Shuen-Fang Lo

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.

Source–Sink Communication: Regulated by Hormone, Nutrient, and Stress Cross-Signaling

Communication between source organs (exporters of photoassimilates) and sink organs (importers of fixed carbon) has a pivotal role in carbohydrate assimilation and partitioning during plant growth and development. Plant productivity is enhanced by sink strength and source activity, which are regulated by a complex signaling network encompassing sugars, hormones, and environmental factors. However, key components underlying the signaling pathways that regulate source-sink communication are only now beginning to be discovered. Here, we discuss recent advances in our understanding of the molecular mechanisms regulating sugar mobilization during seed development and seedling establishment in cereals, which provide the majority of nutrition for humans. Insights into these mechanisms may provide strategies for improving crop productivity.

A late embryogenesis abundant protein HVA1 regulated by an inducible promoter enhances root growth and abiotic stress tolerance in rice without yield penalty

Regulation of root architecture is essential for maintaining plant growth under adverse environment. A synthetic abscisic acid (ABA)/stress-inducible promoter was designed to control the expression of a late embryogenesis abundant protein (HVA1) in transgenic rice. The background of HVA1 is low but highly inducible by ABA, salt, dehydration and cold. HVA1 was highly accumulated in root apical meristem (RAM) and lateral root primordia (LRP) after ABA/stress treatments, leading to enhanced root system expansion. Water-use efficiency (WUE) and biomass also increased in transgenic rice, likely due to the maintenance of normal cell functions and metabolic activities conferred by HVA1 which is capable of stabilizing proteins, under osmotic stress. HVA1 promotes lateral root (LR) initiation, elongation and emergence and primary root (PR) elongation via an auxin-dependent process, particularly by intensifying asymmetrical accumulation of auxin in LRP founder cells and RAM, even under ABA/stress-suppressive conditions. We demonstrate a successful application of an inducible promoter in regulating the spatial and temporal expression of HVA1 for improving root architecture and multiple stress tolerance without yield penalty.

Serotonin accumulation in transgenic rice by over-expressing tryptophan decarboxlyase results in a dark brown phenotype and stunted growth

A mutant M47286 with a stunted growth, low fertility and dark-brown phenotype was identified from a T-DNA-tagged rice mutant library. This mutant contained a copy of the T-DNA tag inserted at the location where the expression of two putative tryptophan decarboxlyase genes, TDC-1 and TDC-3, were activated. Enzymatic assays of both recombinant proteins showed tryptophan decarboxlyase activities that converted tryptophan to tryptamine, which could be converted to serotonin by a constitutively expressed tryptamine 5′ hydroxylase (T5H) in rice plants. Over-expression of TDC-1 and TDC-3 in transgenic rice recapitulated the stunted growth, dark-brown phenotype and resulted in a low fertility similar to M47286. The degree of stunted growth and dark-brown color was proportional to the expression levels of TDC-1 and TDC-3. The levels of tryptamine and serotonin accumulation in these transgenic rice lines were also directly correlated with the expression levels of TDC-1 and TDC-3. A mass spectrometry assay demonstrated that the dark-brown leaves and hulls in the TDC-overexpressing transgenic rice were caused by the accumulation of serotonin dimer and that the stunted growth and low fertility were also caused by the accumulation of serotonin and serotonin dimer, but not tryptamine. These results represent the first evidence that over-expression of TDC results in stunted growth, low fertility and the accumulation of serotonin, which when converted to serotonin dimer, leads to a dark brown plant color.

Genetic resources offer efficient tools for rice functional genomics research

Rice is an important crop and major model plant for monocot functional genomics studies. With the establishment of various genetic resources for rice genomics, the next challenge is to systematically assign functions to predicted genes in the rice genome. Compared with the robustness of genome sequencing and bioinformatics techniques, progress in understanding the function of rice genes has lagged, hampering the utilization of rice genes for cereal crop improvement. The use of transfer DNA (T-DNA) insertional mutagenesis offers the advantage of uniform distribution throughout the rice genome, but preferentially in gene-rich regions, resulting in direct gene knockout or activation of genes within 20-30 kb up- and downstream of the T-DNA insertion site and high gene tagging efficiency. Here, we summarize the recent progress in functional genomics using the T-DNA-tagged rice mutant population. We also discuss important features of T-DNA activation- and knockout-tagging and promoter-trapping of the rice genome in relation to mutant and candidate gene characterizations and how to more efficiently utilize rice mutant populations and datasets for high-throughput functional genomics and phenomics studies by forward and reverse genetics approaches. These studies may facilitate the translation of rice functional genomics research to improvements of rice and other cereal crops.

Increasing Leaf Vein Density by Mutagenesis: Laying the Foundations for C4 Rice

A high leaf vein density is both an essential feature of C4 photosynthesis and a foundation trait to C4 evolution, ensuring the optimal proportion and proximity of mesophyll and bundle sheath cells for permitting the rapid exchange of photosynthates. Two rice mutant populations, a deletion mutant library with a cv. IR64 background (12,470 lines) and a T-DNA insertion mutant library with a cv. Tainung 67 background (10,830 lines), were screened for increases in vein density. A high throughput method with handheld microscopes was developed and its accuracy was supported by more rigorous microscopy analysis. Eight lines with significantly increased leaf vein densities were identified to be used as genetic stock for the global C4 Rice Consortium. The candidate population was shown to include both shared and independent mutations and so more than one gene controlled the high vein density phenotype. The high vein density trait was found to be linked to a narrow leaf width trait but the linkage was incomplete. The more genetically robust narrow leaf width trait was proposed to be used as a reliable phenotypic marker for finding high vein density variants in rice in future screens.

Ectopic expression of specific GA2 oxidase mutants promotes yield and stress tolerance in rice

Summary A major challenge of modern agricultural biotechnology is the optimization of plant architecture for enhanced productivity, stress tolerance and water use efficiency (WUE). To optimize plant height and tillering that directly link to grain yield in cereals and are known to be tightly regulated by gibberellins (GAs), we attenuated the endogenous levels of GAs in rice via its degradation. GA 2‐oxidase (GA2ox) is a key enzyme that inactivates endogenous GAs and their precursors. We identified three conserved domains in a unique class of C20 GA2ox, GA2ox6, which is known to regulate the architecture and function of rice plants. We mutated nine specific amino acids in these conserved domains and observed a gradient of effects on plant height. Ectopic expression of some of these GA2ox6 mutants moderately lowered GA levels and reprogrammed transcriptional networks, leading to reduced plant height, more productive tillers, expanded root system, higher WUE and photosynthesis rate, and elevated abiotic and biotic stress tolerance in transgenic rice. Combinations of these beneficial traits conferred not only drought and disease tolerance but also increased grain yield by 10–30% in field trials. Our studies hold the promise of manipulating GA levels to substantially improve plant architecture, stress tolerance and grain yield in rice and possibly in other major crops.

Candidate regulators of Early Leaf Development in Maize Perturb Hormone Signalling and Secondary Cell Wall Formation When Constitutively Expressed in Rice.

All grass leaves are strap-shaped with a series of parallel veins running from base to tip, but the distance between each pair of veins, and the cell-types that develop between them, differs depending on whether the plant performs C3 or C4 photosynthesis. As part of a multinational effort to introduce C4 traits into rice to boost crop yield, candidate regulators of C4 leaf anatomy were previously identified through an analysis of maize leaf transcriptomes. Here we tested the potential of 60 of those candidate genes to alter leaf anatomy in rice. In each case, transgenic rice lines were generated in which the maize gene was constitutively expressed. Lines grouped into three phenotypic classes: (1) indistinguishable from wild-type; (2) aberrant shoot and/or root growth indicating possible perturbations to hormone homeostasis; and (3) altered secondary cell wall formation. One of the genes in class 3 defines a novel monocot-specific family. None of the genes were individually sufficient to induce C4-like vein patterning or cell-type differentiation in rice. A better understanding of gene function in C4 plants is now needed to inform more sophisticated engineering attempts to alter leaf anatomy in C3 plants.

Lack of Genotype and Phenotype Correlation in a Rice T-DNA Tagged Line Is Likely Caused by Introgression in the Seed Source.

Rice (Oryza sativa) is one of the most important crops in the world. Several rice insertional mutant libraries are publicly available for systematic analysis of gene functions. However, the tagging efficiency of these mutant resources–the relationship between genotype and phenotype–is very low. We used whole-genome sequencing to analyze a T-DNA–tagged transformant from the Taiwan Rice Insertional Mutants (TRIM) resource. The phenomics records for M0028590, one of the TRIM lines, revealed three phenotypes–wild type, large grains, and tillering dwarf–in the 12 T1 plants. Using the sequencing data for 7 plants from three generations of this specific line, we demonstrate that introgression from an indica rice variety might occur in one generation before the seed was used for callus generation and transformation of this line. In addition, the large-grain trait came from the GS3 gene of the introgressed region and the tillering dwarf phenotype came from a single nucleotide change in the D17 gene that occurred during the callus induction to regeneration of the transformant. As well, another regenerant showed completely heterozygous single-nucleotide polymorphisms across the whole genome. In addition to the known sequence changes such as T-DNA integration, single nucleotide polymorphism, insertion, deletion, chromosome rearrangement and doubling, spontaneous outcrossing occurred in the rice field may also explain some mutated traits in a tagged mutant population. Thus, the co-segregation of an integration event and the phenotype should be checked when using these mutant populations.

Large-scale phenomics analysis of a T-DNA tagged mutant population

Abstract Rice, Oryza sativa L., is one of the most important crops in the world. With the rising world population, feeding people in a more sustainable and environmentally friendly way becomes increasingly important. Therefore, the rice research community needs to share resources to better understand the functions of rice genes that are the foundation for future agricultural biotechnology development, and one way to achieve this goal is via the extensive study of insertional mutants. We have constructed a large rice insertional mutant population in a japonica rice variety, Tainung 67. The collection contains about 93 000 mutant lines, among them 85% with phenomics data and 65% with flanking sequence data. We screened the phenotypes of 12 individual plants for each line grown under field conditions according to 68 subcategories and 3 quantitative traits. Both phenotypes and integration sites are searchable in the Taiwan Rice Insertional Mutants Database. Detailed analyses of phenomics data, T-DNA flanking sequences, and whole-genome sequencing data for rice insertional mutants can lead to the discovery of novel genes. In addition, studies of mutant phenotypes can reveal relationships among varieties, cultivation locations, and cropping seasons.