The sweet and the sticky: a new paradigm for divergent phloem function.

Phloem is one of the two major transport systems in all vascular plants, functioning mainly to transport sugars and other nutrients and metabolites throughout the plant. The highly nutritious phloem sap also makes it a very attractive target for pests and pathogens. In addition, many major insect-borne viral diseases spread in the phloem. Knowledge of the functions of the many components of phloem will therefore have wide-reaching consequences for development of strategies in improving plant defence. Protection can, for example, come from antimicrobial or anti-insect proteins, or from small molecules classed as secondary metabolites such as antioxidants or feeding deterrents. Research into phloem has mainly been on species such as members of the pumpkin family (including cucumber and melon) where bleeding phloem sap can be readily collected from cut stems and leaves. In most species a blocking mechanism rapidly seals the wound. Also unusual is the fact that pumpkin sap has a sugar content about 30 times less than in other plants, something that has perplexed researchers for decades. We have now resolved this paradox by showing that the bleeding sap comes from a second phloem system that has long been known as a feature in the pumpkin family but was thought to be a minor contributor. In fact the major phloem does contain high sugar but blocks immediately on cutting (like most species) whereas the minor bleeding phloem has low sugar. The proteins in the two systems are also completely different, something never previously described for any plant. Many questions arise: why is this dual system advantageous? Did it evolve uniquely in the pumpkin family? Which of the two phloems do insects such as aphids feed on? Now that we know the major phloem transports most of the sugar, what functions can be suggested for the minor phloem? Is the sum of functions of the two cucurbit phloems the same as the functions of the single phloem of other plants? Here we address some of these fascinating and important issues. Our long-term aim is to develop a better understanding of phloem function for all plants, with a view, for example, to improving resistance to aphids. To do this we will first compare the proteins in major and minor phloem. These proteins will have diverse functions from wound sealing to carrying signalling information throughout the plant, to acting directly in defence. We will then focus especially on one abundant protein class that we have found to be distributed widely across higher plant families, and is closely related to proteins already known to block phloem wounds in legume species. We will examine the biochemical properties of these proteins to understand the blocking mechanism. We will also express the corresponding gene(s) in the model plant Arabidopsis, allowing us to see if the cucurbit proteins may have the same function in other species. Based on amino acid sequence similarity, we have found relatives of the pumpkin protein in many species, but this is insufficient to judge whether the functions are identical. Finally, we will test aphids that vary in their virulence on melon plants that carry (or lack) an important aphid resistance gene. This will include electrical recordings of aphid behaviour, measurement of aphid growth rates, microscopy and chemical analysis of ingested phloem sap. These experiments will reveal whether aphids prefer to feed on the major sugar phloem or the minor bleeding phloem. The unique dual phloem will allow us to define better what determines success or failure of the pest, from which we can start to design new strategies for insect defence in crops.

We recently discovered that the phloem of cucurbit species exists as two spatially and functionally independent systems, unique amongst plants studied to date. This work resolves a long-standing paradox: why cucurbit bleeding phloem sap has an unusually low sugar content, contrary to requirements for photosynthate transport. The reason is that the major sugar-transporting phloem, contained within vascular bundles, blocks instantly on cutting, whereas it is the minor phloem that bleeds extensively. The proteomes and metabolomes of the two systems differ dramatically, and thus most likely have divergent functions in transport, signalling and defence. Taking advantage of the dual cucurbit phloem as a unique comparative model, we will make substantial advances in phloem biology through three related studies. First, we will extend the knowledge base of the newly defined major phloem compartment, focusing on comparative proteomic approaches. Second, we have identified a dominant protein family in the major phloem that is a strong candidate for acting as the mechanical wound-blocking agent. These proteins have homology to the forisome proteins of legumes which were thought to be unique to that family. In fact, homologs appear in all fully sequenced dicot genomes and may therefore be universal. We will explore the biochemical properties of these blocking proteins, in combination with functional studies on their expression and suppression in Arabidopsis. Third, we will contrast aphid behaviour, performance and preference for the two types of phloem using a suite of analytical, imaging and physiological tools, using compatible and incompatible interactions with resistant and susceptible near-isogenic host lines. The work will provide insights into possible evolutionary advantages of dual phloem systems, and into divergent roles in aphid defence. New strategies and targets for improvement of plant defence against aphids and other stresses may emerge.

This research is on cucurbits, an ideal model system with unique attributes for phloem biology. From this work, generic conclusions will be derived concerning phloem composition and functions in defence, such that translation beyond cucurbits can readily be achieved. Phloem has wide importance in its role as the primary transport highway for photosynthate distribution that directly affects plant productivity. More efficient functioning is therefore one route to increasing crop yields. But phloem also provides plants with many signalling capabilities that allow systemic coordination of development, tuning into environmental cues, and rapid defence responses to diverse forms of stress and biotic attack. The last of these relates especially to phloem feeding insects such as aphids. Cucurbit species are major crop plants worldwide: pumpkin, squash, melon, watermelon, cucumber etc. In the UK, cucumber is the predominant cucurbit industry with 50000 t worth over £60M annually, but there is also an increasing volume of squash/pumpkin. Global cucurbit production exceeds 150 Million tonnes, occupying 9 Million hectares of land. Average global yields are very low (17t/ha) compared with advanced cultivation in UK and Europe (~400-500 t/ha in protected systems and 70t/ha in field crops). UK crop losses due to aphids and associated viruses exceed £100M annually. Global estimates of losses may exceed 30% of food production, translating to a value of several billion dollars. In this context, and in light of increasingly restricted classes of pesticides allowed on food crops, and coupled to uncertainties over impacts of climate change on pest epidemics, the work here sits squarely within several priorities for BBSRC and for the wider community. There are clearly several potential impacts of the research that extend beyond cucurbits to all crop and model species, stemming from using this system to reveal a deeper understanding of phloem function. The opportunities for translation into agricultural or horticultural applications may be long term, but we are engaging with UK industry bodies and with end users around the world. The PI has extensive experience in dealing with the agricultural and horticultural sectors, in the context of developing practical solutions for increasing crop production and quality. We are presently in discussions with horticultural industries, through HDC, on how this research can align with industry needs and HDC have agreed to provide a letter of support for this proposal. National benefits will derive from top quality training of staff engaged on the project, and of research students who will be associated with several components of the work. Employment prospects of Imperial College students and staff are amongst the very top of UK HEIs.