A novel rice gene, NRR responds to macronutrient deficiency and regulates root growth.

To better understand the response of rice to nutrient stress, we have taken a systematic approach to identify rice genes that respond to deficiency of macronutrients and affect rice growth. We report here the expression and biological functions of a previously uncharacterized rice gene that we have named NRR (nutrition response and root growth). NRR is alternatively spliced, producing two 5'-coterminal transcripts, NRRa and NRRb, encoding two proteins of 308 and 223 aa, respectively. Compared to NRRb, NRRa possesses an additional CCT domain at the C-terminus. Expression of NRR in rice seedling roots was significantly influenced by deficiency of macronutrients. Knock-down of expression of NRRa or NRRb by RNA interference resulted in enhanced rice root growth. By contrast, overexpression of NRRa in rice exhibited significantly retarded root growth. These results revealed that both NRRa and NRRb played negative regulatory roles in rice root growth. Our findings suggest that NRRa and NRRb, acting as the key components, modulate the rice root architecture with the availability of macronutrients.

Overexpression of an F-box protein gene reduces abiotic stress tolerance and promotes root growth in rice.

As one of the largest gene families, F-box domain proteins have important roles in regulating various developmental processes and stress responses. In this study, we have investigated a rice F-box domain gene, MAIF1. The MAIF1 protein is mainly localized in the plasma membrane and nucleus. MAIF1 expression is induced rapidly and strongly by abscisic acid (ABA) and abiotic stresses. MAIF1 expression is also induced in root tips by sucrose, independent of its hydrolytic hexose products, glucose and fructose, and the plant hormones auxin and cytokinin. Overexpression of MAIF1 reduces rice ABA sensitivity and abiotic stress tolerance and promotes rice root growth. These results suggest that MAIF1 is involved in multiple signaling pathways in regulating root growth. Growth restraint in plants is an acclimatization strategy against abiotic stress. Our results also suggest that MAIF1 plays the negative role in response to abiotic stress possibly by regulating root growth.

An efficient field screening procedure for identifying transposants for constructing an Ac/Ds-based insertional-mutant library of rice.

An efficient system was developed, and several variables tested, for generating a large-scale insertional-mutagenesis population of rice. The most important feature in this improved Ac/Ds tagging system is that one can conveniently carry out large-scale screening in the field and select transposants at the seedling stage. Rice was transformed with a plasmid that includes a Basta-resistance gene (bar). After the Ds element is excised during transposition, bar becomes adjacent to the ubiquitin promoter, and the rice plant becomes resistant to the herbicide Basta. In principle, one can plant up to one million plants in the field and select those plants that survive after spraying with Basta. To test the utility of this system, 4 Ds starter lines were crossed with 14 different Ac plants, and many transposants were successfully identified after planting 134,285 F2 plants in the field. Over 2,800 of these transposants were randomly chosen for PCR analysis, and the results fully confirmed the reliability of the field screening procedure.

Application of TILLING in plant improvement.

TILLING (Targeting induced local lesions in genomes) is a general reverse-genetic strategy that is used to locate an allelic series of induced point mutations in genes of interest. High-throughput TILLING allows the rapid and cost-effective detection of induced point mutations in populations of chemically mutagenized individuals. The technique can be applied not only to model organisms but also to economically important organisms in plants. Owing to its full of advantages such as simple procedure, high sensitivity, and high efficiency, TILLING provides a powerful approach for gene discovery, DNA polymorphism assessment, and plant improvement. Coupled with other genomic resources, TILLING and EcoTILLING can be used immediately as a haplotyping tool in plant breeding for identifying allelic variation in genes exhibiting expression correlating with phenotypes and establishing an allelic series at genetic loci for the traits of interest in germplasm or induced mutants.

[Ac/Ds tagging system and functional genomics in rice].

With the completion of the rice genome sequence in 2002, the study on functional genomics in rice has become a major task. Establishment of rice mutant library is an essential approach for rice functional genomics study. At present, utilizing maize transposable element Ac/Ds (Activator/Dissociation) is a promising method to construct insertional mutagenesis library of rice. Ac/Ds tagging system has received extensive application in rice during the past several years, but it is still confronted with practical problems. In this paper, constructing rice insertional mutagenesis library using Ac/Ds-tagging system and transpositional behaviors of an Ac/Ds-tagging system, difficulties and advantages are reviewed. The research advances and challenge in rice functional genomics study using Ac/Ds tagging system are also discussed and summarized in the paper.

Isolation and physiological characteristics of a premature senescence mutant in rice (Oryza sativa L.).

A rice pse(t) (premature senescence, tentatively) mutant line, was isolated from 4,500 independent T-DNA inserted transgenic lines. The symptoms of premature senescence appeared more severely than those of the control plants (Zhonghua 11, japonica) at the last development stage. To characterize the mutant and provide basic information on the candidate genes by mapping to a physical region of 220-kb, experiments were carried out in two phytotrons under controlled temperature of 24 degrees C and 28 degrees C, respectively. The content of chlorophyll, soluble protein and MDA (malondialdehyde), net photosynthesis, the antioxidant enzyme activities of SOD (superoxide dismuase) (EC 1.15.1.1) and POD (peroxidase) (EC 1.11.1.7) and the peptidase activities of leaves were measured from top to bottom according to the leaf positions at the flowering stage. Compared with the control plant, the mutant showed the following characteristics: (1) Higher net photosynthesis rate (P(n)) appeared in the 1st and 2nd leaves, contents of chlorophyll and soluble protein were also higher in the 1st leaf; (2) The activities of SOD, POD and peptidase were higher according to the leaf position from top to bottom; (3) The symptom of premature senescence was accelerated in the mutant at 28 degrees C treatment. The MDA content and the SOD and POD activities between the 24 degrees C and 28 degrees C treatment mutants were not significantly different. Content of chlorophyll and soluble protein of leaves mutant decreased rapidly at 28 degrees C treatment. The results show that pse(t) is sensitive to high temperature. The probable function of PSE(T) is discussed.

Enhancing rice resistance to fungal pathogens by transformation with cell wall degrading enzyme genes from Trichoderma atroviride.

Three genes encoding for fungal cell wall degrading enzymes (CWDEs), ech42, nag70 and gluc78 from the biocontrol fungus Trichoderma atroviride were inserted into the binary vector pCAMBIA1305.2 singly and in all possible combinations and transformed to rice plants. More than 1800 independently regenerated plantlets in seven different populations (for each of the three genes and each of the four gene combinations) were obtained. The ech42 gene encoding for an endochitinase increased resistance to sheath blight caused by Rhizoctonia solani, while the exochitinase-encoding gene, nag70, had lesser effect. The expression level of endochitinase but exochitinase was correlated with disease resistance. Nevertheless, exochitinase enhanced the effect of endochitinase on disease resistance when the two genes co-expressed in transgenics. Resistance to Magnaporthe grisea was found in all kinds of regenerated plants including that with single gluc78. A few lines expressing either ech42 or nag70 gene were immune to the disease. Transgenic plants are being tested to further evaluate disease resistance at field level. This is the first report of multiple of expression of genes encoding CWDEs from Trichoderma atroviride that result in resistance to blast and sheath blight in rice.

Preliminary study on a gravity-insensitive rice mutant.

A gravity-insensitive mutant was isolated from rice (Oryza sativa L. cv. Zhonghua 11) transformed by Agrobacterium tumefaciens. The mutant's shoot growth (prostrate growth) was insensitive to gravity; whereas root growth displayed a normal positive gravitropism. Histological observation of root caps and leaf sheaths indicated that there was no significant difference in the number and size of amyloplasts in cells of the mutant and cells of the wild type.

[Observation amyloplasts in the gravity-insensitive mutant of rice under gravity and microgravity conditions].

Using histochemistry and optical microscope, we examined the number and the size of amyloplasts in specialized tissues of gravitropically receptive organs-tissues such as the coleoptile and sheath of rice mutant (insensitive to gravity) and wide type (Oryza sativa L. subsp. japonica) (Zhonghua 11). We found there was no statistical difference between the mutant and wide type, both of which grew on earth or on the clinostat respectively. On earth, it was found that amyloplasts sedimented at the distal end of each cell of the special starch sheath tissues and re-sedimentation of amyloplasts toward the direction of gravity was almost completed in 5 minutes after inverting the seedlings. On the clinostat, amyloplasts dispersed in the starch sheath tissue. Such observations indicated that the mutation was not resulted from the starch-deficiency or starch-absence, the further research is going on.

[Comparison between two methods of establishing DH population for mapping lox-3 gene in rice (Oryza sativa L.)].

Crosses were produced by using a storable rice variety (Daw Dam) and 2070, Ming Hui 63, as well as Dulur/Xie Qing Zao B as parents, among these materials there are abundant polymorphisms of DNA molecular markers. Their F1's were applied to generate DH lines by an improved method of direct induction of pollen plant. The method of the direct induction of pollen plant was used as check treatment to evaluate the improved method. The results showed that the improved method increased the re-differentiation percentage of pollen callus, the percentage of regeneration plantlets, and the percentage of green regeneration plantlets and produced more vigorous seedlings. The improved method was a competent method of establishing DH population for mapping lox-3 gene. It was also showed that the efficiency of the new method was different among genotypes and out of three experimental crosses Daw Dam/2070 and Daw Dam/Minghui 63 showed more efficient. More than 60 DH lines have been obtained in this experiment.