Physiological, Biochemical, and Molecular Analyses Reveal Dark Heartwood Formation Mechanism in Acacia melanoxylon.

Acacia melanoxylon is highly valued for its commercial applications, with the heartwood exhibiting a range of colors from dark to light among its various clones. The underlying mechanisms contributing to this color variation, however, have not been fully elucidated. In an effort to understand the factors that influence the development of dark heartwood, a comparative analysis was conducted on the microstructure, substance composition, differential gene expression, and metabolite profiles in the sapwood (SW), transition zone (TZ), and heartwood (HW) of two distinct clones, SR14 and SR25. A microscopic examination revealed that heartwood color variations are associated with an increased substance content within the ray parenchyma cells. A substance analysis indicated that the levels of starches, sugars, and lignin were more abundant in SP compared to HW, while the concentrations of phenols, flavonoids, and terpenoids were found to be higher in HW than in SP. Notably, the dark heartwood of the SR25 clone exhibited greater quantities of phenols and flavonoids compared to the SR14 clone, suggesting that these compounds are pivotal to the color distinction of the heartwood. An integrated analysis of transcriptome and metabolomics data uncovered a significant accumulation of sinapyl alcohol, sinapoyl aldehyde, hesperetin, 2', 3, 4, 4', 6'-peptahydroxychalcone 4'-O-glucoside, homoeriodictyol, and (2S)-liquiritigenin in the heartwood of SR25, which correlates with the up-regulated expression of CCRs (evm.TU.Chr3.1751, evm.TU.Chr4.654_667, evm.TU.Chr4.675, evm.TU.Chr4.699, and evm.TU.Chr4.704), COMTs (evm.TU.Chr13.3082, evm.TU.Chr13.3086, and evm.TU.Chr7.1411), CADs (evm.TU.Chr10.2175, evm.TU.Chr1.3453, and evm.TU.Chr8.1600), and HCTs (evm.TU.Chr4.1122, evm.TU.Chr4.1123, evm.TU.Chr8.1758, and evm.TU.Chr9.2960) in the TZ of A. melanoxylon. Furthermore, a marked differential expression of transcription factors (TFs), including MYBs, AP2/ERFs, bHLHs, bZIPs, C2H2s, and WRKYs, were observed to be closely linked to the phenols and flavonoids metabolites, highlighting the potential role of multiple TFs in regulating the biosynthesis of these metabolites and, consequently, influencing the color variation in the heartwood. This study facilitates molecular breeding for the accumulation of metabolites influencing the heartwood color in A. melanoxylon, and offers new insights into the molecular mechanisms underlying heartwood formation in woody plants.

Comparative physiological, biochemical, metabolomic, and transcriptomic analyses reveal the formation mechanism of heartwood for Acacia melanoxylon.

Acacia melanoxylon is well known as a valuable commercial tree species owing to its high-quality heartwood (HW) products. However, the metabolism and regulatory mechanism of heartwood during wood development remain largely unclear. In this study, both microscopic observation and content determination proved that total amount of starches decreased and phenolics and flavonoids increased gradually from sapwood (SW) to HW. We also obtained the metabolite profiles of 10 metabolites related to phenolics and flavonoids during HW formation by metabolomics. Additionally, we collected a comprehensive overview of genes associated with the biosynthesis of sugars, terpenoids, phenolics, and flavonoids using RNA-seq. A total of ninety-one genes related to HW formation were identified. The transcripts related to plant hormones, programmed cell death (PCD), and dehydration were increased in transition zone (TZ) than in SW. The results of RT-PCR showed that the relative expression level of genes and transcription factors was also high in the TZ, regardless of the horizontal or vertical direction of the trunk. Therefore, the HW formation took place in the TZ for A. melanoxylon from molecular level, and potentially connected to plant hormones, PCD, and cell dehydration. Besides, the increased expression of sugar and terpenoid biosynthesis-related genes in TZ further confirmed the close connection between terpenoid biosynthesis and carbohydrate metabolites of A. melanoxylon. Furthermore, the integrated analysis of metabolism data and RNA-seq data showed the key transcription factors (TFs) regulating flavonoids and phenolics accumulation in HW, including negative correlation TFs (WRKY, MYB) and positive correlation TFs (AP2, bZIP, CBF, PB1, and TCP). And, the genes and metabolites from phenylpropanoid and flavonoid metabolism and biosynthesis were up-regulated and largely accumulated in TZ and HW, respectively. The findings of this research provide a basis for comprehending the buildup of metabolites and the molecular regulatory processes of HW formation in A. melanoxylon.

Morphological, Anatomical, and Physiological Characteristics of Heteroblastic Acacia melanoxylon Grown under Weak Light.

Acacia melanoxylon is a fast-growing macrophanerophyte with strong adaptability whose leaf enables heteromorphic development. Light is one of the essential environmental factors that induces the development of the heteroblastic leaf of A. melanoxylon, but its mechanism is unclear. In this study, the seedlings of A. melanoxylon clones were treated with weak light (shading net with 40% of regular light transmittance) and normal light (control) conditions for 90 d and a follow-up observation. The results show that the seedlings' growth and biomass accumulation were inhibited under weak light. After 60 days of treatment, phyllodes were raised under the control condition while the remaining compound was raised under weak light. The balance of root, stem, and leaf biomass changed to 15:11:74 under weak light, while it was 40:15:45 under control conditions. After comparing the anatomical structures of the compound leaves and phyllode, they were shown to have their own strategies for staying hydrated, while phyllodes were more able to control water loss and adapt to intense light. The compound leaves exhibited elevated levels of K, Cu, Ca, and Mg, increased antioxidant enzyme activity and proline content, and higher concentrations of chlorophyll a, carotenoids, ABA, CTK, and GA. However, they displayed a relatively limited photosynthetic capacity. Phyllodes exhibited higher levels of Fe, cellulose, lignin, IAA content, and high photosynthetic capacity with a higher maximum net photosynthetic rate, light compensation point, dark respiration rate, and water use efficiency. The comparative analysis of compound leaves and phyllodes provides a basis for understanding the diverse survival strategies that heteroblastic plants employ to adapt to environmental changes.

Correction: Zhang et al. Genotype-Environment Interaction and Horizontal and Vertical Distributions of Heartwood for Acacia melanoxylon R.Br. Genes 2023, 14, 1299.

In the original publication [...].

Profiling of the gene expression and alternative splicing landscapes of Eucalyptus grandis.

Eucalyptus is a widely planted hardwood tree species due to its fast growth, superior wood properties and adaptability. However, the post-transcriptional regulatory mechanisms controlling tissue development and stress responses in Eucalyptus remain poorly understood. In this study, we performed a comprehensive analysis of the gene expression profile and the alternative splicing (AS) landscape of E. grandis using strand-specific RNA-Seq, which encompassed 201 libraries including different organs, developmental stages, and environmental stresses. We identified 10 416 genes (33.49%) that underwent AS, and numerous differentially expressed and/or differential AS genes involved in critical biological processes, such as primary-to-secondary growth transition of stems, adventitious root formation, aging and responses to phosphorus- or boron-deficiency. Co-expression analysis of AS events and gene expression patterns highlighted the potential upstream regulatory role of AS events in multiple processes. Additionally, we highlighted the lignin biosynthetic pathway to showcase the potential regulatory functions of AS events in the KNAT3 and IRL3 genes within this pathway. Our high-quality expression atlas and AS landscape serve as valuable resources for unravelling the genetic control of woody plant development, long-term adaptation, and understanding transcriptional diversity in Eucalyptus. Researchers can conveniently access these resources through the interactive ePlant browser (https://bar.utoronto.ca/eplant_eucalyptus).

Exogenous applications of brassinosteroids promote secondary xylem differentiation in Eucalyptus grandis.

Brassinosteroids (BRs) play many pivotal roles in plant growth and development, especially in cell elongation and vascular development. Although its biosynthetic and signal transduction pathway have been well characterized in model plants, their biological roles in Eucalyptus grandis, a major hardwood tree providing fiber and energy worldwide, remain unclear. Here, we treated E. grandis plantlets with 24-epibrassinolide (EBL), the most active BR and/or BR biosynthesis inhibitor brassinazole. We recorded the plant growth and analyzed the cell structure of the root and stem with histochemical methods; then, we performed a secondary growth, BR synthesis, and signaling-related gene expression analysis. The results showed that the BRs dramatically increased the shoot length and diameter, and the exogenous BR increased the xylem area of the stem and root. In this process, EgrBRI1, EgrBZR1, and EgrBZR2 expression were induced by the BR treatment, and the expressions of HD-ZIPIII and cellulose synthase genes were also altered. To further verify the effect of BRs in secondary xylem development in Eucalyptus, we used six-month-old plants as the material and directly applied EBL to the xylem and cambium of the vertical stems. The xylem area, fiber cell length, and cell numbers showed considerable increases. Several key BR-signaling genes, secondary xylem development-related transcription factor genes, and cellulose and lignin biosynthetic genes were also considerably altered. Thus, BR had regulatory roles in secondary xylem development and differentiation via the BR-signaling pathway in this woody plant.

Physiological and molecular mechanisms of Acacia melanoxylon stem in response to boron deficiency.

Boron is an essential micronutrient for plant growth as it participates in cell wall integrity. The growth and development of Acacia melanoxylon stem can be adversely affected by a lack of boron. To explore the mechanism of boron deficiency in A. melanoxylon stem, the changes in morphological attributes, physiological, endogenous hormone levels, and the cell structure and component contents were examined. In addition, the molecular mechanism of shortened internodes resulting from boron deficiency was elucidated through transcriptome analysis. The results showed that boron deficiency resulted in decreased height, shortened internodes, and reduced root length and surface area, corresponding with decreased boron content in the roots, stems, and leaves of A. melanoxylon. In shortened internodes of stems, oxidative damage, and disordered hormone homeostasis were induced, the cell wall was thickened, hemicellulose and water-soluble pectin contents decreased, while the cellulose content increased under boron deficiency. Furthermore, plenty of genes associated with cell wall metabolism and structural components, including GAUTs, CESAs, IRXs, EXPs, TBLs, and XTHs were downregulated under boron deficiency. Alterations of gene expression in hormone signaling pathways comprising IAA, GA, CTK, ET, ABA, and JA were observed under boron deficiency. TFs, homologous to HD1s, NAC10, NAC73, MYB46s, MYB58, and ERF92s were found to interact with genes related to cell wall metabolism, and the structural components were identified. We established a regulatory mechanism network of boron deficiency-induced shortened internodes in A. melanoxylon based on the above results. This research provides a theoretical basis for understanding the response mechanism of woody plants to boron deficiency.

Genotype-Environment Interaction and Horizontal and Vertical Distributions of Heartwood for Acacia melanoxylon R.Br.

Acacia melanoxylon (blackwood) is a valuable wood with excellent-quality heartwood extensively utilized worldwide. The main aim of this study was to confirm the horizontal and vertical variation and provide estimated values of genetic gains and clonal repeatabilities for improving breeding program of A. melanoxylon. Six blackwood clones at 10 years old were analyzed in Heyuan and Baise cities in China. Stem trunk analysis was conducted for sample trees to explore the differences between heartwood and sapwood. The heartwood radius (HR), heartwood area (HA), and heartwood volume (HV) in heartwood properties decreased as tree height (H) in growth traits increased, and the HV = 1.2502 DBH (diameter at breast height)1.7009 model can accurately estimate the heartwood volume. Furthermore, G × E analysis showed that the heritabilities of the eleven indices, including DBH, DGH (diameter at ground height), H, HR, SW (sapwood width), BT (bark thickness), HA, SA (sapwood area), HV, HRP (heartwood radius percentage), HAP (heartwood area percentage), and HVP (heartwood volume percentage) were between 0.94 and 0.99, and repeatabilities of the eleven indices were between 0.74 and 0.91. Clonal repeatability of DBH (0.91), DGH (0.88), and H (0.90) in growth traits, HR (0.90), HVP (0.90), and HV (0.88) in heartwood properties were slightly higher than for SA (0.74), SW (0.75), HAP (0.75), HRP (0.75), and HVP (0.75). These data also implied that the growth characteristics of heartwood and sapwood of blackwood clones were less affected by the environment and had substantial heritability.

First Report of Twig Anthracnose of Acacia melanoxylon Caused by Colletotrichum siamense in China.

Acacia melanoxylon R. Br. native to Australia, is a high-quality timber tree with wide genetic and phenotypic diversity. In recent years, A. melanoxylon has been extensively cultivated in some provinces in southern China. In December 2019, anthracnose-like symptoms were observed on twigs of A. melanoxylon in China. In certain valleys in south China, the disease incidence on plants and shoots was 60-75% and 80-90%, respectively. The wither rate of disease branches was 30-40% in dry seasons from September to November. The appearance of symptoms occurred in a humid and warm valley. Symptoms were initially observed on the young branches as brown spindle shaped sunken spots. At later stages, the disease spots girdled the whole branch, which became wilted and its leaves turned reddish-brown. For pathogen isolation, diseased branches were sampled and 55 pieces (5× 5 mm) of these branches section were surface-sterilized in 75% ethanol for 30 seconds, followed by 0.5% NaClO for 5 min and then were rinsed three times in distilled water. After drying with sterilized filter paper, the surface-sterilized sections were transferred to potato dextrose agar medium (PDA) and incubated at 25 °C for 7 days in the dark. Three isolates were obtained as representatives for morphological characterization and were labeled as 1A912, 1B912, and 1C912. These specimens were deposited in the Guangdong Province Key Laboratory of Microbial Signals and Disease Control at the South China Agricultural University (China). Purified isolates were initially white, cottony and with dense aerial mycelium on PDA at 25 ℃, ten days later their colonies turned grayish white with orange conidial masses. Conidia were one-celled, hyaline, straight, cylindrical, with round obtuse ends, and measured 11.0 to16.3× 4.0 to 6.0 µm (n= 100), appressoria were 5.86 to 9.07 × 3.55 to 6.96 µm (n= 100). Morphological characteristics of selected isolates matched the Colletotrichum gloeosporioides species complex (Weir et al. 2012). For further identification, the internal transcribed spacer (ITS) region, and partial sequences of the actin (ACT), beta-tubulin (TUB2), and glycerol dehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified by PCR, and sequenced, using primer pairs ITS1/ ITS4 (White et al. 1990), Bt2a/ Bt2b (Donaldson and Glass 1995), ACT512F/ ACT783R, GDF1/ GDR1(Weir et al. 2012). The sequences were deposited in GenBank (ITS: MW228101-MW228103; TUB2: MW250346, MW320707, MW320708; ACT: MW250347, MW320703, MW320704; GAPDH: MW250348, MW320705, MW320706). The multilocus phylogenetic analysis distinguished the isolates 1A912, 1B912, and 1C912 as C. siamense. Pathogenicity of those three isolates of C. siamense was tested on healthy twigs of the one clone of A. melanoxylon. 27 young twigs of nine 1-year-old plants were inoculated with the mycelium of the 7 days-old isolates 1A912, 1B912, and 1C912(Each isolate infected three plants and each infected three young twigs) through an artificial wound. The same nine plants were inoculated with PDA medium alone (each infected three young twigs) as a negative control. Five days after inoculation, brown spindle spots similar to the field disease symptoms were observed on the twigs. No symptoms were observed on the control plants. The experiment was repeated twice. The fungus was successfully reisolated from the symptomatic plants, and had identical morphological and molecular characteristics to the initial isolates, fulfilling Koch´s postulates. To our knowledge, this is the first report of anthracnose caused by C. siamense on A. melanoxylon in China. Twig anthracnose can reduce the growth of A. melanoxylon. Further research on management options for this disease is required.

Agrobacterium-mediated genetic transformation of the most widely cultivated superior clone Eucalyptus urophylla × E. grandis DH32-29 in Southern China.

Eucalyptus, as an economically important species for wood and paper industries, remains a challenge to genetic improvement by transgenic technology owing to the deficiency of a highly efficient and stable genetic transformation system, especially in cultivated superior clones. Eucalyptus urophylla × E. grandis clone DH32-29 is most widely planted in southern China, but it is relatively recalcitrant to adventitious bud regeneration, which blocks the establishment of a genetic transformation system. Here, an efficient adventitious bud regeneration and transformation system of Eucalyptus was established using E. urophylla × E. grandis DH32-29 as material. The in vitro leaves from microshoots that were subcultured for 20-25 days were immersed into liquid Woody Plant Medium supplemented with 0.02 mg·L-1 α-naphthaleneacetic acid (NAA) and 0.24 mg·L-1 forchlorfenuron [callus-inducing medium (CIM)]. After 15 days, explants were transferred to a medium containing 0.10 mg·L-1 NAA and 0.50 mg·L-1 6-benzyladenine (shoot-inducing medium, SIM) for adventitious bud induction. The highest regeneration efficiency of adventitious buds was 76.5%. Moreover, an Agrobacterium tumefaciens-mediated genetic transformation system was optimized. The leaves were precultured for 7 days and infected for 30 min with A. tumefaciens strain EHA105 grown to a bacterial density of 0.3 (OD600). After 72 h of cocultivation in the dark, leaves were transferred to CIM supplemented with 100 mg·L-1 cefotaxime (Cef), 100 mg·L-1 timentin, and 15 mg·L-1 kanamycin (Kan) for 15 days to induce calluses. Then, the explants were transferred to SIM supplemented with the same concentration of antibiotics, and the fresh medium was replaced every 15 days until resistant adventitious buds appeared. After inducing roots in root-inducing medium supplemented with 200 mg·L-1 Cef and 75 mg·L-1 Kan, completely transgenic plants were obtained. Using the aforementioned method, the transformation frequency can reach 1.9%. This provides a powerful approach for genetic improvement of E. urophylla × E. grandis DH32-29 and gene function analysis in Eucalyptus.

Transcriptome and structure analysis in root of Casuarina equisetifolia under NaCl treatment.

BACKGROUND: High soil salinity seriously affects plant growth and development. Excessive salt ions mainly cause damage by inducing osmotic stress, ion toxicity, and oxidation stress. Casuarina equisetifolia is a highly salt-tolerant plant, commonly grown as wind belts in coastal areas with sandy soils. However, little is known about its physiology and the molecular mechanism of its response to salt stress. RESULTS: Eight-week-old C. equisetifolia seedlings grown from rooted cuttings were exposed to salt stress for varying durations (0, 1, 6, 24, and 168 h under 200 mM NaCl) and their ion contents, cellular structure, and transcriptomes were analyzed. Potassium concentration decreased slowly between 1 h and 24 h after initiation of salt treatment, while the content of potassium was significantly lower after 168 h of salt treatment. Root epidermal cells were shed and a more compact layer of cells formed as the treatment duration increased. Salt stress led to deformation of cells and damage to mitochondria in the epidermis and endodermis, whereas stele cells suffered less damage. Transcriptome analysis identified 10,378 differentially expressed genes (DEGs), with more genes showing differential expression after 24 h and 168 h of exposure than after shorter durations of exposure to salinity. Signal transduction and ion transport genes such as HKT and CHX were enriched among DEGs in the early stages (1 h or 6 h) of salt stress, while expression of genes involved in programmed cell death was significantly upregulated at 168 h, corresponding to changes in ion contents and cell structure of roots. Oxidative stress and detoxification genes were also expressed differentially and were enriched among DEGs at different stages. CONCLUSIONS: These results not only elucidate the mechanism and the molecular pathway governing salt tolerance, but also serve as a basis for identifying gene function related to salt stress in C. equisetifolia.

Comprehensive Analysis of SnRK Gene Family and their Responses to Salt Stress in Eucalyptus grandis.

The sucrose non-fermentation-related protein kinase (SnRK) is a kind of Ser/Thr protein kinase, which plays a crucial role in plant stress response by phosphorylating the target protein to regulate the interconnection of various signaling pathways. However, little is known about the SnRK family in Eucalyptus grandis. Thirty-four putative SnRK sequences were identified in E. grandis and divided into three subgroups (SnRK1, SnRK2 and SnRK3) based on phylogenetic analysis and the type of domain. Chromosome localization showed that SnRK family members are unevenly distributed in the remaining 10 chromosomes, with the notable exception of chromosome 11. Gene structure analysis reveal that 10 of the 24 SnRK3 genes contained no introns. Moreover, conserved motif analyses showed that SnRK sequences belonged to the same subgroup that contained the same motif type of motif. The Ka/Ks ratio of 17 paralogues suggested that the EgrSnRK gene family underwent a purifying selection. The upstream region of EgrSnRK genes enriched with different type and numbers of cis-elements indicated that EgrSnRK genes are likely to play a role in the response to diverse stresses. Quantitative real-time PCR showed that the majority of the SnRK genes were induced by salt treatment. Genome-wide analyses and expression pattern analyses provided further understanding on the function of the SnRK family in the stress response to different environmental salt concentrations.

Genome-Wide Characterization, Evolution, and Expression Profiling of VQ Gene Family in Response to Phytohormone Treatments and Abiotic Stress in Eucalyptus grandis.

VQ genes play important roles in plant development, growth, and stress responses. However, little information regarding the functions of VQ genes is available for Eucalyptus grandis. In our study, genome-wide characterization and identification of VQ genes were performed in E. grandis. Results showed that 27 VQ genes, which divided into seven sub-families (I-VII), were found, and all but two VQ genes showed no intron by gene structure and conserved motif analysis. To further identify the function of EgrVQ proteins, gene expression analyses were also developed under hormone treatments (brassinosteroids, methyl jasmonate, salicylic acid, and abscisic acid) and abiotic conditions (salt stress, cold 4 °C, and heat 42 °C). The results of a quantitative real-time PCR analysis indicated that the EgrVQs were variously expressed under different hormone treatments and abiotic stressors. Our study provides a comprehensive overview of VQ genes in E. grandis, which will be beneficial in the molecular breeding of E. grandis to promote its resistance to abiotic stressors; the results also provide a basis from which to conduct further investigation into the functions of VQ genes in E. grandis.

Chlorothalonil: an effective bacteriostatic agent for bud induction of Acacia auriculiformis under open condition (non-axenic).

BACKGROUND: Open tissue culture technique could be simplified by using different bacteriostatic agents. There is a great difference in the bacteriostatic effects of different antimicrobial agents on various explants. However, there is no report about the effective bacteriostatic agent for open tissue culture of Acacia auriculiformis. RESULTS: We carried out the bud induction trials under open conditions to screen out an effective antibacterial agent for open tissue culture of A. auriculiformis. The results showed that the suitable type and concentration of bacteriostatic agent was 0.2 g L-1 Chlorothalonil, and the suitable explant type was middle shoot section with leaves (the shoot section with third to fifth axillary bud). The treatment of 0.8 g L-1 Carbendazim for 3 min was the most suitable strategy for explants disinfection, and October was the best time for explants collection. The suitable bud induction medium was 1/8 MS + agar 7 g L-1 + Chlorothalonil 0.2 g L-1 + 6-BA 1.5 mg L-1, and the bud induction rate was 99.54%. CONCLUSIONS: Our results revealed that Chlorothalonil is an effective bacteriostatic agent for bud induction of A. auriculiformis under open condition. These results would be very helpful for further establishment of open tissue culture technology for A. auriculiformis.

Characterization of Brassinazole resistant (BZR) gene family and stress induced expression in Eucalyptus grandis.

Brassinosteroids (BRs) are a group of plant hormones which play a pivotal role in modulating cell elongation, stress responses, vascular differentiation and senescence. In response to BRs, BRASSINAZOLE-RESISTANT (BZR) transcription factors (TFs) accumulate in the nucleus, where they modulate thousands of target genes and coordinate many biological processes, especially in regulating defense against biotic and abiotic stresses. In this study, 6 BZR TFs of Eucalyptus grandis (EgrBZR) from a genome-wide survey were characterized by sequence analysis and expression profiling against several abiotic stresses. The results showed that BZR gene family in Eucalyptus was slightly smaller compared to Populus and Arabidopsis, but all phylogenetic groups were represented. Various systematic in silico analysis of these TFs validated the basic properties of BZRs, whereas comparative studies showed a high degree of similarity with recognized BZRs of other plant species. In the organ-specific expression analyses, 4 EgrBZRs were expressed in vascular tissue indicating their possible functions in wood formation. Meanwhile, almost all EgrBZR genes showed differential transcript abundance levels in response to exogenously applied BR, MeJA, and SA, and salt and cold stresses. Besides, protein interaction analysis showed that all EgrBZR genes were associated with BR signaling directly or indirectly. These TFs were proposed as transcriptional activators or repressors of abiotic stress response and growth and development pathways of E. grandis by participating in BR signaling processes. These findings would be helpful in resolving the regulatory mechanism of EgrBZRs in stress resistance conditions but require further functional study of these potential TFs in Eucalyptus.

Genome-wide analysis of Eucalyptus grandis WRKY genes family and their expression profiling in response to hormone and abiotic stress treatment.

The WRKY transcription factors, a large family of proteins in plants, are involved in multiple developmental and biological processes including response to phytohormones and abiotic stress. However, little information is available regarding the WRKY family in Eucalyptus, which has been the most widely planted hardwood trees in tropical and subtropical areas. In this study, a total of 79 WRKY genes (named as EgrWRKY1-79) were identified from the Eucalyptus grandis genome and classified into three main groups according to the phylogenetic analysis, which was further supported by their gene structure and conserved motifs. Of which, 28 EgrWRKYs were involved in tandem duplication but none for segmental duplication, indicating that tandem duplication was the main cause for the expansion of WRKY gene family in E. grandis. Subsequently, expression profiles of EgrWRKY genes in eight different tissues and in response to treatments of three hormones (SA, JA, and BR) and two abiotic stresses (salt and cold) were analyzed. The results revealed that the EgrWRKY genes had differential expression in their transcript abundance and they were differentially expressed in response to plant hormones and salt and cold stresses, suggesting their contributions to plant developmental processes as well as abiotic stresses with the involvement of hormone signaling transduction. Taken together, these findings will increase our understanding of EgrWRKY gene family involved in abiotic stresses and hormone signaling transduction, and also will provide some stress-responsive candidate EgrWRKY genes for further characterization of their functions in Eucalyptus.

Exogenous GA3 application altered morphology, anatomic and transcriptional regulatory networks of hormones in Eucalyptus grandis.

Gibberellins (GAs) play a key role in plant growth and development including cell elongation, cell expansion, and xylem differentiation. Eucalyptus are the world's most widely planted hardwood trees providing fiber and energy. However, the roles of GAs in Eucalyptus remain unclear and their effects on xylem development remain to be determined. In this study, E. grandis plants were treated with 0.10 mg L-1 GA3 and/or paclobutrazol (PAC, a GA inhibitor). The growth of shoot and root were recorded, transverse sections of roots and stems were stained using toluidine blue, and expression levels of genes related to hormone response and secondary cell wall biosynthesis were analyzed by quantitative real-time PCR. The results showed that GA3 dramatically promoted the length of shoot and root, but decreased the diameter of root and stem. Exogenous GA3 application also significantly promoted xylem development in both stem and root. Expression analysis revealed that exogenous GA3 application altered the transcript levels of genes related to the GA biosynthetic pathway and GA signaling, as well as genes related to auxin, cytokinin, and secondary cell wall. These findings suggest that GAs may interact with other hormones (such as auxin and cytokinin) to regulate the expression of secondary cell wall biosynthesis genes and trigger xylogenesis in Eucalyptus plants.