Albino Leaf1 That Encodes the Sole Octotricopeptide Repeat Protein Is Responsible for Chloroplast Development.

Chloroplast, the photosynthetic organelle in plants, plays a crucial role in plant development and growth through manipulating the capacity of photosynthesis. However, the regulatory mechanism of chloroplast development still remains elusive. Here, we characterized a mutant with defective chloroplasts in rice (Oryza sativa), termed albino leaf1 (al1), which exhibits a distinct albino phenotype in leaves, eventually leading to al1 seedling lethality. Electronic microscopy observation demonstrated that the number of thylakoids was reduced and the structure of thylakoids was disrupted in the al1 mutant during rice development, which eventually led to the breakdown of chloroplast. Molecular cloning revealed that AL1 encodes the sole octotricopeptide repeat protein (RAP) in rice. Genetic complementation of Arabidopsis (Arabidopsis thaliana) rap mutants indicated that the AL1 protein is a functional RAP. Further analysis illustrated that three transcript variants were present in the AL1 gene, and the altered splices occurred at the 3' untranslated region of the AL1 transcript. In addition, our results also indicate that disruption of the AL1 gene results in an altered expression of chloroplast-associated genes. Consistently, proteomic analysis demonstrated that the abundance of photosynthesis-associated proteins is altered significantly, as is that of a group of metabolism-associated proteins. More specifically, we found that the loss of AL1 resulted in altered abundances of ribosomal proteins, suggesting that RAP likely also regulates the homeostasis of ribosomal proteins in rice in addition to the ribosomal RNA. Taken together, we propose that AL1, particularly the AL1a and AL1c isoforms, plays an essential role in chloroplast development in rice.

Characterization of Rolled and Erect Leaf 1 in regulating leave morphology in rice.

Leaf morphology, particularly in crop, is one of the most important agronomic traits because it influences the yield through the manipulation of photosynthetic capacity and transpiration. To understand the regulatory mechanism of leaf morphogenesis, an Oryza sativa dominant mutant, rolled and erect leaf 1 (rel1) has been characterized. This mutant has a predominant rolled leaf, increased leaf angle, and reduced plant height phenotype that results in a reduction in grain yield. Electron microscope observations indicated that the leaf incurvations of rel1 dominant mutants result from the alteration of the size and number of bulliform cells. Molecular cloning revealed that the rel1 dominant mutant phenotype is caused by the activation of the REL1 gene, which encodes a novel unknown protein, despite its high degree of conservation among monocot plants. Moreover, the downregulation of the REL1 gene in the rel1 dominant mutant restored the phenotype of this dominant mutant. Alternatively, overexpression of REL1 in wild-type plants induced a phenotype similar to that of the dominant rel1 mutant, indicating that REL1 plays a positive role in leaf rolling and bending. Consistent with the observed rel1 phenotype, the REL1 gene was predominantly expressed in the meristem of various tissues during plant growth and development. Nevertheless, the responsiveness of both rel1 dominant mutants and REL1-overexpressing plants to exogenous brassinosteroid (BR) was reduced. Moreover, transcript levels of BR response genes in the rel1 dominant mutants and REL1-overexpressing lines were significantly altered. Additionally, seven REL1-interacting proteins were also identified from a yeast two-hybrid screen. Taken together, these findings suggest that REL1 regulates leaf morphology, particularly in leaf rolling and bending, through the coordination of BR signalling transduction.

An atypical HLH protein OsLF in rice regulates flowering time and interacts with OsPIL13 and OsPIL15.

In plants, flowering as a crucial developmental event is highly regulated by both genetic programs and environmental signals. Genetic analysis of flowering time mutants is instrumental in dissecting the regulatory pathways of flowering induction. In this study, we isolated the OsLF gene by its association with the T-DNA insertion in the rice late flowering mutant named A654. The OsLF gene encodes an atypical HLH protein composed of 419 amino acids (aa). Overexpression of the OsLF gene in wild type rice recapitulated the late flowering phenotype of A654, indicating that the OsLF gene negatively regulates flowering. Flowering genes downstream of OsPRR1 such as OsGI and Hd1 were down regulated in the A654 mutant. Yeast two hybrid and colocalization assays revealed that OsLF interacts strongly with OsPIL13 and OsPIL15 that are potentially involved in light signaling. In addition, OsPIL13 and OsPIL15 colocalize with OsPRR1, an ortholog of the Arabidopsis APRR1 gene that controls photoperiodic flowering response through clock function. Together, these results suggest that overexpression of OsLF might repress expression of OsGI and Hd1 by competing with OsPRR1 in interacting with OsPIL13 and OsPIL15 and thus induce late flowering.

Overexpression of ACL1 (abaxially curled leaf 1) increased Bulliform cells and induced Abaxial curling of leaf blades in rice.

Understanding the genetic mechanism underlying rice leaf-shape development is crucial for optimizing rice configuration and achieving high yields; however, little is known about leaf abaxial curling. We isolated a rice transferred DNA (T-DNA) insertion mutant, BY240, which exhibited an abaxial leaf curling phenotype that co-segregated with the inserted T-DNA. The T-DNA was inserted in the promoter of a novel gene, ACL1 (Abaxially Curled Leaf 1), and led to overexpression of this gene in BY240. Overexpression of ACL1 in wild-type rice also resulted in abaxial leaf curling. ACL1 encodes a protein of 116 amino acids with no known conserved functional domains. Overexpression of ACL2, the only homolog of ACL1 in rice, also induced abaxial leaf curling. RT-PCR analysis revealed high expressions of ACLs in leaf sheaths and leaf blades, suggesting a role for these genes in leaf development. In situ hybridization revealed non-tissue-specific expression of the ACLs in the shoot apical meristem, leaf primordium, and young leaf. Histological analysis showed increased number and exaggeration of bulliform cells and expansion of epidermal cells in the leaves of BY240, which caused developmental discoordination of the abaxial and adaxial sides, resulting in abaxially curled leaves. These results revealed an important mechanism in rice leaf development and provided the genetic basis for agricultural improvement.

Overexpression of Osta-siR2141 caused abnormal polarity establishment and retarded growth in rice.

Small RNAs (smRNAs) including miRNAs and siRNAs are critical for gene regulation and plant development. Among the highly diverse siRNAs, trans-acting siRNAs (ta-siRNAs) have been shown to be plant-specific. In Arabidopsis, eight TAS loci belonging to four families (TAS1, TAS2, TAS3, and TAS4) have been identified, and bioinformatics analysis reveals that the sequence of TAS3 is highly conserved in plants. In this study, the function of TAS3 ta-siRNA (tasiR-ARF) has been revealed in rice (Oryza sativa L.) on polarity establishment and stage transition from vegetative to reproductive development by over-expressing Osta-siR2141. Osta-siR2141 replaced miR390 in the miR390 backbone for ectopic expression in rice, and overexpression of Osta-siR2141 caused disturbed vascular bundle development and adaxialization in polarity establishment. Transgenic lines also displayed abnormal shoot apical meristems (SAMs) and retarded growth at the vegetative stage. Molecular analysis revealed that overexpression of Osta-siR2141 resulted in the down-regulation of miR166 and the up-regulation of class III homeodomain-leucine zipper genes (HD-ZIPIIIs) in the vegetative stage but not in the reproductive stage. Moreover, overexpression of Osta-siR2141 in Arabidopsis disturbed polarity establishment and retarded stage transition, suggesting that tasiR-ARF was functionally conserved in rice and Arabidopsis.

The expression pattern of a rice proteinase inhibitor gene OsPI8-1 implies its role in plant development.

A rice proteinase inhibitor (PI) gene OsPI8-1 was identified. Belonging to the potato inhibitor I family, this gene contains a 201bp coding region with no introns and encodes a deduced protein of 66 amino acids which holds a PI domain. There are two uniform gene copies, OsPI8-1a and OsPI8-1b, with direct-repeat arrangement and an interval span of 13 kb on rice chromosome 8, corresponding to the site of BAC clone P0528B09 (Accession No. AP004703). Reverse transcription polymerase chain reaction (RT-PCR) assays showed that both OsPI8-1a and OsPI8-1b can be expressed in wild-type 'Zhonghua No.11'. To investigate the physiological functions of OsPI8-1 in plant development, we analyzed the expression patterns of the reporter gene beta-glucuronidase (GUS) driven by OsPI8-1 promoter at different developmental stages and tissues. It was demonstrated that no GUS signals were detected in the roots. Despite that very high GUS expression was examined in the shoot apical meristem, no detectable GUS activity in the developmental domains of leaf primordium was observed. OsPI8-1 promoter showed an obvious wound-induced response in mature leaves. Little GUS activity was detected in young nodes and internodes at the seedling stage, but active GUS expression was observed near the nodes on mature culms. In the developing stage of the anther, GUS signal was specifically located in the middle layer and the endothecium between the epidermis and tapetum. In the germinating seed, GUS expression was gradually accumulated in the side of scutellar epithelium close to the embryo. These tissue-specific accumulations suggested that OsPI8-1 has multiple endogenous roles on developmental regulation. In this report, the inhibitor function of OsPI8-1 to proteolytic enzymes and the potential influence of their poise on plant development (such as seed germination, tapetum degeneration, programmed cell death, etc.) were discussed.

Overexpression of transcription factor OsLFL1 delays flowering time in Oryza sativa.

Flowering time is regulated by genetic programs and environment signals in plants. Genetic analysis of flowering time mutants is instrumental in dissecting the regulatory pathways of flower induction. Genotype W378 is a rice (Oryza sativa) late-flowering mutant selected from our collections of T-DNA insertion line. The T-DNA flanking gene in mutant W378 codes OsLFL1 (O. sativa LEC2 and FUSCA3 Like 1), a putative B3 DNA-binding domain-containing transcription factor. In wild-type rice OsLFL1 is expressed exclusively in spikes and young embryos, while in mutant W378 it is ectopically expressed. Introduction of OsLFL1-RNAi into mutant W378 successfully down-regulated OsLFL1 expression and restored flowering to almost normal time, indicating that overexpression of OsLFL1 confers late flowering for mutant W378. The flowering-promoting gene Ehd1 and its downstream genes are all down-regulated in W378. Thus, overexpression of OsLFL1 might delay the flowering of W378 by repressing the expression of Ehd1.

Ectopic expression of OsLFL1 in rice represses Ehd1 by binding on its promoter.

B3 domain was identified as a novel DNA-binding motif specific to higher plant species. The B3 proteins play important roles in plant development. In the mutant W378, the mutant gene coding OsLFL1, a putative B3 transcription factor gene, was ectopically expressed. In this study, it was found that the flowering promoting gene Ehd1 and its putative downstream genes were all repressed by OsLFL1. Electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) analyses suggest that OsLFL1 binds to the RY cis-elements (CATGCATG) in the promoter of the Ehd1 gene. Thus, ectopically expressed OsLFL1 might repress Ehd1 via binding directly to the RY cis-elements in its promoter.

Over-expression of rice OsAGO7 gene induces upward curling of the leaf blade that enhanced erect-leaf habit.

High-yield cultivars are characterized by erect leaf canopies that optimize photosynthesis and thus favor increased biomass. Upward curling of the leaf blade (called rolled leaf) can result in enhanced erect-leaf habit, increase erect duration and promote an overall erect leaf canopy. The rice mutant R05, induced through transferred DNA (T-DNA) insertion, had the rolled-leaf trait. The leaves in the wild type demonstrated natural drooping tendencies, resulting in decreasing leaf erection indices (LEIs) during senescence at the 20th day after flowering. Conversely, LEIs of the leaves in R05 remained high, even 20-day post-flowering. We applied T-DNA tagging and isolated a rolled-leaf gene from rice which, when over-expressed, could induce upward curling of the leaf blade. This gene encodes for a protein of 1,048 amino acids including the PAZ and PIWI conserved domains, belonging to the Argonaute (AGO) family. There are at least 18 members of the AGO family in rice. According to high-sequence conservation, the rolled-leaf gene in rice could be orthologous to the Arabidopsis ZIP/Ago7 gene, so we called it OsAGO7. These results provide a possible opportunity for implementing OsAGO7 gene in crop improvement.

[Transpositional behaviour analysis of Ds element from different insertion sites on chromosome 4 in rice].

Transgenic plants with Ds element distributed over different loci on chromosome 4 (Fig. 1) and the homozygous transformants with Ac transposase gene were established through Agrobacterium-mediated approach. In this study, the plants carrying Ds element from different loci were crossed with the plant carrying Ac transposase individually. The plants of F(1) generation carrying both Ds element and Ac transposase were used to produce the F(2) populations (Table 1). Analysis of the F(2) generation by the PCR method revealed that the excision frequencies of Ds element were higher in the telomeric region of chromosome 4 than in the centromeric region (Fig. 4). These results showed that the insertion site of Ds element has strong effect on its excision frequency. We suggest that the special construct of chromosome near the insertion site of Ds element is related to the excision frequency of the Ds element.

Thermostability of photosynthesis in two new chlorophyll b-less rice mutants.

Leaves of the two new chlorophyll b-less rice mutants VG28-1, VG30-5 and the wild type rice cv. Zhonghua 11 were subjected to temperatures 28, 36, 40, 44 and 48 degrees C in the dark for 30 min or gradually elevated temperature from 30 degrees C to 80 degrees C at 0.5 degrees C/min. The thermostability of photosynthetic apparatus was estimated by the changes in chlorophyll fluorescence parameters, photosynthetic rate and pigment content, chloroplast ultrastructure and tissue location of H2O2 accumulation. There were different patterns of F(o)-temperature curves between the Chl b-less mutants and the wild type plant, and the temperature of F(o) rising threshold was shifted 3 degrees C lower in the Chl b-less mutants (48 degrees C) than in the wild type (51 degrees C). At temperature up to about 45 degrees C, chloroplasts were swollen and thylakoid grana became misty accompanied with the complete loss of photosynthetic oxygen evolution in the two Chl b-less mutants, but chloroplast ultrastructure in the wild type showed no obvious alteration. After 55 degrees C exposure, the disordered thylakoid and significant H2O2 accumulation in leaves were found in the two Chl b-less mutants, whereas in the wild type plant, less H2O2 was accumulated and the swollen thylakoid still maintained a certain extent of stacking. A large extent of the changes in qP, NPQ and Fv/Fm was consistent with the Pn decreasing rate in the Chl b-less mutants during high temperature treatment as compared with the wild type. The results indicated that the Chl b-less mutants showed a tendency for higher thermosensitivity, and loss of Chl b in LHC II could lead to less thermostability of PSII structure and function. Heat damage to photosynthetic apparatus might be partially attributed to the internal oxidative stress produced at severely high temperature.

[Genetic analysis of a heading-delayed mutant by T-DNA insertion in rice (Oryza sativa L.)].

Transposon tagging was used to isolate genes in higher plant. In this study, a delayed heading mutant caused by T-DNA insertion in rice was identified. Genetic analysis of the mutant showed that the three types of phenotype, normal heading, delayed heading and overly delayed heading in the segregating populations derived from the T-DNA heterozygotes fit the ratio of 1:2:1. Test for Basta resistance showed the delayed heading plants were all resistant while the normal heading plants were susceptible, and the ratio of resistant and susceptible plants was 3:1, which indicated that the delayed heading mutant was co-segregated with Basta resistance. The delayed heading mutant caused by T-DNA insertion was confirmed by T-DNA detection using PCR method. This delayed heading mutant will be used for isolation of the tagged gene in rice.

Distribution of T-DNA carrying a Ds element on rice chromosomes.

Over 3000 rice plants with T-DNA carrying a Ds element were constructed by Agrobacterium tumefaciens mediation. Using inverse PCR methodology, 590 unique right flanking sequences of T-DNA (Ds) were retrieved from independent transformants and classified into six main types on the basis of the origin of filler DNA between the right border of T-DNA and flanking sequence of rice genome. Type I sequences were the most common and showed canonical integration that T-DNA right border was followed by rice genome sequence with or without filler DNA of no more than 50 bp, while type II sequences displayed a vector-genome combination that T-DNA right border was followed by a vector fragment and then connected with rice genome sequence. The location and distribution of 340 type I and II flanking sequences on the rice chromosome were determined using BLAST analysis. The 340 Ds insertions at an average interval of 0.8 megabase (Mb) constructed a basic framework of Ds starter points on whole rice chromosomes. The frequency of T-DNA (Ds) inserted into the exons of predicted genes on chromosome one was 21%. Knowledge of T-DNA (Ds) locations on chromosomes will prove to be a useful resource for isolating rice genes by Ds transposon tagging as these Ds insertions can be used as starting lines for further mutagenesis.

[Structure and expression analysis of the gama-glutamylcysteine synthetase gene in rice].

Gama-glutamylcysteine synthetase (GCS) is a rate-limiting enzyme in GSH biosynthesis. The GCS gene has been cloned in Arabidopsis thaliana and other plants, but has still not been reported in rice. From rice mutant population generated from T-DNA insertion, we cloned the rice GCS gene from mutant L395 by T-DNA tag cloning method, and named it OsGCS (Genbank accession No. AJ508915). Full length OsGCS cDNA clones were obtained from a rice cDNA library by the PCR method. A comparison of the genome and cDNA sequence (Genbank accession No. AJ508916) shows that OsGCS gene is composed of 15 exons and 14 introns and coding a 493-amino acid protein. The OsGCS gene is highly homologous with the AtGCS gene in the coding region but completely different in the promoter region. The putative transcription start site (TSS) confirmed by RT-PCR was located 211 bp upstream of the translation start codon "ATG". In mutant L395, a single T-DNA copy was integrated between the second intron and second exon of OsGCS gene, causing one nucleotide deletion in the second exon and two nucleotide deletions in the second intron. No significant differences were found in Cd(2+) stress tolerance, rice GCS gene expression level and GSH content between mutant L395 and Zhonghua 11. It is possible that another GCS gene on chromosome 7 might complement function of OsGCS gene on chromosome 5.

The OsEBP-89 gene of rice encodes a putative EREBP transcription factor and is temporally expressed in developing endosperm and intercalary meristem.

The AP2/EREBP transcription factors play important roles in plant development and in the responses of plants to biotic and abiotic stresses. All members of the EREBP subfamily described to date are from dicotyledonous plants. In this paper, we describe the cloning and characterization of a rice gene, OsEBP-89, encoding a protein 326 amino acids long with a typical EREBP domain; this is the first report of an EREBP transcription factor in a monocotyledonous plant. Except for the EREBP domain, the OsEBP-89 protein does not have substantial sequence similarities to other members of the subfamily. The DNA-binding activity of the EREBP domain was confirmed by electrophoretic mobility-shift assays. An activation domain rich in acidic amino acids was identified by using a yeast one-hybrid system. Two putative nuclear-localization signals were also identified. The results of northern blot hybridization experiments showed that the transcript of the OsEBP-89 gene accumulates primarily in immature seeds, roots, and leaves (low levels). More detailed information about the pattern of OsEBP-89 gene expression was obtained by histochemical studies of transgenic rice plants carrying an OsEBP-89 5'/GUS reporter gene. The reporter gene was expressed in the endosperm starting at 7 days after pollination and in the intercalary meristem of plants. Expression of OsEBP-89 was induced in roots of rice seedlings by treatment with ACC, NaCl, or 2,4-D. Two cis-acting elements, an endosperm motif and a primary PERE, are present upstream of the OsEBP-89 coding region and may be involved in regulating its expression. Collectively, these results suggest that the OsEBP-89 gene is a new member of the EREBP subfamily and may be involved in ethylene-dependent seed maturation and shoot development of rice.