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.

[Analysis on the characters of progenies of the rice transformed with anti-sense waxy gene].

The segregation of exogenous genes was studied by hygromycin-resistant and PCR experiments in the transgenic rice (Oryza sativa L. subsp. japonica and indica) with anti-sense waxy gene, meanwhile the change of amylose and waxy protein contents in progenies of transgenic rice was analyzed. The results showed no matter the rice Guangjing No.1 (O. Sativa L. subsp. japonica) were transformed by p13w4 plasmid carrying anti-sense waxy gene and hygromycin-resistant gene, or in the rice 01Z5202 (O. sativa L. subsp. indica) were co-transformed by p13w8 plasmid carrying anti-sense waxy gene and p1300 plasmid carrying hygromycin-resistant gene, the target gene(s) had been segregated in the progenies; the content of amylose of the transgenic plants was lower than those in non-transgenic ones, and the content of amylose in some of transgenic plants was less than 10.0% (of the weight of grain), which was much lower than those in the control (about 22.04%); and the analysis with SDS-PAGE showed the content of the waxy protein are positively correlated with the con-tent of amylose in the tested transgenic rice materials.

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.

[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.

[Establishment of promoter trapping system mediated by activator/dissociation (beta-glucuronidase) construction in rice].

The coding region of Bar gene, the left border of Ds element, the coding region of GUS gene, the transposase of Ac element, the right border of Ds element and the promoter of Ubi gene were inserted into the T-DNA region of vector pCAMBIA1300 in turn to construct plasmid p13B. The orientations of the ubiquitons' promoter, Ac transposase and Bar are identical but opposite to that of the GUS gene (Fig.1). The plasmid p13B was then introduced into the calli of Oryza sativa subsp. japonica cv. Zhonghua 11 by Agrobacterium tumefaciens-mediated transforming to trap genes in rice. Eighteen independent transgenic lines were obtained and propagated. T(2) generations of 18 independent transgenic lines were screening by herbicide (Basta) (Fig.2) and the herbicide-resistant plants obtained were analyzed by PCR (Fig.3). Ds element transposed in an inheritable manner was found in 37 plants, in which 5 plants showed GUS activity (Fig.4).

[Determination of amylose content in transgenic progenies containing an antisense waxy gene and identifying of pure lines in indica rice].

Through genetic transformation mediated by Agrobacterium tumefaciens, an antisense waxy gene was introduced into Longtefu B line, a male sterile maintainer line in indica (Oryza sativa L.). Thirty transgenic plants showed integration of antisense waxy gene into the genome as determined by PCR assay, and twenty eight were confirmed by Southern blotting. T1 seeds from twenty one transgenic plants showed a marked decrease of amylose content, ranging from 3%-13% less than control, and seeds from some transgenic lines exhibited typical waxy phenotype. Six transgenic lines were selected to examine the amylose content in different generations. In the T4 generation, two homozygous lines, Long 3-1-1-1 and Long 5-8-2-1, were selected, with amylose content of 15.9% and 8.4% respectively. The amylose content in these lines is in consistent with the decrease of the accumulation of Wx protein as determined by SDS-PAGE electrophoresis. Cross and subsequent backcross of Long 3-1-1-1 and Long 5-8-2-1 with the Longtefu mare sterile line were performed to determine the changes of amylose content in F1 and B1F1 seeds. The results showed an average amlyose content of 21.4% in F1 seeds with Long 3-1-1-1 as a parent, while only 13.6% with Long 5-8-2-1 as a parent. In addition, the average amylose contents in B1F1 seeds were 17.1% and 9.3% respectively. Our results indicated that during the fertility transfer in the male sterile line, stable transgenic lines with medium or low contents of amylose had direct effects on amylose content in F1 seeds.

[Rapidly obtaining the markerless transgenic rice with reduced amylose content by co-transformation and anther culture].

The plasmid p13W8 carrying antisense fragment of waxy gene and plasmid pCAMBIA1300 containing hpt gene were introduced into rice by Agrobacterium tumefaciens-mediated co-transformation, and 86 transgenic plants were obtained, 32 of them showed positive bands for antisense waxy gene by PCR analysis, the waxy-positive plant frequency is 37.2%. The segregation of antisense fragment of waxy gene and hpt gene was observed by PCR using hpt gene primers and waxy gene primers respectively in 29 T(1) population. One hundred and eighty-three plants containing only the antisense fragment of waxy gene were identified in 1 264 T(1) plants, the waxy-positive plant frequency is 14.4% (Table 1). The amylose content of seeds derived from transgenic plants with only the antisense fragment of waxy gene were determined, varying degrees of reduction in amylose content were found in some plants (Table 2). Four T(1) plants with reduced amylose content were selected through anther culture. Thirty-four anther culture plants seed normally, 23 of them were shown to contain only the antisense fragment of waxy gene (Table 3) by PCR analysis, and the amylose content was reduced to 5%-12% (Table 4). It took only one and half years to obtain the stably inherited markerless transgenic rice with reduced amylose content by co-transformation and anther culture technique.

[Genetic analysis of a rolled-leaf mutant in rice population of T-DNA insertion].

A rolled-leaf mutant was obtained in a T-DNA(containing bar gene and Ds element) insertion population, which consist of transgenic japonica rice Zhonghua 11 mediated by Agrobacterium tumefaciens. Through self-hybridization of three generations, one of trait-purified mutants (R1-A2) was obtained and used as parent to cross with variety Zhonghua 11. The leaves of 36 F1 plants investigated were rolled and resistant to herbicide Basta. Among 852 F2 plants, the segregation ratio of rolled leaves to normal leaves(645:207) was consistent with 3:1. All rolled-leaf plants were resistant to herbicide Basta, and all normal leaf plants were sensitive to herbicide Basta. These results showed that the trait of rolled-leaf is co-segregated with Basta resistance. The total DNA of 45 rolled-leaf plants and 30 normal leaf plants in F2 population were amplified to test the presence of T-DNA by Ds primers. The results showed that the positive band were amplified in all rolled-leaf plants, but not in every normal leaf plant. In F1B1 progenies, all plants which derived from backcross parent R1-A2 were rolled leaves; while variety Zhonghua 11 was used as backcross parent, the segregation ratio of rolled-leaf to normal leaf was consistent with 1:1. Taking these data together, it indicated that the rolled-leaf mutant was co-segregation with T-DNA and controlled by single dominant gene.

[The morphology observation, mechanics intensity test and genetic analysis of brittle culm mutant bcm581-1 in rice].

A rice brittle culm mutant bcm581-1 which derived from the Ds transposone transformation population was found, but the mutant was identified that it was not to be induced by Ds transposone insertion through PCR. The examination of the vascular bundle and cortical fibre cells in culm under the light and electron microscope showed that, the number of cortical vascular bundle of mutant was much more, the hollow among the cortical vascular bundle was deeper, and the cell walls of cortical fibre cells were thinner than the normal. The test of culm mechanics intensity showed that the load, elongation, strain, and stress of bcm581-1 were 5-9 times lower than normal. The moisture content and the wide fibre content of culm were test, the former was 3.5% higher, but the latter was 8.12% lower than normal. The analysis of genetic segregation in F2 and F1B1 population indicated that the brittle culm mutant was controlled by one recessive gene.