Xylem structure and hydraulic characteristics of deep roots, shallow roots and branches of walnut under seasonal drought.

BACKGROUND: Despite the importance of root hydraulics, there is little research on the in situ dynamic responses of embolism formation and embolism repair of roots distributed in different soil depths in response to different water regimes. RESULTS: The vessel diameter, hydraulic conductivity, and vulnerability to cavitation were in the order of deep root > shallow root > branch. The midday PLC of shallow root was the highest in the dry season, while the midday PLC of deep root slightly higher than that of branch with no significant difference in the two seasons. The capacity of embolism repair of roots was significantly greater than that of branch both in dry season and wet season. The xylem pressure was in the order of deep roots > shallow root > branch, and it was negative in most of the time for the latter two in the dry season, but positive for both of the roots during the observation period in the wet season. The NSC and starch content in roots were significantly higher than those in branches, especially in the dry season. In contrast, roots had lower content of soluble sugar. CONCLUSIONS: The relatively stable water condition in soil, especially in the deep layers, is favorable for the development of larger-diameter vessels in root xylem, however it cannot prevent the root from forming embolism. The mechanism of embolism repair may be different in different parts of plants. Deep roots mainly depend on root pressure to refill the embolized vessels, while branches mainly depend on starch hydrolysis to soluble sugars to do the work, with shallow roots shifted between the two mechanisms in different moisture regimes. There is theoretically an obvious trade-off between conducting efficiency and safety over deep roots, shallow roots and branches. But in natural conditions, roots do not necessarily suffer more severe embolism than branches, maybe due to their root pressure-driven embolism repair and relatively good water conditions.

Poplar aquaporin PIP1;1 promotes Arabidopsis growth and development.

BACKGROUND: Root hydraulic conductance is primarily determined by the conductance of living tissues to radial water flow. Plasma membrane intrinsic proteins (PIPs) in root cortical cells are important for plants to take up water and are believed to be directly involved in cell growth. RESULTS: In this study, we found that constitutive overexpression of the poplar root-specific gene PtoPIP1;1 in Arabidopsis accelerated bolting and flowering. At the early stage of the developmental process, PtoPIP1;1 OE Arabidopsis exhibited faster cell growth in both leaves and roots. The turgor pressure of plants was correspondingly increased in PtoPIP1;1 OE Arabidopsis, and the water status was changed. At the same time, the expression levels of flowering-related genes (CRY1, CRY2 and FCA) and hub genes in the regulatory networks underlying floral timing (FT and SOC1) were significantly upregulated in OE plants, while the floral repressor FLC gene was significantly downregulated. CONCLUSIONS: Taken together, the results of our study indicate that constitutive overexpression of PtoPIP1;1 in Arabidopsis accelerates bolting and flowering through faster cell growth in both the leaf and root at an early stage of the developmental process. The autonomous pathway of flowering regulation may be executed by monitoring developmental age. The increase in turgor and changes in water status with PtoPIP1;1 overexpression play a role in promoting cell growth.

Corticular photosynthesis drives bark water uptake to refill embolized vessels in dehydrated branches of Salix matsudana.

It is well known that xylem embolism can be repaired by bark water uptake and that the sugar required for embolism refilling can be provided by corticular photosynthesis. However, the relationship between corticular photosynthesis and embolism repair by bark water uptake is still poorly understood. In this study, the role of corticular photosynthesis in embolism repair was assessed using Salix matsudana branch segments dehydrated to -1.9 MPa (P50 , water potential at 50% loss of conductivity). The results indicated that corticular photosynthesis significantly promoted water uptake and nonstructural carbohydrate (NSC) accumulation in the bark and xylem during soaking, thereby effectively enhancing the refilling of the embolized vessels and the recovery of hydraulic conductivity. Furthermore, the influence of the extent of dehydration on the embolism refilling enhanced by corticular photosynthesis was investigated. The enhanced refilling effects were much higher in the mildly dehydrated (-1.5 MPa) and moderately dehydrated (-1.9 MPa) branch segments than in the severely dehydrated (-2.2 MPa) branch segments. This study provides evidence that corticular photosynthesis plays a crucial role in xylem embolism repair by bark water uptake for mildly and moderately dehydrated branches.

Daytime and nighttime wind differentially affects hydraulic properties and thigmomorphogenic response of poplar saplings.

This study tested how wind in daytime and nighttime affects hydraulic properties and thigmomorphogenic response of poplar saplings. It shows that wind in daytime interrupted water balance of poplar plants by aggravating cavitation in the stem xylem under high xylem tension in the daytime, reducing water potential in midday and hence reducing gas exchange, including stomatal conductance and CO2 assimilation. The wind blowing in daytime significantly reduced plant growth, including height, diameter, leaf size, leaf area, root and whole biomass, whereas wind blowing in nighttime only caused a reduction in radial and height growth at the early stage compared with the control but decreased height:diameter ratios. In summary, the interaction between wind loading and xylem tension exerted a negative impact on water balance, gas exchanges and growth of poplar plants, and wind in nighttime caused only a small thigmomorphogenic response.

Variation in embolism occurrence and repair along the stem in drought-stressed and re-watered seedlings of a poplar clone.

Root pressure and plasma membrane intrinsic protein (PIP) availability in the xylem have been recognized to participate in the refilling of embolized conduits, yet integration of the two mechanisms has not been reported in the same plant. In this study, 4-month-old seedlings of a hybrid poplar (Populus alba × Populus glandulosa) clone 84K were subjected to two contrasting soil-water treatments, with the drought treatment involving withholding of water for 17 days to reduce the soil-water content to 10% of the saturated field capacity, followed by a re-watering cycle. The percentage loss of stem hydraulic conductance (PLC) sharply increased, and stomatal conductance and photosynthesis declined in response to drought stress; these processes were gradually restored following the subsequent re-watering. Embolism was most severe in the middle portions of the stem, followed by the basal and top portions of the stems of seedlings subjected to drought stress and subsequent re-watering. Although drought stress eliminated root pressure, re-watering partially restored it in a short period of time. The expression of PIP genes in the xylem was activated by drought stress, and some PIP genes were further stimulated in the top portion after re-watering. The dynamics of root pressure and differential expression of PIP genes along the stem coincided with changes in PLC, suggesting that root pressure and PIPs work together to refill the embolized vessels. On the basis of the recovery dynamics in PLC and g(smax) (maximum stomatal conductance) after re-watering, the stomatal closure and xylem cavitation exhibited fatigue due to drought stress.

Maintenance of xylem hydraulic function during winter in the woody bamboo Phyllostachys propinqua McClure.

Background: Frost is a common environmental stress for temperate plants. Xylem embolism occurs in many overwintering plants due to freeze-thaw cycles, so coping with freeze-thaw-induced embolisms is essential for the survival of temperate plants. Methods: This study was conducted on Phyllostachys propinqua McClure, a woody bamboo species that was grown under natural frost conditions to explore its responses to winter embolisms. From autumn to the following spring, the following measurements were recorded: predawn branch and leaf embolism, branch and leaf relative water content (RWC), root pressure and soil temperature, xylem sap osmotic potential, branch and leaf electrolyte leakage (EL), branch nonstructural carbohydrate (NSC) content and leaf net photosynthetic rate. Results: P. propinqua had a mean vessel diameter of 68.95 ±1.27 µm but did not suffer severe winter embolism, peaking around  60% in winter (January), with a distinct reduction in March when root pressure returned. Leaves had a more severe winter embolism, up to 90%. Leaf RWC was much lower in winter, and leaf EL was significantly higher than branch EL in all seasons. Root pressure remained until November when soil temperature reached 9 °C, then appeared again in March when soil temperatures increased from -6 °C (January) to 11 °C. Xylem sap osmotic potential decreased from autumn to winter, reaching a minimum in March, and then increasing again. Soluble sugar (SS) concentration increased throughout the winter, peaked in March, and then decreased. Conclusions: These results suggest that (1) there is a hydraulic segmentation between the stem and leaf, which could prevent stem water loss and further embolization in winter; (2) maintenance of root pressure in early winter played an important role in reducing the effect of freeze-thaw cycles on the winter embolism; (3) the physiological process that resulted in a decrease in xylem sap osmotic potential and tissue water content, and an accumulation of SS associated with cold acclimation also aided in reducing the extent of freeze-thaw-induced embolism. All these strategies could be helpful for the maintenance of xylem hydraulic function of this bamboo species during winter.

Strategies of tree species to adapt to drought from leaf stomatal regulation and stem embolism resistance to root properties.

Considerable evidences highlight the occurrence of increasing widespread tree mortality as a result of global climate change-associated droughts. However, knowledge about the mechanisms underlying divergent strategies of various tree species to adapt to drought has remained remarkably insufficient. Leaf stomatal regulation and embolism resistance of stem xylem serves as two important strategies for tree species to prevent hydraulic failure and carbon starvation, as comprising interconnected physiological mechanisms underlying drought-induced tree mortality. Hence, the physiological and anatomical determinants of leaf stomatal regulation and stems xylem embolism resistance are evaluated and discussed. In addition, root properties related to drought tolerance are also reviewed. Species with greater investment in leaves and stems tend to maintain stomatal opening and resist stem embolism under drought conditions. The coordination between stomatal regulation and stem embolism resistance are summarized and discussed. Previous studies showed that hydraulic safety margin (HSM, the difference between minimum water potential and that causing xylem dysfunction) is a significant predictor of tree species mortality under drought conditions. Compared with HSM, stomatal safety margin (the difference between water potential at stomatal closure and that causing xylem dysfunction) more directly merge stomatal regulation strategies with xylem hydraulic strategies, illustrating a comprehensive framework to characterize plant response to drought. A combination of plant traits reflecting species' response and adaptation to drought should be established in the future, and we propose four specific urgent issues as future research priorities.

Hydraulic traits are coupled with plant anatomical traits under drought-rewatering cycles in Ginkgo biloba L.

Investigating the responses of plant anatomical traits of trees to drought-rewatering cycles helps us to understand their responses to climate change; however, such work has not been adequately reported. In this study, Ginkgo biloba L. saplings were subjected to moderate, severe, extreme and lethal drought conditions by withholding water according to the percentage loss of hydraulic conductivity (PLC) and rewatering on a regular basis. Samples of phloem, cambium and xylem were collected to quantify their cellular properties including cambium and phloem cell vitality, xylem growth ring width, pit aspiration rates and pit membrane thickness using light microscopy and transmission microscopy. The results showed that the mortality rate of G. biloba saplings reached 90% at approximately P88 (xylem water potential inducing 88% loss of hydraulic conductivity). The onset of cambium and phloem cell mortality might be in accordance with that of xylem embolism. Close negative correlations between xylem water potential and PLC and between xylem water potential and cambium and phloem mortality suggested that xylem hydraulic traits are coupled with anatomical traits under declining xylem water potential. Cambium and phloem cell vitality as well as xylem growth ring width decreased significantly with increasing drought conditions. However, xylem pit membrane thickness, cambial zone width and cambial cell geometry were not affected by the drought-rewatering cycles. The tracheid radial diameter, intertracheid cell wall thickness and tracheid density decreased significantly during both drought conditions and rewatering conditions. In addition to hydraulic traits, cambium and phloem cell vitality can be used as anatomical traits to evaluate the mortality of G. biloba under drought. Future work is proposed to observe the dynamics of pit aspiration rates under drought-rewatering cycles in situ to deepen our understanding of the essential role of bordered pits in the 'air-seeding' mechanism.

Coordinated variation in stem and leaf functional traits of temperate broadleaf tree species in the isohydric-anisohydric spectrum.

Stomatal regulation serves as an important strategy for plants to adapt to drought. However, the understanding of how complexes of plant-functional traits vary along the continuum from isohydry to anisohydry remains insufficient. In this study, we investigated a proxy of the degree of iso/anisohydry-the water potential at stomatal closure-and a series of functional traits of leaves and branches in 20 temperate broadleaf species planted in an arid limestone habitat in northern China. The results showed that the water potential at stomatal closure was significantly correlated with many functional traits. At the anisohydric end of the spectrum, species had a higher leaf carbon content and vein density, a greater stomatal length, a thicker lower leaf epidermis, higher embolism resistance, higher wood density, a greater Huber value, a greater ratio of fiber wall thickness to xylem lumen diameter, a larger proportion of total fiber wall area to xylem cross-sectional area, a lower water potential at the turgor loss point (TLP), a smaller relative water content at the TLP, a lower osmotic potential at full turgor and a smaller specific leaf area. It is concluded that a continuum of coordination and trade-offs among co-evolved anatomical and physiological traits gives rise to the spectrum from isohydry to anisohydry spanned by the 20 tree species, and the anisohydric species showed stronger stress resistance, with greater investment in stems and leaves than the isohydric species to maintain stomatal opening under drought conditions.

Osmotic effects of NaCl on cell hydraulic conductivity of corn roots.

In this study, whether the effect of salt (NaCl) stress on cell hydraulic conductivity (L(p)) is via osmotic pressure or ion toxicity and whether abscisic acid (ABA) can release the salt adverse effect were tested. Immediate effects of NaCl and ABA on root cortical cell L(p) of maize (Zea mays L.) were detected by measuring changes in half time of water exchange (T(1/2)) and turgor of individual single cells with a cell pressure probe for at least 1 h. The results showed that stepwise additions of NaCl (50 mM) significantly (P < 0.01) reduced the water permeability. One-step addition of 50 mM NaCl even more drastically decreased L(p). ABA was not able to instantaneously reverse the low water permeability induced by the salt stress. Long-term effects of NaCl, mannitol and sorbitol, and ABA on L(p) were measured for 6 days. Both NaCl and a mixture of mannitol and sorbitol, with the same osmotic strength of 0.25 MPa, significantly reduced L(p) at the early stage of the treatments. The declined L(p) in the salinized cell gradually and partially recovered after 2 days, whereas the L(p) with the mannitol and sorbitol mixture treatment was all time inhibited. With long-time treatment, ABA (500 nM) significantly (P < 0.01) increased turgor and L(p) of the NaCl-treated cells. In general, NaCl reduced water permeability of corn root cortical cells most likely by an osmotic stress. ABA could not instantaneously change water permeability of the corn root cortical cell subjected to NaCl stress; however, with long-time treatment, ABA was able to in part relieve the salt stress likely by osmotic adjustment.

Frost fatigue and its spring recovery of xylem conduits in ring-porous, diffuse-porous, and coniferous species in situ.

Frost-induced embolism and frost fatigue are two major aspects of frost damage to xylem water transport in trees. In this study, three species of each ring-porous, diffuse-porous, and coniferous trees growing in situ were used to explore their differences in winter embolism and frost fatigue. Changes in predawn water potential, predawn native embolism, maximal specific conductivity (Kmax), and cavitation resistance (P50, xylem water potential at 50% loss of conductivity) of current-year branches were measured from autumn to spring. Maximum native embolism of late winter was near 100% for ring-porous species, approximately 80% for diffuse-porous species, and below 50% for conifers. In early spring, there was no significant reduction of native embolism until formation of new vessels in ring-porous trees, while diffuse-porous trees and conifers exhibited a reduction in native embolism before development of new xylem. There was a significant decrease in P50 of ring- and diffuse-porous species over winter; however, in May P50 was markedly reduced along with formation of new vessels. Kmax of ring- and diffuse-porous species significantly decreased from autumn to late winter. The results revealed that vulnerability to cavitation and frost fatigue was related to conduit diameter. The strategies for coping with winter embolism differed among the three wood types: in ring-porous species there was no active embolism refilling; in diffuse-porous species there was refilling associated with positive xylem pressure; and in conifers there was refilling without positive xylem pressure. New vessels could completely restore stem hydraulic conductivity but only partially restore xylem cavitation resistance in spring.

Tradeoff between storage capacity and embolism resistance in the xylem of temperate broadleaf tree species.

Xylem traits are critical plant functional traits associated with water transport, mechanical support, and carbohydrate and water storage. Studies on the xylem hydraulic efficiency-safety tradeoff are numerous; however, the storage function of xylem parenchyma is rarely considered. The effects of a substantial number of xylem traits on water transport, embolism resistance, mechanical support, storage capacity and nonstructural carbohydrate (NSC) content were investigated in 19 temperate broadleaf species planted in an arid limestone habitat in northern China. There was no xylem hydraulic efficiency-safety tradeoff in the 19 broadleaf species. The total parenchyma fraction was negatively correlated with the fiber fraction. Embolism resistance was positively correlated with indicators of xylem mechanical strength such as vessel wall reinforcement, vessel wall thickness and fiber wall thickness, and was negatively related to the axial parenchyma fraction, especially the paratracheal parenchyma fraction. The paratracheal parenchyma fraction was positively correlated with the ratio of the paratracheal parenchyma fraction to the vessel fraction. In addition, the xylem NSC concentration was positively related to the total parenchyma fraction and axial parenchyma fraction. There was a storage capacity-embolism resistance tradeoff in the xylem of 19 broadleaf species in arid limestone habitats. We speculate that the temperate broadleaf species may show a spectrum of xylem hydraulic strategies, from the embolism resistance strategy related to a more negative P50 (the water potential corresponding to 50% loss of xylem conductivity) to the embolization repair strategy based on more paratracheal parenchyma.

Interaction of stomatal behaviour and vulnerability to xylem cavitation determines the drought response of three temperate tree species.

How the mortality and growth of tree species vary with the iso-anisohydric continuum and xylem vulnerability is still being debated. We conducted a precipitation reduction experiment to create a mild drought condition in a forest in the Baotianman Mountains, China, a sub-humid region. Three main sub-canopy tree species in this region were examined. After rainfall reduction, Lindera obtusiloba showed severe dieback, but two other co-occurring species did not show dieback. The water potential at stomatal closure of Dendrobenthamia japonica, L. obtusiloba and Sorbus alnifolia was -1.70, -2.54 and -3.41 MPa, respectively, whereas the water potential at 88 % loss in hydraulic conductivity of the three species was -2.31, -2.11 and -7.01 MPa, respectively. Taken together, near-anisohydric L. obtusiloba with vulnerable xylem was highly susceptible to drought dieback. Anisohydric S. alnifolia had the most negative minimum water potential, and its xylem was the most resistant to cavitation. Isohydric D. japonica conserved water by rapidly closing its stomata. Ultimately, the hydraulic safety margin (HSM) of L. obtusiloba was the smallest among the three species, especially in precipitation-reduced plots. In terms of the stomatal safety margin (SSM), L. obtusiloba was negative, while S. alnifolia and D. japonica were positive. Of the two species without dieback, rainfall reduction decreased growth of D. japonica, but did not influence growth of S. Alnifolia; meanwhile, rainfall reduction led to a decrease of non-structural carbohydrates (NSCs) in D. japonica, but an increase in S. alnifolia. It is concluded that HSM as well as SSM allow interpreting the sensitivity of the three sub-canopy species to drought. The drought-induced dieback of L. obtusiloba is determined by the interaction of stomatal behaviour and xylem vulnerability, and the species could be sensitive to climate change-caused drought although still in sub-humid areas. The isohydric/anisohydric degree is associated with NSCs status and growth of plants.

Prediction of temperate broadleaf tree species mortality in arid limestone habitats with stomatal safety margins.

A growing body of evidence highlights the occurrence of increased widespread tree mortality during climate change-associated severe droughts; however, in situ long-term drought experiments with multispecies communities for the prediction of tree mortality and exploration of related mechanisms are rather limited in natural environments. We conducted a 7-year afforestation trial with 20 drought-resistant broadleaf tree species in an arid limestone habitat in northern China, where the species displayed a broad range of survival rates. The stomatal and xylem hydraulic traits of all the species were measured. We found that species' stomatal closure points were strongly related to their xylem embolism resistance and xylem minimum water potential but not to their survival rates. Hydraulic failure of the vascular system appeared to be the main cause of tree mortality, and the stomatal safety margin was a better predictor of tree mortality than the traditionally considered xylem embolism resistance and hydraulic safety margin. We recommend the stomatal safety margin as the indicator for predicting drought-induced tree mortality and for selecting tree species in future forest restorations in arid regions.

Relative contributions of hydraulic dysfunction and carbohydrate depletion during tree mortality caused by drought.

Drought-induced tree mortality has been observed worldwide. Nevertheless, the physiological mechanisms underlying this phenomenon are still being debated. Potted Robinia pseudoacacia and Platycladus orientalis saplings were subjected to drought and their hydraulic failure and carbon starvation responses were studied. They underwent simulated fast drought (FD) and slow drought (SD) until death. The dynamics of their growth, photosynthesis, water relations and carbohydrate concentration were measured. The results showed that during drought, growth and photosynthesis of all saplings were significantly reduced in both species. The predawn water potential in both species was ~ -8 MPa at mortality. The percentage loss of conductivity (PLC) was at a maximum at mortality under both FD and SD. For R. pseudoacacia and P. orientalis, they were >95 and ~45 %, respectively. At complete defoliation, the PLC of R. pseudoacacia was ~90 % but the trees continued to survive for around 46 days. The non-structural carbohydrate (NSC) concentrations in the stems and roots of both FD and SD R. pseudoacacia declined to a very low level near death. In contrast, the NSC concentrations in the needles, stems and roots of P. orientalis at mortality under FD did not significantly differ from those of the control, whereas the NSC concentrations in SD P. orientalis stems and roots at death were significantly lower than those of the control. These results suggest that the duration of the drought affected NSC at mortality in P. orientalis. In addition, the differences in NSC between FD and SD P. orientalis did not alter mortality thresholds associated with hydraulic failure. The drought-induced death of R. pseudoacacia occurred at 95 % PLC for both FD and SD, indicating that hydraulic failure played an important role in mortality. Nevertheless, the consistent decline in NSC in R. pseudoacacia saplings following drought-induced defoliation may have also contributed to its mortality.

[Application of pressure probe techniques in studies of plant water relations].

This review introduces the pressure probe technique that was originally designed to detect the turgor of a giant algal cell, then adapted to measure the turgor and other water-relations parameters of higher plants, and now has developed into a diverse tool on researches of plant physiology and eco-physiology. This technique can be used to measure in situ the permeability of cell membranes to water and solutes at the resolution of single cells, and hence is a useful tool to study function and regulation of water channels (aquaporins) of intact plant cells. The recently developed xylem-pressure probe technique is the only way to directly measure the negative pressure in xylem conduits. In this review we introduce the basic principles and the theoretical backgrounds underlying the pressure probe. Finally some important achievements and applications of the pressure probe in studies of plant water relations are reviewed and discussed.

Phenology-dependent variation in the non-structural carbohydrates of broadleaf evergreen species plays an important role in determining tolerance to defoliation (or herbivory).

Two broadleaf evergreen canopy species (Schima superba and Engelhardia roxburghiana) with different phenologies in a subtropical region of southern China were used to determine the influence of leaf phenology on the impact of an insect pest attack. S. superba regenerates its leaves in February, while E. roxburghiana regenerates its leaves in May. The moth Thalassodes quadraria attacked the two broadleaf evergreen species in March to April, and the newly produced leaves were removed for S. superba but not for E. roxburghiana. The young trees were artificially defoliated to imitate an insect pest attack during March 2014. Nonstructural carbohydrate (NSC) and growth measurements and a retrospective analysis based on the radial growth of mature trees were conducted in January 2015. The results showed that NSC concentrations decreased in S. superba during canopy rebuilding, and the subsequent defoliation severely inhibited leaf and shoot growth, prevented NSC restoration in roots and stem xylem, and caused high mortality. The insect outbreaks reduced the radial growth of S. superba. In contrast, E. roxburghiana experienced less growth retardation, lower mortality, and normal radial growth. Thus, taking phenology-dependent variation in NSCs into consideration, defoliation and insect pest outbreaks more negatively impacted S. superba than E. roxburghiana.

Genome-Wide Constitutively Expressed Gene Analysis and New Reference Gene Selection Based on Transcriptome Data: A Case Study from Poplar/Canker Disease Interaction.

A number of transcriptome datasets for differential expression (DE) genes have been widely used for understanding organismal biology, but these datasets also contain untapped information that can be used to develop more precise analytical tools. With the use of transcriptome data generated from poplar/canker disease interaction system, we describe a methodology to identify candidate reference genes from high-throughput sequencing data. This methodology will improve the accuracy of RT-qPCR and will lead to better standards for the normalization of expression data. Expression stability analysis from xylem and phloem of Populus bejingensis inoculated with the fungal canker pathogen Botryosphaeria dothidea revealed that 729 poplar transcripts (1.11%) were stably expressed, at a threshold level of coefficient of variance (CV) of FPKM < 20% and maximum fold change (MFC) of FPKM < 2.0. Expression stability and bioinformatics analysis suggested that commonly used house-keeping (HK) genes were not the most appropriate internal controls: 70 of the 72 commonly used HK genes were not stably expressed, 45 of the 72 produced multiple isoform transcripts, and some of their reported primers produced unspecific amplicons in PCR amplification. RT-qPCR analysis to compare and evaluate the expression stability of 10 commonly used poplar HK genes and 20 of the 729 newly-identified stably expressed transcripts showed that some of the newly-identified genes (such as SSU_S8e, LSU_L5e, and 20S_PSU) had higher stability ranking than most of commonly used HK genes. Based on these results, we recommend a pipeline for deriving reference genes from transcriptome data. An appropriate candidate gene should have a unique transcript, constitutive expression, CV value of expression < 20% (or possibly 30%) and MFC value of expression <2, and an expression level of 50-1,000 units. Lastly, when four of the newly identified HK genes were used in the normalization of expression data for 20 differential expressed genes, expression analysis gave similar values to Cufflinks output. The methods described here provide an alternative pathway for the normalization of transcriptome data, a process that is essential for integrating analyses of transcriptome data across environments, laboratories, sequencing platforms, and species.

Signals controlling root suckering and adventitious shoot formation in aspen (Populus tremuloides).

We determined the effects of removal of leaves, stem axillary buds, or the entire shoot on root suckering (adventitious shoot formation by roots) and basal stem sprouts in 3- and 4-year-old potted seedlings of aspen (Populus tremuloides Michx.). The greatest number of root suckers (67.9 +/- 8.5 per plant) emerged after excision of the entire shoot. Defoliated and debudded stems were the major source of inhibitory agents for root suckering, although axillary buds and developing new leaves also exerted a significant inhibitory effect. Removal of mature leaves had only a minor effect on root suckering. Removal of a continuous band of bark (girdling) at the base of the stem consistently stimulated growth of adventitious shoots from the stem below the girdle and occasionally promoted root suckering. Exogenous application of indole-3-acetic acid to excised stumps inhibited root suckering and basal stem sprouting. Naphthylphthalamic acid (NPA), an auxin polar transport inhibitor, had no effect on root suckering or stem sprouting when it was applied to the bark of the basal stem. However, NPA significantly increased root suckering when it was applied to the exposed surface of xylem after girdling. These results suggest that polar transport of auxin in the xylem parenchyma is an important inhibitor of root suckering. On decapitated stems, vacuum extraction of xylem sap from the root system lowered the frequency of root suckering compared with decapitation alone, indicating that substance(s) originating in the root system also play a significant role in controlling root suckering.

Leaf economics of evergreen and deciduous tree species along an elevational gradient in a subtropical mountain.

The ecophysiological mechanisms underlying the pattern of bimodal elevational distribution of evergreen tree species remain incompletely understood. Here we used leaf economics spectrum (LES) theory to explain such patterns. We measured leaf economic traits and constructed an LES for the co-existing 19 evergreen and 15 deciduous species growing in evergreen broad-leaved forest at low elevation, beech-mixed forest at middle elevation and hemlock-mixed forest at high elevation in Mao'er Mountain, Guangxi, Southern China (25°50'N, 110°49'E). Leaf economic traits presented low but significant phylogenetic signal, suggesting trait similarity between closely related species. After considering the effects of phylogenetic history, deciduous species in general showed a more acquisitive leaf strategy with a higher ratio of leaf water to dry mass, higher leaf nitrogen and phosphorous contents, higher photosynthetic and respiratory rates and greater photosynthetic nitrogen-use efficiency. In contrast, evergreen species exhibited a more conservative leaf strategy with higher leaf mass per area, greater construction costs and longer leaf life span. With the elevation-induced decreases of temperature and soil fertility, both evergreen and deciduous species showed greater resource conservation, suggesting the increasing importance of environmental filtering to community assembly with increasing elevation. We found close inter-specific correlations between leaf economic traits, suggesting that there are strong genetic constraints limiting the independent evolution of LES traits. Phylogenetic signal increased with decreasing evolutionary rate across leaf economic traits, suggesting that genetic constraints are important for the process of trait evolution. We found a significantly positive relationship between primary axis species score (PASS) distance and phylogenetic distance across species pairs and an increasing average PASS distance between evergreen and deciduous species with increasing elevation, implying that the frequency of distantly related evergreen and deciduous pairs with wide spreading of leaf economic values increases with increasing elevation. Our findings thus suggest that elevation acts as an environmental filter to both select the locally adapted evergreen and deciduous species with sufficient phylogenetic variation and regulate their distribution along the elevational gradient based on their coordinated spreading of phylogenetic divergence and leaf economic variation.