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Phosphorus is an essential macronutrient for plant growth and development. Plants can respond defensively to phosphorus deficiency by modifying their morphology and metabolic pathways via the differential expression of low phosphate responsive genes. To better understand the mechanisms by which the Masson pine (Pinus massoniana) adapts to phosphorus deficiency, we conducted comparative proteomic analysis using an elite line exhibiting high tolerance to phosphorus deficiency. The selected seedlings were treated with 0.5 mM KH2PO4 (control), 0.01 mM KH2PO4 (P1), or 0.06 mM KH2PO4 (P2) for 48 days. Total protein samples were separated via 2DE. A total of 98 differentially expressed proteins, which displayed at least 1.7-fold change expression compared to the control levels (p ≤ 0.05), were identified by MALDI-TOF/TOF MS. These phosphate starvation responsive proteins were implicated in photosynthesis, defense, cellular organization, biosynthesis, energy metabolism, secondary metabolism, signal transduction etc. Therefore, these proteins might play important roles in facilitating internal phosphorus homeostasis. Additionally, the obtained data may be useful for the further characterization of gene function and may provide a foundation for a more comprehensive understanding of the adaptations of the Masson pine to phosphorus-deficient conditions., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Phosphorus (P) is an essential macronutrient for plant growth and development. Several genes involved in phosphorus deficiency stress have been identified in various plant species. However, a whole genome understanding of the molecular mechanisms involved in plant adaptations to low P remains elusive, and there is particularly little information on the genetic basis of these acclimations in coniferous trees. Masson pine (Pinus massoniana) is grown mainly in the tropical and subtropical regions in China, many of which are severely lacking in inorganic phosphate (Pi). In previous work, we described an elite P. massoniana genotype demonstrating a high tolerance to Pi-deficiency., Methodology/principal Findings: To further investigate the mechanism of tolerance to low P, RNA-seq was performed to give an idea of extent of expression from the two mixed libraries, and microarray whose probes were designed based on the unigenes obtained from RNA-seq was used to elucidate the global gene expression profiles for the long-term phosphorus starvation. A total of 70,896 unigenes with lengths ranging from 201 to 20,490 bp were assembled from 112,108,862 high quality reads derived from RNA-Seq libraries. We identified 1,396 and 943 transcripts that were differentially regulated (P<0.05) under P1 (0.01 mM P) and P2 (0.06 mM P) Pi-deficiency conditions, respectively. Numerous transcripts were consistently differentially regulated under Pi deficiency stress, many of which were also up- or down-regulated in other species under the corresponding conditions, and are therefore ideal candidates for monitoring the P status of plants. The results also demonstrated the impact of different Pi starvation levels on global gene expression in Masson pine., Conclusions/significance: To our knowledge, this work provides the first insight into the molecular mechanisms involved in acclimation to long-term Pi starvation and different Pi availability levels in coniferous trees.

Studies on the dynamics of non-structural carbohydrates (NSCs) play an important role in understanding the mechanisms of plant responses to drought stress. The objective of this study was to assess the influence of ectomycorrhizal fungi (ECMF) on the content and distribution of NSCs in Pinus massoniana seedlings under different drought intensities and to further explore the possible mechanism by which ECMF enhances the stress resistance of host plants. We conducted a pot experiment using P. massoniana seedlings that were inoculated (M) or non-inoculated (NM) with Suillus luteus (Sl) under well-watered, moderate, and severe drought stress conditions. The results showed that drought significantly reduced the photosynthetic capacity of P. massoniana seedlings and inhibited their growth rate. P. massoniana could respond to different degrees of drought stress by increasing the accumulation of NSCs and increasing WUE. However, compared with well-watered treatment, NSCs consumption began to appear in the roots of NM due to the decrease in starch content under severe drought, whereas NSCs content in M seedlings was higher than that in the well-watered treatment, showing that the ability to maintain C balance was higher in M seedlings. Compared with NM, inoculation with Sl increased the growth rate and biomass of roots, stems, and leaves under moderate and severe drought. In addition, Sl can also improve the gas exchange parameters (net photosynthetic rate, transpiration rate, intercellular CO2 concentration and stomatal conductance) of P. massoniana seedlings compared with NM seedlings, which was conducive to the hydraulic regulation of seedlings and improved their C fixation capacity. Meanwhile, the content of NSCs in M seedlings was higher. Moreover, the soluble sugar content and SS/St ratio of leaves, roots, and whole plants were higher under drought stress after Sl inoculation, indicating that Sl could also change the C distribution mode, regulate more soluble sugar to respond to drought stress, which was conducive to improving the osmotic adjustment ability of seedlings, and providing more available C sources for plant growth and defense. Overall, inoculation with Sl could enhance the drought resistance of seedlings and promote their growth under drought stress by improving NSCs storage, increasing soluble sugar distribution, and improving the plant water balance of P. massoniana seedlings. [ABSTRACT FROM AUTHOR]

Aims: Pinus massoniana Lamb. is a widely distributed native species and afforestation pioneer tree in the subtropical forests of China; however, the species' quality and productivity are significantly affected by phosphorus (P) deficiency. Although ectomycorrhizal (ECM) fungi can enhance plant adaptation to nutrient deficiency, the interactive effects of ECM fungi colonization and P availability on physiological responses and nutrient uptake in P. massoniana seedlings remain unknown. Method: We compared the physiological response and nutrient absorption of uninoculated P. massoniana seedlings and seedlings inoculated with Suillus luteus or S. bovinus under three P supplies (P deficiency, 0.01 mM/L; medium P, 0.1 mM/L; and P sufficiency, 1 mM/L) in sand culture. Results: P deficiency limited the seedlings growth and nutrient content but increased the antioxidant enzyme activity. S. luteus and S. bovinus significantly increased the seedlings growth under P deficiency. The activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) as well as the contents of malondialdehyde (MDA) and proline of inoculated and uninoculated seedlings were significantly higher under P deficiency than P sufficiency. Under P deficiency, POD and SOD activities increased with time and were higher in inoculated seedlings; however, proline contents were lower in inoculated seedlings. P deficiency decreased the P and potassium (K) contents in the roots, stems, and leaves of seedlings. Moreover, under P deficiency, S. luteus significantly improved P content in stems and leaves, whereas S. bovinus significantly improved K content in leaves. Conclusion: ECM fungi increased SOD and POD activities and improved P and K absorption in P. massoniana seedlings in response to P deficiency. [ABSTRACT FROM AUTHOR]

A reasonable water and fertilizer supply is key to coordinating nursery stock quality improvement and environmental pollution reduction. Here, the experiment was carried out based on the combinations of four levels of water content (W), substrate available nitrogen (N), phosphorus (P), and potassium (K). The aim was to explore the combining effects of water and fertilizer on the comprehensive growth of Pinus massoniana seedlings, and to obtain the coupling optimization of water-fertilizer. The results indicate that the comprehensive growth index (CGI) of seedlings followed a linear pattern with P and a downward-opening parabola with W, N, and K. The seedling growth was not significantly affected by adjusting the input of other elements when the substrate P ≤ 32.00 mg/kg·dry substrate. Whereas when P was 128.00 mg/kg·dry substrate, the negative effects caused by low water (W was below 45%) could not be alleviated, even if moderate N and K levels were maintained. W and P had direct and significant positive effects on the growth of seedlings, while N and K had negative effects. The combination of 85–91% (W), 65.95–134.27 (N), 128.00 (P), and 215.87–234.00 mg/kg·dry substrate (K) was beneficial to seedling growth. While W of 91% combined with the N, P, and K of 57.39, 88.40, and 123.08 mg/kg·dry substrate coordinated the environmental development and seedling growth. This research showed that P is the key factor affecting the CGI of P. massoniana seedlings. which provides a reference for rational and precise fertilization during the cultivation of P. massoniana seedlings. [Display omitted] • A water-fertilizer multi-factor coupling model for the comprehensive growth of Pinus massoniana seedlings was established. • Substrate effective P is the main driving factor affecting P. massoniana seedling comprehensive growth. • The optimal combination of W, N, P, and K was determined to improve the quality of P. massoniana seedlings. • N-K, P-W, P-N, P-K have synergize effect within a reasonable interval on seeding growth, but W-N have antagonistic effect. [ABSTRACT FROM AUTHOR]

Background: Phosphorus (P) is an essential macronutrient for plant growth and development. Several genes involved in phosphorus deficiency stress have been identified in various plant species. However, a whole genome understanding of the molecular mechanisms involved in plant adaptations to low P remains elusive, and there is particularly little information on the genetic basis of these acclimations in coniferous trees. Masson pine (Pinus massoniana) is grown mainly in the tropical and subtropical regions in China, many of which are severely lacking in inorganic phosphate (Pi). In previous work, we described an elite P. massoniana genotype demonstrating a high tolerance to Pi-deficiency. Methodology/Principal Findings: To further investigate the mechanism of tolerance to low P, RNA-seq was performed to give an idea of extent of expression from the two mixed libraries, and microarray whose probes were designed based on the unigenes obtained from RNA-seq was used to elucidate the global gene expression profiles for the long-term phosphorus starvation. A total of 70,896 unigenes with lengths ranging from 201 to 20,490 bp were assembled from 112,108,862 high quality reads derived from RNA-Seq libraries. We identified 1,396 and 943 transcripts that were differentially regulated (P<0.05) under P1 (0.01 mM P) and P2 (0.06 mM P) Pi-deficiency conditions, respectively. Numerous transcripts were consistently differentially regulated under Pi deficiency stress, many of which were also up- or down-regulated in other species under the corresponding conditions, and are therefore ideal candidates for monitoring the P status of plants. The results also demonstrated the impact of different Pi starvation levels on global gene expression in Masson pine. Conclusions/Significance: To our knowledge, this work provides the first insight into the molecular mechanisms involved in acclimation to long-term Pi starvation and different Pi availability levels in coniferous trees. [ABSTRACT FROM AUTHOR]

In recent years, global forests have been facing an increase in tree mortality owing to increasing droughts. However, the capacity for plants to adjust their physiology and biochemistry during extreme drought and subsequent recovery is still unclear. Here, we used 1.5-year-old Pinus massoniana Lamb. seedlings and simulated drought conditions to achieve three target stress levels (50%, 85%, and 100% loss of stem hydraulic conductivity (PLC)), followed by rehydration. Needle water status, gas exchange, and biochemical parameters were assessed during drought and recovery. The results showed that drought had significantly negative impacts on needle water status and gas exchange parameters, with gas exchange declining to 0 after PLC85 was achieved. Soluble protein concentration (SPC), soluble sugar concentration (SSC), malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, and needle water-use efficiency showed fluctuations. The activity of antioxidant enzymes and the values of osmotic regulators were then gradually decreased as the physiological and biochemical functions of seedlings were disturbed. Seedlings showed a stronger ability to recover from PLC50 than PLC85 and PLC100. We conclude that the physiological and biochemical recovery of P. massoniana seedlings is more likely to be inhibited when plants experience increasing drought stress that induces 85% and greater loss of hydraulic conductance. [ABSTRACT FROM AUTHOR]

Brassinosteroids (BRs) are known to be essential regulators for wood formation in herbaceous plants and poplar, but their roles in secondary growth and xylem development are still not well-defined, especially in pines. Here, we treated Pinus massoniana seedlings with different concentrations of exogenous BRs, and assayed the effects on plant growth, xylem development, endogenous phytohormone contents and gene expression within stems. Application of exogenous BR resulted in improving development of xylem more than phloem, and promoting xylem development in a dosage-dependent manner in a certain concentration rage. Endogenous hormone determination showed that BR may interact with other phytohormones in regulating xylem development. RNA-seq analysis revealed that some conventional phenylpropanoid biosynthesis- or lignin synthesis-related genes were downregulated, but the lignin content was elevated, suggesting that new lignin synthesis pathways or other cell wall components should be activated by BR treatment in P. massoniana. The results presented here reveal the foundational role of BRs in regulating plant secondary growth, and provide the basis for understanding molecular mechanisms of xylem development in P. massoniana. [ABSTRACT FROM AUTHOR]

Light intensity is a critical factor regulating photosynthetic capacity in plants. However, the effects of varying light intensity on morphological and photoprotective mechanisms in Pinus massoniana seedlings have not been explored in depth, especially those in the first seedling growing season. We measured the growth, photosynthetic physiology, biochemistry, and chlorophyll fluorescence of P. massoniana seedlings at four light gradients: 100% relative irradiance (RI, full sunlight), 70% RI, 50% RI, and 20% RI. The seedling height at 70% RI was 9.27% higher than that at 100% RI. However, seedling height was inhibited under low light intensity; at 20% RI, all seedlings died. The decreasing light intensity inhibited ground diameter growth but increased the height-diameter ratio. The secondary needle emergence rate was 53.4% higher at 70% RI than at 100% RI but was only 2% at 50% RI. The chlorophyll and carotenoid contents increased significantly with decreasing light intensity. The increased Chl b and Car contents promoted the photoreceptor potential of the violet (400~420 nm), blue (440~480 nm), and yellow-orange (597~655 nm) regions in leaves. Among the chlorophyll fluorescence parameters, Fv/Fm, Fv′/Fm′, Y(II), qp, and ETR all reached maximum values at 70% RI but were significantly lower at 50% RI than at 100% RI. However, decreasing the light intensity caused a reduction in NPQ. The 70% RI level increased POD and SOD activity and the contents of osmotic regulation substances and slowed MDA accumulation. Seedlings at 70% RI had a higher growth rate, higher photosynthetic activity and potential, and significantly greater stress resistance than the other seedlings. Therefore, appropriate shading measures were beneficial to the cultivation of vigorous seedlings. Furthermore, spectral reflectance indexes were found to be a suitable tool for monitoring the photosynthetic physiological characteristics, stress resistance characteristics, and growth status of P. massoniana seedlings in real time. [ABSTRACT FROM AUTHOR]

Masson pine is an important afforestation species in southern China, where seasonal drought is common. The present study focused on the effects of Suillus placidus, an ectomycorrhizal fungus, inoculation on the growth and physiological and biochemical performance of masson pine seedlings under four different watering treatments (well-watered, mild drought, moderate drought, and severe drought) to evaluate the symbiotic relationship between S. placidus and masson pine seedlings. Ectomycorrhizal-inoculated (ECM) and non-inoculated (NM) seedlings were grown in pots and maintained for 60 days using the weighing method. Results showed that seedlings' growth, dry weight, RWC, chlorophyll content, PSII efficiency, and photosynthesis decreased as drought stress intensified in both ECM and NM plants. This suggests that drought stress significantly limits the growth and photosynthetic performance of masson pine seedlings. Nevertheless, increased An/gs and proline contents in both NM and ECM prevented oxidative damage caused by drought stress. In addition, increased peroxidase (POD) activity is an essential defense mechanism of ECM seedling under drought stress. Compared with NM, ECM seedlings showed faster growth, higher RWC, and photosynthetic performance, and lower lipid peroxidation in cell membranes under drought stress, as indicated by higher POD activity and lower proline and malondialdehyde (MDA). Our experiment found that S. placidus inoculation can enhance the drought resistance of masson pine seedlings by increasing antioxidant enzyme activity, water use efficiency, and proline content, thereby enhancing growth under water-deficiency conditions. S. placidus can be used to cultivate high-quality seedlings and improve their survival in regions that experience seasonal droughts. [ABSTRACT FROM AUTHOR]

Pine seedlings exhibit heteroblastic foliage (primary and secondary needles) during seedling development. However, few trials have studied how heteroblastic foliage influences pine seedling growth by seasonal variation. This study first investigated the anatomical differences between the primary and secondary needles of one-year-old Pinus massoniana seedlings. We measured chlorophyll fluorescence (ChlF) and evaluated the photoprotective mechanisms and light energy partitioning of these heteroblastic leaves from September to November. The results showed that the primary needles, as juvenile foliage, had a greater fraction of mesophyll tissue and stomata. In addition, the primary needles had two vascular bundles, and shorter distance from xylem and phloem to mesophyll cells, exhibiting a luxury growth strategy of rapidly obtaining high returns. The ChlF parameters indicated that the primary needles maintained a relatively high level of photoprotection by thermal dissipation (nonphotochemical quenching (NPQ)) and nonregulated energy dissipation (Y(NO)). The secondary needles, representing mature foliage, had greater area of xylem and phloem tissues. The contents of Chl b and carotenoids (Car) significantly increased in November, promoting φPo and photoprotection, which suggested that the secondary needles were more resistant to low temperatures. During the whole light response process of secondary needles, the increases in the electron transfer rate (ETR) and light energy utilization efficiency (α) helped to increase the actual photosynthetic quantum yield (Y(II)) by reducing energy dissipation by decreasing the proportion of regulated energy dissipation (Y(NPQ)) and Y(NO). Given the sensitivity of this heteroblastic foliage to environmental changes, the practical use and extension of P. massoniana for afforestation purposes should be carried out with caution. • The morphological and photosynthesis-related physiological functions of primary and secondary needles differ significantly. • The primary needles were vulnerable to damage and photosystem II inactivity from intensive light. • The photosynthetic system of secondary needles was relatively stable. • The secondary needles developed greater support and durability tissues, which is more conducive to the growth of seedling. [ABSTRACT FROM AUTHOR]

Gibberellins (GAs) play a crucial role in regulating secondary growth in angiosperms, but their effects on the secondary growth of gymnosperms are rarely reported. In this study, we administered exogenous GA 3 to two-year-old P. massoniana seedlings, and examined its effects on anatomical structure, physiological and biochemical changes, and gene expression in stems. The results showed that exogenous GA 3 could enhance xylem development in P. massoniana by promoting cell division. The content of endogenous hormone (including auxins, brassinosteroids, and gibberellins) were changed and the genes related to phytohormone biosynthesis and signaling pathway, such as GID1 , DELLA , TIR1 , ARF , SAUR , CPD , BR6ox1 , and CYCD3 , were differentially expressed under GA 3 treatment. Furthermore, GA 3 and BR (brassinosteroid) might act synergistically in promoting secondary growth in P. massoniana. Additionally, lignin content was significantly increased after GA 3 treatment accompanied by the express of lignin biosynthesis related genes. PmCAD (TRINITY_DN142116_c0_g1), a crucial gene involved in the lignin biosynthesis, was cloned and overexpressed in Nicotiana benthamiana , significantly promoting the xylem development and enhancing stem lignification. It was regarded as a key candidate gene for improving stem growth of P. massoniana. The findings of this study have demonstrated the impact of GA 3 treatment on secondary growth of stems in P. massoniana , providing a foundation for understanding the molecular regulatory mechanism of stem secondary growth in Pinaceae seedlings and offering theoretical guidance for cultivating new germplasm with enhanced growth and yield. • Exogenous GA 3 enhances xylem development in P. massoniana by promoting cell division. • GA 3 and BR might act synergistically in promoting secondary growth in P. massoniana. • GA 3 changes the expression of lignin biosynthesis related genes and promotes lignin synthesis to regulate the secondary growth of P. massoniana. • PmCAD was regarded as a key candidate gene for improving secondary growth of P. massoniana. [ABSTRACT FROM AUTHOR]