BioMatNet Item: AIR2-CT93-0879 - Pod Shatter in Rape

AIR2-CT93-0879
Pod Shatter in Rape

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AIR Cluster X - Inputs for Non-Food Crops : Biotechnology : Bulk Chemicals : Crops for Bulk Chemicals : Plant Genetics : Vegetable Oil/Fat

	Proposal No:	AIR2-CT93-0879
	Date Prepared:	September 1999, January 1998
	Source:	Final Summary Report December 1996 Second Project Progress Report Summary

Final Summary Report December 1996

Summary

The aim of this project is to produce transgenic rape plants with delayed or impaired pod opening. Growing oilseed rape with a reduced tendency to shatter will lead both to better yield and to better seed quality as plants harvested at full maturity will produce seeds with a lower content of water and chlorophyll. Furthermore, less land will be needed for production at current levels with reduced inputs. This will apply to oilseed rape grown for human consumption and for non-food uses.

In order to propose engineering strategies which would turn out to be sound, careful studies of the biology of pod dehiscence were required. Such studies entailed examination of pod development at the anatomical and biophysical level, and assigning roles to endogenous hormones in involved in regulation of the developmental progression towards pod opening. Finally it entailed assigning roles to gene products which are directly involved in the cell separation events which immediately preceded dehiscence. The three areas are mutually dependent for information.

Based on the knowledge accumulated during this project we two engineering strategies have been proposed. The biochemical processes associated with cell separation in the pod dehiscence zone have been interfered with by inhibiting either the action of enzymes or the hormone-regulated processes which are involved with cell wall degradation. Although apparently separate, the two engineering strategies rely to some extent on the same genetic material. The strategy relying on interference with hormone regulation was formulated followed experiments with synthetic growth regulators combined with measurements of endogenous hormone levels during the course of pod development. Guided by theory alone virtually all classes of plant hormones could play a role in some aspect of pod dehiscence. The picture emanating from these studies shows a direct role of auxin in pod opening with ethylene playing a permissive role. The interaction between these two hormones is more complex than this simple statement may indicate. While there are proven methods of inhibiting ethylene biosynthesis, seed development will be affected by interference with ethylene production as well. From an engineering point of view it is more attractive to interfere with auxin balance in the relevant tissues, i.e. dehiscence zone and not the seeds. The project concluded with transformation of oilseed rape with a construct within which an alteration in auxin biosynthesis in the dehiscence zone is expected.

The second strategy took shape in parallel with previous anatomical studies. Immediately prior to pod opening primary cell walls of the dehiscence zone cells are observed to swell and loose material but never break. The middle lamella between these cells, on the other hand, is completely degraded which is presumably the reason why no cells are broken during pod opening despite the weakened primary walls. The strategy adopted entailed identifying the enzymes which are responsible for these changes and inhibiting their expression through antisensing. One enzyme involved in wall thinning has been identified at the biochemical level while an endo-polygalacturonase involved in middle lamella degradation has been cloned. Its promoter has been characterised (and used, in the 'auxin construct' mentioned above). The project concluded with the production of transgenic oilseed rape which is transgenic for antisense-polygalacturonase run by its native promoter.

Objectives Shattering denotes the process by which the fruit or individual seeds of a plant are released to be shed from the plant. From an agronomic point of view this shattering results in loss of yield and, in the case of oilseed rape, is an indirect cause of both seed quality and environmental problems. Shattering is mostly a problem with cultivation of dicots with an extended flowering period and many-seeded dry fruits; Brassica species, soy bean, sesame and lupin for example. Due to the unsyncronous flowering, and hence seed maturation there is no correct harvest time where all seeds are fully mature, dehydrated and with low residual chlorophyll. Quality problems may stem from harvest of immature seeds. Environmental problems arise from volunteer rape germinating the following year from shattered seeds. Extra herbicide, not always of the most environmentally friendly kind, has to be applied to fight rape as weed in subsequent crop rotations.

Traditions for how best to counter shattering varies from region to region. In some north European regions the rape crop is swathed when half, or slightly less than half of the seeds have turned dark. One or two weeks after swathing the crop is harvested. In other areas direct combining is most often used and the labour intensive swathing is saved while incurring extra loss from shattered seeds. A shatter resistant oilseed rape cultivar would render swathing superfluous. Despite substantial effort, conventional plant breeding has proven incapable of providing a solution to the problem, i.e. so far no cultivar exist carrying the trait 'shatter resistant'. There appears to be very little variation with respect to this trait within Brassica napus. Several attempts to introgress the trait from elsewhere in Brassicaceae have all been unsuccessful. The main objective is thus to solve this problem using the biotechnology, to disclose the underlying biochemistry of pod opening in oilseed rape and delineate the regulation of those genes that are found to be essential to pod opening. Using this information, strategies for interfering with pod opening will be defined in transgenic plants either by directly antisensing individual genes whose products are responsible for the biochemical processes of pod opening, or by interfering with upstream hormonal regulation of expression of these genes, focusing on both known and as yet unknown ones acting in concert to bring about pod shatter.

Background

Dehiscence zone The pod dehiscence zone is a two to four cell layer wide tissue made up of thin-walled cells, located along the margins of the carpels. Detailed anatomical studies have shown that the middle lamella of these cells constitutes a load bearing structure in the dehiscence zone, and once it is degraded the pod may open. Halting or slowing middle lamella depolymerization can be accomplished through the antisensing of key enzymes involved in its degradation or through interference with the hormonal regulation of the expression of these and other key enzymes. The middle lamella is rich in polygalacturonate, a pectic polymer, and it has been demonstrated that a polygalacturonate-degrading enzyme activity is expressed in the dehiscence zone.

Activities Several candidate genes were considered and based on various evidence, one was cloned. Using immunoelectron microscopy with antibodies raised against the polygalacturonase strong evidence for its direct role in the changes in the dehiscence zone cell walls, which have been disclosed by preceding anatomical studies, was provided. As the AIR project closed, first generation transformants in which antisense to the polygalacturonase had been prepared. Their evaluation must await maturation of the primary transformants and another seed generation.

While making detailed developmental studies of pod maturation endogenous hormones in various pod tissues have been mapped. Whenever hormone concentrations appeared to correlate with developmental events in the pod dehiscence zone, attempts were made to substantiate hormonal control of development. A technique has been used to induce the growth of pods without seeds and synthetic growth regulators have been employed to test the functional involvement of endogenous hormones in the regulation of pod opening. These studies have in particular focused on abscisic acid, auxins and ethylene. While abscisic acid has been eliminated as a controlling factor in pod dehiscence, a picture involving a rather complex interaction of ethylene with auxin has emerged. A strategy of using the auxin component of the hormonal regulation to interfere with the weakening of the dehiscence zone prior to pod opening has been formulated. First generation transformants corresponding to this strategy are in vitro as the project closes.

Exploitation results Plant Genetic Systems, the industrial partner of our consortium (since acquired by AgrEvo), has submitted a patent application involving key elements of technology inherent to both the main strategies described above and, in addition, one other major strategy which employs both technology developed by the three academic partners and technology developed by PGS. The long generation time of oilseed rape, some 16 months to produce a secondary transgenic seed generation, has put the evaluation of mature transgenic rape plants beyond the remit of the AIR program, and hence also the potentially very important combinations of different transgenic traits in cultivars of oilseed rape. Provided further financial support for collaboration can be obtained, it will be ensured that the transgenic plants produced so far will be evaluated and, whenever deemed useful, contributed to oilseed rape breeding programs in PGS.

Second Project Progress Report Summary

Introduction

Pod shatter denotes the opening of oilseed rape silques and dispersal of the seeds as they mature. Flowering and seed shatter is not synchronous in rape; hence there is no such thing as the "correct harvest time". Immature seeds contain more moisture and more chlorophyll both of which lead to reduced oil quality. Late harvest dates leading to less immature seeds is often accompanied by loss of yield and problems with fighting run-away rape with chemical herbicides in the following year. Swathing reduces the risk that wind bring about excessive shattering of seeds from the most mature pods while allowing the younger ones to catch up to some extent. Timing of swathing is very critical and it is not always that yields are greater than the potential yield from a crop harvested by direct combining.

It is the purpose of this project to develop the technology required to engineering shatter resistance into oilseed rape using the tools of molecular biology. Transgenic oilseed rape with a reduced tendency to shatter may be prepared for exam-ple by offsetting the initiation of pod opening relative to the maturation of the seeds. In other words, all maturation and senescence processes may be allowed to proceed as in normal plants, only the timely coordination between seed dehydration and pod open-ing is severed and pod opening is delayed relative to seed maturation.

Objective

Two physiologically distinct approaches towards obtaining a selective delay of pod opening relative to seed maturation are being investigated:

interference with the enzymes that bring about the physical separation of the carpels
interference with the hormonal regulation of the pod opening process in broader terms.

These strate-gies are linked in theory by the fact that the enzymes studied in the first strategy are subject to the over-all regulation of the process as studied in strategy two. In practice, when it comes to engineering the traits, they become much more interdependent.

Activities

These include an examination of the roles of endo-polygalacturonase in dissolution of load bearing structures holding the carpels together and a detailed examination of the roles of auxins and ethylene and its precursor on the initia-tion or stimulation of pod opening. Such studies have enabled the possibility of breeding greater shatter resistance in transgenic plants, based on key elements of the pod opening process, based on interference with pod opening.

Strategies

Both main strategies, indicated above, aim to interfere with developmental events in the so-called dehiscence zone, a few layers of thin walled cells along the edges of the two valves of the pod. The cells in this zone eventually separate and allow the pod to open. It is possible to interfere in either late, or early events. Interference may be directly with a processes directly, or be in regulation. In either case the objective is to override develop-mental events in the dehiscence zone. Rape transformation is essential for all strategies. Hence, a rational and efficient transformation procedure, using spectomycin resistance as the selectable marker, has been developed.

Polysaccharide hydrolase-antisense approach
Anatomical studies have shown that the middle lamella between the dehiscence zone cells eventually dissolves while the primary cell walls of the same tissue softens and looses wall material. Biochemical studies have demonstrated the presence or accumu-lation late in development of a range of enzymes that may play a role in the weakening of the tissue so that the pods eventually open. Among these , most research efforts have been applied to endo-polygalacturonase (endo-PG) which is believed to take part in depolymerization of the middle lamella. One endo-PG, among the several that occur in rape has been cloned. It has been found that this isoform is expressed in the dehiscence zone immediately preceding and during pod opening. An antisense strategy, that entails production of transgenic rape plants in which the expression this enzyme is suppressed selectively, has been adopted so that only pod opening and not other processes that depend on related genes are disturbed. The gene in hand will be used to make the first generation of transgenic plants and thus establish the feasibility of the polysaccharide-antisense approach.

Hormonal regulation approach
Investigations of endogenous hormones in various pod tissues in combination with applications of synthetic growth regulators have produced a very interesting picture. Evidence have been produced that the dehiscence zone remains intact as long as the auxin concentration in the dehiscence zone cells is at a sustained high level. The use of par-thenocarpic pods (pods that have been induced to develop without seeds inside), has shown that the seeds is indeed the main source of the auxin influencing the dehiscence zone cells. Dur-ing dehydration auxin transport ceases from the seeds and the dehiscence zone cells respond accordingly. One of the responses may well be that the tissue becomes receptive to ethylene, an accelerator of maturation arid senescence processes. The strategy depends on equipping the dehiscence zone cells with their own auxin bio-synthetic machinery, so that they become independent of seeds as source of auxin, and hence will not acquire sensitivity to ethylene. Removal of the ability to respond to ethylene is expected to delay pod opening.

Exploitation

In a move to ensure exploitation of the results Plant Genetic Systems was made a partner of the project (originally there were no industrial partners in this project). Plant Genetic Systems stepped into the current AlR-project for a number of reasons, the most pertinent being a pressing need to submit a patent-application in order to ascertain the economical viability of the applied aspects of the project. This has now happened with a patent that amply reflects the broadening of the project as it developed during the first two years.

Conclusion

So far a solid foundation for two main approached aimed at engineering shatter resistance has been established. A rape transformation procedure is in place, and the first generation of are being developed.