Yue-Ie C. Hsing

Rice mutant resources for gene discovery

With the completion of genomic sequencing of rice, rice has been firmly established as a model organism for both basic and applied research. The next challenge is to uncover the functions of genes predicted by sequence analysis. Considering the amount of effort and the diversity of disciplines required for functional analyses, extensive international collaboration is needed for this next goal. The aims of this review are to summarize the current status of rice mutant resources, key tools for functional analysis of genes, and our perspectives on how to accelerate rice gene discovery through collaboration.

A Novel Class of Gibberellin 2-Oxidases Control Semidwarfism, Tillering, and Root Development in Rice

Gibberellin 2-oxidases (GA2oxs) regulate plant growth by inactivating endogenous bioactive gibberellins (GAs). Two classes of GA2oxs inactivate GAs through 2β-hydroxylation: a larger class of C19 GA2oxs and a smaller class of C20 GA2oxs. In this study, we show that members of the rice (Oryza sativa) GA2ox family are differentially regulated and act in concert or individually to control GA levels during flowering, tillering, and seed germination. Using mutant and transgenic analysis, C20 GA2oxs were shown to play pleiotropic roles regulating rice growth and architecture. In particular, rice overexpressing these GA2oxs exhibited early and increased tillering and adventitious root growth. GA negatively regulated expression of two transcription factors, O. sativa homeobox 1 and TEOSINTE BRANCHED1, which control meristem initiation and axillary bud outgrowth, respectively, and that in turn inhibited tillering. One of three conserved motifs unique to the C20 GA2oxs (motif III) was found to be important for activity of these GA2oxs. Moreover, C20 GA2oxs were found to cause less severe GA-defective phenotypes than C19 GA2oxs. Our studies demonstrate that improvements in plant architecture, such as semidwarfism, increased root systems and higher tiller numbers, could be induced by overexpression of wild-type or modified C20 GA2oxs.

Mutant resources in rice for functional genomics of the grasses.

Rice ( Oryza sativa ) is the reference genome for the grasses, including cereals. The complete genome sequence lays the foundation for comparative genomics to the other grasses based on genome structure and individual gene function ([Devos, 2005][1]; [International Rice Genome Sequencing Project,

The SnRK1A Protein Kinase Plays a Key Role in Sugar Signaling during Germination and Seedling Growth of Rice

Sugars repress α-amylase expression in germinating embryos and cell cultures of rice (Oryza sativa) through a sugar response complex (SRC) in α-amylase gene promoters and its interacting transcription factor MYBS1. The Snf1 protein kinase is required for the derepression of glucose-repressible genes in yeast. In this study, we explored the role of the yeast Snf1 ortholog in rice, SnRK1, in sugar signaling and plant growth. Rice embryo transient expression assays indicated that SnRK1A and SnRK1B act upstream and relieve glucose repression of MYBS1 and αAmy3 SRC promoters. Both SnRK1s contain N-terminal kinase domains serving as activators and C-terminal regulatory domains as dominant negative regulators of SRC. The accumulation and activity of SnRK1A was regulated by sugars posttranscriptionally, and SnRK1A relieved glucose repression specifically through the TA box in SRC. A transgenic RNA interference approach indicated that SnRK1A is also necessary for the activation of MYBS1 and αAmy3 expression under glucose starvation. Two mutants of SnRK1s, snrk1a and snrk1b, were obtained, and the functions of both SnRK1s were further studied. Our studies demonstrated that SnRK1A is an important intermediate in the sugar signaling cascade, functioning upstream from the interaction between MYBS1 and αAmy3 SRC and playing a key role in regulating seed germination and seedling growth in rice.

Late Embryogenesis Abundant Proteins

ABSTRACT During the late maturation stage of seed development, water content decreases greatly. One of the most striking characteristics of mature orthodox seeds is their ability to withstand severe desiccation. Mechanisms of plant drought/desiccation tolerance have been studied by numerous groups, and a broad range of molecules have been identified to play some roles. Examples are proline, oligosaccharide, and late embryogenesis abundant (LEA) proteins, and so on. LEA proteins were first described from mature cotton seeds decades ago. Since then, many LEA proteins were identified from vascular and nonvascular plants, fungi, algae, and microbes, as well as anhydrobiotic animals such as protozoa, nematodes, insects, and crustaceans, and so on. The extensive distribution of LEA genes among diverse taxa implies that these genes might be primitive yet important and therefore maintained by these species. As a result of evolution, they may have a certain universal function—osmoprotection. Hydrophilic LEA proteins are members of natively unfolded proteins in solution. After the removal of bulk cytoplasmic water, the structures of LEA proteins undergo desiccation-induced folding. These biophysical features suggest that LEA proteins may carry out a bipartite function under different water states. During drought, LEA proteins may establish a water shell and decrease ion strength. After desiccation, they may enhance the bioglass strength and act as a water replacement to stabilize cellular components.

Gene cloning and characterization of a soybean ( Glycine max L.) LEA protein, GmPM16

Late embryogenesis abundant (LEA) proteins, present in abundance in seeds during the late stages of development, are associated with desiccation tolerance. In the present work, we characterize a soybean LEA protein, GmPM16, with low molecular weight, high pI value, and an unusual amino acid residue distribution along the protein. The transcripts were detected in cotyledon mesophyll cells but not in the vascular system of mature or pod-dried soybean seeds. Circular dichroism (CD) analysis and Fourier transfer infrared (FTIR) spectroscopy indicated that the GmPM16 protein in solution was highly unordered, possessing only partial α-helical structures. However, the protein in sodium dodecyl sulfate (SDS) or trifluoroethanol (TFE) solution or in a dry state exhibited a conformation of abundant α-helical structures. As well, the GmPM16 protein interacts with sugar and forms tightly glassy matrixes in the dry state. The protein may play a role in reducing cellular damage in drying seeds by changing the protein conformation and forming tight cellular glasses.

A rice phenomics study--phenotype scoring and seed propagation of a T-DNA insertion-induced rice mutant population.

With the completion of the rice genome sequencing project, the next major challenge is the large-scale determination of gene function. As an important crop and a model organism, rice provides major insights into gene functions important for crop growth or production. Phenomics with detailed information about tagged populations provides a good tool for functional genomics analysis. By a T-DNA insertional mutagenesis approach, we have generated a rice mutant population containing 55,000 promoter trap and gene activation or knockout lines. Approximately 20,000 of these lines have known integration sites. The T0 and T1 plants were grown in net “houses” for two cropping seasons each year since 2003, with the mutant phenotypes recorded. Detailed data describing growth and development of these plants, in 11 categories and 65 subcategories, over the entire four-month growing season are available in a searchable database, along with the genetic segregation information and flanking sequence data. With the detailed data from more than 20,000 T1 lines and 12 plants per line, we estimated the mutation rates of the T1 population, as well the frequency of the dominant T0 mutants. The correlations among different mutation phenotypes are also calculated. Together, the information about mutant lines, their integration sites, and the phenotypes make this collection, the Taiwan Rice Insertion Mutants (TRIM), a good resource for rice phenomics study. Ten T2 seeds per line can be distributed to researchers upon request.

Characterization of Two Soybean (Glycine max L.) LEA IV Proteins by Circular Dichroism and Fourier Transform Infrared Spectrometry

Late embryogenesis-abundant (LEA) proteins, accumulating to a high level during the late stages of seed development, may play a role as osmoprotectants. However, the functions and mechanisms of LEA proteins remained to be elucidated. Five major groups of LEA proteins have been described. In the present study, we report on the characterization of two members of soybean LEA IV proteins, basic GmPM1 and acidic GmPM28, by circular dichroism and Fourier transform infrared spectroscopy. The spectra of both proteins revealed limited defined secondary structures in the fully hydrated state. Thus, the soybean LEA IV proteins are members of ‘natively unfolded proteins’. GmPM1 or GmPM28 proteins showed a conformational change under hydrophobic or dry conditions. After fast or slow drying, the two proteins showed slightly increased proportions of defined secondary structures (α-helix and β-sheet), from 30 to 49% and from 34 to 42% for GmPM1 and GmPm28, respectively. In the dehydrated state, GmPM1 and GmPM28 interact with non-reducing sugars to improve the transition temperature of cellular glass, with poly-l-lysine to prevent dehydration-induced aggregation and with phospholipids to maintain the liquid crystal phase over a wide temperature range. Our work suggests that soybean LEA IV proteins are functional in the dry state. They are one of the important components in cellular glasses and may stabilize desiccation-sensitive proteins and plasma membranes during dehydration.

Unusual sequences of group 3 LEA mRNA inducible by maturation or drying in soybean seeds.

Two cDNA clones, pGmPM8 and pGmPM10, which correspond to two mRNA species in mature or dry soybean seeds, were characterized. The deduced proteins, based on DNA sequence analysis, have a molecular mass of 49 and 51 kDa for pGmPM8 and pGmPM10, respectively. These two cDNA clones share a high homology with an amino acid identity of about 90% between the two deduced proteins. Both proteins appear to be extremely hydrophilic except at their N-termini that contain a 29 amino acid hydrophobic region at the N-terminus and the sizes of proteins decrease after co-incubating with ER membranes. These two proteins contain more than 30 similar, contiguous repeats of 11 amino acids, which is characteristic of group 3 LEA proteins. The mRNAs corresponding to pGmPM8 and pGmPM10 were expressed at high levels in dried or mature soybean seeds, but not in fresh immature seeds. The RNAs were also present in abscisic acid (ABA) treated leaves or cultured cells, and in tissues subjected to water stress or low temperatures.

Tissue- and stage-specific expression of a soybean (Glycine max L.) seed-maturation, biotinylated protein

A cDNA clone GmPM4 which encodes mRNA species in mature or dry soybean seeds was characterized. DNA sequence analysis shows that the deduced polypeptides have a molecular mass of 68 kDa. GmPM4 proteins have a relatively high amino acid sequence homology with a major biotinylated protein isolated from pea seeds, SBP65, but both of these proteins differ markedly from that of presently known biotin enzymes. The accumulation of GmPM4 mRNA is detectable in the leaf primodium and the vascular tissues of the hypocotyl-radicle axis of mature seeds, and the GmPM4 proteins are present at high levels in dry and mature soybean seeds, but not in fresh immature seeds. It degrades rapidly at the early stage of seed germination. These proteins are boiling-soluble and biotinylated when they are present endogenously in soybean seeds; however, the same recombinant protein expressed in Escherichia coli is boiling-soluble, but it is not biotinylated.