Total Flavonoid Contents and the Expression of Flavonoid Biosynthetic Genes in Breadfruit (Artocarpus altilis) Scions Growing on Lakoocha (Artocarpus lakoocha) Rootstocks.

Breadfruit (Artocarpus altilis) is a traditional fruit tree of 15-30 m height in the tropics. The presence of size-controlling rootstock in the species is not known. A small tropical tree species, lakoocha (Artocarpus lakoocha), was recently identified as a potential vigor-controlling rootstock, conferring over a 65% reduction in breadfruit tree height. To better understand the intriguing scion/rootstock interactions involved in dwarfing, we investigate flavonoid accumulation and its regulation in breadfruit scions in response to different rootstocks. To this end, we isolated a chalcone synthase cDNA, AaCHS, and a full-length bifunctional dihydroflavonol 4-reductase cDNA, AaDFR, from breadfruit scion stems. The expression of both AaCHS and AaDFR genes was examined over the period of 16 to 24 months following grafting. During the development of the dwarf phenotype, breadfruit scion stems on lakoocha rootstocks display significant increases in total flavonoid content, and show upregulated AaCHS expression when compared with those on self-grafts and non-grafts. There is a strong, positive correlation between the transcript levels of AaCHS and total flavonoid content in scion stems. The transcript levels of AaDFR are not significantly different across scions on different rootstocks. This work provides insights into the significance of flavonoid biosynthesis in rootstock-induced breadfruit dwarfing.

Differential transcription pathways associated with rootstock-induced dwarfing in breadfruit (Artocarpus altilis) scions.

BACKGROUND: Breadfruit (Artocarpus altilis) is a traditional staple tree crop throughout the tropics. Through interspecific grafting, a dwarf phenotype with over 50% reduction in plant height was identified when marang (Artocarpus odoratissimus) rootstocks were used. However, the molecular mechanism underlying the rootstock-induced breadfruit dwarfing is poorly understood. RESULTS: An RNA-sequencing study of breadfruit scions at 22 months after grafting identified 5409 differentially expressed genes (DEGs) of which 2069 were upregulated and 3339 were downregulated in scion stems on marang rootstocks compared to those on self-graft. The DEGs were predominantly enriched for biological processes involved in carbon metabolism, cell wall organization, plant hormone signal transduction and redox homeostasis. The down-regulation of genes encoding vacuolar acid invertases and alkaline/neutral invertases, was consistent with the decreased activity of both enzymes, accompanying with a higher sucrose but lower glucose and fructose levels in the tissues. Key genes of biosynthetic pathways for amino acids, lipids and cell wall were down regulated, reflecting reduction of sucrose utilisation for stem growth on dwarfing rootstocks. Genes encoding sugar transporters, amino acid transporters, choline transporters, along with large number of potassium channels and aquaporin family members were down-regulated in scion stems on marang rootstocks. Lower activity of plasma membrane H+-ATPase, together with the predominance of genes encoding expansins, wall-associated receptor kinases and key enzymes for biosynthesis and re-modelling of cellulose, xyloglucans and pectins in down-regulated DGEs suggested impairment of cell expansion. Signalling pathways of auxin and gibberellin, along with strigolacton and brassinosteroid biosynthetic genes dominated the down-regulated DEGs. Phenylpropanoid pathway was enriched, with key lignin biosynthetic genes down-regulated, and flavonoid biosynthetic genes upregulated in scions on marang rootstocks. Signalling pathways of salicylic acid, jasmonic acid, ethylene and MAPK cascade were significantly enriched in the upregulated DEGs. CONCLUSIONS: Rootstock-induced disruption in pathways regulating nutrient transport, sucrose utilisation, cell wall biosynthesis and networks of hormone transduction are proposed to impair cell expansion and stem elongation, leading to dwarf phenotype in breadfruit scions. The information provides opportunity to develop screening strategy for rootstock breeding and selection for breadfruit dwarfing.

Expression of Gibberellin Metabolism Genes and Signalling Components in Dwarf Phenotype of Breadfruit (Artocarpus altilis) Plants Growing on Marang (Artocarpus odoratissimus) Rootstocks.

Breadfruit (Artocarpus altilis) is a traditional staple tree crop throughout the tropics. The species is an evergreen tree 15-20 m; there are currently no size-controlling rootstocks within the species. Through interspecific grafting, a dwarf phenotype was identified in breadfruit plants growing on Marang (Artocarpus odoratissimus) rootstocks, which displayed ~60% reduction in plant height with ~80% shorter internodes. To gain insight into the molecular mechanism underlying rootstock-induced dwarfing, we investigated the involvement of gibberellin (GA) in reduction of stem elongation. Expression of GA metabolism genes was analysed in the period from 18 to 24 months after grafting. In comparison to self-graft and non-graft, scion stems on marang rootstocks displayed decrease in expression of a GA biosynthetic gene, AaGA20ox3, and increase in expression of a GA catabolic genes, AaGA2ox1, in the tested 6-month period. Increased accumulation of DELLA proteins (GA-signalling repressors) was found in scion stems growing on marang rootstocks, together with an increased expression of a DELLA gene, AaDELLA1. Exogenous GA treatment was able to restore the stem elongation rate and the internode length of scions growing on marang rootstocks. The possibility that GA deficiency forms a component of the mechanism underlying rootstock-induced breadfruit dwarfing is discussed.

Breadfruit (Artocarpus altilis) gibberellin 2-oxidase genes in stem elongation and abiotic stress response.

Breadfruit (Artocarpus altilis) is a traditional staple tree crop in the Oceania. Susceptibility to windstorm damage is a primary constraint on breadfruit cultivation. Significant tree loss due to intense tropical windstorm in the past decades has driven a widespread interest in developing breadfruit with dwarf stature. Gibberellin (GA) is one of the most important determinants of plant height. GA 2-oxidase is a key enzyme regulating the flux of GA through deactivating biologically active GAs in plants. As a first step toward understanding the molecular mechanism of growth regulation in the species, we isolated a cohort of four full-length GA2-oxidase cDNAs, AaGA2ox1- AaGA2ox4 from breadfruit. Sequence analysis indicated the deduced proteins encoded by these AaGA2oxs clustered together under the C19 GA2ox group. Transcripts of AaGA2ox1, AaGA2ox2 and AaGA2ox3 were detected in all plant organs, but exhibited highest level in source leaves and stems. In contrast, transcript of AaGA2ox4 was predominantly expressed in roots and flowers, and displayed very low expression in leaves and stems. AaGA2ox1, AaGA2ox2 and AaGA2ox3, but not AaGA2ox4 were subjected to GA feedback regulation where application of exogenous GA3 or gibberellin biosynthesis inhibitor, paclobutrazol was shown to manipulate the first internode elongation of breadfruit. Treatments of drought or high salinity increased the expression of AaGA2ox1, AaGA2ox2 and AaGA2ox4. But AaGA2ox3 was down-regulated under salt stress. The function of AaGA2oxs is discussed with particular reference to their role in stem elongation and involvement in abiotic stress response in breadfruit.

Tonoplast lipid composition and proton pump of pineapple fruit during low-temperature storage and blackheart development.

Vacuole represents a major storage organelle playing vital roles in pH homoeostasis and cellular detoxification. The chemical and functional properties of tonoplast in response to chilling temperature and their roles in chilling injury are largely unknown. In the current study, lipid composition of tonoplast and the activities of two vacuolar proton pumps, H?-ATPase (V-ATPase) and H?-pyrophosphatase (V-PPase), were investigated in accordance with the development of blackheart, a form of chilling injury in pineapple fruit (Ananas comosus). Chilling temperature at 10 °C for 1 week induced irreversible blackheart injury in concurrence with a substantial decrease in V-ATPase activity. By contrast, the activity was increased after 1 week at 25 °C. The activity of V-PPase was not changed under both temperatures. Level of total phospholipids of tonoplast decreased at 10 °C, but increased at 25 °C. There was no change at the level of total glycolipids under both temperatures. Thus, low temperature increased the ratio of total glycolipids vs. total phospholipids of tonoplast. Phosphatidylcholine and phosphatidylethanolamine were the predominant phospholipids of tonoplast. Low temperature increased the relative level of phosphatidic acid but decreased the percentage of both phosphatidylcholine and phosphatidylethanolamine. Unsaturated fatty acids accounted for over 60 % of the total tonoplast fatty acids, with C18:1 and C18:2 being predominant. Low temperature significantly decreased the percentage of C18:3. Modification of membrane lipid composition and its effect on the functional property of tonoplast at low temperature were discussed in correlation with their roles in the development of chilling injury in pineapple fruit.

Low temperature alters plasma membrane lipid composition and ATPase activity of pineapple fruit during blackheart development.

Plasma membrane (PM) plays central role in triggering primary responses to chilling injury and sustaining cellular homeostasis. Characterising response of membrane lipids to low temperature can provide important information for identifying early causal factors contributing to chilling injury. To this end, PM lipid composition and ATPase activity were assessed in pineapple fruit (Ananas comosus) in relation to the effect of low temperature on the development of blackheart, a form of chilling injury. Chilling temperature at 10 °C induced blackheart development in concurrence with increase in electrolyte leakage. PM ATPase activity was decreased after 1 week at low temperature, followed by a further decrease after 2 weeks. The enzyme activity was not changed during 25 °C storage. Loss of total PM phospholipids was found during postharvest senescence, but more reduction was shown from storage at 10 °C. Phosphatidylcholine and phosphatidylethanolamine were the predominant PM phospholipid species. Low temperature increased the level of phosphatidic acid but decreased the level of phosphatidylinositol. Both phospholipid species were not changed during storage at 25 °C. Postharvest storage at both temperatures decreased the levels of C18:3 and C16:1, and increased level of C18:1. Low temperature decreased the level of C18:2 and increased the level of C14:0. Exogenous application of phosphatidic acid was found to inhibit the PM ATPase activity of pineapple fruit in vitro. Modification of membrane lipid composition and its effect on the functional property of plasma membrane at low temperature were discussed in correlation with their roles in blackheart development of pineapple fruit.

Dual gene expression cassette vectors with antibiotic selection markers for engineering in Saccharomyces cerevisiae.

BACKGROUND: Manipulations in Saccharomyces cerevisiae classically depend on use of auxotrophy selection markers. There are several disadvantages to this in a microbial cell factory setting: (1) auxotrophies must first be engineered in prototrophic strains, and many industrial strains are polyploid/aneuploid prototrophs (2) available strain auxotrophies must be paired with available repair plasmids (3) remaining auxotrophies must be repaired prior to development of industrial bioprocesses. Use of dominant antibiotic resistance markers can circumvent these problems. However, there are relatively few yeast antibiotic resistance marker vectors available; furthermore, available vectors contain only one expression cassette, and it is often desirable to introduce more than one gene at a time. RESULTS: To overcome these problems, eight new shuttle vectors have been developed. The plasmids are maintained in yeast under a 2 µm ori and in E. coli by a pUC ori. They contain two yeast expression cassettes driven by either (1) the constitutive TEF1 and PGK1 promoters, or (2) the constitutive TEF1 promoter and the inducible GAL10 or HXT7 promoters. Expression strength of these promoters over a typical production time frame in glucose/galactose medium was examined, and identified the TEF1 and HXT7 promoters as preferred promoters over long term fermentations. Selection is provided by either aphA1 (conferring resistance to G418 in yeast and kanamycin/neomycin in E. coli) or ble (conferring resistance to phleomycin in both yeast and E. coli). Selection conditions for these plasmids/antibiotics in defined media were examined, and selection considerations are reviewed. In particular, medium pH has a strong effect on both G418 and phleomycin selection. CONCLUSIONS: These vectors allow manipulations in prototrophic yeast strains with expression of two gene cassettes per plasmid, and will be particularly useful for metabolic engineering applications. The vector set expands the (currently limited) selection of antibiotic marker plasmids available for use in yeast, and in addition makes available dual gene expression cassettes on individual plasmids using antibiotic selection. The resistance gene cassettes are flanked by loxP recognition sites to allow CreA-mediated marker removal and recycling, providing the potential for genomic integration of multiple genes. Guidelines for selection using G418 and phleomycin are provided.

An epidermal-specific ethylene signal cascade regulates trans-differentiation of transfer cells in Vicia faba cotyledons.

*Transfer cells (TCs) trans-differentiate by developing extensive wall ingrowths that facilitate enhanced plasma membrane transport of nutrients. Signal(s) and signalling cascades responsible for initiating this trans-differentiation event are poorly understood. We tested the hypothesis that ethylene functions as a key inductive signal for wall ingrowth formation in epidermal cells of Vicia faba cotyledons. *Scanning electron microscopy of epidermal cells monitored their propensity for wall ingrowth formation. Spatial and temporal expression profiles of ethylene biosynthetic enzymes and key elements of ethylene signalling cascades (ethylene insensitive 3 (EIN3) and ethylene response factors (ERFs)) were determined. *Wall-ingrowth formation responded positively to manipulation of ethylene biosynthesis and perception. It was preceded by a cell-specific burst in ethylene biosynthesis accompanied by a co-localized post-translational up-regulation of VfEIN3-1 and differential expression of three VfERF genes. Blocking ethylene production arrested ongoing wall ingrowth development. Wound-induced ethylene in pod walls and seed coats caused an in planta activation of ethylene biosynthetic genes in adaxial epidermal cells that coincidentally formed wall ingrowths. *A cell-specific burst of ethylene biosynthesis functions as an inductive signal initiating and sustaining trans-differentiation to a TC morphology in vitro. These events are reproduced for developing V. faba seeds in planta.

Early gene expression programs accompanying trans-differentiation of epidermal cells of Vicia faba cotyledons into transfer cells.

Transfer cells (TCs) trans-differentiate from differentiated cells by developing extensive wall ingrowths that enhance plasma membrane transport of nutrients. Here, we investigated transcriptional changes accompanying induction of TC development in adaxial epidermal cells of cultured Vicia faba cotyledons. Global changes in gene expression revealed by cDNA-AFLP were compared between adaxial epidermal cells during induction (3 h) and subsequent building (24 h) of wall ingrowths, and in cells of adjoining storage parenchyma tissue, which do not form wall ingrowths. A total of 5795 transcript-derived fragments (TDFs) were detected; of these, 264 TDFs showed epidermal-specific changes in gene expression and a further 207 TDFs were differentially expressed in both epidermal and storage parenchyma cells. Genes involved in signalling (auxin/ethylene), metabolism (mitochondrial; storage product hydrolysis), cell division, vesicle trafficking and cell wall biosynthesis were specifically induced in epidermal TCs. Blockers of auxin action and vesicle trafficking inhibited ingrowth formation and marked increases in cell division accompanied TC development. Auxin and possibly ethylene signalling cascades induce epidermal cells of V. faba cotyledons to trans-differentiate into TCs. Trans-differentiation is initiated by rapid de-differentiation to a mitotic state accompanied by mitochondrial biogenesis driving storage product hydrolysis to fuel wall ingrowth formation orchestrated by a modified vesicle trafficking mechanism.

Intracellular sucrose communicates metabolic demand to sucrose transporters in developing pea cotyledons.

Mechanistic inter-relationships in sinks between sucrose compartmentation/metabolism and phloem unloading/translocation are poorly understood. Developing grain legume seeds provide tractable experimental systems to explore this question. Metabolic demand by cotyledons is communicated to phloem unloading and ultimately import by sucrose withdrawal from the seed apoplasmic space via a turgor-homeostat mechanism. What is unknown is how metabolic demand is communicated to cotyledon sucrose transporters responsible for withdrawing sucrose from the apoplasmic space. This question was explored here using a pea rugosus mutant (rrRbRb) compromised in starch biosynthesis compared with its wild-type counterpart (RRRbRb). Sucrose influx into cotyledons was found to account for 90% of developmental variations in their absolute growth and hence starch biosynthetic rates. Furthermore, rr and RR cotyledons shared identical response surfaces, indicating that control of transporter activity was likely to be similar for both lines. In this context, sucrose influx was correlated positively with expression of a sucrose/H(+) symporter (PsSUT1) and negatively with two sucrose facilitators (PsSUF1 and PsSUF4). Sucrose influx exhibited a negative curvilinear relationship with cotyledon concentrations of sucrose and hexoses. In contrast, the impact of intracellular sugars on transporter expression was transporter dependent, with expression of PsSUT1 inhibited, PsSUF1 unaffected, and PsSUF4 enhanced by sugars. Sugar supply to, and sugar concentrations of, RR cotyledons were manipulated using in vitro pod and cotyledon culture. Collectively the results obtained showed that intracellular sucrose was the physiologically active sugar signal that communicated metabolic demand to sucrose influx and this transport function was primarily determined by PsSUT1 regulated at the transcriptional level.

Aquaporins and unloading of phloem-imported water in coats of developing bean seeds.

Nutrients are imported into developing legume seeds by mass flow through the phloem, and reach developing embryos following secretion from their symplasmically isolated coats. To sustain homeostasis of seed coat water relations, phloem-delivered nutrients and water must exit seed coats at rates commensurate with those of import through the phloem. In this context, coats of developing French bean seeds were screened for expression of aquaporin genes resulting in cloning PvPIP1;1, PvPIP2;2 and PvPIP2;3. These genes were differentially expressed in all vegetative organs, but exhibited their strongest expression in seed coats. In seed coats, expression was localized to cells of the nutrient-unloading pathway. Transport properties of the PvPIPs were characterized by expression in Xenopus oocytes. Only PvPIP2;3 showed significant water channel activity (Pos = 150-200 microm s(-1)) even when the plasma membrane intrinsic proteins (PIPs) were co-expressed in various combinations. Permeability increases to glycerol, methylamine and urea were not detected in oocytes expressing PvPIPs. Transport active aquaporins in native plasma membranes of seed coats were demonstrated by measuring rates of osmotic shrinkage of membrane vesicles in the presence and absence of mercuric chloride and silver nitrate. The functional significance of aquaporins in nutrient and water transport in developing seeds is discussed.

A suite of sucrose transporters expressed in coats of developing legume seeds includes novel pH-independent facilitators.

A suite of newly discovered sucrose transporter genes, PsSUF1, PsSUF4, PvSUT1 and PvSUF1, were isolated from the coats of developing pea (Pisum sativum L.) and bean (Phaseolus vulgaris L.) seeds. Sequence analysis indicated that deduced proteins encoded by PsSUF1, PvSUT1 and PvSUF1 clustered in a separate sub-group under sucrose transporter Clade I, whereas the deduced protein encoded by PsSUF4 clustered in Clade II. When expressed in yeast, these genes were shown to encode sucrose transporters with apparent Michaelis Menten constant (Km) values ranging from 8.9 to 99.8 mm. PvSUT1 exhibited functional characteristics of a sucrose/H+ symporter. In contrast, PsSUF1, PvSUF1 and PsSUF4 supported the pH- and energy independent transport of sucrose that was shown to be bi-directional. These transport properties, together with that of counter transport, indicated that PsSUF1, PvSUF1 and PsSUF4 function as carriers that support the facilitated diffusion of sucrose. Carrier function was unaffected by diethylpyrocarbonate and by maltose competition, suggesting that the sucrose binding sites of these transporters differed from those of known sucrose/H+ symporters. All sucrose transporters were expressed throughout the plant and, of greatest interest, were co-expressed in cells considered responsible for sucrose efflux from seed coats. The possible roles played by the novel facilitators in sucrose efflux from seed coats are discussed.

Review: Nutrient loading of developing seeds.

Interest in nutrient loading of seeds is fuelled by its central importance to plant reproductive success and human nutrition. Rates of nutrient loading, imported through the phloem, are regulated by transport and transfer processes located in sources (leaves, stems, reproductive structures), phloem pathway and seed sinks. During the early phases of seed development, most control is likely to be imposed by a low conductive pathway of differentiating phloem cells serving developing seeds. Following the onset of storage product accumulation by seeds, and, depending on nutrient species, dominance of path control gives way to regulation by processes located in sources (nitrogen, sulfur, minor minerals), phloem path (transition elements) or seed sinks (sugars and major mineral elements, such as potassium). Nutrients and accompanying water are imported into maternal seed tissues and unloaded from the conducting sieve elements into an extensive post-phloem symplasmic domain. Nutrients are released from this symplasmic domain into the seed apoplasm by poorly understood membrane transport mechanisms. As seed development progresses, increasing volumes of imported phloem water are recycled back to the parent plant by process(es) yet to be discovered. However, aquaporins concentrated in vascular and surrounding parenchyma cells of legume seed coats could provide a gated pathway of water movement in these tissues. Filial cells, abutting the maternal tissues, take up nutrients from the seed apoplasm by membrane proteins that include sucrose and amino acid/H+ symporters functioning in parallel with non-selective cation channels. Filial demand for nutrients, that comprise the major osmotic species, is integrated with their release and phloem import by a turgor-homeostat mechanism located in maternal seed tissues. It is speculated that turgors of maternal unloading cells are sensed by the cytoskeleton and transduced by calcium signalling cascades.

[Rhodamine 6G association particle based spectrophotometric determination of hydroxy free radical and its application to the selection of antioxidant].

In HCl-NaAc buffer solution, the hydroxy free radical from the Fenton reaction is captured by excess KI and releases I3-. The I3- combines with Rhodamine B (RhB, lamdamax=554 nm), Rhodamine 6G(Rh6G, lamdamax=526 nm), Rhodamine S (RhS, lamdamax=526 nm), and butyl Rhodamine B(b -RhB, A,lamdamax56 nm) to form association particles, so the absorbance at max wavelength decreases. The concentration of hydroxy free radical (calculated by the concentration of hydrogen peroxide) is proportional to the decreased absorbance of the systems of RhB, Rh6G, RhS, b-RhB in the range of 0.136-0.680 microg x L(-1), 0.34-0.680 microg x mL(-1), 0.034-0.680 microg x mL(-1), 0.034-0.680 microg c mL(-1), espectively. Based on this fact, a new method for the determination of scavenging percentage of hydroxy free radical with antioxidant was developed. The resistance to oxidation of four substances and six kinds of tea extract were measured with satisfactory results.

Identification of the facilitative glucose transporter 12 gene Glut12 in mouse preimplantation embryos.

In this study we report the cloning and characterisation of the mouse Glut12 gene and examine for the first time its expression pattern in the earliest stages of development. Mouse Glut12 (mGlut12) was cloned from preimplantation embryos by 5'RACE RT-PCR using primers designed from an EST clone corresponding to a human GLUT12 antigenic sequence after positive immunoreactivity was observed in mouse two-cell embryos by western immunoblotting. The mGlut12 gene contains an open reading frame of 1869 base pairs, potentially encoding a polypeptide of 622 amino acids. The predicted mGLUT12 protein bears all the hallmarks of the SLC2A family of hexose transporters and shares an 83% sequence homology to human GLUT12. Consistent with its human homolog mGlut12 mRNA is found highly expressed in skeletal and cardiac muscle and fat. Additionally, it was also found in the uterus and during early embryogenesis. During early development in the mouse, Glut12 expression is clearly apparent in ovulated oocytes and two-cell embryos but declines in day 3 morulae. With the exception of some Glut12 expression apparent in blastocysts, Glut12 mRNA remains at low to undetectable levels until E11.

Transcriptional regulation of a pineapple polyphenol oxidase gene and its relationship to blackheart.

Two genes encoding polyphenol oxidase (PPO) were isolated from pineapple (Ananas comosus[L.] Merr. cv. Smooth Cayenne). Sequence analyses showed that both contained a single intron and encoded typical chloroplast-localized PPO proteins, the sequences of which corresponded to two pineapple PPO cDNAs, PINPPO1 and PINPPO2, recently described by Stewart et al. (2001). Southern blot analyses suggested that pineapple contained only two PPO genes. Analysis of expression of PINPPO1 promoter GUS fusion constructs showed this promoter had a low basal activity and was cold- and wound-inducible, consistent with known mRNA expression profiles. Striking homologies to gibberellin response complexes (GARC) were observed in sequences of both the PINPPO1 and PINPPO2 promoters. Transient assays in mature pineapple fruit and stable expression in transgenic tobacco showed that PINPPO1 promoter-GUS fusions were indeed gibberellin (GA) responsive. A role for the element within the putative GARCs in mediating GA-responsiveness of the PINPPO1 promoter was confirmed by mutational analysis. PINPPO2 was also shown to be GA-responsive by RT-PCR analysis. Mutant PINPPO1 promoter-GUS fusion constructs, which were no longer GA-inducible, showed a delayed response to cold induction in pineapple fruit in transient assays, suggesting a role for GA in blackheart development. This was supported by observations that exogenous GA(3) treatment induced blackheart in the absence of chilling. Sequences showing homology to GARCs are also present in some PPO promoters in tomato, suggesting that GA regulates PPO expression in diverse species.