation of several ripening-related genes in the overexpression lines, providing a link between the expression of this tomato gene and the ripening process. A putative ortholog of the tomato APPR2-Like gene in sweet pepper (Capsicum annuum) was associated with pigment accumulation in fruit tissues. We conclude that the function of this gene is conserved across taxa and that it encodes a protein that has an important role in ripening.

Control of ripening in transgenic tomatoes

Major progress has been made over the last few years in the identification and regulation of tomato ripening genes. At least 25 genes showing elevated expression during ripening have been cloned and several, including polygalacturonase, which modifies fruit textures, have been shown to be ripening-specific. In addition, genes have been cloned for ACC synthase and ACC oxidase, which control the synthesis of ethylene, which plays a critical role in ripening. Inhibition of expression of polygalacturonase, pectinesterase, ACC synthase, ACC oxidase and phytoene synthase has been achieved in transgenic plants, using antisense technology. The expression of several genes has also been inhibited by sense gene suppression. New traits caused by these transgenes are stably inherited. Antisense tomatoes with reduced polygalacturonase have improved textural qualities which are being exploited commercially for the fresh and processed markets. Overexpression of phytoene synthase has been shown to restore carotenoid production in the yellow flesh mutant and can be used to enhance colour in other cultivars. Antisense fruit in which ACC synthase or ACC oxidase are inhibited show slower ripening and reduced over-ripening. ACC oxidase antisense genes have also been shown to delay leaf senescence. It is to be expected that further genes determining other quality traits will be identified and manipulated soon.

Metabolism of Ribonucleic Acid and Protein in Ripening Apple Fruits

Summary In climacteric apples, the incorporation of 3 H-uridine and 3 H-leucine into RNA and protein significantly increased along with increases in total polysomal RNA and Poly(A) +RNA, indicating that increased RNA and protein production plays an important role in ripening. There were substantial differences between the outer and inner tissue zones in soluble-solids content, RNA and protein synthetic rate, and protein content. The relatively high soluble-solids content in the outer tissue was associated with relatively high RNA and protein synthesis as well as high protein content. The formation of the ripening gradient is discussed.

Microbiological and physicochemical characteristics of Gamonedo blue cheese during ripening

Changes in the microflora, physicochemical characteristics and the levels of proteolysis and lipolysis in Gamonedo cheese were examined over a 180 day ripening period. The total microbial count was high throughout manufacture and the ripening period. Growth of lactic acid bacteria was greater in the interior of the cheese than on the surface and among the lactic acid bacteria, lactobacilli predominated. Levels of enterococci and micrococci reached high numbers during the latter stages of maturation which suggests that these organisms may play a significant role in ripening. Of the lactic acid bacteria. Lactobacillus plantarum was present at the highest levels, followed by L. casei spp. casei. Low levels of lactic streptococci and leuconostocs were detected and the species identified included Lactococcus lactis spp. lactis, Leuconostoc mesenteroides spp. mesenteroides and L. paramesenteroides. Levels of proteolysis and lipolysis were high: water soluble nitrogen, NPN and NNH2 levels in the mature cheese were 57·3%, 51·7% and 31·2% of total N, respectively. The degradation of αs- and β-caseins was almost complete at the end of ripening. The level of total FFA was 75 684 mg/kg.