Rice <Emphasis Type="BoldItalic">ZFP15</Emphasis> Gene Encoding for a Novel C2H2-type Zinc Finger Protein Lacking DLN box,is Regulated by Spike Development but not by Abiotic Stresses

A novel C2H2-type zinc finger protein gene, ZFP15, was cloned from rice by RT-PCR approach. The ZFP15 gene encodes a protein of 144 amino acid residues with a predicted molecular mass of 15 kDa. The ZFP15 protein comprises two C2H2-type zinc finger domains, a putative nuclear localization signal (NLS) at its N-terminus but the DLN-box identified in all reported plant C2H2-type zinc finger proteins was not found. A homology search revealed that ZFP15 gene was localized within a cluster of C2H2-type zinc finger genes in BAC clone OJ1754_E06 mapped on chromosome 3. All three members in the cluster encoded proteins showed high identities in amino acids and might contribute to a co-regulation. The RT-PCR assay revealed that ZFP15 mRNA was not regulated by cold, salt, drought and ABA stresses, though CRT/DRE and ABRE elements were found in the promoter region of ZFP15 gene. The expression profiling also showed that ZFP15 mRNA was expressed with a lower level in leaves and roots, but not detected in stems. Besides, ZFP15 was shown to accumulate much more in flowering spike than in immature spike. Thus, ZFP15, as the first characterized C2H2-type zinc finger protein in rice, might play a regulatory role on rice spike development.     

Functional characterization of the stunt lemma palea 1 mutant allele in rice

The rice stunted lemma/palea 1 (slp1) mutant displays a dwarf, shortened panicle length, degenerated lemma and palea, and sterility. A previous study suggested that a missense mutation at the sixth amino acid of the OsSPL16 protein was likely to be responsible for the slp1 mutant phenotype. The current study shows that the overexpression of the wild-type OsSPL16 allele in slp1/slp1 and Slp1/slp1 mutants was unable to convert the slp1 mutant phenotype to normal. However, the introduction of the mutant OsSPL16 allele into a normal rice cultivar led to the slp1 mutant phenotype in transgenic plants. These results indicated that the missense mutation in OsSPL16 creates a neomorphic allele, which affects plant height and the development of the inflorescence and spikelet.     

A Defect in Zinc Finger Protein Double B-box 1a (DBB1a) Causes Abnormal Floral Development in <Emphasis Type="Italic">Arabidopsis</Emphasis>

The double B-box (DBB) type zinc finger protein has thus far been shown to be involved in photomorphegenesis in Arabidopsis thaliana. Here, we show that DBB1a is expressed in the embryo, cytolden, and flower. Misexpression of DBB1a in mutant plants resulted in abnormal numbers and patterns of floral organs. We further show that DBB1a could regulate expression of several floral homeotic genes, including APETALA 2, APETALA 3, PISTILLATA, and AGAMOUS. Interestingly, expression of the microRNA gene MiR172, which is involved in organ boundary establishment, was also misregulated in the dbb1a mutant plants. Our study identified a previously uncharacterized role of DDB1a in regulation of expression of floral homeotic genes and miR172, which is important for understanding of floral pattern formation.     

Poly ADP-ribose polymerase-1 promotes seed-setting rate by facilitating gametophyte development and meiosis in rice (Oryza sativa L.)

Poly(ADP-ribose) polymerases (PARPs), which transfer either monomer or polymer of ADP-ribose from nicotinamide adenine dinucleotide (NAD⁺) onto target proteins, are required for multiple processes in DNA damage repair, cell cycle, development, and abiotic stress in animals and plants. Here, the uncharacterized rice (Oryza sativa) OsPARP1, which has been predicted to have two alternative OsPARP1 mRNA splicing variants, OsPARP1.1 and OsPARP1.2, was investigated. However, bimolecular fluorescence complementation showed that only OsPARP1.1 interacted with OsPARP3 paralog, suggesting that OsPARP1.1 is a functional protein in rice. OsPARP1 was preferentially expressed in the stamen primordial and pollen grain of mature stamen during flower development. The osparp1 mutant and CRISPR plants were delayed in germination, indicating that defective DNA repair machinery impairs early seed germination. The mutant displayed a normal phenotype during vegetative growth but had a lower seed-setting rate than wild-type plants under normal conditions. Chromosome bridges and DNA fragmentations were detected in male meiocytes at anaphase I to prophase II. After meiosis II, malformed tetrads or tetrads with micronuclei were formed. Meanwhile, the abnormality was also found in embryo sac development. Collectively, these results suggest that OsPARP1 plays an important role in mediating response to DNA damage and gametophyte development, crucial for rice yield in the natural environment.     

Mutations in the Dof zinc finger genes DAG2 and DAG1 influence with opposite effects the germination of Arabidopsis seeds

We describe the Arabidopsis gene DAG2 encoding a Dof zinc finger protein and show that it is involved in the control of seed germination. An Arabidopsis mutant line with a T-DNA insertion in DAG2 isolated by reverse genetics produces seeds that are substantially more dependent than the wild type on the physical stimuli-light and cold treatment-that promote germination. Mutant dag2 seeds also are less sensitive to the germination-promotive effect of gibberellins, because a 10-fold higher amount of gibberellins is needed to restore germination when endogenous gibberellin biosynthesis is blocked. The seed germination characteristics of the dag2 mutant are opposite to those of dag1, a knockout mutant of another Dof gene (DAG1) that we showed previously to be involved in the control of seed germination, and are similar to those of plants that overexpress DAG1. The promoter of the DAG2 gene is active specifically in the vascular system of the mother plant but not in the embryo, and segregation analysis indicates that the effect of the dag2 mutation is maternal. Both characteristics are in common with DAG1; additionally, the DAG1 and DAG2 proteins share high sequence homology and an identical zinc finger domain. These data suggest, and the germination phenotype of the double mutant is compatible with, a model whereby the zinc finger proteins DAG1 and DAG2 act on a maternal switch that controls seed germination, possibly by regulating the same gene(s).     

沙冬青RING型锌指蛋白基因AmRFP1的克隆与功能初步分析

利用RT-PCR方法,从强抗逆植物蒙古沙冬青克隆到1个受寒旱诱导的锌指蛋白基因AmRFP1。预测其编码蛋白由366个氨基酸残基组成,其中含有1个C3H2C3型锌指结构域,故属于RING-H2(C3H2C3)型锌指蛋白。该蛋白还含有2个跨膜区,很可能定位于细胞质膜。半定量RT-PCR分析表明,在不同季节野外生长沙冬青植株嫩叶中,AmRFP1在夏季只有低水平表达,进入秋季后表达量明显增加,尤其在秋末冬初其表达量迅速增加并达到全年最高峰,但在进入最寒冷的隆冬后又回落至秋季水平并维持到翌年春季基本未变。将该基因在拟南芥中超表达,则明显降低了转基因拟南芥的抗冻性。     

OsLSD1, a rice zinc finger protein, regulates programmed cell death and callus differentiation

The Arabidopsis LSD1 and LOL1 proteins both contain three conserved zinc finger domains and have antagonistic effects on plant programmed cell death (PCD). In this study, a rice (Oryza sativa) functional homolog of LSD1, designated OsLSD1, was identified. The expression of OsLSD1 was light-induced or dark-suppressed. Overexpression of OsLSD1 driven by the cauliflower mosaic virus 35S promoter accelerated callus differentiation in transformed rice tissues and increased chlorophyll b content in transgenic rice plants. Antisense transgenic rice plants exhibited lesion mimic phenotype, increased expression of PR-1 mRNA, and an accelerated hypersensitive response when inoculated with avirulent isolates of blast fungus. Both sense and antisense transgenic rice plants conferred significantly enhanced resistance against a virulent isolate of blast fungus. Moreover, ectopic overexpression of OsLSD1 in transgenic tobacco (Nicotiana tabacum) enhanced the tolerance to fumonisins B1 (FB1), a PCD-eliciting toxin. OsLSD1 green fluorescent protein fusion protein was located in the nucleus of tobacco cells. Our results suggest that OsLSD1 plays a negative role in regulating plant PCD, whereas it plays a positive role in callus differentiation.     

Mutant and Overexpression Analysis of a C2H2 Single Zinc Finger Gene of Arabidopsis

The goal of this work was to characterize an Arabidopsis C2H2 single zinc finger gene, named AtZFP11, that is similar to SUPERMAN (SUP) and RABBIT EARS (RBE). No altered phenotype was observed in mutants analyzed that were derived through TILLING nor a T-DNA insertion into the exon of AtZFP11. Plants derived by intercrossing AtZFP11 to SUP and RBE alleles did not display any phenotype that could be attributed to AtZFP11. Transgenic plants constitutively overexpressing the AtZFP11 protein displayed severe abnormalities. Phenotypically normal transgenic plants that contained an ecdysone-inducible promoter system fused to the AtZFP11 gene were recovered. These transgenic plants displayed similar abnormalities once induced with methoxyfenozide. Microarray and RT-PCR analysis of gene expression in plants overexpressing AtZFP11 after induction revealed changes in gene expression of a number of genes involved in auxin, jasmonic acid, and stress responses. These results suggest that AtZFP11 plays a role in regulation of gene expression and may be functionally redundant or that conditions needed to observe phenotypic differences in the mutant plants were not provided in the course of these experiments.     

The DC1‐domain protein VACUOLELESS GAMETOPHYTES is essential for development of female and male gametophytes in Arabidopsis

In this work we identified VACUOLELESS GAMETOPHYTES (VLG) as a DC1 domain‐containing protein present in the endomembrane system and essential for development of both female and male gametophytes. VLG was originally annotated as a gene coding for a protein of unknown function containing DC1 domains. DC1 domains are cysteine‐ and histidine‐rich zinc finger domains found exclusively in the plant kingdom that have been named on the basis of similarity with the C1 domain present in protein kinase C (PKC). In Arabidopsis, both male and female gametophytes are characterized by the formation of a large vacuole early in development; this is absent in vlg mutant plants. As a consequence, development is arrested in embryo sacs and pollen grains at the first mitotic division. VLG is specifically located in multivesicular bodies or pre‐vacuolar compartments, and our results suggest that vesicular fusion is affected in the mutants, disrupting vacuole formation. Supporting this idea, AtPVA12 – a member of the SNARE vesicle‐associated protein family and previously related to a sterol‐binding protein, was identified as a VLG interactor. A role for VLG is proposed mediating vesicular fusion in plants as part of the sterol trafficking machinery required for vacuole biogenesis in plants.     

Isolation, characterization and disruption of the areA nitrogen regulatory gene of Gibberella fujikuroi

The gene areA-GF, a homologue of the major nitrogen regulatory genes nit-2, areA, nre and NUT1 of Neurospora crassa, Aspergillus nidulans, Penicillium chrysogenum and Magnaporthe grisea, respectively, was cloned from the gibberellin (GA)-producing rice pathogen Gibberella fujikuroi.areA-GF encodes a protein of 972 amino acid residues which contains a single putative zinc finger DNA-binding domain that is at least 98% identical to the zinc finger domains of the homologous fungal proteins. The areA-GF gene has been shown to be functional in N. crassa by heterologous complementation of a RIP induced nit-2 mutant. The transformation rate was nearly as high as in a homologous complementation control. Transformants were able to utilize nitrate and expressed a normally regulated nitrate reductase activity. To generate areA-GF  ⁻ mutants, gene replacement experiments were performed using a linearized replacement vector carrying the hygromycin B phosphotransferase (hph) gene. The replacement of the zinc finger by the hygromycin cassette resulted in transformants which were unable to utilize nitrogen sources other than ammonium and glutamine, and gave significantly reduced gibberellin production yields. Complementation of such a mutant with the wild-type gene led to the full recovery of gibberellin production. Received: 23 April 1998 / Accepted: 16 October 1998     

The rice <Emphasis Type="Italic">OsLOL2</Emphasis> gene encodes a zinc finger protein involved in rice growth and disease resistance

Arabidopsis LSD1-related proteins that contain LSD1-like zinc finger domains have been identified to be involved in disease resistance and programmed cell death. To investigate the potential role of LSD1-related gene in rice (Oryza sativa L.), we cloned an LSD1 ortholog, OsLOL2, from the rice cDNA plasmid library. The OsLOL2 gene is predicted to encode a polypeptide of 163 amino acids with two LSD1-like zinc finger domains with 74.5% identity to those of LSD1. Southern blot analysis indicated that OsLOL2 was a single-copy gene in the rice genome. Transgenic rice lines carrying the antisense strand of OsLOL2 with decreased expression of OsLOL2 had dwarf phenotypes, and the dwarfism could be restored by exogenous GA₃ treatment, suggesting that the dwarfism was the result of a deficiency in bioactive gibberellin (GA). In agreement with this possibility, the content of endogenous bioactive GA₁ decreased in the antisense transgenic lines. Expression of OsKS1, one of the genes encoding for GA biosynthetic enzymes, was suppressed in the antisense transgenic lines. Sense transgenic lines with increased expression of OsLOL2 were more resistant to rice bacterial blight, while antisense transgenic lines were less resistant to rice bacterial blight. The OsLOL2-GFP (green fluorescence protein) fusion protein was localized in the nucleus of cells of transgenic BY2 tobacco (Nicotiana tabacum L.). These data suggest that OsLOL2 is involved in rice growth and disease resistance.