Antisense RNA in Post Harvesting Improvement of Plants

In this article we will discuss about the antisense RNA in post harvesting improvement of plants.

Engineering of Fruit Ripening Process:

Efficacy of antisense RNA technology in post harvesting has been implicated well in extending shelf life of fruits, vegetables and vase life of flowers. Infact the first genetically modified products commercialized in markets were calgenes flavr savr tomatoes. Antisense RNA technology was employed by calgene Inc in the development of the flavr savr tomato.

The creation of the flavr savr tomato by calgene Inc involved wealth of experimented infor­mation and expertise in the delayed softening technique. Before understanding the implica­tion of biotechnology in extending shelf-life of several fruits and cut flower it is indispensable to understand basics of various physical process such as ripening role of ethylene and its signalling etc.

Basics of Fruit Ripening:

Fruit ripening is an active complex process. Ripening is well controlled by genetically programmed process resulted in various changes like color, flavor, change of texture of the fruit. Both biochemical and genetic level have been studied extensively in view of its economic poten­tial.

Among several fruits, few of them require ethylene burst for normal ripening. Based on fruit ripening mechanism two types of fruits have been considered. One is climacteric fruit in which fruit ripening is accompanied by burst of respiration and high level of ethylene will be produced.

In the second category, non-climacteric, respiration is nominal without attaining peak and ethylene production is kept at low level. Some of the classic examples of climacteric fruits are tomatoes, apple, watermelon and banana. In all climacteric fruits, ethylene produc­tion is highly indispensable for normal fruit ripening.

Softening and ripening are the committed processes responsible for increase in palatability of fruits. Enzymology of fruit ripening has been well recorded. Ripening is a complex and highly coordinated developmental events in plants. Temporal separation between soften­ing and other ripening functions are possible because each ripening process is regulated inde­pendently.

The process is well mediated by separate classes of enzymes and separate genes. During ripening of fruits, number of ripening specific genes is activated. The prominent ones are genes that code for polygalactoronase (PG) and aminocyclopropane carboxylic acid (ACC) synthesis in tomato and cellulases in avocado.

Several tropical fruits soften rapidly during ripening such as banana, mango and papaya. These tropical fruits have shorter shelf-life of about only one to two weeks. Softening of mango and guava and other fruits are characterized by an increase in solubility of cell wall pectin and its solubilization during ripening and which is accompanied by pectin depolymerization.

Extensive softening in mango and papaya is related to extensive modification of the wall pectins. In mango, pectin solubilization in inner and outer mesocarp tissue have been found to very difficult. Depolymerization takes place quite earlier in the inner mesocarp than the outer mesocarp tissue. Similarly, ACC syn­thesis actively in outer and inner mesocarp tissue was found to be more or less equally.

Cell wall hydrolases are implicated in the softening of fruits. Polygalactoronase (PG), the enzyme responsible for degrading (1 – 4) linked galacturon. Activity of PG in different fruits is comparable. In spite of low PG level in mango and papaya, softening process proceeds at much faster rate as tomato indicating that mango and papaya PG functions more efficient than to­mato PG. Pectin esterase (PE), responsible for desterification of ethyl group from acidic protein can be detectable in ripening mango and papaya including banana.

In addition, other cell wall hydrolases detected in ripening fruits are cellulase, xylanase, mannosidase and galactosidase. Both cellulase and xylanase activity get increased during mango. In Mango β-galactosidase is presumed to degrade (1-4) linked galactopyranose. Its activity increases during ripening. β-galactosidase can degrade neutral pectin polymers during ripening of tropical fruits.

When esterification process drop from 90% in mature green fruit to 35% in red ripe fruit and this makes the polyuronide susceptible to degradation by PG. Pectin methyl esterase (PME) is present as a small gene family in tomato. PME protein is found to be distributed in most plant tissues. Suppression of PME in over ripens fruit affect tissue integrity.

Therefore, PME play a little role in ripening but does affect fruit senescence. In addition, expansions are cell wall local­ized enzymes that are thought to cause cell wall loosening by reversible disrupting the hydro­gen bonds between cellulose micro-fibrils and matrix polysaccharides. During tomato development atleast six expansion genes are expressed of which Exp₁ is ethylene regulated.

Climacteric fruit such as tomato is characterized by burst of respiration during ripening followed by high level accumulation of ethylene. As a result, softening and changes to colour and flavour takes place particularly from green to red increases flavour.

Colour changes are due to chlorophyll degradation and the production of red pigment, lycopene. Starch is broken down and sugar accumulation leads to flavour changes. Softening of tomato is mainly due to soften­ing enzymes such as polygalactoronase (PG) and pectin methyl esterase.

Regulation of ethylene synthesis is controlled by two systems, operates in climacteric fruits. In one of the systems ethylene production is at nominal level in all tissues during vegeta­tive growth including those of non-climacteric fruits.

Another system operates during the ripening of climacteric fruit result in senescence of some petals. Ethylene can diffuse from cell to cell result in ripening in one of the regions, spreading to neighbouring region. Fine integration of ethylene in all the regions of the fruit triggers ripening process.

Enzymology and Biosynthesis of Ethylene:

Ethylene hormone is gaseous in nature. The double bond (C = C) in the structure is indispensable for its action and CO₂ made by replacing one carbon with oxygen (C = O), acts as a competitive inhibitor of ethylene. Ethylene biosynthetic pathway is well established. Biosynthesis operates in very simple pathway.

It is formed from amino acid methionine via adomet-S-adenosyl-L methionine and the cyclic non-protein amino acid 1-amino cyclopropane 1-carboxylic acid (ACC). The action of ACC synthase (ACS) transforms adomet (i.e., adenosyl methionine SAM) into Aminocyclopropane-l-carboxylic acid and the conversion of ACC to eth­ylene is done by ACC oxidase (ACO).

Both ACC synthase (ACS) and ACC oxidase (ACO) enzymes are encoded by small multigene families and various factors like environmental and hormonal signal regulate their expression.

(i) ACC synthase:

In climacteric fruit, atleast eight ACS genes have been characterized. They are LEAGSIA, LEAcS-1B and LEAcS-2-7. It has been shown that ACS genes are involved in the production of two system of ethylene. For example, system 1 ethylene is produced by LEAcS1A and LEAcS 6 in green fruit.

System 1 ethylene production continues during fruit development until ripening is attained. LEAcS1A expression increases and LEAcS4 expression induced. The second cat­egory system 2 ethylene synthesis takes place during this transition period and maintained by ethylene dependent induction of LEAcS-2 and LEASc6 (Fig. 22.5).

In some of the mutants like never ripe (Nr) mutants fails to percept ethylene due to mutation in the ethylene binding do­mains of the Nr ethylene receptor. Other mutant ripening inhibitor (rin) mutants do not exhibit ethylene production and fails to transmit ethylene signal downstream to ripening genes due to mutation in the rin transcription factor. These two mutants have shown that ethylene is indispensible for the expression of EACS 2. Whereas LEAcS1A exhibits delayed expression in never ripe mutants clearly shows that the regulation of ripening is independent of ethylene.

(ii) ACC Oxidase (ACO):

Participation of ACO in the regulation of ethylene biosynthesis has become reality dur­ing ripening of fruit. Antisense expression of clone PTOM13 reveals identification of Acogene in ripening tomato fruit and wounded leaves. ACC oxidase enzyme belongs to members of Fec11- dependent family of oxidases.

Invivo expt, shows that the activity of ACO is influenced by CO₂ production during climacteric peak. Subsequent experiment shows further three ACO genes in tomato in response to wounding, during flower development and flower senescence in addition to final ripening.

Ethylene Perception and Signalling in Ripening Fruit:

Climacteric fruit ripening in tomato is induced by burst of ethylene production. This gaseous hormone, for example, cannot alone induce green immature fruit to ripen rapidly but persistant exposure could bring about shortening of green life as in banana.

Signal transduction in ripening fruit is perceived by the presence of ethylene receptors that have a sensor domain. Ethylene receptor contains several different domains and is subdi­vided into GAF domain, transmembrane domain, response domain and histidine kinase do­main. Certain co factors like copper help in the binding of ethylene to the receptor and passes the signal downstream.

In addition, ethylene signal transduction pathway contains another component CTRI a protein kinase component, which is next component of the signal transduction pathway and involve in negative regulator of the signal transduction pathway. Structurally CTRI, shows homology to Raf family of mitogen activated protein kinase kinase kinase (MAP3K).

A protein kinase CTR-1 interacts with two ethylene receptor, ETR1 and ERS1. According to the model on mechanism of ethylene receptor action, in absence of hormone, signal to the negative regulator CTRI and the completely blocked signal pathway (Fig. 22.6). Binding of ethylene by receptor release CTRI (negative regulator) results in ethylene response.

In another model of ethylene signalling, receptor and TCTR2 acts as negative regulator. When ethylene binds with the receptor (A), the receptor and TCTR are inactivated i.e., no phosphorylation, no inhibition of downstream response and kinase inactivation (Fig. 22.7). As a consequence signal transduction takes place results in ripening of fruits.

In the absence of ethylene (B) or failure of ethylene binding due to mutation in the receptor TCTR is activated probably due to phosphorylation. Kinase get activated phosphorylation takes place and as a consequence inhibition of etnylene signalling takes place.

Ethylene signalling pathway shows that a family of six putative ethylene receptor like LeETR1, Le ERT-6 and two CTR₁, homology NR and TCTR2 and ER50 have been characterised in climacteric tomato. Certain receptor like LeETR₁ and LeERT₂ are expressed constitutively in every tissue type during the course of development.

LeETR5 known to express and flowering in fruit during disease attack. Involvement of receptor in ethylene signalling was confirmed by down regulation of receptor gene by antisense expression. It was however, postulates that al­though NR and LeETR as genes are up regulated during ripening, down regulation of NR by antisense have no obvious effect on ethylene signalling and ripening.

But further antisense expression reveals that LeFTR₄ role in ripening is apparent. Down regulation of LeETR4 pro­duces ethylene increases ripening. Other receptors like E1N₂, E1N₃, E1N₅ and E1N₆ acts as
positive regulating downstream in the ethylene signalling pathways. Among this E1N₂ mem­brane protein is required 4 ethylene signalling.

Whereas E1N₃ and E1N₃ like protein transcrip­tion factor that work downstream from E1N₂ and E1N₃. E1L2 binds in a sequence specific manner to the primary ethylene response element of ERF. It is an ethylene inducible gene that belongs to the primary Ethylene Responsive Element Binding Protein (EREBP) family of DNA binding protein.