Lysine-rich rice partially enhanced the growth and development of skeletal system with better skeletal microarchitecture in young rats.

Rice is the primary staple food for half of the world's population but is low in lysine content. Previously, we developed transgenic rice with enhanced free lysine content in rice seeds (lysine-rich rice), which was shown safe for consumption and improved the growth in rats. However, the effects of lysine-rich rice on skeletal growth and development remained unknown. In this study, we hypothesized that lysine-rich rice improved skeletal growth and development in weaning rats. Male weaning Sprague-Dawley rats received lysine-rich rice (HFL) diet, wild-type rice (WT) diet, or wild-type rice with various contents of lysine supplementation diet for 70 days. Bone microarchitectures were examined by microcomputed tomography, bone strength was investigated by mechanical test, and dynamics of bone growth were examined by histomorphometric analysis. In addition, we explored the molecular mechanism of lysine and skeletal growth through biochemical testing of growth hormone, bone turnover marker, and amino acid content of rat serum analysis, as well as in a cell culture system. Results indicated that the HFL diet improved rats' bone growth, strength, and microarchitecture compared with the WT diet group. In addition, the HFL diet increased the serum essential amino acids, growth hormone (insulin-like growth factor-1), and bone formation marker concentrations. The cell culture model showed that lysine deficiency reduced insulin-like growth factor-1 and Osterix expression, Akt/mammalian target of rapamycin signaling, and matrix mineralization, and inhibited osteoblast differentiation associated with bone growth. Our findings showed that lysine-rich rice improved skeletal growth and development in weaning rats. A further increase of rice lysine content is highly desirable to fully optimize bone growth and development.

Lysine biofortification of crops to promote sustained human health in the 21st century.

Crop biofortification is pivotal in preventing malnutrition, with lysine considered the main limiting essential amino acid (EAA) required to maintain human health. Lysine deficiency is predominant in developing countries where cereal crops are the staple food, highlighting the need for efforts aimed at enriching the staple diet through lysine biofortification. Successful modification of aspartate kinase (AK) and dihydrodipicolinate synthase (DHDPS) feedback inhibition has been used to enrich lysine in transgenic rice plants without yield penalty, while increases in the lysine content of quality protein maize have been achieved via marker-assisted selection. Here, we reviewed the lysine metabolic pathway and proposed the use of metabolic engineering targets as the preferred option for fortification of lysine in crops. Use of gene editing technologies to translate the findings and engineer lysine catabolism is thus a pioneering step forward.

Lysine biofortification in rice by modulating feedback inhibition of aspartate kinase and dihydrodipicolinate synthase.

Lysine is the main limiting essential amino acid (EAA) in the rice seeds, which is a major energy and nutrition source for humans and livestock. In higher plants, the rate-limiting steps in lysine biosynthesis pathway are catalysed by two key enzymes, aspartate kinase (AK) and dihydrodipicolinate synthase (DHDPS), and both are extremely sensitive to feedback inhibition by lysine. In this study, two rice AK mutants (AK1 and AK2) and five DHDPS mutants (DHDPS1-DHDPS5), all single amino acid substitution, were constructed. Their protein sequences passed an allergic sequence-based homology alignment. Mutant proteins were recombinantly expressed in Escherichia coli, and all were insensitive to the lysine analog S-(2-aminoethyl)-l-cysteine (AEC) at concentrations up to 12 mm. The AK and DHDPS mutants were transformed into rice, and free lysine was elevated in mature seeds of transgenic plants, especially those expressing AK2 or DHDPS1, 6.6-fold and 21.7-fold higher than the wild-type (WT) rice, respectively. We then engineered 35A2D1L plants by simultaneously expressing modified AK2 and DHDPS1, and inhibiting rice LKR/SDH (lysine ketoglutaric acid reductase/saccharopine dehydropine dehydrogenase). Free lysine levels in two 35A2D1L transgenic lines were 58.5-fold and 39.2-fold higher than in WT and transgenic rice containing native AK and DHDPS, respectively. Total free amino acid and total protein content were also elevated in 35A2D1L transgenic rice. Additionally, agronomic performance analysis indicated that transgenic lines exhibited normal plant growth, development and seed appearance comparable to WT plants. Thus, AK and DHDPS mutants may be used to improve the nutritional quality of rice and other cereal grains.

A Connection between Lysine and Serotonin Metabolism in Rice Endosperm.

Cereal endosperms produce a vast array of metabolites, including the essential amino acid lysine (Lys). Enhanced accumulation of Lys has been achieved via metabolic engineering in cereals, but the potential connection between metabolic engineering and Lys fortification is unclear. In mature seeds of engineered High Free Lysine (HFL) rice (Oryza sativa), the endosperm takes on a characteristic dark-brown appearance. In this study, we use an integrated metabolomic and transcriptomic approach combined with functional validation to elucidate the key metabolites responsible for the dark-brown phenotype. Importantly, we found that serotonin biosynthesis was elevated dramatically and closely linked with dark-brown endosperm color in HFL rice. A functional connection between serotonin and endosperm color was confirmed via overexpression of TDC3, a key enzyme of serotonin biosynthesis. Furthermore, we show that both the jasmonate signaling pathway and TDC expression were strongly induced in the late stage of endosperm development of HFL rice, coinciding with serotonin accumulation and dark-brown pigmentation. We propose a model for the metabolic connection between Lys and serotonin metabolism in which elevated 2-aminoadipate from Lys catabolism may play a key role in the connection between the jasmonate signaling pathway, serotonin accumulation, and the brown phenotype in rice endosperm. Our data provide a deeper understanding of amino acid metabolism in rice. In addition, the finding that both Lys and serotonin accumulate in HFL rice grains should promote efforts to create a nutritionally favorable crop.

Improved growth performance, food efficiency, and lysine availability in growing rats fed with lysine-biofortified rice.

Rice is an excellent source of protein, and has an adequate balance of amino acids with the exception of the essential amino acid lysine. By using a combined enhancement of lysine synthesis and suppression of its catabolism, we had produced two transgenic rice lines HFL1 and HFL2 (High Free Lysine) containing high concentration of free lysine. In this study, a 70-day rat feeding study was conducted to assess the nutritional value of two transgenic lines as compared with either their wild type (WT) or the WT rice supplemented with different concentrations of L-lysine. The results revealed that animal performance, including body weight, food intake, and food efficiency, was greater in the HFL groups than in the WT group. Moreover, the HFL diets had increased protein apparent digestibility, protein efficiency ratio, and lysine availability than the WT diet. Based on the linear relationship between dietary L-lysine concentrations and animal performance, it indicated that the biological indexes of the HFL groups were similar or better than that of the WT20 group, which was supplemented with L-lysine concentrations similar to those present in the HFL diets. Therefore, lysine-biofortified rice contributed to improved growth performance, food efficiency, and lysine availability in growing rats.

Subchronic feeding study of high-free-lysine transgenic rice in Sprague-Dawley rats.

Lysine is considered to be the first essential amino acid in rice. An elite High-Free-Lysine transgenic line HFL1 was previously produced by metabolic engineering to regulate lysine metabolism. In this study, a 90-day toxicology experiment was undertaken to investigate the potential health effect of feeding different doses of HFL1 rice to Sprague-Dawley rats. During the trial, body weight gain, food consumption and food efficiency were recorded, and no adverse effect was observed in rats fed transgenic (T) rice diets compared with non-transgenic (N) or control diets. At both midterm and final assessments, hematological parameters and serum chemistry were measured, and organ weights and histopathology were examined at the end of the trial. There was no diet-related difference in most hematological or serum chemistry parameters or organ weights between rats fed the T diets and those fed the N or control diets. Some parameters were found to differ between T groups and their corresponding N and/or control groups, but no adverse histological effect was observed. Taken together, the data from the current trial demonstrates that high lysine transgenic rice led to no adverse effect in Sprague-Dawley rats given a diet containing up to 70% HFL1 rice in 90 days.

Biofortification of rice with the essential amino acid lysine: molecular characterization, nutritional evaluation, and field performance.

Rice (Oryza sativa L.), a major staple crop worldwide, has limited levels of the essential amino acid lysine. We previously produced engineered rice with increased lysine content by expressing bacterial aspartate kinase and dihydrodipicolinate synthase and inhibiting rice lysine ketoglutarate reductase/saccharopine dehydrogenase activity. However, the grain quality, field performance, and integration patterns of the transgenes in these lysine-enriched lines remain unclear. In the present study, we selected several elite transgenic lines with endosperm-specific or constitutive regulation of the above key enzymes but lacking the selectable marker gene. All target transgenes were integrated into the intragenic region in the rice genome. Two pyramid transgenic lines (High Free Lysine; HFL1 and HFL2) with free lysine levels in seeds up to 25-fold that of wild type were obtained via a combination of the above two transgenic events. We observed a dramatic increase in total free amino acids and a slight increase in total protein content in both pyramid lines. Moreover, the general physicochemical properties were improved in pyramid transgenic rice, but the starch composition was not affected. Field trials indicated that the growth of HFL transgenic rice was normal, except for a slight difference in plant height and grain colour. Taken together, these findings will be useful for the potential commercialization of high-lysine transgenic rice.

Molecular characterization of a tomato purple acid phosphatase during seed germination and seedling growth under phosphate stress.

KEY MESSAGE: SlPAP1 is a phosphate starvation responsive purple acid phosphatase during tomato seed germination. Future research on its family members in tomato might improve the phosphate stress tolerance. Phosphate deficiency is a major constraint upon crop growth and yield. In response to phosphate deficiency, plants secrete acid phosphatases (APases) to scavenge organic phosphate from soil. In this study, we investigated the impact of Pi starvation on germination and seedling growth of tomato, and we then cloned and characterized a phosphate starvation responsive purple APase (SlPAP1) that expressed during tomato seedling growth. Our results showed that phosphate deficiency reduced germination and growth rates of tomato, and also increased intracellular and secretory APase activity in a concentration-dependent manner. An in-gel activity assay found that two APases of 50 and 75 kDa were secreted during conditions of phosphate deficiency. SlPAP1 is a single copy gene belonging to a small gene family. It was expressed as a cDNA of approximately 1.5 kbp encoding a secreted glycoprotein of 470 amino acids. Northern blot analysis showed that SlPAP1 was specifically expressed in root tissue during phosphate deficiency. SlPAP1 had high sequence identity (56-89%) with other plant PAPs and contained highly conserved metal-binding residues. SlPAP1 is the first PAP to be cloned and characterized from tomato. This study provides useful information for future research on PAP family members in tomato, leading to better understanding of phosphate deficiency in this important crop plant.

Food supply and food safety issues in China.

Food supply and food safety are major global public health issues, and are particularly important in heavily populated countries such as China. Rapid industrialisation and modernisation in China are having profound effects on food supply and food safety. In this Review, we identified important factors limiting agricultural production in China, including conversion of agricultural land to other uses, freshwater deficits, and soil quality issues. Additionally, increased demand for some agricultural products is examined, particularly those needed to satisfy the increased consumption of animal products in the Chinese diet, which threatens to drive production towards crops used as animal feed. Major sources of food poisoning in China include pathogenic microorganisms, toxic animals and plants entering the food supply, and chemical contamination. Meanwhile, two growing food safety issues are illegal additives and contamination of the food supply by toxic industrial waste. China's connections to global agricultural markets are also having important effects on food supply and food safety within the country. Although the Chinese Government has shown determination to reform laws, establish monitoring systems, and strengthen food safety regulation, weak links in implementation remain.

A large-scale protein phosphorylation analysis reveals novel phosphorylation motifs and phosphoregulatory networks in Arabidopsis.

Large-scale protein phosphorylation analysis by MS is emerging as a powerful tool in plant signal transduction research. However, our current understanding of the phosphorylation regulatory network in plants is still very limited. Here, we report on a proteome-wide profiling of phosphopeptides in nine-day-old Arabidopsis (Arabidopsis thaliana) seedlings by using an enrichment method combining the titanium (Ti(4+))-based IMAC and the RP-strong cation exchange (RP-SCX) biphasic trap column-based online RPLC. Through the duplicated RPLC-MS/MS analyses, we identified 5348 unique phosphopeptides for 2552 unique proteins. Among the phosphoproteins identified, 41% of them were first-time identified. Further evolutionary conservation and phosphorylation motif analyses of the phosphorylation sites discovered 100 highly conserved phosphorylation residues and identified 17 known and 14 novel motifs specific for Ser/Thr protein kinases. Gene ontology and pathway analyses revealed that many of the new identified phosphoproteins are important regulatory proteins that are involved in diverse biological processes, particularly in central metabolisms and cell signaling. Taken together, our results provided not only new insights into the complex phosphoregulatory network in plants but also important resources for future functional studies of protein phosphorylation in plant growth and development.

A comprehensive differential proteomic study of nitrate deprivation in Arabidopsis reveals complex regulatory networks of plant nitrogen responses.

Nitrogen (N) is an important nutrient and signal for plant growth and development. However, to date, our knowledge of how plants sense and transduce the N signals is very limited. To better understand the molecular mechanisms of plant N responses, we took two-dimensional gel-based proteomic and phosphoproteomic approaches to profile the proteins with abundance and phosphorylation state changes during nitrate deprivation and recovery in the model plant Arabidopsis thaliana. After 7-day-old seedlings were N-deprived for up to 48 h followed by 24 h recovery, a total of 170 and 38 proteins were identified with significant changes in abundance and phosphorylation state, respectively. Bioinformatic analyses implicate these proteins in diverse cellular processes including N and protein metabolisms, photosynthesis, cytoskeleton, redox homeostasis, and signal transduction. Functional studies of the selected nitrate-responsive proteins indicate that the proteasome regulatory subunit RPT5a and the cytoskeleton protein Tubulin alpha-6 (TUA6) play important roles in plant nitrate responses by regulating plant N use efficiency (NUE) and low nitrate-induced anthocyanin biosynthesis, respectively. In conclusion, our study provides novel insights into plant responses to nitrate at the proteome level, which are expected to be highly useful for dissecting the N response pathways in higher plants and for improving plant NUE.

An adjuvant free mouse model of oral allergenic sensitization to rice seeds protein.

BACKGROUND: Rice is commonly known as a staple crop consumed worldwide, though with several rice proteins being reported for allergic properties in clinical studies. Thus, there is a growing need for the development of an animal model to better understand the allergenicity of rice proteins and the immunological and pathophysiological mechanisms underlying the development of food allergy. METHODS: Groups of BALB/c mice were sensitized daily with freshly homogenized rice flour (30 mg or 80 mg) without adjuvant by intragastric gavage. In addition, the mice were challenged with extracted rice flour proteins at several time points intragastrically. Hypersensitivity symptoms in mice were evaluated according to a scoring system. Vascular leakage, ELISA of rice protein-specific IgE, histopathology of small intestine, and passive cutaneous anaphylaxis were conducted on challenged mice. RESULTS: An adjuvant free mouse model of rice allergy was established with sensitized mice showing increased scratching behaviors and increased vascular permeability. Rice protein-specific IgE was detected after eighteen days of sensitization and from the fifth challenge onwards. Inflammatory damage to the epithelium in the small intestine of mice was observed beyond one month of sensitization. Passive cutaneous anaphylaxis results confirmed the positive rice allergy in the mouse model. CONCLUSIONS: We introduced a BALB/c mouse model of rice allergy with simple oral sensitization without the use of adjuvant. This model would serve as a useful tool for further analysis on the immunopathogenic mechanisms of the various rice allergens, for the evaluation of the hypersensitivity of rice or other cereal grains, and to serve as a platform for the development of immunotherapies against rice allergens.

Resequencing of 31 wild and cultivated soybean genomes identifies patterns of genetic diversity and selection.

We report a large-scale analysis of the patterns of genome-wide genetic variation in soybeans. We re-sequenced a total of 17 wild and 14 cultivated soybean genomes to an average of approximately ×5 depth and >90% coverage using the Illumina Genome Analyzer II platform. We compared the patterns of genetic variation between wild and cultivated soybeans and identified higher allelic diversity in wild soybeans. We identified a high level of linkage disequilibrium in the soybean genome, suggesting that marker-assisted breeding of soybean will be less challenging than map-based cloning. We report linkage disequilibrium block location and distribution, and we identified a set of 205,614 tag SNPs that may be useful for QTL mapping and association studies. The data here provide a valuable resource for the analysis of wild soybeans and to facilitate future breeding and quantitative trait analysis.

An ancient P-loop GTPase in rice is regulated by a higher plant-specific regulatory protein.

YchF is a subfamily of the Obg family in the TRAFAC class of P-loop GTPases. The wide distribution of YchF homologues in both eukarya and bacteria suggests that they are descendents of an ancient protein, yet their physiological roles remain unclear. Using the OsYchF1-OsGAP1 pair from rice as the prototype, we provide evidence for the regulation of GTPase/ATPase activities and RNA binding capacity of a plant YchF (OsYchF1) by its regulatory protein (OsGAP1). The effects of OsGAP1 on the subcellular localization/cycling and physiological functions of OsYchF1 are also discussed. The finding that OsYchF1 and OsGAP1 are involved in plant defense response might shed light on the functional roles of YchF homologues in plants. This work suggests that during evolution, an ancestral P-loop GTPase/ATPase may acquire new regulation and function(s) by the evolution of a lineage-specific regulatory protein.

A novel simple extracellular leucine-rich repeat (eLRR) domain protein from rice (OsLRR1) enters the endosomal pathway and interacts with the hypersensitive-induced reaction protein 1 (OsHIR1).

Receptor-like protein kinases (RLKs) containing an extracellular leucine-rich repeat (eLRR) domain, a transmembrane domain and a cytoplasmic kinase domain play important roles in plant disease resistance. Simple eLRR domain proteins structurally resembling the extracellular portion of the RLKs may also participate in signalling transduction and plant defence response. Yet the molecular mechanisms and subcellular localization in regulating plant disease resistance of these simple eLRR domain proteins are still largely unclear. We provided the first experimental evidence to demonstrate the subcellular localization and trafficking of a novel simple eLRR domain protein (OsLRR1) in the endosomal pathway, using both confocal and electron microscopy. Yeast two-hybrid and in vitro pull-down assays show that OsLRR1 interacts with the rice hypersensitive-induced response protein 1 (OsHIR1) which is localized on plasma membrane. The interaction between LRR1 and HIR1 homologs was shown to be highly conserved among different plant species, suggesting a close functional relationship between the two proteins. The function of OsLRR1 in plant defence response was examined by gain-of-function tests using transgenic Arabidopsis thaliana. The protective effects of OsLRR1 against bacterial pathogen infection were shown by the alleviating of disease symptoms, lowering of pathogen titres and higher expression of defence marker genes.

Constitutive expression of a rice GTPase-activating protein induces defense responses.

G-proteins (guanine nucleotide-binding proteins that usually exhibit GTPase activities) and related signal transduction processes play important roles in mediating plant defense responses; here, a rice (Oryza sativa) cDNA clone, OsGAP1, encoding a GTPase-activating protein (GAP) that also contains a protein kinase C conserved region 2 (C2) domain is reported. An interacting G-protein partner for the OsGAP1 protein was identified by yeast two-hybrid library screening and confirmed by co-immunoprecipitation; the GTPase-activation activity of OsGAP1 on this interacting G-protein was demonstrated using in vitro assays. OsGAP1 was induced by wounding in rice and the presence of the R locus Xa14 enhances such induction. Gain-of-function tests in transgenic rice and Arabidopsis thaliana showed that constitutive expression of OsGAP1 led to increased resistance to bacterial pathogens in both monocots and dicots.

Activation of the JNK pathway promotes phosphorylation and degradation of BimEL--a novel mechanism of chemoresistance in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemias (T-ALLs) are highly malignant tumors with 20% of patients continues to fail therapy, in part due to chemoresistance of T-ALL cells via largely unknown mechanisms. Here, we showed that lack of Bcl-2-interacting mediator of cell death (Bim)(EL) protein expression, a BH3-only member of the Bcl-2 family proteins, conferred resistance of a T-ALL cell line, Sup-T1, to etoposide-induced apoptosis. Overexpression of Bim(EL) significantly restored its sensitivity to etoposide-induced caspase activation and poly(ADP-ribose) polymerase cleavage. Surprisingly, we found that constitutive activation of the c-Jun N-terminal kinase (JNK) pathway in Sup-T1 cells promoted phosphorylation and degradation of Bim(EL) via the proteosome. Blocking with a proteosome inhibitor yielded an elevated level of Bim(EL) and accumulation of Bim(EL) species phosphorylated at Ser(69). Pretreatment of Sup-T1 cells with a specific JNK inhibitor, SP600125, also increased the Bim(EL) level and resensitized the cells to etoposide-induced apoptosis. Together, our findings suggest that the JNK activation status may correlate with the Bim(EL) level and in turn can control the sensitivity of T-ALL cells to chemotherapeutic agents.

Expression of a RING-HC protein from rice improves resistance to Pseudomonas syringae pv. tomato DC3000 in transgenic Arabidopsis thaliana.

A cDNA clone (OsRHC1) was obtained, which encodes a novel RING zinc finger protein sharing similar structural features (multiple transmembrane domains at the N-half; a unique RING zinc finger consensus Cys-X(2)-Cys-X(11)-Cys-X-His-X(3)-Cys-X(2)-Cys-X(6)-Cys-X(2)-Cys at the C terminus) to a group of closely related annotated proteins from both monocots and dicots. OsRHC1 was found to be localized on plasma membrane of rice cells and induced by wounding in rice lines containing Xa loci. Ecotopic expression of the OsRHC1 cDNA from rice (a monocot) in transgenic Arabidopsis thaliana (a dicot) enhanced the defence response toward Pseudomonas syringae pv. tomato DC3000, suggesting that OsRHC1 may confer broad-spectrum disease resistance. The protective effects were neutralized in the presence of MG132 or in an npr1-3 mutation background, indicating that the function of OsRHC1 is dependent on the ubiquitin-mediated protein degradation via the 26S proteasome and the presence of the key defence response regulator NPR1.