A New Zealand Perspective on the Application and Regulation of Gene Editing.

New Zealand (NZ) is a small country with an export-led economy with above 90% of primary production exported. Plant-based primary commodities derived from the pastoral, horticultural and forestry sectors account for around half of the export earnings. Productivity is characterized by a history of innovation and the early adoption of advanced technologies. Gene editing has the potential to revolutionize breeding programmes, particularly in NZ. Here, perennials such as tree crops and forestry species are key components of the primary production value chain but are challenging for conventional breeding and only recently domesticated. Uncertainty over the global regulatory status of gene editing products is a barrier to invest in and apply editing techniques in plant breeding. NZs major trading partners including Europe, Asia and Australia are currently evaluating the regulatory status of these technologies and have not made definitive decisions. NZ is one of the few countries where the regulatory status of gene editing has been clarified. In 2014, the NZ Environmental Protection Authority ruled that plants produced via gene editing methods, where no foreign DNA remained in the edited plant, would not be regulated as GMOs. However, following a challenge in the High Court, this decision was overturned such that NZ currently controls all products of gene editing as GMOs. Here, we illustrate the potential benefits of integrating gene editing into plant breeding programmes using targets and traits with application in NZ. The regulatory process which led to gene editing's current GMO classification in NZ is described and the importance of globally harmonized regulations, particularly to small export-driven nations is discussed.

Exogenous cytokinin application to Actinidia chinensis var. deliciosa 'Hayward' fruit promotes fruit expansion through water uptake.

Exogenous application of a cytokinin-like compound forchlorfenuron (CPPU) can promote fruit growth, although often at the expense of dry matter (DM), an important indicator of fruit quality. Actinidia chinensis var. deliciosa 'Hayward' fruit are very responsive to CPPU treatments, but the mechanism underlying the significant fruit weight increase and associated decrease in DM is unclear. In this study, we hypothesised that CPPU-enhanced growth increases fruit carbohydrate demand, but limited carbohydrate supply resulted in decreased fruit DM. During fruit development, CPPU effects on physical parameters, metabolites, osmotic pressure and transcriptional changes were assessed under conditions of both standard and a high carbohydrate supply. We showed that CPPU increased fruit fresh weight but the dramatic DM decrease was not carbohydrate limited. Enhanced glucose and fructose concentrations contributed to an increase in soluble carbohydrate osmotic pressure, which was correlated with increased water accumulation in CPPU-treated fruit and up-regulation of water channel aquaporin gene PIP2.4 at 49 days after anthesis. Transcipt analysis suggested that the molecular mechanism contributing to increased glucose and fructose concentrations was altered by carbohydrate supply. At standard carbohydrate supply, the early glucose increase in CPPU fruit was associated with reduced starch synthesis and increased starch degradation. When carbohydrate supply was high, the early glucose increase in CPPU fruit was associated with a general decrease in starch synthesis but up-regulation of vacuolar invertase and fructokinase genes. We conclude that CPPU affected fruit expansion by increasing the osmotically-driven water uptake and its effect was not carbohydrate supply-limited.

Metabolic analysis of kiwifruit (Actinidia deliciosa) berries from extreme genotypes reveals hallmarks for fruit starch metabolism.

Tomato, melon, grape, peach, and strawberry primarily accumulate soluble sugars during fruit development. In contrast, kiwifruit (Actinidia Lindl. spp.) and banana store a large amount of starch that is released as soluble sugars only after the fruit has reached maturity. By integrating metabolites measured by gas chromatography-mass spectrometry, enzyme activities measured by a robot-based platform, and transcript data sets during fruit development of Actinidia deliciosa genotypes contrasting in starch concentration and size, this study identified the metabolic changes occurring during kiwifruit development, including the metabolic hallmarks of starch accumulation and turnover. At cell division, a rise in glucose (Glc) concentration was associated with neutral invertase (NI) activity, and the decline of both Glc and NI activity defined the transition to the cell expansion and starch accumulation phase. The high transcript levels of ß-amylase 9 (BAM9) during cell division, prior to net starch accumulation, and the correlation between sucrose phosphate synthase (SPS) activity and sucrose suggest the occurrence of sucrose cycling and starch turnover. ADP-Glc pyrophosphorylase (AGPase) is identified as a key enzyme for starch accumulation in kiwifruit berries, as high-starch genotypes had 2- to 5-fold higher AGPase activity, which was maintained over a longer period of time and was also associated with enhanced and extended transcription of the AGPase large subunit 4 (APL4). The data also revealed that SPS and galactinol might affect kiwifruit starch accumulation, and suggest that phloem unloading into kiwifruit is symplastic. These results are relevant to the genetic improvement of quality traits such as sweetness and sugar/acid balance in a range of fruit species.

Is fruit anatomy involved in variation in fruit starch concentration between Actinidia deliciosa genotypes?

The role of anatomical traits in carbohydrate accumulation was investigated in fruit of Actinidia deliciosa (A. Chev.) C. F. Liang et A. R. Ferguson (kiwifruit) var. deliciosa by comparing high and low dry matter (DM) accumulating genotypes. DM was shown previously to be correlated with starch concentration in these fruit. Volume proportions of the three fruit tissues (outer pericarp, inner pericarp and central core) did not vary significantly between genotypes or contribute to variation in total fruit DM. The outer pericarp of the kiwifruit berry contains both small and large cells: the size of these cells was not correlated with final fruit size. In high DM genotypes, the relative volume of outer pericarp tissue occupied by small cells (50%) was significantly greater than that in low DM genotypes (43%). Small cells have a higher starch concentration than large cells: the larger proportion of small cells in the outer pericarp of fruit from high DM genotypes accounted for approximately +25% of the measured differences in fruit starch concentration between high and low DM genotypes. We conclude that, although anatomical traits contribute to variation in fruit starch concentration between kiwifruit genotypes, differences in starch content per small cell are important and worthy of further investigation. This is the first time anatomical investigations have been used to examine differences in fruit carbohydrate accumulation in kiwifruit.

Gene expression studies in kiwifruit and gene over-expression in Arabidopsis indicates that GDP-L-galactose guanyltransferase is a major control point of vitamin C biosynthesis.

Vitamin C (L-ascorbic acid, AsA) is an essential metabolite for plants and animals. Kiwifruit (Actinidia spp.) are a rich dietary source of AsA for humans. To understand AsA biosynthesis in kiwifruit, AsA levels and the relative expression of genes putatively involved in AsA biosynthesis, regeneration, and transport were correlated by quantitative polymerase chain reaction in leaves and during fruit development in four kiwifruit genotypes (three species; A. eriantha, A. chinensis, and A. deliciosa). During fruit development, fruit AsA concentration peaked between 4 and 6 weeks after anthesis with A. eriantha having 3-16-fold higher AsA than other genotypes. The rise in AsA concentration typically occurred close to the peak in expression of the L-galactose pathway biosynthetic genes, particularly the GDP-L-galactose guanyltransferase gene. The high concentration of AsA found in the fruit of A. eriantha is probably due to higher expression of the GDP-mannose-3',5'-epimerase and GDP-L-galactose guanyltransferase genes. Over-expression of the kiwifruit GDP-L-galactose guanyltransferase gene in Arabidopsis resulted in up to a 4-fold increase in AsA, while up to a 7-fold increase in AsA was observed in transient expression studies where both GDP-L-galactose guanyltransferase and GDP-mannose-3',5'-epimerase genes were co-expressed. These studies show the importance of GDP-L-galactose guanyltransferase as a rate-limiting step to AsA, and demonstrate how AsA can be significantly increased in plants.

Analysis of expressed sequence tags from Actinidia: applications of a cross species EST database for gene discovery in the areas of flavor, health, color and ripening.

BACKGROUND: Kiwifruit (Actinidia spp.) are a relatively new, but economically important crop grown in many different parts of the world. Commercial success is driven by the development of new cultivars with novel consumer traits including flavor, appearance, healthful components and convenience. To increase our understanding of the genetic diversity and gene-based control of these key traits in Actinidia, we have produced a collection of 132,577 expressed sequence tags (ESTs). RESULTS: The ESTs were derived mainly from four Actinidia species (A. chinensis, A. deliciosa, A. arguta and A. eriantha) and fell into 41,858 non redundant clusters (18,070 tentative consensus sequences and 23,788 EST singletons). Analysis of flavor and fragrance-related gene families (acyltransferases and carboxylesterases) and pathways (terpenoid biosynthesis) is presented in comparison with a chemical analysis of the compounds present in Actinidia including esters, acids, alcohols and terpenes. ESTs are identified for most genes in color pathways controlling chlorophyll degradation and carotenoid biosynthesis. In the health area, data are presented on the ESTs involved in ascorbic acid and quinic acid biosynthesis showing not only that genes for many of the steps in these pathways are represented in the database, but that genes encoding some critical steps are absent. In the convenience area, genes related to different stages of fruit softening are identified. CONCLUSION: This large EST resource will allow researchers to undertake the tremendous challenge of understanding the molecular basis of genetic diversity in the Actinidia genus as well as provide an EST resource for comparative fruit genomics. The various bioinformatics analyses we have undertaken demonstrates the extent of coverage of ESTs for genes encoding different biochemical pathways in Actinidia.

In vitro and in vivo release of aroma compounds from yellow-fleshed kiwifruit.

Comparisons were made between the aroma volatiles of the yellow-fleshed kiwifruit, "Hort16A", at two different stages of eating ripeness: firm and soft. The firm fruit contained a small number of aroma compounds that the soft fruit did not contain. In general, however, the largest difference between the two firmness categories was in the levels of esters, with the soft fruit containing higher concentrations and a larger number of esters than the firm fruit. In vitro analysis directly after maceration using atmospheric pressure chemical ionization mass spectrometry (APCI-MS) showed the relative importance of the most intense aromas between fruit at the two different firmness stages and was used to compare the release rates of aromas. A comparison of the aroma concentrations from gas chromatography mass spectrometry (GC-MS) and APCI-MS headspace analyses showed that the APCI-MS headspace showed less bias toward enzymatically generated lipid degradation compounds. A GC-sniffing study showed that many of the most intense compounds, acetaldehyde, hexanal, ethyl butanoate, and (E)-2-hexenal but not ethanol, showed odor activity in macerated fruit. In addition, dimethyl sulfide (DMS), a volatile present at very low levels in the fruit, also appeared to be an important contributor to the odor. In vivo analyses also showed much higher levels of aroma compounds in the soft fruit compared to the firm fruit, with evidence of persistence of some compounds, including DMS. There were a number of similarities between the breath profiles of the two panelists, which confirmed the importance of DMS in "Hort16A" aroma.

Extraction of plant RNA.

Optimal sampling procedures for sampling plant tissue for RNA extractions are outlined in this chapter. To extract RNA, kits supplied from biotechnology companies are appropriate, but some procedures will not work with particular plant tissues. Two alternative methods are supplied for troublesome material. Basic methods to check RNA quantity and, more particularly, RNA quality before use are supplied.

Identification and characterisation of acidic and novel basic forms of actinidin, the highly abundant cysteine protease from kiwifruit.

Actinidin is a cysteine protease found in Actinidia Lindl. (kiwifruit) species that affects the nutraceutical properties, processing characteristics and allergenicity of the fruit. Given the increased consumption of kiwifruit worldwide and the release of new varieties from different Actinidia species, the expression of actinidin mRNA and protein in a range of kiwifruit tissues was examined. Ten different actinidin mRNAs were identified encoding mature proteins of similar molecular weight (~24 kDa), but with predicted pIs ranging from acidic (pI 3.9) to basic (pI 9.3). In A. deliciosa 'Hayward' (green-fleshed kiwifruit) and A. chinensis 'Hort16A' and EM4 (gold-fleshed kiwifruit), actinidin mRNAs for acidic and basic proteins were expressed at comparable levels throughout ripening. Actinidin mRNA expression was highest in fruit at harvest, expression decreased as fruit ripened and was much lower in the core compared with outer pericarp tissue. Two-dimensional gel electrophoresis, combined with western analysis and liquid chromatography mass spectrometry (LC-MS) identified low levels of a novel basic actinidin protein in ripe A. deliciosa and A. chinensis fruit. Extremely high levels of an acidic actinidin protein were detected in A. deliciosa fruit and EM4, but this acidic protein appeared to be absent in 'Hort16A', the most important commercial cultivar of A. chinensis. Analyses on native gels indicated that both the basic and acidic actinidin isoforms in A. deliciosa were active cysteine proteases. Immunolocalisation showed that actinidin was present in small cells, but not large cells in the outer pericarp of mature A. deliciosa fruit at harvest. Within the small cells, actinidin was localised diffusely in the vacuole, associated with the plasma membrane, and in a layer in the plastids near starch granules. The presence of multiple forms of actinidin and varying protein levels in fruit will impact on the ability to breed new kiwifruit varieties with altered actinidin levels.

Unusual features of a recombinant apple alpha-farnesene synthase.

A recombinant alpha-farnesene synthase from apple (Malus x domestica), expressed in Escherichia coli, showed features not previously reported. Activity was enhanced 5-fold by K(+) and all four isomers of alpha-farnesene, as well as beta-farnesene, were produced from an isomeric mixture of farnesyl diphosphate (FDP). Monoterpenes, linalool, (Z)- and (E)-beta-ocimene and beta-myrcene, were synthesised from geranyl diphosphate (GDP), but at 18% of the optimised rate for alpha-farnesene synthesis from FDP. Addition of K(+) reduced monoterpene synthase activity. The enzyme also produced alpha-farnesene by a reaction involving coupling of GDP and isoprenyl diphosphate but at <1% of the rate with FDP. Mutagenesis of active site aspartate residues removed sesquiterpene, monoterpene and prenyltransferase activities suggesting catalysis through the same active site. Phylogenetic analysis clusters this enzyme with isoprene synthases rather than with other sesquiterpene synthases, suggesting that it has evolved differently from other plant sesquiterpene synthases. This is the first demonstration of a sesquiterpene synthase possessing prenyltransferase activity.

Characterisation of Mal d 1-related genes in Malus.

It has been suggested that there are at least 15 Mal d 1-related (PR10) genes in one genotype of apple (Malus x domestica Borkh). We sequenced cDNA libraries of cultivar 'Royal Gala' and identified 12 members of the Mal d 1 family, including the previously reported Mal d 1b and Mal d 1d, an allelic variant of the previously reported Mal d 1a. Eight Mal d 1 gene products were expressed in tree-ripened fruit, in either the cortex or the skin, and most of these were also expressed in leaves in response to challenge with Venturia inaequalis -a fungal disease of apple. Mal d 1 gene products were identified from a large number of different tissues. Degree of ripeness as measured by standard parameters was shown not to predict either the amount of protein able to bind to a specific monoclonal antibody 5H8, previously shown to bind to an allergenic epitope in Mal d 1b and a/d, or the amount of Mal d 1 mRNA present. Mal d 1d and Mal d 1b were the most highly expressed isoforms in 'Royal Gala', particularly in the skin of fruit, and these isoforms were also predominant in other cultivars and species of apple. Genotypes, however, differed in relative predominance of Mal d 1b and Mal d 1d. The predominantly expressed Mal d 1 genes in ripe apple fruit were translated in vivo into proteins and proteins binding to the antibody were found in all cultivars and species examined. New Mal d 1 proteins were identified that bound to the 5H8 antibody. At least two new subfamilies have been identified, and while some structural differences are predicted between groups of isoforms, the P-loop motif is identical in all except two isoforms. A role in intracellular signalling in plants is suggested and in vitro expression of the isoforms should help in assessing their relative roles in disease, allergic responses, senescence and nucleotide-, cytokinin- and brassinosteroid-binding.

A highly specific L-galactose-1-phosphate phosphatase on the path to ascorbate biosynthesis.

Ascorbate is a critical compound in plants and animals. Humans are unable to synthesize ascorbate, and their main source of this essential vitamin are plants. However, the pathway of synthesis in plants is yet to be established, and several unknown enzymes are only postulated to exist. We describe a specific L-galactose-1-phosphate (L-gal-1-P) phosphatase that we partially purified from young kiwifruit (Actinidia deliciosa) berries. The enzyme had a native molecular mass of approximately 65 kDa, was completely dependent on Mg2+ for activity and was very specific in its ability to hydrolyze L-gal-1-P. The activity had a pH optimum of 7.0, a K(-M(L-gal-1-P) of 20-40 microM and a Ka(Mg2+) of 0.2 mM. The activity was inhibited by Mg2+ at concentrations >2 mM. The enzyme from Arabidopsis thaliana shoots showed similar properties to the kiwifruit enzyme. The Arabidopsis thaliana enzyme preparation was digested with trypsin, and proteins present were identified by using liquid chromatography-MS. One of 24 proteins present in our preparation was an Arabidopsis thaliana protein, At3g02870, annotated myo-inositol-1-phosphate phosphatase in GenBank, that matched the characteristics of the purified l-gal-1-phosphate phosphatase. We then expressed a kiwifruit homologue of this gene in Escherichia coli and found that it showed 14-fold higher maximum velocity for l-gal-1-P than myo-inositol-1-P. The expressed enzyme showed very similar properties to the enzyme purified from kiwifruit and Arabidopsis, except that its KM(L-gal-1-P) and Ka(Mg2+) were higher in the expressed enzyme. The data are discussed in terms of the pathway to ascorbate biosynthesis in plants.

Evolution and function of the sucrose-phosphate synthase gene families in wheat and other grasses.

Suc-phosphate synthase (SPS) is a key regulatory enzyme in the pathway of Suc biosynthesis and has been linked to quantitative trait loci controlling plant growth and yield. In dicotyledonous plants there are three SPS gene families: A, B, and C. Here we report the finding of five families of SPS genes in wheat (Triticum aestivum) and other monocotyledonous plants from the family Poaceae (grasses). Three of these form separate subfamilies within the previously described A, B, and C gene families, but the other two form a novel and distinctive D family, which on present evidence is only found in the Poaceae. The D-type SPS proteins lack the phosphorylation sites associated with 14-3-3 protein binding and osmotic stress activation, and the linker region between the N-terminal catalytic glucosyltransferase domain and the C-terminal Suc-phosphatase-like domain is 80 to 90 amino acid residues shorter than in the A, B, or C types. The D family appears to have arisen after the divergence of mono- and dicotyledonous plants, with a later duplication event resulting in the two D-type subfamilies. Each of the SPS gene families in wheat showed different, but overlapping, spatial and temporal expression patterns, and in most organs at least two different SPS genes are expressed. Analysis of expressed sequence tags indicated similar expression patterns to wheat for each SPS gene family in barley (Hordeum vulgare) but not in more distantly related grasses. We identified an expressed sequence tag from rice (Oryza sativa) that appears to be derived from an endogenous antisense SPS gene, and this might account for the apparently low level of expression of the related OsSPS11 sense gene, adding to the already extensive list of mechanisms for regulating the activity of SPS in plants.

Pectins from the albedo of immature lemon fruitlets have high water binding capacity.

The white part of citrus peel, the albedo, has a special role in water relations of both fruit and leaves from early on in fruit development. In times of drought, this tissue acts as a water reservoir for juice sacs, seeds and leaves. When water was injected into the albedo, free water was undetectable using magnetic resonance imaging. Microscopy showed tightly packed cells with little intercellular space, and thick cell walls. Cell wall material comprised 21% of the fresh albedo weight, and contained 26.1% galacturonic acid, the main constituent of pectin. From this, we postulated that pectin of the cell wall was responsible for the high water-binding capacity of the immature lemon albedo. Cell wall material was extracted using mild procedures that keep polymers intact, and four pectic fractions were recovered. Of these fractions, the SDS and chelator-soluble fractions showed viscosities ten and twenty times higher than laboratory-grade citrus pectin or the other albedo-derived pectins. The yield of these two pectins represented 28% of the cell walls and 62% of the galacturonic acid content of immature lemon albedo. We concluded that, from viscosity and abundance, these types of pectin account for the high water-binding capacity of this tissue. Compositional analyses showed that the two highly viscous pectic fractions differ in galacturonic acid content, degree of branching and length of side chains from the less viscous albedo-derived pectins. The most striking feature of these highly viscous pectins, however, was their high molecular weight distribution compared to the other pectic fractions.

Kiwifruit L-galactose dehydrogenase: molecular, biochemical and physiological aspects of the enzyme.

l-Galactose dehydrogenase, an enzyme in the pathway of ascorbate biosynthesis, was purified to homogeneity from leaves of kiwifruit [Actinidia deliciosa (A.Chev.) CF Liang et AR Ferguson var. deliciosa 'Hayward']. The enzyme had a molecular mass of 34.2 kD, and behaved as a monomer during gel filtration. The Km(galactose) and Km(NAD) decreased as pH increased from 6.5 to 9, while the Vmax increased over this range. A number of related sugars were tested as alternative substrates or inhibitors, but these were ineffective. Ascorbate caused slow inactivation of the enzyme, possibly through metal catalysed generation of oxygen radicals. Inactivation appeared to be active-site directed as it was protected by the substrate NAD, and not by NADH or l-galactose. This is not likely to be physiologically significant. Through partially sequencing the protein, the gene was identified in the HortResearch Actinidia EST database, and the translation of the full length sequence of this cDNA showed a high homology (80% identity, 90% similarity) to the translation of an Arabidopsis gene (accession CAD10386) and to translations of other genes identified in GenBank. Levels of l-GalDH activity decreased during fruit and leaf development, and levels of mRNA showed a similar reduction. Activity varied between flower parts, with ovaries and styles showing equivalent activity to young fruitlets and sink leaves. Nucleotide sequences reported are available in the Genbank database under the accession number AY176585 (kiwifruit) and AY264803 (apple).

Turgor, solute import and growth in maize roots treated with galactose.

It has been observed that extension growth in maize roots is almost stopped by exposure to 5 mm d-galactose in the root medium, while the import of recent photoassimilate into the entire root system is temporarily promoted by the same treatment. The aim of this study was to reconcile these two apparently incompatible observations. We examined events near the root tip before and after galactose treatment since the tip region is the site of elongation and of high carbon deposition in the root. The treatment rapidly decreased root extension along the whole growing zone. In contrast, turgor pressure, measured directly with the pressure probe in the cortical cells of the growing zone, rapidly increased by 0.15 MPa within the first hour following treatment, and the increase was maintained over the following 24 h. Both tensiometric measurements and a comparison of turgor pressure with local growth rate demonstrated that a rapid tightening of the cell wall caused the reduction in growth. Single cell sampling showed cell osmotic pressure increased by 0.3 MPa owing to accumulation of both organic and inorganic solutes. The corresponding change in cell water potential was a rise from -0.18 MPa to approximately zero. More mature cells at 14 mm from the root tip (just outside the growing region) showed a qualitatively similar response.

Planteose is a short-term storage carbohydrate in Actinidia leaves.

The polyol myo-inositol constitutes 10-20% of soluble carbohydrates in mature leaves of Actinidia deliciosa (A.Chev.) C.F. Liang et A.R. Ferguson var. deliciosa 'Hayward' and A. arguta (Sieb. et Zucc.) Planch. ex Miq. var. arguta. In contrast with other non-structural carbohydrates, myo-inositol concentrations in A. deliciosa leaves increase only slightly during development from sink to source, and are not affected in source leaves by increased sink demand upon fruit set. In mature fruit-bearing leaves myo-inositol concentrations fluctuate diurnally, increasing during the night and declining towards morning, but in plants with less sink demand a diurnal pattern is not observed. In potted A. arguta seedlings subjected to extended dark periods, leaf concentrations of sugars and starch decline rapidly while myo-inositol concentrations are maintained. Labelling studies with 14CO2 revealed that myo-inositol in leaves is not a primary photosynthetic product and is turned over more slowly than other soluble carbohydrates. A suggested role of myo-inositol as a precursor in mucilage synthesis was not substantiated, as radioactivity was incorporated into mucilage more rapidly than into free myo-inositol. Planteose, a trisaccharide comprising sucrose and galactose, incorporated substantial amounts of radioactivity and accumulated to high levels, indicating a role in short-term storage of sucrose. Planteose was synthesised during the day and degraded during the night in a manner that was opposite to that of sucrose while starch and myo-inositol levels remained relatively constant. Planteose has been reported in Cyclamen persicum, ash and sesame seed. This is the first report of planteose in Actinidia, and the first time it has been identified as a major short-term storage carbohydrate in Actinidia leaves.

New complexities in the synthesis of sucrose.

Sucrose is universal in plants and fulfils many roles: transport sugar, storage reserve, compatible solute and signal compound. Consequently, sucrose synthesis is highly regulated, with much of the control operating at the first step in the committed pathway, which is catalysed by sucrose-phosphate synthase (SPS). The discovery of at least three SPS gene families in plants has added a further layer of complexity to an already complicated picture involving transcriptional, allosteric and post-translational control of this enzyme's activity. After years of neglect, the gene encoding the last enzyme in the pathway, sucrose-phosphatase (SPP), has finally been cloned, revealing that SPS contains an SPP-like domain at the carboxy-terminus, to which SPP might bind. This has reinvigorated the search for an SPS-SPP complex, and has hinted at further complexities to be unravelled in the control of sucrose synthesis in plants.

Actinidia arguta: volatile compounds in fruit and flowers.

More than 240 compounds were detected when the volatile components of the flowers and the fruit from several Actinidia arguta genotypes were investigated. Around 60-70 different compounds were extracted from individual tissues of each genotype. Two different methods of volatile sampling (headspace and solvent) favoured different classes of compounds, dependent upon their volatilities and solubilities in the flower or fruit matrices. The compounds extracted from flowers largely comprised linalool derivatives including the lilac aldehydes (12a-d) and alcohols (13a-d), 2,6-dimethyl-6-hydroxyocta-2,7-dienal (8), 8-hydroxylinalool (9), sesquiterpenes, and benzene compounds that are presumed metabolites of phenylalanine and tyrosine. Extracts of fruit samples contained some monoterpenes, but were dominated by esters such as ethyl butanoate, hexanoate, 2-methylbutanoate and 2-methylpropanoate, and by the aldehydes hexanal and hex-E2-enal. A number of unidentified compounds were also detected, including 8 from flowers that are so closely related that they are either isomers of one compound or two or more closely related compounds. This is the first report of the presence of a range of linalool derivatives in Actinidia.