Effects of exogenous GA3 on stem secondary growth of Pinus massoniana seedlings.

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 GA3 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 GA3 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 GA3 treatment. Furthermore, GA3 and BR (brassinosteroid) might act synergistically in promoting secondary growth in P. massoniana. Additionally, lignin content was significantly increased after GA3 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 GA3 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 Brassinosteroid Facilitates Xylem Development in Pinus massoniana Seedlings.

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.

Proteomic analyses provide new insights into the responses of Pinus massoniana seedlings to phosphorus deficiency.

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.

The temporal transcriptomic response of Pinus massoniana seedlings to phosphorus deficiency.

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.