SummarySumoylation is a posttranslational regulatory process in higher eukaryotes modifying substrate proteins through conjugation of small ubiquitin-related modifiers (SUMOs). Sumoylation modulates protein stability, subcellular localization and activity; thus, it regulates most cellular functions including response to environmental stress in plants. To study the feasibility of manipulating SUMO E3 ligase, one of the important components in the sumoylation pathway in transgenic (TG) crop plants for improving overall plant performance under adverse environmental conditions, we have analysed TG creeping bentgrass (Agrostis stolonifera L.) plants constitutively expressing OsSIZ1, a rice SUMO E3 ligase. Overexpression of OsSIZ1 led to increased photosynthesis and overall plant growth. When subjected to water deficiency and heat stress, OsSIZ1 plants exhibited drastically enhanced performance associated with more robust root growth, higher water retention and cell membrane integrity than wild-type (WT) controls. OsSIZ1 plants also displayed significantly better growth than WT controls under phosphatestarvation conditions, which was associated with a higher uptake of phosphate (Pi) and other minerals, such as potassium and zinc. Further analysis revealed that overexpression of OsSIZ1 enhanced stress-induced SUMO conjugation to substrate in TG plants, which was associated with modified expression of stress-related genes. This strongly supports a role sumoylation plays in regulating multiple molecular pathways involved in plant stress response, establishing a direct link between sumoylation and plant response to environmental adversities. Our results demonstrate the great potential of genetic manipulation of sumoylation process in TG crop species for improved resistance to broad abiotic stresses.

Ethylene-responsive element binding factors (ERFs) are involved in regulation of various stress responses in plants, but their biological functions in waterlogging stress are largely unclear. In this study, we identified a petunia (Petunia × hybrida) ERF gene, PhERF2, that was significantly induced by waterlogging in wild-type (WT). To study the regulatory role of PhERF2 in waterlogging responses, transgenic petunia plants with RNAi silencing and overexpression of PhERF2 were generated. Compared with WT plants, PhERF2 silencing compromised the tolerance of petunia seedlings to waterlogging, shown as 96% mortality after 4 days waterlogging and 14 days recovery, while overexpression of PhERF2 improved the survival of seedlings subjected to waterlogging. PhERF2-RNAi lines exhibited earlier and more severe leaf chlorosis and necrosis than WT, whereas plants overexpressing PhERF2 showed promoted growth vigor under waterlogging. Chlorophyll content was dramatically lower in PhERF2-silenced plants than WT or overexpression plants. Typical characteristics of programmed cell death (PCD), DNA condensation, and moon-shaped nuclei were only observed in PhERF2-overexpressing lines but not in PhERF2-RNAi or control lines. Furthermore, transcript abundances of the alcoholic fermentation-related genes ADH1-1, ADH1-2, ADH1-3, PDC1, and PDC2 were reduced in PhERF2-silenced plants, but increased in PhERF2-overexpressing plants following exposure to 12-h waterlogging. In contrast, expression of the lactate fermentation-related gene LDH was up-regulated in PhERF2-silenced plants, but down-regulated in its overexpressing plants. Moreover, PhERF2 was observed to directly bind to the ADH1-2 promoter bearing ATCTA motifs. Our results demonstrate that PhERF2 contributes to petunia waterlogging tolerance through modulation of PCD and alcoholic fermentation system.

Real-time imaging of the GH gene promoter linked to luciferase in living pituitary cells has revealed surprising heterogeneity and variety of dynamic patterns of gene expression. Cells treated with either forskolin or thyroid hormone generated a consistent and characteristic temporal response from cell populations, but detailed analysis of individual cells revealed different patterns. Approximately 25-26% of cells displayed no response, 25-33% of cells exhibited a sustained progressive rise in luciferase activity, and 41-50% showed a transient phasic, or oscillatory response, after given stimuli. In cells treated consecutively with the two stimuli, the population response to the second stimulus was augmented. Single-cell analysis revealed that this was partly due to an increased number of cells responding, but also that the prevalence of response patterns changed: cells that responded to an initial stimulus were more likely to respond subsequently in a progressive sustained manner. In conclusion, these studies have indicated that GH promoter activity in individual living pituitary cells is unstable and possibly stochastic, with dynamic variations from hour to hour. The prevalence of different temporal patterns of response to hormonal stimulation among a population of cells is altered by the endocrine history of those cells.

Although analysis of luciferase activity using luminescence imaging has provided new insights into the dynamic regulation of gene expression in living tissues, studies in vitro have relied on stably transfected clonal cell lines, limiting the choice of cell type and species, or DNA microinjection, which is arduous and highly selective. We report here the first use of a recombinant adenovirus in which the firefly luciferase reporter gene was regulated by the prolactin gene promoter, to study temporal dynamics of promoter activity. This vector was used to infect the pituitary GH3 cell line, and also primary cultures of Syrian hamster pituitary cells. We show that adenovirally transduced cells retained normal regulation of the promoterreporter transgene by appropriate signals. Furthermore, microscopic imaging studies indicated that both clonal and primary pituitary cells were transduced efficiently, giving readily detectable luminescence signals in real-time over long periods. Finally, analysis of single-cell expression patterns indicated that prolactin promoter activity was highly dynamic with pulses in gene expression, revealing that the transcriptional instability seen in clonal cells is a feature of normal pituitary cells. Adenoviral vectors offer a valuable tool for studies of gene regulation where conventional transgenesis and clonal cell lines are not available.