Cloning and characterization of an allene oxide cyclase,PpAOC3, in <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

Allene oxide cyclase (AOC) is a key enzyme in the octadecanoid pathway of flowering plants that synthesizes 12-oxo-phytodienoic acid (OPDA), which is a biosynthetic precursor of the signal molecule jasmonic acid (JA). A database search of the Physcomitrella patens genome revealed the presence of an AOC gene unique from the two previously reported AOC genes, PpAOC1 and PpAOC2. After cloning the identified AOC gene, designated PpAOC3, the obtained cDNA sequence (897 bp) was larger than the predicted AOC gene (765 bp) in the database because a speculated intron was not fully deleted. Although PpAOC3 did not display high similarity with AOC proteins from other species, recombinant PpAOC3 exhibited AOC activity and translocated to chloroplasts, as is observed for other AOC proteins. Notably, the expression profile of PpAOC3 differed from the other PpAOCs, as its expression in protonemata was higher than that in gametophores. Although the function of oxylipins such as OPDA and JA remains elusive in P. patens, further characterization of the enzymes in the octadecanoid pathway and the role of oxylipin will aid in the elucidation of physiological processes in this model bryophyte.     

Jasmonate biosynthesis and the allene oxide cyclase family of Arabidopsis thaliana

In biosynthesis of octadecanoids and jasmonate (JA), the naturally occurring enantiomer is established in a step catalysed by the gene cloned recently from tomato as a single-copy gene (Ziegler et al., 2000). Based on sequence homology, four full-length cDNAs were isolated from Arabidopsis thaliana ecotype Columbia coding for proteins with AOC activity. The expression of AOCgenes was transiently and differentially up-regulated upon wounding both locally and systemically and was induced by JA treatment. In contrast, AOC protein appeared at constitutively high basal levels and was slightly increased by the treatments. Immunohistochemical analyses revealed abundant occurrence of AOC protein as well as of the preceding enzymes in octadecanoid biosynthesis, lipoxygenase (LOX) and allene oxide synthase (AOS), in fully developed tissues, but much less so in 7-day old leaf tissues. Metabolic profiling data of free and esterified polyunsaturated fatty acids and lipid peroxidation products including JA and octadecanoids in wild-type leaves and the jasmonate-deficient mutant OPDA reductase 3 (opr3) revealed preferential activity of the AOS branch within the LOX pathway. 13-LOX products occurred predominantly as esterified derivatives, and all 13-hydroperoxy derivatives were below the detection limits. There was a constitutive high level of free 12-oxo-phytodienoic acid (OPDA) in untreated wild-type and opr3 leaves, but an undetectable expression of AOC. Upon wounding opr3 leaves exhibited only low expression of AOC, wounded wild-type leaves, however, accumulated JA and AOC mRNA. These and further data suggest regulation of JA biosynthesis by OPDA compartmentalization and a positive feedback by JA during leaf development.     

Overexpression of a NTR1 in transgenic soybean confers tolerance to water stress

Methyl jasmonate (MeJA) is an important plant regulator that involves in plant development and regulates the expression of plant defense genes in response to various stresses such as wounding, drought, and pathogens. In order to determine the physiological role of endogenous MeJA in plants, a NTR1 from Brassica campestris encoding a jasmonic acid carboxyl methyltransferase that produces methyl jasmonate was constructed under the control of CaMV 35S promoter and transformed into soybean [Glycine max (L) Merrill]. The transgenic soybean plants constitutively expressed the NTR1 and accumulated more MeJA levels than wild type plants. Overexpression of the gene in transgenic soybean conferred tolerance to dehydration during seed germination and seedling growth as reflected by the percentage of the fresh weight of seedlings. In addition, the transgenic soybean plants also conferred better capacity to retain water than wild type plants when drought tolerance was tested using detached leaves.     

Molecular cloning and expression profile of a jasmonate biosynthetic pathway gene for allene oxide cyclase from <Emphasis Type="Italic">Hyoscyamus niger</Emphasis>

Hyoscyamus niger L. is a medicinal plant which produces a class of jasmonate-responsive pharmaceutical secondary metabolites named tropane alkaloids. As a family of signaling phytohormones, jasmonates play significant roles in the biosynthesis of many plant secondary metabolites. In the jasmonate biosynthetic pathway of plants, allene oxide cyclase (AOC, EC 5.3.99.6) catalyzes the most important step. Here we cloned a cDNA from H. niger, named HnAOC (GenBank accession no.: AY708383), which was 1044 bp long, with a 747-bp open reading frame (ORF) encoding a polypeptide of 248 amino acid residues. Southern blot analysis indicated that it was a multicopy gene. RT-PCR analysis revealed that the expression of HnAOC was regulated by various stresses and elicitors, with methyl-jasmonate showing the most prominent inducement. The characterization of HnAOC would be helpful for improving the production of valuable secondary metabolites by regulating the biosynthesis of jasmonates. The text was submitted by the authors in English.     

Arabidopsis thaliana Atvsp is homologous to soybean VspA and VspB,genes encoding vegetative storage protein acid phosphatases,and is regulated similarly by methyl jasmonate,wounding, sugars,light and phosphate

The soybean vegetative storage proteins, VSP and VSP, are acid phosphatases that accumulate to very high levels in hypocotyls, young leaves and flowers and pods. The genes encoding the soybean VSP are activated by jasmonate, wounding, sugars and light and down regulated by phosphate and auxin. In this study, expression of an Arabidopsis thaliana gene (Atvsp) encoding a protein homologous to soybean Vsp and Vsp, was examined and compared to expression of the soybean Vsp genes. Atvsp mRNA was present at high levels in flowers and buds and at low levels in roots, stems, leaves and siliques. Expression of Atvsp in leaves could be induced by wounding or by treatment of illuminated plants with methyl jasmonate and sucrose. Roots of plants with wounded leaves also accumulated Atvsp mRNA indicating that this gene can be regulated by a transmissible wound signal. Phosphate partially inhibited expression of Atvsp. Arabidopsis proteins of 29 and 30 kDa crossreacted with antibodies against soybean VSP. These proteins were very abundant in flowers and the proteins accumulated in leaves and roots of plants treated with methyl jasmonate. The level of these proteins in flowers was similar to the levels of soybean VSP in young soybean leaves. Overall, these data indicate that Arabidopsis Atvsp and soybean VspA/B genes are regulated similarly and that in both plants, the gene products can accumulate to high levels. This suggests that genes homologous to VspA/B may be of greater general significance than previously recognized.     

玉米萜类植保素代谢关键基因对小斑病侵染的防御响应分析

该研究选用小斑病差异抗性的玉米自交系Mo17（较抗）、‘郑58’（中抗）和‘吉419’（感）,分别于接种小斑病病原菌玉蜀黍平脐孺胞（Cochliobolus heterostrophus）0、6、12、24、36和48 h时采集接种叶片为材料,采用半定量和实时荧光定量PCR技术,检测倍半萜植保素Zealexin生物合成关键基因（TPS6、TPS11）、二萜植保素Kauralexin代谢关键基因An2以及茉莉酸合成关键基因AOC的表达模式,为阐明不同抗性品种对玉米小斑病的差异防御机制提供理论依据。结果显示：接种小斑病病原菌后,抗病自交系Mo17中TPS6、TPS11基因表达诱导不显著,An2基因表达迅速增加,AOC基因于接种12 h后表达明显上调。‘郑58’中TPS6、TPS11基因被快速诱导,在接种24 h时表达量达到最大,An2基因表达逐渐增加但差异不显著,AOC基因表达量于接种6 h后显著增加;感病自交系‘吉419’中TPS6、TPS11、An2基因在接种24 h后才显著上调,明显比抗性自交系缓慢,AOC基因表达先呈现递减的趋势然后上升,在24 h表达量最高并持续到48 h。研究表明,两类植保素代谢在玉米小斑病防御中具有不同的时间应答模式,但都受到茉莉酸介导。感病自交系中植保素代谢防御响应较慢,而抗病自交系中响应较快,符合其抗性差异。     

Overexpression of jasmonic acid carboxyl methyltransferase increases tuber yield and size in transgenic potato

Jasmonates control diverse plant developmental processes, such as seed germination, flower, fruit and seed development, senescence and tuberization in potato. To understand the role of methyl jasmonate (MeJA) in potato tuberization, the Arabidopsis JMT gene encoding jasmonic acid carboxyl methyltransferase was constitutively overexpressed in transgenic potato plants. Increases in tuber yield and size as well as in vitro tuberization frequency were observed in transgenic plants. These were correlated with JMT mRNA level––the higher expression level, the higher the tuber yield and size. The levels of jasmonic acid (JA), MeJA and tuberonic acid (TA) were also higher than those in control plants. Transgenic plants also exhibited higher expression of jasmonate-responsive genes such as those for allene oxide cyclase (AOC) and proteinase inhibitor II (PINII). These results indicate that JMT overexpression induces jasmonate biosynthesis genes and thus JA and TA pools in transgenic potatoes. This results in enhanced tuber yield and size in transgenic potato plants.     

An (E,E)‐α‐farnesene synthase gene of soybean has a role in defence against nematodes and is involved in synthesizing insect‐induced volatiles

Plant terpene synthase genes (TPSs) have roles in diverse biological processes. Here, we report the functional characterization of one member of the soybean TPS gene family, which was designated GmAFS. Recombinant GmAFS produced in Escherichia coli catalysed the formation of a sesquiterpene (E,E)‐α‐farnesene. GmAFS is closely related to (E,E)‐α‐farnesene synthase gene from apple, both phylogenetically and structurally. GmAFS was further investigated for its biological role in defence against nematodes and insects. Soybean cyst nematode (SCN) is the most important pathogen of soybean. The expression of GmAFS in a SCN‐resistant soybean was significantly induced by SCN infection compared with the control, whereas its expression in a SCN‐susceptible soybean was not changed by SCN infection. Transgenic hairy roots overexpressing GmAFS under the control of the CaMV 35S promoter were generated in an SCN‐susceptible soybean line. The transgenic lines showed significantly higher resistance to SCN, which indicates that GmAFS contributes to the resistance of soybean to SCN. In soybean leaves, the expression of GmAFS was found to be induced by Tetranychus urticae (two‐spotted spider mites). Exogenous application of methyl jasmonate to soybean plants also induced the expression of GmAFS in leaves. Using headspace collection combined with gas chromatography–mass spectrometry analysis, soybean plants that were infested with T. urticae were shown to emit a mixture of volatiles with (E,E)‐α‐farnesene as one of the most abundant constituents. In summary, this study showed that GmAFS has defence roles in both below‐ground and above‐ground organs of soybean against nematodes and insects, respectively.     

Molecular characterization of the <Emphasis Type="Italic">GmAMS1</Emphasis> gene encoding β-amyrin synthase in soybean plants

Suppression subtractive hybridization was used to isolate wound-induced cDNAs from soybean plants. One of wound-induced cDNA, gmwi33 designated GmAMS1, showed a high homology with genes encoding β-amyrin synthase. GmAMS1 protein showed 89% identity with licorice GgbAS1. In five-day-old dark-grown seedlings, RNA expression of GmAMS1 was strongly induced by light, weakly induced by methyl jasmonate and low temperature, and was not induced by elicitor or UV-B treatment. Genomic blot and FISH analyses confirmed that the soybean genome has two copies of the GmAMS1 gene. To our knowledge, this is the first report on β-amyrin synthase from soybean. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 4, pp. 584–589. The text was submitted by the authors in English.     

Lipoxygenase gene expression is modulated in plants by water deficit, wounding, and methyl jasmonate

Summary Two classes of lipoxygenase (LOX) cDNAs, designated loxA and loxB, were isolated from soybean. A third lipoxygenase cDNA, loxP1, was isolated from pea. The deduced amino acid sequences of loxA and loxB show 61–74% identity with those of soybean seed LOXs. loxA and loxB mRNAs are abundant in roots and non-growing regions of seedling hypocotyls. Lower levels of these mRNAs are found in hypocotyl growing regions. Exposure of soybean seedlings to water deficit causes a rapid increase in loxA and loxB mRNAs in the elongating hypocotyl region. Similarly, loxP1 mRNA levels increase rapidly when pea plants are wilted. loxA and loxB mRNA levels also increase in wounded soybean leaves, and these mRNAs accumulate in soybean suspension cultures treated with 20 M methyl jasmonate. These results demonstrate that LOX gene expression is modulated in response to water deficit and wounding and suggest a role for lipoxygenase in plant responses to these stresses.     

JA but not JA‐Ile is the cell‐nonautonomous signal activating JA mediated systemic defenses to herbivory in Nicotiana attenuata

The whole‐plant activation of defense responses to wounding and herbivory requires systemic signaling in which jasmonates (JAs) play a pivotal role. To examine the nature of the slower cell‐nonautonomous as compared to the rapid cell‐autonomous signal in mediating systemic defenses in Nicotiana attenuata, reciprocal stem grafting‐experiments were used with plants silenced for the JA biosynthetic gene ALLENE OXIDE CYCLASE (irAOC) or plants transformed to create JA sinks by ectopically expressing Arabidopsis JA‐O‐methyltransferase (ovJMT). JA‐impaired irAOC plants were defective in the cell‐nonautonomous signaling pathway but not in JA transport. Conversely, ovJMT plants abrogated the production of a graft‐transmissible JA signal. Both genotypes displayed unaltered cell‐autonomous signaling. Defense responses (17‐hydroxygeranyllinalool diterpene glycosides, nicotine, and proteinase inhibitors) and metabolite profiles were differently induced in irAOC and ovJMT scions in response to graft‐transmissible signals from elicited wild type stocks. The performance of Manduca sexta larvae on the scions of different graft combinations was consistent with the patterns of systemic defense metabolite elicitations. Taken together, we conclude that JA and possibly MeJA, but not JA‐Ile, either directly functions as a long‐distance transmissible signal or indirectly interacts with long distance signal(s) to activate systemic defense responses.     

GmHKT1;4, a novel soybean gene regulating Na+/K+ ratio in roots enhances salt tolerance in transgenic plants

Salt is an important factor affecting the growth and development of soybean in saline or alkaline soil. The aims of the present study were to identify and functionally analyse the soybean GmHKTs gene family, and to explore their roles under NaHCO₃ and NaCl stresses. The GmHKTs gene family were isolated from soybean using genome sequence information. The GmHKTs gene family were further analysed for the structure and phylogenetic relationship. The expression patterns of soybean GmHKTs genes under NaHCO₃ and NaCl stresses were analysed via quantitative real-time PCR. As a result, the expression level of GmHKT1;4 was extremely up regulated in root under each treatment. Overexpression of GmHKT1;4 significantly enhanced the tolerance of transgenic tobacco plants to NaHCO₃ and NaCl stresses, compared with null plants. The overexpressed transgenic plants of this gene accomulated more K⁺ and less Na⁺ under salt stress, compaired with null plants. Our findings suggest that GmHKT1;4 plays an important role for regulation Na⁺/K⁺ ratio in roots under alkaline (NaHCO₃) and saline (NaCl) stresses.     

<Emphasis Type="Italic">GmFtsH9</Emphasis> expression correlates with in vivo photosystem II function: chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence transient analysis and eQTL mapping in soybean

Filamentation temperature-sensitive H (FtsH) is an ATP-dependent zinc metalloprotease involved in diverse biological functions. There are 12 FtsH proteins in Arabidopsis, among which AtFtsH2 plays an important role in regulating the turnover of photosystem II (PSII) reaction center D1 protein and the development of the photosynthetic apparatus. Here, we have identified 11 FtsH genes in the soybean genome by a bioinformatics approach. These soybean FtsH genes corresponded to seven Arabidopsis FtsH genes, suggesting that the main characteristics of soybean FtsH genes were formed before the evolutionary split of soybean and Arabidopsis. Phylogenetic analyses allowed us to clone a soybean AtFtsH2-like gene designated as GmFtsH9. The predicted protein of GmFtsH9 consists of 690 amino acids and contains three typical FtsH proteins conserved domains. The expression level of GmFtsH9 was determined in a soybean recombinant inbred line population under a pot experiment conducted for measuring chlorophyll a fluorescence transient parameters, photosynthetic CO₂ fixation rate (P _N), and seed yield. Expression quantitative trait loci (eQTL) mapping revealed two trans-acting eQTLs for GmFtsH9. The significant correlation of gene expression level with chlorophyll a fluorescence transient parameters and the presence of overlapping eQTL (QTL) between gene expression level and chlorophyll a fluorescence transient parameters indicated that GmFtsH9 could be involved in regulating PSII function. These results further lead to the understanding of the mechanism underlying FtsH gene expression, and contribute to the development of marker-assisted selection breeding programs for modulating soybean FtsH gene expression.