Tissue specific aquaporin expression for seedling water stress resistance

This plant science project combines cell biology and plant physiology to understand how seeds take up water during germination and post-germination seedling growth and how we can control this process to develop crops that can germinate and establish in water stress conditions.

Rapid plant tissue expansion after seed germination is crucial for crop establishment and plant uniformity and it directly impacts on quality and yield at the end of the crop cycle. For these reasons early vigour is an important target for crop improvement. Most cells (including seed cells) take up water through protein water channels called aquaporins (AQP). While a lot is known about AQP structure and function in plant roots and leaves, very little is known about their role in seed germination and post-germination seedling growth. Our project directly addresses this question.

AQP exist on different cellular membranes and water intake is usually controlled by AQP on the outer cell membrane (the plasma membrane). Remarkably, we have found that in seeds the AQP that are normally present on the plasma membrane are NOT produced until AFTER the seed has germinated. So what is controlling water entry during germination? We have discovered that AQP that normally reside on the membrane of the vacuole (the tonoplast) are also found at the plasma membrane during seed maturation and germination. Therefore it seems that seeds have a unique way of regulating water intake by sending tonoplast AQP to the plasma membrane.

We have also discovered that plants modified to produce very high amounts of seed AQP are able to germinate and grow in conditions of water stress, performing significantly better than wild-type plants. Therefore we hypothesise that seed AQP are major controllers of water flow in seeds and early seedling growth that can be used to generate plants that cope better with drought conditions.

Our project aims at understanding the role of AQP by studying the way the tonoplast AQP are targeted to the plasma membrane. We also propose to manipulate both the amount and type of AQP present on the seed cells' plasma membrane and correlate it with the ability of seeds to germinate and the vigour of post-germination seedling growth using a range of water stress conditions. We will perform the basic experiments in the model plant Arabidopsis but, in parallel we will also perform pilot experiments in Brassica oleracea, a crop for which co-ordinated germination and early vigour can have a direct, major impact on final yield.

By the end of the project we aim to have improved understanding of the regulation of AQP during germination and early sedling growth and to have established strategies to manipulate AQP in order to maximise early vigour and water stress resistance.

This project addresses three closely linked topics: 1) the intracellular trafficking of vacuolar aquaporins (AQP) in seeds, 2) their role in mediating water intake during germination and post-germination seedling growth and 3) their ability of AQP to improve seedling performance in water stress conditions.

Aquaporins are important during germination. However, the most abundant AQP during that phase in Arabidopsis are tonoplast (TIP), rather than plasma membrane intrinsic proteins (PIP). This is surprising as PIP are normally the main gateway for water intake. We have found that seed-specific TIP3 isoforms have a dual localisation - both at the tonoplast and the plasma membrane (PM). This suggests that in seeds TIPs may be recruited to the PM in lieu of PIP. This dual localisation also raises the issue of how TIP targeting is achieved - a long-standing question in plant cell biology. We have recently discovered that plasma membrane sorting information is present in the C-terminal region of a seed TIP but we do not know the nature of the sorting signal.

We have also found that seed TIP - overexpressing seedlings grow significantly better than wild-type in conditions of water stress.

We propose to elucidate the sorting signal(s) and the sorting route of TIP3. We will selectively downregulate every seed TIP and then replace them with exclusively tonoplast- or plasma membrane- targeted AQP. We will study the effect of these modifications on germination and post-germination seedling growth. We will also selectively upregulate distinct TIP at different stages of seed germination using an inducible promoter and determine whether up-regulation of TIP activity can be used to boost early vigour under conditions of water stress. In parallel we will test constitutive TIP upregulation in transgenic Brassica oleracea, in order to correlate AQP modulation with efficiency of seed water uptake and the establishment of early vigour in this crop model.

For direct-drilled crops, where seed is sown into highly variable field environments, co-ordinated germination and early vigour have major crop-specific impacts on the size and uniformity of harvestable produce and on final yield. These traits are highly desired by both seed companies and growers. By studying the process of water uptake in germinating seeds and early seedling growth, our project directly addresses the optimisation of early vigour - and how this can be optimised in water-stress conditions - both in a model plant and in the crop plant Brassica oleracea. Therefore in the medium to long term beneficiaries of this work will include the agricultural sector, the biotechnology and the biofuels sector through the optimisation of oilseed productivity.

Warwick has an active technology transfer office, Warwick Ventures. Both applicants already hold international patents. Therefore, in the event of any exploitable IP emerging from the project they are well placed for exploitation. Both applicants are extremely active in public outreach and engagement, giving Schools talks, hosting school and public open days and participating in local science fairs etc. These activities will continue and incorporate interesting and relevant findings from this research programme. They also interact regularly with seed companies and farmer representatives.

The two applicants have over the years had major success in training both graduate and postdoctoral researchers. It is expected that the postdoctoral researcher will be trained to such a level that, at the end of the grant period, they will be able to function as a successful independent scientist and thus contribute both scientifically and economically to the wider community.