Is expression of aquaporins (plasma membrane intrinsic protein 2s, PIP2s) associated with thermonasty (leaf-curling) in Rhododendron?

It is postulated that leaf thermonasty (leaf curling) in rhododendrons under sub-freezing temperatures is caused by water redistribution due to extracellular freezing. We hypothesize that aquaporins (AQPs), the transmembrane water-channels, may be involved in regulating water redistribution and thus leaf curling. Our experimental system includes two Rhododendron species with contrasting leaf curling behavior whereby it was observed in R. catawbiense but not in R. ponticum. We compared leaf movements and the expression of two AQPs, i.e. R. catawbiense/ponticum plasma-membrane intrinsic protein 2 (Rc/RpPIP2;1 and Rc/RpPIP2;2), in the two species under freezing-rewarming and dehydration-rehydration cycles. To determine the relationship between extracellular freezing and leaf-curling, we monitored leaf-curling in R. catawbiense with or without controlled ice-nucleation. Our data indicate that extracellular freezing may be required for leaf curling. Moreover, in both species, PIP2s were up-regulated at temperatures that fell in ice-nucleation temperature range. Such up-regulation could be associated with the bulk-water efflux caused by extracellular freezing. When leaves were frozen beyond the ice-nucleation temperature range, PIP2s were continuously down-regulated in R. catawbiense along with the progressive leaf curling, as also observed for RcPIP2;2 in dehydrated leaves; as leaves uncurled during re-warming/rehydration, RcPIP2 expression was restored. On the other hand, R. ponticum, a non-curling species, exhibited substantial up-regulation of RpPIP2s during freezing/dehydration. Taken together, our data suggest that RcPIP2 down-regulation was associated with leaf curling. Moreover, the contrasting PIP2 expression patterns combined with leaf behavior of R. catawbiense and R. ponticum under these two cycles may reflect different strategies employed by these two species to tolerate/resist cellular dehydration.

Dehydrin metabolism is altered during seed osmopriming and subsequent germination under chilling and desiccation in Spinacia oleracea L. cv. Bloomsdale: possible role in stress tolerance.

Osmopriming improves seed germination performance as well as stress tolerance. To understand the biochemistry of osmopriming-induced seed stress tolerance, we investigated dehydrin (DHN) accumulation patterns at protein and transcript level (determined by immunoblotting and qPCR) during priming, and subsequent germination under optimal and stress conditions (i.e. chilling and desiccation) in spinach (Spinacia oleracea L. cv. Bloomsdale) seeds. Our data indicate enhanced germination performance of primed seeds is accompanied by increased accumulation of three dehydrin-like proteins (DLPs): 30, 26, and 19-kD. Moreover, 30, 26 and 19-kD DLPs that first only transiently accumulated during priming re-accumulated in response to stresses, suggesting an evidence for 'cross-tolerance', which is initially induced by priming and later recruited during post-priming germination under stresses. Study with CAP85, a spinach DHN, corroborates above observations at the gene-expression and protein accumulation level. Additionally, our results suggest that during seed germination and seedling establishment, CAP85 expression may be regulated by the interplay of two factors: seedling development and stress responses. In conclusion, our data suggest that 30, 26, and 19-kD dehydrin-like proteins and CAP85 may be used as potential biochemical/molecular markers for priming-induced stress tolerance in 'Bloomsdale' spinach.

Interaction of anammox bacteria and inactive methanogenic granules under high nitrogen selective pressure.

Granular anammox reactors usually adopted anaerobic/aerobic granules as source sludge, in which the washout of other species and enrichment of anammox biomass were very slow because of the competition of the coexisting bacteria. In this study, inactive methanogenic granules were proved to be suitable for rapid anammox granulation under high nitrogen concentrations by investigating their interaction with anammox bacteria. The start-up nitrite concentration was significantly higher than the published toxic level for anammox bacteria and other lab-scale studies. The nitrogen loading rate increased from 141 to 480 mg/L/d in 120 days operation with a total nitrogen removal efficiency of 96.0+/-0.6%. Anammox granules with a diameter of 1.3+/-0.4mm were observed over the course of three months. Molecular analysis showed that over 67% of the cells in the anammox granules were anammox bacteria after 90 days. The accommodations and proliferations of anammox bacteria in the inactive methanogenic granules might be the main reason for the high anammox purity in a short period. The important role of the extracellular polymer in the granule structure was observed via morphological observation.

Enrichment and biofilm formation of Anammox bacteria in a non-woven membrane reactor.

An innovative reactor configuration for Anammox enrichment by connecting a non-woven membrane module with an anaerobic reactor was developed in this study. The Anammox non-woven membrane reactor (ANMR) exhibited high biomass retention ability through the formation of aggregates in the reactor and biofilm on the interior surface of the non-woven membrane. No fouling problems occurred on the membrane after the development of mature biofilms. After 8 months of operation, the nitrogen loading rate (NLR) and nitrogen removal rate (NRR) reached 1263 mg N/l/d and 1047.5 mg N/l/d, respectively, with a maximum specific ammonium consumption (SAC) of 51 nmol/mg protein/min. At steady state, the average ammonium and nitrite removal efficiencies were 90.9% and 95.0%, respectively. Morphological observation of Anammox aggregates and biofilm showed a high degree of compactness. Also, enrichment of Anammox bacteria was quantified by real-time polymerase chain reaction (PCR) analysis as 97.7%.

RcDhn5, a cold acclimation-responsive dehydrin from Rhododendron catawbiense rescues enzyme activity from dehydration effects in vitro and enhances freezing tolerance in RcDhn5-overexpressing Arabidopsis plants.

Dehydrins (DHNs) are typically induced in response to abiotic stresses that impose cellular dehydration. As extracellular freezing results in cellular dehydration, accumulation of DHNs and development of desiccation tolerance are believed to be key components of the cold acclimation (CA) process. The present study shows that RcDhn5, one of the DHNs from Rhododendron catawbiense leaf tissues, encodes an acidic, SK(2) type DHN and is upregulated during seasonal CA and downregulated during spring deacclimation (DA). Data from in vitro partial water loss assays indicate that purified RcDhn5 protects enzyme activity against a dehydration treatment and that this protection is comparable with acidic SK(n) DHNs from other species. To investigate the contribution of RcDhn5 to freezing tolerance (FT), Arabidopsis plants overexpressing RcDhn5 under the control of 35S promoter were generated. Transgenic plants exhibited improved 'constitutive' FT compared with the control plants. Furthermore, a small but significant improvement in FT of RcDhn5-overexpressing plants was observed after 12 h of CA; however, this gained acclimation capacity was not sustained after a 6-day CA. Transcript profiles of cold-regulated native Arabidopsis DHNs (COR47, ERD10 and ERD14) during a CA time-course suggests that the apparent lack of improvement in cold-acclimated FT of RcDhn5-overexpressing plants over that of wild-type controls after a 6-day CA might have been because of the dilution of the effect of RcDhn5 overproduction by a strong CA-induced expression of native Arabidopsis DHNs. This study provides evidence that RcDhn5 contributes to freezing stress tolerance and that this could be, in part, because of its dehydration stress-protective ability.

Rhododendron catawbiense plasma membrane intrinsic proteins are aquaporins, and their over-expression compromises constitutive freezing tolerance and cold acclimation ability of transgenic Arabidopsis plants.

Extracellular freezing results in cellular dehydration caused by water efflux, which is likely regulated by aquaporins (AQPs). In a seasonal cold acclimation (CA) study of Rhododendron catawbiense, two AQP cDNAs, RcPIP2;1 and RcPIP2;2, were down-regulated as the leaf freezing tolerance (FT) increased from -7 to approximately -50 degrees C. We hypothesized this down-regulation to be an adaptive component of CA process allowing cells to resist freeze-induced dehydration. Here, we characterize full-length cDNAs of the two Rhododendron PIPs, and demonstrate that RcPIP2s have water channel activity. Moreover, RcPIP2s were over-expressed in Arabidopsis, and FT of transgenic plants was compared with that of wild-type (WT) controls. Data indicated a significantly lower constitutive FT and CA ability of RcPIP2-OXP plants (compared with WT) due, presumably, to their lower ability to resist freeze desiccation. A relatively higher dehydration rate of RcPIP2-OXP leaves (than WT) supports this notion. Phenotypic and microscopic observations revealed bigger leaf size and mesophyll cells of RcPIP2-OXP plants than WT. It is proposed that lower FT of transgenic plants may be associated with their leaf cells' propensity to greater mechanical stress, that is, volume strain per unit surface, during freeze-thaw-induced contraction or expansion. Additionally, greater freeze injury in RcPIP2-OXP plants could also be attributed to their susceptibility to potentially faster rehydration (than WT) during a thaw.

An oligonucleotide array for the identification and differentiation of bacteria pathogenic on potato.

ABSTRACT Oligonucleotides, 16 to 24 bases long, were selected from the 3' end of the 16S gene and the 16S-23S intergenic spacer regions of bacteria pathogenic on potato, including Clavibacter michiganensis subsp. sepedonicus, Ralstonia solanacearum, and the pectolytic erwinias, including Erwinia carotovora subsp. atroseptica and carotovora and E. chrysanthemi. Oligonucleotides were designed and formatted into an array by pin spotting on nylon membranes. Genomic DNA from bacterial cultures was amplified by polymerase chain reaction using conserved ribosomal primers and labeled simultaneously with digoxigenin-dUTP. Hybridization of amplicons to the array and subsequent serological detection of digoxigenin label revealed different hybridization patterns that were distinct for each species and subspecies tested. Hybridization of amplicons generally was restricted to appropriate homologous oligonucleotides and cross-hybridization with heterologous oligonucleotides was rare. Hybridization patterns were recorded as separate gray values for each hybridized spot and revealed a consistent pattern for multiple strains of each species or subspecies isolated from diverse geographical regions. In preliminary tests, bacteria could be correctly identified and detected by hybridizing to the array amplicons from mixed cultures and inoculated potato tissue.