Characterization of HAK protein family in Casuarina equisetifolia and the positive regulatory role of CeqHAK6 and CeqHAK11 genes in response to salt tolerance.

The potassium transporter group of the HAK/KUP/KT (high-affinity K+)/KUP (K+ uptake)/KT (K+ transporter) family plays a crucial role in plant growth and development as well as in environmental adaptation such as tolerance to salt stress. HAK/KUP/KT genes and their functions have been characterized for a number of plant species, but they remain unknown for Casuarina equisetifolia, an important tree species for coastal protection in southern China and many other countries. In this study, 25 HAK genes were identified in the C. equisetifolia genome. Their gene structure, conserved motif, phylogeny, and expression were comprehensively and systematically analyzed to understand their functions. All HAK genes were relatively conserved and could be divided into four clusters. The expression level of two particular genes, CeqHAK11 and CeqHAK6, increased significantly with the duration of salt treatment. To further elucidated their function in response to salt stress, subcellular localization, and their functional analysis were developed. Results revealed that CeqHAK11 and CeqHAK6 were localized on the plasma membrane, which mainly mediated high-affinity K+ uptake. Overexpression of CeqHAK6 or CeqHAK11 in Arabidopsis showed higher germination and survival rates and longer root length than wild-type (WT) under salt stress, suggesting that both genes improve tolerance to salt stress. Moreover, CeqHAK6 and CeqHAK11 improved their ability to tolerate salt stress by increasing the K+/Na+ ratio and antioxidant enzyme activities (CAT, POD, and SOD), and decreasing reactive oxygen species (ROS) accumulation. Consequently, CeqHAK6 and CeqHAK11 were verified as potassium transport proteins and could be applied for further molecular breeding for salt tolerance in C. equisetifolia or other crops to increasing salt tolerance.

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.

Genome-wide analysis of MYB transcription factors and their responses to salt stress in Casuarina equisetifolia.

BACKGROUND: MYB transcription factors are a kind of DNA binding protein that can specifically interact with the promoter region. Members of MYB TFs are widely involved in plant growth and development, secondary metabolism, stress response, and hormone signal transduction. However, there is no report of comprehensive bioinformatics analysis on the MYB family of Casuarina equisetifolia. RESULTS: In this study, bioinformatics methods were used to screen out 182 MYB transcription factors from the Casuarina equisetifolia genome database, including 69 1R-MYB, 107 R2R3-MYB, 4 R1R2R3-MYB, and 2 4R-MYB. The C. equisetifolia R2R3-MYB genes were divided into 29 groups based on the phylogenetic topology and the classification of the MYB superfamily in Arabidopsis thaliana, while the remaining MYB genes (1R-MYB, R1R2R3-MYB, and 4R-MYB) was divided into 19 groups. Moreover, the conserved motif and gene structure analysis shown that the members of the CeqMYBs were divided into the same subgroups with mostly similar gene structures. In addition, many conserved amino acids in the R2 and R3 domains of CeqMYBs by WebLogo analysis, especially tryptophan residues (W), with 3 conserved W in R2 repeat and 2 conserved W in R3 repeat. Combining promoter and GO annotation analysis, speculated on the various biological functions of CeqMYBs, thus 32 MYB genes were selected to further explore its response to salt stress by using qPCR analysis technique. Most CeqMYB genes were differentially regulated following multiple salt treatments. CONCLUSIONS: Seven genes (CeqMYB164, CeqMYB4, CeqMYB53, CeqMYB32, CeqMYB114, CeqMYB71 and CeqMYB177) were assigned to the "response to salt stress" by GO annotation. Among them, the expression level of CeqMYB4 was up-regulated under various salt treatments, indicating CeqMYB4 might participated in the response to salt stress. Our results provide important information for the biological function of C. equisetifolia, as well as offer candidate genes for further study of salt stress mechanism.

Transcriptome and metabolome profiling in naturally infested Casuarina equisetifolia clones by Ralstonia solanacearum.

Casuarina equisetifolia is an important pioneer tree and suffers from bacterial wilt caused by Ralstonia solanacearum. We collected resistant (R) and susceptible (S) C. equisetifolia clones naturally infected by R. solanacearum and compared their transcriptome and metabolome with a clone (CK) from a non-infested forest, in order to study their response and resistance to bacterial wilt. We identified 18 flavonoids differentially accumulated among the three clonal groups as potential selection biomarkers against R. solanacearum. Flavonoid synthesis-related genes were up-regulated in the resistant clones, probably enhancing accumulation of flavonoids and boosting resistance against bacterial wilt. The down-regulation of auxin/indoleacetic acid-related genes and up-regulation of brassinosteroid, salicylic acid and jasmonic acid-related differentially expressed genes in the R vs CK and R vs S clonal groups may have triggered defense signals and increased expression of defense-related genes against R. solanacearum. Overall, this study provides an important insight into pathogen-response and resistance to bacterial wilt 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.

The role of Frankia inoculation in casuarina plantations in China.

Casuarina trees are planted along the coast from Hainan province in South China to the Zhoushan Islands of Zhejiang province in Southeastern China. Three key species, Casuarina equisetifolia, Casuarina cunninghamiana and Casuarina glauca, are used as windbreaks, in agroforestry systems, and for the production of timber and fuel wood. Frankia have been studied in China since 1984. Today, Frankia research fields are very wide, and cover morphology, physiology and genetic diversity, and the application of inocula for specific purposes on poor quality sites. In this paper, we review the role of Frankia inoculations in nurseries and casuarina plantations in China and discuss the benefits of inoculation.

Casuarina glauca: a model tree for basic research in actinorhizal symbiosis.

Casuarina glauca is a fast-growing multipurpose tree belonging to the Casuarinaceae family and native to Australia. It requires limited use of chemical fertilizers due to the symbiotic association with the nitrogen-fixing actinomycete Frankia and with mycorrhizal fungi, which help improve phosphorous and water uptake by the root system. C. glauca can grow in difficult sites, colonize eroded lands and improve their fertility, thereby enabling the subsequent growth of more demanding plant species. As a result, this tree is increasingly used for reforestation and reclamation of degraded lands in tropical and subtropical areas such as China and Egypt. Many tools have been developed in recent years to explore the molecular basis of the interaction between Frankia and C. glauca. These tools include in vitro culture of the host and genetic transformation with Agrobacterium, genome sequencing of Frankia and related studies, isolation of plant symbiotic genes combined with functional analyses (including knock-down expression based on RNA interference), and transcriptome analyses of roots inoculated with Frankia or Rhizophagus irregularis. These efforts have been fruitful since recent results established that many common molecular mechanisms regulate the nodulation process in actinorhizal plants and legumes, thus providing new insights into the evolution of nitrogen-fixing symbioses.

Post-transcriptional gene silencing in the root system of the actinorhizal tree Allocasuarina verticillata.

In recent years, RNA interference has been exploited as a tool for investigating gene function in plants. We tested the potential of double-stranded RNA interference technology for silencing a transgene in the actinorhizal tree Allocasuarina verticillata. The approach was undertaken using stably transformed shoots expressing the beta-glucuronidase (GUS) gene under the control of the constitutive promoter 35S; the shoots were further transformed with the Agrobacterium rhizogenes A4RS containing hairpin RNA (hpRNA) directed toward the GUS gene, and driven by the 35S promoter. The silencing and control vectors contained the reporter gene of the green fluorescent protein (GFP), thus allowing a screening of GUS-silenced composite plantlets for autofluorescence. With this rapid procedure, histochemical data established that the reporter gene was strongly silenced in both fluorescent roots and actinorhizal nodules. Fluorometric data further established that the level of GUS silencing was usually greater than 90% in the hairy roots containing the hairpin GUS sequences. We found that the silencing process of the reporter gene did not spread to the aerial part of the composite A. verticillata plants. Real-time quantitative polymerase chain reaction showed that GUS mRNAs were substantially reduced in roots and, thereby, confirmed the knock-down of the GUS transgene in the GFP(+) hairy roots. The approach described here will provide a versatile tool for the rapid assessment of symbiotically related host genes in actinorhizal plants of the Casuarinaceae family.