Casein kinase 1 AELs promote senescence by enhancing ethylene biosynthesis through phosphorylating WRKY22 transcription factor.

Leaf senescence is a complex process regulated by developmental and environmental factors, and plays a pivotal role in the development and life cycle of higher plants. Casein kinase 1 (CK1) is a highly conserved serine/threonine protein kinase in eukaryotes and functions in various cellular processes including cell proliferation, light signaling and hormone effects of plants. However, the biological function of CK1 in plant senescence remains unclear. Through systemic genetic and biochemical studies, we here characterized the function of Arabidopsis EL1-like (AEL), a CK1, in promoting leaf senescence by stimulating ethylene biosynthesis through phosphorylating transcription factor WRKY22. Seedlings lacking or overexpressing AELs presented delayed or accelerated leaf senescence, respectively. AELs interact with and phosphorylate WRKY22 at Thr57, Thr60 and Ser69 residues to enhance whose transactivation activity. Being consistent, increased or suppressed phosphorylation of WRKY22 resulted in the promoted or delayed leaf senescence. WRKY22 directly binds to promoter region and stimulates the transcription of 1-amino-cyclopropane-1-carboxylate synthase 7 gene to promote ethylene level and hence leaf senescence. Our studies demonstrated the crucial role of AEL-mediated phosphorylation in regulating ethylene biosynthesis and promoting leaf senescence by enhancing WRKY22 transactivation activity, which helps to elucidate the fine-controlled ethylene biosynthesis and regulatory network of leaf senescence.

A novel prognostic index for sporadic Burkitt lymphoma in adult patients: a real-word multicenter study.

BACKGROUND: Adult sporadic Burkitt lymphoma (BL) is a rare but highly aggressive subtype of lymphoma which lacks its own unique prognostic model. Systemic inflammatory biomarkers have been confirmed as prognostic markers in several types of malignancy. Our objective was to explore the predictive value of pretreatment inflammatory biomarkers and establish a novel, clinically applicable prognostic index for adult patients with sporadic BL. METHODS: We surveyed retrospectively 336 adult patients with newly diagnosed sporadic BL at 8 Chinese medical centers and divided into training cohort (n = 229) and validation cohort (n = 107). The pretreatment inflammatory biomarkers were calculated for optimal cut-off value. The association between serum biomarkers and overall survival (OS) was analyzed by Kaplan-Meier curves and Cox proportional models. The risk stratification was defined based on normal LDH level, Ann Arbor stage of I and completely resected abdominal lesion or single extra-abdominal mass < 10 cm. RESULTS AND CONCLUSIONS: Univariate and multivariate analyses revealed that platelets< 254 × 109/L, albumin< 40 g/L, lactate dehydrogenase≥334 U/L independently predicted unfavorable OS. We used these data as the basis for the prognostic index, in which patients were stratified into Group 1 (no or one risk factor), Group 2 (two risk factors), or Group 3 (three risk factors), which were associated with 5-year OS rates of 88.1, 72.4, and 45%, respectively. In the subgroup analysis for high-risk patients, our prognostic model results showed that high-risk patients with no more than one adverse factor presented a 5-year survival rate of 85.9%, but patients with three adverse factors had a 5-year survival rate of 43.0%. Harrell's concordance index (C-index) of the risk group score was 0.768. Therefore, the new prognostic model could be used to develop risk-adapted treatment approaches for adult sporadic BL.

Two tonoplast proton pumps function in Arabidopsis embryo development.

Two types of tonoplast proton pumps, H+ -pyrophosphatase (V-PPase) and the H+ -ATPase (V-ATPase), establish the proton gradient that powers molecular traffic across the tonoplast thereby facilitating turgor regulation and nutrient homeostasis. However, how proton pumps regulate development remains unclear. In this study, we investigated the function of two types of proton pumps in Arabidopsis embryo development and pattern formation. While disruption of either V-PPase or V-ATPase had no obvious effect on plant embryo development, knocking out both resulted in severe defects in embryo pattern formation from the early stage. While the first division in wild-type zygote was asymmetrical, a nearly symmetrical division occurred in the mutant, followed by abnormal pattern formation at all stages of embryo development. The embryonic defects were accompanied by dramatic differences in vacuole morphology and distribution, as well as disturbed localisation of PIN1. The development of mutant cotyledons and root, and the auxin response of mutant seedlings supported the hypothesis that mutants lacking tonoplast proton pumps were defective in auxin transport and distribution. Taking together, we proposed that two tonoplast proton pumps are required for vacuole morphology and PIN1 localisation, thereby controlling vacuole and auxin-related developmental processes in Arabidopsis embryos and seedlings.

Casein kinase 1 regulates ethylene synthesis by phosphorylating and promoting the turnover of ACS5.

The casein kinase 1 (CK1) family participates in various cellular processes in eukaryotes, but CK1 function in higher plants remains largely unknown. Here, we characterize the function of Arabidopsis CK1 in the regulation of ethylene biosynthesis. Etiolated seedlings of a CK1.8-deficient mutant, ck1.8-1, showed characteristic ethylene-specific constitutive responses due to overaccumulation of ethylene. Biochemical and physiological studies showed that CK1.8 phosphorylates ACS5, a key enzyme of ethylene biosynthesis, at threonine 463 to promote its interaction with the E3 ubiquitin ligase Ethylene Overproduction 1 (ETO1). Deficiency of CK1.8 leads to the accumulation of ACS5, and transgenic plants harboring a dephosphorylation-mimic ACS5(T463A) showed constitutive ethylene responses, confirming the role of CK1.8 in regulating ACS5 stability by phosphorylation and demonstrating that CK1.8 is an important regulator of ethylene biosynthesis. CK1.8 expression is feedback regulated by ethylene. Our studies provide insight into the regulation of ACS5 and ethylene biosynthesis.

Inositol polyphosphate 5-phosphatase-controlled Ins(1,4,5)P3/Ca2+ is crucial for maintaining pollen dormancy and regulating early germination of pollen.

Appropriate pollen germination is crucial for plant reproduction. Previous studies have revealed the importance of dehydration in maintaining pollen dormancy; here, we show that phosphatidylinositol pathway-controlled Ins(1,4,5)P(3)/Ca(2+) levels are crucial for maintaining pollen dormancy in Arabidopsis thaliana. An interesting phenotype, precocious pollen germination within anthers, results from a disruption of inositol polyphosphate 5-phosphatase 12 (5PT12). The knockout mutant 5pt12 has normal early pollen development and pollen dehydration, and exhibits hypersensitive ABA responses, indicating that precocious pollen germination is not caused either by abnormal dehydration or by suppressed ABA signaling. Deficiency of 5PT13 (a close paralog of 5PT12) synergistically enhances precocious pollen germination. Both basal Ins(1,4,5)P(3) levels and endogenous Ca(2+) levels are elevated in pollen from 5pt12 mutants, and 5pt12 5pt13 double mutants show an even higher precocious germination rate along with much higher levels of Ins(1,4,5)P(3)/Ca(2+). Strikingly, exogenous Ca(2+) stimulates the germination of wild-type pollen at floral stage 12, even in very low humidity, both in vitro and in vivo, and treatment with BAPTA, a [Ca(2+)](cyt) inhibitor, reduces the precocious pollen germination rates of 5pt12, 5pt13 and 5pt12 5pt13 mutants. These results indicate that the increase in the levels of Ins(1,4,5)P(3)/Ca(2+) caused by deficiency of inositol polyphosphate 5-phosphatases is sufficient to break pollen dormancy and to trigger early germination. The study reveals that independent of dehydration, the control of Ins(1,4,5)P(3)/Ca(2+) levels by Inositol polyphosphate 5-phosphatases is crucial for maintaining pollen dormancy.

Overexpression of the phosphatidylinositol synthase gene from Zea mays in tobacco plants alters the membrane lipids composition and improves drought stress tolerance.

Phosphatidylinositol (PtdIns) is an important lipid because it serves as a key membrane constituent and is the precursor of the inositol-containing lipids that are found in all plants and animals. It is synthesized from cytidine-diphosphodiacylglycerol (CDP-DG) and myo-inositol by PtdIns synthase (PIS). We have previously reported that two putative PIS genes from maize (Zea mays L.), ZmPIS and ZmPIS2, are transcriptionally up-regulated in response to drought (Sui et al., Gene, 426:47-56, 2008). In this work, we report on the characterization of ZmPIS in vitro and in vivo. The ZmPIS gene successfully complemented the yeast pis mutant BY4743, and the determination of PIS activity in the yeast strain further confirmed the enzymatic function of ZmPIS. An ESI-MS/MS-based lipid profiling approach was used to identify and quantify the lipid species in transgenic and wild-type tobacco plants before and after drought treatment. The results show that the overexpression of ZmPIS significantly increases lipid levels in tobacco leaves under drought stress compared to those of wild-type tobacco, which correlated well with the increased drought tolerance of the transgenic plants. Further analysis showed that, under drought stress conditions, ZmPIS overexpressors were found to exhibit increased membrane integrity, thereby enabling the retention of more solutes and water compared with the wild-type and the vector control transgenic lines. Our findings give us new insights into the role of the ZmPIS gene in the response of maize to drought/osmotic stress and the mechanisms by which plants adapt to drought stress.

Inositol trisphosphate-induced Ca2+ signaling modulates auxin transport and PIN polarity.

The phytohormone auxin is an important determinant of plant development. Directional auxin flow within tissues depends on polar localization of PIN auxin transporters. To explore regulation of PIN-mediated auxin transport, we screened for suppressors of PIN1 overexpression (supo) and identified an inositol polyphosphate 1-phosphatase mutant (supo1), with elevated inositol trisphosphate (InsP(3)) and cytosolic Ca(2+) levels. Pharmacological and genetic increases in InsP(3) or Ca(2+) levels also suppressed the PIN1 gain-of-function phenotypes and caused defects in basal PIN localization, auxin transport and auxin-mediated development. In contrast, the reductions in InsP(3) levels and Ca(2+) signaling antagonized the effects of the supo1 mutation and disrupted preferentially apical PIN localization. InsP(3) and Ca(2+) are evolutionarily conserved second messengers involved in various cellular functions, particularly stress responses. Our findings implicate them as modifiers of cell polarity and polar auxin transport, and highlight a potential integration point through which Ca(2+) signaling-related stimuli could influence auxin-mediated development.

The role of Arabidopsis 5PTase13 in root gravitropism through modulation of vesicle trafficking.

Inositol polyphosphate 5-phosphatases (5PTases) are enzymes of phosphatidylinositol metabolism that affect various aspects of plant growth and development. Arabidopsis 5PTase13 regulates auxin homeostasis and hormone-related cotyledon vein development, and here we demonstrate that its knockout mutant 5pt13 has elevated sensitivity to gravistimulation in root gravitropic responses. The altered responses of 5pt13 mutants to 1-N-naphthylphthalamic acid (an auxin transport inhibitor) indicate that 5PTase13 might be involved in the regulation of auxin transport. Indeed, the auxin efflux carrier PIN2 is expressed more broadly under 5PTase13 deficiency, and observations of the internalization of the membrane-selective dye FM4-64 reveal altered vesicle trafficking in 5pt13 mutants. Compared with wild-type, 5pt13 mutant seedlings are less sensitive to the inhibition by brefeldin A of vesicle cycling, seedling growth, and the intracellular cycling of the PIN1 and PIN2 proteins. Further, auxin redistribution upon gravitropic stimulation is stimulated under 5PTase13 deficiency. These results suggest that 5PTase13 may modulate auxin transport by regulating vesicle trafficking and thereby play a role in root gravitropism.

An inositol polyphosphate 5-phosphatase functions in PHOTOTROPIN1 signaling in Arabidopis by altering cytosolic Ca2+.

Inositol polyphosphate 5-phosphatase (5PTase) is a key enzyme in the phosphatidylinositol metabolic pathway, which plays critical roles in a number of cellular processes in plants. Our previous work implicated the role of 5PTase13, which encodes a WD40-containing type II 5PTase, in hormone-mediated cotyledon vein development. Here, we show that 5PTase13 is also involved in blue light responses in Arabidopsis thaliana. Compared with that in darkness, the expression of 5PTase13 was suppressed by blue light irradiation, and disruption of the gene resulted in shortened hypocotyls and expanded cotyledons. Genetic analysis showed that 5PTase13 acted independently from CRYPTOCHROME1 and CONSTITUTIVE PHOTOMORPHOGENIC1 but interacted functionally with PHOTOTROPIN1 (PHOT1). The expression level of 5PTase13 was significantly enhanced in phot1 single or phot1 phot2 double mutants under blue light, and suppression of 5PTase13 expression rescued the elongated hypocotyls in the phot1 or phot1 phot2 mutants. Further analysis showed that the blue light-induced elevation of cytosolic Ca2+ was inhibited in the phot1 mutant but enhanced in the 5pt13 mutant, suggesting that 5PTase13 antagonizes PHOT1-mediated effects on calcium signaling under blue light.

Low dose radiation increased the therapeutic efficacy of cyclophosphamide on S(180) sarcoma bearing mice.

We examined whether low dose radiation (LDR) exposure (75 mGy) could increase the therapeutic efficacy of cyclophosphamide (CTX) by comparing the effects of tumor suppression, tumor cell apoptosis, cell cycle and proliferation of bone marrow in vivo. Kunming mice implanted with S(180) sarcoma cells were given 75 mGy whole body gamma-ray radiation exposure and CTX (300 mg/kg) by intraperitoneal injection 36 hours after LDR. Proliferation of bone marrow and tumor cells was analyzed by flow cytometry. Cytochrome c leakage from the tumor was measured by Western-blot. We discovered that tumor growth was significantly reduced in the group exposed to CTX add to LDR. The apoptosis of tumor cells increased significantly after LDR. The tumor cells were arrested in G(1) phase in the groups treated with CTX and CTX + LDR, but cell cycle was more significantly arrested in mice exposed to LDR followed by CTX than in mice exposed only to LDR or CTX chemotherapy. Concentration of bone marrow cells and proliferation index in CTX + LDR mice were higher than those in the untreated mice. LDR or CTX + LDR could induce greater cytochrome c levels and caspase-3 activity in tumors. These results suggest that low dose radiation can enhance the anti-tumor effect of the chemotherapy agent CTX markedly. Furthermore, LDR significantly protects hematopoetic function of the bone marrow, which is of practical significance on adjuvant chemotherapy.

Auxin flow in anther filaments is critical for pollen grain development through regulating pollen mitosis.

It was well known that auxin is critical for anther/pollen grain development, however, the clear distribution and detailed effects of auxin during floral development are still unclear. We have shown here that, through analyzing GUS activities of Arabidopsis lines harboring auxin response elements DR5-GUS, auxin was mainly accumulated in the anther during flower stages 10-12. Further studies employing the indoleacetic acid-lysine synthetase (iaaL) coding gene from Pseudomonas syringae subsp. savastanoi under control of the promoter region of Arabidopsis phosphatidylinositol monophosphate 5-kinase 1 gene, which conducts the anther filament-specific expression, showed that block of auxin flow of filaments resulted in shortened filaments and significantly defective pollen grains. Similar phenotype was observed in tobacco plants transformed with the same construct, confirming the effects of auxin flow in filaments on anther development. Detailed studies further revealed that the meiosis process of pollen grain was normal while the mitosis at later stage was significantly defected, indicating the effects of auxin flow in filaments on pollen grain mitosis process. Analysis employing [(14)C]IAA, as well as the observation on the expression of AtPIN1, coding for auxin efflux carrier, demonstrated the presence of polar auxin transport in anther filaments and pollen grains.

The highly charged region of plant beta-type phosphatidylinositol 4-kinase is involved in membrane targeting and phospholipid binding.

In Arabidopsis thaliana and Oryza sativa, two types of PI 4-kinase (PI4Ks) have been isolated and functionally characterized. The alpha-type PI4Ks (approximately 220 kDa) contain a PH domain, which is lacking in beta-type PI4Ks (approximately 120 kDa). Beta-type PI4Ks, exemplified by Arabidopsis AtPI4Kbeta and rice OsPI4K2, contain a highly charged repetitive segment designated PPC (Plant PI4K Charged) region, which is an unique domain only found in plant beta-type PI4Ks at present. The PPC region has a length of approximately 300 amino acids and harboring 11 (AtPI4Kbeta) and 14 (OsPI4K2) repeats, respectively, of a 20-aa motif. Studies employing a modified yeast-based "Sequence of Membrane-Targeting Detection" system demonstrate that the PPC(OsPI4K2) region, as well as the former 8 and latter 6 repetitive motifs within the PPC region, are able to target fusion proteins to the plasma membrane. Further detection on the transiently expressed GFP fusion proteins in onion epidermal cells showed that the PPC(OsPI4K2) region alone, as well as the region containing repetitive motifs 1-8, was able to direct GFP to the plasma membrane, while the regions containing less repetitive motifs, i.e. 6, 4, 2 or single motif(s) led to predominantly intracellular localization. Agrobacterium-mediated transient expression of PPC-GFP fusion protein further confirms the membrane-targeting capacities of PPC region. In addition, the predominant plasma membrane localization of AtPI4Kbeta was mediated by the PPC region. Recombinant PPC peptide, expressed in E. coli, strongly binds phosphatidic acid, PI and PI4P, but not phosphatidylcholine, PI5P, or PI(4,5)P2 in vitro, providing insights into potential mechanisms for regulating sub-cellular localization and lipid binding for the plant beta-type PI4Ks.

MORN motifs in plant PIPKs are involved in the regulation of subcellular localization and phospholipid binding.

Multiple repeats of membrane occupation and recognition nexus (MORN) motifs were detected in plant phosphatidylinositl monophosphate kinase (PIPK), a key enzyme in PI-signaling pathway. Structural analysis indicates that all the MORN motifs (with varied numbers at ranges of 7-9), which shared high homologies to those of animal ones, were located at N-terminus and sequentially arranged, except those of OsPIPK1 and AtPIPK7, in which the last MORN motif was separated others by an approximately 100 amino-acid "island" region, revealing the presence of two kinds of MORN arrangements in plant PIPKs. Through employing a yeast-based SMET (sequence of membrane-targeting) system, the MORN motifs were shown being able to target the fusion proteins to cell plasma membrane, which were further confirmed by expression of fused MORN-GFP proteins. Further detailed analysis via deletion studies indicated the MORN motifs in OsPIPK1, together with the 104 amino-acid "island" region are involved in the regulation of differential subcellular localization, i.e. plasma membrane or nucleus, of the fused proteins. Fat Western blot analysis of the recombinant MORN polypeptide, expressed in Escherichia coli, showed that MORN motifs could strongly bind to PA and relatively slightly to PI4P and PI(4,5)P2. These results provide informative hints on mechanisms of subcellular localization, as well as regulation of substrate binding, of plant PIPKs.

At5PTase13 modulates cotyledon vein development through regulating auxin homeostasis.

Phosphatidylinositol signaling pathway and the relevant metabolites are known to be critical to the modulation of different aspects of plant growth, development, and stress responses. Inositol polyphosphate 5-phosphatase is a key enzyme involved in phosphatidylinositol metabolism and is encoded by an At5PTase gene family in Arabidopsis thaliana. A previous study shows that At5PTase11 mediates cotyledon vascular development probably through the regulation of intracellular calcium levels. In this study, we provide evidence that At5PTase13 modulates the development of cotyledon veins through its regulation of auxin homeostasis. A T-DNA insertional knockout mutant, At5pt13-1, showed a defect in development of the cotyledon vein, which was rescued completely by exogenous auxin and in part by brassinolide, a steroid hormone. Furthermore, the mutant had reduced auxin content and altered auxin accumulation in seedlings revealed by the DR5:beta-glucuronidase fusion construct in seedlings. In addition, microarray analysis shows that the transcription of key genes responsible for auxin biosynthesis and transport was altered in At5pt13-1. The At5pt13-1 mutant was also less sensitive to auxin inhibition of root elongation. These results suggest that At5PTase13 regulates the homeostasis of auxin, a key hormone controlling vascular development in plants.

Control of root cap formation by MicroRNA-targeted auxin response factors in Arabidopsis.

The plant root cap mediates the direction of root tip growth and protects internal cells. Root cap cells are continuously produced from distal stem cells, and the phytohormone auxin provides position information for root distal organization. Here, we identify the Arabidopsis thaliana auxin response factors ARF10 and ARF16, targeted by microRNA160 (miR160), as the controller of root cap cell formation. The Pro(35S):MIR160 plants, in which the expression of ARF10 and ARF16 is repressed, and the arf10-2 arf16-2 double mutants display the same root tip defect, with uncontrolled cell division and blocked cell differentiation in the root distal region and show a tumor-like root apex and loss of gravity-sensing. ARF10 and ARF16 play a role in restricting stem cell niche and promoting columella cell differentiation; although functionally redundant, the two ARFs are indispensable for root cap development, and the auxin signal cannot bypass them to initiate columella cell production. In root, auxin and miR160 regulate the expression of ARF10 and ARF16 genes independently, generating a pattern consistent with root cap development. We further demonstrate that miR160-uncoupled production of ARF16 exerts pleiotropic effects on plant phenotypes, and miR160 plays an essential role in regulating Arabidopsis development and growth.

A role of Arabidopsis inositol polyphosphate kinase, AtIPK2alpha, in pollen germination and root growth.

Inositol polyphosphates, such as inositol trisphosphate, are pivotal intracellular signaling molecules in eukaryotic cells. In higher plants the mechanism for the regulation of the type and the level of these signaling molecules is poorly understood. In this study we investigate the physiological function of an Arabidopsis (Arabidopsis thaliana) gene encoding inositol polyphosphate kinase (AtIPK2alpha), which phosphorylates inositol 1,4,5-trisphosphate successively at the D-6 and D-3 positions, and inositol 1,3,4,5-tetrakisphosphate at D-6, resulting in the generation of inositol 1,3,4,5,6-pentakisphosphate. Semiquantitative reverse transcription-PCR and promoter-beta-glucuronidase reporter gene analyses showed that AtIPK2alpha is expressed in various tissues, including roots and root hairs, stem, leaf, pollen grains, pollen tubes, the flower stigma, and siliques. Transgenic Arabidopsis plants expressing the AtIPK2alpha antisense gene under its own promoter were generated. Analysis of several independent transformants exhibiting strong reduction in AtIPK2alpha transcript levels showed that both pollen germination and pollen tube growth were enhanced in the antisense lines compared to wild-type plants, especially in the presence of nonoptimal low Ca(2+) concentrations in the culture medium. Furthermore, root growth and root hair development were also stimulated in the antisense lines, in the presence of elevated external Ca(2+) concentration or upon the addition of EGTA. In addition, seed germination and early seedling growth was stimulated in the antisense lines. These observations suggest a general and important role of AtIPK2alpha, and hence inositol polyphosphate metabolism, in the regulation of plant growth most likely through the regulation of calcium signaling, consistent with the well-known function of inositol trisphosphate in the mobilization of intracellular calcium stores.

DNA chip-based expression profile analysis indicates involvement of the phosphatidylinositol signaling pathway in multiple plant responses to hormone and abiotic treatments.

The phosphatidylinositol (PI) metabolic pathway is considered critical in plant responses to many environmental factors, and previous studies have indicated the involvement of multiple PI-related gene families during cellular responses. Through a detailed analysis of the Arabidopsis thaliana genome, 82 polypeptides were identified as being involved in PI signaling. These could be grouped into different families including PI synthases (PIS), PI-phosphate kinases (PIPK), phospholipases (PL), inositol polyphosphate phosphatases (IPPase), inositol polyphosphate kinases (IPK), PI transfer proteins and putative inositol polyphosphate receptors. The presence of more than 10 isoforms of PIPK, PLC, PLD and IPPase suggested that these genes might be differentially expressed during plant cellular responses or growth and development. Accordingly, DNA chip technology was employed to study the expression patterns of various isoforms. In total, 79 mRNA clones were amplified and used for DNA chip generation. Expression profile analysis was performed using samples that represented multiple tissues or cellular responses. Tested samples included normal leaf, stem and flower tissues, and leaves from plants treated with various hormones (auxin, cytokinin, gibberellin, abscisic acid and brassinosteroid) or environmental factors (temperature, calcium, sodium, drought, salicylic acid and jasmonic acid). Results showed that many PI pathway-related genes were differentially expressed under these experimental conditions. In particular, the different isoforms of each family were specifically expressed in many cases, suggesting their involvement in tissue specificity and cellular responses to environmental conditions. This work provides a starting point for functional studies of the relevant PI-related proteins and may help shed light onto the role of PI pathways in development and cellular responses.

[Isolation and expression pattern analysis of rice auxin-induced eIF3a coding gene].

Eukaryotic initiation factors (eIFs) are key elements in protein synthesis. Among identified 13 initiation factors, eIF3, composed of 8 or more subunits, is the largest one and plays a central role in the initiation of translation. As the largest subunit, eIF3a mediates most functions of eIF3. Based on the experiments that rice eIF3a gene could be induced by auxin (through fluorescent differential display PCR), a cDNA coding for rice eIF3a, named OseIF3a1, was isolated. OseIF3a1 cDNA, with a length of 3459 bp (including 5' and 3'-UTR) and encoding a 986-aa polypeptide, shared sequence identity of 82.4% and 70.1% with that in maize and tobacco. Comparison of cDNA and genomic sequence revealed the presence of 13 exons and 12 introns in OseIF3a1 gene. Reverse transcript PCR (RT-PCR) analysis indicated the expression of OseIF3a1 in root, shoots, young spike, stem and leaf tissues, promoter-GUS fusion studies further confirmed its expression in root tip, leaf, glume and stigma. The induction of OseIF3a1 by auxin suggested that function of auxin on growth may involve the regulation on translation level.

Isolation and functional characterization of the C-terminus of rice phosphatidylinositol 4-kinase in vitro.

A partial rice (Oryza sativa L.) cDNA clone, OsPI4K1c, was isolated through screening of a cDNA library constructed from tillering materials. OsPI4K1c encoded a peptide of 608 amino acids with a calculated molecular mass of 68.4 kDa. The OsPI4K1c peptide shared high homology and possessed the highly conserved domains present in most isolated cloned PI4-kinases, i.e. a lipid kinase unique (LKU) domain and a catalytic (CAT) domain. A region with similarity to pleckstrin homology (PH) domain was present in OsPI4K1c as well. Further comparison with genomic sequences in databases revealed that OsPI4K1c is located at the 3'-end of a putative rice PI 4-kinase coding gene OsPI4K1, and its coding region corresponded to the C-terminal half of OsPI4K1 protein. Twelve exons (49-562 bp in size) and 11 introns (77-974 bp in size) were identified in OsPI4K1c. The recombinant protein expressed in Escherichia coli phosphorylates phosphatidylinositol at the D4 position of the inositol ring. OsPI4K1 transcript levels were detected in a low but constitutive manner in shoot, stem, leaf, spike and root tissues and did not change upon treatment with different hormones, calcium and jasmonic acid (JA). However, treatment with salicylic acid (SA) elevated the mRNA level of the OsPI4K1 gene, which suggested the involvement of OsPI4K1 in wounding responses.

A pin gene families encoding components of auxin efflux carriers in Brassica juncea.

Based on the sequence information of Arabidopsis PIN1, two cDNAs encoding PIN homologues from Brassica juncea, Bjpin2 and Bjpin3, were isolated through cDNA library screening. Bjpin2 and Bjpin3 encoded proteins containing 640 and 635 amino acid residues, respectively, which shared 97.5% identities with each other and were highly homologous to Arabidopsis PIN1, PIN2 and other putative PIN proteins. BjPIN2 and BjPIN3 had similar structures as AtPIN proteins. Northern blot analysis indicated that Bjpin2 was expressed in stem, leaf and floral tissues, while Bjpin3 was expressed predominantly in stem and hypocotyls. Two promoter fragments of pin genes, Bjpin-X and Bjpin-Z, were isolated by 'genome walking' technique using primers at 5'-end of pin cDNA. Promoter-gus fusion studies revealed the GUS activities driven by Bjpin-X were at internal side of xylem and petal; while those driven by Bjpin-Z were detected at leaf vein, epidermal cell and cortex of stem, vascular tissues and anther. Results of the pin genes with different expression patterns in B. juncea suggested the presence of a gene family.