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Sandra B. Wilson and Dennis R. Decoteau

Similarities exist between the effects of phytochrome and cytokinins on plant growth and development (e.g., chloroplast development, amaranthin synthesis, seed germination). It is unclear, however, if and how these two systems interact. The coaction between phytochrome and cytokinins was investigated by using Nicotiana plumbaginifolia plants transformed with the isopentenyl transferase (ipt) cytokinin gene and treated with end-of-day (EOD) red (R) and far-red (FR) light. The ipt gene was under control of either a constitutive cauliflower mosaic virus promoter (35S-plants) or an inducible, heat shock promoter (HS-plants). When treated with EOD FR light, whole plants were characterized by decreased chlorophyll concentrations and increased fresh weights. When treated with EOD R light, 35S-plants contained high concentrations of zeatin riboside (ZR) compared to plants treated with EOD FR light. When treated with EOD FR light, HS-plants contained high concentrations of ZR compared to plants treated with EOD R light. Both cytokinin responses were photoreversible. The reasons for the differences between the 35S- and HS-plant responses are not known. Results appear to implicate interactions between phytochrome and cytokinins in plant growth and development.

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A.R. Kuehnle, T. Fujii, F.C. Chen, A. Alvarez, N. Sugii, R. Fukui, S.L. Aragon, and J.M. Jaynes

Two cultivars of Anthurium andraeanum Hort. hybrids, `Paradise Pink' and `Tropic Flame', were transformed by Agrobacterium to contain gene sequences for Shiva-1, a cecropin-based lytic peptide. The antibacterial gene was driven by a 35-35S cauliflower mosaic viral (35-35S CaMV) promoter and the construct included the secretory signal sequence for pathogenesis-related protein 1b (PR1b). Blight tolerance of regenerated plants was tested by inoculation with a virulent strain of Xanthomonas axonopodis (formerly campestris) pv. dieffenbachiae (Xad) that is bioluminescent to allow detection of symptomless infections in Shiva-1 transformants. Primary regenerants for two Shiva-1 transgenic lines of `Paradise Pink' displayed significantly enhanced tolerance to bacterial blight over blight susceptible `Rudolph' and even the blight tolerant `Kalapana'. Two Shiva-1 transgenic lines of `Tropic Flame' showed no improved resistance when compared to the control at the mean percent leaf infection level. One Shiva-1 transgenic line of `Tropic Flame' was unexpectedly more susceptible to blight than the nontransgenic control. Low expression of Shiva-1 observed in this line is hypothesized to be the cause of its increased susceptibility to Xad.

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Hong Y. Yang and Schuyler S. Korban

Developing an efficient gene transfer system for apple (Malus ×domestica L.) remains a major objective in genetic engineering efforts of this fruit crop. Transient expression of the uidA gene coding for β-glucuronidase (GUS) and driven by the cauliflower mosaic virus 35S promoter (CaMV35S) has been induced in apple cotyledonary explants of mature seeds by tungsten particle bombardment using the Particle Inflow Gun (PIG). Several factors that affect transient expression of the GUS gene in apple cotyledons were investigated. The gene transfer efficiency was monitored by recording the number of blue spots observed on explants two days following bombardment. Precultivation of cotyledons for 18 hours before bombardment significantly increased the number of blue foci. Of the three different precipitation methods tested including water, 25% PEG, and 60% glycerol, the latter was the most effective for coating DNA onto tungsten particles. Washing DNA-coated tungsten particles with 70% ethanol and resuspending in 100% ethanol significantly enhanced gene delivery to cotyledons. The amount of particles used for each bombardment also influenced GUS expression. About 0.5 mg of particles per shot resulted in the highest number of blue foci. Using larger quantity of particles (i.e., 2 mg) drastically decreased GUS expression probably due to the toxicity of tungsten particles.

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Carol J. Lovatt

The goal of this research was to identify the role essential nutrients play in the physiology of tree crops, and then to apply the nutrient as a foliar fertilizer to stimulate a specific metabolic process at phenological stages when nutrient demand is high. This approach has proven successful. A single winter prebloom foliar application of nitrogen as low-biuret urea [0.16 kg N/tree (0.35 lb N/tree)] to 30-year-old `Washington' navel orange (Citrus sinensis L. Osbeck) trees during flower initiation significantly increased yield and fruit number per tree for each of 3 consecutive years (P ≤ 0.05). The number of commercially valuable largesize fruit also increased significantly with yield increases (r 2 = 0.88). Sodium tetraborate applied foliarly to `Hass' avocado (Persea americana Mill.) trees at the cauliflower stage of inflorescence development (elongation of inflorescence secondary axes, pollen and ovule development) increased the number of pollen tubes reaching the ovule, ovule viability and cumulative yield (P ≤ 0.05). Additional examples are presented.

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Guo-qing Song, Hideo Honda, and Ken-ichi Yamaguchi

Leaves are usually the target tissue for expressing transgenes conferring resistances to herbicides, pests, and diseases. To achieve leaf-specific expression, a light-harvest chlorophyll a/b binding protein (CAB) of photosystem-II (CAB2) promoter (CAB2-p) from rice (Oryza sativa L.) and the cauliflower mosaic virus 35S promoter were fused to the β-glucuronidase (GUS) reporter and subsequently evaluated in transgenic sweetpotato [Ipomoea batatas L. (Lam.)]. The 35S promoter-directed GUS activities varied from 46.0 to 61.2 nmol 4-methyl-umbelliferyl-β-D-glucuronide (4-MU) per minute per milligram of protein in leaf, stem, primary, and storage roots. In contrast, the CAB2-p directed an uneven distribution of GUS activities (4-MU at 1.1 to 12.6 nmol·min−1·mg−1 protein); GUS activity in mature leaves was ≈12-fold as high as that in storage roots. In addition, GUS assay in leaf tissues revealed that CAB2-p enabled a developmentally controlled and light-regulated GUS expression. These results indicate that the rice CAB2-p could be used to drive leaf-specific expression of linked genes in sweetpotato.

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Jiao Chen, De-bao Yuan, Chao-zheng Wang, Yi-xing Li, Fen-fang Li, Ke-qian Hong, and Wang-jin Lu

Many reports indicate that an abundance of really interesting new gene (RING) play key roles in regulating defense responses against abiotic and biotic stresses in plants. In this study, the cloning and functional characterization of a RING gene, MaRING2, in banana (Musa acuminata) fruit are reported. MaRING2 belongs to the NEP1-interacting protein (NIP) RING-H2 finger protein family. Gene expression profiles revealed that MaRING2 was cold responsive and induced by abscisic acid (ABA) treatment during cold storage. In this study, the MaRING2 under control of the Cauliflower mosaic virus 35S (CaMV 35S) promoter was transformed to tobacco (Nicotiana benthamiana) using agrobacterium (Agrobacterium tumefaciens)-mediated transformation. The resultant MaRING2-overexpressing transgenic plants (35S:MaRING2) exhibited significantly increased tolerance to low temperatures and were hypersensitive to exogenous ABA in terms of germination and early seedling growth. In addition, overexpression of MaRING2 enhanced the expression of stress-responsive genes under normal (before cold stress) or cold conditions. These results demonstrate the biological role of MaRING2 in conferring cold tolerance. Taken together, these results suggest that MaRING2, a C3H2C3-type RING protein, is a positive regulator of the ABA-dependent stress response.

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Yasutaka Kubo, Akitsugu Inaba, and Reinosuke Nakamura

The respiration rate (O2 uptake) and the rate of C2H4. production were measured before, during, and after 24 hours of treatment with 60% CO2 (20% O2) in 18 kinds of fruits and vegetables by use of an automated system connected to a microcomputer. High CO2 decreased respiration only in climacteric fruit and broccoli, which were producing C2H4. Ethylene production decreased with CO, treatment of peaches, tomatoes, and broccoli, but that of bananas increased. In five nonclimacteric fruits (three citrus species, grapes, and Japanese pears) and several vegetables (carrots, onions, cauliflower, and cabbage), in which C2H4 production was not detected, high CO2 affected respiration little, if at all. When eggplants, cucumbers, podded peas, spinach, and lettuce were treated with high CO2, C2H4 production began and respiration increased. These results indicate that the respiratory responses of harvested horticultural crops to high CO2 might be mediated by the effects of CO2 on the action and/or synthesis of C2H4.

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Kisung Ko, John L. Norelli, Jean-Paul Reynoird, Herb S. Aldwinckle, and Susan K. Brown

Genes encoding lysozyme (T4L) from T4 bacteriophage and attacin E (attE) from Hyalophora cecropia were used, either singly or in combination, to construct plant binary vectors, pLDB15, p35SAMVT4, and pPin2Att35SAMVT4, respectively, for Agrobacterium-mediated transformation of `Galaxy' apple, to enhance resistance to Erwinia amylovora. In these plasmids, the T4L gene was controlled by the cauliflower mosaic virus 35S promoter with duplicated upstream domain and the untranslated leader sequence of alfalfa mosaic virus RNA 4, and the attE gene was controlled by the potato proteinase inhibitor II (Pin2) promoter. All transgenic lines were screened by polymerase chain reaction (PCR) for T4L and attE genes, and a double-antibody sandwich enzyme-linked immunosorbent assay for neomycin phosphotransferase II. Amplification of T4L and attE genes was observed in reverse transcriptase-PCR, indicating that these genes were transcribed in all tested transgenic lines containing each gene. The attacin protein was detected in all attE transgenic lines. The expression of attE under the Pin2 promoter was constitutive but higher levels of expression were observed after mechanical wounding. Some T4L or attE transgenic lines had significant disease reduction compared to nontransgenic `Galaxy'. However, transgenic lines containing both attE and T4L genes were not significantly more resistant than nontransgenic `Galaxy', indicating that there was no in planta synergy between attE and T4L with respect to resistance to E. amylovora.

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Manjul Dutt, Zhijian T. Li, Sadanand Dhekney, and Dennis J. Gray

Genetic transformation of plants necessitates the use of promoters to control transgene expression. Numerous promoters have been isolated from a wide range of organisms for use in plants. However, many of these natural promoters exhibit relatively low activity and/or have limited use. To provide an alternative, we constructed a composite promoter (EP) using a genomic DNA sequence and a 35 bp TATA-containing fragment from the 2S albumin (VvAlb1) gene core promoter of grapevine. The 0.9-kb genomic sequence was identified after TAIL-PCR, based on the presence of several unique cis-acting elements. The sequence showed no homology to any known plant gene, enhancer, and promoter. Two binary vectors, pEP-EGFP/NPT and pEP-GUS, containing a bifunctional EGFP/NPTII fusion gene and a GUS gene, respectively, were constructed to test transcriptional activity of the composite promoter both qualitatively and quantitatively. Transient GFP expression was observed in somatic embryos (SE) of Vitis vinifera `Thompson Seedless' after Agrobacterium-mediated transformation using pEP-EGFP/NPT. Quantitative GUS assay of stably transformed SE containing pEP-GUS indicated that the EP composite promoter was capable of producing GUS activity as high as 12% of that from a doubly enhanced Cauliflower Mosaic Virus 35S promoter or eight times higher than that from a doubly enhanced Cassava Vein Mosaic Virus promoter. In addition, transformation of Arabidopsis with pEP-GUS yielded comparable GUS activity throughout the plant. These data indicate that the EP composite promoter can be used in transformation studies to provide sustained constitutive gene expression in plants.

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Jessica L. Boldt, James E. Barrett, and David G. Clark

Petunia × hybrida `Electric Purple' plants, genetically transformed (Selecta Klemm Co.) via Agrobacterium tumefaciens to constitutively express the Cauliflower Mosaic Virus 35S promoter (CaMV35S) fused to two separate Arabidopsis c-repeat binding factor cDNAs (CBF3 & CBF4), were utilized to evaluate water relations. Non-stressed plants followed a classical stomatal conductance pattern, with maximum conductance between 1000 hr and 1400 hr. CBF3 and CBF4 plants showed an increase in transpiration rates and a decrease in stomatal resistance at 1230 hr, compared to `Electric Purple'. Transpiration rates (per unit leaf area) were similar in CBF3 and `Electric Purple' plants, but CBF4 plants were 12% less than `Electric Purple'. Xylem water potentials at visible wilt were between –1.4 and –1.5 MPa and there were no significant differences between line or irrigation treatment. A fourth experiment observed differential plant responses to stress cycles. Under non-stress irrigation conditions, CBF4 plants showed an increase in stomatal resistance and a decrease in transpiration rate compared to `Electric Purple' plants. There were no differences in the xylem water potential at visible wilt for the first and third stress cycles, but, for the second cycle, xylem water potentials at wilt were –1.9, –1.7 and –1.4 Mpa for CBF4, `Electric Purple' and CBF3 plants, respectively. CBF3 and CBF4 plants showed small differences in performance as compared to `Electric Purple' and under mild stress conditions as imposed in these experiments apparent heterologous overexpression of the Arabidopsis CBF3 & 4 transgenes may not be sufficient for conferring drought tolerance in petunia.