Abscission

Abscission is common in the life-cycle of herbaceous plants, involving the process of organ shedding of leaves, stems and reproductive structures (eg. flower buds) such as floral organs and fruits (Sexton & Roberts, 1982). It occurs at pre-determined sites, with the loss of cell-to-cell cohesion within the abscission zone (AZ). Abscission has two distinct roles, one enables propagation (active shedding of structures which can become independent individuals) and the other is the shedding of organs that have reached the end of their requirement (Osborne, 1989). They exhibit a characteristic, temporal shedding pattern with regards to the various tissues involved. Pod dehiscence (shatter) is therefore classed as a form of reproductive abscission as it shares many similarities with the general mechanism of abscission.

The process of abscission is highly co-ordinated and once induced (see diagram below) a series of physiological and molecular changes occur that culminate with the dissolution of the middle lamella and cell walls at precise locations (ie. abscission zones, AZ). There are two main enzymes which show increased hydrolytic activity and are tightly linked both temporally and spatially to the abscission event. These are a cellulase (Beta-1,4-glucanase) and polygalacturonase (PG), both of which have been isolated during dehiscence.

Abscission has been studied for many years as crop yield can be seriously affected by premature reproductive abscission. For crops such as broad bean (Vicia faba), premature reproductive abscission can drastically reduce yields to a non-profitable state (Baylis & Clifford, 1991) and is often responsible for extreme yield variability. Clifford et al (1992) explained the genetic basis for reproductive abscission patterns, as well identifying an environmental factor (eg. stress, by chemical or physical induction). Therefore the understanding of abscission at the molecular level is crucial so that genetic engineering can be utilised to prevent or reduce this premature abscission. Pod shatter was initially believed to be an abscission event as it too involves cell separation after cell wall and middle lamella dissolution (now shown to only be similar), thus prompting further investigations because of the associated significant yield losses.

Determinating The Biochemistry Of Abscission

The process of abscission occurs due to the co-ordinated action of enzymes which cause the degradation of the cell walls and middle lamella of cells within the abscission zone (AZ). The first enzyme to be identified that acts to breakdown cell walls of the AZ was a pectin methyl esterase (PME) (Sexton & Roberts, 1982). PME causes the solubilisation of cell wall pectic material, however this initial evidence is subjective as to whether PME actually plays a role in abscission.

Cellulase (Beta-1,4-glucanase) was the next enzyme to be isolated, but unlike PME it is clearly involved as it has been temporally and spatially correlated to the AZ (Horton & Osborne, 1967; Lewis & Varner, 1970). Two types of cellulase were characterised in the AZ of bean (Phaseolus vulgaris), separated by their isoelectric points. They have both been shown to increase within the AZ, although they have different temporal relationships. This led to the hypothesis of enzymic activation at the onset of abscission, but further enzyme analysis revealed that they actually correspond to two different genes (del Campillo et al, 1988). This discovery was very important as it provided the first real evidence about gene regulation during abscission, prompting Tucker et al (1988) to investigate. They isolated a cDNA that encoded an AZ cellulase from bean and used the cDNA as a probe to illustrate that ethylene can induce abscission and the associated rise in cellulase gene expression. Antagonistically, auxin (indole acetic acid, IAA) acts to repress both abscission and cellulase gene expression. From cellulase antibody and mRNA hybridisations, cellulase accumulation within the AZ cortical cells was visibly confirmed (del Campillo et al, 1990; Tucker et al, 1991). Equivalent studies in elder (Sambucus nigra) have also located a cellulase accumulation specifically within the AZ and is also up-regulated by ethylene exposure (Webb et al, 1993; Roberts et al, 1993).

Ethylene is renowned for its promotion of abscission and can be produced by tissues adjacent to the AZ, or contained in the atmosphere as a result of pollutants or other local plants and micro-organisms. The cells in the AZ that respond to ethylene by enlargement (and inhibited by auxin) are classed as type II, as opposed to type I (ethylene inhibits and auxin stimulates elongation) cells which comprise the main body of the plant.

As well as cellulase, polygalacturonase (PG, pectinase) is also involved in cell wall degradation within the AZ, although early studies had been unsuccessful in ascertaining any relationship (Sexton & Roberts, 1982). The role of PG in fruit ripening has been studied for many years as a potential means of shelf-life enhancement and uniform ripening. Therefore many PG analytical techniques were developed, enabling Tucker et al (1984) to identify the up-regulation of AZ PG during tomato flower abscission. PG expression during tomato leaf abscission was studied by Taylor et al (1990), who transformed plants with an antisense gene for the previously identified flower abscission PG. An antisense gene copy is used to down-regulate (to less than 1% of normal) the endogenous gene expression by sequestering its mRNA (complementary mRNA binding renders it untranslatable). However this failed to affect leaf abscission, later explained after leaf and flower abscission PG mRNA hybridisation studies revealed that the two PG's were coded by two different genes (ie. tissue specific PGs).

Ethylene has already been considered to be involved but its mechanism of action is still unclear, as it may induce or merely accelerate abscission. Initial studies have utilised the information from both tomatoes and Arabidopsis thaliana, as they have had genes identified for regulating ethylene sensitivity. A. thaliana has an ethylene receptor, ETR1, mutants of which cause delayed petal shedding coupled with unaltered dehiscence. The ethylene insensitive 'Never ripe' tomato has a homologous gene which when mutated also causes delayed, but not inhibited abscission. Therefore such genes may be responsible for modifying the abscission process, in conjunction with numerous other genes yet to be characterised in the signal-transduction-response pathway. The research into the direct effect of ethylene is important, but other chemical and physical factors that might further influence the ethylene biosynthetic pathway may play a crucial role in understanding abscission, and even dehiscence! For example, polyamines and abscissic acid both stimulate abscission (see diagram) but their mode of action is unknown (Gonzalez-Carranza et al, 1998).

The two main enzymes, cellulase and PG play a key role in abscission, but other proteins have also been shown to increase (amount or activity) in association with abscission. Peroxidases are postulated to act by either oxidating IAA (auxin), thereby removing its retarding effect or by regulating cell wall rigidity as changes in activity could influence cell wall loosening. Another protein shows homology with metallothioneins and may act to stimulate pathogenesis-related (PR) protein synthesis as a means of protecting the newly exposed fracture surface against bacterial and fungal pathogens (Gonzalez-Carranza et al, 1998).