Genome-wide analysis of the invertase gene family from maize

Key message

The recent release of the maize genome (AGPv4) contains annotation errors of invertase genes and therefore the enzymes are bestly curated manually at the protein level in a comprehensible fashion

Abstract

The synthesis, transport and degradation of sucrose are determining factors for biomass allocation and yield of crop plants. Invertase (INV) is a key enzyme of carbon metabolism in both source and sink tissues. Current releases of the maize genome correctly annotates only two vacuolar invertases (ivr1 and ivr2) and four cell wall invertases (incw1, incw2 (mn1), incw3, and incw4). Our comprehensive survey identified 21 INV isogenes for which we propose a standard nomenclature grouped phylogenetically by amino acid similarity: three vacuolar (INVVR), eight cell wall (INVCW), and ten alkaline/neutral (INVAN) isogenes which form separate dendogram branches due to distinct molecular features. The acidic enzymes were curated for the presence of the DPN tripeptide which is coded by one of the smallest exons reported in plants. Particular attention was placed on the molecular role of INV in vascular tissues such as the nodes, internodes, leaf sheath, husk leaves and roots. We report the expression profile of most members of the maize INV family in nine tissues in two developmental stages, R1 and R3. INVCW7, INVVR2, INVAN8, INVAN9, INVAN10, and INVAN3 displayed the highest absolute expressions in most tissues. INVVR3, INVCW5, INVCW8, and INVAN1 showed low mRNA levels. Expressions of most INVs were repressed from stage R1 to R3, except for INVCW7 which increased significantly in all tissues after flowering. The mRNA levels of INVCW7 in the vegetative stem correlated with a higher transport rate of assimilates from leaves to the cob which led to starch accumulation and growth of the female reproductive organs.

     

The inhibition of sphingomyelin synthase 1 activity induces collecting duct cells to lose their epithelial phenotype

Epithelial tissue requires that cells attach to each other and to the extracellular matrix by the assembly of adherens junctions (AJ) and focal adhesions (FA) respectively. We have previously shown that, in renal papillary collecting duct (CD) cells, both AJ and FA are located in sphingomyelin (SM)-enriched plasma membrane microdomains. In the present work, we investigated the involvement of SM metabolism in the preservation of the epithelial cell phenotype and tissue organization. To this end, primary cultures of renal papillary CD cells were performed. Cultured cells preserved the fully differentiated epithelial phenotype as reflected by the presence of primary cilia. Cells were then incubated for 24 h with increasing concentrations of D609, a SM synthase (SMS) inhibitor. Knock-down experiments silencing SMS 1 and 2 were also performed. By combining biochemical and immunofluorescence studies, we found experimental evidences suggesting that, in CD cells, SMS 1 activity is essential for the preservation of cell-cell adhesion structures and therefore for the maintenance of CD tissue/tubular organization. The inhibition of SMS 1 activity induced CD cells to lose their epithelial phenotype and to undergo an epithelial-mesenchymal transition (EMT) process.     

Somatic embryogenesis and plant regeneration in cotton (Gossypium hirsutum L.)

Tissue culture methods for improvement of cotton has lagged seriously compared to other major crops. A method for regeneration of cotton which includes a morphogenetically competent cell suspension was needed to facilitate selection of stress-resistant variants and gene manipulation. Preliminary screening of eight strains of Gossypium hirsutum L. for embryogenic potential resulted in the production of somatic embryos in all strains. Coker 312 was selected for use in the development of a model regeneration system for G. hirsutum. Calli were initiated from hypocotyl tissues of 3-day-old-seedlings. Globular embryos were present after six weeks in culture. Calli were subcultured to liquid suspension in growth regulator-free medium. After three to four weeks, suspensions were sieved to collect globular and heart stage embryos. Collected embryos developed further when plated onto semi-solid medium. To induce germination and plantlet growth, mature embryos were placed on sterile vermiculite saturated with medium. Upon development of roots and two true leaves, plantlets were potted in peat and sand, and hardened. Mature plants and progeny have been obtained with this procedure. A high percentage of infertile plants was observed among the regenerants.Abbreviations NAA 1 naphthaleneacetic acid - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - MS Murashige and Skoog - BA 6 benzylamino purine - 2i P N⁶-(²-isopentenyladenine     

The development of gender-related behavior in females following prenatal exposure to diethylstilbestrol (DES)

Animal research has shown that diethylstilbestrol (DES) present during the sensitive developmental periods of the hypothalamus and adjacent areas of the brain affects the development of sex-dimorphic brain structures and subsequent behavior. To test for corresponding behavioral effects in humans, 30 women with a history of prenatal DES exposure were contrasted with 30 unexposed women who had been referred to the same clinic for a colposcopic examination because of an abnormal Pap smear. Gender-role behavior of childhood, adolescence, and adulthood was assessed by means of a semistructured interview, the Gender Role Assessment Schedule-Adult, and the Bem Sex Role Inventory. The mothers of these women were interviewed about their daughters with the "mother form" of the same interview schedule. The results suggest that DES women show less orientation toward parenting than the controls. There were no consistent group differences in other domains of gender-role behavior.     

Dna Synthetic Activity in Tumor-Bearing Mice

The rate of DNA synthesis in normal tissues exhibits circadian rhythmicity. However, there have been conflicting reports of the effects of tumor burden on the circadian rhythm of DNA synthesis in non-cancer tissues. We have developed a mouse colon cancer (MC-26) that exhibits different growth under different photoperiods. The purpose of this study was to analyze DNA synthetic activity in tissues removed from tumor-bearing and tumor-free mice maintained under two different photoperiods. Two groups each of approximately 80 male Balb/c mice were acclimated to one of two light-dark cycles, 12L:12D or 6L:18D. Half of each group were injected with 5.0 × 104 MC-26 cells. Twenty-two days later, all mice were killed in subgroups at 4-6 hr intervals over one 24-hr period. Colons and tumors were removed for measurement of DNA synthesis. Results were analyzed by means of one-way analysis of variance (ANO VA) in order to determine whether DNA synthesis varied significantly within groups over the 24-hr period. The DNA synthetic activity, as measured by uptake of tritiated thymidine, exhibited significant temporal variation in the colons of control (tumor-free) mice under both the 12L:12D and 6L:18D photoperiods. The colons of tumor-bearing mice failed to exhibit a fluctuation under a 12L:12D photoperiod but did show a significant 24-hr rhythm under the 6L:18D photoperiod. The subcutaneously growing cancers did not exhibit a circadian variation in DNA synthetic activity under either photoperiod. Both photoperiod and the presence of cancer appear to affect the DNA synthetic activity observed in mice bearing the MC-26 colon cancer.     

Somatic embryogenesis in cotton (Gossypium). II. Requirements for embryo development and plant regeneration

Calli of cotton (Gossypium hirsutum L.) initiated from seedling hypocotyl tissue were placed in liquid suspension and maintained by serial subculture in hormone-free Murashige and Skoog (MS) medium. Suspensions were sieved and globular embryos collected, washed, resuspended in basal medium and plated onto various semi-solid media. High inorganic salts (MS), low salt (2/3 MS), excess KNO₃, and the growth regulators napthaleneacetic acid (NAA), gibberellic acid (GA₃) and kinetin were tested for their effects on somatic embryo maturation. Long-term embryo proliferation and maturation were best on medium containing MS plus 1.9g/l KNO₃. Embryos 3 mm to 10 mm in size were removed from this plating medium and placed on sterile vermiculite saturated with Stewart and Hsu's medium plus 0.1 mg/l indoleacetic acid (IAA). Plants were recovered from 10.6% of the embryos. When 5 mm embryos were placed on this medium, 30% of the embryos formed plants within six weeks. Smaller embryos required a longer period of development on the vermiculite and the addition of fresh medium supplemented with 0.1 mg/l GA₃. Plants with an extensive root system and two true leaves were removed from sterile culture and potted in either one-to-one peat and sand, or vermiculite. Eighty percent of the regenerants were successfully hardened when glass beakers of increasing size (10 to 150 ml) were sequentially placed over the young plants during a two-week period.     

25-Hydroxysterols increase the permeability of liposomes to Ca2+ and other cations

25-Hydroxycholesterol and 25-hydroxy vitamin D-3 increased the permeability of liposomes to Ca²⁺ measured by the arsenazo III encapsulation technique. This effect was sensitive to the lipid composition of the membrane, with changes that decreased the motional freedom of phospholipid acyl chains decreasing Ca²⁺ permeability. The greatest permeability was observed with the zwitter-ionic phospholipids, phosphatidylcholine and phosphatidylethanolamine, whereas the acidic phospholipids, phosphatidylinositol and phosphatidylserine, depressed Ca²⁺ permeability. The effect was not specific for Ca²⁺. Other divalent cations were translocated in the order Mn²⁺ > Mg²⁺  Ca²⁺ ? Sr²⁺  Ba²⁺. The permeability of liposomes to the monovalent cation, Na⁺, was also substantially increased. The effect did not appear to be due to ionophoretic properties of the sterols, and it is suggested that perturbation of the membranes by the polar 25-hydroxyl group may play a role in increasing membrane permeability.     

Identification and localization of G-proteins in the clonal adipocyte cell lines HGFu and Ob17

HGFu and Ob17 are cell lines derived from adipose tissue of lean (+/?) and ob/ob mice, respectively. Neither adenylyl cyclase activity nor G protein abundance and subcellular distribution have been assessed previously in these cells. Cyclase activity was low and resistant to catecholamine stimulation in both cell lines. However, the enzyme could be stimulated to high levels by forskolin and Mn²⁺. G_sα (largely the long isoform), G_iα2, and Gβ were the major G protein subunits identified. The levels of G protein mRNA expression were similar in both cell lines and, unlike actin expression, did not change as a result of differentiation. Immunoblotting and ADP-ribosylation of the G peptides corroborated these results. Assessment of the subcellular localization of the subunits by indirect epifluorescence and scanning confocal microscopy showed that each of the subunits had a characteristic subcellular pattern. G_sα showed vesicular cytoplasmic and nuclear staining; G_iα2 colocalized with actin stress fibers and disruption of these structures altered the distribution of G_iα2; β subunits showed some colocalization with the stress fibers as well as a cytoplasmic vesicular and nuclear pattern. As a result of differentiation, there was reorganization of the actin, together with the G_iα2 and β fibrous patterns. Both cell lines showed similar modifications. The induction of differentiation in these cells is therefore not associated with changes in adenylyl cyclase activity nor of the abundance of G-protein subunits, although reorganization of some of these subunits does accompany actin reorganization.     

Cell cycle regulated expression of nucleolar antigen P120 in normal and transformed human fibroblasts

Normal and SV40 virus-transformed WI-38 human lung fibroblasts were serum starved and refed, or synchronized by double thymidine block and released from the block. At different time points in the cell cycle, steady state levels of P120 mRNA and P120 protein content of the cells were determined by densitometric scans of Northern and Western blots. At the same time points, [³H]thymidine uptake was measured and flow cytometric analysis performed for DNA content and P120 antigen staining. Levels of P120 protein and P120 mRNA were approximately 4 times greater in non-synchronous, exponentially growing transformed cells than in similarly growing normal cells. Early G₁-cells, synchronized either with serum deprivation or with metabolic block, contained only a trace amount of P120 protein and mRNA. The P120 gene was transcribed early in G₁ and P120 protein synthesis initiated in middle G₁. A dramatic increase of P120 protein level occurred in S-phase with a corresponding mRNA peak preceding the P120 protein peak. These results indicate that P120 is overexpressed in transformed WI-38 cells and that P120 is temporally regulated during the cell cycle of both transformed and normal fibroblasts. The dramatic increase in P120 protein expression at the G₁ to S boundary suggests that P120 may play a role in the regulation of cell cycle and increased nucleolar activity that is associated with cell proliferation. © 1993 Wiley-Liss, Inc.