Viable circadian clocks help organisms to synchronize their development with daily and seasonal changes, thereby providing both evolutionary fitness and advantage from an agricultural perspective. A ...high-resolution mapping approach combined with mutant analysis revealed a cereal ortholog of Arabidopsis thaliana LUX ARRHYTHMO/PHYTOCLOCK 1 (LUX/PCL1) as a promising candidate for the earliness per se 3 (Eps-3A(m)) locus in einkorn wheat (Triticum monococcum L.). Using delayed fluorescence measurements it was shown that Eps-3A(m) containing einkorn wheat accession KT3-5 had a distorted circadian clock. The hypothesis was subsequently confirmed by performing a time course study on central and output circadian clock genes, which showed arrhythmic transcript patterns in KT3-5 under constant ambient conditions, i.e., constant light and temperature. It was also demonstrated that variation in spikelet number between wild-type and mutants is sensitive to temperature, becoming negligible at 25°. These observations lead us to propose that the distorted clock is causative for both early flowering and variation in spike size and spikelet number, and that having a dysfunctional LUX could have neutral, or even positive, effects in warmer climates. To test the latter hypothesis we ascertained sequence variation of LUX in a range of wheat germplasm. We observed a higher variation in the LUX sequence among accessions coming from the warmer climate and a unique in-frame mutation in early-flowering Chinese T. turgidum cultivar 'Tsing Hua no. 559.' Our results emphasize the importance of the circadian clock in temperate cereals as a promising target for adaptation to new environments.
Powdery mildew fungi are obligate biotrophic pathogens that only grow on living hosts and cause damage in thousands of plant species. Despite their agronomical importance, little direct functional ...evidence for genes of pathogenicity and virulence is currently available because mutagenesis and transformation protocols are lacking. Here, we show that the accumulation in barley (Hordeum vulgare) and wheat (Triticum aestivum) of double-stranded or antisense RNA targeting fungal transcripts affects the development of the powdery mildew fungus Blumeria graminis. Proof of concept for host-induced gene silencing was obtained by silencing the effector gene Avra10, which resulted in reduced fungal development in the absence, but not in the presence, of the matching resistance gene Mla10. The fungus could be rescued from the silencing of Avra10 by the transient expression of a synthetic gene that was resistant to RNA interference (RNAi) due to silent point mutations. The results suggest traffic of RNA molecules from host plants into B. graminis and may lead to an RNAi-based crop protection strategy against fungal pathogens.
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has contributed to the improvement of various agronomic traits in cereal crops, ...including plant stature, inflorescence architecture, grain yield, grain chemical content, seed shattering, seed dormancy, and tolerance to environmental stresses.New advancements in CRISPR/Cas technology have focused on delivering preassembled editing systems, enabling multiplex editing, editing of miRNAs and E3 ligases, and utilizing artificial intelligence-based tools specifically tailored for cereals.The combination of CRISPR/Cas technology with innovative speed-breeding strategies holds promise for the efficient and timely development of new cereal germplasm.
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has become the most important tool for targeted genome editing in many plant and animal species over the past decade. The CRISPR/Cas9 technology has also sparked a flood of applications and technical advancements in genome editing in the key cereal crops, including rice, wheat, maize, and barley. Here, we review advanced uses of CRISPR/Cas9 and derived systems in genome editing of cereal crops to enhance a variety of agronomically important features. We also highlight new technological advances for delivering preassembled Cas9-gRNA ribonucleoprotein (RNP)-editing systems, multiplex editing, gain-of-function strategies, the use of artificial intelligence (AI)-based tools, and combining CRISPR with novel speed breeding (SB) and vernalization strategies.
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has become the most important tool for targeted genome editing in many plant and animal species over the past decade. The CRISPR/Cas9 technology has also sparked a flood of applications and technical advancements in genome editing in the key cereal crops, including rice, wheat, maize, and barley. Here, we review advanced uses of CRISPR/Cas9 and derived systems in genome editing of cereal crops to enhance a variety of agronomically important features. We also highlight new technological advances for delivering preassembled Cas9-gRNA ribonucleoprotein (RNP)-editing systems, multiplex editing, gain-of-function strategies, the use of artificial intelligence (AI)-based tools, and combining CRISPR with novel speed breeding (SB) and vernalization strategies.
Main conclusion
Genome editing offers revolutionized solutions for plant breeding to sustain food production to feed the world by 2050. Therefore, genome-edited products are increasingly recognized ...via more relaxed legislation and community adoption.
The world population and food production are disproportionally growing in a manner that would have never matched each other under the current agricultural practices. The emerging crisis is more evident with the subtle changes in climate and the running-off of natural genetic resources that could be easily used in breeding in conventional ways. Under these circumstances, affordable CRISPR-Cas-based gene-editing technologies have brought hope and charged the old plant breeding machine with the most energetic and powerful fuel to address the challenges involved in feeding the world. What makes CRISPR-Cas the most powerful gene-editing technology? What are the differences between it and the other genetic engineering/breeding techniques? Would its products be labeled as "conventional" or "GMO"? There are so many questions to be answered, or that cannot be answered within the limitations of our current understanding. Therefore, we would like to discuss and answer some of the mentioned questions regarding recent progress in technology development. We hope this review will offer another view on the role of CRISPR-Cas technology in future of plant breeding for food production and beyond.
WHIRLIES are plant-specific proteins binding to DNA in plastids, mitochondria, and nucleus. They have been identified as significant components of nucleoids in the organelles where they regulate the ...structure of the nucleoids and diverse DNA-associated processes. WHIRLIES also fulfil roles in the nucleus by interacting with telomers and various transcription factors, among them members of the WRKY family. While most plants have two WHIRLY proteins, additional WHIRLY proteins evolved by gene duplication in some dicot families. All WHIRLY proteins share a conserved WHIRLY domain responsible for ssDNA binding. Structural analyses revealed that WHIRLY proteins form tetramers and higher-order complexes upon binding to DNA. An outstanding feature is the parallel localization of WHIRLY proteins in two or three cell compartments. Because they translocate from organelles to the nucleus, WHIRLY proteins are excellent candidates for transducing signals between organelles and nucleus to allow for coordinated activities of the different genomes. Developmental cues and environmental factors control the expression of WHIRLY genes. Mutants and plants with a reduced abundance of WHIRLY proteins gave insight into their multiple functionalities. In chloroplasts, a reduction of the WHIRLY level leads to changes in replication, transcription, RNA processing, and DNA repair. Furthermore, chloroplast development, ribosome formation, and photosynthesis are impaired in monocots. In mitochondria, a low level of WHIRLIES coincides with a reduced number of cristae and a low rate of respiration. The WHIRLY proteins are involved in the plants' resistance toward abiotic and biotic stress. Plants with low levels of WHIRLIES show reduced responsiveness toward diverse environmental factors, such as light and drought. Consequently, because such plants are impaired in acclimation, they accumulate reactive oxygen species under stress conditions. In contrast, several plant species overexpressing WHIRLIES were shown to have a higher resistance toward stress and pathogen attacks. By their multiple interactions with organelle proteins and nuclear transcription factors maybe a comma can be inserted here? and their participation in organelle-nucleus communication, WHIRLY proteins are proposed to serve plant development and stress resistance by coordinating processes at different levels. It is proposed that the multifunctionality of WHIRLY proteins is linked to the plasticity of land plants that develop and function in a continuously changing environment.
Abstract Establishment of final leaf size in plants relies on the precise regulation of 2 interconnected processes, cell division and cell expansion. The barley (Hordeum vulgare) protein BROAD LEAF1 ...(BLF1) limits cell proliferation and leaf growth in the width direction. However, how the levels of this potent repressor of leaf growth are controlled remains unclear. Here, we used a yeast 2-hybrid screen to identify the BLF1-INTERACTING RING/U-BOX 1 (BIR1) E3 ubiquitin ligase that interacts with BLF1 and confirmed the interaction of the 2 proteins in planta. Inhibiting the proteasome caused overaccumulation of a BLF1-eGFP fusion protein when co-expressed with BIR1, and an in vivo ubiquitination assay in bacteria confirmed that BIR1 can mediate ubiquitination of BLF1 protein. Consistent with regulation of endogenous BLF1 in barley by proteasomal degradation, inhibition of the proteasome in BLF1-vYFP–expressing barley plants caused an accumulation of the BLF1 protein. The BIR1 protein co-localized with BLF1 in nuclei and appeared to reduce BLF1 protein levels. Analysis of bir1-1 knockout mutants suggested the involvement of BIR1 in leaf growth control, although mainly on leaf length. Together, our results suggest that proteasomal degradation, in part mediated by BIR1, helps fine-tune BLF1 protein levels in barley.
Methods of the Agrobacterium-mediated transformation of bread wheat (Triticum aestivum L.) have been improved in recent years so that genetic engineering can be routinely used for functional genomics ...as well as for wheat breeding. In the protocol described here, immature embryos of the spring-type model genotype Bobwhite SH 98 26 have been used. Preculture and temperature pretreatment of these explants have led to the reproducible generation of transgenic plants at efficiencies between 5 and 15%. Whereas primary transgenic plants regenerated in vitro commonly show reduced fitness and fertility, no apparent variations with regard to morphology and grain set in their transgenic progeny as compared to wild-type counterparts were observed.
Vascular patterning is intimately related to plant form and function. Here, using barley (Hordeum vulgare) as a model, we studied the vascular anatomy of the spike-type inflorescence. The main aim of ...the present work was to clarify the relationship between rachis (spike axis) vasculature and spike size, to define vascular dynamics and to discuss the implications for transport capacity and its interaction with the spikelets.
We used serial transverse internode sections to determine the internode area, vascular area and number of veins along the rachis of several barley lines.
Internode area and total vascular area show a clear positive correlation with spike size, whereas the number of veins is only weakly correlated. The lateral periphery of the rachis contains large mature veins of constant size, whereas the central part is occupied by small immature veins. Spikelet-derived veins entering the rachis often merge with the immature rachis veins but never merge with the mature veins. An increase in floret fertility through the conversion of a two-rowed barley into an isogenic six-rowed line, in addition to a decrease in floret fertility owing to enhanced pre-anthesis tip degeneration caused by the mutation tip sterile 2.b (tst2.b), significantly affected vein size but had limited to no effects on the number of veins or internode area.
The rachis vasculature is the result of a two-step process involving an initial layout followed by size adjustment according to floret fertility/spike size. The restriction of large mature vessels to the periphery and that of small immature vessels to the centre of the rachis suggests that long-distance transport and local supply to spikelets are spatially separated processes. The identification of spikelet-derived veins entering the rachis without fusing with its vasculature indicates that a vascular continuity between rachis and spikelets might be non-essential.
SUMMARY
Cereal endosperm represents the most important source of the world’s food; nevertheless, the molecular mechanisms underlying cell and tissue differentiation in cereal grains remain poorly ...understood. Endosperm cellularization commences at the maternal–filial intersection of grains and generates endosperm transfer cells (ETCs), a cell type with a prominent anatomy optimized for efficient nutrient transport. Barley HISTIDINE KINASE1 (HvHK1) was identified as a receptor component with spatially restricted expression in the syncytial endosperm where ETCs emerge. Here, we demonstrate its function in ETC fate acquisition using RNA interference‐mediated downregulation of HvHK1. Repression of HvHK1 impairs cell specification in the central ETC region and the development of transfer cell morphology, and consecutively defects differentiation of adjacent endosperm tissues. Coinciding with reduced expression of HvHK1, disturbed cell plate formation and fusion were observed at the initiation of endosperm cellularization, revealing that HvHK1 triggers initial cytokinesis of ETCs. Cell‐type‐specific RNA sequencing confirmed loss of transfer cell identity, compromised cell wall biogenesis and reduced transport capacities in aberrant cells and elucidated two‐component signaling and hormone pathways that are mediated by HvHK1. Gene regulatory network modeling was used to specify the direct targets of HvHK1; this predicted non‐canonical auxin signaling elements as the main regulatory links governing cellularization of ETCs, potentially through interaction with type‐B response regulators. This work provides clues to previously unknown molecular mechanisms directing ETC specification, a process with fundamental impact on grain yield in cereals.
Significance Statement
Although endosperm transfer cells (ETCs) have a substantial impact on grain yield in cereals, the molecular basis and regulatory networks triggering differentiation of ETCs remain poorly understood. This work demonstrates that barley HISTIDINE KINASE1 directs ETC specification in the young endosperm by orchestrating distinct phosphorelay and hormone signaling modules.
The secreted fungal effector Pep1 is essential for penetration of the host epidermis and establishment of biotrophy in the Ustilago maydis–maize pathosystem. Previously, Pep1 was found to be an ...inhibitor of apoplastic plant peroxidases, which suppresses the oxidative burst, a primary immune response of the host plant and enables fungal colonization. To investigate the conservation of Pep1 in other pathogens, genomes of related smut species were screened for pep1 orthologues. Pep1 proteins were produced in Escherichia coli for functional assays. The biological function of Pep1 was tested by heterologous expression in U. maydis and Hordeum vulgare. Pep1 orthologues revealed a remarkable degree of sequence conservation, indicating that this effector might play a fundamental role in virulence of biotrophic smut fungi. Pep1 function and its role in virulence are conserved in different pathogenic fungi, even across the monocot–dicot border of host plants. The findings described in this study classify Pep1 as a phylogenetically conserved fungal core effector. Furthermore, we documented the influence of Pep1 on the disease caused by Blumeria graminis f. sp. hordei which is a non‐smut‐related pathosystem.
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