Transcriptomic and Phenotypic Analyses Reveal the Molecular Mechanism of Dwarfing in Tetraploid Robinia pseudoacacia L.

Polyploid breeding techniques aid in the cultivation of new forestry cultivars, thus expanding the suite of strategies for the improvement of arboreal traits and innovation within the field of forestry. Compared to diploid Robinia pseudoacacia L. (black locust) 'D26-5①' (2×), its dwarfed homologous tetraploid 'D26-5②' (4×) variety has better application prospects in garden vegetation guardrails and urban landscape. However, the molecular mechanism of the generation and growth of this dwarf variety is still unclear. Here, plant growth and development as well as histological differences between the diploid and its autotetraploid were investigated. Levels of endogenous hormones at three different developmental stages (20, 40, and 70 days) of 2× and homologous 4× tissue culture plantlets were assessed, and it was found that the brassinosteroid (BR) contents of the former were significantly higher than the latter. Transcriptome sequencing data analysis of 2× and homologous 4× showed that differentially expressed genes (DEGs) were significantly enriched in plant hormone synthesis and signal transduction, sugar and starch metabolism, and the plant circadian rhythm pathway, which are closely related to plant growth and development. Therefore, these biological pathways may be important regulatory pathways leading to dwarfism and slow growth in tetraploids. Additionally, utilizing weighted gene coexpression network analysis (WGCNA), we identified three crucial differentially expressed genes (DEGs)-PRR5, CYP450, and SPA1-that potentially underlie the observed ploidy variation. This study provides a new reference for the molecular mechanism of dwarfism in dwarfed autotetraploid black locusts. Collectively, our results of metabolite analysis and comparative transcriptomics confirm that plant hormone signaling and the circadian rhythm pathway result in dwarfism in black locusts.

Identification of Robinia pseudoacacia target proteins responsive to Mesorhizobium amphore CCNWGS0123 effector protein NopT.

Nodulation outer proteins secreted via type 3 secretion systems are involved in the process of symbiosis between legume plants and rhizobia. To study the function of NopT in symbiosis, we mutated nopT in Mesorhizobium amphore CCNWGS0123 (GS0123), which can nodulate black locust (Robinia pseudoacacia). The nopT mutant induced higher levels of jasmonic acid, salicylic acid, and hydrogen peroxide accumulation in the roots of R. pseudoacacia compared with wild-type GS0123. The ΔnopT mutant induced higher disease-resistant gene expression 72 hours post-inoculation (hpi), whereas GS0123 induced higher disease-resistant gene expression earlier, at 36 hpi. Compared with the nopT mutant, GS0123 induced the up-regulation of most genes at 36 hpi and the down-regulation of most genes at 72 hpi. Proteolytically active NopT_GS0123 induced hypersensitive responses when expressed transiently in tobacco leaves (Nicotiana benthamiana). Two NopT_GS0123 targets in R. pseudoacacia were identified, ATP-citrate synthase alpha chain protein 2 and hypersensitive-induced response protein. Their interactions with NopT_GS0123 triggered resistance by the plant immune system. In conclusion, NopT_GS0123 inhibited the host plant immune system and had minimal effect on nodulation in R. pseudoacacia. Our results reveal the underlying molecular mechanism of NopT function in plant-symbiont interactions.

The Rpf84 gene, encoding a ribosomal large subunit protein, RPL22, regulates symbiotic nodulation in Robinia pseudoacacia.

MAIN CONCLUSION: A homologue of the ribosomal protein L22e, Rpf84, regulates root nodule symbiosis by mediating the infection process of rhizobia and preventing bacteroids from degradation in Robinia pseudoacacia. Ribosomal proteins (RPs) are known to have extraribosomal functions, including developmental regulation and stress responses; however, the effects of RPs on symbiotic nodulation of legumes are still unclear. Ribosomal protein 22 of the large 60S subunit (RPL22), a non-typical RP that is only found in eukaryotes, has been shown to function as a tumour suppressor in animals. Here, a homologue of RPL22, Rpf84, was identified from the leguminous tree R. pseudoacacia. Subcellular localization assays showed that Rpf84 was expressed in the cytoplasm and nucleus. Knockdown of Rpf84 by RNA interference (RNAi) technology impaired the infection process and nodule development. Compared with the control, root and stem length, dry weight and nodule number per plant were drastically decreased in Rpf84-RNAi plants. The numbers of root hair curlings, infection threads and nodule primordia were also significantly reduced. Ultrastructure analyses showed that Rpf84-RNAi nodules contained fewer infected cells with fewer bacteria. In particular, remarkable deformation of bacteroids and fusion of multiple symbiosomes occurred in infected cells. By contrast, overexpression of Rpf84 promoted nodulation, and the overexpression nodules maintained a larger infection/differentiation region and had more infected cells filled with bacteroids than the control at 45 days post inoculation, suggesting a retarded ageing process in nodules. These results indicate for the first time that RP regulates the symbiotic nodulation of legumes and that RPL22 may function in initiating the invasion of rhizobia and preventing bacteroids from degradation in R. pseudoacacia.

Comparative mitochondrial proteomic, physiological, biochemical and ultrastructural profiling reveal factors underpinning salt tolerance in tetraploid black locust (Robinia pseudoacacia L.).

BACKGROUND: Polyploidy is an important phenomenon in plants because of its roles in agricultural and forestry production as well as in plant tolerance to environmental stresses. Tetraploid black locust (Robinia pseudoacacia L.) is a polyploid plant and a pioneer tree species due to its wide ranging adaptability to adverse environments. To evaluate the ploidy-dependent differences in leaf mitochondria between diploid and tetraploid black locust under salinity stress, we conducted comparative proteomic, physiological, biochemical and ultrastructural profiling of mitochondria from leaves. RESULTS: Mitochondrial proteomic analysis was performed with 2-DE and MALDI-TOF-MS, and the ultrastructure of leaf mitochondria was observed by transmission electron microscopy. According to 2-DE analysis, 66 proteins that responded to salinity stress significantly were identified from diploid and/or tetraploid plants and classified into 9 functional categories. Assays of physiological characters indicated that tetraploids were more tolerant to salinity stress than diploids. The mitochondrial ultrastructure of diploids was damaged more severely under salinity stress than that of tetraploids. CONCLUSIONS: Tetraploid black locust possessed more tolerance of, and ability to acclimate to, salinity stress than diploids, which may be attributable to the ability to maintain mitochondrial structure and to trigger different expression patterns of mitochondrial proteins during salinity stress.

De novo sequencing and comparative transcriptome analysis of adventitious root development induced by exogenous indole-3-butyric acid in cuttings of tetraploid black locust.

BACKGROUND: Indole-3-butyric acid (IBA) is applied to the cuttings of various plant species to induce formation of adventitious roots (ARs) in commercial settings. Tetraploid black locust is an attractive ornamental tree that is drought resistant, sand tolerant, can prevent sand erosion and has various commercial uses. To further elucidate the mechanisms of AR formation, we used Illumina sequencing to analyze transcriptome dynamics and differential gene expression at four developmental stages in control (CK) and IBA-treated groups. RESULTS: The short reads were assembled into 127,038 unitranscripts and 101,209 unigenes, with average lengths of 986 and 852 bp. In total, 10,181 and 14,924 differentially expressed genes (DEGs) were detected in the CK and IBA-treated groups, respectively. Comparison of the four consecutive developmental stages showed that 282 and 260 DEGs were shared between IBA-treated and CK, suggesting that IBA treatment increased the number of DEGs. We observed 1,721 up-regulated and 849 down-regulated genes in CI vs. II, 849 up-regulated and 836 down-regulated genes in CC vs. IC, 881 up-regulated and 631 down-regulated genes in CRP vs. IRP, and 5,626 up-regulated and 4,932 down-regulated genes in CAR vs. IAR, of which 25 up-regulated DEGs were common to four pairs, and these DEGs were significantly up-regulated at AR. These results suggest that substantial changes in gene expression are associated with adventitious rooting. GO functional category analysis indicated that IBA significantly up- or down-regulated processes associated with regulation of transcription, transcription of DNA dependent, integral to membrane and ATP binding during the development process. KEGG pathway enrichment indicated that glycolysis/gluconeogenesis, cysteine and methionine metabolism, photosynthesis, nucleotide sugar metabolism, and lysosome were the pathways most highly regulated by IBA. We identified a number of differentially regulated unigenes, including 12 methionine-related genes and 12 ethylene-related genes, associated with the KEGG pathway cysteine and methionine metabolism. The GO enrichment, pathway mapping, and gene expression profile analyses revealed molecular traits for root induction and initiation. CONCLUSION: Our study presents a global view of the transcriptomic profiles of tetraploid black locust cuttings in response to IBA treatment and provides new insights into the fundamental mechanisms associated with auxin-induced adventitious rooting.

Molecular cloning and expression analysis of the MTN gene during adventitious root development in IBA-induced tetraploid black locust.

5'-Methylthioadenosine (MTA) nucleosidase (MTN) plays a key role in the methionine (Met) recycling pathway of plants. Here, we report the isolation of the 1158 bp full-length, cDNA sequence encoding tetraploid black locust (Robinia pseudoacacia L.) MTN (TrbMTN), which contains an open reading frame of 810 bp that encodes a 269 amino acid protein. The amino acid sequence of TrbMTN has more than 88% sequence identity to the MTNs from other plants, with a closer phylogenetic relationship to MTNs from legumes than to MTNs from other plants. Subcellular localization analysis revealed that the TrbMTN gene localizes mainly to the cell membrane and cytoplasm of onion epidermal cells. Indole-3-butyric acid (IBA)-treated cuttings showed higher TrbMTN transcript levels than untreated control cuttings during root primordium and adventitious root formation. TrbMTN and key Met cycle genes showed differential expression in shoots, leaves, stems, and roots, with the highest expression observed in stems. IBA-treated cuttings also showed higher TrbMTN activity than control cuttings during root primordium and adventitious root formation. These results indicate that TrbMTN gene might play an important role in the regulation of IBA-induced adventitious root development in tetraploid black locust cuttings.

Molecular cloning, characterization and expression analysis of the SAMS gene during adventitious root development in IBA-induced tetraploid black locust.

S-Adenosylmethionine synthetase (SAMS) catalyzes the synthesis of S-adenosylmethionine (SAM), a precursor for ethylene and polyamine biosynthesis. Here, we report the isolation of the 1498 bp full-length cDNA sequence encoding tetraploid black locust (Robinia pseudoacacia L.) SAMS (TrbSAMS), which contains an open reading frame of 1179 bp encoding 392 amino acids. The amino acid sequence of TrbSAMS has more than 94% sequence identity to SAMSs from other plants, with a closer phylogenetic relationship to SAMSs from legumes than to SAMS from other plants. The TrbSAMS monomer consists of N-terminal, central, and C-terminal domains. Subcellular localization analysis revealed that the TrbSAMS protein localizes mainly to in the cell membrane and cytoplasm of onion epidermal cells and Arabidopsis mesophyll cell protoplasts. Indole-3-butyric acid (IBA)-treated cuttings showed higher levels of TrbSAMS transcript than untreated control cuttings during root primordium and adventitious root formation. TrbSAMS and its downstream genes showed differential expression in shoots, leaves, bark, and roots, with the highest expression observed in bark. IBA-treated cuttings also showed higher SAMS activity than control cuttings during root primordium and adventitious root formation. These results indicate that TrbSAMS might play an important role in the regulation of IBA-induced adventitious root development in tetraploid black locust cuttings.

Physiological and proteomic responses of diploid and tetraploid black locust (Robinia pseudoacacia L.) subjected to salt stress.

Tetraploid black locust (Robinia pseudoacacia L.) is adaptable to salt stress. Here, we compared morphological, physiological, ultrastructural, and proteomic traits of leaves in tetraploid black locust and its diploid relatives under salt stress. The results showed that diploid (2×) plants suffered from greater negative effects than those of tetraploid (4×) plants. After salt treatment, plant growth was inhibited, photosynthesis was reduced, reactive oxygen species, malondialdehyde content, and relative electrolyte leakage increased, and defense-related enzyme activities decreased in 2× compared to those in 4×. In addition, salt stress resulted in distorted chloroplasts, swollen thylakoid membranes, accumulation of plastoglobules, and increased starch grains in 2× compared to those in 4×. However, 4× developed diverse responses under salt stress. A comparative proteomic analysis revealed that 41 and 37 proteins were differentially expressed in 2× and 4×, respectively. These proteins were mainly involved in photosynthesis, stress and defense, energy, metabolism, transcription/translation, and transportation. Distinct patterns of protein changes between 2× and 4× were analyzed. Collectively, our results suggest that the plants showed significantly different responses to salt stress based on ploidy level of the plant. The 4× possessed a better salt protection mechanism than that of 2×, suggesting salt tolerance in the polyploid plant.

A novel family of lectins evolutionarily related to class V chitinases: an example of neofunctionalization in legumes.

A lectin has been identified in black locust (Robinia pseudoacacia) bark that shares approximately 50% sequence identity with plant class V chitinases but is essentially devoid of chitinase activity. Specificity studies indicated that the black locust chitinase-related agglutinin (RobpsCRA) preferentially binds to high-mannose N-glycans comprising the proximal pentasaccharide core structure. Closely related orthologs of RobpsCRA could be identified in the legumes Glycine max, Medicago truncatula, and Lotus japonicus but in no other plant species, suggesting that this novel lectin family most probably evolved in an ancient legume species or possibly an earlier ancestor. This identification of RobpsCRA not only illustrates neofunctionalization in plants, but also provides firm evidence that plants are capable of developing a sugar-binding domain from an existing structural scaffold with a different activity and accordingly sheds new light on the molecular evolution of plant lectins.

High somatic instability of a microsatellite locus in a clonal tree, Robinia pseudoacacia.

Robinia pseudoacacia L. is a clonal tree species. To investigate a mutation within eight microsatellite loci of R. pseudoacacia, we analyzed DNA samples obtained from different leaf samples within each ramet, leaves from ramets within the genet, and seeds. Of the eight loci, locus Rops15 (AG motif) displayed hypermutability. The mutation rates of Rops15 within each ramet, among ramets within the genet, and offspring were 6.27% (ranging from 0 to 31.1%), 6.11% (from 0 to 25.0%) and 3.78% (from 0 to 10.9%), respectively. The mutation rate increased with allele size (13-71 repeat units). The mutation patterns observed in Rops15 were distinctive in two ways. First, there was a significant bias toward additions over deletions, and both addition and deletion of single repeats were dominant at alleles with lengths less than 232 bp (63 repeats). Second, for the longest allele of 248 bp (71 repeats), the number of losses was higher than the number of gains. These observations suggest that the mutation patterns of microsatellites in R. pseudoacacia may follow a generalized stepwise mutation model, and that the tendency of long alleles to mutate to shorter lengths acts to prevent infinite growth. Finally, the observation of somatic hypermutability at locus Rops15 highlights the need for caution when using highly polymorphic microsatellites for population genetic structure and paternity analysis in tree species.

An effective method for axillary bud culture and RAPD analysis of cloned plants in tetraploid black locust.

An efficient micropropagation technique by axillary bud multiplication was established for cloning tetraploid black locust tree (Robinia pseudoacacia L.). The result showed that the optimal medium for shoot multiplication and elongation was Murashige and Skoog (MS) medium supplemented with 0.5 mg/l 6-benzylaminopurine in combination with 0.5 mg/l kinetin and 0.1 mg/l 1-naphthaleneacetic acid. The best medium for rooting was half-strength MS medium with 0.25 mg/l indole-3-butyric acid. In the present report, we examined the genetic fidelity of the micropropagated plants by the randomly amplified polymorphic DNA (RAPD) method with 25 primers. The cloned plants of tetraploid black locust showed complete stability.