Expression analysis and functional characterization of a novel cold-responsive gene <Emphasis Type="Italic">CbCOR15a</Emphasis> from <Emphasis Type="Italic">Capsella bursa</Emphasis>-<Emphasis Type="Italic">pastoris</Emphasis>

The cold-responsive (COR) genes involved in C-repeat binding factor signaling pathway function essentially in cold acclimation of higher plants. A novel COR gene CbCOR15a from shepherd’s purse (Capsella bursa-pastoris) was predicted to be a homolog of COR15 in Arabidopsis. The analysis of tissue specific expression pattern as well as characterization of the CbCOR15a promoter revealed that the expression of CbCOR15a was induced by coldness not only in leaves and stem but also in roots. Sequence analysis showed that a 909 bp promoter region of CbCOR15a contained two CRT/DRE elements, two ABRE elements, one auxin-responsive TGA-element and one MeJA-responsive CGTCA-motif. In young seedlings the expression of CbCOR15a could be apparently increased by SA, ABA, MeJA and IAA, and transiently increased by GA₃ accompanied by obvious feedback suppression. According to the altered physiological index values in tobacco under cold treatments, the overexpression of CbCOR15a significantly increased the cold tolerance of transgenic tobacco plants. It can be suggested that CbCOR15a was involved in cold response of Capsella bursa-pastoris associated with SA, ABA, MeJA, IAA and GA₃ regulation and confers enhanced cold acclimation in transgenic plants.     

<Emphasis Type="Italic">Ptcorp</Emphasis> gene induced by cold stress was identified by proteomic analysis in leaves of <Emphasis Type="Italic">Poncirus trifoliata</Emphasis> (L.) Raf.

A proteomic approach was employed to investigate the cold stress-responsive proteins in trifoliate orange (Poncirus trifoliata (L.) Raf.), which is a well-known cold tolerant citrus relative and widely used as rootstock in China. Two-year-old potted seedlings were exposed to freezing temperature (−6°C) for 50 min (nonlethal) and 80 min (lethal), and the total proteins were isolated from leaves of the treated plants. Nine differentially accumulated proteins over 2-fold changes in abundance were identified by two-dimensional gel electrophoresis and mass spectrometry. Among these proteins, a resistance protein induced by the nonlethal cold treatment (protein spot #2 from P. trifoliata) was selected as target sequence for degenerated primer design. By using the designed primers, a PCR product of about 700 bp size was amplified from P. trifoliata genomic DNA, which was further cloned and sequenced. A nucleotide sequence of 676 bp was obtained and named Ptcorp. Blast retrieval showed that Ptcorp shared 88% homology with an EST of cold acclimated Bluecrop (Vaccinium corymbosum) library (Accession number: CF811080), indicating that Ptcorp had association with cold acclimation. Semiquantitative RT-PCR analysis demonstrated that Ptcorp gene was up-regulated by cold stress which was consistent with the former result of protein expression profile. As the resistance protein (NBS-LRR disease resistance protein family) gene was up-regulated by cold stress in trifoliate orange and satsuma mandarin, it may imply that NBS-LRR genes might be associated with cold resistance in citrus.     

Cloning of Ten Peroxidase (<Emphasis Type="Italic">POD</Emphasis>) Genes from <Emphasis Type="Italic">Tamarix Hispida</Emphasis> and Characterization of their Responses to Abiotic Stress

Plant peroxidases (PODs) have been ascribed a variety of biological functions, including hydrogen peroxide detoxification, lignin biosynthesis, hormonal signaling, and stress response. In the present study, ten POD genes, including three ascorbate peroxidases (class I PODs) and seven secretory peroxidases (class III PODs), were cloned from Tamarix hispida. The roles of the ten POD genes were addressed under different abiotic stress conditions, and gene expression profiles in roots, stems, and leaves were evaluated using real-time quantitative reverse-transcribed polymerase chain reaction. Our results showed that the relative abundance of the PODs was markedly different in roots, stems, and leaves, indicating that POD activity differs in these three organs. ThPOD1 and ThPOD8 were the most and least abundant, respectively, in all organs. The expression profiles in response to abiotic stresses were organ specific. All of the genes were highly induced by drought, salt, salt–alkaline, CdCl₂, and abscisic acid (ABA) treatments in at least one organ. Five ThPOD genes were induced in roots, stems, and leaves under all of the studied stress conditions, indicating that they are closely associated with abiotic stress. Our results demonstrate that the ten plant peroxidases are all expressed in leaves, stems, and roots, that they are involved in different abiotic stress responses, and that they are controlled by an ABA-dependent stress signaling pathway.     

Ectopic Expression of an <Emphasis Type="Italic">FT</Emphasis> Homolog from <Emphasis Type="Italic">Citrus</Emphasis> Confers an Early Flowering Phenotype on Trifoliate Orange (<Emphasis Type="Italic">Poncirus trifoliata</Emphasis> L. Raf.)

Citrus FT (CiFT) cDNA, which promoted the transition from the vegetative to the reproductive phase in Arabidopsis thaliana, when constitutively expressed was introduced into trifoliate orange (Poncirus trifoliata L. Raf.). The transgenic plants in which CiFT was expressed constitutively showed early flowering, fruiting, and characteristic morphological changes. They started to flower as early as 12 weeks after transfer to a greenhouse, whereas wild-type plants usually have a long juvenile period of several years. Most of the transgenic flowers developed on leafy inflorescences, apparently in place of thorns; however, wild-type adult trifoliate orange usually develops solitary flowers in the axils of leaves. All of the transgenic lines accumulated CiFT mRNA in their shoots, but there were variations in the accumulation level. The transgenic lines showed variation in phenotypes, such as time to first flowering and tree shape. In F₁ progeny obtained by crossing ‘Kiyomi’ tangor (C. unshiu × sinensis) with the pollen of one transgenic line, extremely early flowering immediately after germination was observed. The transgene segregated in F₁ progeny in a Mendelian fashion, with complete co-segregation of the transgene and the early flowering phenotype. These results showed that constitutive expression of CiFT can reduce the generation time in trifoliate orange.     

Early low-temperature responsive mitogen activated protein kinases <Emphasis Type="Italic">RaMPK1</Emphasis> and <Emphasis Type="Italic">RaMPK2</Emphasis> from <Emphasis Type="Italic">Rheum australe</Emphasis> D. Don respond differentially to diverse stresses

Rheum grows luxuriantly in a niche of low temperature (LT) at high altitudes in Himalayan belt. The plant is expected to harbor novel genes particularly for tolerance to LT. Using differential display, two cDNAs RaMPK1 and RaMPK2, showing homology to mitogen-activated protein kinases (MAPKs) were isolated. As compared to RaMPK1, RaMPK2 exhibited strong up-regulation in response to LT. RaMPK1 was novel in terms of possessing a small glutamine and proline rich region at the N-terminal end. Secondly, though RaMPK1 showed homology with salicylic acid (SA) responsive MAPKs, the gene was down-regulated by SA but activated by jasmonate (JA). Abscisic acid (ABA) and polyethylene glycol (PEG) also down-regulated RaMPK1. RaMPK2 showed down-regulation within 5 min of exposure to JA and SA treatments, followed by gradual increase in expression. Expression of RaMPK2 was wavy in response to ABA and PEG treatment. Results are discussed in light of the novelty of these MAPKs.     

Identification and expression analysis of <Emphasis Type="Italic">CjLTI</Emphasis>, a novel low temperature responsive gene from <Emphasis Type="Italic">Caragana jubata</Emphasis>

Using rapid amplification of cDNA ends, a full length cDNA (CjLTI) was cloned from apical buds of Caragana jubata, a plant species that grows under extreme cold. The cDNA obtained was 573 bp long consisting of an open reading frame of 351 bp encoding 116 amino acids. Homology analysis did not exhibit significant similarity with any sequence at NCBI database, therefore it was deduced as a novel gene. Secondary structure analysis suggested that the deduced CjLTI contained 25.86% α-helices, 4.31% β-turns, 6.90% extended strands, and 62.93% random coils. The hydropathy profile suggested CjLTI to be a hydrophobic protein having characteristic features of signal peptides at N-terminus. The gene exhibited down-regulation at 5 min of exposure to low temperature (LT, 4 ± 3°C) followed by a strong up-regulation after 15 min and onwards. Methyl jasmonate (MJ) lead to up-regulation of CjLTI starting at 5 min onwards. The gene exhibited up- and down-regulation of expression pattern in response to abscisic acid (ABA) and salicylic acid (SA). Mild drought stress slightly up-regulated gene expression and at severe drought (up to 115% reduction in leaf water potential) slight down-regulation of gene expression was observed. These results suggested CjLTI to be a LT responsive gene wherein MJ, ABA and SA pathways might be involved in regulating the gene expression.     

Cloning of a 9-<Emphasis Type="Italic">cis</Emphasis>-epoxycarotenoid dioxygenase gene (<Emphasis Type="Italic">SgNCED1</Emphasis>) from <Emphasis Type="Italic">Stylosanthes guianensis </Emphasis>and its expression in response to abiotic stresses

Abscisic acid (ABA) regulates plant adaptive responses to various environmental stresses. Oxidative cleavage of cis-epoxycarotenoids catalyzed by 9-cis-epoxycarotenoid dioxygenase (NCED) is the main regulatory step in the biosynthesis of ABA in higher plants. A NCED gene, SgNCED1, was cloned from the dehydrated leaves of Stylosanthes guianensis. The 2,241-bp full-length SgNCED1 had a 1,809-bp ORF, which encodes a peptide of 602 amino acids. The deduced amino acid sequence of SgNCED1 protein shared high identity with other NCEDs. At the N-terminus of the SgNCED1 located a chloroplast transit peptide sequence. DNA blot analysis revealed that SgNCED1 was a single copy gene in the genome of S. guianensis. The relationship between expression of SgNCED1 and endogenous ABA level was investigated. The expression of SgNCED1 was induced in both leaves and roots of S. guianensis under drought stress. Dehydration and salt stress induced the expression of SgNCED1 strongly and rapidly. The ABA accumulation was coincidently induced with the SgNCED1 mRNA under drought, dehydration and salt stress. The expression of SgNCED1 and ABA accumulation were also induced under chilling condition.     

Validation and application of reference genes for quantitative gene expression analyses in various tissues of <Emphasis Type="Italic">Bupleurum chinense</Emphasis>

It is crucial to select stable references in gene expression analyses using quantitative real-time PCR (qRT-PCR). In this work, seven frequently used reference genes, 18S, Actin, EF1α, α-tubulin, β-tubulin, Cyclophilin and Cytoplasmic ribosomal protein L2 (L2), from Bupleurum chinense DC. were evaluated as the internal control in five tissues, roots, stems, leaves, flowers and fruits, before tissue specific gene expression assays. The results showed that β-tubulin was the most stable and reliable reference gene among the seven candidate genes in the measured tissues. The expression levels of four genes involved in saikosaponins (the pharmacological active compounds of B. chinense) biosynthesis, HMGR, IPPI, FPS and β-AS, were assayed with β-tubulin as the internal control in the five tissues. All the four genes were expressed in the five tissues with different profiles and HMGR in the order of roots > flowers, stems and leaves > fruits, IPPI of stems > leaves and fruits > roots and flowers, FPS of flowers > fruits > stems and roots > leaves and β-AS of roots > flowers, stems and fruits > leaves. The genes of FPS and β-AS were expressed predominantly in flowers and roots, respectively. This study may provide a suitable internal control for quantitative gene expression assays in various tissues and give insight into the tissue expression profiles of four saikosaponins biosynthesis-involved genes of medicinal B. chinense.