A newly evolved rice-specific gene JAUP1 regulates jasmonate biosynthesis and signalling to promote root development and multi-stress tolerance.

Root architecture and function are critical for plants to secure water and nutrient supply from the soil, but environmental stresses alter root development. The phytohormone jasmonic acid (JA) regulates plant growth and responses to wounding and other stresses, but its role in root development for adaptation to environmental challenges had not been well investigated. We discovered a novel JA Upregulated Protein 1 gene (JAUP1) that has recently evolved in rice and is specific to modern rice accessions. JAUP1 regulates a self-perpetuating feed-forward loop to activate the expression of genes involved in JA biosynthesis and signalling that confers tolerance to abiotic stresses and regulates auxin-dependent root development. Ectopic expression of JAUP1 alleviates abscisic acid- and salt-mediated suppression of lateral root (LR) growth. JAUP1 is primarily expressed in the root cap and epidermal cells (EPCs) that protect the meristematic stem cells and emerging LRs. Wound-activated JA/JAUP1 signalling promotes crosstalk between the root cap of LR and parental root EPCs, as well as induces cell wall remodelling in EPCs overlaying the emerging LR, thereby facilitating LR emergence even under ABA-suppressive conditions. Elevated expression of JAUP1 in transgenic rice or natural rice accessions enhances abiotic stress tolerance and reduces grain yield loss under a limited water supply. We reveal a hitherto unappreciated role for wound-induced JA in LR development under abiotic stress and suggest that JAUP1 can be used in biotechnology and as a molecular marker for breeding rice adapted to extreme environmental challenges and for the conservation of water resources.

Proof of concept and early development stage of market-oriented high iron and zinc rice expressing dicot ferritin and rice nicotianamine synthase genes.

Micronutrient deficiencies such as iron (Fe), zinc (Zn), and vitamin A, constitute a severe global public health phenomenon. Over half of preschool children and two-thirds of nonpregnant women of reproductive age worldwide have micronutrient deficiencies. Biofortification is a cost-effective strategy that comprises a meaningful and sustainable means of addressing this issue by delivering micronutrients through staple foods to populations with limited access to diverse diets and other nutritional interventions. Here, we report on the proof-of-concept and early development stage of a collection of biofortified rice events with a high density of Fe and Zn in polished grains that have been pursued further to advance development for product release. In total, eight constructs were developed specifically expressing dicot ferritins and the rice nicotianamine synthase 2 (OsNAS2) gene under different combinations of promoters. A large-scale transformation of these constructs to Bangladesh and Philippines commercial indica cultivars and subsequent molecular screening and confined field evaluations resulted in the identification of a pool of ten events with Fe and Zn concentrations in polished grains of up to 11 µg g-1 and up to 37 µg g-1, respectively. The latter has the potential to reduce the prevalence of inadequate Zn intake for women of childbearing age in Bangladesh and in the Philippines by 30% and 50%, respectively, compared to the current prevalence. To our knowledge, this is the first potential biotechnology public-sector product that adopts the product cycle phase-gated approach, routinely applied in the private sector.

Effect of seed biopriming with selected endophytes on the growth and chilling tolerance of rice plants.

The aim of this study was to develop an efficient bioinoculant for amelioration of adverse effects from chilling stress (10°C), which are frequently occurred during rice seedling stage. Seed germination bioassay under chilling condition with rice (Oryza sativa L.) cv. Tainan 11 was performed to screen for plant growth-promoting (PGP) bacteria among 41 chilling-tolerant rice endophytes. And several agronomic traits were used to evaluate the effects of bacterial inoculation on rice seedling, which were experienced for 7-d chilling stress in walk-in growth chamber. The field trials were further used to verify the performance of potential PGP endophytes on rice growth. A total of three endophytes with multiple PGP traits were obtained. It was demonstrated that Pseudomonas sp. CC-LS37 inoculation led to 18% increase of maximal efficiency of Photosystem II (PSII) after 7-d chilling stress and 7% increase of chlorophyll a content, and 64% decline of malondialdehyde content in shoot after 10-d recovery at normal temperature in walk-in growth chamber. In field trial, biopriming of seeds with strain CC-LS37 caused rice plants to increase shoot chlorophyll soil plant analysis development values (by 2.9% and 2.5%, respectively) and tiller number (both by 61%) under natural climate and chilling stress during the end of tillering stage, afterward 30% more grain yield was achieved. In conclusion, strain CC-LS37 exerted its function in increase of tiller number of chilling stress-treated rice seedlings via improvement of photosynthetic characteristics, which in turn increases the rice grain yield. This study also proposed multiple indices used in the screening of potential endophytes for conferring chilling tolerance of rice plants.

A unique self-truncation of bacterial GH5 endoglucanases leads to enhanced activity and thermostability.

BACKGROUND: ß-1,4-endoglucanase (EG) is one of the three types of cellulases used in cellulose saccharification during lignocellulosic biofuel/biomaterial production. GsCelA is an EG secreted by the thermophilic bacterium Geobacillus sp. 70PC53 isolated from rice straw compost in southern Taiwan. This enzyme belongs to glycoside hydrolase family 5 (GH5) with a TIM-barrel structure common among all members of this family. GsCelA exhibits excellent lignocellulolytic activity and thermostability. In the course of investigating the regulation of this enzyme, it was fortuitously discovered that GsCelA undergoes a novel self-truncation/activation process that appears to be common among GH5 enzymes. RESULTS: Three diverse Gram-positive bacterial GH5 EGs, but not a GH12 EG, undergo an unexpected self-truncation process by removing a part of their C-terminal region. This unique process has been studied in detail with GsCelA. The purified recombinant GsCelA was capable of removing a 53-amino-acid peptide from the C-terminus. Natural or engineered GsCelA truncated variants, with up to 60-amino-acid deletion from the C-terminus, exhibited higher specific activity and thermostability than the full-length enzyme. Interestingly, the C-terminal part that is removed in this self-truncation process is capable of binding to cellulosic substrates of EGs. The protein truncation, which is pH and temperature dependent, occurred between amino acids 315 and 316, but removal of these two amino acids did not stop the process. Furthermore, mutations of E142A and E231A, which are essential for EG activity, did not affect the protein self-truncation process. Conversely, two single amino acid substitution mutations affected the self-truncation activity without much impact on EG activities. In Geobacillus sp. 70PC53, the full-length GsCelA was first synthesized in the cell but progressively transformed into the truncated form and eventually secreted. The GsCelA self-truncation was not affected by standard protease inhibitors, but could be suppressed by EDTA and EGTA and enhanced by certain divalent ions, such as Ca2+, Mg2+, and Cu2+. CONCLUSIONS: This study reveals novel insights into the strategy of Gram-positive bacteria for directing their GH5 EGs to the substrate, and then releasing the catalytic part for enhanced activity via a spontaneous self-truncation process.

A single promoter-TALE system for tissue-specific and tuneable expression of multiple genes in rice.

In biological discovery and engineering research, there is a need to spatially and/or temporally regulate transgene expression. However, the limited availability of promoter sequences that are uniquely active in specific tissue-types and/or at specific times often precludes co-expression of multiple transgenes in precisely controlled developmental contexts. Here, we developed a system for use in rice that comprises synthetic designer transcription activator-like effectors (dTALEs) and cognate synthetic TALE-activated promoters (STAPs). The system allows multiple transgenes to be expressed from different STAPs, with the spatial and temporal context determined by a single promoter that drives expression of the dTALE. We show that two different systems-dTALE1-STAP1 and dTALE2-STAP2-can activate STAP-driven reporter gene expression in stable transgenic rice lines, with transgene transcript levels dependent on both dTALE and STAP sequence identities. The relative strength of individual STAP sequences is consistent between dTALE1 and dTALE2 systems but differs between cell-types, requiring empirical evaluation in each case. dTALE expression leads to off-target activation of endogenous genes but the number of genes affected is substantially less than the number impacted by the somaclonal variation that occurs during the regeneration of transformed plants. With the potential to design fully orthogonal dTALEs for any genome of interest, the dTALE-STAP system thus provides a powerful approach to fine-tune the expression of multiple transgenes, and to simultaneously introduce different synthetic circuits into distinct developmental contexts.

Knockdown expression of a MYB-related transcription factor gene, OsMYBS2, enhances production of recombinant proteins in rice suspension cells.

BACKGROUND: Transgenic plant suspension cells show economic potential for the production of valuable bioproducts. The sugar starvation-inducible rice αAmy3 promoter, together with its signal peptide, is widely applied to produce recombinant proteins in rice suspension cells. The OsMYBS2 transcription factor was shown recently to reduce activation of the αAmy3 promoter by competing for the binding site of the TA box of the αAmy3 promoter with the potent OsMYBS1 activator. In this study, rice suspension cells were genetically engineered to silence OsMYBS2 to enhance the production of recombinant proteins. RESULTS: The mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) gene was controlled by the αAmy3 promoter and expressed in OsMYBS2-silenced transgenic rice suspension cells. Transcript levels of the endogenous αAmy3 and the transgene mGM-CSF were increased in the OsMYBS2-silenced suspension cells. The highest yield of recombinant mGM-CSF protein attained in the OsMYBS2-silenced transgenic suspension cells was 69.8 µg/mL, which is 2.5-fold that of non-silenced control cells. The yield of recombinant mGM-CSF was further increased to 118.8 µg/mL in cultured cells derived from homozygous F5 seeds, which was 5.1 times higher than that of the control suspension cell line. CONCLUSIONS: Our results demonstrate that knockdown of the transcription factor gene OsMYBS2 increased the activity of the αAmy3 promoter and improved the yield of recombinant proteins secreted in rice cell suspension cultures.

Improvements of the productivity and saccharification efficiency of the cellulolytic ß-glucosidase D2-BGL in Pichia pastoris via directed evolution.

BACKGROUND: ß-Glucosidases are essential for cellulose hydrolysis by catalyzing the final cellulolytic degradation of cello-oligomers and cellobiose to glucose. D2-BGL is a fungal glycoside hydrolase family 3 (GH3) ß-glucosidase isolated from Chaetomella raphigera with high substrate affinity, and is an efficient ß-glucosidase supplement to Trichoderma reesei cellulase mixtures for the saccharification of lignocellulosic biomass. RESULTS: We have carried out error-prone PCR to further increase catalytic efficiency of wild-type (WT) D2-BGL. Three mutants, each with substitution of two amino acids on D2-BGL, exhibited increased activity in a preliminary mutant screening in Saccharomyces cerevisiae. Effects of single amino acid replacements on catalysis efficiency and enzyme production have been investigated by subsequent expression in Pichia pastoris. Substitution F256M resulted in enhancing the tolerance to substrate inhibition and specific activity, and substitution D224G resulted in increasing the production of recombinant enzyme. The best D2-BGL mutant generated, Mut M, was constructed by combining beneficial mutations D224G, F256M and Y260D. Expression of Mut M in Pichia pastoris resulted in 2.7-fold higher production of recombinant protein, higher Vmax and greater substrate inhibition tolerance towards cellobiose relative to wild-type enzyme. Surprisingly, Mut M overexpression induced the ER unfolded protein response to a level lower than that with WT D2 overexpression in P. pastoris. When combined with the T. reesei cellulase preparation Celluclast 1.5L, Mut M hydrolyzed acid-pretreated sugarcane bagasse more efficiently than WT D2. CONCLUSIONS: D2-BGL mutant Mut M was generated successfully by following directed evolution approach. Mut M carries three mutations that are not reported in other directed evolution studies of GH3 ß-glucosidases, and this mutant exhibited greater tolerance to substrate inhibition and higher Vmax than wild-type enzyme. Besides the enhanced specific activity, Mut M also exhibited a higher protein titer than WT D2 when it was overexpressed in P. pastoris. Our study demonstrates that both catalytic efficiency and productivity of a cellulolytic enzyme can be enhanced via protein engineering.

From simple and specific zymographic detections to the annotation of a fungus Daldinia caldariorum D263 that encodes a wide range of highly bioactive cellulolytic enzymes.

BACKGROUND: Lignocellulolytic enzymes are essential for agricultural waste disposal and production of renewable bioenergy. Many commercialized cellulase mixtures have been developed, mostly from saprophytic or endophytic fungal species. The cost of complete cellulose digestion is considerable because a wide range of cellulolytic enzymes is needed. However, most fungi can only produce limited range of highly bioactive cellulolytic enzymes. We aimed to investigate a simple yet specific method for discovering unique enzymes so that fungal species producing a diverse group of cellulolytic enzymes can be identified. RESULTS: The culture medium of an endophytic fungus, Daldinia caldariorum D263, contained a complete set of cellulolytic enzymes capable of effectively digesting cellulose residues into glucose. By taking advantage of the unique product inhibition property of ß-glucosidases, we have established an improved zymography method that can easily distinguish ß-glucosidase and exoglucanase activity. Our zymography method revealed that D263 can secrete a wide range of highly bioactive cellulases. Analyzing the assembled genome of D263, we found over 100 potential genes for cellulolytic enzymes that are distinct from those of the commercially used fungal species Trichoderma reesei and Aspergillus niger. We further identified several of these cellulolytic enzymes by mass spectrometry. CONCLUSIONS: The genome of Daldinia caldariorum D263 has been sequenced and annotated taking advantage of a simple yet specific zymography method followed by mass spectrometry analysis, and it appears to encode and secrete a wide range of bioactive cellulolytic enzymes. The genome and cellulolytic enzyme secretion of this unique endophytic fungus should be of value for identifying active cellulolytic enzymes that can facilitate conversion of agricultural wastes to fermentable sugars for the industrial production of biofuels.

How does rice cope with too little oxygen during its early life?

Most crops cannot germinate underwater. Rice exhibits certain degrees of tolerance to oxygen deficiency for anaerobic germination (AG) and anaerobic seedling development (ASD). Direct rice seeding, whereby seeds are sown into soil rather than transplanting seedlings from the nursery, becomes an increasingly popular cultivation method due to labor shortages and opportunities for sustainable cultivation. Flooding is common under direct seeding, but most rice varieties have poor capability of AG/ASD, which is a major obstacle to broad adoption of direct seeding. A better understanding of the physiological basis and molecular mechanisms regulating AG/ASD should facilitate rice breeding for enhanced seedling vigor under flooding. This review highlights recent advances on molecular and physiological mechanisms and future breeding strategies of rice AG/ASD.

Rice Big Grain 1 promotes cell division to enhance organ development, stress tolerance and grain yield.

Grain/seed yield and plant stress tolerance are two major traits that determine the yield potential of many crops. In cereals, grain size is one of the key factors affecting grain yield. Here, we identify and characterize a newly discovered gene Rice Big Grain 1 (RBG1) that regulates grain and organ development, as well as abiotic stress tolerance. Ectopic expression of RBG1 leads to significant increases in the size of not only grains but also other major organs such as roots, shoots and panicles. Increased grain size is primarily due to elevated cell numbers rather than cell enlargement. RBG1 is preferentially expressed in meristematic and proliferating tissues. Ectopic expression of RBG1 promotes cell division, and RBG1 co-localizes with microtubules known to be involved in cell division, which may account for the increase in organ size. Ectopic expression of RBG1 also increases auxin accumulation and sensitivity, which facilitates root development, particularly crown roots. Moreover, overexpression of RBG1 up-regulated a large number of heat-shock proteins, leading to enhanced tolerance to heat, osmotic and salt stresses, as well as rapid recovery from water-deficit stress. Ectopic expression of RBG1 regulated by a specific constitutive promoter, GOS2, enhanced harvest index and grain yield in rice. Taken together, we have discovered that RBG1 regulates two distinct and important traits in rice, namely grain yield and stress tolerance, via its effects on cell division, auxin and stress protein induction.

Chaetomella raphigera ß-glucosidase D2-BGL has intriguing structural features and a high substrate affinity that renders it an efficient cellulase supplement for lignocellulosic biomass hydrolysis.

BACKGROUND: To produce second-generation biofuels, enzymatic catalysis is required to convert cellulose from lignocellulosic biomass into fermentable sugars. ß-Glucosidases finalize the process by hydrolyzing cellobiose into glucose, so the efficiency of cellulose hydrolysis largely depends on the quantity and quality of these enzymes used during saccharification. Accordingly, to reduce biofuel production costs, new microbial strains are needed that can produce highly efficient enzymes on a large scale. RESULTS: We heterologously expressed the fungal ß-glucosidase D2-BGL from a Taiwanese indigenous fungus Chaetomella raphigera in Pichia pastoris for constitutive production by fermentation. Recombinant D2-BGL presented significantly higher substrate affinity than the commercial ß-glucosidase Novozyme 188 (N188; K m = 0.2 vs 2.14 mM for p-nitrophenyl ß-d-glucopyranoside and 0.96 vs 2.38 mM for cellobiose). When combined with RUT-C30 cellulases, it hydrolyzed acid-pretreated lignocellulosic biomasses more efficiently than the commercial cellulase mixture CTec3. The extent of conversion from cellulose to glucose was 83% for sugarcane bagasse and 63% for rice straws. Compared to N188, use of D2-BGL halved the time necessary to produce maximal levels of ethanol by a semi-simultaneous saccharification and fermentation process. We upscaled production of recombinant D2-BGL to 33.6 U/mL within 15 days using a 1-ton bioreactor. Crystal structure analysis revealed that D2-BGL belongs to glycoside hydrolase (GH) family 3. Removing the N-glycosylation N68 or O-glycosylation T431 residues by site-directed mutagenesis negatively affected enzyme production in P. pastoris. The F256 substrate-binding residue in D2-BGL is located in a shorter loop surrounding the active site pocket relative to that of Aspergillus ß-glucosidases, and this short loop is responsible for its high substrate affinity toward cellobiose. CONCLUSIONS: D2-BGL is an efficient supplement for lignocellulosic biomass saccharification, and we upscaled production of this enzyme using a 1-ton bioreactor. Enzyme production could be further improved using optimized fermentation, which could reduce biofuel production costs. Our structure analysis of D2-BGL offers new insights into GH3 ß-glucosidases, which will be useful for strain improvements via a structure-based mutagenesis approach.

Sugar starvation-regulated MYBS2 and 14-3-3 protein interactions enhance plant growth, stress tolerance, and grain weight in rice.

Autotrophic plants have evolved distinctive mechanisms for maintaining a range of homeostatic states for sugars. The on/off switch of reversible gene expression by sugar starvation/provision represents one of the major mechanisms by which sugar levels are maintained, but the details remain unclear. α-Amylase (αAmy) is the key enzyme for hydrolyzing starch into sugars for plant growth, and it is induced by sugar starvation and repressed by sugar provision. αAmy can also be induced by various other stresses, but the physiological significance is unclear. Here, we reveal that the on/off switch of αAmy expression is regulated by 2 MYB transcription factors competing for the same promoter element. MYBS1 promotes αAmy expression under sugar starvation, whereas MYBS2 represses it. Sugar starvation promotes nuclear import of MYBS1 and nuclear export of MYBS2, whereas sugar provision has the opposite effects. Phosphorylation of MYBS2 at distinct serine residues plays important roles in regulating its sugar-dependent nucleocytoplasmic shuttling and maintenance in cytoplasm by 14-3-3 proteins. Moreover, dehydration, heat, and osmotic stress repress MYBS2 expression, thereby inducing αAmy3 Importantly, activation of αAmy3 and suppression of MYBS2 enhances plant growth, stress tolerance, and total grain weight per plant in rice. Our findings reveal insights into a unique regulatory mechanism for an on/off switch of reversible gene expression in maintaining sugar homeostatic states, which tightly regulates plant growth and development, and also highlight MYBS2 and αAmy3 as potential targets for crop improvement.

Enhancement of laccase activity by pre-incubation with organic solvents.

Laccases that are tolerant to organic solvents are powerful bio-catalysts with broad applications in biotechnology. Most of these uses must be accomplished at high concentration of organic solvents, during which proteins undergo unfolding, thereby losing enzyme activity. Here we show that organic-solvent pre-incubation provides effective and reversible 1.5- to 4.0-fold enhancement of enzyme activity of fungal laccases. Several organic solvents, including acetone, methanol, ethanol, DMSO, and DMF had an enhancement effect among all laccases studied. The enhancement was not substrate-specific and could be observed by using both phenolic and non-phenolic substrates. Laccase preincubated with organic solvents was sensitive to high temperature but remained stable at 25 °C, for an advantage for long-term storage. The acetone-pre-incubated 3-D structure of DLac, a high-efficiency fungal laccase, was determined and confirmed that the DLac protein structure remains intact and stable at a high concentration of organic solvent. Moreover, the turnover rates of fungal laccases were improved after organic-solvent pre-incubation, with DLac showing the highest enhancement among the fungal laccases examined. Our investigation sheds light on improving fungal laccase usage under extreme conditions and extends opportunities for bioremediation, decolorization, and organic synthesis.

The Nucleotide-Dependent Interactome of Rice Heterotrimeric G-Protein α -Subunit.

The rice heterotrimeric G-protein complex, a guanine-nucleotide-dependent on-off switch, mediates vital cellular processes and responses to biotic and abiotic stress. Exchange of bound GDP (resting state) for GTP (active state) is spontaneous in plants including rice and thus there is no need for promoting guanine nucleotide exchange in vivo as a mechanism for regulating the active state of signaling as it is well known for animal G signaling. As such, a master regulator controlling the G-protein activation state is unknown in plants. Therefore, an ab initio approach is taken to discover candidate regulators. The rice Gα subunit (RGA1) is used as bait to screen for nucleotide-dependent protein partners. A total of 264 proteins are identified by tandem mass spectrometry of which 32 were specific to the GDP-bound inactive state and 22 specific to the transition state. Approximately, 10% are validated as previously identified G-protein interactors.

Kinetic analysis and structural studies of a high-efficiency laccase from Cerrena sp. RSD1.

A high-efficiency laccase, DLac, was isolated from Cerrena sp. RSD1. The kinetic studies indicate that DLac is a diffusion-limited enzyme. The crystal structure of DLac was determined to atomic resolution, and its overall structure shares high homology to monomeric laccases, but displays unique substrate-binding loops from those in other laccases. The substrate-binding residues with small side chain and the short substrate-binding loop IV broaden the substrate-binding cavity and may facilitate large substrate diffusion. Unlike highly glycosylated fungal laccases, the less-glycosylated DLac contains one highly conserved glycosylation site at N432 and an unique glycosylation site at N468. The N-glycans stabilize the substrate-binding loops and the protein structure, and the first N-acetylglucosamine is crucial for the catalytic efficiency. Additionally, a fivefold increase in protein yield is achieved via the submerged culture method for industrial applications. DATABASE: The atomic coordinates of the structure of DLac from Cerrena sp. RSD1 and structural factors have been deposited in the RCSB Protein Data Bank (PDB ID: 5Z1X).

Ectopic Expression of WINDING 1 Leads to Asymmetrical Distribution of Auxin and a Spiral Phenotype in Rice.

Ectopic expression of the rice WINDING 1 (WIN1) gene leads to a spiral phenotype only in shoots but not in roots. Rice WIN1 belongs to a specific class of proteins in cereal plants containing a Bric-a-Brac/Tramtrack/Broad (BTB) complex, a non-phototropic hypocotyl 3 (NPH3) domain and a coiled-coil motif. The WIN1 protein is predominantly localized to the plasma membrane, but is also co-localized to plasmodesmata, where it exhibits a punctate pattern. It is observed that WIN1 is normally expressed in roots and the shoot-root junction, but not in the rest of shoots. In roots, WIN1 is largely localized to the apical and basal sides of cells. However, upon ectopic expression, WIN1 appears on the longitudinal sides of leaf sheath cells, correlated with the appearance of a spiral phenotype in shoots. Despite the spiral phenotype, WIN1-overexpressing plants exhibit a normal phototropic response. Although treatments with exogenous auxins or a polar auxin transport inhibitor do not alter the spiral phenotype, the excurvature side has a higher auxin concentration than the incurvature side. Furthermore, actin filaments are more prominent in the excurvature side than in the incurvature side, which correlates with cell size differences between these two sides. Interestingly, ectopic expression of WIN1 does not cause either unequal auxin distribution or actin filament differences in roots, so a spiral phenotype is not observed in roots. The action of WIN1 appears to be different from that of other proteins causing a spiral phenotype, and it is likely that WIN1 is involved in 1-N-naphthylphthalamic acid-insensitive plasmodesmata-mediated auxin transport.

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

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.

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.

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

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.

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.