Origin and evolution of the bread wheat D genome.

Bread wheat (Triticum aestivum) is a globally dominant crop and major source of calories and proteins for the human diet. Compared with its wild ancestors, modern bread wheat shows lower genetic diversity, caused by polyploidisation, domestication and breeding bottlenecks1,2. Wild wheat relatives represent genetic reservoirs, and harbour diversity and beneficial alleles that have not been incorporated into bread wheat. Here we establish and analyse extensive genome resources for Tausch's goatgrass (Aegilops tauschii), the donor of the bread wheat D genome. Our analysis of 46 Ae. tauschii genomes enabled us to clone a disease resistance gene and perform haplotype analysis across a complex disease resistance locus, allowing us to discern alleles from paralogous gene copies. We also reveal the complex genetic composition and history of the bread wheat D genome, which involves contributions from genetically and geographically discrete Ae. tauschii subpopulations. Together, our results reveal the complex history of the bread wheat D genome and demonstrate the potential of wild relatives in crop improvement.

Compensatory Modulation of Seed Storage Protein Synthesis and Alteration of Starch Accumulation by Selective Editing of 13 kDa Prolamin Genes by CRISPR-Cas9 in Rice.

Rice prolamins are categorized into three groups by molecular size (10, 13, or 16 kDa), while the 13 kDa prolamins are assigned to four subgroups (Pro13a-I, Pro13a-II, Pro13b-I, and Pro13b-II) based on cysteine residue content. Since lowering prolamin content in rice is essential to minimize indigestion and allergy risks, we generated four knockout lines using CRISPR-Cas9, which selectively reduced the expression of a specific subgroup of the 13 kDa prolamins. These four mutant rice lines also showed the compensatory expression of glutelins and non-targeted prolamins and were accompanied by low grain weight, altered starch content, and atypically-shaped starch granules and protein bodies. Transcriptome analysis identified 746 differentially expressed genes associated with 13 kDa prolamins during development. Correlation analysis revealed negative associations between genes in Pro13a-I and those in Pro13a-II and Pro13b-I/II subgroups. Furthermore, alterations in the transcription levels of 9 ER stress and 17 transcription factor genes were also observed in mutant rice lines with suppressed expression of 13 kDa prolamin. Our results provide profound insight into the functional role of 13 kDa rice prolamins in the regulatory mechanisms underlying rice seed development, suggesting their promising potential application to improve nutritional and immunological value.

Gene editing of authentic Brassica rapa flavonol synthase 1 generates dihydroflavonol-accumulating Chinese cabbage.

Flavonols are the major class of flavonoids of green Chinese cabbage (Brassica rapa subsp. pekinensis). The B. rapa genome harbors seven flavonol synthase genes (BrFLSs), but they have not been functionally characterized. Here, transcriptome analysis showed four BrFLSs mainly expressed in Chinese cabbage. Among them, only BrFLS1 showed major FLS activity and additional flavanone 3ß-hydroxylase (F3H) activity, while BrFLS2 and BrFLS3.1 exhibited only marginal F3H activities. We generated BrFLS1-knockout (BrFLS1-KO) Chinese cabbages using CRISPR/Cas9-mediated genome editing and obtained transgene-free homozygous plants without off-target mutation in the T1 generation, which were further advanced to the T2 generation showing normal phenotype. UPLC-ESI-QTOF-MS analysis revealed that flavonol glycosides were dramatically decreased in the T2 plants, while dihydroflavonol glycosides accumulated concomitantly to levels corresponding to the reduced levels of flavonols. Quantitative PCR analysis revealed that the early steps of phenylpropanoid and flavonoid biosynthetic pathway were upregulated in the BrFLS1-KO plants. In accordance, total phenolic contents were slightly enhanced in the BrFLS1-KO plants, which suggests a negative role of flavonols in phenylpropanoid and flavonoid biosynthesis in Chinese cabbage. Phenotypic surveys revealed that the BrFLS1-KO Chinese cabbages showed normal head formation and reproductive phenotypes, but subtle morphological changes in their heads were observed. In addition, their seedlings were susceptible to osmotic stress compared to the controls, suggesting that flavonols play a positive role for osmotic stress tolerance in B.rapa seedling. In this study, we showed that CRISPR/Cas9-mediated BrFLS1-KO successfully generated a valuable breeding resource of Chinese cabbage with distinctive metabolic traits and that CRISPR/Cas9 can be efficiently applied in functional Chinese cabbage breeding.

Down-Regulation of Rice Glutelin by CRISPR-Cas9 Gene Editing Decreases Carbohydrate Content and Grain Weight and Modulates Synthesis of Seed Storage Proteins during Seed Maturation.

The glutelins are a family of abundant plant proteins comprised of four glutelin subfamilies (GluA, GluB, GluC, and GluD) encoded by 15 genes. In this study, expression of subsets of rice glutelins were suppressed using CRISPR-Cas9 gene-editing technology to generate three transgenic rice variant lines, GluA1, GluB2, and GluC1. Suppression of the targeted glutelin genes was confirmed by SDS-PAGE, Western blot, and q-RT-PCR. Transgenic rice variants GluA1, GluB2, and GluC1 showed reduced amylose and starch content, increased prolamine content, reduced grain weight, and irregularly shaped protein aggregates/protein bodies in mature seeds. Targeted transcriptional profiling of immature seeds was performed with a focus on genes associated with grain quality, starch content, and grain weight, and the results were analyzed using the Pearson correlation test (requiring correlation coefficient absolute value ≥ 0.7 for significance). Significantly up- or down-regulated genes were associated with gene ontology (GO) and KEGG pathway functional annotations related to RNA processing (spliceosomal RNAs, group II catalytic introns, small nucleolar RNAs, microRNAs), as well as protein translation (transfer RNA, ribosomal RNA and other ribosome and translation factors). These results suggest that rice glutelin genes may interact during seed development with genes that regulate synthesis of starch and seed storage proteins and modulate their expression via post-transcriptional and translational mechanisms.

Molecular mechanism controlling anthocyanin composition and content in radish plants with different root colors.

Radish (Raphanus sativus) roots exhibit various colors that reflect their anthocyanin compositions and contents. However, the details of the mechanism linking the expression of anthocyanin biosynthesis and their transcriptional regulators to anthocyanin composition in radish roots remained unknown. Here, we characterized the role of the anthocyanin biosynthetic enzyme flavonoid 3'-hydroxylase (RsF3'H), together with the R2R3 MYB transcription factor (TF) RsMYB1 and the basic helix-loop-helix (bHLH) TF TRANSPARENT TESTA 8 (RsTT8), in four radish plants with different root colors: white (W), deep red (DR), dark purple (DP), and dark greyish purple (DGP). The DR plant contained heterozygous for RsF3'H with low expression level and accumulated a large amount of pelargonidin, resulting in deep red color. While, the DP and DGP plants accumulated the cyanidin due to the higher expression level of functional RsF3'H. Notably, RsMYB1 and RsTT8 transcripts were abundant in all pigmented roots, but not in white roots. To investigate the differential expression of RsMYB1 and RsTT8, we compared the sequences of their promoter regions among the four radish plants, revealing variations in the numbers of cis-elements and in promoter architecture. Promoter activation assays demonstrated that variation in the RsMYB1 and RsTT8 promoters may contribute to the expression level of these genes, and RsMYB1 can activate its own expression as well as promote the RsTT8 expression. These results suggested that RsF3'H plays a vital role in anthocyanin composition and the expression level of both RsMYB1 and RsTT8 are crucial determinants for anthocyanin content in radish roots. Overall, these findings provide insight into the molecular basis of anthocyanin composition and level in radish roots.

PROTEIN PHOSPHATASE 2C08, a Negative Regulator of Abscisic Acid Signaling, Promotes Internode Elongation in Rice.

Clade A protein phosphatase 2Cs (PP2CAs) negatively regulate abscisic acid (ABA) signaling. Here, we investigated the functions of OsPP2CAs and their crosstalk with ABA and gibberellic acid (GA) signaling pathways in rice (Oryza sativa). Among the nine OsPP2CAs, OsPP2C08 had the highest amino acid sequence similarity with OsPP2C51, which positively regulates GA signaling in rice seed germination. However, OsPP2C08 was expressed in different tissues (internodes, sheaths, and flowers) compared to OsPP2C51, which was specifically expressed in seeds, and showed much stronger induction under abiotic stress than OsPP2C51. Transgenic rice lines overexpressing OsPP2C08 (OsPP2C08-OX) had a typical ABA-insensitive phenotype in a post-germination assay, indicating that OsPP2C08, as with other OsPP2CAs, negatively regulates ABA signaling. Furthermore, OsPP2C08-OX lines had longer stems than wild-type (WT) plants due to longer internodes, especially between the second and third nodes. Internode cells were also longer in OsPP2C08-OX lines than in the WT. As GA positively regulates plant growth, these results suggest that OsPP2C08 might positively regulate GA biosynthesis. Indeed, the expression levels of GA biosynthetic genes including gibberellin 20-oxidase (OsGA20ox4) and Ent-kaurenoic acid oxidase (OsKAO) were increased in OsPP2C08-OX lines, and we observed that GIBBERELLIN 2-OXIDASE 4 (OsGA2ox4), encoding an oxidase that catalyzes the 2-beta-hydroxylation of several biologically active GAs, was repressed in the OsPP2C08-OX lines based on a transcriptome deep sequencing and RT-qPCR analysis. Furthermore, we compared the accumulation of SLENDER RICE 1 (SLR1), a DELLA protein involved in GA signaling, in OsPP2C08-OX and WT plants, and observed lower levels of SLR1 in the OsPP2C08-OX lines than in the WT. Taken together, our results reveal that OsPP2C08 negatively regulates ABA signaling and positively regulates GA signaling in rice. Our study provides valuable insight into the molecular mechanisms underlying the crosstalk between GA and ABA signaling in rice.

Expression of RsPORB Is Associated with Radish Root Color.

Radish (Raphanus sativus) plants exhibit varied root colors due to the accumulation of chlorophylls and anthocyanins compounds that are beneficial for both human health and visual quality. The mechanisms of chlorophyll biosynthesis have been extensively studied in foliar tissues but remain largely unknown in other tissues. In this study, we examined the role of NADPH:protochlorophyllide oxidoreductases (PORs), which are key enzymes in chlorophyll biosynthesis, in radish roots. The transcript level of RsPORB was abundantly expressed in green roots and positively correlated with chlorophyll content in radish roots. Sequences of the RsPORB coding region were identical between white (948) and green (847) radish breeding lines. Additionally, virus-induced gene silencing assay with RsPORB exhibited reduced chlorophyll contents, verifying that RsPORB is a functional enzyme for chlorophyll biosynthesis. Sequence comparison of RsPORB promoters from white and green radishes showed several insertions and deletions (InDels) and single-nucleotide polymorphisms. Promoter activation assays using radish root protoplasts verified that InDels of the RsPORB promoter contribute to its expression level. These results suggested that RsPORB is one of the key genes underlying chlorophyll biosynthesis and green coloration in non-foliar tissues, such as roots.

Toward reducing the immunogenic potential of wheat flour: identification and characterization of wheat lines missing omega-5 gliadins encoded by the 1D chromosome.

KEY MESSAGE: Eleven wheat lines that are missing genes for the 1D-encoded omega-5 gliadins will facilitate breeding efforts to reduce the immunogenic potential of wheat flour for patients susceptible to wheat allergy. Efforts to reduce the levels of allergens in wheat flour that cause wheat-dependent exercise-induced anaphylaxis are complicated by the presence of genes encoding omega-5 gliadins on both chromosomes 1B and 1D of hexaploid wheat. In this study, we screened 665 wheat germplasm samples using gene specific DNA markers for omega-5 gliadins encoded by the genes on 1D chromosome that were obtained from the reference wheat Chinese Spring. Eleven wheat lines missing the PCR product corresponding to 1D omega-5 gliadin gene sequences were identified. Two of the lines contained the 1BL·1RS translocation. Relative quantification of gene copy numbers by qPCR revealed that copy numbers of 1D omega-5 gliadins in the other nine lines were comparable to those in 1D null lines of Chinese Spring, while copy numbers of 1B omega-5 gliadins were like those of Chinese Spring. 2-D immunoblot analysis of total flour proteins from the selected lines using a specific monoclonal antibody against the N-terminal sequence of omega-5 gliadin showed no reactivity in regions of the blots containing previously identified 1D omega-5 gliadins. Interestingly, RP-UPLC analysis of the gliadin fractions of the selected lines indicated that the expression of omega-1,2 gliadins was also significantly reduced in seven of the lines, implying that 1D omega-5 gliadin and 1D omega-1,2 gliadin genes are tightly linked on the Gli-D1 loci of chromosome 1D. Wheat lines missing the omega-5 gliadins encoded by the genes on 1D chromosome should be useful in future breeding efforts to reduce the immunogenic potential of wheat flour.

RsTTG1, a WD40 Protein, Interacts with the bHLH Transcription Factor RsTT8 to Regulate Anthocyanin and Proanthocyanidin Biosynthesis in Raphanus sativus.

MBW complexes, consisting of MYB, basic helix-loop-helix (bHLH), and WD40 proteins, regulate multiple traits in plants, including anthocyanin and proanthocyanidin (PA) biosynthesis and the determination of epidermal cell fate. Here, a WD40 gene from Raphanus sativus, designated TRANSPARENT TESTA GLABRA 1 (RsTTG1), was cloned and functionally characterized. Heterologous expression of RsTTG1 in the Arabidopsis thaliana mutant ttg1-22 background restored accumulation of anthocyanin and PA in the mutant and rescued trichome development. In radish, RsTTG1 was abundantly expressed in all root and leaf tissues, independently of anthocyanin accumulation, while its MBW partners RsMYB1 and TRANSPARENT TESTA 8 (RsTT8) were expressed at higher levels in pigment-accumulating tissues. In yeast two-hybrid analysis, the full-length RsTTG1 protein interacted with RsTT8. Moreover, transient protoplast co-expression assays demonstrated that RsTTG1, which localized to both the cytoplasm and nucleus, moves from the cytoplasm to the nucleus in the presence of RsTT8. When co-expressed with RsMYB1 and RsTT8, RsTTG1 stably activated the promoters of the anthocyanin biosynthesis genes CHALCONE SYNTHASE (RsCHS) and DIHYDROFLAVONOL 4-REDUCTASE (RsDFR). Transient expression of RsTTG1 in tobacco leaves exhibited an increase in anthocyanin accumulation due to activation of the expression of anthocyanin biosynthesis genes when simultaneously expressed with RsMYB1 and RsTT8. These results indicate that RsTTG1 is a vital regulator of pigmentation and trichome development as a functional homolog of AtTTG1.

Silencing of Dihydroflavonol 4-reductase in Chrysanthemum Ray Florets Enhances Flavonoid Biosynthesis and Antioxidant Capacity.

Flavonoid biosynthesis requires the activities of several enzymes, which form weakly-bound, ordered protein complexes termed metabolons. To decipher flux regulation in the flavonoid biosynthetic pathway of chrysanthemum (Chrysanthemum morifolium Ramat), we suppressed the gene-encoding dihydroflavonol 4-reductase (DFR) through RNA interference (RNAi)-mediated post-transcriptional gene silencing under a floral-specific promoter. Transgenic CmDFR-RNAi chrysanthemum plants were obtained by Agrobacterium-mediated transformation. Genomic PCR analysis of CmDFR-RNAi chrysanthemums propagated by several rounds of stem cuttings verified stable transgene integration into the genome. CmDFR mRNA levels were reduced by 60-80% in CmDFR-RNAi lines compared to those in wild-type (WT) plants in ray florets, but not leaves. Additionally, transcript levels of flavonoid biosynthetic genes were highly upregulated in ray florets of CmDFR-RNAi chrysanthemum relative to those in WT plants, while transcript levels in leaves were similar to WT. Total flavonoid contents were high in ray florets of CmDFR-RNAi chrysanthemums, but flavonoid contents of leaves were similar to WT, consistent with transcript levels of flavonoid biosynthetic genes. Ray florets of CmDFR-RNAi chrysanthemums exhibited stronger antioxidant capacity than those of WT plants. We propose that post-transcriptional silencing of CmDFR in ray florets modifies metabolic flux, resulting in enhanced flavonoid content and antioxidant activity.

Chili Pepper AN2 (CaAN2): A Visible Selection Marker for Nondestructive Monitoring of Transgenic Plants.

Selecting transformed plants is generally time consuming and laborious. To develop a method for transgenic plant selection without the need for antibiotics or herbicides, we evaluated the suitability of the R2R3 MYB transcription factor gene CaAN2 from purple chili pepper (Capsicum annuum) for use as a visible selection marker. CaAN2 positively regulates anthocyanin biosynthesis. Transient expression assays in tobacco (Nicotiana tabacum) leaves revealed that CaAN2 actively induced sufficient pigment accumulation for easy detection without the need for a basic helix-loop-helix (bHLH) protein as a cofactor; similar results were obtained for tobacco leaves transiently co-expressing the anthocyanin biosynthesis regulators bHLH B-Peru from maize and R2R3 MYB mPAP1D from Arabidopsis. Tobacco plants harboring CaAN2 were readily selected based on their red color at the shoot regeneration stage due to anthocyanin accumulation without the need to impose selective pressure from herbicides. Transgenic tobacco plants harboring CaAN2 showed strong pigment accumulation throughout the plant body. The ectopic expression of CaAN2 dramatically promoted the transcription of anthocyanin biosynthetic genes as well as regulators of this process. The red coloration of tobacco plants harboring CaAN2 was stably transferred to the next generation. Therefore, anthocyanin accumulation due to CaAN2 expression is a useful visible trait for stable transformation, representing an excellent alternative selection system for transgenic plants.

The R3-Type MYB Transcription Factor BrMYBL2.1 Negatively Regulates Anthocyanin Biosynthesis in Chinese Cabbage (Brassica rapa L.) by Repressing MYB-bHLH-WD40 Complex Activity.

Chinese cabbage (Brassica rapa L.) leaves are purple in color due to anthocyanin accumulation and have nutritional and aesthetic value, as well as antioxidant properties. Here, we identified the R3 MYB transcription factor BrMYBL2.1 as a key negative regulator of anthocyanin biosynthesis. A Chinese cabbage cultivar with green leaves harbored a functional BrMYBL2.1 protein, designated BrMYBL2.1-G, with transcriptional repressor activity of anthocyanin biosynthetic genes. By contrast, BrMYBL2.1 from a Chinese cabbage cultivar with purple leaves carried a poly(A) insertion in the third exon of the gene, resulting in the insertion of multiple lysine residues in the predicted protein, designated BrMYBL2.1-P. Although both BrMYBL2.1 variants localized to the nucleus, only BrMYBL2.1-G interacted with its cognate partner BrTT8. Transient infiltration assays in tobacco leaves revealed that BrMYBL2.1-G, but not BrMYBL2.1-P, actively represses pigment accumulation by inhibiting the transcription of anthocyanin biosynthetic genes. Transient promoter activation assay in Arabidopsis protoplasts verified that BrMYBL2.1-G, but not BrMYBL2.1-P, can repress transcriptional activation of BrCHS and BrDFR, which was activated by co-expression with BrPAP1 and BrTT8. We determined that BrMYBL2.1-P may be more prone to degradation than BrMYBL2.1-G via ubiquitination. Taken together, these results demonstrate that BrMYBL2.1-G blocks the activity of the MBW complex and thus represses anthocyanin biosynthesis, whereas the variant BrMYBL2.1-P from purple Chinese cabbage cannot, thus leading to higher anthocyanin accumulation.

Functional Characterization of BrF3'H, Which Determines the Typical Flavonoid Profile of Purple Chinese Cabbage.

Flavonols and anthocyanins are the two major classes of flavonoids in Brassica rapa. To elucidate the flavonoid biosynthetic pathway in Chinese cabbage (B. rapa L. subsp. pekinensis), we analyzed flavonoid contents in two varieties of Chinese cabbage with normal green (5546) and purple (8267) leaves. The 8267 variety accumulates significantly higher levels of quercetin, isorhamnetin, and cyanidin than the 5546 variety, indicating that 3'-dihydroxylated flavonoids are more prevalent in the purple than in the green variety. Gene expression analysis showed that the expression patterns of most phenylpropanoid pathway genes did not correspond to the flavonoid accumulation patterns in 5546 and 8267 varieties, except for BrPAL1.2 while most early and late flavonoid biosynthetic genes are highly expressed in 8267 variety. In particular, the flavanone 3'-hydroxylase BrF3'H (Bra009312) is expressed almost exclusively in 8267. We isolated the coding sequences of BrF3'H from the two varieties and found that both sequences encode identical amino acid sequences and are highly conserved with F3'H genes from other species. An in vitro enzymatic assay demonstrated that the recombinant BrF3'H protein catalyzes the 3'-hydroxylation of a wide range of 4'-hydroxylated flavonoid substrates. Kinetic analysis showed that kaempferol is the most preferred substrate and dihydrokaempferol (DHK) is the poorest substrate for recombinant BrF3'H among those tested. Transient expression of BrF3'H in Nicotiana benthamiana followed by infiltration of naringenin and DHK as substrates resulted in eriodictyol and quercetin production in the infiltrated leaves, demonstrating the functionality of BrF3'H in planta. As the first functional characterization of BrF3'H, our study provides insight into the molecular mechanism underlying purple coloration in Chinese cabbage.

An OsKala3, R2R3 MYB TF, Is a Common Key Player for Black Rice Pericarp as Main Partner of an OsKala4, bHLH TF.

Rice (Oryza sativa) pericarp exhibits various colors due to the accumulation of anthocyanins and/or proanthocyanidins. Previous work revealed that the two basic helix-loop-helix (bHLH) transcription factors OsKala4 and OsRc are key regulators for the black and red pericarp traits, respectively, and their inactivation results in rice with white pericarp. However, their pericarp-specific R2R3 MYB partner remained unknown. Here, we characterized the role of the R2R3 MYB gene OsKala3 in rice pericarp pigmentation through genetic and molecular approaches. A rice protoplast transfection assay showed that OsKala3 is a nuclear-localized protein. Furthermore, OsKala3 physically interacted with OsKala4 in a yeast two-hybrid analysis. Co-transfection assays in rice protoplasts revealed that OsKala3 and OsKala4 mediate the activation of anthocyanin biosynthetic genes. Notably, the OsKala3 promoter region exhibited an insertion polymorphism specifically in rice cultivars with black pericarp, creating two tandem repeats while red and white varieties harbor only one. The number of repeats within the OsKala3 promoter correlated with increased transactivation by OsKala3, thus providing a rationale for the black pericarp characteristic of cultivars with two repeats. These results thus provide evidence for the molecular basis of anthocyanin biosynthesis in rice pericarp and may facilitate the introduction of this beneficial trait to other rice cultivars through marker-assisted breeding.

Loss of the R2R3 MYB Transcription Factor RsMYB1 Shapes Anthocyanin Biosynthesis and Accumulation in Raphanus sativus.

The red or purple color of radish (Raphanus sativus L.) taproots is due to anthocyanins, which have nutritional and aesthetic value, as well as antioxidant properties. Moreover, the varied patterns and levels of anthocyanin accumulation in radish roots make them an interesting system for studying the transcriptional regulation of anthocyanin biosynthesis. The R2R3 MYB transcription factor RsMYB1 is a key positive regulator of anthocyanin biosynthesis in radish. Here, we isolated an allele of RsMYB1, named RsMYB1Short, in radish cultivars with white taproots. The RsMYB1Short allele carried a 4 bp insertion in the first exon causing a frame-shift mutation of RsMYB1, generating a truncated protein with only a partial R2 domain at the N-terminus. Unlike RsMYB1Full, RsMYB1Short was localized to the nucleus and the cytoplasm and failed to interact with their cognate partner RsTT8. Transient expression of genomic or cDNA sequences for RsMYB1Short in radish cotyledons failed to induce anthocyanin accumulation, but that for RsMYB1Full activated it. Additionally, RsMYB1Short showed the lost ability to induce pigment accumulation and to enhance the transcript level of anthocyanin biosynthetic genes, while RsMYB1Full promoted both processes when co-expressed with RsTT8 in tobacco leaves. As the result of the transient assay, co-expressing RsTT8 and RsMYB1Full, but not RsMYB1Short, also enhanced the promoter activity of RsCHS and RsDFR. We designed a molecular marker for RsMYB1 genotyping, and revealed that the RsMYB1Short allele is common in white radish cultivars, underscoring the importance of variation at the RsMYB1 locus in anthocyanin biosynthesis in the radish taproot. Together, these results indicate that the nonsense mutation of RsMYB1 generated the truncated protein, RsMYB1Short, that had the loss of ability to regulate anthocyanin biosynthesis. Our findings highlight that the frame shift mutation of RsMYB1 plays a key role in anthocyanin biosynthesis in the radish taproot.

A Rapid, Reliable RP-UPLC Method for Large-Scale Analysis of Wheat HMW-GS Alleles.

High-molecular-weight glutenin subunits (HMW-GS) account for only 10% of total wheat storage proteins, but play an important role in the processing quality of wheat flour. Therefore, identifying HMW-GS alleles associated with good end-use quality provides important information for wheat breeders. To rapidly, accurately and reproducibly identify HMW-GS, we established an optimized reversed-phase ultra-performance liquid chromatography (RP-UPLC) method. Separation parameters were optimized using an ACQUITY UPLC Protein BEH C4 column and stepwise ACN gradient, and the separation patterns and retention times (RTs) of 22 subunits were comparatively analyzed in 16 standard wheat cultivars. All HMW-GS proteins were well separated within about 5.5 min, and all analyses were complete within 12 min. We distinguished the 16 subunits based on RT, although three subunits in 1Bx (1Bx7/1Bx7OE and 1Bx17) and three subunits in 1By (1By8*, 1By9 and 1By15) had overlapping RTs; these were differentiated by SDS-PAGE. To distinguish 1Bx7 and 1Bx7OE, which differ in protein abundance, RP-UPLC was combined with PCR analysis of DNA junction markers. The optimized method was successfully applied to determine HMW-GS alleles in a large collection of bread wheat germplasm (1787 lines). This protocol is an appropriate option for selecting lines harboring favorable HMW-GS alleles in wheat breeding.

Proteomic Determination of Low-Molecular-Weight Glutenin Subunit Composition in Aroona Near-Isogenic Lines and Standard Wheat Cultivars.

The low-molecular weight glutenin subunit (LMW-GS) composition of wheat (Triticum aestivum) flour has important effects on end-use quality. However, assessing the contributions of each LMW-GS to flour quality remains challenging because of the complex LMW-GS composition and allelic variation among wheat cultivars. Therefore, accurate and reliable determination of LMW-GS alleles in germplasm remains an important challenge for wheat breeding. In this study, we used an optimized reversed-phase HPLC method and proteomics approach comprising 2-D gels coupled with liquid chromatography-tandem mass spectrometry (MS/MS) to discriminate individual LMW-GSs corresponding to alleles encoded by the Glu-A3, Glu-B3, and Glu-D3 loci in the 'Aroona' cultivar and 12 'Aroona' near-isogenic lines (ARILs), which contain unique LMW-GS alleles in the same genetic background. The LMW-GS separation patterns for 'Aroona' and ARILs on chromatograms and 2-D gels were consistent with those from a set of 10 standard wheat cultivars for Glu-3. Furthermore, 12 previously uncharacterized spots in 'Aroona' and ARILs were excised from 2-D gels, digested with chymotrypsin, and subjected to MS/MS. We identified their gene haplotypes and created a 2-D gel map of LMW-GS alleles in the germplasm for breeding and screening for desirable LMW-GS alleles for wheat quality improvement.

Alternative Splicing of the Basic Helix-Loop-Helix Transcription Factor Gene CmbHLH2 Affects Anthocyanin Biosynthesis in Ray Florets of Chrysanthemum (Chrysanthemum morifolium).

Chrysanthemum is an important ornamental crop worldwide. Some white-flowered chrysanthemum cultivars produce red ray florets under natural cultivation conditions, but little is known about how this occurs. We compared the expression of anthocyanin biosynthetic and transcription factor genes between white ray florets and those that turned red based on cultivation conditions to comprehend the underlying mechanism. Significant differences in the expression of CmbHLH2 were detected between the florets of different colors. CmbHLH2 generated two alternatively spliced transcripts, designated CmbHLH2Full and CmbHLH2Short . Compared with CmbHLH2Full , CmbHLH2Short encoded a truncated protein with only a partial MYB-interaction region and no other domains normally present in the full-length protein. Unlike the full-length form, the splicing variant protein CmbHLH2Short localized to the cytoplasm and the nucleus and could not interact with CmMYB6. Additionally, CmbHLH2Short failed to activate anthocyanin biosynthetic genes and induce pigment accumulation in transiently transfected tobacco leaves, whereas CmbHLH2Full promoted both processes when simultaneously expressed with CmMYB6. Co-expressing CmbHLH2Full and CmMYB6 also enhanced the promoter activities of CmCHS and CmDFR. Notably, the Arabidopsis tt8-1 mutant, which lacks red pigmentation in the leaves and seeds, could be complemented by the heterologous expression of CmbHLH2Full, which restored red pigmentation and resulted in red pigmentation in high anthocyanin and proanthocyanidin contents in the leaves and seeds, respectively, whereas expression of CmbHLH2Short did not. Together, these results indicate that CmbHLH2 and CmMYB6 interaction plays a key role in the anthocyanin pigmentation changes of ray florets in chrysanthemum. Our findings highlight alternative splicing as a potential approach to modulate anthocyanin biosynthesis in specific tissues.

Reconstitution of Cytokinin Signaling in Rice Protoplasts.

The major components of the cytokinin (CK) signaling pathway have been identified from the receptors to their downstream transcription factors. However, since signaling proteins are encoded by multigene families, characterizing and quantifying the contribution of each component or their combinations to the signaling cascade have been challenging. Here, we describe a transient gene expression system in rice (Oryza sativa) protoplasts suitable to reconstitute CK signaling branches using the CK reporter construct TCSn:fLUC, consisting of a synthetic CK-responsive promoter and the firefly luciferase gene, as a sensitive readout of signaling output. We used this system to systematically test the contributions of CK signaling components, either alone or in various combinations, with or without CK treatment. The type-B response regulators (RRs) OsRR16, OsRR17, OsRR18, and OsRR19 all activated TCSn:fLUC strongly, with OsRR18 and OsRR19 showing the strongest induction by CK. Cotransfecting the reporter with OsHP01, OsHP02, OsHP05, or OsHK03 alone resulted in much weaker effects relative to those of the type-B OsRRs. When we tested combinations of OsHK03, OsHPs, and OsRRs, each combination exhibited distinct CK signaling activities. This system thus allows the rapid and high-throughput exploration of CK signaling in rice.

High-throughput analysis of high-molecular weight glutenin subunits in 665 wheat genotypes using an optimized MALDI-TOF-MS method.

Gluten protein composition determines the rheological characteristics of wheat dough and is influenced by variable alleles with distinct effects on processing properties. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), we determined the high-molecular weight glutenin subunit (HMW-GS) composition of 665 wheat genotypes employed in breeding programs in South Korea. We identified 22 HMW-GS alleles, including 3 corresponding to the Glu-A1 locus, 14 to Glu-B1, and 5 to Glu-D1. The Glu-1 quality score, which is an important criterion for high-quality wheat development, was found to be 10 for 105/665 (15.79%) of the studied genotypes, and included the following combinations of HMW-GS: 2*, 7 + 8, 5 + 10; 2*, 17 + 18, 5 + 10; 1, 7 + 8, 5 + 10; and 1, 17 + 18, 5 + 10. To select wheat lines with the 1Bx7 overexpression (1Bx7OE) subunit, which is known to have a positive effect on wheat quality, we used a combination of MALDI-TOF-MS and published genotyping markers and identified 6 lines carrying 1Bx7OE out of the 217 showing a molecular weight of 83,400 Da, consistent with 1Bx7G2 and 1Bx7OE. This study demonstrates that the MALDI-TOF-MS method is fast, accurate, reliable, and effective in analyzing large numbers of wheat germplasms or breeding lines in a high-throughput manner. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-020-02637-z.