Integrative transcriptomic and metabolomic analyses provide insight into the long-term submergence response mechanisms of young Salix variegata stems.

MAIN CONCLUSION: The mechanisms underlying long-term complete submergence tolerance in S. variegata involve enhanced oxidative stress responses, strengthened ethylene and ABA signaling, synthesis of raffinose family oligosaccharides, unsaturated fatty acids, and specific stress-related amino acids. Salix variegata Franch. is a riparian shrub species that can tolerate long-term complete submergence; however, the molecular mechanisms underlying this trait remain to be elucidated. In this study, we subjected S. variegata plants to complete submergence for 60 d and collected stems to perform transcriptomic and metabolomic analyses, as well as quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays. Results revealed that photosynthesis and the response to light stimulus were inhibited during submergence and recovered after desubmergence. Ethylene and abscisic acid (ABA) signaling could be important for the long-term submergence tolerance of S. variegata. Jasmonic acid (JA) signaling also participated in the response to submergence. Raffinose family oligosaccharides, highly unsaturated fatty acids, and specific stress-related amino acids accumulated in response to submergence, indicating that they may protect plants from submergence damage, as they do in response to other abiotic stressors. Several organic acids were produced in S. variegata plants after submergence, which may facilitate coping with the toxicity induced by submergence. After long-term submergence, cell wall reorganization and phenylpropanoid metabolic processes (the synthesis of specific phenolics and flavonoids) were activated, which may contribute to long-term S. variegata submergence tolerance; however, the detailed mechanisms require further investigation. Several transcription factors (TFs), such as MYB, continuously responded to submergence, indicating that they may play important roles in the responses and adaption to submergence. Genes related to oxidative stress tolerance were specifically expressed after desubmergence, potentially contributing to recovery of S. variegata plants within a short period of time.

Isolation and promoter analysis of a chalcone synthase gene PtrCHS4 from Populus trichocarpa.

As perennial plants, Populus species are constantly exposed to environmental stresses, such as wounding and pathogen attack, which lead to production of compounds including lignin, flavonoids and phytoalexins. Chalcone synthase (CHS) is a key enzyme in the flavonoid biosynthesis pathway. In this study, a cDNA clone encoding CHS was isolated from Populus trichocarpa by reverse transcription-polymerase chain reaction (RT-PCR). The full-length cDNA, named PtrCHS4, was 1,314 bp with a 1,173 bp open reading frame that corresponded to a deduced protein of 391 amino acid residues. Multiple sequence alignments showed that PtrCHS4 shared high homology with CHS proteins from other plants. Phylogenetic analysis revealed that PtrCHS4 was most closely related to PhCHS from Petunia hybrida and NaCHS from Nicotiana attenuata. Semi-quantitative RT-PCR analysis identified that the PtrCHS4 gene was abundantly expressed in the leaves and stems, while its expression was drastically reduced in the roots. Transcript abundance of PtrCHS4 was stimulated by 2.5-fold within 24 h of wounding treatment. Promoter analysis confirmed that the PtrCHS4 promoter was capable of directing expression of the GUS reporter in both wounded and unwounded leaves of transgenic Chinese white poplar (P. tomentosa Carr.), indicating that the PtrCHS4 promoter is systemically responsive to wounding stimuli. Furthermore, promoter deletion analysis showed that the proximal 1,592 bp from the transcription start site were required for promoter activation by jasmonic acid and the -1,096 to -148 region was proved to be necessary for establishing wound-induced pattern of expression.

[Induction and culture of hairy-root by Agrobacteriun rhizogenes in Dendrobium nobile].

OBJECTIVE: To obtain the hairy-roots of Dendrobium nobile. METHODS: The hairy-root were obtained by infecting different explants with Agrobacteriun rhizogenes A4 and culture conditions of hariy root were optimized. RESULT: The best conditions of hairy root culture were 1/2MS medium with sucrose (3%) as carbon material and lactalbumin hydrolyze (1g/L) as nitron material.

[Agrobacterium-mediated transformation of LJAMP2 gene into 'Red Sun' kiwifruit and its molecular identification].

Bacterial canker caused by Pseudomonas syringae pv. Actinidiae is one of the most important diseases of kiwifruit (Actinidia chinensis) and leads to considerable yield losses. In order to obtain transgenic plants with resistance for 'Red Sun' kiwifruit to canker disease, a non-specific lipid transfer protein-like antimicrobial protein gene (LJAMP2) from motherwort (Leonurus japonicus) was introduced into 'Red Sun' kiwifruit through Agrobacterium-mediated transformation. After two days of co-cultivation with A. tumefaciens strain LBA4404 harboring 35S:LJAMP2, the transformed explants were transferred to the selection medium containing 25 mg/L kanamycin+3.0 mg/L BA+1.0 mg/L NAA. The regeneration efficiency of kanamycin-resistant shoots reached to 85%. All (100%) of kanamycin-resistant shoots rooted on half-strength MS medium supplemented with 0.8 mg/L IBA and a total of 40 regenerated plantlets were obtained. PCR and histochemical GUS activity analysis show that 23 of 40 lines (57.50%) were positive, suggesting that the LJAMP2 gene was integrated into the genome of 'Red Sun' kiwifruit. Taken together, we established an efficient genetic transformation method for 'Red Sun' kiwifruit using A. tumefaciens and the transformation frequency reached 5.11%. This protocol will be useful for the genetic breeding of 'Red Sun' kiwifruit for improvement of disease resistance.

[Establishment of high frequency regeneration via leaf explants of 'Red Sun' kiwifruit (Actinidia chinensis)].

A high efficient in vitro regeneration protocol was developed from leaf explants of the female 'Red Sun' kiwifruit (Actinidia chinensis) and the multiplication coefficient and rooting rate of adventitious buds were also optimized. This method does not require formation of callus tissues which leads to somaclonal variations. The results show that the adventitious buds developing directly from explants tissue were noticed after 30 d of culture. The maximum regeneration frequency of adventitious buds is 100% and 18.67 shoots was observed in each leaf explants when MS medium was supplemented with 3.0 mg/L BA+1.0 mg/L NAA. The optimal culture medium for bud multiplication is MS+2.0 mg/L BA+1.0 mg/L NAA+0.1 mg/L GA3 and the multiplication coefficient reached 8.63. On the rooting medium with 1/2 MS+0.8 mg/L IBA for 15 d, the adventitious plantlets were transferred into matrix perlite supplied with 1/2 MS liquid medium for 15 d and the rooting rate reached 100%. 95 out of 98 plantlets (96.94%) survived acclimatization, producing healthy plants in the greenhouse. Taken together, a highly efficient regeneration method via leaf explants of 'Red Sun' kiwifruit was successfully established. This protocol may be useful for micropropagation and genetic transformation studies of 'Red Sun' kiwifruit.

Molecular cloning and expression profiling of a chalcone synthase gene from hairy root cultures of Scutellaria viscidula Bunge.

A cDNA encoding chalcone synthase (CHS), the key enzyme in flavonoid biosynthesis, was isolated from hairy root cultures of Scutellaria viscidula Bunge by rapid amplification of cDNA ends (RACE). The full-length cDNA of S. viscidula CHS, designated as Svchs (GenBank accession no. EU386767), was 1649 bp with a 1170 bp open reading frame (ORF) that corresponded to a deduced protein of 390 amino acid residues, a calculated molecular mass of 42.56 kDa and a theoretical isoelectric point (pI) of 5.79. Multiple sequence alignments showed that SvCHS shared high homology with CHS from other plants. Functional analysis in silico indicated that SvCHS was a hydrophilic protein most likely associated with intermediate metabolism. The active sites of the malonyl-CoA binding motif, coumaroyl pocket and cyclization pocket in CHS of Medicago sativa were also found in SvCHS. Molecular modeling indicated that the secondary structure of SvCHS contained mainly α-helixes and random coils. Phylogenetic analysis showed that SvCHS was most closely related to CHS from Scutellaria baicalensis. In agreement with its function as an elicitor-responsive gene, the expression of Svchs was induced and coordinated by methyl jasmonate. To our knowledge, this is the first report to describe the isolation and expression of a gene from S. viscidula.