Dormancy characteristics of lammas-growth seedlings of subtropical trees and their phenological responses to experimental warming.

Lammas growth of trees means the additional growth of the shoot after the growth cessation and bud set in late summer. In temperate tree species, lammas growth occurs irregularly and is often regarded as abnormal, disturbed growth. In subtropical tree species, however, lammas growth is a prevalent phenomenon, possibly due to the prolonged occurrence of high temperatures in the autumn. The occurrence of lammas growth extends the growing season of trees, but its influence on subsequent dormancy phenomena and bud burst phenology remains largely unexplored. By comparing seedlings showing lammas growth with others not showing it, we carried out an experimental study of how lammas growth affects the bud burst phenology and the underlying dormancy phenomena under both ambient and controlled chilling, forcing, and warming conditions in four subtropical tree species: Carya illinoinensis, Cinnamomum japonicum, Phoebe chekiangensis, and Torreya grandis. With the exception of C. illinoinensis, lammas growth delayed bud burst in all the species under ambient conditions. In a chilling experiment, the delay appeared to be due to higher minimum forcing requirement, higher dormancy depth, and in T. grandis, also to lower chilling sensitivity in the lammas-growth seedlings than in the non-lammas-growth ones. However, a spring warming experiment showed that the sensitivity of bud burst to spring temperatures was higher in the lammas-growth seedlings than in the non-lammas-growth ones. Because of this, the difference between the two phenotypes in the timing of bud burst vanished with increasing warming. Our findings elucidate the significant impact of lammas growth on the dormancy dynamics of subtropical tree species, highlighting the necessity to better understand how the physiological phenomena causing lammas growth change the trees' subsequent environmental responses under changing climatic conditions.

Immediate and carry-over effects of late-spring frost and growing season drought on forest gross primary productivity capacity in the Northern Hemisphere.

Forests are increasingly exposed to extreme global warming-induced climatic events. However, the immediate and carry-over effects of extreme events on forests are still poorly understood. Gross primary productivity (GPP) capacity is regarded as a good proxy of the ecosystem's functional stability, reflecting its physiological response to its surroundings. Using eddy covariance data from 34 forest sites in the Northern Hemisphere, we analyzed the immediate and carry-over effects of late-spring frost (LSF) and growing season drought on needle-leaf and broadleaf forests. Path analysis was applied to reveal the plausible reasons behind the varied responses of forests to extreme events. The results show that LSF had clear immediate effects on the GPP capacity of both needle-leaf and broadleaf forests. However, GPP capacity in needle-leaf forests was more sensitive to drought than in broadleaf forests. There was no interaction between LSF and drought in either needle-leaf or broadleaf forests. Drought effects were still visible when LSF and drought coexisted in needle-leaf forests. Path analysis further showed that the response of GPP capacity to drought differed between needle-leaf and broadleaf forests, mainly due to the difference in the sensitivity of canopy conductance. Moreover, LSF had a more severe and long-lasting carry-over effect on forests than drought. These results enrich our understanding of the mechanisms of forest response to extreme events across forest types.

Effect of the photoperiod on bud dormancy in Liriodendron chinense.

Bud dormancy and its release are complex physiological phenomena in plants. The molecular mechanisms of bud dormancy in Liriodendron chinense are mainly unknown. Here, we studied bud dormancy and the related physiological and molecular phenomena in Liriodendron under long-day (LD) and short-day (SD). Bud burst was released faster under LD than under SD. Abscisic acid (ABA), superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GR) activities were increased significantly under LD in Liriodendron buds. In contrast, the contents of gibberellic acid (GA3), ascorbic acid (AsA), glutathione (GSH), malondialdehyde (MDA), and ascorbate peroxidase (APX) activity decreased under LD but increased under SD. Differentially expressed genes (DEGs) were up-regulated under LD and down-regulated under SD and these changes correspondingly promoted (LD) or repressed (SD) cell division and the number and/or size of cells in the bud. Transcriptomic analysis of Liriodendron buds under different photoperiods identified 187 DEGs enriched in several pathways such as flavonoid biosynthesis and phenylpropanoid biosynthesis, plant hormone and signal transduction, etc. that are associated with antioxidant enzymes, non-enzymatic antioxidants, and subsequently promote the growth of the buds. Our findings provide novel insights into regulating bud dormancy via flavonoid and phenylpropanoid biosynthesis, plant hormone and signal transduction pathways, and ABA content. These physiological and biochemical traits would help detect bud dormancy in plants.

Climate warming leads to advanced fruit development period of temperate woody species but divergent changes in its length.

Climate warming has significantly altered the phenology of plants in recent decades. However, in contrast to the widely reported warming-induced extension of vegetative growing season, the response of fruit development period (FDP) from flowering to fruiting remains largely unexplored, particularly for woody plants. Analyzing >560,000 in situ observations of both flowering and fruiting dates for six temperate woody species across 2958 European phenological observations sites during 1980-2013, we found that in all species both flowering and fruiting phenology, that is, the FDP, advanced with climate warming. However, the advancing rates of the two events were not necessarily equal for any given species, resulting in divergent changes in the length of FDP among species with climate warming. During 1980-2013, not only the temperature during FDP but also the forcing requirement for fruit development increased, both affecting the length of FDP. The shortened FDP was mainly due to elevated temperature, thus accelerating the accumulation of forcing, whereas the prolonged FDP was primarily caused by the substantial increase of the forcing requirement of fruiting, which could be fulfilled only in a longer time and thus slowed down the advance of fruiting. This study provides large-scale empirical evidence of warming-induced advances of FDP but divergent changes in its length in temperate woody species. Our findings demonstrate the contrasting reproductive phenological strategies among temperate woody species under the pressure of warming climate, contrary to the lengthening of vegetative growing season, which is by and largely similar with different woody species.

Extending the Cultivation Area of Pecan (Carya illinoinensis) Toward the South in Southeastern Subtropical China May Cause Increased Cold Damage.

Pecan (Carya illinoinensis) is an important nut tree species in its native areas in temperate and subtropical North America, and as an introduced crop in subtropical southeastern China as well. We used process-based modeling to assess the effects of climatic warming in southeastern China on the leaf-out phenology of pecan seedlings and the subsequent risk of "false springs," i.e., damage caused by low temperatures occurring as a result of prematurely leafing out. In order to maximize the biological realism of the model used in scenario simulations, we developed the model on the basis of experiments explicitly designed for determining the responses modeled. The model showed reasonable internal accuracy when calibrated against leaf-out observations in a whole-tree chamber (WTC) experiment with nine different natural-like fluctuating temperature treatments. The model was used to project the timing of leaf-out in the period 2022-2099 under the warming scenarios RCP4.5 and RCP8.5 in southeastern China. Two locations in the main pecan cultivation area in the northern subtropical zone and one location south of the main cultivation area were addressed. Generally, an advancing trend of leaf-out was projected for all the three locations under both warming scenarios, but in the southern location, a delay was projected under RCP8.5 in many years during the first decades of the 21st century. In the two northern locations, cold damage caused by false springs was projected to occur once in 15-26 years at most, suggesting that pecan cultivation can be continued relatively safely in these two locations. Paradoxically, more frequent cold damage was projected for the southern location than for the two northern locations. The results for the southern location also differed from those for the northern locations in that more frequent cold damage was projected under the RCP4.5 warming scenario (once in 6 years) than under the RCP8.5 scenario (once in 11 years) in the southern location. Due to the uncertainties of the model applied, our conclusions need to be re-examined in an additional experimental study and further model development based on it; but on the basis of our present results, we do not recommend starting large-scale pecan cultivation in locations south of the present main pecan cultivation area in southeastern subtropical China.

A hundred years after: endodormancy and the chilling requirement in subtropical trees.

Endodormancy and the related chilling requirement synchronize the seasonal development of trees from the boreal and temperate regions under the climatic conditions prevailing at their native growing sites. The phenomenon of endodormancy has been known at the whole-plant level for 100 years, and in the last couple of decades, insights into the physiological and molecular basis of endodormancy and its release have also been obtained. Intriguingly, recent studies have shown experimentally that subtropical trees also show endodormancy and a chilling requirement. Motivated by the climatic differences between the subtropical and more northern zones, here we address the similarities and differences in endodormancy between trees growing in the subtropical zone and those growing in more northern zones.

Endodormancy release in Norway spruce grafts representing trees of different ages.

Studies addressing endodormancy release in adult trees are usually carried out using twigs detached from the trees in the experiments. Potential problems caused by cutting the root-shoot connection when detaching the twigs can be avoided by using grafts as the experimental material. We studied the effects of chilling on the endodormancy release in Norway spruce (Picea abies (L.) Karst.) grafts where twigs of 16-, 32- and 80-year-old trees were used as the scions. The grafts were first exposed to chilling in natural conditions and then samples of them were transferred at intervals to a regrowth test in forcing conditions in a greenhouse. The bud burst percentage, BB%, in the forcing conditions generally increased from zero to near 100% with increasing previous chilling accumulation from mid-October until mid-November, indicating that endodormancy was released in almost all of the grafts by mid-November. The days to bud burst, DBB, decreased in the forcing conditions with successively later transfers until the next spring. Neither BB% nor DBB was dependent on the age of the scion. However, in the early phase of ecodormancy release, the microscopic internal development of the buds was more advanced in the grafts representing the 16-year-old than in those representing the 32- or 80-year-old trees. In conclusion, our findings suggest that no major change in the environmental regulation of endodormancy release in Norway spruce takes place when the trees get older. Taken together with earlier findings with Norway spruce seedlings, our results suggest that regardless of the seedling or tree age, the chilling requirement of endodormancy release is met in late autumn. The implications of our findings for Norway spruce phenology under climatic warming and the limitations of our novel method of using grafts as a proxy of trees of different ages are discussed.

Photosynthetic Mechanisms of Metaxenia Responsible for Enlargement of Carya cathayensis Fruits at Late Growth Stages.

Fruits of hickory (Carya cathayensis) are larger and their peel is greener after interspecific pollination by pecan (Carya illinoinensis; later pp fruits) than after intraspecific pollination by hickory (later ph fruits). Previous studies have found little genetic differences between offspring and their maternal parent, indicating that the observed trait differences between pp and ph fruits are due to metaxenia. Fruit development depends on the amount of photosynthetic assimilate available. Since there is no difference in photosynthesis of the associated leaves between pp and ph fruits, the larger size of the pp fruits might be attributed to changes in fruit photosynthesis caused by the different pollen sources. To elucidate to the photosynthetic mechanisms behind the metaxenia effect on fruit development in hickory, the effects of intraspecific and interspecific pollination regimes were examined in the present study. We observed the photosynthetic capacity in the peel of fruits and the related ecophysiological and morphological traits of both ph and pp fruits over a period of 120 days after pollination. Significant differences in the appearance and dry weight between ph and pp fruits were observed at 50 days after pollination (DAP). More than 70% of dry matter accumulation of the fruits was completed during 60-120 DAP, while the true photosynthetic rate of the associated leaves significantly decreased by about 50% during the same period. In several cell layers of the peel, the number of chloroplasts per cell was significantly higher in pp than in ph fruits. Similarly, the ribulose 1, 5-bisphosphate carboxylase (Rubisco) activity, the total chlorophyll content, and the nitrogen content were all significantly higher in pp than in ph fruits during all growth stages; and all of these physiological quantities were positively correlated with the gross photosynthetic rate of the fruits. We conclude that the enhanced photosynthetic capacity of pp fruits contributes to their fast dry matter accumulation and oil formation. This result will provide a theoretical basis for improving hickory fruit yields in practical cultivation.

Beyond rest and quiescence (endodormancy and ecodormancy): A novel model for quantifying plant-environment interaction in bud dormancy release.

Bud dormancy of plants has traditionally been explained either by physiological growth arresting conditions in the bud or by unfavourable environmental conditions, such as non-growth-promoting low air temperatures. This conceptual dichotomy has provided the framework also for developing process-based plant phenology models. Here, we propose a novel model that in addition to covering the classical dichotomy as a special case also allows the quantification of an interaction of physiological and environmental factors. According to this plant-environment interaction suggested conceptually decades ago, rather than being unambiguous, the concept of "non-growth-promoting low air temperature" depends on the dormancy status of the plant. We parameterized the model with experimental results of growth onset for seven boreal plant species and found that based on the strength of the interaction, the species can be classified into three dormancy types, only one of which represents the traditional dichotomy. We also tested the model with four species in an independent experiment. Our study suggests that interaction of environmental and physiological factors may be involved in many such phenomena that have until now been considered simply as plant traits without any considerations of effects of the environmental factors.

Nuclear export signal (NES) of transposases affects the transposition activity of mariner-like elements Ppmar1 and Ppmar2 of moso bamboo.

Ppmar1 and Ppmar2 are two active mariner-like elements (MLEs) cloned from moso bamboo (Phyllostachys edulis (Carrière) J. Houz) genome possessing transposases that harbour nuclear export signal (NES) domain, but not any nuclear localization signal (NLS) domain. To understand the functions of NES in transposon activity, we have conducted two experiments, fluorescence and excision frequency assays in the yeast system. For this, by site-directed mutagenesis, three NES mutants were developed from each of the MLE. In the fluorescence assay, the mutants, NES-1, 2 and 3 along with the wild types (NES-0) were fused with fluorescent proteins, enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP) were co-transformed into yeast system. To differentiate protein localisation under the NES influence, ECFP alone was fused to wild and mutant NES domains either on N- or C-terminal and not to EYFP. Fluorescence assay revealed that blue fluorescence of ECFP was more intense than the red fluorescence of the EYFP in the yeast cell matrix. Further, ECFP had a wider localisation in the cellular matrix, but EYFP was largely located in the nucleus. The NES-1 domain was related to the comparatively high spread of ECFP, while NES-2 and NES-3 indicated a low spread, implying that NES activity on nuclear export increased when the NES is made leucine-rich, while the signalling activity was reduced when the leucine content was lowered in the NES domain. In the transposon excision assay, the mutant and wild type NES of both the Ppmar elements were integrated into an Ade2 vector, and within the Ade2 gene. Co-transformation of the vector together with non-autonomous Ppmar transposons and NES-lacking transposases was used to assess the differential excision frequencies of the mutants NES domains. In both the MLEs, NES-1 had the highest excision suppression, which was less than half of the excision frequency of the wild type. NES-2 and NES-3 elements showed, up to three times increase in transposon excision than the wild types. The results suggested that NES is an important regulator of nuclear export of transposase in Ppmar elements and the mutation of the NES domains can either increase or decrease the export signalling. We speculate that in moso bamboo, NESs regulates the transposition activity of MLEs to maintain the genome integrity.

Affinities of Terminal Inverted Repeats to DNA Binding Domain of Transposase Affect the Transposition Activity of Bamboo Ppmar2 Mariner-Like Element.

Mariner-like elements (MLE) are a super-family of DNA transposons widespread in animal and plant genomes. Based on their transposition characteristics, such as random insertions and high-frequency heterogeneous transpositions, several MLEs have been developed to be used as tools in gene tagging and gene therapy. Two active MLEs, Ppmar1 and Ppmar2, have previously been identified in moso bamboo (Phyllostachys edulis). Both of these have a preferential insertion affinity to AT-rich region and their insertion sites are close to random in the host genome. In Ppmar2 element, we studied the affinities of terminal inverted repeats (TIRs) to DNA binding domain (DBD) and their influence on the transposition activity. We could identify two putative boxes in the TIRs which play a significant role in defining the TIR's affinities to the DBD. Seven mutated TIRs were constructed, differing in affinities based on similarities with those of other plant MLEs. Gel mobility shift assays showed that the TIR mutants with mutation sites G669A-C671A had significantly higher affinities than the mutants with mutation sites C657T-A660T. The high-affinity TIRs indicated that their transposition frequency was 1.5-2.0 times higher than that of the wild type TIRs in yeast transposition assays. The MLE mutants with low-affinity TIRs had relatively lower transposition frequency from that of wild types. We conclude that TIR affinity to DBD significantly affects the transposition activity of Ppmar2. The mutant MLEs highly active TIRs constructed in this study can be used as a tool for bamboo genetic studies.

Nutrient availability alters the correlation between spring leaf-out and autumn leaf senescence dates.

Leaf senescence (LS) affects tree fitness, species distribution and ecosystem structure and functioning. The drivers of LS and the processes underlying it have been studied, but the studies have mainly focused on environmental cues and have mainly been based on statistical analyses using in situ data sets. Experimental investigation and field verification of the processes and drivers are thus urgently needed. We conducted a nutrient-addition experiment after a spring-warming experiment in which an ~40-day range of leaf-out (LO) dates was induced in horse chestnut (Aesculus hippocastanum) and beech (Fagus sylvatica) saplings. We found that both increased nutrient supply and advanced LO date significantly affected the timing of LS, but their effects were opposite, as the former delayed and the latter advanced the senescence. The effects of nutrient supply and LO interacted species specifically. In chestnut, the delay of senescence caused by fertilization increased with the delay of LO and was thus stronger for individuals that flushed late in the spring. On the contrary, in beech the delay of senescence caused by fertilization decreased with the delay of LO and was insignificant for individuals with the latest LO. The experimental findings for beech were confirmed with mature trees at a regional scale. The interactive effect between nutrients and LO on senescence may be associated with variable sensitivity to photoperiod, growth sink limitation and/or direct effect of foliar nutrition on the timing of senescence. Our novel results show that the interactive effects of LO and nutrient supply on the timing of LS should be further addressed experimentally in forthcoming studies. It would also be interesting to consider our results in the further development of phenological models used in assessing the effects of climatic change. The differences found in the present study between horse chestnut and beech suggest that the results found for one species cannot necessarily be generalized to other species, so studies with different temperate tree species are called for.

Divergent trends in the risk of spring frost damage to trees in Europe with recent warming.

Frost events during the active growth period of plants can cause extensive frost damage with tremendous economic losses and dramatic ecological consequences. A common assumption is that climate warming may bring along a reduction in the frequency and severity of frost damage to vegetation. On the other hand, it has been argued that rising temperature in late winter and early spring might trigger the so called "false spring", that is, early onset of growth that is followed by cold spells, resulting in increased frost damage. By combining daily gridded climate data and 1,489 k in situ phenological observations of 27 tree species from 5,565 phenological observation sites in Europe, we show here that temporal changes in the risk of spring frost damage with recent warming vary largely depending on the species and geographical locations. Species whose phenology was especially sensitive to climate warming tended to have increased risk of frost damage. Geographically, compared with continental areas, maritime and coastal areas in Europe were more exposed to increasing occurrence of frost and these late spring frosts were getting more severe in the maritime and coastal areas. Our results suggest that even though temperatures will be elevated in the future, some phenologically responsive species and many populations of a given species will paradoxically experience more frost damage in the future warming climate. More attention should be paid to the increased frost damage in responsive species and populations in maritime areas when developing strategies to mitigate the potential negative impacts of climate change on ecosystems in the near future.

Long-term changes in the impacts of global warming on leaf phenology of four temperate tree species.

Contrary to the generally advanced spring leaf unfolding under global warming, the effects of the climate warming on autumn leaf senescence are highly variable with advanced, delayed, and unchanged patterns being all reported. Using one million records of leaf phenology from four dominant temperate species in Europe, we investigated the temperature sensitivities of spring leaf unfolding and autumn leaf senescence (ST , advanced or delayed days per degree Celsius). The ST of spring phenology in all of the four examined species showed an increase and decrease during 1951-1980 and 1981-2013, respectively. The decrease in the ST during 1981-2013 appears to be caused by reduced accumulation of chilling units. As with spring phenology, the ST of leaf senescence of early successional and exotic species started to decline since 1980. In contrast, for late successional species, the ST of autumn senescence showed an increase for the entire study period from 1951 to 2013. Moreover, the impacts of rising temperature associated with global warming on spring leaf unfolding were stronger than those on autumn leaf senescence. The timing of leaf senescence was positively correlated with the timing of leaf unfolding during 1951-1980. However, as climate warming continued, the differences in the responses between spring and autumn phenology gradually increased, so that the correlation was no more significant during 1981-2013. Our results further suggest that since 2000, due to the decreased temperature sensitivity of leaf unfolding the length of the growing season has not increased any more. These finding needs to be addressed in vegetation models used for assessing the effects of climate change.

Experiments Are Necessary in Process-Based Tree Phenology Modelling.

In boreal and temperate trees, air temperature is a major environmental factor regulating the timing of spring phenological events, such as vegetative bud burst, through underlying physiological processes. This has been established by experimental research, and mathematical process-based tree phenology models have been developed based on the results. The models have often been applied when assessing the effects of climate change. Currently, there is an increasing trend to develop process-based tree phenology models using only observational phenological records from natural conditions. We point out that this method runs a high risk of producing models that do not simulate the real physiological processes in the trees and discuss experimental designs facilitating the development of biologically realistic process-based models for tree spring phenology.

Spring phenology at different altitudes is becoming more uniform under global warming in Europe.

Under current global warming, high-elevation regions are expected to experience faster warming than low-elevation regions. However, due to the lack of studies based on long-term large-scale data, the relationship between tree spring phenology and the elevation-dependent warming is unclear. Using 652k records of leaf unfolding of five temperate tree species monitored during 1951-2013 in situ in Europe, we discovered a nonlinear trend in the altitudinal sensitivity (SA , shifted days per 100 m in altitude) in spring phenology. A delayed leaf unfolding (2.7 ± 0.6 days per decade) was observed at high elevations possibly due to decreased spring forcing between 1951 and 1980. The delayed leaf unfolding at high-elevation regions was companied by a simultaneous advancing of leaf unfolding at low elevations. These divergent trends contributed to a significant increase in the SA (0.36 ± 0.07 days 100/m per decade) during 1951-1980. Since 1980, the SA started to decline with a rate of -0.32 ± 0.07 days 100/m per decade, possibly due to reduced chilling at low elevations and improved efficiency of spring forcing in advancing the leaf unfolding at high elevations, the latter being caused by increased chilling. Our results suggest that due to both different temperature changes at the different altitudes, and the different tree responses to these changes, the tree phenology has shifted at different rates leading to a more uniform phenology at different altitudes during recent decades.

A transposition-active Phyllostachys edulis long terminal repeat (LTR) retrotransposon.

Due to infrequent sexual reproduction, moso bamboo breeding by hybridization is extremely technically difficult. Insertional mutagenesis based on endogenous active transposons may thus serve as an alternative method to create new germplasm of moso bamboo. In the present study, using LTR-STRUC, a full-length intact long terminal repeat (LTR) retrotransposon was identified in the moso bamboo genome and was named PHRE2 (Phyllostachys edulis retrotransposon 2). The 5' and 3' LTR sequences of PHRE2 were highly (98.39%) similar. PHRE2 contains all domains necessary for transposition such as gag, pr, rt, rh, and int. The coding frames of these essential domains were complete and had no apparent mutations. In addition, PHRE2 possessed a prime binding site (PBS), a polypurine tract (PPT), and two typical sequences of LTR retrotransposons. A genome-wide scan showed that the moso bamboo genome has only one full-length sequence of PHYRE2. After its transfer to Arabidopsis thaliana, an increase in PHRE2 copy number occurred in the T3 plants compared to in the T2 plants. After moso bamboo seedlings were grown in tissue culture or treated by irradiation or plant hormones, the copy number of PHRE2 significantly increased. These findings indicate that PHRE2 has the capacity for transposition, which can be induced by environmental conditions.

Larger temperature response of autumn leaf senescence than spring leaf-out phenology.

Climate warming is substantially shifting the leaf phenological events of plants, and thereby impacting on their individual fitness and also on the structure and functioning of ecosystems. Previous studies have largely focused on the climate impact on spring phenology, and to date the processes underlying leaf senescence and their associated environmental drivers remain poorly understood. In this study, experiments with temperature gradients imposed during the summer and autumn were conducted on saplings of European beech to explore the temperature responses of leaf senescence. An additional warming experiment during winter enabled us to assess the differences in temperature responses of spring leaf-out and autumn leaf senescence. We found that warming significantly delayed the dates of leaf senescence both during summer and autumn warming, with similar temperature sensitivities (6-8 days delay per °C warming), suggesting that, in the absence of water and nutrient limitation, temperature may be a dominant factor controlling the leaf senescence in European beech. Interestingly, we found a significantly larger temperature response of autumn leaf senescence than of spring leaf-out. This suggests a possible larger contribution of delays in autumn senescence, than of the advancement in spring leaf-out, to extending the growing season under future warmer conditions.

Nitrogen Deposition Enhances Photosynthesis in Moso Bamboo but Increases Susceptibility to Other Stress Factors.

Atmospheric nitrogen (N) deposition can increase the susceptibility of vascular plants to other stresses, but the physiological basis of such a response remains poorly understood. This study was designed to clarify the physiological mechanisms and to evaluate bioindicators of N deposition impact on vascular plants. We evaluate multiple physiological responses to ~4 years of simulated additional N deposition (30-90 kg N ha-1 year-1) on three age-classes (1a, 3a, and 5a) of Moso bamboo. A saturating response to the additional N deposition was found both in foliar N concentration and in Pn. However, 3- and 5-year-old bamboo seemed to be less tolerant to extremely high N deposition than 1-year-old bamboo since they were saturated at a lower N addition. Furthermore, C/N/P stoichiometric ratios were very sensitive to N deposition in all three-age classes of bamboo, but the responses to N deposition in the various age-classes were diverse. We also found that the highest additional N deposition suppressed stomatal conductance and transpiration rate, suggesting an induced water stress. The stress induced by the high N load was also seen in photochemistry, where it reduced potential and actual photosynthetic use of light energy, diminished photo-protection capacity, and increased risk of the photo-damage. High additional N deposition contributed to a decrease in the foliar soluble protein contents and to an increase in the peroxidase activity (POD). Our study suggested, for the first time, that although the photosynthetic rate was enhanced by the increased N deposition in Moso bamboo, long-term high N load causes negative effects, such as damage to photosystem II. In Moso bamboo photochemical parameters are more sensitive to N deposition than photosynthetic rate or foliar N concentration. Furthermore, plant age should be taken into account when assessing plants' susceptibility to changes in global change drivers, such as N deposition. These findings facilitate the revealing of the risks potentially caused to vascular plants by increased N deposition before any visible symptoms of injury are seen.