Genome-wide identification of the plant homeodomain-finger family in rye and ScPHD5 functions in cold tolerance and flowering time.

KEY MESSAGE: 111 PHD genes were newly identified in rye genome and ScPHD5's role in regulating cold tolerance and flowering time was suggested. Plant homeodomain (PHD)-finger proteins regulate the physical properties of chromatin and control plant development and stress tolerance. Although rye (Secale cereale L.) is a major winter crop, PHD-finger proteins in rye have not been studied. Here, we identified 111 PHD genes in the rye genome that exhibited diverse gene and protein sequence structures. Phylogenetic tree analysis revealed that PHDs were genetically close in monocots and diverged from those in dicots. Duplication and synteny analyses demonstrated that ScPHDs have undergone several duplications during evolution and that high synteny is conserved among the Triticeae species. Tissue-specific and abiotic stress-responsive gene expression analyses indicated that ScPHDs were highly expressed in spikelets and developing seeds and were responsive to cold and drought stress. One of these genes, ScPHD5, was selected for further functional characterization. ScPHD5 was highly expressed in the spike tissues and was localized in the nuclei of rye protoplasts and tobacco leaves. ScPHD5-overexpressing Brachypodium was more tolerant to freezing stress than wild-type (WT), with increased CBF and COR gene expression. Additionally, these transgenic plants displayed an extremely early flowering phenotype that flowered more than two weeks earlier than the WT, and vernalization genes, rather than photoperiod genes, were increased in the WT. RNA-seq analysis revealed that diverse stress response genes, including HSPs, HSFs, LEAs, and MADS-box genes, were also upregulated in transgenic plants. Our study will help elucidate the roles of PHD genes in plant development and abiotic stress tolerance in rye.

TaMAPK3 phosphorylates TaCBF and TaICE and plays a negative role in wheat freezing tolerance.

Freezing temperature during overwintering often kills plants; plants have thus, developed a defense mechanism called 'cold acclimation', in which a number of genes are involved in increasing cell protection and gene expression. Mitogen-activated protein kinase (MAPK) controls proteins' activities by phosphorylation and is involved in numerous metabolic pathways. In this study, we identified the protein interaction between TaMAPK3 and the proteins in the cold response pathway, ICE41, ICE87, and CBFIVd-D9. The subcellular localization and bimolecular fluorescence complement (BiFC) assays revealed that these proteins interact in the nucleus or in the plasma membrane. Furthermore, MAPK3-mediated phosphorylation of ICE41, ICE87, and CBFIVd-D9 was verified using an in vitro phosphorylation assay. TaMAPK3-overexpressing transgenic Brachypodium showed a lower survival rate upon freezing stress and lower proline content during cold acclimation, compared to wild-type plants. Furthermore, cold response gene expression analysis revealed that the expression of these genes was suppressed in the transgenic lines under cold treatment. It was further elucidated that MAPK3 mediates the degradation of ICE and CBF proteins, which implies the negative impact of MAPK3 on the freezing tolerance of plants. This study will help to elucidate the molecular mechanisms of cold tolerance and the activity of MAPK3 in wheat.

The wheat TaF-box3, SCF ubiquitin ligase component, participates in the regulation of flowering time in transgenic Arabidopsis.

Histone methylation is actively involved in plant flowering time and is regulated by a myriad of genetic pathways that integrate endogenous and exogenous signals. We identified an F-box gene from wheat (Triticum aestivum L.) and named it TaF-box3. Transcript expression analysis showed that TaF-box3 expression was gradually induced during the floret development and anthesis stages (WS2.5-10). Furthermore, ubiquitination assays have shown that TaF-box3 is a key component of the SCF ubiquitin ligase complex. TaF-box3 overexpression in Arabidopsis resulted in an early flowering phenotype and different cell sizes in leaves compared to the WT. Furthermore, the transcript level of a flowering time-related gene was significantly reduced in TaF-box3 overexpressing plants, which was linked with lower histone H3 Lys4 trimethylation (H3K4me3) and H3 Lys36 trimethylation (H3K36me3). Overexpression of TaF-box3 in Arabidopsis was shown to be involved in the regulation of flowering time by demethylating FLC chromatin, according to ChIP experiments. Protein analysis confirmed that TaMETS interacts with TaF-box3 and is ubiquitinated and degraded in a TaF-box3-dependnent manner. Based on these findings, we propose that TaF-box3 has a positive role in flowering time, which leads to a better understanding of TaF-box3 physiological mechanism in Arabidopsis.

Identification and molecular characterization of novel sucrose transporters in the hexaploid wheat (Triticum aestivum L.).

Common wheat (Triticum aestivum) is a major cereal crop grown and consumed globally. Recent advances in sequencing technology have facilitated the exploration of large and repetitive genomes. Plant sucrose transporter (SUT) genes are vital components of energy transport systems that play prominent roles in various plant functions, such as signaling and stress regulation. In this study, we identified and analyzed five novel sucrose transporter genes in wheat. The wheat sucrose transporter genes were divided into five clades based on their phylogenetic relationships. Synteny analysis revealed that synteny in the genome is highly conserved between wheat and rye, barley, and Brachypodium. Furthermore, the cis-element analysis indicated that sucrose transporter genes might be regulated by light and some phytohormone-related transcriptional factors. Overall, plant tissue-specific gene expression revealed enhanced expression of the transporter genes in the root and stem, whereas they were differentially expressed under abiotic stress treatments (cold, heat, NaCl, PEG-6000, and sucrose). These results indicate that each TaSUT gene may play a crucial role in stabilizing plants under stress by actively regulating the energy demands of cells. The findings of this study may provide a basis for further research on sucrose transporters and their significant roles in plant energy metabolism as well as in abiotic stress response, signaling, and regulation.

Genome-wide identification and characterization of the lettuce GASA family in response to abiotic stresses.

BACKGROUND: Lettuce is one of the most extensively farmed vegetables in the world, and it prefers cool growing conditions. High temperatures promote premature bolt formation, reducing quality and yield. The gibberellic acid-stimulated Arabidopsis (GASA) family genes play critical roles in plant growth, development, and stress responses. However, the biological functions of GASA proteins in lettuce have yet to be thoroughly investigated. RESULTS: Using genome-wide analysis, 20 GASAs were identified in lettuce including, three groups of LsGASA proteins based on the phylogenetic analysis. Except for one, all GASA proteins included a conserved GASA domain with 12 cysteine residues. Cis-element analysis showed that LsGASAs were closely associated with light, phytohormones, and stress resistance. Five segmental and three tandem duplication events were observed in the LsGASA family based on duplication analysis. GASA synteny analysis among lettuce, Arabidopsis, tobacco, and rice revealed that LsGASA5 is highly collinear with all species. Six of the 20 LsGASA showed increased expression patterns at specific time points in the shoot apical meristem when subjected to heat stress. According to gene expression analysis, the majority of GASA were highly expressed in flowers compared to other organs, and six GASA exhibited highly increased expression levels in response to NaCl, abscisic acid, and gibberellin treatment. Furthermore, LsGASA proteins are predominantly found in the plasma membrane and/or the cytosol. CONCLUSIONS: This study provides a comprehensive characterization of LsGASA genes for their diversity and biological functions. Moreover, our results will be useful for further studies on the function of lettuce GASA in abiotic stress- and heat-induced bolting signaling.

Transcriptome and Proteome Co-Profiling Offers an Understanding of Pre-Harvest Sprouting (PHS) Molecular Mechanisms in Wheat (Triticum aestivum).

While wheat (Triticum aestivum L.) is a widely grown and enjoyed crop, the diverse and complex global situation and climate are exacerbating the instability of its supply. In particular, pre-harvest sprouting (PHS) is one of the major abiotic stresses that frequently occurs due to irregular climate conditions, causing serious damage to wheat and its quality. In this study, transcriptomic analysis with RNA-seq and proteomic analysis with LC-MS/MS were performed in PHS-treated spikes from two wheat cultivars presenting PHS sensitivity and tolerance, respectively. A total of 13,154 differentially expressed genes (DEGs) and 706 differentially expressed proteins (DEPs) were identified in four comparison groups between the susceptible/tolerant cultivars. Gene function and correlation analysis were performed to determine the co-profiled genes and proteins affected by PHS treatment. In the functional annotation of each comparative group, similar functions were confirmed in each cultivar under PHS treatment; however, in Keumgang PHS+7 (K7) vs. Woori PHS+7 (W7), functional annotations presented clear differences in the "spliceosome" and "proteasome" pathways. In addition, our results indicate that alternative splicing and ubiquitin-proteasome support the regulation of germination and seed dormancy. This study provides an advanced understanding of the functions involved in transcription and translation related to PHS mechanisms, thus enabling specific proposals for the further analysis of germination and seed dormancy mechanisms and pathways in wheat.

Molecular characterization of wheat floret development-related F-box protein (TaF-box2): Possible involvement in regulation of Arabidopsis flowering.

In wheat (Triticum aestivum L.), the floret development stage is an important step in determining grain yield per spike; however, the molecular mechanisms underlying floret development remain unclear. In this study, we elucidated the role of TaF-box2, a member of the F-box-containing E3 ubiquitin protein ligases, which is involved in floret development and anthesis of wheat. TaF-box2 was transiently expressed in the plasma membrane and cytoplasm of both tobacco and wheat. We also found that the SCFF-box2 (Skp1-Cul1-Rbx1-TaF-box2) ubiquitin ligase complex mediated self-ubiquitination activity. Transgenic Arabidopsis plants that constitutively overexpressed TaF-box2 showed markedly greater hypocotyl and root length than wild-type plants, and produced early flowering phenotypes. Flowering-related genes were significantly upregulated in TaF-box2-overexpressing Arabidopsis plants. Further protein interaction analyses such as yeast two-hybrid, in vitro pull-down, and bimolecular fluorescence complementation assays confirmed that TaF-box2 physically interacted with TaCYCL1 (Triticum aestivum cyclin-L1-1). Ubiquitination and degradation assays demonstrated that TaCYCL1 was ubiquitinated by SCFF-box2 and degraded through the 26S proteasome complex. The physiological functions of the TaF-box2 protein remain unclear; however, we discuss several potential routes of involvement in various physiological mechanisms which counteract flowering in transgenic Arabidopsis plants.

Identification of genetic loci associated with major agronomic traits of wheat (Triticum aestivum L.) based on genome-wide association analysis.

BACKGROUND: Bread wheat (Triticum aestivum L.) is one of the most widely consumed cereal crops, but its complex genome makes it difficult to investigate the genetic effect on important agronomic traits. Genome-wide association (GWA) analysis is a useful method to identify genetic loci controlling complex phenotypic traits. With the RNA-sequencing based gene expression analysis, putative candidate genes governing important agronomic trait can be suggested and also molecular markers can be developed. RESULTS: We observed major quantitative agronomic traits of wheat; the winter survival rate (WSR), days to heading (DTH), days to maturity (DTM), stem length (SL), spike length (SPL), awn length (AL), liter weight (LW), thousand kernel weight (TKW), and the number of seeds per spike (SPS), of 287 wheat accessions from diverse country origins. A significant correlation was observed between the observed traits, and the wheat genotypes were divided into three subpopulations according to the population structure analysis. The best linear unbiased prediction (BLUP) values of the genotypic effect for each trait under different environments were predicted, and these were used for GWA analysis based on a mixed linear model (MLM). A total of 254 highly significant marker-trait associations (MTAs) were identified, and 28 candidate genes closely located to the significant markers were predicted by searching the wheat reference genome and RNAseq data. Further, it was shown that the phenotypic traits were significantly affected by the accumulation of favorable or unfavorable alleles. CONCLUSIONS: From this study, newly identified MTA and putative agronomically useful genes will help to study molecular mechanism of each phenotypic trait. Further, the agronomically favorable alleles found in this study can be used to develop wheats with superior agronomic traits.

Molecular characterization of the wheat putative proline-rich protein TaELF7 and its involvement in the negative regulation of Arabidopsis flowering.

Late stages of floret development, such as booting, heading, and anthesis stages, are important steps for determining grain setting and for filling in wheat. Herein, we report the molecular function of Triticum aestivum ELF7 encoding RNA polymerase II-associated factor 1 (PAF1), which may act as a negative regulator in floret development and anthesis stages. Among the six TaELF7-like genes isolated from wheat, TaELF7 like1-A and TaELF7 like2-B showed contrasting expression levels during the late stage of floret development stages, with observation of decreased expression level of TaELF7 like1-A compared to that of TaELF7 like2-B. The full-length TaELF7 like1-A has a 1038-bp open reading frame that contains a proline-rich domain in the N-terminal region and a nuclear localization signal domain in the C-terminal region. TaELF7 like1-A was found to be localized in the nucleus in both tobacco and wheat. Direct interaction of TaELF7 with the RING-type E3 ligase TaHUB2 was confirmed using a yeast two-hybrid system, an in vitro pull-down assay, and a bimolecular fluorescence complementation assay. The flowering time was delayed in TaELF7-overexpressing plants compared to that in the control plants. Expression levels of few floral repressor genes were markedly increased in TaELF7-overexpressing Arabidopsis plants.

Identification of novel C-repeat binding factor (CBF) genes in rye (Secale cereale L.) and expression studies.

Although rye is one of the most cold-tolerant species among temperate cereals, its huge and complex genome has prevented us from identifying agronomically useful genes. However, advances in high-throughput sequencing technology are making it increasingly possible to investigate its genome. The C-repeat binding factor (CBF) gene family controls cold tolerance in plants and its members are well conserved among eudicots and monocots, among which there are diverse homologs. Despite its large genome, only a small number of CBF genes have been identified in rye. In this study, we explored high-throughput sequencing data of the rye genome and identified 12 novel CBF genes. Sequence analyses revealed that these genes contain signature sequences of the CBF family. Chromosomal localization of the genes by PCR using wheat-rye addition lines showed that most of these are located on the long arm of chromosome 5, but also on the long arm of chromosomes 2 and 6. On the basis of comparative analyses of CBF family members in the Triticeae, CBF proteins were divided into several groups according to phylogenetic relationship and conserved motifs. Light is essential to fully induce CBF gene expression and there is specificity in the response to different types of abiotic stresses in ScCBF genes. The results of our study will assist investigations of CBF genes in the Triticeae and the mechanism of cold tolerance through the CBF-dependent pathway in plants.

Robot-assisted anterior lumbar interbody fusion (ALIF) using retroperitoneal approach.

BACKGROUND: Over the past few years, robot-assisted surgery has become increasingly popular, affecting virtually all surgical fields. It has been proven to overcome pitfalls of laparoscopic procedures, such as high complication rates and steep learning curve. We have, therefore, performed experimental anterior lumbar interbody fusion (ALIF) using retroperitoneal approach in swine model to test the feasibility of robot-assisted surgery in spinal surgery. METHOD: In this report, we describe the setup with the da Vinci surgical system, operative method, result and discuss technical aspects and the future of robot-assisted ALIF. FINDINGS: Experimental retroperitoneal dissection using robotic surgical system was successfully performed with great visual cue, minimal retraction and minimal bleeding. CONCLUSION: Although retroperitoneal approach for spinal fusion has never been attempted with robotic surgical system, we could demonstrate the possibility with swine model. Further studies and development of appropriate instruments will bring minimally invasive spine surgery to a new era.