Abscisic acid inhibits germination of Striga seeds and is released by them likely as a rhizospheric signal supporting host infestation.

Seeds of the root parasitic plant Striga hermonthica undergo a conditioning process under humid and warm environments before germinating in response to host-released stimulants, particularly strigolactones (SLs). The plant hormone abscisic acid (ABA) regulates different growth and developmental processes, and stress response; however, its role during Striga seed germination and early interactions with host plants is under-investigated. Here, we show that ABA inhibited Striga seed germination and that hindering its biosynthesis induced conditioning and germination in unconditioned seeds, which was significantly enhanced by treatment with the SL analog rac-GR24. However, the inhibitory effect of ABA remarkably decreased during conditioning, confirming the loss of sensitivity towards ABA in later developmental stages. ABA measurement showed a substantial reduction of its content during the early conditioning stage and a significant increase upon rac-GR24-triggered germination. We observed this increase also in released seed exudates, which was further confirmed by using the Arabidopsis ABA-reporter GUS marker line. Seed exudates of germinated seeds, containing elevated levels of ABA, impaired the germination of surrounding Striga seeds in vitro and promoted root growth of a rice host towards germinated Striga seeds. Application of ABA as a positive control caused similar effects, indicating its function in Striga/Striga and Striga/host communications. In summary, we show that ABA is an essential player during seed dormancy and germination processes in Striga and acts as a rhizospheric signal likely to support host infestation.

Zaxinone Synthase overexpression modulates rice physiology and metabolism, enhancing nutrient uptake, growth and productivity.

The rice Zaxinone Synthase (ZAS) gene encodes a carotenoid cleavage dioxygenase (CCD) that forms the apocarotenoid growth regulator zaxinone in vitro. Here, we generated and characterized constitutive ZAS-overexpressing rice lines, to better understand ZAS role in determining zaxinone content and regulating growth and architecture. ZAS overexpression enhanced endogenous zaxinone level, promoted root growth and increased the number of productive tillers, leading to about 30% higher grain yield per plant. Hormone analysis revealed a decrease in strigolactone (SL) content, which we confirmed by rescuing the high-tillering phenotype through application of a SL analogue. Metabolomics analysis revealed that ZAS overexpressing plants accumulate higher amounts of monosaccharide sugars, in line with transcriptome analysis. Moreover, transgenic plants showed higher carbon (C) assimilation rate and elevated root phosphate, nitrate and sulphate level, enhancing the tolerance towards low phosphate (Pi). Our study confirms ZAS as an important determinant of rice growth and architecture and shows that ZAS regulates hormone homoeostasis and a combination of physiological processes to promote growth and grain yield, which makes this gene an excellent candidate for sustainable crop improvement.

Chromosome-scale pearl millet genomes reveal CLAMT1b as key determinant of strigolactone pattern and Striga susceptibility.

The yield of pearl millet, a resilient cereal crop crucial for African food security, is severely impacted by the root parasitic weed Striga hermonthica, which requires host-released hormones, called strigolactones (SLs), for seed germination. Herein, we identify four SLs present in the Striga-susceptible line SOSAT-C88-P10 (P10) but absent in the resistant 29Aw (Aw). We generate chromosome-scale genome assemblies, including four gapless chromosomes for each line. The Striga-resistant Aw lacks a 0.7 Mb genome segment containing two putative CARLACTONOIC ACID METHYLTRANSFERASE1 (CLAMT1) genes, which may contribute to SL biosynthesis. Functional assays show that P10CLAMT1b produces the SL-biosynthesis intermediate methyl carlactonoate (MeCLA) and that MeCLA is the precursor of P10-specific SLs. Screening a diverse pearl millet panel confirms the pivotal role of the CLAMT1 section for SL diversity and Striga susceptibility. Our results reveal a reason for Striga susceptibility in pearl millet and pave the way for generating resistant lines through marker-assisted breeding or direct genetic modification.

A Fast and Cost-Effective Genotyping Method for CRISPR-Cas9-Generated Mutant Rice Lines.

With increasing throughput in both the generation and phenotyping of mutant lines in plants, it is important to have an efficient and reliable genotyping method. Traditional workflows, still commonly used in many labs, have time-consuming and expensive steps, such as DNA purification, cloning and growing E. coli cultures. We propose an alternative workflow where these steps are bypassed, using Phire polymerase on fresh plant tissue, and ExoProStar treatment as preparation for sequencing. We generated CRISPR-Cas9 mutants for ZAS (ZAXINONE SYNTHASE) in rice with two guide RNAs. Using both a traditional workflow and our proposed workflow, we genotyped nine T1 plants. To interpret the sequencing output, which is often complex in CRISPR-generated mutants, we used free online automatic analysis systems and compared the results. Our proposed workflow produces results of the same quality as the old workflow, but in 1 day instead of 3 days and about 35 times cheaper. This workflow also consists of fewer steps and reduces the risk of cross contamination and mistakes. Furthermore, the automated sequence analysis packages are mostly accurate and could easily be used for bulk analysis. Based on these advantages, we encourage academic and commercial labs conducting genotyping to consider switching over to our proposed workflow.

Installing the neurospora carotenoid pathway in plants enables cytosolic formation of provitamin A and its sequestration in lipid droplets.

Vitamin A deficiency remains a severe global health issue, which creates a need to biofortify crops with provitamin A carotenoids (PACs). Expanding plant cell capacity for synthesis and storing of PACs outside the plastids is a promising biofortification strategy that has been little explored. Here, we engineered PAC formation and sequestration in the cytosol of Nicotiana benthamiana leaves, Arabidopsis seeds, and citrus callus cells, using a fungal (Neurospora crassa) carotenoid pathway that consists of only three enzymes converting C5 isopentenyl building blocks formed from mevalonic acid into PACs, including ß-carotene. This strategy led to the accumulation of significant amounts of phytoene and γ- and ß-carotene, in addition to fungal, health-promoting carotenes with 13 conjugated double bonds, such as the PAC torulene, in the cytosol. Increasing the isopentenyl diphosphate pool by adding a truncated Arabidopsis hydroxymethylglutaryl-coenzyme A reductase substantially increased cytosolic carotene production. Engineered carotenes accumulate in cytosolic lipid droplets (CLDs), which represent a novel sequestering sink for storing these pigments in plant cytosol. Importantly, ß-carotene accumulated in the cytosol of citrus callus cells was more light stable compared to compared with plastidial ß-carotene. Moreover, engineering cytosolic carotene formation increased the number of large-sized CLDs and the levels of ß-apocarotenoids, including retinal, the aldehyde corresponding to vitamin A. Collectively, our study opens up the possibility of exploiting the high-flux mevalonic acid pathway for PAC biosynthesis and enhancing carotenoid sink capacity in green and non-green plant tissues, especially in lipid-storing seeds, and thus paves the way for further optimization of carotenoid biofortification in crops.

Carotenoid Biofortification of Crops in the CRISPR Era.

Carotenoids are micronutrients important for human health. The continuous improvements in clustered regularly interspaced short palindromic repeats (CRISPR)-based genome-editing techniques make rapid, DNA/transgene-free and targeted multiplex genetic modification a reality, thus promising to accelerate the breeding and generation of 'golden' staple crops. We discuss here the progress and future prospects of CRISPR/Cas9 applications for carotenoid biofortification.

Transcript Profiling of MIKCc MADS-Box Genes Reveals Conserved and Novel Roles in Barley Inflorescence Development.

MADS-box genes have a wide range of functions in plant reproductive development and grain production. The ABCDE model of floral organ development shows that MADS-box genes are central players in these events in dicotyledonous plants but the applicability of this model remains largely unknown in many grass crops. Here, we show that transcript analysis of all MIKCc MADS-box genes through barley (Hordeum vulgare L.) inflorescence development reveals co-expression groups that can be linked to developmental events. Thirty-four MIKCc MADS-box genes were identified in the barley genome and single-nucleotide polymorphism (SNP) scanning of 22,626 barley varieties revealed that the natural variation in the coding regions of these genes is low and the sequences have been extremely conserved during barley domestication. More detailed transcript analysis showed that MADS-box genes are generally expressed at key inflorescence developmental phases and across various floral organs in barley, as predicted by the ABCDE model. However, expression patterns of some MADS genes, for example HvMADS58 (AGAMOUS subfamily) and HvMADS34 (SEPALLATA subfamily), clearly deviate from predicted patterns. This places them outside the scope of the classical ABCDE model of floral development and demonstrates that the central tenet of antagonism between A- and C-class gene expression in the ABC model of other plants does not occur in barley. Co-expression across three correlation sets showed that specifically grouped members of the barley MIKCc MADS-box genes are likely to be involved in developmental events driving inflorescence meristem initiation, floral meristem identity and floral organ determination. Based on these observations, we propose a potential floral ABCDE working model in barley, where the classic model is generally upheld, but that also provides new insights into the role of MIKCc MADS-box genes in the developing barley inflorescence.

MADS1 maintains barley spike morphology at high ambient temperatures.

Temperature stresses affect plant phenotypic diversity. The developmental stability of the inflorescence, required for reproductive success, is tightly regulated by the interplay of genetic and environmental factors. However, the mechanisms underpinning how plant inflorescence architecture responds to temperature are largely unknown. We demonstrate that the barley SEPALLATA MADS-box protein HvMADS1 is responsible for maintaining an unbranched spike architecture at high temperatures, while the loss-of-function mutant forms a branched inflorescence-like structure. HvMADS1 exhibits increased binding to target promoters via A-tract CArG-box motifs, which change conformation with temperature. Target genes for high-temperature-dependent HvMADS1 activation are predominantly associated with inflorescence differentiation and phytohormone signalling. HvMADS1 directly regulates the cytokinin-degrading enzyme HvCKX3 to integrate temperature response and cytokinin homeostasis, which is required to repress meristem cell cycle/division. Our findings reveal a mechanism by which genetic factors direct plant thermomorphogenesis, extending the recognized role of plant MADS-box proteins in floral development.