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.     

Identification of cold acclimation-responsive Rhododendron genes for lipid metabolism, membrane transport and lignin biosynthesis: importance of moderately abundant ESTs in genomic studies

We have previously analysed expressed sequence tags (ESTs) from non-acclimated (NA) and cold-acclimated (CA) Rhododendron leaves, and identified highly abundant complementary DNAs (cDNAs) possibly involved in cold acclimation. A potentially significant, but relatively unexplored, application of these EST data sets is the study of moderately abundant cDNAs, such as those picked only 1-3 times from each Rhododendron EST library containing approximately 430 ESTs. Using statistical tests and Northern blots, we established that the probability of differential expression of moderately abundant cDNAs based on the EST data is, indeed, a reasonably accurate predictor of their 'true' upregulation or downregulation as 11 out of 13 cDNAs (85%) studied fit this criterion. The analyses also revealed four aspects of cold acclimation in Rhododendron leaf tissues. Firstly, the concomitant upregulation of long-chain acyl-coenzyme A (acyl-CoA) synthetase, CTP:cholinephosphate cytidylyltransferase and delta-12 fatty acid desaturase in CA leaf tissues suggests that phospholipid biosynthesis and desaturation are important components of cold hardening in Rhododendron. Secondly, upregulation of plastidic nicotinamide adenine dinucleotide phosphatemalic enzyme (NADP-ME) in CA tissues suggests that malate is an important source of acetyl-CoA used for fatty acid biosynthesis during cold acclimation. Thirdly, down-regulation of plasma membrane intrinsic protein (PIP)2-1 aquaporin and upregulation of gated outward rectifying K+ channel (GORK) in CA tissues may be associated with the protection of overwintering leaves from freeze-induced cellular dehydration. Fourthly, upregulation of coumarate 3-hydroxylase may be associated with cell wall thickening in CA tissues. Physiological implications of these results, which reveal potentially novel regulations of cold acclimation in overwintering woody evergreens, are discussed. This work highlights the importance of also investigating low/moderately abundant ESTs (in addition to highly abundant ones) in genomic studies, in that it offers an effective strategy for identifying stress-related genes, especially when large-scale cDNA sequencing/microarray studies are not possible.     

Mesophyll freezing and effects of freeze dehydration visualized by simultaneous measurement of IDTA and differential imaging chlorophyll fluorescence

Infrared differential thermal analysis (IDTA) and differential imaging chlorophyll fluorescence (DIF) were employed simultaneously to study the two-dimensional pattern of ice propagation in leaves and mesophyll freeze dehydration as detected by a significant increase of basic chlorophyll fluorescence (F(0)). IDTA and DIF technique gave different insights into the freezing process of leaves that was highly species-specific. IDTA clearly visualized the freezing process consisting of an initial fast spread of ice throughout the vascular system followed by mesophyll freezing. While mesophyll freezing was homogeneously in Poa alpina, Rhododendron ferrugineum and Senecio incanus as determined by IDTA, DIF showed a distinct pattern only in S. incanus, with the leaf tips being affected earlier. In Cinnamomum camphora, a mottled freezing pattern of small mesophyll compartments was observed by both methods. In IDTA images, a random pattern predominated, while in DIF images, compartments closer to lower order veins were affected earlier. The increase of F(0) following mesophyll freezing started after a species-specific time lag of up to 26 min. The start of the F(0) increase and its slope were significantly enhanced at lower temperatures, which suggest a higher strain on mesophyll protoplasts when freezing occurs at lower temperatures.     

Cold hardiness and water relations parameters in Rhododendron cv. Catawbiense Boursault subjected to drought episodes

We determined whether increase in cold hardiness of Rhododendron cv. Catawbiense Boursault induced by water stress was correlated with changes in tissue water relations. Water content of the growing medium was either maintained near field capacity for the duration of the study or plants were subjected to drought episodes at different times between 15 July and 19 February. Watering during a drought episode was delayed until soil water content decreased below 0.4 m³ m⁻³ then watering was resumed at a level to maintain soil water content between 0.3 and 0.4 m³ m⁻³. Cold hardiness was evaluated in the laboratory with freeze tolerance tests on detached leaves. Water relations parameters were determined using pressure-volume analysis. Exposure to drought episodes increased cold hardiness during the cold acclimation stage in late summer and fall but not during the winter. When water-stressed plants were re-watered to field capacity, the previous gain in cold hardiness gradually disappeared. Water relations parameters correlating with seasonal changes of cold hardiness included dry matter content (r =−0.67). apoplastic water content (r =−0.60), and water potential at the turgor loss point (r = 0.40). Changes of cold hardiness in water-stressed plants in reference to well-watered plants were correlated with changes of all water relations parameters, except for osmotic potential at full turgor (r = 0.13). It is proposed that water stress reduced the hydration of cell walls, thereby increasing their rigidity. Increased rigidity of cell walls could result in a development of greater negative turgor pressures at subfreezing temperatures and therefore increased resistance to freeze dehydration.     

Manipulation of Catalase Levels Produces Altered Photosynthesis in Transgenic Tobacco Plants

Constructs containing the cDNAs encoding the primary leaf catalase in Nicotiana or subunit 1 of cottonseed (Gossypium hirsutum) catalase were introduced in the sense and antisense orientation into the Nicotiana tabacum genome. The N. tabacum leaf cDNA specifically overexpressed CAT-1, the high catalytic form, activity. Antisense constructs reduced leaf catalase specific activities from 0.20 to 0.75 times those of wild type (WT), and overexpression constructs increased catalase specific activities from 1.25 to more than 2.0 times those of WT. The NADH-hydroxypyruvate reductase specific activity in transgenic plants was similar to that in WT. The effect of antisense constructs on photorespiration was studied in transgenic plants by measuring the CO₂ compensation point (Γ) at a leaf temperature of 38°C. A significant linear increase was observed in Γ with decreasing catalase (at 50% lower catalase activity Γ increased 39%). There was a significant temperature-dependent linear decrease in Γ in transgenic leaves with elevated catalase compared with WT leaves (at 50% higher catalase Γ decreased 17%). At 29°C, Γ also decreased with increasing catalase in transgenic leaves compared with WT leaves, but the trend was not statistically significant. Rates of dark respiration were the same in WT and transgenic leaves. Thus, photorespiratory losses of CO₂ were significantly reduced with increasing catalase activities at 38°C, indicating that the stoichiometry of photorespiratory CO₂ formation per glycolate oxidized normally increases at higher temperatures because of enhanced peroxidation.     

Overexpression of a <Emphasis Type="Italic">Panax ginseng</Emphasis> tonoplast aquaporin alters salt tolerance,drought tolerance and cold acclimation ability in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis> plants

Water movement across cellular membranes is regulated largely by a family of water channel proteins called aquaporins (AQPs). Since several abiotic stresses such as, drought, salinity and freezing, manifest themselves via altering water status of plant cells and are linked by the fact that they all result in cellular dehydration, we overexpressed an AQP (tonoplast intrinsic protein) from Panax ginseng, PgTIP1, in transgenic Arabidopsis thaliana plants to test its role in plant’s response to drought, salinity and cold acclimation (induced freezing tolerance). Under favorable conditions, PgTIP1 overexpression significantly increased plant growth as determined by the biomass production, and leaf and root morphology. PgTIP1 overexpression had beneficial effect on salt-stress tolerance as indicated by superior growth status and seed germination of transgenic plants under salt stress; shoots of salt-stressed transgenic plants also accumulated greater amounts of Na⁺ compared to wild-type plants. Whereas PgTIP1 overexpression diminished the water-deficit tolerance of plants grown in shallow (10 cm deep) pots, the transgenic plants were significantly more tolerant to water stress when grown in 45 cm deep pots. The rationale for this contrasting response, apparently, comes from the differences in the root morphology and leaf water channel activity (speed of dehydration/rehydration) between the transgenic and wild-type plants. Plants overexpressed with PgTIP1 exhibited lower (relative to wild-type control) cold acclimation ability; however, this response was independent of cold-regulated gene expression. Our results demonstrate a significant function of PgTIP1 in growth and development of plant cells, and suggest that the water movement across tonoplast (via AQP) represents a rate-limiting factor for plant vigor under favorable growth conditions and also significantly affect responses of plant to drought, salt and cold stresses.     

Overexpression of Loquat Dehydrin Gene <Emphasis Type="Italic">EjDHN1</Emphasis> Promotes Cold Tolerance in Transgenic Tobacco

Dehydrins (DHNs) play vital roles in response to dehydration stress in plants. To examine the contribution of EjDHN to low-temperature stress in loquat (Eriobotrya japonica Lindl.), EjDHN1 was overexpressed in tobacco (Nicotiana tabacum L.). The plant growth of transgenic lines was significantly better than wild type (WT) after 4 d of recovery from cold stress. Cold stress led to membrane lipid peroxidation and reduced photosystem II (PSII) activity in leaves, and these were less severe in transgenic lines. To examine oxidative stress tolerance, the plants were treated with different concentrations of methyl viologen (MV), which inhibited plant growth both in WT and transgenic lines. After exposure to 2.0 μM MV for 10 d, the WT plants had a dramatically lower survival rate. MV treatment in leaf disks confirmed that transgenic lines accumulated less reactive oxygen species (ROS) and suffered less lipid peroxidation. The results suggested that the tolerance of the transgenic plants to cold was increased, and EjDHN1 could protect cells against oxidative damage caused by ROS production under cold stress. It also provided evidences that the enhanced cold tolerance resulted from EjDHN1 overexpression could be partly due to their protective effect on membranes by alleviating oxidative stresses.     

Calcium-Dependent Freezing Tolerance in Arabidopsis Involves Membrane Resealing via Synaptotagmin SYT1

Plant freezing tolerance involves the prevention of lethal freeze-induced damage to the plasma membrane. We hypothesized that plant freezing tolerance involves membrane resealing, which, in animal cells, is accomplished by calcium-dependent exocytosis following mechanical disruption of the plasma membrane. In Arabidopsis thaliana protoplasts, extracellular calcium enhanced not only freezing tolerance but also tolerance to electroporation, which typically punctures the plasma membrane. However, calcium did not enhance survival when protoplasts were exposed to osmotic stress that mimicked freeze-induced dehydration. Calcium-dependent freezing tolerance was also detected with leaf sections in which ice crystals intruded into tissues. Interestingly, calcium-dependent freezing tolerance was inhibited by extracellular addition of an antibody against the cytosolic region of SYT1, a homolog of synaptotagmin known to be a calcium sensor that initiates exocytosis. This inhibition indicates that the puncture allowing the antibody to flow into the cytoplasm occurs during freeze/thawing. Thus, we propose that calcium-dependent freezing tolerance results from resealing of the punctured site. Protoplasts or leaf sections isolated from Arabidopsis SYT1-RNA interference (RNAi) plants lost calcium-dependent freezing tolerance, and intact SYT1-RNAi plants had lower freezing tolerance than control plants. Taken together, these findings suggest that calcium-dependent freezing tolerance results from membrane resealing and that this mechanism involves SYT1 function.     

Effect of cold exposure on cortical microtubules of rye (Secale cereale) as observed by immunocytochemistry

The responses of cortical microtubules to sub-zero temperatures were examined in non-acclimated (NA) and cold-acclimated (CA) rye ( Secale cereale L. cv. Voima) leaf and root cells, and in protoplasts isolated enzymatically from leaves. Responses of leaf and root cells to hypertonic solutions equivalent to the dehydration response of freezing (P. L. Steponkus and D. V. Lynch 1989. J. Bioenerg. Biomembr. 21: 21–41) were also examined. At the respective growth temperatures both NA and CA leaf and root cells had typical organization and abundance of cortical microtubules as observed by indirect immunofluorescence (IIF) staining. Unchanged microtubule arrays were still present in CA leaf cells after -4°C treatment, while in leaf cells of NA plants and in the root cells of both NA and CA plants microtubules were shorter and less abundant. After -10°C treatment the cortical microtubules were almost totally depolymerized in both types of root cells and in leaf cells of NA plants, while CA leaf cells still had abundant cortical microtubule arrays. Semiquantitative analyses of cortical microtubules (MTs) of protoplasts confirmed the findings with intact leaf cells. Hypertonic treatment of NA and CA leaf cells gave similar effects as exposure of cells to sub-zero temperatures. However, after the hypertonic treatment, more microtubules remained present in the CA root cells than in the NA root cells, suggesting that also in root cells cold acclimation increases the dehydration stability of MTs. In conclusion, cold acclimation induces both greater frost stability and greater osmotic tolerance in the cortical microtubules of the leaf cells, and greater osmotic tolerance in the microtubules of the root cells in winter rye.     

Exposure to ultraviolet-B radiation increases cold hardiness in Rhododendron

The change in the cold hardiness of Rhododendron (cv. English Roseum) following chronic exposure to ultraviolet-B (UV-B) radiation (280–320 nm) was studied. Leaf disks removed from UV-B-exposed plants exbibited a greater tolerance to freezing temperatures than plants that received no UV-B exposure. Visual browning and percent phenolic leakage indicated that UV-B-exposed leaf disks were killed al -11°C. while control disks were killed at -8°C. Ultraviolet-induced production of phenolic compounds may be involved in increasing cold hardiness of Rhododendron leaf tissues.     

Protein, leucine aminopeptidase, esterase, acid phosphatase and photosynthetic responses of oleander (Nerium oleander L.) during cold acclimation and freezing treatments

Six-month-old oleander (Nerium oleander L.) pot plants, derived from vegetative propagation by cuttings, were tested for their ability to cold hardening. Damage of the non-acclimated (NA) plants was visible when treated by low freezing temperatures (below -2 degrees C). The responses of total proteins, leucine aminopeptidase (LAP), esterase (EST) and acid phosphatase (ACP) isoforms of NA and cold-acclimated (CA; 4 degrees C for 14 days) plants were compared using polyacrylamide gel electrophoresis. These molecular markers were also compared in NA and CA plants which received for 2h temperatures of 0, -2, -4, -6 and -8 degrees C. A new 38-kDa polypeptide appeared from day 7 to 14 during the acclimation treatment in the bark extracts and on day 14 in the leaf extracts. The above-mentioned polypeptide band (38 kDa) strongly appeared in all freezing treatments (0, -2, -4, -6 and -8 degrees C) in both bark and leaf extracts of the CA plants. Alterations in the number and the intensity of LAP and EST isoforms as well as in the intensity of ACP isoforms were observed in both bark and leaf of the CA oleander plants. A newly expressed EST isoform is proposed as biochemical marker for the cold acclimation treatment. CO2 assimilation rates (A) as well as transpiration rates (E) in NA plants were positive in 0 degrees C and negative in all temperatures below zero in the freezing treatments. In contrast, CO2 assimilation rates (A) and transpiration rates (E) were positive in CA plants in all temperatures of freezing treatment. A significant decrease (P<0.05) in chlorophyll (Chl) a, Chl a+b concentration and Chl a/b ratio were noticed in oleander plants during the acclimation treatment (from day 0 to 14), while Chl b concentration was unchanged at the respective time. On the other hand, no significant (P<0.05) differences were observed in the freezing treatments.     

植物冻害和氧化胁迫——甘蓝叶冻-融循环中超氧的产生

为检验植物冻害的发生和氧化胁迫这一假说,在冰冻前把氮蓝四唑（NBT）真空渗入到甘蓝叶圆片中,在叶圆片冻－融循环中NBT被还原为甲Zan,把其中的单甲Zan用乙醇提取出来,在分光光度计上比色,可作为冻融循环中产生的氧化胁迫的定量指标,NBT本身作为氧化剂,使冻害稍有增加,作为冰冻保护剂的二甲基亚砜真空渗入叶圆片使其抗冻性显著增加,而NBT还原则显著减少,表明二甲基亚砜在保护叶组织免受冻害上的作用和它减缓植物组织氧化胁迫的作用有关。实验结果支持植物冻害的发生和氧化胁迫有关这一假说。实验还表明还原NBT的还原剂很可能是超氧阴离子自由基。     

Altered physiology in trehalose-producing transgenic tobacco plants: Enhanced tolerance to drought and salinity stresses

Transgenic tobaccoNicotiana tabacum L. var. SR1) plants that over-express theEscherichia coli trehalose-6-phosphate synthase (TPS) gene(otsA) synthesized small amounts of trehalose (<400 μg g^-1 leaf) while non-transformants produced no detectable trehalose. Some transgenic plants expressing a high level ofotsA exhibited stunted growth and morphologically altered leaves. We tested F₂2 homozygous plants devoid of phenotypic changes to determine their physiological responses to dehydration and salinity stresses. All transgenic plants maintained better leaf turgidity under a limited water supply or after treatment with polyethylene glycol (PEG). Furthermore, fresh weight was maintained at higher levels after either treatment. The initial leaf water potential was higher in transgenic plants than non-transformants, but, in both plant types, was decreased to a comparable degree following dehydration. When grown with 250 mM NaCl, transgenic plants exhibited a significant delay in leaf withering and chlorosis, as well as more efficient seed germination. Our results suggest that either trehalose or trehalose-6-phosphate can act as an osmoprotective molecule without maintaining water potential, in contrast to other osmolytes. Furthermore, both appear to protect young embryos under unfavorable water status to ensure subsequent germination.     

Transgenic barley expressing the Arabidopsis AKR4C9 aldo-keto reductase enzyme exhibits enhanced freezing tolerance and regenerative capacity

Aldo-keto reductase (AKR) enzymes contribute to reactive aldehyde detoxifying capacity and to osmotic stress protection of various plant species. The protective effects of these enzymes have already been well characterised but only limited information has been gained on the role of AKRs in frost tolerance. The approach to study frost tolerance was based on transgenic barley (Hordeum vulgare L.) carrying and expressing the Arabidopsis thaliana At2g37770.2 gene (coding AKR4C9) under constitutive regulation. We demonstrated that the osmoprotective sugar alcohol sorbitol was present in both non-transgenic (WT) and transgenic barley plants. The increase of the sorbitol concentration was around 2- and 4-fold higher in the low (line C2) and in the high (line C1) AKR4C9-expressing transgenic line, respectively, compared to WT. Furthermore by applying three subsequent, identical − 20 °C treatments 94.7% of all the leaves of WT plants had died, but only 80% in the case of transgenic line C1. The average electrolyte leakage of the transgenic plants (line C1) was lower than that of WT plants. Line C1 plant also had significantly higher fresh weight than the WT after 6 days of recovery following frost-treatment. Transgenic line C2 had an intermediate freezing tolerance based on the same physiological parameters. The results indicate that AKR-overexpression may lead to higher frost tolerance and higher post-frost regenerative capacity.     

Functional significance of shade-induced leaf senescence in dense canopies: an experimental test using transgenic tobacco

Canopy photosynthesis models have predicted an optimal leaf area index (LAI; leaf area per unit surface area) and leaf nitrogen distribution at which whole-plant carbon gain per unit N is maximized. In this study we experimentally tested these models, using transgenic P(SAG12)-IPT tobacco (SAG; Nicotiana tabacum L.) plants with delayed leaf senescence and therefore a greater LAI and more uniform N distribution than the wild type (WT). In a competition experiment, the increased density of surrounding WT plants caused a greater reduction in dry mass of mature SAG target plants than in that of WT target plants, indicating negative effects of delayed leaf senescence on performance at high canopy density. Vegetative SAG plants achieved a lower calculated daily carbon gain than competing WT plants because the former retained leaves with a negative carbon gain in the shaded, lower part of the canopy. Sensitivity analyses showed that the carbon gain of SAG plants would increase if these lower leaves were shed and the N reallocated from these leaves were used to form additional leaf area at the canopy top. This strategy, which is adopted by the WT, is most advantageous because it results in the shading of competing neighbors.