mRNA-Seq and miRNA-Seq Analyses Provide Insights into the Mechanism of Pinellia ternata Bulbil Initiation Induced by Phytohormones.

Pinellia ternata (Thunb.) Breit (abbreviated as P. ternata) is a plant with an important medicinal value whose yield is restricted by many factors, such as low reproductive efficiency and continuous cropping obstacles. As an essential breeding material for P. ternata growth and production, the bulbils have significant advantages such as a high survival rate and short breeding cycles. However, the location effect, influencing factors, and molecular mechanism of bulbil occurrence and formation have not been fully explored. In this study, exogenously applied phytohormones were used to induce in vitro petiole of P. ternata to produce bulbil structure. Transcriptome sequencing of mRNA and miRNA were performed in the induced petiole (TCp) and the induced bulbil (TCb). Gene Ontology (GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed for the identification of key genes and pathways involved in bulbil development. A total of 58,019 differentially expressed genes (DEGs) were identified. The GO and KEGG analysis indicated that DEGs were mainly enriched in plant hormone signal transduction and the starch and sucrose metabolism pathway. The expression profiles of miR167a, miR171a, and miR156a during bulbil induction were verified by qRT-PCR, indicating that these three miRNAs and their target genes may be involved in the process of bulbil induction and play an important role. However, further molecular biological experiments are required to confirm the functions of the identified bulbil development-related miRNAs and targets.

GhMYB44 enhances stomatal closure to confer drought stress tolerance in cotton and Arabidopsis.

MYB genes play crucial roles in plant response to abiotic stress. However, the function of MYB genes in cotton during abiotic stress is less well elucidated. Here, we found an R2R3-type MYB gene, GhMYB44, was induced by simulated drought (PEG6000) and ABA in three cotton varieties. After drought stress, the GhMYB44-silenced plants showed substantial changes at the physiological level, including significantly increased malondialdehyde content and decreased SOD activity. Silencing the GhMYB44 gene increased stomatal aperture and water loss rate, reduced plant drought tolerance. Transgenic Arabidopsis thaliana over-expressed GhMYB44 (GhMYB44-OE) enhanced resistance to mannitol-simulated osmotic stress. The stomatal aperture of the GhMYB44-OE Arabidopsis was significantly smaller than those of the wild type (WT), and the GhMYB44-OE Arabidopsis increased tolerance to drought stress. Transgenic Arabidopsis had higher germination rate under ABA treatment compared to WT, and the transcript levels of AtABI1, AtPP2CA and AtHAB1 were suppressed in GhMYB44-OE plants, indicating a potential role of GhMYB44 in the ABA signal pathway. These results showed that GhMYB44 acts as a positive regulator in plant response to drought stress, potentially useful for engineering drought-tolerant cotton.

Selection of suitable candidate genes for mRNA expression normalization in bulbil development of Pinellia ternata.

Pinellia ternata (Thunb.) Breit. (Abbreviated as P. ternata). It is a commonly prescribed Chinese traditional medicinal herb for the treatment of phlegm, cough, and morning sick. Bulbil reproduction is one of the main reproductive methods of P. ternata. The accurate quantification of gene expression patterns associated with bulbil development might be helpful to explore the molecular mechanism involved in P. ternata reproduction. Quantitative real-time PCR was the most preferred method for expression profile and function analysis of mRNA. However, the reference genes in different tissues of P. ternata in different periods of bulbil development have not been studied in detail. In present study, the expression stability of eight candidate reference genes were determined with programs: geNorm, NormFinder, BestKeeper, and refFinder. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as the top- rated reference gene in all samples of P. ternata, while different combinations of reference gene proved to be the most stable depending on development stage and tissue type. Furthermore, the reliability of GAPDH expression was verified by six P. ternata related genes in hormone and nutrient biosynthesis pathways, and the expression profiles of these genes were agreed with the results of RNA-seq digital gene expression analysis. These results can contribute to studies of gene expression patterns and functional analysis of P. ternata involved in bulbil development.

IDD16 negatively regulates stomatal initiation via trans-repression of SPCH in Arabidopsis.

In Arabidopsis, the initiation and proliferation of stomatal lineage cells is controlled by SPEECHLESS (SPCH). Phosphorylation of SPCH at the post-translational level has been reported to regulate stomatal development. Here we report that IDD16 acts as a negative regulator for stomatal initiation by directly regulating SPCH transcription. In Arabidopsis, IDD16 overexpression decreased abaxial stomatal density in a dose-dependent manner. Time course analysis revealed that the initiation of stomatal precursor cells in the IDD16-OE plants was severely inhibited. Consistent with these findings, the transcription of SPCH was greatly repressed in the IDD16-OE plants. In contrast, IDD16-RNAi transgenic line resulted in enhanced stomatal density, suggesting that IDD16 is an intrinsic regulator of stomatal development. ChIP analysis indicated that IDD16 could directly bind to the SPCH promoter. Furthermore, Arabidopsis plants overexpressing IDD16 exhibited significantly increased drought tolerance and higher integrated water use efficiency (WUE) due to reduction in leaf transpiration. Collectively, our results established that IDD16 negatively regulates stomatal initiation via trans-repression of SPCH, and thus provide a practical tool for increasing plant WUE through the manipulation of IDD16 expression.

Arabidopsis AMINO ACID PERMEASE1 Contributes to Salt Stress-Induced Proline Uptake from Exogenous Sources.

Stress-induced proline accumulation in plants is thought to result primarily from enhanced proline biosynthesis and decreased proline degradation. To identify regulatory components involved in proline transport, we screened for Arabidopsis thaliana T-DNA mutants with enhanced tolerance to toxic levels of exogenous proline (45 mM). We isolated the proline resistant 1-1 (pre1-1) mutant and map-based cloning identified PRE1 as AMINO ACID PERMEASE1 (AAP1, At1g58360), which encodes a plasma membrane-localized amino acid permease. AAP1 expression is induced by salt stress and abscisic acid, but not by proline. In pre1-1 mutants, a 19-nucleotide deletion in the AAP1 coding region produced a premature stop codon. When grown on proline-containing medium, pre1-1 mutants accumulated significantly less proline than did the wild type. Under salt stress, proline uptake decreased significantly in pre1-1 mutants. By contrast, proline uptake increased significantly in the wild type. These results suggest that AAP1 functions in the increase of proline uptake during salt stress. In addition, proline uptake promotes salt tolerance in Arabidopsis seedlings. We conclude that plants can increase proline accumulation by AtAAP1-mediated proline uptake from exogenous source, which help to improve the salt tolerance of seedlings.

The RING Finger E3 Ligase SpRing is a Positive Regulator of Salt Stress Signaling in Salt-Tolerant Wild Tomato Species.

Protein ubiquitination in plants plays critical roles in many biological processes, including adaptation to abiotic stresses. Previously, RING finger E3 ligase has been characterized during salt stress response in several plant species, but little is known about its function in tomato. Here, we report that SpRing, a stress-inducible gene, is involved in salt stress signaling in wild tomato species Solanum pimpinellifolium 'PI365967'. In vitro ubiquitination assay revealed that SpRing is an E3 ubiquitin ligase and the RING finger conserved region is required for its activity. SpRing is expressed in all tissues of wild tomato and up-regulated by salt, drought and osmotic stresses, but repressed by low temperature. Green fluorescent protein (GFP) fusion analysis showed that SpRing is localized at the endoplasmic reticulum. Silencing of SpRing through a virus-induced gene silencing approach led to increased sensitivity to salt stress in wild tomato. Overexpression of SpRing in Arabidopsis thaliana resulted in enhanced salt tolerance during seed germination and early seedling development. The expression levels of certain key stress-related genes are altered both in SpRing-overexpressing Arabidopsis plants and virus-induced gene silenced tomato seedlings. Taken together, our results indicate that SpRing is involved in salt stress and functions as a positive regulator of salt tolerance.

Genome-wide analysis of phylogeny, expression profile and sub-cellular localization of SKP1-Like genes in wild tomato.

SKP1 is a core component of SCF complex, a major type of E3 ubiquitin ligase catalyzing the last step in ubiquitin-mediated protein degradation pathway. In present study, SKP1 gene family in Solanum pimpinellifolium (SSK), a wild species of tomato, was investigated. A total of 19 SSK genes were identified through homologous search. Their chromosomal locations, gene structures, phylogeny, expression profiles, sub-cellular localizations and protein-protein interaction patterns with putative F-box proteins were analyzed in detail. The high homology and similar expression patterns among clustered SSK genes in chromosome suggested that they may have evolved from duplication events and are functionally redundant. Sub-cellular localization indicated that most of the SSK proteins are distributed in both cytosol and nucleus, except for SSK8, which is detected in cytosol only. Tissue-specific expression patterns suggested that many SSK genes may be involved in tomato fruit development. Furthermore, several SSK genes were found to be responsive to heat stress and salicylic acid treatment. Based on phylogenetic analysis, expression profiles and protein interaction property, we proposed that tomato SSK1 and SSK2 might have similar function to ASK1 and ASK2 in Arabidopsis.

A tetratricopeptide repeat domain-containing protein SSR1 located in mitochondria is involved in root development and auxin polar transport in Arabidopsis.

Auxin polar transport mediated by a group of Pin-formed (PIN) transporters plays important roles in plant root development. However, the mechanism underlying the PIN expression and targeting in response to different developmental and environmental stimuli is still not fully understood. Here, we report a previously uncharacterized gene SSR1, which encodes a mitochondrial protein with tetratricopeptide repeat (TPR) domains, and show its function in root development in Arabidopsis thaliana. In ssr1-2, a SSR1 knock-out mutant, the primary root growth was dramatically inhibited due to severely impaired cell proliferation and cell elongation. Significantly lowered level of auxin was found in ssr1-2 roots by auxin measurement and was further supported by reduced expression of DR5-driven reporter gene. As a result, the maintenance of the root stem cell niche is compromised in ssr1-2. It is further revealed that the expression level of several PIN proteins, namely, PIN1, PIN2, PIN3, PIN4 and PIN7, were markedly reduced in ssr1-2 roots. In particular, we showed that the reduced protein level of PIN2 on cell membrane in ssr1-2 is due to impaired retrograde trafficking, possibly resulting from a defect in retromer sorting system, which destines PIN2 for degradation in vacuoles. In conclusion, our results indicated that SSR1 is functioning in root development in Arabidopsis, possibly by affecting PIN protein expression and subcellular targeting.

Metabolic engineering of the phenylpropanoid pathway enhances the antioxidant capacity of Saussurea involucrata.

The rare wild species of snow lotus Saussurea involucrata is a commonly used medicinal herb with great pharmacological value for human health, resulting from its uniquely high level of phenylpropanoid compound production. To gain information on the phenylpropanid biosynthetic pathway genes in this critically important medicinal plant, global transcriptome sequencing was performed. It revealed that the phenylpropanoid pathway genes were well represented in S. involucrata. In addition, we introduced two key phenylpropanoid pathway inducing transcription factors (PAP1 and Lc) into this medicinal plant. Transgenic S. involucrata co-expressing PAP1 and Lc exhibited purple pigments due to a massive accumulation of anthocyanins. The over-expression of PAP1 and Lc largely activated most of the phenylpropanoid pathway genes, and increased accumulation of several phenylpropanoid compounds significantly, including chlorogenic acid, syringin, cyanrine and rutin. Both ABTS (2,2'-azinobis-3-ethylbenzotiazo-line-6-sulfonic acid) and FRAP (ferric reducing anti-oxidant power) assays revealed that the antioxidant capacity of transgenic S. involucrata lines was greatly enhanced over controls. In addition to providing a deeper understanding of the molecular basis of phenylpropanoid metabolism, our results potentially enable an alternation of bioactive compound production in S. involucrata through metabolic engineering.

Molecular characterization and expression analysis of dihydroflavonol 4-reductase (DFR) gene in Saussurea medusa.

Dihydroflavonol 4-reductase (DFR), which catalyzes the reduction of dihydroflavonols to leucoanthocyanins, is a key enzyme in the biosynthesis of anthocyanidins, proanthocyanidins, and other flavonoids of importance in plant development and human nutrition. This study isolated a full length cDNA encoding DFR, designated as SmDFR (GenBank Accession No. EF600682), by screening a cDNA library from a red callus line of Saussurea medusa, which is an endangered, traditional Chinese medicinal plant with high pharmacological value. SmDFR was functionally expressed in yeast (Saccharomyces cerevisiae) to confirm that SmDFR can readily reduce dihydroquercetin (DHQ) and dihydrokampferol (DHK), but it could not reduce dihydromyricetin (DHM). The deduced SmDFR structure shared extensive sequence similarity with previously characterized plant DFRs and phylogenetic analysis showed that it belonged to the plant DFR super-family. SmDFR also possessed flavanone 4-reductase (FNR) activity and can catalyze the conversion of eridictyol to luteoforol. Real-time PCR analysis showed that the expression level of SmDFR was higher in flowers compared with both leaves and roots. This work greatly enhances our knowledge of flavonoid biosynthesis in S. medusa and marks a major advance that could facilitate future genetic modification of S. medusa.

Proline accumulation is inhibitory to Arabidopsis seedlings during heat stress.

The effect of proline (Pro) accumulation on heat sensitivity was investigated using transgenic Arabidopsis (Arabidopsis thaliana) plants ectopically expressing the Δ(1)-pyrroline-5-carboxylate synthetase 1 gene (AtP5CS1) under the control of a heat shock protein 17.6II gene promoter. During heat stress, the heat-inducible expression of the AtP5CS1 transgene was capable of enhancing Pro biosynthesis. Twelve-day-old seedlings were first treated with heat at 37 °C for 24 h to induce Pro and then were stressed at 50 °C for 4 h. After recovery at 22 °C for 96 h, the growth of Pro-overproducing plants was significantly more inhibited than that of control plants that do not accumulate Pro, manifested by lower survival rate, higher ion leakage, higher reactive oxygen species (ROS) and malondialdehyde levels, and increased activity of the Pro/P5C cycle. The activities of antioxidant enzymes superoxide dismutase, guaiacol peroxidase, and catalase, but not those of glutathione reductase and ascorbate peroxidase, increased in all lines after heat treatment, but the increase was more significant in Pro-overproducing seedlings. Staining with MitoSox-Red, reported for being able to specifically detect superoxide formed in mitochondria, showed that Pro accumulation during heat stress resulted in elevated levels of ROS in mitochondria. Interestingly, exogenous abscisic acid (ABA) and ethylene were found to partially rescue the heat-sensitive phenotype of Pro-overproducing seedlings. Measurement of ethylene and ABA levels further confirmed that these two hormones are negatively affected in Pro-overproducing seedlings during heat stress. Our results indicated that Pro accumulation under heat stress decreases the thermotolerance, probably by increased ROS production via the Pro/P5C cycle and inhibition of ABA and ethylene biosynthesis.

Net sodium fluxes change significantly at anatomically distinct root zones of rice (Oryza sativa L.) seedlings.

Casparian bands of endodermis and exodermis play crucial roles in blocking apoplastic movement of ions and water into the stele of roots through the cortex. These apoplastic barriers differ considerably in structure and function along the developing root. The present study assessed net Na+ fluxes in anatomically distinct root zones of rice seedlings and analyzed parts of individual roots showing different Na+ uptake. The results indicated that anatomically distinct root zones contributed differently to the overall uptake of Na+. The average Na+ uptake in root zones in which Casparian bands of the endo- and exo-dermis were interrupted by initiating lateral root primordia (root zone III) was significantly greater than that at the root apex, where Casparian bands were not yet formed (root zone I), or in the region where endo- and exo-dermis with Casparian bands were well developed (root zone II). The measurement of net Na+ fluxes using a non-invasive scanning ion-selective electrode technique (SIET) demonstrated that net Na+ flux varied significantly in different positions along developing rice roots, and a net Na+ influx was obvious at the base of young lateral root primordia. Since sodium fluxes changed significantly along developing roots of rice seedlings, we suggest that the significantly distinct net Na+ flux profile may be attributed to different apoplastic permeability due to lateral root primordia development for non-selective apoplastic bypass of ions along the apoplast.

Development of Casparian strip in rice cultivars.

The development of Casparian strips (CSs) on the endo- and exodermis and their chemical components in roots of three cultivars of rice (Oryza sativa) with different salt tolerance were compared using histochemistry and Fourier transform infrared (FTIR) spectroscopy. The development and deposition of suberin lamellae of CSs on the endo- and exodermis in the salt-tolerant cultivar Liaohan 109 was earlier than in the moderately tolerant cultivar Tianfeng 202 and the sensitive cultivar Nipponbare. The detection of chemical components indicated major contributions to the structure of the outer part from aliphatic suberin, lignin, and cell wall proteins and carbohydrates to the rhizodermis, exodermis, sclerenchyma, and one layer of cortical cells in series (OPR) and the endodermal Casparian strip. Moreover, the amounts of these major chemical components in the outer part of the Liaohan 109 root were higher than in Tianfeng 202 and Nipponbare, but there was no distinct difference in endodermal CSs among the three rice cultivars. The results suggest that the exodermis of the salt-tolerant cultivar Liaohan 109 functions as a barrier for resisting salt stress.

Proline induces calcium-mediated oxidative burst and salicylic acid signaling.

Although free proline accumulation is a well-documented phenomenon in many plants in response to a variety of environmental stresses, and is proposed to play protective roles, high intracellular proline content, by either exogenous application or endogenous over-production, in the absence of stresses, is found to be inhibitory to plant growth. We have shown here that exogenous application of proline significantly induced intracellular Ca(2+) accumulation in tobacco and calcium-dependent ROS production in Arabidopsis seedlings, which subsequently enhanced salicylic acid (SA) synthesis and PR genes expression. This suggested that proline can promote a reaction similar to hypersensitive response during pathogen infection. Other amino acids, such as glutamate, but not arginine and phenylalanine, were also found to be capable of inducing PR gene expression. In addition, proline at concentration as low as 0.5 mM could induce PR gene expression. However, proline could not induce the expression of PDF1.2 gene, the marker gene for jasmonic acid signaling pathway. Furthermore, proline-induced SA production is mediated by NDR1-dependent signaling pathway, but not that mediated by PAD4. Our data provide evidences that exogenous proline, and probably some other amino acids can specifically induce SA signaling and defense response.

Comparative transcriptomic profiling of a salt-tolerant wild tomato species and a salt-sensitive tomato cultivar.

Wild halophytic tomato has long been considered as an ideal gene donor for improving salt tolerance in tomato cultivars. Extensive research has been focused on physiological and quantitative trait locus (QTL) characterization of wild tomato species in comparison with cultivated tomato. However, the global gene expression modification of wild tomato in response to salt stress is not well known. A wild tomato genotype, Solanum pimpinellifolium 'PI365967' is significantly more salt tolerant than the cultivar, Solanum lycopersicum 'Moneymaker', as evidenced by its higher survival rate and lower growth inhibition at the vegetative stage. The Affymetrix Tomato Genome Array containing 9,200 probe sets was used to compare the transcriptome of PI365967 and Moneymaker. After treatment with 200 mM NaCl for 5 h, PI365967 showed relatively fewer responsive genes compared with Moneymaker. The salt overly sensitive (SOS) pathway was found to be more active in PI365967 than in Moneymaker, coinciding with relatively less accumulation of Na(+) in shoots of PI365967. A gene encoding salicylic acid-binding protein 2 (SABP2) was induced by salinity only in PI365967, suggesting a possible role for salicylic acid signaling in the salt response of PI365967. The fact that two genes encoding lactoylglutathione lyase were salt inducible only in PI365967, together with much higher basal expression of several glutathione S-transferase genes, suggested a more effective detoxification system in PI365967. The specific down-regulation in PI365967 of a putative high-affinity nitrate transporter, known as a repressor of lateral root initiation, may explain the better root growth of this genotype during salt stress.

Proline accumulation and transcriptional regulation of proline biosynthesis and degradation in Brassica napus.

To understand the molecular mechanism underlying proline accumulation in Brassica napus, cDNAs encoding Delta(1)-pyrroline-5-carboxylate synthetase (BnP5CS), ornithine delta-aminotransferase (BnOAT) and proline dehydrogenase (BnPDH) were isolated and characterized. Southern blot analysis of BnP5CSs in B. napus and its diploid ancestors suggested a gene loss may have occurred during evolution. The expression of BnP5CS1 and BnP5CS2 was induced, while the expression of BnPDH was inhibited under salt stress, ABA treatment and dehydration, prior to proline accumulation. The upregulation of BnOAT expression was only detected during prolonged severe osmotic stress. Our results indicate that stress-induced proline accumulation in B. napus results from the reciprocal action of activated biosynthesis and inhibited proline degradation. Whether the ornithine pathway is activated depends on the severity of stress. During development, proline content was high in reproductive organs and was accompanied by markedly high expression of BnP5CS and BnPDH, suggesting possible roles of proline during flower development. [BMB reports 2009; 42(1): 28-34].

GMCHI, cloned from soybean [Glycine max (L.) Meer.], enhances survival in transgenic Arabidopsis under abiotic stress.

Plants respond to cold stress by modifying the expression of a battery of cold-responsive genes. Using cDNA-AFLP techniques, GMCHI (G lycine m ax chilling-inducible) (accession no. EU699765) was isolated from the embryonic axis of a chilling-resistant cultivar of soybean seed imbibed at 4 degrees C for 24 h. The full-length GMCHI cDNA which consisted of a single open reading frame (ORF) encoded a putative polypeptide of 129 amino acids. Sequence analysis revealed neither significant similarity of GMCHI to known proteins, nor any conserved domains found. Soybean seed imbibed at 4 degrees C dramatically enhanced transcript level of GMCHI after 1 h, and reached a maximum at 18 h, while the expression was only detected in the embryonic axis. GMCHI expression was strongly induced by treatment with ABA and PEG, but weakly by 250 mM NaCl which suggests that GMCHI is probably regulated by ABA-dependent signal transduction pathway during cold acclimation. Overexpression of GMCHI in Arabidopsis under the control of CaMV35S promoter enhanced the tolerance to cold, drought and NaCl stresses. Therefore, GMCHI may play an important role in the adaptation of chilling-resistant soybean seed to chilling imbibition.

Enhanced tolerance to drought stress in transgenic tobacco plants overexpressing VTE1 for increased tocopherol production from Arabidopsis thaliana.

Tocopherol cyclase (VTE1, encoded by VTE1 gene) catalyzes the penultimate step of tocopherol synthesis. Transgenic tobacco plants overexpressing VTE1 from Arabidopsis were exposed to drought conditions during which transgenic lines had decreased lipid peroxidation, electrolyte leakage and H(2)O(2) content, but had increased chlorophyll compared with the wild type. Thus VTE1 can be used to increase vitamin E content of plants and also to enhance tolerance to environmental stresses.