Metabolic Control of Tobacco Pollination by Sugars and Invertases.

Pollination in flowering plants is initiated by germination of pollen grains on stigmas followed by fast growth of pollen tubes representing highly energy-consuming processes. The symplastic isolation of pollen grains and tubes requires import of Suc available in the apoplast. We show that the functional coupling of Suc cleavage by invertases and uptake of the released hexoses by monosaccharide transporters are critical for pollination in tobacco (Nicotiana tabacum). Transcript profiling, in situ hybridization, and immunolocalization of extracellular invertases and two monosaccharide transporters in vitro and in vivo support the functional coupling in supplying carbohydrates for pollen germination and tube growth evidenced by spatiotemporally coordinated expression. Detection of vacuolar invertases in maternal tissues by these approaches revealed metabolic cross talk between male and female tissues and supported the requirement for carbohydrate supply in transmitting tissue during pollination. Tissue-specific expression of an invertase inhibitor and addition of the chemical invertase inhibitor miglitol strongly reduced extracellular invertase activity and impaired pollen germination. Measurements of (competitive) uptake of labeled sugars identified two import pathways for exogenously available Suc into the germinating pollen operating in parallel: direct Suc uptake and via the hexoses after cleavage by extracellular invertase. Reduction of extracellular invertase activity in pollen decreases Suc uptake and severely compromises pollen germination. We further demonstrate that Glc as sole carbon source is sufficient for pollen germination, whereas Suc is supporting tube growth, revealing an important regulatory role of both the invertase substrate and products contributing to a potential metabolic and signaling-based multilayer regulation of pollination by carbohydrates.

Transcriptome Analysis in Chinese Cabbage (Brassica rapa ssp. pekinensis) Provides the Role of Glucosinolate Metabolism in Response to Drought Stress.

Although drought stress is one of the most limiting factors in growth and production of Chinese cabbage (Brassica rapa L. ssp. pekinensis), the underlying biochemical and molecular causes are poorly understood. In the present study, to address the mechanisms underlying the drought responses, we analyzed the transcriptome profile of Chinese cabbage grown under drought conditions. Drought stress transcriptionally activated several transcription factor genes, including AP2/ERFs, bHLHs, NACs and bZIPs, and was found to possibly result in transcriptional variation in genes involved in organic substance metabolic processes. In addition, comparative expression analysis of selected BrbZIPs under different stress conditions suggested that drought-induced BrbZIPs are important for improving drought tolerance. Further, drought stress in Chinese cabbage caused differential acclimation responses in glucosinolate metabolism in leaves and roots. Analysis of stomatal aperture indicated that drought-induced accumulation of glucosinolates in leaves directly or indirectly controlled stomatal closure to prevent water loss, suggesting that organ-specific responses are essential for plant survival under drought stress condition. Taken together, our results provide information important for further studies on molecular mechanisms of drought tolerance in Chinese cabbage.

The Arabidopsis PLAT domain protein1 promotes abiotic stress tolerance and growth in tobacco.

Plant growth and consequently crop yield can be severely compromised by abiotic and biotic stress conditions. Transgenic approaches that resulted in increased tolerance against abiotic stresses often were typically accompanied by adverse effects on plant growth and fitness under optimal growing conditions. Proteins that belong to the PLAT-plant-stress protein family harbour a single PLAT (Polycystin, Lipoxygenase, Alpha-toxin and Triacylglycerol lipase) domain and are ubiquitously present in monocot and dicot plant species. Until now, only limited data is available for PLAT-plant-stress family members, which suggested that these proteins in general could promote tolerance towards stress responses. We studied the function of the Arabidopsis PLAT-plant-stress protein AtPLAT1 employing heterologous gain-of-function analysis in tobacco. AtPLAT1 conferred increased abiotic stress tolerance in tobacco, evident by improved tolerance towards cold, drought and salt stresses, and promoted growth, reflected by a faster development under non-stressed conditions. However, the overexpression of AtPLAT1 in tobacco reduced the tolerance towards biotic stress conditions and, therefore, could be involved in regulating the crosstalk between abiotic and biotic stress responses. Thus, we showed that heterologously expressed AtPLAT1 functions as positive regulator of abiotic stress tolerance and plant growth, which could be an important new asset for strategies to develop plants with improved abiotic stress tolerance, without growth and subsequent yield penalties under optimal growth conditions.

Overexpression of the Brassica rapa bZIP Transcription Factor, BrbZIP-S, Increases the Stress Tolerance in Nicotiana benthamiana.

In higher plants, S1-basic region-leucine zipper (S1-bZIP) transcription factors fulfill crucial roles in the physiological homeostasis of carbon and amino acid metabolisms and stress responses. However, very little is known about the physiological role of S1-bZIP in cruciferous vegetables. Here, we analyzed the physiological function of S1-bZIP from Brassica rapa (BrbZIP-S) in modulating proline and sugar metabolism. Overexpression of BrbZIP-S in Nicotiana benthamiana resulted in delayed chlorophyll degradation during the response to dark conditions. Under heat stress or recovery conditions, the transgenic lines exhibited a lower accumulation of H2O2, malondialdehyde, and protein carbonyls compared to the levels in transgenic control plants. These results strongly indicate that BrbZIP-S regulates plant tolerance against dark and heat stress. We propose that BrbZIP-S is a modulator of proline and sugar metabolism, which are required for energy homeostasis in response to environmental stress conditions.

Plant Response to Cold Stress: Cold Stress Changes Antioxidant Metabolism in Heading Type Kimchi Cabbage (Brassica rapa L. ssp. Pekinensis).

Cold stress is known as the important yield-limiting factor of heading type Kimchi cabbage (HtKc, Brassica rapa L. ssp. pekinensis), which is an economically important crop worldwide. However, the biochemical and molecular responses to cold stress in HtKc are largely unknown. In this study, we conducted transcriptome analyses on HtKc grown under normal versus cold conditions to investigate the molecular mechanism underlying HtKc responses to cold stress. A total of 2131 genes (936 up-regulated and 1195 down-regulated) were identified as differentially expressed genes and were significantly annotated in the category of "response to stimulus". In addition, cold stress caused the accumulation of polyphenolic compounds, including p-coumaric, ferulic, and sinapic acids, in HtKc by inducing the phenylpropanoid pathway. The results of the chemical-based antioxidant assay indicated that the cold-induced polyphenolic compounds improved the free-radical scavenging activity and antioxidant capacity, suggesting that the phenylpropanoid pathway induced by cold stress contributes to resistance to cold-induced reactive oxygen species in HtKc. Taken together, our results will serve as an important base to improve the cold tolerance in plants via enhancing the antioxidant machinery.

Molecular Cloning and Functional Characterization of Heat Stress-Responsive Superoxide Dismutases in Garlic (Allium sativum L.).

Superoxide dismutases (SODs) are key antioxidant enzymes that can detoxify the superoxide radicals generated by various stresses. Although various plant SODs have been suggested to improve stress tolerance, SODs in garlic, an economically important vegetable grown worldwide, remain relatively unknown. In this study, we found that heat stress strongly induced the activities of Cu/ZnSODs, FeSODs, and MnSODs in garlic leaves. In addition, we cloned four garlic SODs (AsSODs) and suggest that heat stress-increased SOD activity was reflected at least by the induction of these AsSODs. The results of the agro-infiltration assay suggested that the cloned AsSODs encoded functional SOD enzymes belonging to the Cu/ZnSOD and MnSOD families. As a first step toward understanding the enzymatic antioxidant system in garlic plants, our results provide a solid foundation for an in-depth analysis of the physiological functions of the AsSOD family.

Hovenia dulcis--an Asian traditional herb.

Hovenia dulcis Thunb., known as Japanese raisin tree, is commonly found in East Asia. It has a long history as a food supplement and traditional medicine in Japan, China and Korea, but is little known and used in Western countries so far. This minireview summarizes traditional uses and current knowledge on the pharmacology and phytochemistry of H. duclcis and covers, in particular, literature from specialized Asian journals that are not readily accessible. Extracts from H. dulcis accelerate detoxification of ethanol, and possess hepatoprotective, antioxidative, antimicrobial and antidiabetic properties. Although the underlying molecular mechanisms are not fully understood, free radical scavenging and enhancement of ethanol catabolism have been reported.

Enhanced biosynthesis of saponins by coronatine in cell suspension culture of Kalopanax septemlobus.

Kalopanax septemlobus is a medicinal woody species of the family Araliaceae, and the pharmaceutical properties of saponins obtained from K. septemlobus suggest that K. septemlobus has the potential to be a crude drug and dietary health supplement. In this study, we established cell suspension culture of K. septemlobus to develop a sustainable source of natura-ceuticals. Friable calli were used for establishing cell suspension culture. The maximum amount of total saponins (1.56 mg/60 ml suspension) was obtained during the 15th day of incubation, whereas the maximum capacity of saponin production was reached after day 6 (0.42 µg/mg of fresh weight). The total saponin production in the cell suspension of K. septemlobus was significantly increased by coronatine (COR) at 160% at a dose of 1 µM compared with the mock-treated control, whereas methyl jasmonate treated cells exhibited less increase in total saponin level as compared to the COR-treated cells. In addition, the elicitation of COR strongly induced the expression of beta-amyrin synthase, thus resulting in the accumulation of oleanolic acid (2.369 ± 0.98 µg/mg of extract), a precursor for oleanane-type triterpene saponins. These results indicate that COR is an efficient elicitor for inducing phytochemicals in cell suspension culture and that it provides the possibility for producing saponins of K. septemlobus using cell suspension culture.

Evolution and expression analysis of the soybean glutamate decarboxylase gene family.

Glutamate decarboxylase (GAD) is an enzyme that catalyses the conversion of L-glutamate into gamma-aminobutyric acid (GABA), which is a four-carbon non-protein amino acid present in all organisms. Although plant GAD plays important roles in GABA biosynthesis, our knowledge concerning GAD gene family members and their evolutionary relationship remains limited. Therefore, in this study, we have analysed the evolutionary mechanisms of soybean GAD genes and suggested that these genes expanded in the soybean genome partly due to segmental duplication events. The approximate dates of duplication events were calculated using the synonymous substitution rate, and we suggested that the segmental duplication of GAD genes in soybean originated 9.47 to 11.84 million years ago (Mya). In addition, all segmental duplication pairs (GmGAD1/3 and GmGAD2/4) are subject to purifying selection. Furthermore, GmGAD genes displayed differential expression either in their transcript abundance or in their expression patterns under abiotic stress conditions like salt, drought, and cold. The expression pattern of paralogous pairs suggested that they might have undergone neofunctionalization during the subsequent evolution process. Taken together, our results provide valuable information for the evolution of the GAD gene family and represent the basis for future research on the functional characterization of GAD genes in higher plants.

The Arabidopsis PLAT domain protein1 is critically involved in abiotic stress tolerance.

Despite the completion of the Arabidopsis genome sequence, for only a relatively low percentage of the encoded proteins experimental evidence concerning their function is available. Plant proteins that harbour a single PLAT (Polycystin, Lipoxygenase, Alpha-toxin and Triacylglycerol lipase) domain and belong to the PLAT-plant-stress protein family are ubiquitously present in monocot and dicots. However, the function of PLAT-plant-stress proteins is still poorly understood. Therefore, we have assessed the function of the uncharacterised Arabidopsis PLAT-plant-stress family members through a combination of functional genetic and physiological approaches. PLAT1 overexpression conferred increased abiotic stress tolerance, including cold, drought and salt stress, while loss-of-function resulted in opposite effects on abiotic stress tolerance. Strikingly, PLAT1 promoted growth under non-stressed conditions. Abiotic stress treatments induced PLAT1 expression and caused expansion of its expression domain. The ABF/ABRE transcription factors, which are positive mediators of abscisic acid signalling, activate PLAT1 promoter activity in transactivation assays and directly bind to the ABRE elements located in this promoter in electrophoretic mobility shift assays. This suggests that PLAT1 represents a novel downstream target of the abscisic acid signalling pathway. Thus, we showed that PLAT1 critically functions as positive regulator of abiotic stress tolerance, but also is involved in regulating plant growth, and thereby assigned a function to this previously uncharacterised PLAT domain protein. The functional data obtained for PLAT1 support that PLAT-plant-stress proteins in general could be promising targets for improving abiotic stress tolerance without yield penalty.