Deciphering the Transcriptional Regulatory Network Governing Starch and Storage Protein Biosynthesis in Wheat for Breeding Improvement.

Starch and seed storage protein (SSP) composition profoundly impact wheat grain yield and quality. To unveil regulatory mechanisms governing their biosynthesis, transcriptome, and epigenome profiling is conducted across key endosperm developmental stages, revealing that chromatin accessibility, H3K27ac, and H3K27me3 collectively regulate SSP and starch genes with varying impact. Population transcriptome and phenotype analyses highlight accessible promoter regions' crucial role as a genetic variation resource, influencing grain yield and quality in a core collection of wheat accessions. Integration of time-serial RNA-seq and ATAC-seq enables the construction of a hierarchical transcriptional regulatory network governing starch and SSP biosynthesis, identifying 42 high-confidence novel candidates. These candidates exhibit overlap with genetic regions associated with grain size and quality traits, and their functional significance is validated through expression-phenotype association analysis among wheat accessions and loss-of-function mutants. Functional analysis of wheat abscisic acid insensitive 3-A1 (TaABI3-A1) with genome editing knock-out lines demonstrates its role in promoting SSP accumulation while repressing starch biosynthesis through transcriptional regulation. Excellent TaABI3-A1Hap1 with enhanced grain weight is selected during the breeding process in China, linked to altered expression levels. This study unveils key regulators, advancing understanding of SSP and starch biosynthesis regulation and contributing to breeding enhancement.

A k-mer-based pangenome approach for cataloging seed-storage-protein genes in wheat to facilitate genotype-to-phenotype prediction and improvement of end-use quality.

Wheat is a staple food for more than 35% of the world's population, with wheat flour used to make hundreds of baked goods. Superior end-use quality is a major breeding target; however, improving it is especially time-consuming and expensive. Furthermore, genes encoding seed-storage proteins (SSPs) form multi-gene families and are repetitive, with gaps commonplace in several genome assemblies. To overcome these barriers and efficiently identify superior wheat SSP alleles, we developed "PanSK" (Pan-SSP k-mer) for genotype-to-phenotype prediction based on an SSP-based pangenome resource. PanSK uses 29-mer sequences that represent each SSP gene at the pangenomic level to reveal untapped diversity across landraces and modern cultivars. Genome-wide association studies with k-mers identified 23 SSP genes associated with end-use quality that represent novel targets for improvement. We evaluated the effect of rye secalin genes on end-use quality and found that removal of ω-secalins from 1BL/1RS wheat translocation lines is associated with enhanced end-use quality. Finally, using machine-learning-based prediction inspired by PanSK, we predicted the quality phenotypes with high accuracy from genotypes alone. This study provides an effective approach for genome design based on SSP genes, enabling the breeding of wheat varieties with superior processing capabilities and improved end-use quality.

The maize transcription factor CCT regulates drought tolerance by interacting with Fra a 1, E3 ligase WIPF2, and auxin response factor Aux/IAA8.

Plants are commonly exposed to abiotic stressors, which can affect their growth, productivity, and quality. Previously, the maize transcription factor ZmCCT was shown to be involved in the photoperiod response, delayed flowering, and quantitative resistance to Gibberella stalk rot. In this study, we demonstrate that ZmCCT can regulate plant responses to drought. ZmCCT physically interacted with ZmFra a 1, ZmWIPF2, and ZmAux/IAA8, which localized to the cell membrane, cytoplasm, and nucleus, respectively, both in vitro and in vivo in a yeast two-hybrid screen in response to abiotic stress. Notably, ZmCCT recruits ZmWIPF2 to the nucleus, which has strong E3 self-ubiquitination activity dependent on its RING-H2 finger domain in vitro. When treated with higher indole-3-acetic acid/abscisic acid ratios, the height and root length of Y331-ΔTE maize plants increased. Y331-ΔTE plants exhibited increased responses to exogenously applied auxin or ABA compared to Y331 plants, indicating that ZmCCT may be a negative regulator of ABA signalling in maize. In vivo, ZmCCT promoted indole-3-acetic acid biosynthesis in ZmCCT-overexpressing Arabidopsis. RNA-sequencing and DNA affinity purification-sequencing analyses showed that ZmCCT can regulate the expression of ZmRD17, ZmAFP3, ZmPP2C, and ZmARR16 under drought. Our findings provide a detailed overview of the molecular mechanism controlling ZmCCT functions and highlight that ZmCCT has multiple roles in promoting abiotic stress tolerance.

Type I MADS-box transcription factor TaMADS-GS regulates grain size by stabilizing cytokinin signalling during endosperm cellularization in wheat.

Grain size is one of the important traits in wheat breeding programs aimed at improving yield, and cytokinins, mainly involved in cell division, have a positive impact on grain size. Here, we identified a novel wheat gene TaMADS-GS encoding type I MADS-box transcription factor, which regulates the cytokinins signalling pathway during early stages of grain development to modulate grain size and weight in wheat. TaMADS-GS is exclusively expressed in grains at early stage of seed development and its knockout leads to delayed endosperm cellularization, smaller grain size and lower grain weight. TaMADS-GS protein interacts with the Polycomb Repressive Complex 2 (PRC2) and leads to repression of genes encoding cytokinin oxidase/dehydrogenases (CKXs) stimulating cytokinins inactivation by mediating accumulation of the histone H3 trimethylation at lysine 27 (H3K27me3). Through the screening of a large wheat germplasm collection, an elite allele of the TaMADS-GS exhibits higher ability to repress expression of genes inactivating cytokinins and a positive correlation with grain size and weight, thus representing a novel marker for breeding programs in wheat. Overall, these findings support the relevance of TaMADS-GS as a key regulator of wheat grain size and weight.

Mutation of a highly conserved amino acid in RPM1 causes leaf yellowing and premature senescence in wheat.

KEY MESSAGE: A point mutation of RPM1 triggers persistent immune response that induces leaf premature senescence in wheat, providing novel information of immune responses and leaf senescence. Leaf premature senescence in wheat (Triticum aestivum L.) is one of the most common factors affecting the plant's development and yield. In this study, we identified a novel wheat mutant, yellow leaf and premature senescence (ylp), which exhibits yellow leaves and premature senescence at the heading and flowering stages. Consistent with the yellow leaves phenotype, ylp had damaged and collapsed chloroplasts. Map-based cloning revealed that the phenotype of ylp was caused by a point mutation from Arg to His at amino acid 790 in a plasma membrane-localized protein resistance to Pseudomonas syringae pv. maculicola 1 (RPM1). The point mutation triggered excessive immune responses and the upregulation of senescence- and autophagy-associated genes. This work provided the information for understanding the molecular regulatory mechanism of leaf senescence, and the results would be important to analyze which mutations of RPM1 could enable plants to obtain immune activation without negative effects on plant growth.

A comparison of micro-flocculation and ozonation as pretreatments for ultrafiltration: organic removal and membrane fouling.

Micro-flocculation and ozone were applied as pretreatments of ultrafiltration to treat sodium alginate (SA) and humic acid (HA) simulated water, respectively, to investigate the effects of different pretreatments of ultrafiltration (UF) on filtration flux and removal of organic matters. Regarding the SA simulated water, micro-flocculation helped to improve the dissolved organic carbon (DOC) removal efficiency highly, maximum DOC removal efficiency reached to 79.77%, due to the rejection of gel layer introduced by the alginate-aluminum complexes, but the gel layer had a negative impact on membrane flux. Compared with micro-flocculation, ozone as pretreatments had better ability to enhance the membrane specific flux, the maximum final specific flux remained as 0.786, larger than that of MF-UF process (0.574). Ozonation oxidizing SA into small organic molecules significantly reduced membrane fouling and filtration resistance, but also produced some dissolved organic matters hindering DOC removal of effluent. As for HA simulated water, both the micro-flocculation and ozone could effectively improve the specific flux, the final specific flux of MF-UF and ozone-UF were about 0.930, but MF-UF exhibited better DOC removal than ozone-UF, which avoided the introduction of additional dissolved organic matters.

Thermosensitive SUMOylation of TaHsfA1 defines a dynamic ON/OFF molecular switch for the heat stress response in wheat.

Dissecting genetic components in crop plants associated with heat stress (HS) sensing and adaptation will facilitate the design of modern crop varieties with improved thermotolerance. However, the molecular mechanisms underlying the ON/OFF switch controlling HS responses (HSRs) in wheat (Triticum aestivum) remain largely unknown. In this study, we focused on the molecular action of TaHsfA1, a class A heat shock transcription factor, in sensing dynamically changing HS signals and regulating HSRs. We show that the TaHsfA1 protein is modified by small ubiquitin-related modifier (SUMO) and that this modification is essential for the full transcriptional activation activity of TaHsfA1 in triggering downstream gene expression. During sustained heat exposure, the SUMOylation of TaHsfA1 is suppressed, which partially reduces TaHsfA1 protein activity, thereby reducing the intensity of downstream HSRs. In addition, we demonstrate that TaHsfA1 interacts with the histone acetyltransferase TaHAG1 in a thermosensitive manner. Together, our findings emphasize the importance of TaHsfA1 in thermotolerance in wheat. In addition, they define a highly dynamic SUMOylation-dependent "ON/OFF" molecular switch that senses temperature signals and contributes to thermotolerance in crops.

Reducing brassinosteroid signalling enhances grain yield in semi-dwarf wheat.

Modern green revolution varieties of wheat (Triticum aestivum L.) confer semi-dwarf and lodging-resistant plant architecture owing to the Reduced height-B1b (Rht-B1b) and Rht-D1b alleles1. However, both Rht-B1b and Rht-D1b are gain-of-function mutant alleles encoding gibberellin signalling repressors that stably repress plant growth and negatively affect nitrogen-use efficiency and grain filling2-5. Therefore, the green revolution varieties of wheat harbouring Rht-B1b or Rht-D1b usually produce smaller grain and require higher nitrogen fertilizer inputs to maintain their grain yields. Here we describe a strategy to design semi-dwarf wheat varieties without the need for Rht-B1b or Rht-D1b alleles. We discovered that absence of Rht-B1 and ZnF-B (encoding a RING-type E3 ligase) through a natural deletion of a haploblock of about 500 kilobases shaped semi-dwarf plants with more compact plant architecture and substantially improved grain yield (up to 15.2%) in field trials. Further genetic analysis confirmed that the deletion of ZnF-B induced the semi-dwarf trait in the absence of the Rht-B1b and Rht-D1b alleles through attenuating brassinosteroid (BR) perception. ZnF acts as a BR signalling activator to facilitate proteasomal destruction of the BR signalling repressor BRI1 kinase inhibitor 1 (TaBKI1), and loss of ZnF stabilizes TaBKI1 to block BR signalling transduction. Our findings not only identified a pivotal BR signalling modulator but also provided a creative strategy to design high-yield semi-dwarf wheat varieties by manipulating the BR signal pathway to sustain wheat production.

An elite γ-gliadin allele improves end-use quality in wheat.

Gluten is composed of glutenins and gliadins and determines the viscoelastic properties of dough and end-use quality in wheat (Triticum aestivum L.). Gliadins are important for wheat end-use traits, but the contribution of individual gliadin genes is unclear, since gliadins are encoded by a complex, multigenic family, including many pseudogenes. We used CRISPR/Cas9-mediated gene editing and map-based cloning to investigate the contribution of the γ-gliadin genes annotated in the wheat cultivar 'Fielder', showing that Gli-γ1-1D and Gli-γ2-1B account for most of the γ-gliadin accumulation. The impaired activity of only two γ-gliadin genes in knockout mutants improved end-use quality and reduced gluten epitopes associated with celiac disease (CD). Furthermore, we identified an elite haplotype of Gli-γ1-1D linked to higher end-use quality in a wheat germplasm collection and developed a molecular marker for this allele for marker-assisted selection. Our findings provide information and tools for biotechnology-based and classical breeding programs aimed at improving wheat end-use quality.

Fault detection and analysis for wheelset bearings via improved explicit shift-invariant dictionary learning.

The wheelset bearing is an indispensable part of the high-speed train, and monitoring its service performance is a concern of many researchers. Effective extraction of those impulse signals induced by the defects on the bearing elements is the key to fault detection and behaviour analysis. However, the presence of considerable noise and irrelevant components brings difficulties to extracting the wheelset bearing fault impulse signals from the measured vibration signals. This paper proposes an improved explicit shift-invariant dictionary learning (IE-SIDL) method to address this issue. Based on the shift-invariant characteristics of the wheelset bearing fault impulse signal in the time-domain, the circulant matrix is used to construct a shift-invariant dictionary and explicitly characterize the fault impulses at any time. To improve the efficiency of dictionary learning, a method of three flips is introduced to realize fast dictionary construction, and the frequency-domain reconstruction property of the circulant matrix is employed to quickly update the dictionary. Besides, an indicator-guided subspace pursuit (SP) method based on the sparsity of envelope spectrum (SES) is adopted for the sparse coding to improve sparse solution accuracy and adaptation. The effectiveness of the IE-SIDL method is proved through the simulated and experimental signals. The results demonstrate that the improved dictionary learning method has an excellent capacity in extracting fault impulse signal of the wheelset bearings, and the good time- and frequency-domain characteristics of the processed signals facilitate fault detection and behaviour analysis.

Multi-Omics Analysis Reveals a Regulatory Network of ZmCCT During Maize Resistance to Gibberella Stalk Rot at the Early Stage.

Gibberella stalk rot (GSR) caused by Fusarium graminearum is one of the most devastating diseases in maize; however, the regulatory mechanism of resistance to GSR remains largely unknown. We performed a comparative multi-omics analysis to reveal the early-stage resistance of maize to GSR. We inoculated F. graminearum to the roots of susceptible (Y331) and resistant (Y331-ΔTE) near-isogenic lines containing GSR-resistant gene ZmCCT for multi-omics analysis. Transcriptome detected a rapid reaction that confers resistance at 1-3 hpi as pattern-triggered immunity (PTI) response to GSR. Many key properties were involved in GSR resistance, including genes in photoperiod and hormone pathways of salicylic acid and auxin. The activation of programmed cell death-related genes and a number of metabolic pathways at 6 hpi might be important to prevent further colonization. This is consistent with an integrative analysis of transcriptomics and proteomics that resistant-mediated gene expression reprogramming exhibited a dynamic pattern from 3 to 6 hpi. Further metabolomics analysis revealed that the amount of many chemical compounds was altered in pathways associated with the phenylpropanoid biosynthesis and the phenylalanine metabolism, which may play key roles to confer the GSR resistance. Taken together, we generated a valuable resource to interpret the defense mechanism during early GSR resistance.

Unprocessed wheat γ-gliadin reduces gluten accumulation associated with the endoplasmic reticulum stress and elevated cell death.

Along with increasing demands for high yield, elite processing quality and improved nutrient value in wheat, concerns have emerged around the effects of gluten in wheat-based foods on human health. However, knowledge of the mechanisms regulating gluten accumulation remains largely unexplored. Here we report the identification and characterization of a wheat low gluten protein 1 (lgp1) mutant that shows extremely low levels of gliadins and glutenins. The lgp1 mutation in a single γ-gliadin gene causes defective signal peptide cleavage, resulting in the accumulation of an excessive amount of unprocessed γ-gliadin and a reduced level of gluten, which alters the endoplasmic reticulum (ER) structure, forms the autophagosome-like structures, leads to the delivery of seed storage proteins to the extracellular space and causes a reduction in starch biosynthesis. Physiologically, these effects trigger ER stress and cell death. This study unravels a unique mechanism that unprocessed γ-gliadin reduces gluten accumulation associated with ER stress and elevated cell death in wheat. Moreover, the reduced gluten level in the lgp1 mutant makes it a good candidate for specific diets for patients with diabetes or kidney diease.

Ectopic expression of VRT-A2 underlies the origin of Triticum polonicum and Triticum petropavlovskyi with long outer glumes and grains.

Polish wheat (Triticum polonicum) is a unique tetraploid wheat species characterized by an elongated outer glume. The genetic control of the long-glume trait by a single semi-dominant locus, P1 (from Polish wheat), was established more than 100 years ago, but the underlying causal gene and molecular nature remain elusive. Here, we report the isolation of VRT-A2, encoding an SVP-clade MADS-box transcription factor, as the P1 candidate gene. Genetic evidence suggests that in T. polonicum, a naturally occurring sequence rearrangement in the intron-1 region of VRT-A2 leads to ectopic expression of VRT-A2 in floral organs where the long-glume phenotype appears. Interestingly, we found that the intron-1 region is a key ON/OFF molecular switch for VRT-A2 expression, not only because it recruits transcriptional repressors, but also because it confers intron-mediated transcriptional enhancement. Genotypic analyses using wheat accessions indicated that the P1 locus is likely derived from a single natural mutation in tetraploid wheat, which was subsequently inherited by hexaploid T. petropavlovskyi. Taken together, our findings highlight the promoter-proximal intron variation as a molecular basis for phenotypic differentiation, and thus species formation in Triticum plants.

FRIZZY PANICLE defines a regulatory hub for simultaneously controlling spikelet formation and awn elongation in bread wheat.

Grain yield in bread wheat (Triticum aestivum L.) is largely determined by inflorescence architecture. Zang734 is an endemic Tibetan wheat variety that exhibits a rare triple spikelet (TRS) phenotype with significantly increased spikelet/floret number per spike. However, the molecular basis underlying this specific spike morphology is completely unknown. Through map-based cloning, the causal genes for TRS trait in Zang734 were isolated. Furthermore, using CRISPR/Cas9-based gene mutation, transcriptome sequencing and protein-protein interaction, the downstream signalling networks related to spikelet formation and awn elongation were defined. Results showed that the null mutation in WFZP-A together with deletion of WFZP-D led to the TRS trait in Zang734. More interestingly, WFZP plays a dual role in simultaneously repressing spikelet formation gene TaBA1 and activating awn development genes, basically through the recruitments of chromatin remodelling elements and the Mediator complex. Our findings provide insights into the molecular bases by which WFZP suppresses spikelet formation but promotes awn elongation and, more importantly, define WFZP-D as a favourable gene for high-yield crop breeding.

Size- and pathotropism-driven targeting and washout-resistant effects of boronic acid-rich protein nanoparticles for liver cancer regression.

40nm-sized boronic acid-rich protein nanoparticles composed of bovine serum albumin and poly(N-3-acrylamidophenylboronic acid) were prepared by polymerizing N-3-acrylamidophenylboronic acid in the presence of albumin. The content of boronic acid-containing poly(N-3-acrylamidophenylboronic acid) in the nanoparticles can be tuned from 80% to 32% at constant nanoparticle size. When used to deliver doxorubicin in vivo, such sized nanoparticles show dominantly liver-targeting, and significant washout-resistant ability compared to those boronic acid-absent nanoparticles due to the interaction between sialic acid residues in the liver and boronic acid groups in the nanoparticles. The sialic acid overexpression on hepatic H22 tumor cells is demonstrated to be much higher than that on hepatocytes, resulting in the preferential accumulation of boronic acid-rich nanoparticles in liver cancer cells. In vivo antitumor examination in orthotopic liver cancer model shows that these doxorubicin-loaded nanoparticles not only have significantly superior ability in impeding tumor growth, but also induce distinct tumor regression with no hepatic and cardiac toxicities.