Transcription Factor ANAC074 Binds to NRS1, NRS2, or MybSt1 Element in Addition to the NACRS to Regulate Gene Expression

NAC (NAM, ATAF1/2, and CUC2) transcription factors play important roles in many biological processes, and mainly bind to the NACRS with core sequences “CACG” or “CATGTG” to regulate gene expression. However, whether NAC proteins can bind to other motifs without these core sequences remains unknown. In this study, we employed a Transcription Factor-Centered Yeast one Hybrid (TF-Centered Y1H) screen to study the motifs recognized by ANAC074. In addition to the NACRS core cis-element, we identified that ANAC074 could bind to MybSt1, NRS1, and NRS2. Y1H and GUS assays showed that ANAC074 could bind the promoters of ethylene responsive genes and stress responsive genes via the NRS1, NRS2, or MybSt1 element. ChIP study further confirmed that the bindings of ANAC074 to MybSt1, NRS1, and NRS2 actually occurred in Arabidopsis. Furthermore, ten NAC proteins from different NAC subfamilies in Arabidopsis thaliana were selected and confirmed to bind to the MybSt1, NRS1, and NRS2 motifs, indicating that they are recognized commonly by NACs. These findings will help us to further reveal the functions of NAC proteins.

The maize Activator/Dissociation system is functional in hexaploid wheat through successive generations

The aim of the present study was to provide useful background information and evidence of the functionality of the maize Activator/Dissociation (Ac/Ds) system in hexaploid wheat. Two transgenic parental wheat lines, one harbouring the immobilised Ac element (iAc) and the other the Ds element (pUbi[Ds-uidA]bar), were crossed. Transient GUS assays confirmed that the iAc transposase is active in hexaploid wheat. Selected F1 and F2 lines were analysed by PCR using primers specific to Ac, uidA and bar genes. The primer pair Ubi/bar-tag was used to detect excision of the Ds-uidA sequence, which occurred at a frequency of 39% in the F1 generation. Lines free of Ac and showing evidence of Ds excision were subject to Southern analysis, which indicated that at least one transposition event might have occurred in these lines. Although more evidence is required to unequivocally support the reintegration of the Ds element in the wheat genome, the evidence presented here nevertheless demonstrates the effectiveness and potential value of using this system to tag genes in wheat.

Tracking Papaya Pollen Movement with the GUS Transgene Marker

Genetically engineered (GE), virus-resistant papaya cultivars in Hawaii are easily identified by a colorimetric assay for the β-glucuronidase (GUS) marker transgene. We used GUS to track pollen movement from a central 1-acre plot of gynodioecious GE `Rainbow' plants into seeds on surrounding border rows of non-GE `Sunrise' papaya. GUS evidence of cross-pollination occurred in 70% of female plants (43% of assayed seeds), compared with only 13% of the predominantly self-pollinating hermaphrodite plants (7% of seeds) segregating in the gynodioecious `Sunrise' border rows. The percentage of GUS+ seeds in border row plants showed a weak negative correlation (r = –0.32) with distance from the nearest GE tree (30 m maximum). In a non-GE papaya field located less than a mile downwind from the `Rainbow' source, no evidence of GUS was found in 1000 assayed seeds. In a separate study, the origin of GUS+ seed discovered in papaya fruits from an organic farm was investigated. Leaf GUS assays revealed that 70% of trees were GE, indicating that the grower had planted GE seed. The impact of pollen drift from GE trees in the same field was determined by screening seed samples from 20 non-GE hermaphrodites for GUS expression. Only three hermaphrodites (15%) showed GUS+ seeds, at low levels ranging from 3% to 6% of contaminated samples. These data indicate that the major source of GE contamination in organic fields is seeds of unverified origin, rather than pollen drift from neighboring GE fields. Organic growers are advised to: 1) plant only seed that is known to be non-GE, preferably obtained by manual self-pollination of selected non-GE hermaphrodites; 2) avoid open-pollinated seed; and 3) grow only hermaphrodite (self-pollinating) trees, removing any female or male plants from production fields.

MsEnod12A Expression Is Linked to Meristematic Activity During Development of Indeterminate and Determinate Nodules and Roots

During development of nodules and roots, the alfalfa early nodulin gene MsEnod12A is expressed adjacent to the meristem. Using transgenic alfalfa carrying a MsEnod12A promoter-gusA fusion, we investigated the regulation of MsEnod12A expression in mature nodules and roots by GUS assays and reverse transcription-PCR. We found that in alfalfa indeterminate nodules induced by various Fix- Rhizobium meliloti mutants and in spontaneous nodules devoid of rhizobia, MsEnod12A was expressed at the distal end when a persistent meristem was present. However, this gene was not expressed when the meristem was lacking in nodules arrested in development. The MsEnod12A-gusA fusion was introduced into Lotus corniculatus plants that form determinate nodules devoid of a persistent meristem. Using these plants we found MsEnod12A-gusA expression only in young nodules and a disappearance in mature nodules. Moreover, when alfalfa roots were treated with auxins a lateral band of MsEnod12A expression was observed surrounding the club-shaped root apex and coinciding with induced lateral meristematic activities. Thus, in all cases MsEnod12A expression was associated with meristematic activities, suggesting that MsEnod12A plays a role in the differentiation processes of nodule and root cells and that it may serve as molecular tool for analyzing meristem establishment during nodule development.

Tumor Formation and β-Glucuronidase Expression in Phaseolus vulgaris Inoculated with Agrobacterium tumefaciens

Ten common bean (Phaseolus vulgaris L.) lines—including cultivars, breeding lines, and one wild line—were evaluated for susceptibility to Agrobacterium tumefaciens strain C58 by stab-inoculating intact shoot tips of germinating seeds. Significant differences for tumor frequency and size were found on the resulting 3-week-old seedlings. UW 325, a wild bean, had the highest rate of tumorigenesis; `Olathe', a dry bean cultivar, had the lowest. Uninoculated excised shoot tips cultured in media with BA or BA plus NAA exhibited differences in phytohormone sensitivity, as evidenced by callusing and root initiation. The cultivar Montcalm seemed to be highly sensitive, while `Olathe' was relatively insensitive. Fluorometric GUS assays of shoot tips from germinating seeds inoculated with the disarmed GUS-containing A. tumefaciens strain C58C1(pGV3850/pKIWI105) showed that UW 325 had the highest level of GUS activity. `Montcalm' had a high rate of tumorigenesis but a low level of GUS activity; this anomaly was attributed to its high phytohormone sensitivity. The use of the virulence-inducing compound acetosyringone in the inoculum culture medium did not alter genotypic differences (ranks) in susceptibility. Histochemical GUS assays of inoculated UW 325 shoot tips showed that 60% of the apexes exhibited one or more transformation events. Chemical names used: β-glucuronidase (GUS); α-naphthaleneacetic acid (NAA); N-(phenylmethyl)-1H-purin-6-amine (BA).

HORTICULTURAL CHARACTERISTICS OF TRANSGENIC TOBACCO EXPRESSING THE ROL C GENE FROM AGROBACTERIUM RHIZOGENES

Tobacco (Nicotiana tabacum cv Wisconsin 38) leaf discs were transformed with the disarmed Agrobacterium tumefaciens strain EHA101 carrying the Rol C gene from A. rhizogenes (Oono et al., Jpn. J. Genet. 62:501-505, 1987), NPT II and GUS. Shoots that regenerated on kanamycin-containing medium were confirmed transgenic through GUS assays, Southern analyses and transmission of foreign genes through the sexual cycle. Transgenic plants were as short as half the height of control plants, earlier flowering by up to 35 days, had smaller leaves, smaller seed capsules, fewer seeds, smaller flowers and reduced pollen viability. The number of seed capsules, leaf number and root density were similar between transgenic and control plants. Transgenic clones varied in the expression of the Rol C gene and transgenic plants similar or only slightly different from controls were identified. Transformation with the Rol C gene presents a potentially useful method of genetically modifying horticultural crops, particularly for flowering date, height, and leaf and flower size.