Arabidopsis thaliana

Arabidopsis thaliana is a model system for the genetic study of cruciferous crop plants because it shows a wide genetic diversity and in comparison to oilseeds, it is small, with a rapid growth cycle, possesses novel genetic characteristics and importantly shows significant genetic homology to the related B. napus. Arabidopsis has a low chromosome number and simple structure (almost identical to B. napus growth and development), subsequently it has been used as a subject for experiments in classical plant genetics for 50 years and over the past few years has been adopted for investigations into many problems in plant physiology, biochemistry and development, with the potential to understand how to manipulate such plant processes as pod dehiscence to improve yield.

Physiology And Biochemistry Of Fruit Development

There are a number of different ecotypes of Arabidopsis that show natural variation due to their broad range of habitat colonisation. These adaptations and variations have been identified in all areas of its development physiology, including flower timing, fruit set and leaf morphology, but unfortunately no significant variations have been observed for fruit dehiscence.

At present the majority of investigations utilise the Columbia or Landsberg erecta (erecta mutation causes short and erect growth) phenotype. Gene expression analysis within shoot apical meristems has shown that a characteristic change corresponds to the essential switch from vegetative to reproductive growth, whereby the meristem becomes transformed from its vegetative state to an inflorescence state. The subsequent forming stem and floral meristems show a species dependent, determinate or indeterminate growth pattern (Weberling, 1989). Arabidopsis and Brassica species (eg. B. napus) are indeterminate and follow monocarpic growth, so they rarely produce a terminal flower, growth terminates with the senescence of the shoot apical meristem and the entire plant senesces after reproductive growth.

The gynoecium is the female part of the flower that develops into the fruit, consisting of two carpels (one or more in other plants) that form a stigma to which pollen binds for germination. The ovules (within ovary) lie in locules and each locule is separated by a septum. The fruit wall (pericarp) is differentiated into three layers:

Post-fertilisation events for fruit ripening mainly involve seed and fruit wall development (Fisher & Bennett, 1991). The physical and biochemical changes that occur during fruit maturation have been extensively studied for B. napus, with regards to the dehiscence zone (DZ) formation and carpel wall differentiation (Meakin & Roberts, 1990a,b; Whitelaw et al, 1999 & references there in). Fruit maturation culminates in dehiscence (see pod shatter in B. napus), which is similar to both senescence and abscission. Senescence relates to the programmed cell death within the DZ that facilitates cell separation and/or rupture, whilst abscission merely refers to the shedding of plant organs by the enzymic degradation of AZ middle lamella and cell walls that results in the loss of cellular cohesion. As well as the physical comparison, there is also biochemical homology with regards to the enzymes and plant hormones that have been identified, such as the stimulatory/enhancing effect of ethylene, auxin inhibition and cellulase & polygalacturonase cell wall degradation (Thimann, 1980).

Pod shatter is a significant agronomic trait in terms of seed loss, so its study in Arabidopsis could potentially provide lines of shatter resistant oilseed crops. There are two main areas of investigation:

The process of synchronisation is considered very difficult as it would require the understanding of many of the plants developmental pathways, including knowledge about the indeterminate nature of the inflorescence meristem. Therefore most interest is concentrated on methods (ie. genetic engineering) for modifying the pod tissues, to produce for example, pods with reduced levels of exocarp desiccation (reduces physical stress on pod) or altered enzyme activity within the DZ (prevent middle lamella degradation). However, although it is important to develop these new shatter resistant varieties, we also need to understand the physiological and chemical patterns of the tissues associated with pod shatter.

Spence et al (1995) provided evidence as to the areas of development which are intrinsically linked to the shattering characteristic of Arabidopsis thaliana. The developmental differentiation of the inner most layer of the pod wall (enb of endocarp) was shown to be significantly different in shatter susceptible Arabidopsis and B. napus varieties when compared to the shatter reduced/resistant varieties of B. juncea. The cells of the enb layer undergo substantial elongation, followed by the formation of a heavily lignified, single-celled secondary wall adjacent to ena and stretches around the DZ to the exocarp. This physical isolation of the ena from the other tissues causes its rapid senescence, but the effect of these ena cells doesn't seem to be important.

Dehiscence requires both adequate pod maturation and mechanical stimulation (pod desiccation induced tensions and other factors [eg. wind]) to cause the cells of the DZ to separate (Meakin & Roberts, 1990a,b). Spence et al (1995) also investigated the occurrence of random, explosive shattering events. They believe that the enb cells of the endocarp (previously shown to have characteristic differentiation patterns depending on their line of shatter susceptibility) are responsible for the generation of the tensions associated with the explosive shattering. The heavy lignification of enb cells of shatter susceptible varieties, form a continuous rigid wall arcing convexly around the pod. Therefore during desiccation, only the mesocarp and exocarp tissues contract to stress the inflexible enb layer, so that it becomes 'sprung'. This force concentrates at the thin layer of parenchymatous cells of the DZ which have simultaneously undergone reduced cell-to-cell cohesion. Shatter resistant lines of B. juncea show both reduced lignification of the secondary walls of the enb layer and no lignification of the primary walls and middle lamella regions between the cells so that they remain pectin rich. Pectin would increase flexibility of the layer so that the desiccation induced tensions within the carpel valve would be dramatically reduced, as would be the shatter susceptibility. The breeding of pod shatter resistant varieties in both Arabidopsis and B. napus may therefore lie in the genetic engineering the enb reduced lignin deposition.

Arabidopsis thaliana has also played a role in the determination and characterisation of the mRNA's and their proteins that are differentially expressed in cells of the DZ during the latter stages of pod development. Over past years the enzymes involved in dehiscence have been identified, such as cellulase and polygalacturonase (PG), but the latest research has focused on understanding the intricate signal transduction pathways that regulate such gene expression. Arabidopsis has had components identified from their pod DZ's that show close homology to genes of bacterial, two-component-like proteins which are responsible for the reception and transduction of external signals (Chang et al, 1993; Hua et al, 1995; Kakimoto, 1996; Imamaura et al, 1998; Urao et al, 1998; Brandstatter & Kieber, 1998; Sakakibara et al, 1998). There are three main genes that have been associated with the DZ gene regulation, these being:

Both ETR1 and CKI1 have been implicated with a plant response to the phytohormones, ethylene (Chang et al, 1993) and cytokinin (Kakimoto, 1996) respectively. Both of these proteins consist of a variable N-terminus and a histidine kinase domain which is linked to a response regulator (ie. self-contained sensor/regulator). The ERS gene encodes another type of ethylene receptor that doesn't contain a receiver domain, so it is assumed that it interacts with a different upstream component as for bacterial systems (Hua et al, 1995). Jenkins et al (1999) characterised the SAC29 gene in B. napus by comparisons with such bacterial two-component-like proteins, identifying it as just encoding a receiver domain of a response regulator. Thus the SAC29 protein could interact with an equivalent protein to the ERS in Arabidopsis, thereby creating the first step of an ethylene mediated dehiscence transduction mechanism in B. napus.

ETR1 and ERS interact directly with CTR1 (a Raf-related kinase) to induce the ethylene response (Clark et al, 1998). It is under the control of the response regulator, CheY, which is similar to the well research Ras protein in structure, binding and function (Chang et al, 1993). ETR1 and CKI1 both consist of a sensor, thus specifying the proteins to be membrane-boun, thus indicating the need for a cytoplasmic element for complete signal transduction.

The information provided by the research into Arabidopsis has enabled such homologous studies of the genes expressed in the DZ of B. napus to ascertain the molecular mechanisms involved in stimulation and/or tuning of the dehiscence process for further manipulative investigations into shatter resistance.