Secondary Needles Research Articles

Comparative analysis of differential gene expression reveals novel insights into the heteroblastic foliage functional traits of Pinus massoniana seedlings

Pinus massoniana needles, rich in medicinal polysaccharides and flavonoids, undergo heteroblastic foliage, transitioning from primary needles (PN) to secondary needles (SN) during growth, resulting in altered functional traits. Despite its significance, the molecular regulatory mechanisms governing these traits remain unclear. This study employs Iso-Seq and RNA-Seq analyses to explore differentially expressed genes (DEGs) associated with functional traits throughout the main growth season of heteroblastic foliage. Co-expression network analysis identified 34 hub genes and 17 key transcription factors (TFs) influencing light-harvesting antenna, photosystem I and II, crucial in photosynthesis regulation. Additionally, 14 genes involved in polysaccharide metabolism pathways, synthesizing sucrose, glucose, UDP sugars, and xylan, along with four genes in flavonoid biosynthesis pathways, regulating p-coumaroyl-CoA, quercetin, galangin, and myricetin production, exhibited differential expression between PN and SN. Further analysis unveils a highly interconnected network among these genes, forming a pivotal cascade of TFs and DEGs. Therefore, heteroblastic changes significantly impact needle functional traits, potentially affecting the pharmacological properties of PN and SN. Thus, these genomic insights into understanding the molecular-level differences of heteroblastic foliage, thereby establishing a foundation for advancements in the pharmaceutical industry related to needle-derived products.

Transcriptomic and targeted metabolomic unravelling the molecular mechanism of sugar metabolism regulating heteroblastic changes in Pinus massoniana seedlings

Pine seedling leaf characteristics show a distinct transition from primary to secondary needles, known as heteroblastic change. However, the underlying regulatory mechanism is poorly understood. The molecular mechanism of sugar metabolism involved in regulating heteroblastic changes in Pinus massoniana seedlings was investigated via transcriptomics and targeted metabolomics. The results identified 12 kinds of sugar metabolites in the foliage. Three types of sugar accumulated at the highest levels: sucrose, glucose and fructose. Compared to seedlings with only primary needles (PN), the contents of these soluble sugars were lower in seedlings with developing secondary needle buds (SNB). RNA-seq analysis highlighted 1086 DEGs between PN and SNB seedlings, revealing significant enrichment in KEGG pathways including starch and sucrose metabolism, plant hormone signal transduction and amino sugar and nucleic acid sugar metabolism. Combined transcriptomic and metabolomic analysis revealed that HK, MDH, and ATPase could potentially enhance sugar availability by stimulating the glycolytic/TCA cycle and oxidative phosphorylation. These processes may lead to a reduced sugar content in the foliage of SNB seedlings. We also identified 72 transcription factors, among which the expression levels of MYB, WRKY, NAC and C2H2 family genes were closely related to those of DEGs in the sugar metabolism pathway. In addition, we identified alternative splicing (AS) events in one NAC gene leading to two isoforms, PmNAC5L and PmNAC5S. PmNAC5L was significantly upregulated, while PmNAC5S was significantly downregulated in SNB seedlings. Overall, our results provide new insights into how sugar metabolism is involved in regulating heteroblastic changes in pine seedlings.

Structure and chlorophyll fluorescence of heteroblastic foliage affect first-year growth in Pinus massoniana Lamb. seedlings

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.

Heteroblastic Foliage Affects the Accumulation of Non-Structural Carbohydrates and Biomass in Pinus massoniana (Lamb.) Seedlings

Pines have heteroblastic foliage (primary and secondary needles) during seedling stage, but how heteroblastic foliage affects carbon storage and biomass accumulation, contributing to seedling quality, is unclear. We investigated the influences of heteroblastic foliage on photosynthetic physiological characteristics, non-structural carbohydrate (NSC) and biomass accumulation in current-year seedlings; the key factors determining biomass accumulation were mainly determined by principal component screening, Spearman correlation, and path analysis. The results indicated that (1) primary needles have high photosynthetic pigments (chlorophyll a and total chlorophyll), net photosynthetic rates (Pn), the potential maximum photochemical efficiency (Fv/Fm), and leaf instantaneous water use efficiency (WUEi), whereas higher non-photochemical quenching (NPQ) suggested that sudden light increases induce the initiation of quenching mechanism in primary needles; additionally, secondary needles had a lower transpiration rate (Tr), limiting stomata (Ls), and light saturation point. (2) Secondary needles promoted soluble sugar (fructose and glucose) increases in leaves compared to that of primary needles and increased the leaf biomass accumulation (from 47.06% to 54.30%), enhancing the overall ability of photosynthetic organs; additionally, secondary needles can enhance the proportion of starch storage in the roots, and NSC accumulation was significantly increasing in the seedling leaves and roots. (3) Photosynthetic pigments (carotenoids, chlorophyll a, and total chlorophyll) had direct positive effects on primary needle seedling (PNS) biomass and promoted biomass by indirectly increasing soluble sugar synthesis in the stems. The Pn was the main physiological factor determining PNS biomass accumulation. In addition, the WUEi, Ls, and NPQ had direct negative effects on PNS biomass accumulation, inhibiting photosynthesis to limit seedling growth. Considering the functional traits in heteroblastic foliage is necessary when assessing different leaf types of Pinus massoniana (Lamb.) seedlings, in particular those threats implicated in light, water, and temperature relations. Our results can be beneficial to guide the establishment of seedling management and afforestation measures.

Coexistent Heteroblastic Needles of Adult Pinus canariensis C.Sm. ex DC. in Buch Trees Differ Structurally and Physiologically

Great variation in shape and size between primary (juvenile) and secondary (adult) needles, so-called leaf-heteroblasty, occurs in several Pinus species. Most of them loss primary needles during the juvenile-to-adult transition of the tree. An exception to this is Pinus canariensis (a Canary Islands endemism) in which basal resprouting twigs of adult trees frequently wear both primary and secondary needles. Taking advantage of this extraordinary study-case-species, we conducted an exhaustive comparison of both needle types through quantitative analyses of needle anatomy, photochemical performance, gas exchange, and resistance to extreme dehydration and to extreme needle temperature. We hypothesized that primary needles would show lower investment to leaf structure but higher photosynthetical efficiency. Primary needles had less stomatal density and thicker and less wettable cuticles. In cross section, primary needles showed smaller structural fraction (e.g., percent of hypodermis, endodermis and vascular tissue) and higher fraction of photosynthetic parenchyma. Significant differences between primary and secondary needles were not found in net carbon assimilation not in their leaf mass area values. Interestingly, secondary needles showed higher electron transport rate, and they were additionally much more efficient in retaining water under severe and controlled desiccant conditions. When subjected to extreme temperatures (−10° to +50 °C), primary needles recovered better their photochemical efficiency than secondary needles, after +46° and +48 °C heat-shock treatments. Our results indicate that both needle types broaden the diversity of physiological responses against environmental constrains in basal twigs of adult P. canariensis trees. Considering that this is a fire-resistant and resprouting species, this advantage could be particularly useful after a drastic environmental change such a fire or a gap opening in the forest.

Early Diagenetic Imprint on Temperature Proxies in Holocene Corals: A Case Study From French Polynesia

Coral-based reconstructions of sea surface temperatures (SSTs) using Sr/Ca, U/Ca and d18O are important tools for quantitative analysis of past climate variabilities. However, post-depositional alteration of coral aragonite, particularly early diagenesis, restrict the accuracy of calibrated proxies even on young corals. Considering the diagenetic effects, we present new Mid to Late Holocene SST reconstructions on well-dated (U/Th: ca. 70 yr to 5.4 ka) fossil Porites sp. collected from the Society Islands, French Polynesia. For few corals, quality pre-screening routines revealed the presence of secondary aragonite needles inside primary pore space, resulting in a mean increase in Sr/Ca ratios between 5-30%, in contrast to the massive skeletal parts. Characterized by a Sr/Ca above 10 mmol/mol, we interpret this value as the threshold between diagenetically altered and unaltered coral material. At a high-resolution, observed intra-skeletal variability of 5.4 to 9.9 mmol/mol probably reflects the physiological control of corals over their trace metal uptake, and individual variations controlled by CaCO3- precipitation rates. Overall, the Sr/Ca, U/Ca and d18O trends are well correlated, but we observed a significant offset up to +/- 7°C among the proxies on derived palaeo-SST estimates. It appears that the related alteration process tends to amplify temperature extremes, resulting in increased SST-U/Ca and SST-Sr/Ca gradients, and consequently their apparent temperature sensitivities. A relative SST reconstruction is still feasible by normalizing our records to their individual mean value defined as 1SST. This approach shows that 1SST records derived from different proxies agree with an amplitudinal variability of up to +/- 2°C with respect to their Holocene mean value. Higher 1SST values than the mean SSTs (Holocene warm periods) were recorded from ca. 1.8 to ca. 2.8 ka (Interval I), ca. 3.7 to 4.0 ka (Interval III) and before ca. 5 ka, while lower 1SST values (Holocene cold periods, Interval II and IV) were recorded in between. The ensuing SST periodicity of ca. 1.5 ka in the Society Islands record is in line with the solar activity reconstructed from 10Be and 14C production (Vonmoos et al., 2006), emphasizing the role of solar activity on climate variability during the Late Holocene.

Transient gene expression in western white pine using agroinfiltration

A method for transient gene expression was developed for western white pine (WWP, Pinus monticola Dougl. ex D.Don) using reporter gene uidA encoding β-glucuronidase (GUS). GUS was transiently expressed in cross sections of primary and secondary needles, cotyledons, and current and second year stems of WWP via vacuum-infiltration with Agrobacterium tumefaciens. Histochemical assays of cross sections of secondary needles showed stronger blue color indicating GUS expression at day 1 and 2 than on other days post agroinfiltration (dpa). GUS activity expressed inside WWP cells was confirmed using light microscopy. In fluorometric assays, GUS expression was high at 1 dpa and lasted until 4 dpa in detached secondary needles, while similarly high expression levels only lasted until 2 dpa in attached secondary needles then dropped significantly. Although the length of GUS-staining zones varied among different WWP organs and between growth and dormant seasons, all tested WWP tissues using the protocol had high levels of transient GUS expression. Thus, heterologous candidate genes or endogenous silencing can be expressed in various WWP tissues or organs using this agroinfiltration approach. The current protocol for efficient transient gene expression will aid functional genomics study of WWP and its pathogens and related conifer species.

Structural controls on photosynthetic capacity through juvenile‐to‐adult transition and needle ageing in Mediterranean pines

AbstractNeedle photosynthetic potentials strongly vary among primary (juvenile) and secondary (adult) needles (heteroblasty) inPinusspecies, but there is limited understanding of the underlying structural, diffusional and chemical controls.We studied differences in needle photosynthetic characteristics among current‐year juvenile and adult needles and among different‐aged adult needles in Mediterranean pinesPinus halepensisMill.,P. pineaL. andP. nigraJ. F. Arnold subsp.salzmannii(Dunal) Franco, hypothesizing that needle anatomical modifications upon juvenile‐to‐adult transition lead to reduced photosynthetic capacity due to greater limitation of photosynthesis by mesophyll conductance and due to an increase in the share of support tissues at the expense of photosynthetic tissues. We also hypothesized that such alterations occur with needle ageing, but to a lower degree.Photosynthetic capacity per dry mass was 2.4‐ to 2.7‐fold higher in juvenile needles, and this was associated with 3.4‐ to 3.7‐fold greater mesophyll diffusion conductance, 2‐ to 2.5‐fold greater maximum carboxylase activity of Rubisco (Vcmax) and 2.2‐ to 3‐fold greater capacity for photosynthetic electron transport (Jmax). The latter differences were driven by modifications in mesophyll volume fraction and changes in the share of nitrogen between structural and photosynthetic functions. Analogous changes in photosynthetic characteristics occurred with needle ageing, but their extent was less.These results indicate that conifer foliage photosynthetic machinery undergoes a profound change from a fast return strategy in juveniles to slow return stress‐resistant strategy in adults and that this strategy shift is driven by modifications in foliage biomass investments in support and photosynthetic functions as well as by varying mesophyll diffusional controls on photosynthesis. Changes in needle morphophysiotype during tree and needle ageing need consideration in predicting changes in tree photosynthetic potentials through tree ontogeny and during and among growing seasons.Aplain language summaryis available for this article.

Lophodermium resinosum sp. nov. from red pine (Pinus resinosa) in Eastern Canada

A morphologically distinct Lophodermium species was collected from fallen secondary needles of Pinus resinosa Aiton over two consecutive years in Eastern Ontario; subsequent herbarium studies confirmed its presence in Nova Scotia, Quebec, and Maine. Symptomatic needles frequently exhibited red bands and completely subepidermal ascomata and conidiomata. Ascospore isolates from specimens were used to reconstruct phylogenies inferred from internal transcribed spacer rDNA and partial actin gene sequences. Both phylogenetic analyses delineated the specimens from other sequenced Lophodermium species. Phylogenetic evidence combined with morphological characters of ascomata and conidiomata supported the distinctiveness of this species, described here as Lophodermium resinosum sp. nov.

Erratum to: Transcriptional and post-translational control of chlorophyll biosynthesis by dark-operative protochlorophyllide oxidoreductase in Norway spruce.

Unlike angiosperms, gymnosperms use two different enzymes for the reduction of protochlorophyllide to chlorophyllide: the light-dependent protochlorophyllide oxidoreductase (LPOR) and the dark-operative protochlorophyllide oxidoreductase (DPOR). In this study, we examined the specific role of both enzymes for chlorophyll synthesis in response to different light/dark and temperature conditions at different developmental stages (cotyledons and needles) of Norway spruce (Picea abies Karst.). The accumulation of chlorophyll and chlorophyll-binding proteins strongly decreased during dark growth in secondary needles at room temperature as well as in cotyledons at low temperature (7 °C) indicating suppression of DPOR activity. The levels of the three DPOR subunits ChlL, ChlN, and ChlB and the transcripts of their encoding genes were diminished in dark-grown secondary needles. The low temperature had minor effects on the transcription and translation of these genes in cotyledons, which is suggestive for post-translational control in chlorophyll biosynthesis. Taking into account the higher solubility of oxygen at low temperature and oxygen sensitivity of DPOR, we mimicked low-temperature condition by the exposure of seedlings to higher oxygen content (33%). The treatment resulted in an etiolated phenotype of dark-grown seedlings, confirming an oxygen-dependent control of DPOR activity in spruce cotyledons. Moreover, light-dependent suppression of mRNA and protein level of DPOR subunits indicates that more efficiently operating LPOR takes over the DPOR function under light conditions, especially in secondary needles.

Transcriptional and post-translational control of chlorophyll biosynthesis by dark-operative protochlorophyllide oxidoreductase in Norway spruce.

Unlike angiosperms, gymnosperms use two different enzymes for the reduction of protochlorophyllide to chlorophyllide: the light-dependent protochlorophyllide oxidoreductase (LPOR) and the dark-operative protochlorophyllide oxidoreductase (DPOR). In this study, we examined the specific role of both enzymes for chlorophyll synthesis in response to different light/dark and temperature conditions at different developmental stages (cotyledons and needles) of Norway spruce (Picea abies Karst.). The accumulation of chlorophyll and chlorophyll-binding proteins strongly decreased during dark growth in secondary needles at room temperature as well as in cotyledons at low temperature (7°C) indicating suppression of DPOR activity. The levels of the three DPOR subunits ChlL, ChlN, and ChlB and the transcripts of their encoding genes were diminished in dark-grown secondary needles. The low temperature had minor effects on the transcription and translation of these genes in cotyledons, which is suggestive for post-translational control in chlorophyll biosynthesis. Taking into account the higher solubility of oxygen at low temperature and oxygen sensitivity of DPOR, we mimicked low-temperature condition by the exposure of seedlings to higher oxygen content (33%). The treatment resulted in an etiolated phenotype of dark-grown seedlings, confirming an oxygen-dependent control of DPOR activity in spruce cotyledons. Moreover, light-dependent suppression of mRNA and protein level of DPOR subunits indicates that more efficiently operating LPOR takes over the DPOR function under light conditions, especially in secondary needles.

Content of far-red light in supplemental light modified characteristics related to drought tolerance and post-planting growth in Scots pine seedlings

Light quality affects the morphological development of a plant. The aim of the experiment was to examine the effect of far-red radiation (FR, 700–800nm) on growth, morphology, and root growth capacity (RGC) in Scots pine seedlings originating from latitudes 61°N and 67°N, and on gas exchange and subsequent field growth in seedlings originating from 61°N. The seedlings were grown in a greenhouse under natural daylight, receiving either FR-rich (+FR) or FR-deficient (−FR) supplemental light for 20h per day, provided using light-emitting diodes (LEDs). The seedlings were outplanted and grown in the field for two growing seasons. Growth under +FR hastened terminal bud formation in seedings originating from 61°N, and delayed it in seedlings from 67°N. At the end of the growth period under the LEDs, +FR seedlings were dominated by secondary needles and had a higher needle mass than −FR seedlings, whose foliage comprised mainly primary needles. −FR improved root growth capacity in the seedlings originating from 61°N, but not in seedlings originating from 67°N. In seedlings from 61°N, photosynthesis (Pn) at the needle area level was higher and stomatal conductance lower under −FR, resulting in higher water use efficiency than under +FR. Due to the greater amount of photosynthetic tissue under +FR, the whole-seedling-level Pn was higher under +FR. Height and diameter growth were greater in −FR seedlings than in +FR seedlings during the second growing season. Growth habit and growth rate of Scots pine seedlings can be manipulated by controlling the FR content in the supplemental light, enabling production of seedlings targeted for outplanting on sites with specific characteristics.

A new species of Lophodermium on needles of mountain pine (Pinus mugo) from the Giant Mountains in Poland

A fungus tentatively identified as Lophodermium sp. was repeatedly isolated from living secondary needles of mountain pine (Pinus mugo) in the Giant Mountains of Poland. Needles showed symptoms of yellow spots, gradual discolouration and premature falling in July. Thin black zone lines, subepidermal conidiomata and partially subepidermal ascomata morphologically similar to L. pinastri occurred on fallen needles in litter below the same trees. Evidence from internal transcribed spacers of rDNA and the gene for actin showed that strains isolated from symptom-bearing needles and fruiting structures were identical, and differed from other Lophodermium species known from pine, including L. pinastri. The fungus differs subtly from L. pinastri, for example, in the lengths of its conidiomata, ascomata and asci, but can be reliably distinguished only by molecular data. Known only on mountain pine from the Giant Mountains, it is described here as L. corconticum sp. nov.

A new species of <I>Leptostroma</I> on <I>Pinus henryi</I> from China

A coelomycetous fungus found on dead fallen secondary needles of Pinus henryi from Shennongjia forestry region of Hubei Province, China, is described, illustrated, and discussed. Habitat, morphology, and conidial development place it in the Rhytismataceae. It differs from other members of this family on the same substratum by having conidiomata with upper walls more than one cell thick, and is formally named as Leptostroma magnum sp. nov. Its conservation status is evaluated as Data Deficient. The type specimen is deposited in the Reference Collection of Forest Fungi of Anhui Agricultural University, China (AAUF).

The influence of nitrogen nutrition on the dynamics of growth and metabolism of endogenous growth regulators in Scotch pine (Pinus silvestris L.) seedlings

Pine seedlings were cultivated in sterile agar cultures containing nitrogen as NH4Cl. The most pronounced positive effect on the growth of seedlings was affected by N used at a concentration of 50 ppm. After 4 months was stated that nitrogen had only a slight effect on elongation, of .shoots but decreased the length of roots. Nitrogen increased the lenght and number of primary and secondary needles as well as the fresh and dry matter of sboots. It stimulated also the number of lateral roots and the fresh and dry matter of the root system. Stimulation of shoot growth and differentiation as a result of nitrogen treatment was correlated with the increase -of free gibberellins and auxins and decrease of the amount of bound gibberellins and ABA-like inhibitor in shoots. However the effect of N on growth of roots was connected with the increase of auxins, cytokinins and ABA-like inhibitor in these organs.

The effect of potassium nutrition on growth and on plant hormones content in Scots pine (Pinus silvestris L.) seedlings

Pine seedlings were cultivated in Ingestad nutrient solution containing potassium as KCl on a 16-hr day, under light intensity of about 4000 lx and temperature of about 23° C. K+ used in a concentration of 50 ppm exerted a most pronounced positive effect on the growth of seedlings. After 4 months it was found that potassium used in this concentration increased elongation of hypocotyls, shoots, roots, primary and secondary needles as well as fresh weight and dry matter of tissues. In leafy shoots K+ increased the level of free gibberelirns (GAs) and auxins and decreased the amount of bound GAs and an ABA-like inhibitor. In the roots K+ nutrition increased the amount of free GAs and of the growth inhibitor and decreased the level of bound GAs and auxins. No evident influence of K+ on the level of cytokinins in plant tissues was stated.

Clonal variation for shoot ontogenetic heteroblasty in maritime pine (Pinus pinaster Ait.)

Pine seedling shoots undergo sharp heteroblastic changes during the early ontogenetic stages. The rate of these changes has been seen to vary between species and provenances within species, but there is a marked lack of information about its genetic control at the lower hierarchical levels. We used clonal replicates of maritime pine to determine broad-sense heritability of shoot ontogenetic heteroblasty and its correlation to rooting ability. We applied a simple ontogenetic index based on the proportion of basal nodes with secondary needles in rooted cuttings of 15 clones from 9 environmentally contrasting origins. We found a high clonal heritability for shoot ontogenetic index and a moderately high heritability for rooting ability, but both genetic and phenotypic correlations between these two traits were weak and non-significant. These results indicate that both developmental phenomena are genetically controlled, but not strictly associated in this species.

Effect of microstructure on cutting processability of porcelain tile subjected to different firing cycles

Abstract The fast firing technique is one of the most important ways to save energy consumption and improve production efficiency in the porcelain tile industry. In the actual production, excessively short firing cycle time easily causes the cutting edge defects. This work examines the effect of microstructure on cutting processability of a representative composition of a commercial porcelain tile fired at 1200 °C with two different firing cycles as follows: 40 min and 60 min. The phase composition and microstructure were investigated by using a combination of techniques such as X-ray diffraction (XRD) and scanning electron microscopy (SEM). The result indicated that it was beneficial to extend firing cycle (from 40 min to 60 min) for the cutting processability of porcelain tiles, which was due to the formation of positive microstructures such as secondary mullite needles and small-volume residual quartz.

Early season short-day treatment did not affect the field performance of Pinus sylvestris container seedlings in summer planting

When Scots pine (Pinus sylvestris L.) seedlings are short-day (SD) treated in May or early June, height growth ceases but seedlings reflush within a few weeks and grow secondary needles (later referred to as early SD treatment). These “2-year-old-like” seedlings have been thought to be more stress tolerant than traditional 1-year-old pine seedlings, and thus suitable for summer and autumn plantings. To test this, seedlings sown in April were early SD treated over a 3-week period from the beginning of June and the seedlings were planted at 10-day intervals from the beginning of July until the end of September, and also during the following spring. Neither survival nor height growth of early SD-treated seedlings differed from untreated (1-year-old) seedlings. Moreover, for both early SD-treated seedlings and 1-year-old seedlings, those planted in summer grew and survived better after planting but had more multiple leaders than seedlings planted in autumn or spring.

Biomass allocation and foliage heteroblasty in hard pine species respond differentially to reduction in rooting volume

Rooting space is considered as a resource in plants, but comparative studies on the biomass allocation plasticity in response to rooting volume (RV) are rare. We compared responses in growth, biomass allocation and ontogenetic heteroblasty in nine hard pine species of contrasted ecology. Seedlings were cultivated in containers of 0.2, 0.5, 1.0, 2.8 and 7 L for two growing seasons (425 days). Reduction in RV caused a reduction in plant absolute and relative growth rate and biomass allocated to stems but it increased biomass allocated to roots. RV affected to a lesser extent and in a less consistent direction allocation to leaves. Species that grew faster (higher relative growth rate) had a steeper decrease in growth with the reduction in RV. Ontogenetic heteroblasty, evaluated as the proportion of secondary needles in the needle biomass, showed highly different plasticity patterns in response to RV. Decrease in RV caused negligible or no change either in the most ontogenetically delayed Mediterranean pines or in the most ontogenetically advanced pines, the mesic Pinus sylvestris and P. uncinata. By contrast, ontogenetically intermediate species showed steep reaction norms in response to reduction in RV. While P. pinaster and P. brutia showed marked rejuvenation, P. nigra accelerated the development of adult foliage.