Identification and characterization of the CRK gene family in the wheat genome and analysis of their expression profile in response to high temperature-induced male sterility.

Cysteine-rich receptor-like kinases (CRKs) play many important roles during plant development, including defense responses under both biotic and abiotic stress, reactive oxygen species (ROS) homeostasis, callose deposition and programmed cell death (PCD). However, there are few studies on the involvement of the CRK family in male sterility due to heat stress in wheat (Triticum aestivum L.). In this study, a genome-wide characterization of the CRK family was performed to investigate the structural and functional attributes of the wheat CRKs in anther sterility caused by heat stress. A total of 95 CRK genes were unevenly distributed on 18 chromosomes, with the most genes distributed on chromosome 2B. Paralogous homologous genes with Ka/Ks ratios less than 1 may have undergone strong purifying selection during evolution and are more functionally conserved. The collinearity analysis results of CRK genes showed that wheat and Arabidopsis (A. thaliana), foxtail millet, Brachypodium distachyon (B. distachyon), and rice have three, 12, 15, and 11 pairs of orthologous genes, respectively. In addition, the results of the network interactions of genes and miRNAs showed that five miRNAs were in the hub of the interactions map, namely tae-miR9657b-5p, tae-miR9780, tae-miR9676-5p, tae-miR164, and tae-miR531. Furthermore, qRT-PCR validation of the six TaCRK genes showed that they play key roles in the development of the mononuclear stage anthers, as all six genes were expressed at highly significant levels in heat-stressed male sterile mononuclear stage anthers compared to normal anthers. We hypothesized that the TaCRK gene is significant in the process of high-temperature-induced sterility in wheat based on the combination of anther phenotypes, paraffin sections, and qRT-PCR data. These results improve our understanding of their relationship.

Corrigendum: Genome-wide identification of genes encoding cystathionine beta synthase domain-containing proteins in wheat and its relationship with anther male sterility under heat stress.

[This corrects the article DOI: 10.3389/fpls.2022.1061472.].

Genome-wide identification of cystathionine beta synthase genes in wheat and its relationship with anther male sterility under heat stress.

Cystathionine beta synthase (CBS) domains containing proteins (CDCPs) plays an important role in plant development through regulation of the thioredoxin system, as well as its ability to respond to biotic and abiotic stress conditions. Despite this, no systematic study has examined the wheat CBS gene family and its relation to high temperature-induced male sterility. In this study, 66 CBS family members were identified in the wheat genome, and their gene or protein sequences were used for subsequent analysis. The TaCBS gene family was found to be unevenly distributed on 21 chromosomes, and they were classified into four subgroups according to their gene structure and phylogeny. The results of collinearity analysis showed that there were 25 shared orthologous genes between wheat, rice and Brachypodium distachyon, and one shared orthologous gene between wheat, millet and barley. The cis-regulatory elements of the TaCBS were related to JA, IAA, MYB, etc. GO and KEGG pathway analysis identified these TaCBS genes to be associated with pollination, reproduction, and signaling and cellular processes, respectively. A heatmap of wheat plants based on transcriptome data showed that TaCBS genes were expressed to a higher extent in spikelets relative to other tissues. In addition, 29 putative tae-miRNAs were identified, targeting 41 TaCBS genes. Moreover, qRT-PCR validation of six TaCBS genes indicated their critical role in anther development, as five of them were expressed at lower levels in heat-stressed male sterile anthers than in Normal anthers. Together with anther phenotypes, paraffin sections, starch potassium iodide staining, and qRT-PCR data, we hypothesized that the TaCBS gene has a very important connection with the heat-stressed sterility process in wheat, and these data provide a basis for further insight into their relationship.

Fusion-PCR generates attL recombination site adaptors and allows Rapid One-Step Gateway (ROG) cloning.

Gateway recombination-based cloning, which eliminates the use of restriction endonucleases and ligase, has been widely used for the construction of high-throughput (HTP) vectors. However, this approach is very expensive and its two-stage reaction process is laborious and time consuming. Therefore, we developed a Gateway cloning method that uses fusion-PCR to generate attL recombination site adaptors, and the PCR products, which can be directly cloned into destination vectors, giving rise to Rapid One-Step Gateway (ROG) Cloning. 100% of cloning efficiencies were obtained by this ROG method. This method has no BP reaction/entry clone step, thus halving the cost and time consumed. Overall, this work provides a highly efficient, rapid, low-cost method for directional recombination cloning.

An easily-performed high-throughput method for plant genomic DNA extraction.

Genomic DNA isolation is a crucial technique for researchers studying plant molecular biology. A current widely-used protocol for DNA extraction needs a pestle and mortal for each sample and consumes a large amount of liquid nitrogen in grinding the samples. Most high-throughput methods depend on expensive machines for tissue homogenization. Here we developed a CTAB-based DNA extraction method using 2.0 mL microcentrifuge tubes for sample processing. This protocol has the advantages that it is suitable for a variety of plants, easily-performed without special equipment, and high-throughput; it effectively avoids sample cross-contamination, and is inexpensive, rapid and safe.

Overexpression of RcLEC1-B, a HAP3 transcription factor of PLB from Rosa canina, increases the level of endogenous gibberellin and alters the development of cuticle and floral organs in Arabidopsis.

The HAP3 subfamily gene RcLEC1-B, was isolated from protocorm-like body (PLB) of Rosa canina, encodes 213 amino acid residues. It was shown that RcLEC1-B was specifically expressed in PLB of R. canina and its subcellular localization is in the nucleus. Overexpression of RcLEC1-B in Arabidopsis resulted in a decrease in endogenous ABA level, an increase in GA, IAA and CTK contents, and an increased number of branches. RcLEC1-B promotes the formation of spontaneous embryoids, suggesting that it may be a homolog of the Arabidopsis LEC1 gene. RcLEC1-B-OE changed the number and morphology of flower organs and resulted in open carpels and exposed ovules, along with a reduced percentage of fertile fruit. This is the first observation that overexpression of a homolog of LEC1 in Arabidopsis can lead to morphological changes in floral organs, cuticle defects, and adhesions between organs; this may result from the increased level of gibberellin in the transgenic plants.

Development of a Gateway-compatible pCAMBIA binary vector for RNAi-mediated gene knockdown in plants.

RNA interference (RNAi), based on hairpin RNA (hpRNA) expression, plays an important role in functional analysis of plant genes. Traditional methods for making RNAi constructs usually involve multiple time-consuming cloning steps. We have developed a Gateway-compatible binary vector for RNAi-mediated gene knockdown in plants from pCAMBIA2301 and pHANNIBAL vectors. The new plant RNAi binary vector, named pCAMBIA2301-GW-RNAi, has two inverted repeated Gateway cassettes driven by the cauliflower mosaic virus 35S (CaMV 35S) promoter. This enables site-specific recombination at two sites by one Gateway LR reaction without restriction enzymes and ligases. The pCAMBIA2301-GW-RNAi vector's effectiveness was evaluated by Agrobacterium-mediated transient co-expression assays of overexpression and silencing constructs of HvCEBiP in Nicotiana benthamiana followed by western blot analysis. Obtained results show that the developed RNAi vector successfully knocked down 35S-driven expression of HvCEBiP, as expression levels of the encoded HvCEBiP protein were significantly reduced.

A novel, easy and rapid method for constructing yeast two-hybrid vectors using In-Fusion technology.

Yeast two-hybrid systems are powerful tools for analyzing interactions between proteins. Vector construction is an essential step in yeast two-hybrid experiments, which require bait and prey plasmids. In this study, we modified the multiple cloning site sequence of the yeast plasmid pGADT7 by site-directed mutagenesis PCR to generate the pGADT7-In vector, which resulted in an easy and rapid method for constructing yeast two-hybrid vectors using the In-Fusion cloning technique. This method has three key advantages: only one pair of primers and one round of PCR are needed to generate bait and prey plasmids for each gene, it is restriction endonuclease- and ligase-independent, and it is fast and easily performed.

Vacuum and Co-cultivation Agroinfiltration of (Germinated) Seeds Results in Tobacco Rattle Virus (TRV) Mediated Whole-Plant Virus-Induced Gene Silencing (VIGS) in Wheat and Maize.

Tobacco rattle virus (TRV)-mediated virus-induced gene silencing (VIGS) has been frequently used in dicots. Here we show that it can also be used in monocots, by presenting a system involving use of a novel infiltration solution (containing acetosyringone, cysteine, and Tween 20) that enables whole-plant level VIGS of (germinated) seeds in wheat and maize. Using the established system, phytoene desaturase (PDS) genes were successfully silenced, resulting in typical photo-bleaching symptoms in the leaves of treated wheat and maize. In addition, three wheat homoeoalleles of MLO, a key gene repressing defense responses to powdery mildew in wheat, were simultaneously silenced in susceptible wheat with this system, resulting in it becoming resistant to powdery mildew. The system has the advantages generally associated with TRV-mediated VIGS systems (e.g., high-efficiency, mild virus infection symptoms, and effectiveness in different organs). However, it also has the following further advantages: (germinated) seed-stage agroinfiltration; greater rapidity and convenience; whole-plant level gene silencing; adequately stable transformation; and suitability for studying functions of genes involved in seed germination and early plant development stages.

Rorippa indica Regeneration via Somatic Embryogenesis Involving Frog Egg-like Bodies Efficiently Induced by the Synergy of Salt and Drought Stresses.

Frog egg-like bodies (FELBs), novel somatic embryogenesis (SE) structures first observed in Solanum nigrum, were induced in Rorippa indica. NaCl-mediated salt and mannitol-mimicked drought stresses induced FELBs in R. indica, which is very different from the induction by plant growth regulators (PGRs) under low light condition that was used in S. nigrum FELB induction. It demonstrated that NaCl or mannitol supplements alone could induce FELBs in R. indica, but with low induction rates, while the synergy of NaCl and mannitol significantly increased the FELB induction rates. For the combination of 5.0 g/L mannitol and 10.0 g/L NaCl the highest FELB induction rate (100%) was achieved. It suggests that the synergy of drought and salt stresses can replace PGRs to induce FELBs in R. indica. On medium supplemented with 1.0 mg/L gibberellic acid all the inoculated in vitro FELBs developed into multiple plantlets. Morphological and histological analyses confirmed the identity of FELBs induced in R. indica and revealed that FELBs originate from root cortex cells.

Transcriptome analysis of the compatible interaction of tomato with Verticillium dahliae using RNA-sequencing.

Tomato Verticillium wilt is a soil-borne vascular disease caused by the necrotrophic fungus Verticillium dahliae. Although some understanding of plant defense mechanisms against V. dahliae infection has been gained for incompatible interactions, including identification of inducible resistant genes and defense signaling pathways, the genes and signaling pathways involved in the compatible interaction remain unclear. To investigate the molecular basis of the compatible interaction between tomato and V. dahliae, transcriptomes of V. dahliae infected tomatoes were compared to those of a control group. A total of approximately 25 million high-quality reads were generated by means of the RNA sequencing (RNA-seq) method. The sequence reads were aligned to the tomato reference genome and analyzed to measure gene expression levels, and to identify alternative splicing events. Comparative analysis between the two samples revealed 1,953 significantly differentially expressed genes (DEGs), including 1,281 up-regulated and 672 down-regulated genes. The RNA-Seq output was confirmed using RT-qPCR for 10 selected genes. The Nr, Swiss-Prot, Gene Ontology (GO), Clusters of Orthologous Groups (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were used to annotate DEG functions. Of the 1,953 DEGs identified, 1,953, 1,579, 1,739, 862, and 380 were assigned by Nr, Swiss-Prot, GO, COG, and KEGG, respectively. The important functional groups identified via GO and COG enrichment were those responsible for fundamental biological regulation, secondary metabolism, and signal transduction. Of DEGs assigned to 87 KEGG pathways, most were associated with phenylpropanoid metabolism and plant-pathogen interaction pathways. Most of the DEGs involved in these two pathways were up-regulated, and may be involved in regulating the tomato-V. dahliae compatible interaction. The results will help to identify key susceptible genes and contribute to a better understanding of the mechanisms of tomato susceptible response to V. dahliae.

A lower pH value benefits regeneration of Trichosanthes kirilowii by somatic embryogenesis, involving rhizoid tubers (RTBs), a novel structure.

A new approach was established for the regeneration of Trichosanthes kirilowii from root, stem, and leaf explants by somatic embryogenesis (SE), involving a previously unreported SE structure, rhizoid tubers (RTBs). During SE, special rhizoids were first induced from root, stem, and leaf explants with average rhizoid numbers of 62.33, 40.17, and 11.53 per explant, respectively, on Murashige and Skoog (MS) medium (pH 4.0) supplemented with 1.0 mg/L 1-naphthaleneacetic acid (NAA) under dark conditions. Further, one RTB was formed from each of the rhizoids on MS medium (pH 4.0) supplemented with 20 mg/L thidiazuron (TDZ) under light conditions. In the suitable range (pH 4.0-9.0), a lower pH value increased the induction of rhizoids and RTBs. Approximately 37.77, 33.47, and 31.07% of in vivo RTBs from root, stem, and leaf explants, respectively, spontaneously developed into multiple plantlets on the same MS medium (supplemented with 20 mg/L TDZ) for induction of RTBs, whereas >95.00% of in vitro RTBs from each kind of explant developed into multiple plantlets on MS medium supplemented with 5.0 mg/L 6-benzylaminopurine (BAP). Morphological and histological analyses revealed that RTB is a novel type of SE structure that develops from the cortex cells of rhizoids.

Efficient soybean regeneration and Agrobacterium-mediated transformation using a whole cotyledonary node as an explant.

An optimized regeneration and Agrobacterium-mediated transformation protocol based on whole cotyledonary node explants was developed in soybean (Glycine max) cultivar Zhong Huang 13. Adding 6-benzylaminopurine (BAP) in a germinating medium could significantly increase regeneration efficiency; the optimal BAP concentration for shoot formation was 0.5 mg/L. The concentrations of plant growth regulators in a shoot induction medium were optimized by the orthogonal test [L9 (3(3))]. The best combination for shoot regeneration was a medium of Murashige & Skoog salts with B5 vitamins (MSB) supplemented with 3.5 mg/L BAP, 0.2 mg/L indole-3-butyric acid (IBA), and 0.2 mg/L kinetin (KT). Under this favorable condition, one node could regenerate 28-30 shoots. Soybean whole cotyledonary nodes were transformed by inoculation with A. tumefaciens strain EHA105 harboring a vector pBI121 containing a ß-glucuronidase gene (gus). GUS assay, polymerase chain reaction, and Southern blot analysis indicated that the gus gene was transformed into soybean plants with 23.1% transformation efficiency. Transgenic plants could be obtained within 5-6 weeks, which was about 4 weeks less than that of a traditional single cotyledonary node method.

Regeneration of Solanum nigrum by somatic embryogenesis, involving frog egg-like body, a novel structure.

A new protocol was established for the regeneration of Solanum nigrum by frog egg-like bodies (FELBs), which are novel somatic embryogenesis (SE) structures induced from the root, stem, and leaf explants. The root, stem, and leaf explants (93.33%, 85.10%, and 100.00%, respectively) were induced to form special embryonic calli on Murashige and Skoog (MS) medium containing 1.0 mg/L 2,4-dichlorophenoxyacetic acid, under dark condition. Further, special embryonic calli from the root, stem, and leaf explants (86.97%, 83.30%, and 99.47%, respectively) were developed into FELBs. Plantlets of FELBs from the three explants were induced in vitro on MS medium supplemented with 5.0 mg/L 6-benzylaminopurine and 0.1 mg/L gibberellic acid, and 100.00% plantlet induction rates were noted. However, plantlet induction in vivo on MS medium supplemented with 20 mg/L thidiazuron showed rates of 38.63%, 15.63%, and 61.30% for the root, stem, and leaf explants, respectively, which were lower than those of the in vitro culture. Morphological and histological analyses of FELBs at different development stages revealed that they are a novel type of SE structure that developed from the mesophyll (leaf) or cortex (stem and root) cells of S. nigrum.

A rapid, highly efficient and economical method of Agrobacterium-mediated in planta transient transformation in living onion epidermis.

Transient transformation is simpler, more efficient and economical in analyzing protein subcellular localization than stable transformation. Fluorescent fusion proteins were often used in transient transformation to follow the in vivo behavior of proteins. Onion epidermis, which has large, living and transparent cells in a monolayer, is suitable to visualize fluorescent fusion proteins. The often used transient transformation methods included particle bombardment, protoplast transfection and Agrobacterium-mediated transformation. Particle bombardment in onion epidermis was successfully established, however, it was expensive, biolistic equipment dependent and with low transformation efficiency. We developed a highly efficient in planta transient transformation method in onion epidermis by using a special agroinfiltration method, which could be fulfilled within 5 days from the pretreatment of onion bulb to the best time-point for analyzing gene expression. The transformation conditions were optimized to achieve 43.87% transformation efficiency in living onion epidermis. The developed method has advantages in cost, time-consuming, equipment dependency and transformation efficiency in contrast with those methods of particle bombardment in onion epidermal cells, protoplast transfection and Agrobacterium-mediated transient transformation in leaf epidermal cells of other plants. It will facilitate the analysis of protein subcellular localization on a large scale.

Curcumin decreases the expression of Pokemon by suppressing the binding activity of the Sp1 protein in human lung cancer cells.

Pokemon, which stands for POK erythroid myeloid ontogenic factor, can regulate expression of many genes and plays an important role in tumorigenesis. Curcumin, a natural and non-toxic yellow compound, has capacity for antioxidant, free radical scavenger, anti-inflammatory properties. Recent studies shows it is a potential inhibitor of cell proliferation in a variety of tumour cells. To investigate whether curcumin can regulate the expression of Pokemon, a series of experiments were carried out. Transient transfection experiments demonstrated that curcumin could decrease the activity of the Pokemon promoter. Western blot analysis suggested that curcumin could significantly decrease the expression of the Pokemon. Overexpression of Sp1 could enhance the activity of the Pokemon promoter, whereas knockdown of Sp1 could decrease its activity. More important, we also found that curcumin could decrease the expression of the Pokemon by suppressing the stimulation of the Sp1 protein. Therefore, curcumin is a potential reagent for tumour therapy which may target Pokemon.

[Molecular identification on Waxy genes in wheat using multiple-PCR].

Multiple-PCR was conducted to establish a stable PCR system for identifying the three Wx genes in wheat. Two pairs of primers were employed to amplify Wx-A1, Wx-B1, and Wx-D1 genes of wheat, with the target sequences of 230 bp/265 bp, 854 bp, and 204 bp, respectively. The results showed that Wx-A1, Wx-B1, and Wx-D1 can be detected simultaneously in a single reaction. This method proved to be repeatable and low cost for evaluation of wheat quality properties in breeding program. This multiple-PCR technique can be efficiently used in marker-assisted selection for Wx genes, which will improve selection procedure for waxy wheat.

Alterations in cytosine methylation and species-specific transcription induced by interspecific hybridization between Oryza sativa and O. officinalis.

Interspecific hybridization and polyploidization may involve programmed genetic and epigenetic changes. In this study, we used the methylation-sensitive amplified polymorphism (MSAP) method to survey cytosine methylation alterations that occurred in F(1) hybrid and BC(1) progeny following interspecific hybridization between Oryza sativa and O. officinalis. Across all 316 parental methylated sites, 25 (7.9%) cytosine methylation alterations were detected in the F(1) and/or BC(1) progeny. Thirty additional cytosine methylation alterations were detected at parental non-methylated or novel sites. In total, 55 cytosine methylation alterations (90.9% of all alterations) were detected in the F(1) hybrid, which were maintained in the BC(1) progeny. The alterations in cytosine methylation were biased toward the O. officinalis parent and were in some cases repeatable in independent hybridizations between O. sativa and O. officinalis. Twelve fragments showing cytosine methylation alterations were isolated, sequenced and subsequently validated by methylation-sensitive Southern blot analysis. Where possible, we designed species-specific primers to amplify the polymorphic transcripts from either the O. sativa or the O. officinalis parent using reverse transcription (RT)-PCR in combination with single-strand conformation polymorphism (SSCP) analysis. In four of five cases, modified gene expression could be correlated with the altered cytosine methylation pattern. Our results demonstrated cytosine methylation alterations induced by interspecific hybridization between a rice cultivar and its wild relative, and indicated a direct relationship between cytosine methylation alteration and gene expression variation.

[Monosomic alien addition lines: a new tool for studying and using plant genomics].

Interspecies hybridization and backcrossing is a means to transfer desirable genes from one species to another in breeding programs of higher plants. Monosomic alien addition lines (MAALs) can be produced via addition of single chromosome of an alien donor species to the entire chromosome complement of the recipient species. MAALs are powerful tools for elucidating genome structure and transferring genes. Backcrossing of MAALs to the recipient parent results in plants containing short overlapping introgressions, which cover the entire donor genome. These introgression lines can be used as vectors of alien genomic libraries in a recipient genetic background. In addition, a complete set of MAALs also serves as a library of the donor genome dissected into individual chromosome entities, which facilitates high-throughput marker allocation to individual donor chromosomes, and marker assignments and syntenic relationships can be compared between the donor chromosomes and the respective orthologous recipient chromosomes. Meanwhile, MAALs can be used to study the introgression mechanism and the pairing status of homologous chromosomes. In this review, we presented the production and properties of MAALs and highlighted their advantages for genetic breeding and fundamental researches.