SUMMARYPolyamines (PAs) are ubiquitous, polycationic biogenic amines that are implicated in many biological processes, including plant growth and development, but their precise roles remain to be determined. Most of the previous studies have involved three biogenic amines: putrescine (Put), spermidine (Spd) and spermine (Spm), and their derivatives. We have expressed a yeast spermidine synthase (ySpdSyn) gene under constitutive (CaMV35S) and fruit-ripening specific (E8) promoters in Solanum lycopersicum (tomato), and determined alterations in tomato vegetative and fruit physiology in transformed lines compared with the control. Constitutive expression of ySpdSyn enhanced intracellular levels of Spd in the leaf, and transiently during fruit development, whereas E8-ySpdSyn expression led to Spd accumulation early and transiently during fruit ripening. The ySpdSyn transgenic fruits had a longer shelf life, reduced shriveling and delayed decay symptom development in comparison with the wild-type (WT) fruits. An increase in shelf life of ySpdSyn transgenic fruits was not facilitated by changes in the rate of water loss or ethylene evolution. Additionally, the expression of several cell wall and membrane degradation-related genes in ySpdSyn transgenic fruits was not correlated with an extension of shelf life, indicating that the Spd-mediated increase in fruit shelf life is independent of the above factors. Crop maturity, indicated by the percentage of ripening fruits on the vine, was delayed in a CaMV35S-ySpdSyn genotype, with fruits accumulating higher levels of the antioxidant lycopene. Notably, whole-plant senescence in the transgenic plants was also delayed compared with WT plants. Together, these results provide evidence for a role of PAs, particularly Spd, in increasing fruit shelf life, probably by reducing post-harvest senescence and decay.

Cell production is an essential facilitator of fruit growth and development. Cell production during carpel/floral-tube growth, fruit set, and fruit growth, and its regulation by cell cycle genes were investigated in apple (Malus×domestica Borkh.). Cell production was inhibited during late carpel/floral-tube development, resulting in growth arrest before bloom. Fruit set re-activated cell production between 8 d and 11 d after full bloom (DAFB) and triggered fruit growth. The early phase of fruit growth involved rapid cell production followed by exit from cell proliferation at ∼24 DAFB. Seventy-one cell cycle genes were identified, and expression of 59 genes was investigated using quantitative RT-PCR. Changes in expression of 19 genes were consistently associated with transitions in cell production during carpel/floral-tube growth, fruit set, and fruit growth. Fourteen genes, including B-type cyclin-dependent kinases (CDKs) and A2-, B1-, and B2-type cyclins, were positively associated with cell production, suggesting that availability of G2/M phase regulators of the cell cycle is limiting for cell proliferation. Enhanced expression of five genes including that of the putative CDK inhibitors, MdKRP4 and MdKRP5, was associated with reduced cell production. Exit from cell proliferation at G0/G1 during fruit growth was facilitated by multiple mechanisms including down-regulation of putative regulators of G1/S and G2/M phase progression and up-regulation of KRP genes. Interestingly, two CDKA genes and several CDK-activating factors were up-regulated during this period, suggesting functions for these genes in mediating exit from cell proliferation at G0/G1. Together, the data indicate that cell cycle genes are important facilitators of cell production during apple fruit development.

Fruit size regulation was studied in the apple cultivar ‘Gala’ and a large fruit size spontaneous mutant of ‘Gala’, ‘Grand Gala’ (GG). GG fruits were 15% larger in diameter and 38% heavier than ‘Gala’ fruits, largely due to an increase in size of the fruit cortex. The mutation in GG altered growth prior to fruit set and during fruit development. Prior to fruit set, the carpel/floral-tube size was enhanced in GG and was associated with higher cell number, larger cell size, and increased ploidy through endoreduplication, an altered form of the cell cycle normally absent in apple. The data suggest that the mutation in GG promotes either cell production or endoreduplication in the carpel/floral-tube cells depending on their competence for division. Ploidy was not altered in GG leaves. During fruit growth, GG fruit cells exited cell production earlier, and with a DNA content of 4C suggesting G2 arrest. Cell size was higher in GG fruits during exit from cell production and at later stages of fruit growth. Final cell diameter in GG fruit cortex cells was 15% higher than that in ‘Gala’ indicating that enhanced fruit size in GG was facilitated by increased cell size. The normal progression of cell expansion in cells arrested in G2 may account for the increase in cell size. Quantitative RT-PCR analysis indicated higher MdCDKA1 expression and reduced MdCYCA2 expression during early fruit development in GG fruits. Together, the data indicate an important role for cell expansion in regulating apple fruit size.

Application of transcriptomics approaches can greatly enhance our understanding of blueberry physiology. The success of transcriptomics approaches is dependent on the extraction of high-quality RNA which is complicated by the abundance of polyphenolics and polysaccharides in blueberry. Additionally, transcriptomics requires the accurate quantification of transcript abundance. Quantitative real-time polymerase chain reaction (qRT-PCR) is a robust method to determine transcript abundance. Normalization of gene expression using stably expressed reference genes is essential in qRT-PCR. An evaluation of the stability of expression of reference genes has not yet been reported in blueberry. The objectives of this study were to develop an effective procedure for extracting RNA from different organs and to evaluate potential reference genes for qRT-PCR analyses in blueberry. RNA of high quality and yield was extracted from eight and six organs of rabbiteye and southern highbush blueberry, respectively, using a modified cetyltrimethyl ammonium bromide-based method. The expression stability of 12 reference genes was evaluated. UBIQUITIN-CONJUGATING ENZYME (UBC28), RNA HELICASE-LIKE (RH8), CLATHRIN ADAPTER COMPLEXES MEDIUM SUBUNIT FAMILY PROTEIN (CACSa), and POLYUBIQUITIN (UBQ3b) were the most stably expressed genes across multiple organs in both blueberry species. Further, the expression stability of the reference genes in the branch abscission zone following treatment with fruit abscission-inducing compounds was analyzed. CACSa, RH8, and UBC28 were the most stably expressed genes in the abscission zone under abscission-inducing conditions. We suggest a preliminary evaluation of UBC28, CACSa, RH8, and UBQ3b to identify the most suitable reference genes for the experimental conditions under consideration in blueberry.

BackgroundFruit growth in apple (Malus × domestica Borkh.) is mediated by cell production and expansion. Genes involved in regulating these processes and thereby fruit growth, are not well characterized. We hypothesized that the apple homolog(s) of AINTEGUMENTA (ANT), an APETALA2–repeat containing transcription factor, regulates cell production during fruit growth in apple.ResultsTwo ANT genes, MdANT1 and MdANT2, were isolated from apple and their expression was studied during multiple stages of fruit development. MdANT1 and MdANT2 expression was high during early fruit growth coincident with the period of cell production, rapidly declined during exit from cell production, and remained low during the rest of fruit development. The effects of increase in carbohydrate availability during fruit growth were characterized. Increase in carbohydrate availability enhanced fruit growth largely through an increase in cell production. Expression of MdANT1 and MdANT2 increased sharply by up to around 5-fold in response to an increase in carbohydrate availability. Expression of the ANT genes was compared across two apple genotypes, ‘Gala’ and ‘Golden Delicious Smoothee’ (GS), which differ in the extent of fruit growth, largely due to differences in cell production. In comparison to ‘Gala’, the larger fruit-size genotype, GS, displayed higher levels and a longer duration of MdANT1 and MdANT2 expression. Expression of the ANTs and cell cycle genes in the fruit core and cortex tissues isolated using laser capture microdissection was studied. During early fruit growth, expression of the MdANTs was higher within the cortex, the tissue that constitutes the majority of the fruit. Additionally, MdANT1 and MdANT2 expression was positively correlated with that of A- and B-type CYCLINS, B-type CYCLIN-DEPENDENT-KINASES (CDKBs) and MdDEL1.ConclusionsMultiple lines of evidence from this study suggest that MdANT1 and MdANT2 regulate cell production during fruit growth in apple. ANTs may coordinate the expression of cell proliferation genes and thereby affect the competence of cells for cell production during fruit growth. Together, data from this study implicate MdANT1 and MdANT2 in the regulation of fruit growth in apple.

Plant responses to drought stress vary depending on the severity of stress and the stage of drought progression. To improve the understanding of such responses, the leaf physiology, abscisic acid (ABA) concentration, and expression of genes associated with ABA metabolism and signalling were investigated in Petunia × hybrida. Plants were exposed to different specific substrate water contents (θ = 0.10, 0.20, 0.30, or 0.40 m3·m–3) to induce varying levels of drought stress. Plant responses were investigated both during the drying period (θ decreased to the θ thresholds) and while those threshold θ were maintained. Stomatal conductance (gs) and net photosynthesis (A) decreased with decreasing midday leaf water potential (Ψleaf). Leaf ABA concentration increased with decreasing midday Ψleaf and was negatively correlated with gs (r = –0.92). Despite the increase in leaf ABA concentration under drought, no significant effects on the expression of ABA biosynthesis genes were observed. However, the ABA catabolism-related gene CYP707A2 was downregulated, primarily in plants under severe drought (θ = 0.10 m3∙m–3), suggesting a decrease in ABA catabolism under severe drought. Expression of phospholipase Dα (PLDα), involved in regulating stomatal responses to ABA, was enhanced under drought during the drying phase, but there was no relationship between PLDα expression and midday Ψleaf after the θ thresholds had been reached. The results show that drought response of plants depends on the severity of drought stress and the phase of drought progression.

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Fruit size is a valuable commercial trait in blueberry. The cellular basis of variation in fruit size among rabbiteye blueberry (Vaccinium ashei) genotypes was investigated. Twenty genotypes, including cultivars and advanced selections from the University of Georgia blueberry breeding program, were analyzed. Among the 20 genotypes, fruit weight and fruit diameter varied by over threefold and 1.6-fold, respectively. Regression analysis indicated a linear relationship between fruit weight and fruit diameter (R2 = 0.97, P < 0.001), suggesting that fruit diameter is a good predictor of fruit weight. Among the 20 genotypes, mesocarp cell number and cell area varied by almost 2.5-fold and 1.5-fold, respectively. Although fruit diameter and cell number were significantly related (R2 = 0.79, P < 0.001), no relationship could be established between fruit diameter and cell area. These data indicate that variation in fruit size among rabbiteye blueberry genotypes is primarily facilitated by variation in cell number. Two small and two large fruit size genotypes were further analyzed. Differences in cell number among some of these genotypes were apparent at bloom suggesting that cell production before bloom is an important mechanism contributing to variation in final cell number. Differences in final cell number among other genotypes were manifested during fruit development, indicating that cell production during fruit development was also instrumental in determining variation in final cell number. This study suggests that fruit size variation in rabbiteye blueberry genotypes is determined by mechanisms that regulate cell production before bloom and during fruit development.