Nitric oxide participates in plant flowering repression by ascorbate.

In Oncidium, redox homeostasis involved in flowering is mainly due to ascorbic acid (AsA). Here, we discovered that Oncidium floral repression is caused by an increase in AsA-mediated NO levels, which is directed by the enzymatic activities of nitrate reductase (NaR) and nitrite reducatase (NiR). Through Solexa transcriptomic analysis of two libraries, 'pseudobulb with inflorescent bud' (PIB) and 'pseudobulb with axillary bud' (PAB), we identified differentially expressed genes related to NO metabolism. Subsequently, we showed a significant reduction of NaR enzymatic activities and NO levels during bolting and blooming stage, suggesting that NO controlled the phase transition and flowering process. Applying AsA to Oncidium PLB (protocorm-like bodies) significantly elevated the NO content and enzyme activities. Application of sodium nitroprusside (-NO donor) on Arabidopsis vtc1 mutant caused late flowering and expression level of flowering-associated genes (CO, FT and LFY) were reduced, suggesting NO signaling is vital for flowering repression. Conversely, the flowering time of noa1, an Arabidopsis NO-deficient mutant, was not altered after treatment with L-galacturonate, a precursor of AsA, suggesting AsA is required for NO-biosynthesis involved in the NO-mediated flowering-repression pathway. Altogether, Oncidium bolting is tightly regulated by AsA-mediated NO level and downregulation of transcriptional levels of NO metabolism genes.

Prolonged exposure to elevated temperature induces floral transition via up-regulation of cytosolic ascorbate peroxidase 1 and subsequent reduction of the ascorbate redox ratio in Oncidium hybrid orchid.

The bolting time of the Oncidium hybrid orchid is not season dependent and so it is a useful year-round model system to study thermal-induced flowering mechanisms in planta. Previously, we reported that a low ascorbate (AsA) content is essential for floral transition in Oncidium; however, the environmental factors governing initiation of the flowering process remained to be elucidated. The current study revealed that a prolonged elevated temperature treatment (30°C over a 14 d period) induces floral transition. This floral induction in response to thermal stress was associated with a significantly increased reactive oxygen species (ROS) level and a lowered AsA redox ratio, as well as prominently up-regulated expression of cytosolic ascorbate peroxidase (cytAPX1). Transcriptome analysis confirmed that increased temperature affected the differential expression of genes involved in antioxidant metabolism. Likewise, transgenic Arabidopsis ectopically overexpressing Oncidium cytAPX1 displayed an early-flowering phenotype and low AsA redox ratio under thermal stress, while cytAPX1 mutants, apx1-1 and apx1-2, exhibited a delayed-flowering phenotype and a high AsA redox ratio. Our present data illustrate that the floral transition response to thermal stress is mediated by the AsA redox ratio, and that CytAPX plays a pivotal role in modulating the AsA redox ratio in Oncidium hybrid orchid. Taken together, the results from this investigation of the thermal-induced flowering mechanism indicated that the AsA redox ratio is a master switch to mediate phase transition from the vegetative to reproductive stage.

RNA interference-based gene silencing of phytoene synthase impairs growth, carotenoids, and plastid phenotype in Oncidium hybrid orchid.

Phytoene synthase (PSY) is the first rate-limiting regulatory enzyme in the carotenoid biosynthesis pathway. In order to modify the floral color pattern by reducing carotenoid contents, a phytoene synthase-RNAi construct was delivered into protocorm-like body (PLB) of Oncidium hybrid orchid. The transgenic orchids show down-regulated level of PSY and geranyl synthase gene. They displayed semi-dwarf phenotype and brilliant green leaves. The microscopic anatomy revealed development-arrested plastids with rare grana. The total carotenoid content was decreased and the efficiency of the photosynthetic electron transport was declined. The chlorophyll level and the expression of chlorophyll biosynthetic genes, such as OgGLUTR and OgCS were dramatically reduced. HPLC analysis showed that the endogenous level of gibberellic acid and abscisic acid in the dwarf transformants are 4-fold lower than in wild type plants. In addition, chilling tolerance of the transgenic Oncidium plants was reduced. The data showed that down-regulation of PSY resulted in alterations of gene expression in enzymes involved in many metabolic pathways, such as carotenoid, gibberellic acid, abscisic acid and chlorophyll biosynthetic pathway as well as causes predominant defects in plant growth and development.

Growth promotion-related miRNAs in Oncidium orchid roots colonized by the endophytic fungus Piriformospora indica.

Piriformospora indica, an endophytic fungus of Sebacinales, colonizes the roots of a wide range of host plants and establishes various benefits for the plants. In this work, we describe miRNAs which are upregulated in Oncidium orchid roots after colonization by the fungus. Growth promotion and vigorous root development were observed in Oncidium hybrid orchid, while seedlings were colonized by P. indica. We performed a genome-wide expression profiling of small RNAs in Oncidium orchid roots either colonized or not-colonized by P. indica. After sequencing, 24,570,250 and 24744,141 clean reads were obtained from two libraries. 13,736 from 17,036,953 unique sequences showed homology to either 86 miRNA families described in 41 plant species, or to 46 potential novel miRNAs, or to 51 corresponding miRNA precursors. The predicted target genes of these miRNAs are mainly involved in auxin signal perception and transduction, transcription, development and plant defense. The expression analysis of miRNAs and target genes demonstrated the regulatory functions they may participate in. This study revealed that growth stimulation of the Oncidium orchid after colonization by P. indica includes an intricate network of miRNAs and their targets. The symbiotic function of P. indica on Oncidium orchid resembles previous findings on Chinese cabbage. This is the first study on growth regulation and development of Oncidium orchid by miRNAs induced by the symbiotic fungus P. indica.

The maturation zone is an important target of Piriformospora indica in Chinese cabbage roots.

The mutualistic symbiont Piriformospora indica exhibits a great potential in agriculture. The interaction between P. indica and Chinese cabbage (Brassica campestris cv. Chinensis) results in growth and biomass promotion of the host plant and in particular in root hair development. The resulting highly bushy root phenotype of colonized Chinese cabbage seedlings differs substantially from reports of other plant species, which prompted the more detailed study of this symbiosis. A large-scale expressed sequence tag (EST) data set was obtained from a double-subtractive EST library, by subtracting the cDNAs of Chinese cabbage root tissue and of P. indica mycelium from those of P. indica-colonized root tissue. The analysis revealed ~700 unique genes rooted in 141 clusters and 559 singles. A total of 66% of the sequences could be annotated in the NCBI GenBank. Genes which are stimulated by P. indica are involved in various types of transport, carbohydrate metabolism, auxin signalling, cell wall metabolism, and root development, including the root hair-forming phosphoinositide phosphatase 4. For 20 key genes, induction by fungal colonization was confirmed kinetically during the interaction by real-time reverse transcription-PCR. Moreover, the auxin concentration increases transiently after exposure of the roots to P. indica. Microscopic analyses demonstrated that the development of the root maturation zone is the major target of P. indica in Chinese cabbage. Taken together, the symbiotic interaction between Chinese cabbage and P. indica is a novel model to study root growth promotion which, in turn, is important for agriculture and plant biotechnology.

Proteomic profiling of proteins associated with the rejuvenation of Sequoia sempervirens (D. Don) Endl.

BACKGROUND: Restoration of rooting competence is important for rejuvenation in Sequoia sempervirens (D. Don) Endl and is achieved by repeatedly grafting Sequoia shoots after 16 and 30 years of cultivation in vitro. RESULTS: Mass spectrometry-based proteomic analysis revealed three proteins that differentially accumulated in different rejuvenation stages, including oxygen-evolving enhancer protein 2 (OEE2), glycine-rich RNA-binding protein (RNP), and a thaumatin-like protein. OEE2 was found to be phosphorylated and a phosphopeptide (YEDNFDGNSNVSVMVpTPpTDK) was identified. Specifically, the protein levels of OEE2 increased as a result of grafting and displayed a higher abundance in plants during the juvenile and rejuvenated stages. Additionally, SsOEE2 displayed the highest expression levels in Sequoia shoots during the juvenile stage and less expression during the adult stage. The expression levels also steadily increased during grafting. CONCLUSION: Our results indicate a positive correlation between the gene and protein expression patterns of SsOEE2 and the rejuvenation process, suggesting that this gene is involved in the rejuvenation of Sequoia sempervirens.

The signal network of ascorbate homeostasis.

Ascorbate is a crucial antioxidant for scavenging hydrogen peroxide generated from the physiological processes and environmental stresses. Besides, the endo/exogenous factors that influence ascorbate level, our knowledge of the methanol stimulation is relatively less. Methanol, a byproduct from the demethylation of pectin during the enlargement of plant cell, is effective in enhancing the expression of ascorbate-biosynthetic genes of the Smirnoff-Wheeler and galacturonate (GalUA) pathways. In our previous work, hydrogen peroxide is a product of methanol detoxification through alcohol oxidase and NADPH oxidase activation, and acts as a secondary messenger for the activation of ascorbate-related genes. In this addendum, we propose a working model of the signaling network for ascorbate homeostasis in association with the apoplastic factors, such as methanol and oligogalacturonide during the growth and development of plant cell.

Hydrogen peroxide mediates the expression of ascorbate-related genes in response to methanol stimulation in Oncidium.

We investigated the signaling role of hydrogen peroxide (H(2)O(2)) in regulating the ascorbate (AsA) level after exogenous methanol (MeOH) application. The endogenous H(2)O(2) and AsA levels as well as the expression of related genes were monitored after MeOH treatment of cultures of Oncidium protocorm-like bodies (PLB). A high MeOH concentration was deleterious and caused irreversible consumption of endogenous AsA. However, a low MeOH concentration (50mM) triggered the synthesis of H(2)O(2) and was effective in enhancing the expression of AsA-biosynthetic genes of the Smirnoff-Wheeler and galacturonate (GalUA) pathways. The increased expression of these genes could be blocked by the addition of hydroxylamine, an inhibitor of alcohol oxidase (EC: 1.1.3.13), and diphenyleneiodonium chloride (DPI), an inhibitor of NADPH oxidase (EC: 1.6.3.1). Thus, the H(2)O(2) generated by MeOH application is a product of MeOH detoxification through alcohol oxidase and NADPH oxidase activation. In this chain, H(2)O(2) acts as a secondary messenger for the activation of AsA-related genes. Our results reveal the signaling function of H(2)O(2) and cellular AsA homeostasis in Oncidium orchids in response to MeOH stimulation. A mechanism for the MeOH effect on AsA production is suggested.

Decreased L-ascorbate content mediating bolting is mainly regulated by the galacturonate pathway in Oncidium.

We investigated the alteration in l-ascorbate (AsA, reduced form) content and the expression pattern of its related genes during the phase transition in Oncidium orchid. During the vegetative growth, a high H2O2 level was associated with a high content of the reduced form of AsA. During the bolting period, the AsA content and H2O2 level were greatly reduced in parallel with increased expression of OgLEAFY, the gene encoding a key transcription factor integrating different flowering-inducing pathways. This observation suggests that reduced AsA content, due to it having been consumed in scavenging H2O2, is a prerequisite for mediating the phase transition in Oncidium. A survey of the AsA biosynthetic pathway revealed that the gene expression and enzymatic activities of the products of relevant genes of the galacturonate (GalUA) pathway, such as polygalacturonase (OgPG), pectin methylesterase (OgPME) and galacturonate reductase (OgGalUAR), were markedly decreased during the bolting period, as compared with during the vegetative stage. However, the genes whose products were involved in the Smirnoff-Wheeler pathway retained a similar expression level in the two growth stages. The data suggested that OgPME of the GalUA pathway was the pivotal gene in regulating AsA biosynthesis during the bolting period. Further elucidation by overexpressing OgPME in Arabidopsis demonstrated a considerable increase in AsA content, as well as a resulting delayed-flowering phenotype. Our results strongly imply that the reduced level of AsA, regulating bolting for phase transition, resulting in part from its consumption by scavenging H2O2, was mainly caused by the down-regulation of the GalUA pathway, not the Smirnoff-Wheeler pathway.