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Plant height is an important agronomic trait with a significant impact on grain yield, as demonstrated by the positive effect of the REDUCED HEIGHT (RHT) dwarfing alleles (Rht1b) on lodging and harvest index in the "Green Revolution" wheat varieties. However, these gibberellic acid (GA)-insensitive alleles also reduce coleoptile length, biomass production, and yield potential in some environments, triggering the search for alternative GA-sensitive dwarfing genes. Here we report the identification, validation, and characterization of the gene underlying the GA-sensitive dwarfing locus RHT25 in wheat. This gene, designated as PLATZ-A1 (TraesCS6A02G156600), is expressed mainly in the elongating stem and developing spike and encodes a plant-specific AT-rich sequence- and zinc-binding protein (PLATZ). Natural and induced loss-of-function mutations in PLATZ-A1 reduce plant height and its overexpression increases plant height, demonstrating that PLATZ-A1 is the causative gene of RHT25. PLATZ-A1 and RHT1 show a significant genetic interaction on plant height, and their encoded proteins interact with each other in yeast and wheat protoplasts. These results suggest that PLATZ1 can modulate the effect of DELLA on wheat plant height. We identified four natural truncation mutations and one promoter insertion in PLATZ-A1 that are more frequent in modern varieties than in landraces, suggesting positive selection during wheat breeding. These mutations can be used to fine-tune wheat plant height and, in combination with other GA-sensitive dwarfing genes, to replace the GA-insensitive Rht1b alleles and search for grain yield improvements beyond those of the Green Revolution varieties.

The photoperiodic response is critical for plants to adjust their reproductive phase to the most favorable season. Wheat heads earlier under long days (LD) than under short days (SD) and this difference is mainly regulated by the PHOTOPERIOD1 (PPD1) gene. Tetraploid wheat plants carrying the Ppd-A1a allele with a large deletion in the promoter head earlier under SD than plants carrying the wildtype Ppd-A1b allele with an intact promoter. Phytochromes PHYB and PHYC are necessary for the light activation of PPD1, and mutations in either of these genes result in the downregulation of PPD1 and very late heading time. We show here that both effects are reverted when the phyB mutant is combined with loss-of-function mutations in EARLY FLOWERING 3 (ELF3), a component of the Evening Complex (EC) in the circadian clock. We also show that the wheat ELF3 protein interacts with PHYB and PHYC, is rapidly modified by light, and binds to the PPD1 promoter in planta (likely as part of the EC). Deletion of the ELF3 binding region in the Ppd-A1a promoter results in PPD1 upregulation at dawn, similar to PPD1 alleles with intact promoters in the elf3 mutant background. The upregulation of PPD1 is correlated with the upregulation of the florigen gene FLOWERING LOCUS T1 (FT1) and early heading time. Loss-of-function mutations in PPD1 result in the downregulation of FT1 and delayed heading, even when combined with the elf3 mutation. Taken together, these results indicate that ELF3 operates downstream of PHYB as a direct transcriptional repressor of PPD1, and that this repression is relaxed both by light and by the deletion of the ELF3 binding region in the Ppd-A1a promoter. In summary, the regulation of the light mediated activation of PPD1 by ELF3 is critical for the photoperiodic regulation of wheat heading time.

Improving our understanding of the genes regulating grain yield can contribute to the development of more productive wheat varieties. Previously, a highly significant QTL affecting spikelet number per spike (SNS), grain number per spike (GNS) and grain yield was detected on chromosome arm 7AL in multiple genome-wide association studies. Using a high-resolution genetic map, we established that the A-genome homeolog of WHEAT ORTHOLOG OF APO1 (WAPO-A1) was a leading candidate gene for this QTL. Using mutants and transgenic plants, we demonstrate in this study that WAPO-A1 is the causal gene underpinning this QTL. Loss-of-function mutants wapo-A1 and wapo-B1 showed reduced SNS in tetraploid wheat, and the effect was exacerbated in wapo1 combining both mutations. By contrast, spikes of transgenic wheat plants carrying extra copies of WAPO-A1 driven by its native promoter had higher SNS, a more compact spike apical region and a smaller terminal spikelet than the wild type. Taken together, these results indicate that WAPO1 affects SNS by regulating the timing of terminal spikelet formation. Both transgenic and wapo1 mutant plants showed a wide range of floral abnormalities, indicating additional roles of WAPO1 on wheat floral development. Previously, we found three widespread haplotypes in the QTL region (H1, H2 and H3), each associated with particular WAPO-A1 alleles. Results from this and our previous study show that the WAPO-A1 allele in the H1 haplotype (115-bp deletion in the promoter) is expressed at significantly lower levels in the developing spikes than the alleles in the H2 and H3 haplotypes, resulting in reduced SNS. Field experiments also showed that the H2 haplotype is associated with the strongest effects in increasing SNS and GNS (H2>H3>H1). The H2 haplotype is already present in most modern common wheat varieties but is rare in durum wheat, where it might be particularly useful to improve grain yield.

Plants possess regulatory mechanisms that allow them to flower under conditions that maximize reproductive success. Selection of natural variants affecting those mechanisms has been critical in agriculture to modulate the flowering response of crops to specific environments and to increase yield. In the temperate cereals, wheat and barley, the photoperiod and vernalization pathways explain most of the natural variation in flowering time. However, other pathways also participate in fine-tuning the flowering response. In this work, we integrate the conserved microRNA miR172 and its targets APETALA2-like (AP2L) genes into the temperate grass flowering network involving VERNALIZATION 1 (VRN1), VRN2 and FLOWERING LOCUS T 1 (FT1 = VRN3) genes. Using mutants, transgenics and different growing conditions, we show that miR172 promotes flowering in wheat, while its target genes AP2L1 (TaTOE1) and AP2L5 (Q) act as flowering repressors. Moreover, we reveal that the miR172-AP2L pathway regulates FT1 expression in the leaves, and that this regulation is independent of VRN2 and VRN1. In addition, we show that the miR172-AP2L module and flowering are both controlled by plant age through miR156 in spring cultivars. However, in winter cultivars, flowering and the regulation of AP2L1 expression are decoupled from miR156 downregulation with age, and induction of VRN1 by vernalization is required to repress AP2L1 in the leaves and promote flowering. Interestingly, the levels of miR172 and both AP2L genes modulate the flowering response to different vernalization treatments in winter cultivars. In summary, our results show that conserved and grass specific gene networks interact to modulate the flowering response, and that natural or induced mutations in AP2L genes are useful tools for fine-tuning wheat flowering time in a changing environment.

Inflorescence architecture is an important determinant of crop productivity. The number of spikelets produced by the wheat inflorescence meristem (IM) before its transition to a terminal spikelet (TS) influences the maximum number of grains per spike. Wheat MADS-box genes VERNALIZATION 1 (VRN1) and FRUITFULL 2 (FUL2) (in the SQUAMOSA-clade) are essential to promote the transition from IM to TS and for spikelet development. Here we show that SQUAMOSA genes contribute to spikelet identity by repressing MADS-box genes VEGETATIVE TO REPRODUCTIVE TRANSITION 2 (VRT2), SHORT VEGETATIVE PHASE 1 (SVP1), and SVP3 in the SVP clade. Constitutive expression of VRT2 resulted in leafy glumes and lemmas, reversion of spikelets to spikes, and downregulation of MADS-box genes involved in floret development, whereas the vrt2 mutant reduced vegetative characteristics in spikelets of squamosa mutants. Interestingly, the vrt2 svp1 mutant showed similar phenotypes to squamosa mutants regarding heading time, plant height, and spikelets per spike, but it exhibited unusual axillary inflorescences in the elongating stem. We propose that SQUAMOSA-SVP interactions are important to promote heading, formation of the TS, and stem elongation during the early reproductive phase, and that downregulation of SVP genes is then necessary for normal spikelet and floral development. Manipulating SVP and SQUAMOSA genes can contribute to engineering spike architectures with improved productivity.

In Arabidopsis, CONSTANS (CO) integrates light and circadian clock signals to promote flowering under long days (LD). In the grasses, a duplication generated two paralogs designated as CONSTANS1 (CO1) and CONSTANS2 (CO2). Here we show that in tetraploid wheat plants grown under LD, combined loss-of-function mutations in the A and B-genome homeologs of CO1 and CO2 (co1 co2) result in a small (3 d) but significant (P60 d relative to the co1 mutant allele under LD. We detected significant genetic interactions among CO1, CO2 and PPD1 genes on heading time, which were reflected in complex interactions at the transcriptional and protein levels. Loss-of-function mutations in PPD1 delayed heading more than combined co1 co2 mutations and, more importantly, PPD1 was able to perceive and respond to differences in photoperiod in the absence of functional CO1 and CO2 genes. Similarly, CO1 was able to accelerate heading time in response to LD in the absence of a functional PPD1. Taken together, these results indicate that PPD1 and CO1 are able to respond to photoperiod in the absence of each other, and that interactions between these two photoperiod pathways at the transcriptional and protein levels are important to fine-tune the flowering response in wheat.

The spikelet is the basic unit of the grass inflorescence. In this study, we show that wheat MADS-box genes VRN1, FUL2 and FUL3 play critical and redundant roles in spikelet and spike development, and also affect flowering time and plant height. In the vrn1ful2ful3-null triple mutant, the inflorescence meristem formed a normal double-ridge structure, but then the lateral meristems generated vegetative tillers subtended by leaves instead of spikelets. These results suggest an essential role of these three genes in the fate of the upper spikelet ridge and the suppression of the lower leaf ridge. Inflorescence meristems of vrn1ful2ful3-null and vrn1ful2-null remained indeterminate and single vrn1-null and ful2-null mutants showed delayed formation of the terminal spikelet and increased number of spikelets per spike. Moreover, the ful2-null mutant showed more florets per spikelet, which together with a higher number of spikelets, resulted in a significant increase in the number of grains per spike in the field. Our results suggest that a better understanding of the mechanisms underlying wheat spikelet and spike development can inform future strategies to improve grain yield in wheat.

FLOWERING LOCUS T2 (FT2) is the closest paralog of the FT1 flowering gene in the temperate grasses. Here we show that overexpression of FT2 in Brachypodium distachyon and barley results in precocious flowering and reduced spikelet number, while down-regulation by RNA interference results in delayed flowering and a reduced percentage of filled florets. Similarly, truncation mutations of FT2 homeologs in tetraploid wheat delayed flowering (2-4 d) and reduced fertility. The wheat ft2 mutants also showed a significant increase in the number of spikelets per spike, with a longer spike development period potentially contributing to the delayed heading time. In the wheat leaves, FT2 was expressed later than FT1, suggesting a relatively smaller role for FT2 in the initiation of the reproductive phase. FT2 transcripts were detected in the shoot apical meristem and increased during early spike development. Transversal sections of the developing spike showed the highest FT2 transcript levels in the distal part, where new spikelets are formed. Our results suggest that, in wheat, FT2 plays an important role in spike development and fertility and a limited role in the timing of the transition between the vegetative and reproductive shoot apical meristem.

Introduction: The pathogenesis of diabetic neuropathic pain (DNP) is complex involving various processes, which need exploring reliable biomarkers for its early detection and severity prediction. Methods: Study enrolled 181 patients diagnosed with diabetes, among which 74 patients developed DNP. Serum miR-34a-5p levels were compared between DNP patients and non-DNP patients by polymerase chain reaction (PCR), and the potential of miR-34a-5p in predicting the risk and discriminating patients with DNP was evaluated. The regulatory effect of miR-34a-5p on the inflammation, proliferation, and polarization of microglia was evaluated in HMC3 cells treated with high glucose. Results: Upregulated miR-34a-5p was identified as a risk factor and discriminated DNP patients miR-34a-5p was positively correlated with the levels of triglyceride (r = 0.797), fasting blood glucose (r = 0.840), and glycated hemoglobin (r = 0.894) of DNP patients. In HMC3 cells, the high-glucose-induced inflammation, promoted cell growth and caused polarization. The knockdown of miR-34a-5p showed the significant protective effect of microglia activation by high glucose, which was reversed by silencing ENPP3. Discussion: miR-34a-5p served as a biomarker for the prediction and early detection of DNP and mediated microglia inflammation caused by DNP via modulating ENPP3. [ABSTRACT FROM AUTHOR]

Plants utilize variation in day length (photoperiod) to anticipate seasonal changes. They respond by modulating their growth and development to maximize seed production, which in cereal crops is directly related to yield. In wheat (Triticum aestivum), the acceleration of flowering under long days (LD) is dependent on the light induction of PHOTOPERIOD1 (PPD1) by phytochromes. Under LD, PPD1 activates FLOWERING LOCUS T1 (FT1), a mobile signaling protein that travels from the leaves to the shoot apical meristem to promote flowering. Here, we show that the interruption of long nights by short pulses of light ("night-break" [NB]) accelerates wheat flowering, suggesting that the duration of the night is critical for wheat photoperiodic response. PPD1 transcription was rapidly upregulated by NBs, and the magnitude of this induction increased with the length of darkness preceding the NB Cycloheximide abolished the NB up-regulation of PPD1, suggesting that this process is dependent on active protein synthesis during darkness. While one NB was sufficient to induce PPD1, more than 15 NBs were required to induce high levels of FT1 expression and a strong acceleration of flowering. Multiple NBs did not affect the expression of core circadian clock genes. The acceleration of flowering by NB disappeared in ppd1-null mutants, demonstrating that this response is mediated by PPD1 The acceleration of flowering was strongest when NBs were applied in the middle of the night, suggesting that in addition to PPD1, other circadian-controlled factors are required for the up-regulation of FT1 expression and the acceleration of flowering.

The FLOWERING LOCUS T (FT) protein is a central component of a mobile flowering signal (florigen) that is transported from leaves to the shoot apical meristem (SAM). Two FT monomers and two DNA-binding bZIP transcription factors interact with a dimeric 14-3-3 protein bridge to form a hexameric protein complex. This complex, designated as the 'florigen activation complex' (FAC), plays a critical role in flowering. The wheat homologue of FT, designated FT1 (= VRN3), activates expression of VRN1 in the leaves and the SAM, promoting flowering under inductive long days. In this study, we show that FT1, other FT-like proteins, and different FD-like proteins, can interact with multiple wheat and barley 14-3-3 proteins. We also identify the critical amino acid residues in FT1 and FD-like proteins required for their interactions, and demonstrate that 14-3-3 proteins are necessary bridges to mediate the FT1-TaFDL2 interaction. Using in vivo bimolecular fluorescent complementation (BiFC) assays, we demonstrate that the interaction between FT1 and 14-3-3 occurs in the cytoplasm, and that this complex is then translocated to the nucleus, where it interacts with TaFDL2 to form a FAC. We also demonstrate that a FAC including FT1, TaFDL2 and Ta14-3-3C can bind to the VRN1 promoter in vitro. Finally, we show that relative transcript levels of FD-like and 14-3-3 genes vary among tissues and developmental stages. Since FD-like proteins determine the DNA specificity of the FACs, variation in FD-like gene expression can result in spatial and temporal modulation of the effects of mobile FT-like signals.

Phytochromes are dimeric proteins that function as red and far-red light sensors influencing nearly every phase of the plant life cycle. Of the three major phytochrome families found in flowering plants, phytochrome C (PHYC) is the least understood. In Arabidopsis and rice, PHYC is unstable and functionally inactive unless it heterodimerizes with another phytochrome. However, when expressed in an Arabidopsis phy-null mutant, wheat PHYC forms signaling active homodimers that translocate into the nucleus in red light to mediate photomorphogenic responses. Tetraploid wheat plants homozygous for loss-of-function mutations in all PHYC copies (phyC(AB)) flower on average 108 d later than wild-type plants under long days but only 19 d later under short days, indicating a strong interaction between PHYC and photoperiod. This interaction is further supported by the drastic down-regulation in the phyC(AB) mutant of the central photoperiod gene photoperiod 1 (PPD1) and its downstream target flowering locus T1, which are required for the promotion of flowering under long days. These results implicate light-dependent, PHYC-mediated activation of PPD1 expression in the acceleration of wheat flowering under inductive long days. Plants homozygous for the phyC(AB) mutations also show altered profiles of circadian clock and clock-output genes, which may also contribute to the observed differences in heading time. Our results highlight important differences in the photoperiod pathways of the temperate grasses with those of well-studied model plant species.

The transition from vegetative to reproductive development in the temperate cereals is mainly regulated by seasonal cues including vernalization (determined mainly by VRN1 and VRN2 genes) and photoperiod (determined mainly by PPD1 and CO2 genes). The wheat VRN3 gene, which is similar to Arabidopsis FT, plays a central role in the integration of the competing signals from these two pathways. Under long days, VRN3 transcription is down-regulated by VRN2, a unique flowering repressor in cereals, and up-regulated by CO2. Overexpression of VRN3 overcomes VRN2 repression and promotes VRN1 transcription and flowering initiation. Using yeast two- and three-hybrid assays we show here that the CCT domains present in VRN2 and CO2 proteins interact with the same subset of eight NF-Y proteins, and that these CCT proteins compete with NF-YA for interactions with NF-YB proteins. We have confirmed all these interactions in vitro, and the interactions between VRN2 and two of the three NF-YB proteins were further confirmed in planta. In addition, we show that mutations in the CCT domain of VRN2 that eliminate the vernalization requirement in winter wheat also reduce the strength of the interactions between VRN2 and NF-Y proteins, and the ability of VRN2 to compete with CO2. Taken together, our results suggest that the interactions between CCT and NF-Y proteins play an important role in the integration of the vernalization and photoperiod seasonal signals, and provide a flexible combinatorial system to integrate multiple developmental and environmental signals in the regulation of flowering initiation in the temperate cereals.

In winter wheat (Triticum spp.) and barley (Hordeum vulgare) varieties, long exposures to nonfreezing cold temperatures accelerate flowering time (vernalization) and improve freezing tolerance (cold acclimation). However, when plants initiate their reproductive development, freezing tolerance decreases, suggesting a connection between the two processes. To better understand this connection, we used two diploid wheat (Triticum monococcum) mutants, maintained vegetative phase (mvp), that carry deletions encompassing VRN-1, the major vernalization gene in temperate cereals. Homozygous mvp/mvp plants never flower, whereas plants carrying at least one functional VRN-1 copy (Mvp/-) exhibit normal flowering and high transcript levels of VRN-1 under long days. The Mvp/- plants showed reduced freezing tolerance and reduced transcript levels of several cold-induced C-REPEAT BINDING FACTOR transcription factors and COLD REGULATED genes (COR) relative to the mvp/mvp plants. Diploid wheat accessions with mutations in the VRN-1 promoter, resulting in high transcript levels under both long and short days, showed a significant down-regulation of COR14b under long days but not under short days. Taken together, these studies suggest that VRN-1 is required for the initiation of the regulatory cascade that down-regulates the cold acclimation pathway but that additional genes regulated by long days are required for the down-regulation of the COR genes. In addition, our results show that allelic variation in VRN-1 is sufficient to determine differences in freezing tolerance, suggesting that quantitative trait loci for freezing tolerance previously mapped on this chromosome region are likely a pleiotropic effect of VRN-1 rather than the effect of a separate closely linked locus (FROST RESISTANCE-1), as proposed in early freezing tolerance studies.

Winter wheat (Triticum spp.) varieties require long exposures to low temperatures to flower, a process called vernalization. The VRN2 locus includes two completely linked zinc finger-CCT domain genes (ZCCT1 and ZCCT2) that act as flowering repressors down-regulated during vernalization. Deletions or mutations in these two genes result in the elimination of the vernalization requirement in diploid wheat (Triticum monococcum). However, natural allelic variation in these genes has not been described so far in polyploid wheat (tetraploid Triticum turgidum and hexaploid Triticum aestivum). A tetraploid wheat population segregating for both VRN-A2 and VRN-B2 loci facilitated the characterization of different alleles. Comparisons between functional and nonfunctional alleles revealed that both ZCCT1 and ZCCT2 genes are able to confer vernalization requirement and that different ZCCT genes are functional in different genomes. ZCCT1 and ZCCT2 proteins from nonfunctional vrn2 alleles have mutations at arginine amino acids at position 16, 35, or 39 of the CCT domain. These positions are conserved between CCT and HEME ACTIVATOR PROTEIN2 (HAP2) proteins, supporting a model in which the action of CCT domains is mediated by their interactions with HAP2/HAP3/HAP5 complexes. This study also revealed natural variation in gene copy number, including a duplication of the functional ZCCT-B2 gene and deletions or duplications of the complete VRN-B2 locus. Allelic variation at the VRN-B2 locus was associated with a partially dominant effect, which suggests that variation in the number of functional ZCCT genes can be used to expand allelic diversity for heading time in polyploid wheat and, hopefully, improve its adaptation to different environments.

Recent progress in the characterization of two groups of genes responsible for natural differences in frost tolerance in wheat and barley is reviewed here. The first group includes the vernalization genes that delay flowering until the end of the winter and protect sensitive floral primordia. This process is regulated mainly by differences in the regulatory regions of VRN1 and VRN3 genes or in the coding regions of VRN2. The second group includes a set of tandemly duplicated CBF (C-repeat Binding Factors) transcription factors at the FR2 (Frost Resistance 2) locus. CBF transcription factors are known regulators of the COR genes (COld Regulated genes) which are induced by cold and confer tolerance to subsequent freezing temperatures (acclimation). Natural differences in frost tolerance in both wheat and barley have been mapped to the FR2 locus, and are associated with differences in threshold induction temperatures and/or transcript levels of several CBF genes. Higher threshold induction temperatures result in earlier up-regulation of COR genes during the fall, whereas higher induction levels by cold are associated with faster cold acclimation rates. Both processes result in longer acclimation periods and improved frost tolerance. Increases in VRN1 transcript levels in the leaves are associated with reduced responsiveness of CBF and COR genes to cold and with the end of the acclimation period. Therefore, delays in the induction of VRN1 and in the transition to the reproductive stage can extend the acclimation period and improve frost tolerance. These observations suggest that the vernalization and cold acclimation regulatory gene networks are interconnected.

SummaryA precise regulation of flowering time is central to plant species survival. Therefore, mechanisms have evolved in plants to integrate various environmental cues to optimize flowering time. In this study, we show that the product of the wheat gene TaFT, which integrates photoperiod and vernalization signals promoting flowering, interacts with bZIP proteins TaFDL2 and TaFDL6. We also show that TaFDL2 can interact in vitro with five ACGT elements in the promoter of the meristem identity gene VRN1, suggesting that TaFDL2 is a functional homologue of Arabidopsis FD. No direct interactions between the TaFT protein and the VRN1 promoter were detected. Transgenic wheat plants over-expressing TaFT showed parallel increases in VRN1 transcripts, suggesting that TaFT provides transcriptional activation of VRN1, possibly through interactions with the TaFDL2 protein. The same transgenic plants also showed increased transcript levels of TaFT2 (a TaFT paralogue), indicating that TaFT2 acts downstream of TaFT. The fact that TaFT2 interacts with another bZIP protein TaFDL13, which lacks the ability to interact with the VRN1 promoter, suggests that TaFT and TaFT2 have different gene targets. This observation agrees with the functional divergence observed for the TaFT and TaFT2 orthologous genes in rice. The temperate cereals analyzed so far show VRN1 transcripts in the leaves, a characteristic not observed in Arabidopsis or rice. The high levels of TaFDL2 transcripts observed in wheat leaves provide a simple explanation for this difference. We present a hypothesis to explain the conservation of VRN1 expression in the leaves of temperate cereals.

A cluster of eleven CBF genes was recently mapped to the Frost resistance-2 (Fr-Am2) locus on chromosome 5 of diploid wheat (Triticum monococcum) using a cross between frost tolerant accession G3116 and frost sensitive DV92. The Fr-Am2 locus was mapped at the peak of two overlapping quantitative trait loci (QTL), one for frost survival and the other for differential expression of the cold regulated gene COR14b. Seven lines with recombination events within the CBF cluster were used to identify CBF candidate genes for these QTL. The lines carrying the critical recombination events were tested for whole plant frost survival and for differential transcript levels of cold induced COR14b and DHN5 genes. The strongest effect for these traits was associated to the linked TmCBF12, TmCBF14 and TmCBF15 genes, with the G3116 allele conferring improved frost tolerance and higher levels of COR14b and DHN5 transcript at mild cold temperatures (12-15 degrees C) than the DV92 allele. Comparison of CBF protein sequences revealed that the DV92 TmCBF12 protein contains a deletion of five amino acids in the AP2 DNA binding domain. Electrophoretic Mobility Shift Assays (EMSA) confirmed that the protein encoded by this allele cannot bind to the CRT/DRE (C-repeat/ dehydration-responsive element) motif present in the promoters of several cold induced genes. A smaller effect on frost tolerance was mapped to the distal group of CBF genes including TmCBF16. Transcript levels of TmCBF16, as well as those of TmCBF12 and TmCBF15 were up-regulated at mild cold temperatures in G3116 but not in DV92. Higher threshold induction temperatures can result in earlier initiation of the cold acclimation process and better resistance to subsequent freezing temperatures. The non-functional TmCBF12 allele in DV92 can also contribute to its lower frost tolerance.

Summary: Heterosis utilization is the most effective way to improve rice yields. The cytoplasmic male‐sterility (CMS) and photoperiod/thermosensitive genic male‐sterility (PTGMS) systems have been widely used in rice production. However, the rate of resource utilization for the CMS system hybrid rice is low, and the hybrid seed production for the PTGMS system is affected by the environment. The technical limitations of these two breeding methods restrict the rapid development of hybrid rice. The advantages of the genic male‐sterility (GMS) rice, such as stable sterility and free combination, can fill the gaps of the first two generations of hybrid rice technology. At present, the third‐generation hybrid rice breeding technology is being used to realize the application of GMS materials in hybrid rice. This study aimed to use an artificial CMS gene as a pollen killer to create a smart sterile line for hybrid rice production. The clustered regularly interspaced short palindromic repeats/CRISPR‐associated 9 (CRISPR/Cas9) technology was used to successfully obtain a CYP703A3‐deficient male‐sterile mutant containing no genetically modified component in the genetic background of indica 9311. Through young ear callus transformation, this mutant was transformed with three sets of element‐linked expression vectors, including pollen fertility restoration gene CYP703A3, pollen‐lethality gene orfH79 and selection marker gene DsRed2. The maintainer 9311‐3B with stable inheritance was obtained, which could realize the batch breeding of GMS materials. Further, the sterile line 9311‐3A and restorer lines were used for hybridization, and a batch of superior combinations of hybrid rice was obtained. [ABSTRACT FROM AUTHOR]

Introduction: Anti-epidermal growth factor receptor (EGFR)-targeted nanoparticles can be used to deliver a therapeutic and imaging agent to EGFR-overexpressing tumor cells. I-labeled anti-EGFR nanoparticles derived from cetuximab were used as a tumor-targeting vehicle in radionuclide therapy. Methods: This paper describes the construction of the anti-EGFR nanoparticle EGFR-BSA-PCL. This nanoparticle was characterized for EGFR-targeted binding and cellular uptake in EGFR-overexpressing cancer cells by using flow cytometry and confocal microscopy. Anti-EGFR and non-targeted nanoparticles were labeled with I using the chloramine-T method. Analyses of cytotoxicity and targeted cell killing with I were performed using the MTT assay. The time-dependent cellular uptake of I-labeled anti-EGFR nanoparticles proved the slow-release effects of nanoparticles. A radioiodine therapy study was also performed in mice. Results: The EGFR-targeted nanoparticle EGFR-BSA-PCL and the non-targeted nanoparticle BSA-PCL were constructed; the effective diameters were approximately 100 nm. The results from flow cytometry and confocal microscopy revealed significant uptake of EGFR-BSA-PCL in EGFR-overexpressing tumor cells. Compared with EGFR-BSA-PCL, BSA-PCL could also bind to cells, but tumor cell retention was minimal and weak. In MTT assays, the EGFR-targeted radioactive nanoparticle I-EGFR-BSA-PCL showed greater cytotoxicity and targeted cell killing than the non-targeted nanoparticle I-BSA-PCL. The radioiodine uptake of both I-labeled nanoparticles, I-EGFR-BSA-PCL and I-BSA-PCL, was rapid and reached maximal levels 4 h after incubation, but the I uptake of I-EGFR-BSA-PCL was higher than that of I-BSA-PCL. On day 15, the average tumor volumes of the I-EGFR-BSA-PCL and I-BSA-PCL groups showed a slow growth relationship compared with that of the control group. Conclusion: The EGFR-targeted nanoparticle EGFR-BSA-PCL demonstrated superior cellular binding and uptake compared with those of the control BSA-PCL. The EGFR-targeted radioactive nanoparticle I-EGFR-BSA-PCL exhibited favorable intracellular retention of I. Radionuclide therapy using I-EGFR-BSA-PCL, which showed excellent targeted cell killing, suppressed cancer cell growth caused by EGFR overexpression. [ABSTRACT FROM AUTHOR]