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Robert E. Rouse, Monica Ozores-Hampton, Fritz M. Roka, and Pamela Roberts

with real-time PCR for detection of HLB bacteria. Total DNA was extracted from HLB-symptomatic leaves in which the petioles chopped and processed by the modified Tris-Base β-mercaptoethanol method of Li et al. (2006) . Reactions were carried out in a

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Denise V. Duclos* and Thomas Björkman

Cauliflower (Brassica oleracea var. italica) and Broccoli (Brassica oleracea var. botrytis) differ mainly in the stage of reproductive arrest. Cauliflower curd is an inflorescence meristem, while broccoli arrests just before anthesis. Arabidopsis studies led to the hypothesis that a mutant BoCAL allele arrested cauliflower earlier. Later, a mutant in BoAP1 was found to have similar effects. These partially redundant genes, and several identified since, are present in multiple copies in B. oleracea. Understanding their role in the arrest requires quantification of transcript abundance analysis by real-time PCR. Designing selective PCR primers is a critical first step in the process. Designs were based on alignment among the genes of interest (MADS-box genes BoCAL, BoAP1, FUL, and the non MADS-box genes LFY and TFL1) and their paralogs. The high sequence similarity (some over 95%) makes the target transcripts difficult to distinguish. Therefore, primers were designed mostly for targets in the 3'UTR region in order to gain specificity. Short amplicons, 68bp to 200bp, were required for the high PCR efficiency required to quantify these low-abundance transcripts. Primers were evaluated by conventional RT-PCR and real-time PCR. By altering temperature, Total RNA was isolated from plants that were arrested at three developmental stages, inflorescence meristem (cauliflower), floral meristem (intermediate), and floral bud (broccoli) by varying temperature. RT-PCR products were single bands of the expected size, despite the high homology between genes under study. Real-time melting curve analysis (fluorescence derivative vs. melting temperature) corroborated the presence of a single amplicon. The identity of products was confirmed by sequencing and restriction enzyme digestion.

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Xiaoxu Yang, Chang Liu, Zhishan Yan, Youjun Fan, Guojun Feng, and Dajun Liu

Environmental factors (daylength and temperature) and internal signals (gibberellins and autonomous pathways) simultaneously regulate flowering time in plants ( Bhakta et al., 2017 ; Putterill et al., 2004 ). Higher plants detect fluctuations in

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Ping Lang, Can-kui Zhang, Fenny Dane, Shasha Meng, Robert Ebel, and Narendra Singh

Commercial citrus species, some of the most important fruit crops worldwide, are sensitive to sub-freezing temperatures. Poncirus trifoliata, a species closely related to commercial citrus and tolerant to –30 °C, has been used in breeding programs or as a rootstock to impart greater freeze tolerance. Gene expression of P. trifoliata and C. unshiu (Satsuma mandarin) were investigated and compared under slow and fast cold-acclimation regimes. The mRNA differential display-polymerase chain reaction (DDRT-PCR) and cDNA-AFLP, coupled with quantitative relative RT-PCR or real-time PCR were used. Many unique gene fragments were isolated and found to be up- or down–regulated as a result of exposure to low temperature. The up-regulated fragments in Poncirus show high similarities to genes involved in osmotic regulation (betaine/proline transporter, water channel protein, and nitrate transporter), oxidative stress (aldoketo reductase, early light induced protein), and protein interaction (tetratricopeptide-repeat protein, F-box protein, and ribosomal protein L15). In C. unshiu the up-regulated genes show high similarities to genes involved in transcription (zinc finger and GTP-binding protein-related), signal transduction (14–3–3 protein and extension-like protein), protein synthesis and amino acid translocation (permease and ribosomal proteins), chromosome folding (chromosome condensation, structural maintenance of chromosomes-like protein), and carbohydrate metabolism (glycosyl transferase). Several genes involved in photosynthesis, defense and cell wall metabolism were down regulated. Characterization of cold responsive genes will be discussed.

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Suping Zhou, Roger Sauve, Tingting Chen, Sara Bhatti, and Debrah Long

Identification of low temperature–regulated gene expression in Pachysandra terminalis: Pachysandra terminalis is a cold-hardy, evergreen plant species. In order to identify molecular mechanism of cold tolerance of this plant species, seedlings with four fully expanded leaves were subjected to 4, 0, and –1 °C low temperature treatments. Low temperature–induced genes were identified from treated plants using cDNA differential display. The cDNA fragments were cloned onto PCR-trap vectors. Low temperature regulation of these genes was confirmed by reverse-northern blot. Sequence analysis has identified that these genes can be classified into three groups, stress-related, photosystem-related. Most of the genes cannot find matching sequences in the database. To further study the regulation of these genes by temperature fluctuation, the plants were treated at 4, 0, and 40 °C. Northern blot analysis showed that several clones showed increased expression after cold and heat shock. Previous cold treatment at 4 °C can negate the effect of heat shock on expression of these genes. Complete sequence of these genes is cloned from the cDNA library and their temporal regulation by environmental stresses is analyzed using real-time PCR.

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H.R. Pappu, S.D. Wyatt, and K.L. Druffel

Dahlia is an important ornamental crop in the U.S. The economic value of the crop is often affected by viral diseases. Of several viruses that infect dahlia, dahlia mosaic virus (DMV) is of most concern. However, little or no information is available about its distribution. A survey of dahlias in several states in the U.S. was carried out during 2003 and 2004. Samples from CA, GA, MD, ME, MT, NM, PA, OR, and WA were tested for DMV. To develop a molecular detection assay, the viral genome was cloned and sequenced and based on the sequence information, DMV-specific primers were used in a PCR-based assay. DMV was detected in >90% of the samples tested. Based on the detection of DMV, a wide range of symptoms were found to be associated with DMV infection. A real-time PCR assay was adapted for rapid detection of DMV. Considering its widespread occurrence, steps are needed to limit its further spread. An effective intervention program would include use of virus-free material to minimize its impact. Availability of a rapid and sensitive detection method such as the once described should facilitate not only production of virus-free dahlias but elimination of virus infected material from breeding and propagating stocks. This is the first report of a survey to determine the extent of DMV incidence in dahlias.

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Denise V. Duclos and Thomas N. Björkman

Brassica oleracea species differ in the developmental stage of their reproductive meristems at harvest. The stage that characterizes each variety depends on its genetic makeup, environment and the interaction between them. We tested a model of arrest in B. oleracea to determine functional redundancy among the paralogous genes CAL, AP1a, AP1c, FULa, FULb, FULc, and FULd; and to resolve the immediate effect of temperature on gene expression in meristems whose developmental fate is temperature regulated. By varying temperature during reproductive development, three stages of arrest were obtained: inflorescence meristem (cauliflower), floral meristem (intermediate) and floral bud (broccoli), the latter initiated by low temperature. Gene expression was measured by quantitative real time PCR (qRT-PCR). The LFY/TFL1 ratio increased as the reproductive development advanced, mainly due to decreased TFL1 expression; influenced by a dramatic increase in AP1c toward floral bud formation. The expression patterns of the FUL paralogs indicate different roles in reproductive development. FULa was more abundant in the floral primordia, while FULb, FULc, and FULd were associated with earlier arrest at the inflorescence meristem stage. The high expression of FULc and FULd at all stages of arrest differs from their homolog in Arabidopsis. High temperature reduced AP1 and LFY expression but the meristem did not revert from reproductive to vegetative. Floral bud formation in plants recessive for AP1a and CAL reaffirm that functional redundancy among some of these genes can complement the mutations. Varying temperature alone, at a fixed developmental stage, caused little variation in the expression of genes studied, causing small significant differences in TFL1 and AP1c.

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Sheetal Rao, Scott Finlayson, Chuanjiu He, Ronald Lacey, Raymond Wheeler, and Fred T. Davies

The NASA Advanced Life Support (ALS) System for space habitation will likely operate under reduced atmospheric pressure (hypobaria). There are engineering, safety, and plant growth advantages in growing crops under low pressure. In closed production environments, such as ALS, excessive plant-generated ethylene may negatively impact plant growth. Growth of lettuce (Lactuca sativa) in the Low Pressure Plant Growth (LPPG) system was enhanced under low pressure (25kPa), due in part to decreased ethylene production. Under reduced pO2, ethylene production decreased under low as well as ambient conditions (He et al., 2003). During hypobaria, the expression of genes encoding ethylene biosynthesis enzymes, namely ACC synthase (ACS) and ACC oxidase (ACO), is not known. The primary objective of this research was to characterize the expression of ACS and ACO genes in response to hypobaria. Three-week-old Arabidopsis was used to determine the effects of hypobaria (25 kPa) and reduced O2 (12 kPa pO2) at the molecular level. Candidate gene expression was tested using quantitative real-time PCR at different times after treatment. Under low pressure, ACO1 expression is induced in the initial 12 hours of treatment, gradually decreasing with increased exposure. At 12 kPa pO2, ACO1 was induced under ambient conditions, suggesting that plants under low pressure may be more tolerant to hypoxic stress. The mechanism for enhanced growth of lettuce under hypobaric conditions will be studied further by analysis of the ACS and ACO gene families, and stress-responsive genes, namely late-embryogenesis abundant (LEA) proteins and dehydrins.

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Gordon J. Lightbourn, Robert J. Griesbach, and John R. Stommel

Color observed in plants is due to several pigments, in particular chlorophylls, carotenoids, flavonoids, and betalains. The many hues can be attributed to a number of biochemical factors, inclusive of pigment concentration, pigment combinations and their ratios, and vacuolar pH. Shades of violet to black pigmentation in pepper (Capsicum annuum L.) are attributed to anthocyanin accumulation. The color of unripe pepper fruit varies from green and yellow to ivory, through varying shades of violet and purple to nearly black. Whereas pepper fruit color is important for culinary product quality, foliar pigmentation is also an important aspect of ornamental variety appeal. Foliage and stem color may vary from green to varying shades of green/purple to nearly black. HPLC analysis of violet and black pepper fruit revealed a single anthocyanidin that was identified as delphinidin. Black fruit contained five-fold higher chlorophyll concentrations in comparison to violet fruit, which contained relatively little chlorophyll. Differences in fruit pH were not statistically significant. Similar to fruit, black pepper leaf tissue contained delphinidin as the predominant anthocyanidin, but in higher concentration relative to that found in fruit. The results demonstrate that high concentrations of delphinidin in combination with chlorophyll account for black pigmentation. Real-time PCR analysis of tissues that varied in pigmentation intensity due to varying anthocyanin concentration revealed functional, but differentially expressed, structural genes in the anthocyanin biosynthetic pathway. Analysis of regulatory gene expression identified a MYB transcription factor that was differentially expressed in response to varying anthocyanin concentration.

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Ruigang Wu, Yi Wang, Ting Wu, Xuefeng Xu, and Zhenhai Han

of MdMYB4 were analyzed in detail, and the effects of abiotic stress at different time spans on the expression of MdMYB4 was analyzed using qRT-PCR. We identified a variety of hormone-related response elements ( Supplemental Table 3 ), including