Clonal variation in growth, PSII photochemical activity and polar metabolites in Pinus elliottii × P.caribaea

Wenbing Guo · Fencheng Zhao · Yang Liu · Zhen Li · Huishan Wu · Yiliang Li · Fangyan Liao

Abstract Pinus elliottii × P.caribaea is a widely planted commercial tree hybrid in South China.To investigate the potential of physiological parameters for clonal selection, cuttings of three clones (EH3, EH4 and EH5) and a control were grown in phosphorus treated pots.Significant variations to phosphorous, photosystem II activity and polar metabolite abundance in needles were noted among clones.Clone EH5 was the most P-responsive and had maximum height growth.The highest maximum quantum yield of PSII in the dark (Fv/Fm), maximum quantum yield of PSII in the light and the actual PSII efficiency (ΦPSII) values were also found in EH5.A total of 64 polar metabolites were identified, 21 were regulated by phosphorous, while 28 accumulated differentially among the three clones.There were few overlapping responsive metabolites to phosphorous fertilization.In EH5, the abundance of maltotriitol, neohesperidin and raffinose were highest, whereas galactinol and shikimic acid were lower compared with EH3 and EH4.The results reveal that physiological traits were genetically controlled and might be useful for selecting clones with high phosphorous utilization efficiency.

Keywords Carbohydrates · Chlorophyll fluorescence parameters · Metabolic profiling · Phosphorus fertilization · Physiological traits

Introduction

The hybrid betweenPinus elliottiiEngelm.andP.caribaeaMorelet is one of the most important commercial forest species in the world (Dieters et al.2007).In China, rooted cuttings from superior families have been used for afforestation and the plantations are regularly used as a main source of oleoresin.However, information on the nutrient demand of the hybrid in China is scarce.Previous studies on the parent species indicated that phosphorous (P) is the limiting factor for growth.A pot culture experiment withP.caribaeavar.hondurensis(Sénécl) Barrett & Golfari showed that P rather than nitrogen (N) and potassium (K) was benefciial for height growth (Srivastava et al.1979).Phosphorous application to one-year-oldP.elliottiisignificantly increased height and diameter (Pritchett et al.1966).Volume differences of two to three times have been reported following P or a combination of P and N fertilization on typical soils in the southeastern United States (Jokela 2004).Similar results were observed in a plantation ofP.elliottiiin subtropical Australia (Xu et al.1995).Therefore, it was necessary to determine whether phosphate fertilizers also have a positive effect on hybrid pine productivity.

Nutrient demands differ across different genotypes and the ones producing higher yields per unit of nutrients have been identified for numerous crop species (Fageria et al.2008).With tree species, research has focused on genotypic variations in growth response to fertilization.InPseudotsuga menziesii(Mirbel) Franco seedlings, N uptake and utilization contributed to higher growth rates in high-ranking families (Hawkins 2007).InP.elliottii, family variations were reported for N, P, K and Ca demands and improved families had higher nutrient requirements than unimproved ones (Xiao et al.2003).InPinus taedaL., some varieties were more responsive to intensive silviculture than other varieties (Yáñez et al.2015).Hence, matching high nutrientdemanding genotypes to intensive silvicultural operations may be necessary to increase stand yields (Yáñez et al.2017).Instead of screening hundreds of clones at sites, using physiological traits to select high nutrient-demanding genotypes is a better and less time-consuming choice.An ideal physiological trait is not only related to nutrient demand but it is also genetically controlled.

Chlorophyll fluorescence is a non-invasive physiological parameter to measure photosystem II (PSII) activity (Murchie and Lawson 2013).Since PSII operating efficiency is related to CO2assimilation, chlorophyll fluorescence measurements could be used to monitor photosynthetic performance (Seaton et al.1990; Baker 2008).It has been mainly used to monitor environmental effects, including nutrient supply and deficiency (Kalaji et al.2014, 2018; Mauromicale et al.2006).In most crop species, P deficiency causes the reduction of photochemical efficiency of PSII.InPinus pinasterAit.seedlings, maximum photochemical efficiency of PSII (estimated fromFv/Fm, a normalized ratio determined by dividing the variable florescence by the maximum florescence) was significantly reduced due to low P concentrations (Loustau et al.1999).However, in someEucalyptusspecies (e.g.,E.globulusLabill.andE.microcorysF.Muell.), theFv/Fmand effective quantum yield of PSII (ΦPSII) was reduced significantly in seedlings supplied with phosphorous (Bulgarelli et al.2019).Previous studies have indicated that chlorophyll fluorescence parameters are genetically controlled.Chlorophyll fluorescence analysis inP.elliottiifamilies at the seedling stage indicated that photochemical quenching ( qp) and yield of PSII were influenced by genetics, whereas non-photochemical quenching ( qN) was more strongly influenced by environmental conditions (Koehn et al.2003).Population and family effects were significant for photosystem II activity inP.pinasterand interior and coastal populations could be distinguished using theFv/Fmratio in current year needles (Corcuera et al.2011).The estimated clonal repeatability for needleFv/Fmamong 40Pinus radiataD.Don clones was 0.2, similar to that of the growth traits.Fv/Fmwas positively correlated to stem volume and annual increments of height and DBH for unimproved clones of 4-year-old radiata pine on serpentine soil, while for improve clones, it was positively correlated only to increments of height (Xue et al.2013).These results indicate the potential use of chlorophyll fluorescence traits, includingFv/Fm,in genotype discrimination.

Since chlorophyll fluorescence quenching is related to carbon metabolism (Maxwell et al.2000; Baker 2008), changes in chlorophyll fluorescence may lead to metabolic changes.Plant metabolism is basis of many important biological processes, including development (Diaz et al.2005; Fait et al.2006; Lombardo et al.2011) and in response to nutrients (Nikiforova et al.2005; Okazaki et al.2008; Luo et al.2019).The environment strongly influences metabolism.For example, plants coping with P limitation, the synthesis of sulfolipids can be increased, the production of amino acids and organic acids can be intensified and alternative types of carbon metabolism can be activated (Péret et al.2011; Pant et al.2015; Esfahani et al.2016; Ziegler et al.2016).Recent studies inPopulus cathayanaRehder revealed that P deprivation caused changes to phospholipids and phosphorylated metabolites, (e.g.fructose-6-phosphate, glycerol-3-phosphate, glucose-6-phosphate, phosphoric acid and inositol-1-phosphate), in roots and leaves and the changing metabolites, especially in amino acid metabolism, revealed the differences of nutrient utilizing strategies in male and female woody plants (Zhang et al.2019).Temperate beech also has a set of seasonal survival strategies for P-deficient areas.In autumn, phospholipids, glucosamine-6-phosphate (GlcN6P) or N-acetyl-D-glucosamine-6-phosphate (GlcNAc6P) are accumulated in the bark or wood for phosphorous storage, while in summer, their leaves use galactolipids and sulfolipids to replace phospholipids (Netzer et al.2018).It was indicated that characteristics of primary and secondary metabolites were associated with the nutrient status of the plants.

There are also numerous metabolic features genetically controlled and showing remarkable potential in genotype discrimination (Soltis et al.2015).InCitrus spp.,using untargeted liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry metabolite profiling can assist to discriminate plant genotypes (Arbona et al.2009).Metabolite profiling of wild-typePopulus tremula×albaand two transgenic lines indicated that metabolic traits could be used to select genotypes differentiated in wood traits (Robinson et al.2005).InPinus, monoterpene emissions from phloem samples could be used to distinguish four species (Santos et al.2006).InP.halepensisMill., certain terpenoids were found to differentially accumulate between the oleoresin of high-and low-yielding populations (Karanikas et al.2010).Metabolite features varied in different genotypes.

In this study, we hypothesized that chlorophyll fluorescence and metabolic profiles followed a specific pattern in high nutrient-demand or high nutrient-utilization efficiency genotypes.Three clones, propagated from cuttings from different families ofP.elliottii×P.caribaeaF1hybrids were tested.The aims of this study were: (1) to detect any clonal variation in growth, chlorophyll fluorescence and metabolite accumulation as affected by phosphorous fertilization; (2) to evaluate the availability of physiological traits in selecting high nutrient-demand or high nutrient-utilization efficiency genotypes; and, (3) to examine the mechanism underlying genotypic variations in nutrient requirements.

Materials and methods

Plant materials and growing conditions

Three genetically unrelated commercialP.elliottii×P.caribaeaclones (EH3, EH4 and EH5) were used in this study.In May 2011, healthy, half-woody 8-cm shoots were collected from the three clones from the Hedge Garden, immediately planted in a clay subsoil medium and placed in a greenhouse under an intermittent misting system.The cuttings were fertilized with KH2PO4(0.1%) after one month.In September 2011, the rooted cuttings were moved outdoors and fertilized with NPK fertilizer (N30P30K30, 0.1%) monthly.The experiments were conducted at a nursery in Yunfu City, Guangdong Province (112°11ʹE, 22°53ʹN).

Treatments

In March 2012, each rooted cutting was cultivated in a nonwoven bag (40 cm diameter × 50 cm deep) filled with loam subsoil sifted using a 5 mm mesh sieve (see Table S1 for soil nutrient status), at a 1 m × 1 m spacing.For each bag, 250 g fused calcium magnesium phosphate containing 12% P2O5was mixed with the medium one month before planting as the P-fertilizer treatment.The control group did not have any fertilizer applied.The experiment was conducted in a split-plot randomized complete block design with four replications, where fertilization treatments + control were considered as main plots and clones were assigned to subplots.The pot trial was 4 blocks × 2 treatments × 3 clones × 4 ramets=96 individuals.

Growth and measurement of N, P and K

Shoot heights (H) and ground-level diameters (D) were measured on all cuttings after two months (May 2012), nine months (December 2012), 16 months (July 2013) and 25 months (April 2014).

In February 2014, four upper, mature fascicles were collected from one of the four ramets in each treatment to determine N, P and K concentrations.The needles were dried for 72 h at 70 °C, ground to powder and digested in concentrated sulfuric acid, followed by oxidation in hydrogen peroxide.Total N levels were measured using the Kjeldahl auto-analyzer method.Total P was determined using the colorimetric molybdenum blue method and total K determined using inductively coupled plasma emission spectroscopy.

Chlorophyll fluorescence measurements

One ramet of each clone from the four blocks was selected for chlorophyll fluorescence measurement using a leaf chamber fluorometer (6400-40) attached to a LI-6400 portable photosynthetic system (LI-COR Biosciences, Lincoln, NE, USA) in September-October 2014.The fascicles were dark-adapted for 2 h and their minimum fluorescence (Fo) and maximum fluorescence (Fm) following saturating flashes (＞ 7000 μmol photons m-2·s-1) were measured.The fascicles were then acclimated to 1500 μmol photons m-2·s-1of continuous actinic light for 25 min until the absolute value of dF/dt was ＜ 5 and their minimum fluorescence, steady state fluorescence (Fs) and maximum fluorescencewere measured.The maximum quantum yield of PSII in the dark (Fv/Fm) was given by (Fm-Fo)/Fm.The maximum quantum yield of PSII in the lightwas given byʹ.The actual PSII efficiency (ΦPSII) was computed asʹ.

Analysis of polar metabolites by gas chromatography-mass spectrometry

The same four ramets of clones, EH3, EH4 and EH5 used for chlorophyll fluorescence measurements and two other ramets from blocks 1 and 2 were selected for metabolite analysis.A bundle of upper, mature fascicles was collected separately from the clonal ramets.The needle tissue was ground into powder in liquid nitrogen and freeze-dried and a 60-mg aliquot was mixed with 360 μL of pre-cooled methanol and 40 μL internal standards (0.1 mg mL-1ribitol) and incubated at 70 °C for 15 min.After the mixture was centrifuged for 10 min, the supernatant was transferred to a new tube and 200 μL chloroform and 400 μL water added.The mixture was vortexed for 2 min and centrifuged for 15 min.A 400 μL of the upper phase was transferred to a new tube for vacuum-drying at room temperature.For derivatization, 80 μL of methoxyamine (15 mg·mL-1in pyridine) was added to the tube and shaken for 90 min at 37 °C.Finally, 80 μL of BSTFA (1% TMCS) and 20 μL ofn-hexane were added and the tube was shaken for 60 min at 70 °C.The repeatability of the analytical runs was assessed by preparing quality control (QC) samples by mixing equal volumes of extracts from the above 36 samples.

Each 1-μL aliquot of the derivatized sample was injected into a gas chromatograph system using a mass spectrometer (Agilent 7890A-5975C) in the split mode with a split ratio of 30:1.A QC was injected after every nine samples.The injector temperature was 260 °C.Separation was performed using a DB-5 capillary column (30 m × 250 μm I.D.; J&W Scientific, Folsom, CA, USA).High-purity helium was the carrier gas at a constant flow rate of 1.0 mL min-1.The ion source was adjusted to 230 °C.The column temperature was held for 5 min at 70 °C, followed by an 8 °C min-1oven temperature increased to 125 °C, a 15 °C min-1to 170 °C, a 4 °C min-1to 210 °C, a 10 °C min-1to 270 °C and a 5 °C min-1to 305 °C and maintained at 305 °C for 5 min.The column eluent was ionized by electron impact (-70 eV) and the mass spectra were recorded at 20 scans·s-1with an m/z range of 30-600.The raw data were acquired and processed using ChromaTOF software version 4.34 (LECO, St.Joseph, MI, USA).The chromatograms were manually checked after automatic peak deconvolution.The mass spectra were used for identification by matching to the National Institute of Standards and Technology 05 databases, Feinh databases and an in-house constructed database.A total of 351 peaks with signal-to-noise ratio of more than 10 were detected in the samples.After peaks that were ions of the same compounds were combined, a total of 65 compounds were reliably identified with a similarity score of ＞ 600 (maximum match, 1000).The metabolite abundance was normalized per milligram of fresh weight and to the peak area of the internal standard ribitol.A partial least squares discrimination analysis (PLS-DA) and volcano analysis were performed using the web-based tool MetaboAnalyst 4.0 (Chong et al.2019).The heatmaps and the Venn diagrams were constructed by using the java-based tool TBtools 1.0 (Chen et al.2020).

Statistical analysis

Statistical analysis was performed using SAS and the MIXED procedure for all variables (Littell et al.2006).The effect of clonal genotypes and treatments on the growth and physiological parameters were analyzed using the following mixed model:

Yjkl=μ+Tj+Gk+Bl+TGjk+TBjl+ε

where Yjklis the corresponding variable (height, diameter, nutrient concentration and chlorophyll fluorescence); μ is the overall mean;Tj, Gkand Blare the effects ofjth fertilization treatment,kth clonal genotype andlth block, respectively; TGjkand TBjlare the corresponding interactions effects; and ε is the experimental random error.The model included the random effect of block Bland the interaction between fertilization treatment and block(TBjl) , as well as the other fixed effects.Multiple comparison tests were conducted for the differences of the least-squares means using Fisher’s LSD test.

Results

Clonal variation in growth

Two months after planting, cuttings of the three clones had similar heights and ground-level diameters.After nine months, the effects of P fertilization on height and groundlevel diameters were significant.Multiple comparison analysis showed that the EH5 clone was most responsive to phosphorous, with mean height and diameter increasing by 32.4% and 25.8%, respectively, compared to the controls.After 25 months, P fertilizer effect on height was the maximum for EH5.No significant differences were noted among treatments, possibly because of limited space for root growth in the bag.Overall, the EH5 clone grew more rapidly with phosphorous fertilization than the other clones (Table 1; Fig.1).

Fig.1 Height and diameter growth in the EH3, EH4 and EH5 clones in control and P-fertilized treatments at 2-25 months after planting.a height at 2 months; b diameter at 2 months; c height at 9 months; d diameter at 9 months; e height at 14 months; f diameter at 14 months; g height at 25 months; h diameter at 25 months.Asterisks indicate LSD (p ＜ 0.05) for comparisons across treatments and clones in the same plot.Data are mean ± SE

Nutrient concentration and chlorophyll fluorescence

After 25 months, neither treatment nor clonal effects significantly affected N, P and K concentrations.Nitrogen decreased with phosphorous fertilization, especially in the EH3 and EH4 clones, although the difference was insignificant (Table S2).However, chlorophyll fluorescence parameters were also affected by phosphorous fertilization in the EH3 and EH4 clones.Compared to the controls, maximum chlorophyll fluorescence (Fv/Fm) was significantly decreased in EH3 and the maximum quantum yield of PSII in lightin EH4.Significant clonal effects influenced chlorophyll fluorescence parameters.Fv/Fmandʹweresignificantly higher in the EH5 clone than in EH3 and EH4.The differences ofʹ in EH5 were greater with phosphorous fertilization.No significant variations were found for the effective quantum yield (ΦPSII) among clones (Table 1; Fig.2).

Fig.2 Maximum chlorophyll fluorescence (Fv/Fm), maximum quantum yield of PSII in light and effective quantum yield (ΦPSII) of the needles of EH3, EH4 and EH5 clones in control and P-fertilized treatments.Letters indicate LSD (p ＜ 0.05) for comparison across treatments and clones in the same plot.Data are mean ± SE

Table 1 The p value of the main effects of treatment (T), clone and their interaction on height (H) and ground diameter (D) at 2, 9, 16 and 25 months after planting, as well as the chlorophyll fluorescence parameters

Metabolite variation between P treatments

Sixty-four compounds were identified in the needles of the three clones (Table S3).Only a small portion of metabolites differentially accumulated between fertilized treatments and controls (Fig.3).In EH3, two compounds (sucrose and vanillylmandelic acid) increased significantly by more than twice with phosphorous fertilization, while two compounds (tartaric acid and xylitol) significantly decreased.In the EH4 clone, 11 compounds varied significantly between phosphorous treatments.All the compounds increased with fertilization; d-glyceric acid increased by twice gentiobiose by more than four times.The pattern of metabolite response was different in the EH5 clone.Seven compounds varied significantly between treatments, six of them decreased significantly.For example, gentiobiose and glycerol levels decreased by more than twice.There were almost no overlapping responsive metabolites to fertilization in the three clones (Fig.S1).Only gentiobiose levels responded to fertilization in the EH4 and EH5 clones, increasing in EH4 and decreasing in EH5.

Fig.3 Metabolic changes of three clones between P-(Control) and P + (P-fertilized).Volcano plots show significantly changed metabolites with P-value threshold 0.05 (Six repetitions for each group).Values in pink with metabolite names were significantly different

Metabolite variation among clones

Partial least squares discrimination analysis (PLS-DA) was applied to obtain an overview of the accumulation patterns of the metabolites (Fig.4).PLS-DA showed separation among the three clones in the controls (Fig.4a and b) and phosphorous fertilized clones (Fig.4c and d).The metabolites, which had the highest weight (values of variable importance in projection, VIP ＞ 1.0) to discriminate between the three clones, differed in two treatments (Fig.5).Of the 28 differentially accumulated compounds among the clones, 15 were carbohydrates, implying that carbohydrate accumulation might be an important metabolic trait for genotype discriminate.Ten and seven compounds were differentially accumulated among three clones in controls and fertilized treatments without overlapping.Without fertilization, glucose, gluconic alctone, citric acid, hesperitin and melezitose were highest in the EH5 clone, while epigallocatechin and tartaric acid were lowest.With fertilization, gentiobiose, beta-mannosylglycerate, digalacturonic acid, d-glucoheptose and D-glyceric acid were lowest in EH5, while maltotriitol was highest.Eleven compounds accumulated differentially among the clones regardless of phosphorous applications.Most of these compounds accumulated in the EH3 clone, while neohesperidin and raffinose accumulated in EH5.The abundance of galactinol and shikimic acid were lower in EH4 and lowest in EH5.

Fig.4 Partial least squares discrimination analysis (PLS-DA) score plot and cross validation of the three clones on P-(Control) and P + (P-fertilized).a, c Principal component analysis (PCA) scores plot between the two first principal components.The explained variances of each PCs are in brackets and color shaded areas indicate 95% confidence regions.b, d Performance of PLS-DA model classification using different number of components.The red star indicates the best class

Fig.5 Important features identified by PLS-DA among three clones on P-(Control) and P + (P-fertilized), respectively.a The important features which correspond to the metabolites are indicated on the left and are organized by descending order of their scores of variable importance in projection (VIP) in the component 2 (P-) or component 1 (P +) with VIP score threshold 1.0.The colored boxes on the right indicate the relative concentrations of the corresponding metabolite in each group under study.b Venn diagram shows the overlap of the important metabolites among three clones on P-and P + condition.Heatmaps showed relative content change patterns of these metabolites

Discussion

Phosphorous fertilizers had a positive effect onP.elliottii×P.caribaeagrowth in the current study but this depended on genotypes.Variations in the effect of fertilizer were found among the three clones, with EH5 the most responsive, suggesting that phosphorous addition should be considered when establishingP.elliottii×P.caribaeaplantations.An analysis of mechanisms underlying high phosphorous-utilization efficiency of EH5 can help clone selection and increase silvicultural prescriptions to increase productivity (Yáñez et al.2017).In this study, it was hypothesized that physiological characteristics related to metabolism followed a specific pattern in genotypes that had higher phosphorous utilization efficiency, which might be useful in clone selection.

Previous studies indicate that chlorophyll fluorescence traits are genetically controlled inPinusspp.(Koehn et al.2003; Corcuera et al.2011; Čepl et al.2016).There were positive relationships between growth traits and chlorophyll fluorescence parameters in 4-year-oldP.radiataclones (Xue et al.2013) and in first-yearP.elliottiiseedlings (Koehn et al.2003).Studies suggest using chlorophyll fluorescence traits might assist in breeding programs (Prinzenberg et al.2018).In this study, there were significant clonal variations in chlorophyll fluorescence parameters.Relatively higher values ofʹand ΦPSIIwere found in fertilized EH5 which were consistent with its higher growth rate.Thus, chlorophyll fluorescence traits might be used to select fast-growing clones under P-supply conditions.Studies have indicated that chlorophyll a fluorescence could be used to detect plant nutrient status (Mauromicale et al.2006; Kalaji et al.2018).The application of phosphorous changed the chlorophyll fluorescence inPistacia veraandPistacia atlanticaand lower concentration of phosphorous increased chlorophyll fulorescence, while high concentrations damaged plant cells and reduced chlorophyll fluorescence (Hamed et al.2019).When poplar plants were deficient in phosphorous, or grown under high nitrogen levels, phosphorous application diminishedphotosynthesis in mature leaves in order to meet the growth needs of sufficient phosphorous (Netzer et al.2019).These results suggest that plant phosphorous demand status and application amount would be reflected in photosynthesis from multiple aspects and the supply status of other nutrients should also be considered comprehensively.In the EH3 and EH4 clones,Fv/Fmandʹdecreased with phosphorous fertilization, while nitrogen levels also decreased.Bulgarelli et al.(2019) reported the similar findings inEucalyptusspecies.It was suggested that phosphorous limitations would result in excess nitrogen not being effectively used by plants, which would be reflected by higherFv/Fmand.This would change after phosphorous limitation was relieved and decreased chlorophyll fluorescence would be observed.Although no significant interaction were found between clone × P fertilization, different patterns were found with EH5 in response to fertilization.Fv/Fmandwere more stable in EH5 than in the other clones, with theFv/Fmvalues mostly above 0.80.Metabolic profiles reflect physiological mechanisms in response to nutrient changes (Nikiforova et al.2005; Okazaki et al.2008; Luo et al.2019).However, metabolic changes in woody plants in response to phosphorous regimes might be small.InEucalyptus globulusLabill., phosphorous deficiency for 2.5 months had little or no significant effects on carbohydrates, organic acids and most amino acids (Warren 2011).In this study, only a small portion of metabolites significantly changed in response to phosphorous fertilization, mainly carbohydrates, sugar alcohols and organic acids.Almost no changes in phosphorylated sugars and lipids, except for glycerol, were detected.

In these results, performances of the three clones correspond to the three response states to phosphorous.The EH3 clone had the smallest metabolite change after fertilization with no significant differences in height and groundlevel diameters, indicating that the response to phosphorous was ineffective.In the EH4 clone, sugars, sugar alcohols and organic acids increased after fertilization.Although no changes in height and ground-level diameters occurred, metabolic changes showed that EH4 benefited from fertilization.This suggests that EH4 may be a high P demand clone and the amount applied only changed its metabolic level which may not be enough to change its phenotype.EH5 was a P-utilization efficient clone.After fertilization, height and ground-level diameter increased and some sugars (except glucose) and organic acid metabolites decreased significantly, showing that when phosphorous was sufficient, growth was more rapid.Sugars significantly increased following fertilization in the three clones, but the type of sugars were different.Sucrose, gentianose and glucose increased in EH3, EH4 and EH5, respectively.Changes in glucose might be a general response, since a decrease in glucose and fructose under low N and P treatments were detected in maize (Schlüter et al.2013).A study of the genusGentianaindicated that gentianose, a main storage carbohydrate, could be converted to sucrose and glucose, thus providing the energy required for development (Takahashi et al.2014).The abundance of gentianose decreased and glucose was highest in EH5 following phosphorous fertilization, implying that gentianose might also be converted to glucose in EH5.Conversely, gentianose increased in EH4 in response to phosphorous and might explain why EH5 grew rapidly after P-fertilized treatment.

Although the differentially accumulated metabolites between treatments were in the minority, the results indicate that metabolite profiles might be an effective tool to differentiatePinusclones.Previous studies of forest plants indicated that metabolic traits have a remarkable potential to discriminate genotypes exhibiting excellent economic characteristics such as wood traits (Robinson et al.2005) and oleoresin-yielding traits (Karanikas et al.2010).In this study, 28 compounds had the highest weight (VIP ＞ 1.0) to distinguish the three clones.The majority of the differentially accumulated compounds were carbohydrates, indicating that C assimilation and metabolism had different levels amongst the clones.Studies of the genusArabidopsissuggested that high growth rates caused a depletion of central metabolite pools and increased stress response-related compounds in the vegetative growth phase (Meyer et al.2007).In this study, a high growth rate was detected in the EH5 clone following fertilization.Levels of maltotriitol, raffinose and neohesperidin were higher in EH5 than in the other clones while levels of most of the other compounds were lower.Maltotriitol is a sugar alcohol synthesized from maltose and accumulates in salt-treated plants (Llanes et al.2016; Li et al.2017).During freezing inArabidopsis, maltose might act as a protective solute and contribute to the protection of the photosynthetic electron transport chain (Kaplan et al.2005).The higher abundance of maltotriitol in EH5 might also related to the protection of the photosynthetic electron transport chain, since the chlorophyll fluorescence parameters were more stable in EH5.Raffinose is known to be a stress-responsive carbohydrate and compatible solutes activated when exposed to stress conditions (Zhang et al.2016).Neohesperidin is a flavonoid metabolite belonging to the flavanone subclass (Saxena et al.2012).A study of soybeans showed that neohesperidin was enhanced by low nitrogen stress and it is accumulated in low nitrogen-tolerant genotypes (Li et al.2018).With phosphorous fertilization, carbohydrates, including gentiobiose, beta-mannosylglycerate, digalacturonic acid, d-glucoheptose and D-glyceric acid were lowest in the EH5 clone.In addition, levels of galactinol and shikimic acid were lowest in EH5, regardless of fertilization.Thus, low levels of galactinol and shikimic acid and high amounts of maltotriitol, neohesperidin and raffinose might be ideal metabolic traits to select high P-utilization efficiency clones in hybrid pine.

Conclusion

Investigation of the effects of phosphorous fertilizers on the growth of threePinus elliottii×P.caribaeaclones shows that clone EH5 had the maximum growth following fertilization and it was the most responsive to phosphorous.Chlorophyll fluorescence measurements and metabolic profiles revealed that the physiological characteristics of EH5 followed specific patterns which could have been responsible for its higher phosphorous utilization efficiency.The relatively higher values of the maximum quantum yield of PSII in the dark (Fv/Fm) and the maximum quantum yield of PSII in the light, the higher levels of stress responserelated compounds and less accumulation of galactinol and shikimic acid might be the key features of phosphorousresponsive clones ofPinus elliottii×P.caribaea.These physiological features were consistent regardless of phosphorous supply and thus might be used for selecting clones for silviculture.

AcknowledgmentsWe thank the staff of the Taishan Hongling Seed Orchard for assistance in pine breeding and the Guangdong Datang Agro-Technology Co.Ltd.for assistance in clone culturing.