Robert E. Paull

The draft genome of the transgenic tropical fruit tree papaya (Carica papaya Linnaeus)

Papaya, a fruit crop cultivated in tropical and subtropical regions, is known for its nutritional benefits and medicinal applications. Here we report a 3× draft genome sequence of ‘SunUp’ papaya, the first commercial virus-resistant transgenic fruit tree to be sequenced. The papaya genome is three times the size of the Arabidopsis genome, but contains fewer genes, including significantly fewer disease-resistance gene analogues. Comparison of the five sequenced genomes suggests a minimal angiosperm gene set of 13,311. A lack of recent genome duplication, atypical of other angiosperm genomes sequenced so far, may account for the smaller papaya gene number in most functional groups. Nonetheless, striking amplifications in gene number within particular functional groups suggest roles in the evolution of tree-like habit, deposition and remobilization of starch reserves, attraction of seed dispersal agents, and adaptation to tropical daylengths. Transgenesis at three locations is closely associated with chloroplast insertions into the nuclear genome, and with topoisomerase I recognition sites. Papaya offers numerous advantages as a system for fruit-tree functional genomics, and this draft genome sequence provides the foundation for revealing the basis of Carica’s distinguishing morpho-physiological, medicinal and nutritional properties.

Heat treatment and fruit ripening

Abstract Postharvest heat treatments lead to an alteration of gene expression and fruit ripening can sometimes be either delayed or disrupted. The extent of the alternation of fruit ripening is a function of the exposure temperature and duration and how quickly the commodity is cooled following the heat treatment. The most commonly measured components of fruit ripening affected by heat treatments include fruit softening, membrane and flavor changes, respiration rate, ethylene production, and volatile production. Cell wall degrading enzymes and ethylene production are frequently the most disrupted and are sometimes not produced or their appearance is delayed following heating. Other processes associated with ripening are not altered to the same extent or soon recover. Fruit sensitivity to heat treatments is modified by preharvest weather conditions, cultivar, rate of heating, and subsequent storage conditions. The amount of sensitivity or tolerance to heat stress of a commodity is related to the level of heat protective proteins at harvest and the postharvest production of heat shock proteins. Two types of heat responses are seen. The first is a normal cellular response ( 45°C and is modified by the pre-stress environmental conditions, the cellular response to stress and cellular recovery. Loss of membrane integrity appears to be an effect and not a cause of injury. The site of the injury lesion is still unknown and could be associated with transcription, translation and cellular recovery capacity after an injury threshold has been exceeded.

The pineapple genome and the evolution of CAM photosynthesis

Pineapple (Ananas comosus (L.) Merr.) is the most economically valuable crop possessing crassulacean acid metabolism (CAM), a photosynthetic carbon assimilation pathway with high water-use efficiency, and the second most important tropical fruit. We sequenced the genomes of pineapple varieties F153 and MD2 and a wild pineapple relative, Ananas bracteatus accession CB5. The pineapple genome has one fewer ancient whole-genome duplication event than sequenced grass genomes and a conserved karyotype with seven chromosomes from before the ρ duplication event. The pineapple lineage has transitioned from C3 photosynthesis to CAM, with CAM-related genes exhibiting a diel expression pattern in photosynthetic tissues. CAM pathway genes were enriched with cis-regulatory elements associated with the regulation of circadian clock genes, providing the first cis-regulatory link between CAM and circadian clock regulation. Pineapple CAM photosynthesis evolved by the reconfiguration of pathways in C3 plants, through the regulatory neofunctionalization of preexisting genes and not through the acquisition of neofunctionalized genes via whole-genome or tandem gene duplication.

SOME ASPECTS OF LEAD IN PLANT NUTRITION

SummaryAccretion of lead by abscised barley roots from a lead nitrate solution was metabolically obtained, complemented by a fraction acquired physico-chemically. Added calcium supply did not significantly modify lead accretion. A ten-fold increase in supply concentration produced approximately a two-fold increase in lead accretion.During growth of bean, barley, and tomato plants in solution cultures, a substantial part of the lead supplied became associated with the roots; correspondingly little was present in the tops. A very large fraction of the lead associated with the roots of barley was readily extracted by various solvents, probably resident on the root surface or from free spaces therein; the remainder of the lead acquired may have been that sorbed within the root cells. A possible relationship of lead to phosphate accretion was not evident. There was no apparent effect of applied lead on the sorption of the other elements supplied. Plant dry weights, their top to root ratios and dry-weight percentages were constant under various lead supply concentrations in solution. Where lead supplies were advertently withheld, lead contents in plants were 5 to 25 times that which could have been expected under the solution culture conditions in a greenhouse with carbon filtered ambient air supply. Restriction of advertent lead supply caused no limitation of growth.If lead is essential to growth of these plant species, the critical concentration will probably be less than 2 to 6 ng atoms/g dry weight in plant tops.

Changes in papaya cell walls during fruit ripening

Abstract The apparent molecular mass range of different extractable fractions of papaya fruit pectin and hemicellulose during fruit ripening was determined. The pectin molecular mass declined and the solubility of pectin in cyclohexane-diaminotetraacetic acid and Na 2 CO 3 solutions increased during ripening. The molecular mass decreased and the solubility of hemicellulose in KOH increased during ripening. Water soluble uronic acid increased 6-fold during ripening as the yield of cell wall material declined. The loss of high molecular mass pectins decreased throughout ripening while the demethylation rate was greater early in ripening. Changes in pectin molecular size did not parallel loss of fruit firmness during early ripening. The pectin fractions were mainly composed of rhamnose, glucose, xylose, galactose, mannose and arabinose, in decreasing order of concentration. Non-cellulosic glucose and xylose were the main neutral sugars in the hemicellulose fraction followed by mannose and galactose and traces of rhamnose and arabinose. These results suggested that pectin hydrolysis and the modification of hemicellulose both were involved in papaya fruit softening. Pectin hydrolysis was apparently more important during the late phase of fruit softening.

Metabolic engineering of invertase activities in different subcellular compartments affects sucrose accumulation in sugarcane cells

Transgenic sugarcane (Saccharum officinarum L.) lines were created to express altered invertase isoform activity to elucidate the role(s) of invertase in the sucrose accumulation process. A sugarcane soluble acid invertase cDNA (SCINVm, AF062734) in the antisense orientation was used to decrease invertase activity. The Saccharomyces cerevisiae invertase gene (SUC2), fused with appropriate targeting elements, was used to increase invertase activity in the apoplast, cytoplasm and vacuole. A callus/liquid culture system was established to evaluate change in invertase activity and sugar concentration in the transgenic lines. Increased invertase activity in the apoplast led to rapid hydrolysis of sucrose and rapid increase of hexose in the medium. The cellular hexose content increased dramatically and the sucrose level decreased. Cells with higher cytoplasmic invertase activity did not show a significant change in the sugar composition in the medium, but did significantly reduce the sucrose content in the cells. Transformation with the sugarcane antisense acid invertase gene produced a cell line with moderate inhibition of soluble acid invertase activity and a 2-fold increase in sucrose accumulation. Overall, intracellular and extracellular sugar composition was very sensitive to the change in invertase activities. Lowering acid invertase activity increased sucrose accumulation.

Construction of a Sequence-Tagged High-Density Genetic Map of Papaya for Comparative Structural and Evolutionary Genomics in Brassicales

A high-density genetic map of papaya (Carica papaya L.) was constructed using microsatellite markers derived from BAC end sequences and whole-genome shot gun sequences. Fifty-four F2 plants derived from varieties AU9 and SunUp were used for linkage mapping. A total of 707 markers, including 706 microsatellite loci and the morphological marker fruit flesh color, were mapped into nine major and three minor linkage groups. The resulting map spanned 1069.9 cM with an average distance of 1.5 cM between adjacent markers. This sequence-based microsatellite map resolved the very large linkage group 2 (LG 2) of the previous high-density map using amplified fragment length polymorphism markers. The nine major LGs of our map represent papayas haploid nine chromosomes with LG 1 of the sex chromosome being the largest. This map validates the suppression of recombination at the male-specific region of the Y chromosome (MSY) mapped on LG 1 and at potential centromeric regions of other LGs. Segregation distortion was detected in a large region on LG 1 surrounding the MSY region due to the abortion of the YY genotype and in a region of LG6 due to an unknown cause. This high-density sequence-tagged genetic map is being used to integrate genetic and physical maps and to assign genome sequence scaffolds to papaya chromosomes. It provides a framework for comparative structural and evolutional genomic research in the order Brassicales.

Minimal processing of papaya (Carica papaya L.) and the physiology of halved fruit

Abstract The stage of papaya fruit ripening suitable for minimal processing and the effect of processing on physiology were determined. Wounding during slicing and deseeding led to an increase in ethylene production and respiration, earlier skin degreening and flesh softening. Fruit at the 10–50% skin yellow ripening stage showed higher wound-induced ethylene production and respiration when sliced and deseeded than riper fruit. Fruit with 60–80% skin yellowing when halved and deseeded had higher initial ethylene production and respiration than other ripening stages. When halved and deseeded fruit were stored at ca. 4°C, ethylene production and respiration declined rapidly within 1 day. Fruit with 55–80% skin yellowing and less than 50 N flesh firmness had more than 50% edible flesh and easily removed seeds. These fruit were suitable for minimal processing when combined with low storage temperature (ca. 4°C) to inhibit ethylene production and respiration.

Cloning of the Papaya Chromoplast-Specific Lycopene β-Cyclase, CpCYC-b, Controlling Fruit Flesh Color Reveals Conserved Microsynteny and a Recombination Hot Spot

Carotenoid pigments in fruits are indicative of the ripening process and potential nutritional value. Papaya (Carica papaya) fruit flesh color is caused by the accumulation of lycopene or β-carotenoids in chromoplasts. It is a distinct feature affecting nutritional composition, fruit quality, shelf life, and consumer preference. To uncover the molecular basis of papaya flesh color, we took map-based cloning and candidate gene approaches using integrated genetic and physical maps. A DNA marker tightly linked to flesh color colocalized on a contig of the physical map with a cDNA probe of the tomato (Solanum lycopersicum) chromoplast-specific lycopene β-cyclase, CYC-b. Candidate gene sequences were obtained from amplified fragments and verified by sequencing two bacterial artificial chromosomes containing the two alleles. Sequence comparison revealed a 2-bp insertion in the coding region of the recessive red flesh allele resulting in a frame-shift mutation and a premature stop codon. A color complementation test in bacteria confirmed that the papaya CpCYC-b is the gene controlling fruit flesh color. Sequence analysis of wild and cultivated papaya accessions showed the presence of this frame-shift mutation in all red flesh accessions examined. Evaluation of DNA markers near CpCYC-b revealed a recombination hot spot, showing that CpCYC-b is located in a gene-rich region with a recombination rate at 3.7 kb per centimorgan, more than 100-fold higher than the genome average at 400 kb per centimorgan. Conserved microsynteny of the CpCYC-b region is indicated by colinearity of two to four genes between papaya, Arabidopsis (Arabidopsis thaliana), grape (Vitis vinifera), and tomato. Our results enhanced our understanding of papaya flesh color inheritance and generated new tools for papaya improvement.

Effect of gamma-irradiation on ripening papaya pectin

Abstract Papaya ( Carica papaya , L., var. Sunset) at three initial ripeness stages were irradiated with 0.25, 0.50, 0.75, 1.0, or 1.5 kGy gamma-irradiation and pectin changes during ripening determined. A significant linear relationship was found between irradiation dose and firmness immediately after irradiation. Irradiation had no effect on fruit skin or flesh color of papaya fruit irradiated at the 5 to 30% yellow stage and allowed to ripen. Papaya irradiated when 5 to 30% yellow showed no significant changes in pectin methylesterase activity when ripe. Immediately after irradiation, the pectin in 10 to 30% yellow papaya showed depolymerization and demethoxylation, though no effect on pectin methylesterase activity was detected. There was an increase in water soluble pectin (WSP), while chelator soluble (CSP) and alkali soluble pectin (ASP) decreased, with a significant decline in the methanol content of the ASP fraction. After the 25 to 30% yellow ripeness stage, fruit irradiated at 0.50 to 1.0 kGy had less pectic depolymerization, and had a firmer texture than nonirradiated when ripe. A lower level of WSP and higher levels of CSP and ASP were found in ripe fruit that had been irradiated at 0.5 to 1.0 kGy when 25 to 30% yellow skin with a significant quadratic relationship between irradiation dose and the three pectin fractions. The firmness of these irradiated fruit were retained for two days longer than the nonirradiated control.