Transformation of broccoli (Brassica oleracea var. italica) with isopentenyltransferase gene via Agrobacterium tumefaciens for post-harvest yellowing retardation

Transgenic plantlets with a retarding effect on post-harvest yellowing in broccoli have been generated via Agrobacterium tumefaciens-mediated transformation of cytokinin synthesizing ipt (isopentenyltransferase) gene. The ipt gene is constructed under the control of senescence-associated gene promoters from Arabidopsis in the forms of pSG529(+) and pSG766A, which were the gifts from Dr R.M. Amasino at University of Wisconsin, Madison. Evidence of transgene integration was confirmed by assays on neomycin phosphotransferase II (NPTII) activity of selection markers, PCR and Southern hybridization. Based on the chlorophyll retention rate (>50%) after 4 days of post-harvest storage at 25 °C, it was found that 31% of transformants exhibited the effect of retarding yellowing in detached leaves, with 16% having the effect on florets and 7.2% on both leaves and florets. RT-PCR revealed that ipt gene expression occurred early on the day of detachment. Factors such as vacuum aid infiltration, plasmid differences, explant types, seedling ages and kanamycin concentrations were also studied. Putative transformation frequencies tended to vary with plasmids and explant types. The advantage of vacuum aid infiltration depended on explant types. The optimal kanamycin concentration should be determined experimentally for each study to avoid the high escape rate of kanamycin selection. Flow cytometric analysis of explant nuclear DNA phases was found to be helpful for selecting suitable explants for transformation and minimizing the polyploid transformants. A reproducible transformation protocol without any pre-culture was established for explants of hypocotyl, cotyledon, and peduncle. Most of the ipt transformants with a retarding effect on yellowing had a chimeric nature but showed little or no serious morphological abnormality in comparison with their parental line. Through proper selection, transformation lines with the capability of retarding post-harvest yellowing in broccoli should be feasible.     

Transposable elements ofXanthomonas campestris pv.citri originating from indigenous plasmids

Summary After introduction of the broad host range plasmid RP4 inXanthomonas campestris pv.citri strain XAS4501 twoXanthomonas transposable elements, ISXC4 and ISXC5, were isolated. These elements were found to be capable of transposition inEscherichia coli. Restriction analysis, DNA hybridization and heteroduplex experiments revealed that ISXC4 and ISXC5 are about 5.55 and 6.95 kb in size, respectively, possess inverted repeats about 50±18 bp in length and share DNA homology in their left (5.0 kb) and right (0.6 kb) ends. ISXC4 and ISXC5 were found to originate from plasmids pXW45N and pXW45J, which are indigenous replicons inX. campestris pv.citri strain XW45.     

Tomato plants ectopically expressing Arabidopsis CBF1 show enhanced resistance to water deficit stress

A DNA cassette containing an Arabidopsis C repeat/dehydration-responsive element binding factor 1 (CBF1) cDNA and a nos terminator, driven by a cauliflower mosaic virus 35S promoter, was transformed into the tomato (Lycopersicon esculentum) genome. These transgenic tomato plants were more resistant to water deficit stress than the wild-type plants. The transgenic plants exhibited growth retardation by showing dwarf phenotype, and the fruit and seed numbers and fresh weight of the transgenic tomato plants were apparently less than those of the wild-type plants. Exogenous gibberellic acid treatment reversed the growth retardation and enhanced growth of transgenic tomato plants, but did not affect the level of water deficit resistance. The stomata of the transgenic CBF1 tomato plants closed more rapidly than the wild type after water deficit treatment with or without gibberellic acid pretreatment. The transgenic tomato plants contained higher levels of Pro than those of the wild-type plants under normal or water deficit conditions. Subtractive hybridization was used to isolate the responsive genes to heterologous CBF1 in transgenic tomato plants and the CAT1 (CATALASE1) was characterized. Catalase activity increased, and hydrogen peroxide concentration decreased in transgenic tomato plants compared with the wild-type plants with or without water deficit stress. These results indicated that the heterologous Arabidopsis CBF1 can confer water deficit resistance in transgenic tomato plants.     

Maintenance of abiotic stress memory in plants: Lessons learned from heat acclimation

Plants acquire enhanced tolerance to intermittent abiotic stress by employing information obtained during prior exposure to an environmental disturbance, a process known as acclimation or defense priming. The capacity for stress memory is a critical feature in this process. The number of reports related to plant stress memory (PSM) has recently increased, but few studies have focused on the mechanisms that maintain PSM. Identifying the components involved in maintaining PSM is difficult due in part to the lack of clear criteria to recognize these components. In this review, based on what has been learned from genetic studies on heat acclimation memory, we propose criteria for identifying components of the regulatory networks that maintain PSM. We provide examples of the regulatory circuits formed by effectors and regulators of PSM. We also highlight strategies for assessing PSMs, update the progress in understanding the mechanisms of PSM maintenance, and provide perspectives for the further development of this exciting research field.

A review of the strategies and principles for assessing abiotic stress memory in plants, the roles of components that maintain stress memory, and the regulatory circuits they form.     

The Retromer Complex Is Required for Rhodopsin Recycling and Its Loss Leads to Photoreceptor Degeneration

Rhodopsin mistrafficking can cause photoreceptor (PR) degeneration. Upon light exposure, activated rhodopsin 1 (Rh1) in Drosophila PRs is internalized via endocytosis and degraded in lysosomes. Whether internalized Rh1 can be recycled is unknown. Here, we show that the retromer complex is expressed in PRs where it is required for recycling endocytosed Rh1 upon light stimulation. In the absence of subunits of the retromer, Rh1 is processed in the endolysosomal pathway, leading to a dramatic increase in late endosomes, lysosomes, and light-dependent PR degeneration. Reducing Rh1 endocytosis or Rh1 levels in retromer mutants alleviates PR degeneration. In addition, increasing retromer abundance suppresses degenerative phenotypes of mutations that affect the endolysosomal system. Finally, expressing human Vps26 suppresses PR degeneration in Vps26 mutant PRs. We propose that the retromer plays a conserved role in recycling rhodopsins to maintain PR function and integrity.