1,25(OH)2 D3 inhibits Lewis lung cancer cell migration via NHE1-sensitive metabolic reprograming.

High prevalence and metastasis rates are characteristics of lung cancer. Glycolysis provides energy for the development and metastasis of cancer cells. The 1,25-dihydroxy vitamin D3 (1,25(OH)2 D3 ) has been linked to reducing cancer risk and regulates various physiological functions. We hypothesized that 1,25(OH)2 D3 could be associated with the expression and activity of Na+ /H+ exchanger isoform 1 (NHE1) of Lewis lung cancer cells, thus regulating glycolysis as well as migration by actin reorganization. Followed by online public data analysis, Vitamin D3 receptor, the receptor of 1,25(OH)2 D3 has been proved to be abundant in lung cancers. We demonstrated that 1,25(OH)2 D3 treatment suppressed transcript levels, protein levels, and activity of NHE1 in LLC cells. Furthermore, 1,25(OH)2 D3 treatment resets the metabolic balance between glycolysis and OXPHOS, mainly including reducing glycolytic enzymes expression and lactate production. In vivo experiments showed the inhibition effects on tumor growth as well. Therefore, we concluded that 1,25(OH)2 D3 could amend the NHE1 function, which leads to metabolic reprogramming and cytoskeleton reconstruction, finally inhibits the cell migration.

A two-locus interaction causes interspecific hybrid weakness in rice.

Reproductive barriers perform a vital role during speciation. Hybrid weakness, the poorer development of hybrids compared with their parents, hinders gene exchange between different species at the postzygotic stage. Here we show that two incompatible dominant loci (Hwi1 and Hwi2) involving three genes are likely to determine the high temperature-dependent expression of hybrid weakness in interspecific hybrids of rice. Hwi1 comprises two leucine-rich repeat receptor-like kinase (LRR-RLK) genes, 25L1 and 25L2, which are specific to wild rice (Oryza rufipogon) and induce hybrid weakness. Hwi2, a rare allele that is predominantly distributed in indica rice (Oryza sativa), encodes a secreted putative subtilisin-like protease. Functional analysis indicated that pyramiding of Hwi1 and Hwi2 activates the autoimmune response in the basal nodes of hybrids, interrupting root formation and then impairing shoot growth. These findings bring new insights into our understanding of reproductive isolation and may benefit rice breeding.

Genetic and physiological analysis of a novel type of interspecific hybrid weakness in rice.

Hybrid weakness is an important reproductive barrier that hinders genetic exchange between different species at the post-zygotic stage. However, our understanding of the molecular mechanisms underlying hybrid weakness is limited. In this study, we report discovery of a novel interspecific hybrid weakness in a rice chromosome segment substitution line (CSSL) library derived from a cross between the indica variety Teqing (Oryza sativa) and common wild rice (O. rufipogon). The dominant Hybrid weakness i1 (Hwi1) gene from wild rice is genetically incompatible with Teqing and induced a set of weakness symptoms, including growth suppression, yield decrease, impaired nutrient absorption, and the retardation of crown root initiation. Phytohormone treatment showed that salicylic acid (SA) could restore the height of plants expressing hybrid weakness, while other phytohormones appear to have little effect. Fine mapping indicated that Hwi1 is located in a tandem leucine-rich repeat receptor-like kinase (LRR-RLK) gene cluster. Within the 13.2-kb candidate region on the short arm of chromosome 11, there are two annotated LRR-RLK genes, LOC_Os11g07230 and LOC_Os11g07240. The Teqing allele of LOC_Os11g07230 and the wild rice allele of LOC_Os11g07240 encode predicted functional proteins. Based on the genetic inheritance of hybrid weakness, LOC_Os11g07240 is implicated as the candidate gene for Hwi1. Functional analysis of Hwi1 will expand our knowledge of the regulation of hybrid weakness in rice.

The novel quantitative trait locus GL3.1 controls rice grain size and yield by regulating Cyclin-T1;3.

Increased crop yields are required to support rapid population growth worldwide. Grain weight is a key component of rice yield, but the underlying molecular mechanisms that control it remain elusive. Here, we report the cloning and characterization of a new quantitative trait locus (QTL) for the control of rice grain length, weight and yield. This locus, GL3.1, encodes a protein phosphatase kelch (PPKL) family - Ser/Thr phosphatase. GL3.1 is a member of the large grain WY3 variety, which is associated with weaker dephosphorylation activity than the small grain FAZ1 variety. GL3.1-WY3 influences protein phosphorylation in the spikelet to accelerate cell division, thereby resulting in longer grains and higher yields. Further studies have shown that GL3.1 directly dephosphorylates its substrate, Cyclin-T1;3, which has only been rarely studied in plants. The downregulation of Cyclin-T1;3 in rice resulted in a shorter grain, which indicates a novel function for Cyclin-T in cell cycle regulation. Our findings suggest a new mechanism for the regulation of grain size and yield that is driven through a novel phosphatase-mediated process that affects the phosphorylation of Cyclin-T1;3 during cell cycle progression, and thus provide new insight into the mechanisms underlying crop seed development. We bred a new variety containing the natural GL3.1 allele that demonstrated increased grain yield, which indicates that GL3.1 is a powerful tool for breeding high-yield crops.

Dissecting the genetic basis of extremely large grain shape in rice cultivar 'JZ1560'.

Rice grain shape, grain length (GL), width (GW), thickness (GT) and length-to-width ratio (LWR), are usually controlled by multiple quantitative trait locus (QTL). To elucidate the genetic basis of extremely large grain shape, QTL analysis was performed using an F(2) population derived from a cross between a japonica cultivar 'JZ1560' (extremely large grain) and a contrasting indica cultivar 'FAZ1' (small grain). A total number of 24 QTLs were detected on seven different chromosomes. QTLs for GL, GW, GT and LWR explained 11.6%, 95.62%, 91.5% and 89.9% of total phenotypic variation, respectively. Many QTLs pleiotropically controlled different grain traits, contributing complex traits correlation. GW2 and qSW5/GW5, which have been cloned previously to control GW, showed similar chromosomal locations with qGW2-1/qGT2-1/qLWR2-2 and qGW5-2/qLWR5-1 and should be the right candidate genes. Plants pyramiding GW2 and qSW5/GW5 showed a significant increase in GW compared with those carrying one of the two major QTLs. Furthermore, no significant QTL interaction was observed between GW2 and qSW5/GW5. These results suggested that GW2 and qSW5/GW5 might work in independent pathways to regulate grain traits. 'JZ1560' alleles underlying all QTLs contributed an increase in GW and GT and the accumulation of additive effects generates the extremely large grain shape in 'JZ1560'.

Rice carotenoid ß-ring hydroxylase CYP97A4 is involved in lutein biosynthesis.

Lutein is the most abundant plant carotenoid and plays essential roles in photosystem assembly and stabilization, as well as protection against photostress. To date, only a few lutein biosynthesis genes have been identified in crop plants. In this study, the rice Cyt P450 gene CYP97A4 encoding a carotenoid ß-ring hydroxylase was shown to be involved in lutein biosynthesis. The results revealed that CYP97A4 was preferentially expressed in leaf compared with spikelet, sheath, stalk and root, and encoded a protein localized at the subcellular level to the chloroplasts. Compared with the wild type, the three allelic mutants of CYP97A4 displayed lutein reductions of 12-24% with substantially increased α-carotene, while Chl a/b levels were unaltered. The increased α-carotene in the mutants led to greater sensitivity under high light stress. Similarly, reactive oxygen species (ROS) imaging of leaves treated with intense light showed that the mutants generally accumulated greater levels of ROS compared with wild-type plants, which probably caused detrimental effects to the plant photosystem. In conclusion, this study demonstrated the important role of CYP97A4 in α-carotene hydroxylation in rice, and knock-out of the gene reduced lutein and increased α-carotene, contributing to sensitivity to intense light.

Identification of quantitative trait Loci for lipid metabolism in rice seeds.

Plant seed oil is important for human dietary consumption and industrial application. The oil trait is controlled by quantitative trait loci (QTLs), but no QTLs for fatty acid composition are known in rice, the monocot model plant. QTL analysis was performed using F(2) and F(2:3) progeny from a cross of an indica variety and a japonica variety. Gas chromatography-mass spectrometry (GC-MS) analysis revealed significant differences between parental lines in fatty acid composition of brown rice oil, and 29 associated QTLs in F(2) and/or F(2:3) populations were identified throughout the rice genome, except chromosomes 9 and 10. Eight QTLs were repeatedly identified in both populations across different environments. Five loci pleiotropically controlled different traits, contributing to complex interactions of oil with fatty acids and between fatty acids. Nine rice orthologs of Arabidopsis genes encoding key enzymes in lipid metabolism co-localized with 11 mapped QTLs. A strong QTL for oleic (18:1) and linoleic (18:2) acid were associated with a rice ortholog of a gene encoding acyl-CoA:diacylglycerol acyltransferase (DGAT), and another for palmitic acid (16:0) mapped similarly to the acyl-ACP thioesterase (FatB) gene ortholog. Our approach rapidly and efficiently identified candidate genes for mapped QTLs controlling fatty acid composition and oil concentration, providing information for improving rice grain quality by marker assisted selection.

[Comparison of whole genome sequences and replication ability in cell cultures between two avian leukosis viruses of subgroup B].

The purpose of this study was to compare the whole genome sequences and replication dynamics in cell cultures of two Avian leukosis viruses of subgroup B (ALV) isolates, SDAU09E3 and SDAU09C2. Comparison of the amino acid sequences indicated that the gp85 identity of these two subgroup B isolates was 95.4%, the identity with other three ALV-B reference strains was 91.0%-94.9%, and less than 87.9% with ALV subgroup A, C, D, E and J. Comparison of the nucleotide sequence of gag and pol genes indicated that homologies of gag gene and pol gene of these two ALV-B isolates with all compared reference strains of different subgroups were above 93%. Homologies of LTR sequence of these two ALV-B isolates with other exogenous ALVs subgroups A, B, C, D and J were 72.6%-88.3%, but only 51.5% when compared with endogenous ALV subgroup E. The identity of LTR between these two ALV-B strains was only 74.8%, which was far lower than the identity of other genes. The identity of U3 region of LTR between these two ALV-B isolates was only 68.8% and there were obvious differences in the number CAAT Boxes. Replication dynamics in DF-1 cell indicated that the value of TCID50 was similar between 2 isolates but the concentration of nucleocapsid protein p27 antigen of SDAU09E3 was significantly higher than SDAU09C2 in cell culture supernatant, which indicated there was no parallel relationship between p27 antigen concentration and infectious virus particles. Whether such difference was resulted from the diversity of U3 region of LTR, further studies with their recombinant infectious clones is necessary.

Mediodorsal thalamic stimulation is not protective against seizures induced by amygdaloid kindling in rats.

Deep brain stimulation (DBS) is now emerging as a new option for treating intractable epilepsy. Cumulative studies suggest that the mediodorsal thalamic nucleus (MD) is involved in limbic seizure activity. This study aims to investigate whether DBS of the MD can protect against seizures induced by amygdaloid kindling. We studied the effect of low-frequency stimulation (LFS, 1 Hz) or high-frequency stimulation (HFS, 100 Hz) in the MD on amygdaloid kindling seizures. During the kindling acquisition, DBS in the MD was daily administered immediately after the kindling stimulus or before the kindling stimulus (preemptive DBS). The effects of both post-treatment of DBS and preemptive DBS in the MD on the expression of amygdaloid kindling seizures were evaluated. We found the DBS or preemptive DBS in the MD, either LFS or HFS, did not significantly change the rate of amygdaloid kindling. Similarly, DBS or preemptive DBS in the MD did not significantly change any parameters representing the expression of amygdaloid kindling. Our study suggests that DBS in the MD may have no significant effect on limbic seizures.

A previously unknown zinc finger protein, DST, regulates drought and salt tolerance in rice via stomatal aperture control.

Abiotic stresses, such as drought and salinity, lead to crop growth damage and a decrease in crop yields. Stomata control CO(2) uptake and optimize water use efficiency, thereby playing crucial roles in abiotic stress tolerance. Hydrogen peroxide (H(2)O(2)) is an important signal molecule that induces stomatal closure. However, the molecular pathway that regulates the H(2)O(2) level in guard cells remains largely unknown. Here, we clone and characterize DST (drought and salt tolerance)-a previously unknown zinc finger transcription factor that negatively regulates stomatal closure by direct modulation of genes related to H(2)O(2) homeostasis-and identify a novel pathway for the signal transduction of DST-mediated H(2)O(2)-induced stomatal closure. Loss of DST function increases stomatal closure and reduces stomatal density, consequently resulting in enhanced drought and salt tolerance in rice. These findings provide an interesting insight into the mechanism of stomata-regulated abiotic stress tolerance, and an important genetic engineering approach for improving abiotic stress tolerance in crops.

Fine mapping and candidate gene analysis of spd6, responsible for small panicle and dwarfness in wild rice (Oryza rufipogon Griff.).

Identification of genes in rice that affect production and quality is necessary for improving the critical global food source. CSSL58, a chromosome segment substitution line (CSSL) containing a chromosome segment of Oryza rufipogon in the genetic background of the indica cultivar Teqing showed significantly smaller panicles, fewer grains per panicle, smaller grains and dwarfness compared with the recurrent parent Teqing. Genetic analysis of the BC(4)F(1) and BC(4)F(2) generations, derived from a cross between CSSL58 and Teqing, showed that these traits are controlled by the recessive gene spd6, which mapped to the short arm of chromosome 6. Fine mapping and high-resolution linkage analysis using 24,120 BC(4)F(3) plants and markers flanking spd6 were carried out, and the gene was localized to a 22.4 kb region that contains four annotated genes according to the genome sequence of japonica Nipponbare. Phenotypic evaluation of the nearly isogenic line NIL(spd6) revealed that spd6 from wild rice has pleiotropic effects on panicle number per plant, grain size, grain weight, grain number per panicle and plant height, suggesting that this gene might play an important role in the domestication of rice. The discovery of spd6 may ultimately be useful for the design and breeding of crops with high grain yield and quality.

Identification of quantitative trait loci for rice quality in a population of chromosome segment substitution lines.

The demand for high quality rice represents a major issue in rice production. The primary components of rice grain quality include appearance, eating, cooking, physico-chemical, milling and nutritional qualities. Most of these traits are complex and controlled by quantitative trait loci (QTLs), so the genetic characterization of these traits is more difficult than that of traits controlled by a single gene. The detection and genetic identification of QTLs can provide insights into the genetic mechanisms underlying quality traits. Chromosome segment substitution lines (CSSLs) are effective tools used in mapping QTLs. In this study, we constructed 154 CSSLs from backcross progeny (BC(3)F(2)) derived from a cross between 'Koshihikari' (an Oryza sativa L. ssp. japonica variety) as the recurrent parent and 'Nona Bokra' (an O. sativa L. ssp. indica variety) as the donor parent. In this process, we carried out marker-assisted selection by using 102 cleaved amplified polymorphic sequence and simple sequence repeat markers covering most of the rice genome. Finally, this set of CSSLs was used to identify QTLs for rice quality traits. Ten QTLs for rice appearance quality traits were detected and eight QTLs concerned physico-chemical traits. These results supply the foundation for further genetic studies and breeding for the improvement of grain quality.

OsHAL3 mediates a new pathway in the light-regulated growth of rice.

Plants show distinct morphologies in different light conditions through a process called photomorphogenesis. A predominant feature of photomorphogenesis is the reduced growth of seedlings under light conditions compared with darkness. For this adaptive event, the most well-known molecular mechanism involves photoreceptor-mediated inhibition of cell elongation. However, it is not known whether additional pathways exist. Here, we describe a newly discovered pathway of light-modulated plant growth mediated by the halotolerance protein HAL3, a flavin mononucleotide (FMN)-binding protein involved in cell division. We found that light, especially blue light, suppresses growth of rice seedlings by reducing the activity of Oryza sativa (Os) HAL3. Both in vitro and in vivo studies showed that OsHAL3 is structurally inactivated by light through photo-oxidation and by direct interaction with photons. In addition, the transcriptional expression of OsHAL3 is synergistically regulated by different light conditions. Further investigation suggested that OsHAL3 promotes cell division by recruiting a ubiquitin system, rather than by its 4'-phosphopantothenoylcysteine (PPC) decarboxylase activity. Our results uncover a new mechanism for light-regulated plant growth, namely, light not only inhibits cell elongation but also suppresses cell division through HAL3 and E3 ubiquitin ligase. This study thus brings new insights into our understanding of plant photomorphogenesis.

Genetic control of rice plant architecture under domestication.

The closely related wild rice species Oryza rufipogon is considered the progenitor of cultivated rice (Oryza sativa). The transition from the characteristic plant architecture of wild rice to that of cultivated rice was one of the most important events in rice domestication; however, the molecular basis of this key domestication transition has not been elucidated. Here we show that the PROG1 gene controls aspects of wild-rice plant architecture, including tiller angle and number of tillers. The gene encodes a newly identified zinc-finger nuclear transcription factor with transcriptional activity and is mapped on chromosome 7. PROG1 is predominantly expressed in the axillary meristems, the site of tiller bud formation. Rice transformation experiments demonstrate that artificial selection of an amino acid substitution in the PROG1 protein during domestication led to the transition from the plant architecture of wild rice to that of domesticated rice.

Overexpression of the trehalose-6-phosphate phosphatase gene OsTPP1 confers stress tolerance in rice and results in the activation of stress responsive genes.

Trehalose plays a protective role in yeast and microorganisms under abiotic stresses. However, little is known about its role in higher plants when subjected to environmental challenges. A systematic search of rice databases discovered a large TPS/TPP gene family in the rice genome, which is similar to that found in Arabidopsis thaliana, especially in the gene family structure. Expression analysis demonstrated that OsTPP1 was initially and transiently up-regulated after salt, osmotic and abscisic acid (ABA) treatments but slowly up-regulated under cold stress. OsTPP1 overexpression in rice enhanced tolerance to salt and cold stress. Analysis of the overexpression lines revealed that OsTPP1 triggered abiotic stress response genes, which suggests a possible transcriptional regulation pathway in stress induced reprogramming initiated by OsTPP1. The current study revealed the mechanism of an OsTPP gene involved in stress tolerance in rice and also suggested the use of OsTPP1 in abiotic stress engineering of crops.

A QTL for rice grain width and weight encodes a previously unknown RING-type E3 ubiquitin ligase.

Grain weight is one of the most important components of grain yield and is controlled by quantitative trait loci (QTLs) derived from natural variations in crops. However, the molecular roles of QTLs in the regulation of grain weight have not been fully elucidated. Here, we report the cloning and characterization of GW2, a new QTL that controls rice grain width and weight. Our data show that GW2 encodes a previously unknown RING-type protein with E3 ubiquitin ligase activity, which is known to function in the degradation by the ubiquitin-proteasome pathway. Loss of GW2 function increased cell numbers, resulting in a larger (wider) spikelet hull, and it accelerated the grain milk filling rate, resulting in enhanced grain width, weight and yield. Our results suggest that GW2 negatively regulates cell division by targeting its substrate(s) to proteasomes for regulated proteolysis. The functional characterization of GW2 provides insight into the mechanism of seed development and is a potential tool for improving grain yield in crops.

[Construction of chromosome segment substitution lines carrying overlapping chromosome segments of the whole wild rice genome and identification of quantitative trait loci for rice quality].

Rice is one of the major staple cereal grains. Most of the important traits of crops are complex traits controlled by quantitative trait loci (QTL). Detection and genetic identification of QTLs can provide insights into molecular and biological mechanisms of development and physiology. Chromosome segment substitution lines (CSSLs) have been proposed as a simple and powerful way to identify QTLs. The demand for rice grain quality becomes increasingly important. The primary components of rice grain quality include appearance, milling and physico-chemical, cooking and eating quality. Most of these traits are complex and controlled by QTLs, so genetic characterization of these traits is more difficult than that of traits each controlled by a singular gene such as Waxy, which controls glutinousness of rice grain. We constructed 133 chromosome segment substitution lines (CSSLs) from backcross progenies (BC3F2) derived from a cross between Teqing (an O. sativa L. ssp. indica variety) as the recurrent parent and wild rice (O. rufipogon Griff.) as the donor parent. In this process, we carried out marker-assisted selection (MAS) by using 118 CAPS or SSR markers covering the whole rice genome (Figs.1, 2). Because phenotypic values of quality trait were significantly different between cultivar Teqing and wild rice, the CSSLs were used to identify QTLs for rice quality traits. Three appearance quality traits (head rice percentage, HRP; percentage of chalky rice grains, PCRG; transparency, TP) and five physico-chemical properties (gelatinization temperature, GT; gel consistency, GC; amylose content, AC; protein content, PC; fat content, FC) were scored using 133 CSSLs which substituted segments covering the whole genome of wild rice. A total of 15 QTLs for five of the eight traits mentioned above (HRP, PCRG, TP, PC and FC) were identified on nine chromosomes, and several QTLs affecting different quality traits were mapped in the same regions (Fig.3, Table 1).

A rice quantitative trait locus for salt tolerance encodes a sodium transporter.

Many important agronomic traits in crop plants, including stress tolerance, are complex traits controlled by quantitative trait loci (QTLs). Isolation of these QTLs holds great promise to improve world agriculture but is a challenging task. We previously mapped a rice QTL, SKC1, that maintained K(+) homeostasis in the salt-tolerant variety under salt stress, consistent with the earlier finding that K(+) homeostasis is important in salt tolerance. To understand the molecular basis of this QTL, we isolated the SKC1 gene by map-based cloning and found that it encoded a member of HKT-type transporters. SKC1 is preferentially expressed in the parenchyma cells surrounding the xylem vessels. Voltage-clamp analysis showed that SKC1 protein functions as a Na(+)-selective transporter. Physiological analysis suggested that SKC1 is involved in regulating K(+)/Na(+) homeostasis under salt stress, providing a potential tool for improving salt tolerance in crops.

[Study on correlationship of TCM syndrome type and expression of ICAM-1, CD62P in patients with ischemic stroke].

OBJECTIVE: To study the correlationship between TCM Syndrome type with changes of neutrophil surface intercellular adhesion molecule-1 (ICAM-1) and platelet membrane P selectin (CD62P) in patients with ischemic stroke for exploring the pathogenesis of the disease. METHODS: Seventy-two patients with ischemic stroke were dividcd into 3 typing groups according to TCM syndrome-differentiation, the Meridian-phlegm stagnancy group (MPS), the visceral phlegm-heat accumulation group (VPHA) and the qi-deficiency with blood stasis group (QDBS), 24 in each group. Besides, a control group consisted of 24 healthy subjects was set up. Blood levels of ICAM-1 and CDP62 expression were monitored by flow cytometry. RESULTS: Blood levels of ICAM-1 and CD62P expression in ischemic stroke patients were significantly higher than those in healthy subjects (P < 0.01). Among the three type groups, ICAM-1 expression was significantly higher in MPS than that in the VPHA and the QDBS group (P<0.01), and CD62P expression in the MPS and the QDBS group was significantly higher than that in the VPHA (P <0.01). CONCLUSION: Blood levels of ICAM-1 and CD62P expression in different typing of patients with ischemic stroke are different. ICAM-1 expression reflects the pathological state of phlegm retention or phlegm-stasis mutual bindings, CD62P expression reflected the blood stasis state in organism, these evidences suggest that MPS may be the key pathogenic factor of ischemic stroke.