Fruit development and ripening are unique to plants and represent an important component of human and animal diets. Recent discoveries have shed light on the molecular basis of developmental ripening control, suggested common regulators of climacteric and nonclimacteric ripening physiology, and

Genetic regulation of fruit development and ripening

Pigments are present in all living matter and provide attractive colors and play basic roles in the development of organisms. Human beings, like most animals, come in contact with their surroundings through color, and things can or cannot be acceptable based on their color characteristics. This review presents the basic information about pigments focusing attention on the natural ones; it emphasizes the principal plant pigments: carotenoids, anthocyanins, and betalains. Special considerations are given to their salient characteristics; to their biosynthesis, taking into account the biochemical and molecular biology information generated in their elucidation; and to the processing and stability properties of these compounds as food colorants.

Natural Pigments: Carotenoids, Anthocyanins, and Betalains — Characteristics, Biosynthesis, Processing, and Stability

Excessive softening is the main factor limiting fruit shelf life and storage. Transgenic plants modified in the expression of cell wall modifying proteins have been used to investigate the role of particular activities in fruit softening during ripening, and in the manufacture of processed fruit products. Transgenic experiments show that polygalacturonase (PG) activity is largely responsible for pectin depolymerization and solubilization, but that PG-mediated pectin depolymerization requires pectin to be de-methyl-esterified by pectin methylesterase (PME), and that the PG β-subunit protein plays a role in limiting pectin solubilization. Suppression of PG activity only slightly reduces fruit softening (but extends fruit shelf life), suppression of PME activity does not affect firmness during normal ripening, and suppression of β-subunit protein accumulation increases softening. All these pectin-modifying proteins affect the integrity of the middle lamella, which controls cell-to-cell adhesion and thus influences fruit texture. Diminished accumulation of either PG or PME activity considerably increases the viscosity of tomato juice or paste, which is correlated with reduced polyuronide depolymerization during processing. In contrast, suppression of β-galactosidase activity early in ripening significantly reduces fruit softening, suggesting that the removal of pectic galactan side-chains is an important factor in the cell wall changes leading to ripening-related firmness loss. Suppression or overexpression of endo-(1\to4)β-d-glucanase activity has no detectable effect on fruit softening or the depolymerization of matrix glycans, and neither the substrate nor the function for this enzyme has been determined. The role of xyloglucan endotransglycosylase activity in softening is also obscure, and the activity responsible for xyloglucan depolymerization during ripening, a major contributor to softening, has not yet been identified. However, ripening-related expansin protein abundance is directly correlated with fruit softening and has additional indirect effects on pectin depolymerization, showing that this protein is intimately involved in the softening process. Transgenic work has shown that the cell wall changes leading to fruit softening and textural changes are complex, and involve the coordinated and interdependent activities of a range of cell wall-modifying proteins. It is suggested that the cell wall changes caused early in ripening by the activities of some enzymes, notably β-galactosidase and ripening-related expansin, may restrict or control the activities of other ripening-related enzymes necessary for the fruit softening process.

Cell wall metabolism in fruit softening and quality and its manipulation in transgenic plants

Embryonic development in many angiosperms occurs concomitantly with the development of the ovary into a specialized organ, the fruit, which provides a suitable environment for seed maturation and often a mechanism for the dispersa1 of mature seeds, as Darwin observed. Despite centuries of intensive genetic selection of agriculturally valuable fruit, we still lack most information about how fruits develop, how this development is coordinated with embryonic development and seed formation, and the molecular, cellular, and physiological events that control fruit growth and differentiation. The last 10 years have seen a rapid surge of information on one commercially important aspect of fruit development, fruit ripening, including the genetic control of temporal events during the ripening phase (Theologis, 1992; Theologis et al., 1992). However, fewer advances have been made on temporal and spatial controls of fruit set and growth, although from the agricultura1 point of view, these aspects are of equally critical importance. We will provide a perspective on the molecular, cellular, and physiological mechanisms that must be considered as integral parts of the fruit developmental process. The discussion below will illustrate that fruit development is a potentially useful system to learn more about complex regulatory mechanisms that control the division, growth, and differentiation of plant cells.

Fruits: A Developmental Perspective.

Recombinant polynucleotides and recombinant polypeptides useful for improvement of plants are provided. The disclosed recombinant polynucleotides and recombinant polypeptides find use in production of transgenic plants to produce plants having improved properties.

Nucleic acid molecules and other molecules associated with plants and uses thereof for plant improvement

Regulation of expression of specific genes by antisense RNA is a naturally occurring mechanism in bacteria1,2, although gene regulation by this mechanism has not yet been observed in higher eukaryotes. However, antisense RNA has been shown to reduce expression of specific genes when injected into frog oocytes3 and Drosophila embryos4. Inhibition of expression of artificially introduced genes has been demonstrated by transient expression of antisense RNA constructs in mammalian cells5,6, and plant protoplasts7, and by stable expression in transgenic plants8. Here, we report a striking inhibition of expression of the endogenous, developmentally regulated gene for polygalacturonase in stably transformed tomato expressing antisense RNA.

Antisense RNA inhibition of polygalacturonase gene expression in transgenic tomatoes

Tomato plants were transformed with a chimaeric polygalacturonase (PG) gene, designed to produce a truncated PG transcript constitutively. In these plants expression of the endogenous PG gene was inhibited during ripening, resulting in a substantial reduction in PG mRNA and enzyme accumulation. This inhibition was comparable to that achieved previously using antisense genes. The expression of the truncated gene in ripe fruit was substantially lower than its expression in green fruit. Thus expression of both the endogenous and truncated genes is reduced in ripe fruit in which both are active. The implication of this observation is discussed in relation to the possible mechanism whereby sense constructs inhibit gene expression.

Expression of a truncated tomato polygalacturonase gene inhibits expression of the endogenous gene in transgenic plants.

Polygalacturonase (PG) is the major cell wall degrading enzyme of tomato fruit It is developmentally regulated and is synthesised de novo in ripening fruit Genomic clones encoding a PG gene of tomato (Lycopersicon esculentum Mill cv Ailsa Craig) have been isolated, mapped and sequenced The sequence of the protein-coding region is identical to that of a PG cDNA [20] Comparison of the cloned restriction fragments with genomic Southern data suggests that there may only be one gene for PG per haploid genome The PG gene, which covers approximately 7 kb, is interrupted by 8 intervening sequences ranging in size from 99 bp to 953 bp The transcription start point was identified by S1 mapping and primer extension analysis About 14 kb of 5′ flanking DNA has been sequenced This contains putative TATA and CAAT boxes and also direct repeat sequences A transcriptional fusion has been constructed between the putative 14 kb promoter fragment and the chloramphenicol acetyl transferase (CAT) gene Constructs containing this gene have been transferred to tomato using binary vectors Regenerated transgenic plants express CAT in ripe tomato fruit, but not in unripe tomatoes, leaves, or roots

The tomato polygalacturonase gene and ripening-specific expression in transgenic plants.

The 2a isoenzyme of tomato polygalacturonase was purified from ripe fruit and characterised. The N-terminal amino acid sequence of the protein was determined in order to identify polygalacturonase cDNA clones. The nucleotide sequence of a ripening-related cDNA (pTOM 6) was determined and found to encode the N-terminal sequence of mature polygalacturonase 2a. The complete open reading frame encodes a polypeptide of molecular weight 50,051, including a putative pre-sequence of 71 amino acids.

Sequencing and identification of a cDNA clone for tomato polygalacturonase.

Transgenic tomato plants expressing antisense RNA to a ripening related gene (pTOM5) have yellow ripening fruit and pale colored flowers. The yellow fruit color is correlated with a severe reduction in the level of the pTOMS gene mRNA during fruit ripening. The level of carotenoids in ripening fruit from selected transgenic plants showing yellow fruit was reduced by more than 97 percent. In addition, the carotenoid lycopene, which is primarily responsible for the red color of ripening fruit, was reduced to undetectable levels (<0.1%). These data indicate that the pTOM5 gene is crucial to tomato fruit carotenoid biosynthesis.

John A. Ray

Papers

Antisense RNA inhibition of polygalacturonase gene expression in transgenic tomatoes

Expression of a truncated tomato polygalacturonase gene inhibits expression of the endogenous gene in transgenic plants.

The tomato polygalacturonase gene and ripening-specific expression in transgenic plants.

Sequencing and identification of a cDNA clone for tomato polygalacturonase.

Using Antisense RNA to Study Gene Function: Inhibition of Carotenoid Biosynthesis in Transgenic Tomatoes