The HD-ZIP class I transcription factor, HvHOX1 (Homeobox 1) or VRS1 (Vulgare Row-type Spike 1 or Six-rowed Spike 1), regulates lateral spikelet fertility in barley (Hordeum vulgare L.). It was shown that HvHOX1 has a high expression only in lateral spikelets, while its paralog HvHOX2 was found to be expressed in different plant organs. Yet, the mechanistic function of HvHOX1 and HvHOX2 during spikelet development is still fragmentary. Here, we show that compared to HvHOX1, HvHOX2 is more highly conserved across different barley genotypes and Hordeum species, hinting at a possibly vital but still unclarified biological role. Using bimolecular fluorescence complementation, DNA-binding, and transactivation assays, we validate that HvHOX1 and HvHOX2 are bona fide transcriptional activators that may potentially heterodimerize. Accordingly, both genes exhibit similar spatiotemporal expression patterns during spike development and growth, albeit their mRNA levels differ quantitatively. We show that HvHOX1 delays the lateral spikelet meristem differentiation and affects fertility by aborting the reproductive organs. Interestingly, the ancestral relationship of these genes inferred from their co-expressed gene networks suggested that HvHOX1 and HvHOX2 might play a similar role during barley spikelet development. However, CRISPR-derived mutants of HvHOX1 and HvHOX2 demonstrated the suppressive role of HvHOX1 on lateral spikelets, while the loss of HvHOX2 does not influence spikelet development. Collectively, our study shows that through the suppression of reproductive organs, lateral spikelet fertility is regulated by HvHOX1, whereas HvHOX2 is dispensable for spikelet development in barley.

Plant phytohormone pathways are regulated by an intricate network of signaling components and modulators, many of which still remain unknown. Here, we report a forward chemical genetics approach for the identification of functional SA agonists in Arabidopsis thaliana that revealed Neratinib (Ner), a covalent pan-HER kinase inhibitor drug in humans, as a modulator of SA signaling. Instead of a protein kinase, chemoproteomics unveiled that Ner covalently modifies a surface-exposed cysteine residue of Arabidopsis epoxide hydrolase isoform 7 (AtEH7), thereby triggering its allosteric inhibition. Physiologically, the Ner application induces jasmonate metabolism in an AtEH7-dependent manner as an early response. In addition, it modulates PATHOGENESIS RELATED 1 (PR1) expression as a hallmark of SA signaling activation as a later effect. AtEH7, however, is not the exclusive target for this physiological readout induced by Ner. Although the underlying molecular mechanisms of AtEH7-dependent modulation of jasmonate signaling and Ner-induced PR1-dependent activation of SA signaling and thus defense response regulation remain unknown, our present work illustrates the powerful combination of forward chemical genetics and chemical proteomics for identifying novel phytohormone signaling modulatory factors. It also suggests that marginally explored metabolic enzymes such as epoxide hydrolases may have further physiological roles in modulating signaling.

Enzyme-based synthetic chemistry provides a green way to synthesize industrially important chemical scaffolds and provides incomparable substrate specificity and unmatched stereo-, regio-, and chemoselective product formation. However, using biocatalysts at an industrial scale has its challenges, like their narrow substrate scope, limited stability in large-scale one-pot reactions, and low expression levels. These limitations can be overcome by engineering and fine-tuning these biocatalysts using advanced protein engineering methods. A detailed understanding of the enzyme structure and catalytic mechanism and its structure–function relationship, cooperativity in binding of substrates, and dynamics of substrate–enzyme–cofactor complexes is essential for rational enzyme engineering for a specific purpose. This Review covers all these aspects along with an in-depth categorization of various industrially and pharmaceutically crucial bisubstrate enzymes based on their reaction mechanisms and their active site and substrate/cofactor-binding site structures. As the bisubstrate enzymes constitute around 60% of the known industrially important enzymes, studying their mechanism of actions and structure–activity relationship gives significant insight into deciding the targets for protein engineering for developing industrial biocatalysts. Thus, this Review is focused on providing a comprehensive knowledge of the bisubstrate enzymes’ structure, their mechanisms, and protein engineering approaches to develop them into industrial biocatalysts.

Abstract The comparison of transcriptome time-courses of the first 2 h of the cold or highlight response of 24 h cold primed and naive Arabidopsis thaliana showed that priming quickly modifies gene expression in a trigger-specific manner. It dampened up- as well as down-regulation of genes in the cold and in the light. 1/3 of the priming-regulated genes were jasmonate sensitive, including the full set of genes required for oxylipin biosynthesis. qPCR-based analysis in wildtype plants and mutants demonstrated that OPDA (12-oxo phytenoic acid) biosynthesis relative to the jasmonic acid (JA) availability controls dampening of the genes for oxylipin biosynthetic enzymes: Gene regulation in oxylipin biosynthesis mutants more strongly depended on the biosynthesis of the JA precursor OPDA than on its conversion to JA. Additionally, priming-dependent dampening during triggering was more linked to OPDA than to JA level regulation and spray application of OPDA prior to triggering counteracted gene dampening. In contrast to cold-priming induced dampening of ZAT10, priming regulation of the oxylipin hub was insensitive to priming-induced accumulation of thylakoid ascorbate peroxidase and mediated by modulation of the oxylipin sensitivity of genes for OPDA biosynthesis.

Infection of Arabidopsis thaliana by the ascomycete fungus Colletotrichum higginsianum is characterised by an early symptomless biotrophic phase followed by a destructive necrotrophic phase. The fungal genome contains 77 secondary metabolism-related biosynthetic gene clusters (BGCs), and their expression during the infection process is tightly regulated. Deleting CclA, a chromatin regulator involved in repression of some BGCs through H3K4 trimethylation, allowed overproduction of 3 families of terpenoids and isolation of 12 different molecules. These natural products were tested in combination with methyl jasmonate (MeJA), an elicitor of jasmonate responses, for their capacity to alter defence gene induction in Arabidopsis. Higginsianin B inhibited MeJA-triggered expression of the defence reporter VSP1p:GUS, suggesting it may block bioactive JA-Ile synthesis or signalling in planta. Using the JA-Ile sensor Jas9-VENUS, we found that higginsianin B, but not three other structurally-related molecules, suppressed JA-Ile signalling by preventing degradation of JAZ proteins, the repressors of JA responses. Higginsianin B likely blocks the 26S proteasome-dependent degradation of JAZ proteins because it inhibited chymotrypsin- and caspase-like protease activities. The inhibition of target degradation by higginsianin B also extended to auxin signalling, as higginsianin B treatment reduced IAA-dependent expression of DR5p:GUS. Overall, our data indicate that specific fungal secondary metabolites can act similarly to protein effectors to subvert plant immune and developmental responses.

Long-lasting and broad-spectrum disease resistance throughout plants is an ever-important objective in basic and applied plant and crop research. While the recent identification of N-hydroxpipecolic acid (NHP) and its central role in systemic plant immunity in the model Arabidopsis thaliana provides a conceptual framework toward this goal, Schnake et al. (2020) quantify levels of NHP and its direct precursor in six mono- and dicotyledonous plant species subsequent to attacks by their natural pathogens, thereby implicating (phloem-mobile) NHP as a general and conserved activator of disease resistance.

Plant microtubules form a highly dynamic intracellular network with important roles for regulating cell division, cell proliferation and cell morphology. Its organization and dynamics are coordinated by various microtubule-associated proteins (MAPs) that integrate environmental and developmental stimuli to fine-tune and adjust cytoskeletal arrays. IQ67 DOMAIN (IQD) proteins recently emerged as a class of plant-specific MAPs with largely unknown functions. Here, using a reverse genetics approach, we characterize Arabidopsis IQD5 in terms of its expression domains, subcellular localization and biological functions. We show that IQD5 is expressed mostly in vegetative tissues, where it localizes to cortical microtubule arrays. Our phenotypic analysis of iqd5 loss-of-function lines reveals functions of IQD5 in pavement cell (PC) shape morphogenesis. Histochemical analysis of cell wall composition further suggests reduced rates of cellulose deposition in anticlinal cell walls, which correlate with reduced anisotropic expansion. Lastly, we demonstrate IQD5-dependent recruitment of calmodulin calcium sensors to cortical microtubule arrays and provide first evidence for important roles of calcium in regulation of PC morphogenesis. Our work thus identifies IQD5 as a novel player in PC shape regulation, and, for the first time, links calcium signaling to developmental processes that regulate anisotropic growth in PCs.

Plant growth and development are a genetically predetermined series of events but can change dramatically in response to environmental stimuli, involving perpetual pattern formation and reprogramming of development. The rate of growth is determined by cell division and subsequent cell expansion, which are restricted and controlled by the cell wall–plasma membrane–cytoskeleton continuum, and are coordinated by intricate networks that facilitate intra- and intercellular communication. An essential role in cellular signaling is played by calcium ions, which act as universal second messengers that transduce, integrate, and multiply incoming signals during numerous plant growth processes, in part by regulation of the microtubule cytoskeleton. In this review, we highlight recent advances in the understanding of calcium-mediated regulation of microtubule-associated proteins, their function at the microtubule cytoskeleton, and their potential role as hubs in crosstalk with other signaling pathways.

Dynamic regulation of protein function and abundance plays an important role in virtually every aspect of plant life. Diversifying mechanisms at the RNA and protein level result in many protein molecules with distinct sequence and modification, termed proteoforms, arising from a single gene. Distinct protein termini define proteoforms arising from translation of alternative transcripts, use of alternative translation initiation sites, and different co- and post-translational modifications of the protein termini. Also site-specific proteolytic processing by endo- and exoproteases generates truncated proteoforms, defined by distinct protease-generated neo-N- and neo-C-termini, that may exhibit altered activity, function, and localization compared with their precursor proteins. In eukaryotes, the N-degron pathway targets cytosolic proteins, exposing destabilizing N-terminal amino acids and/or destabilizing N-terminal modifications for proteasomal degradation. This enables rapid and selective removal not only of unfolded proteins, but also of substrate proteoforms generated by proteolytic processing or changes in N-terminal modifications. Here we summarize current protocols enabling proteome-wide analysis of protein termini, which have provided important new insights into N-terminal modifications and protein stability determinants, protein maturation pathways, and protease–substrate relationships in plants.

Conjugates between polyamines and hydroxycinnamic acids are found on the pollen surface of all higher plants, both mono- and dicots. But we don’t know why they are there. Delporte et al. (2018) have now shown that in the tapetum of the Asteraceae (sunflower family) a new type of BAHD-acyltransferase is expressed, able to transfer coenzyme A-activated coumaric acid to all four primary and secondary amine groups of the polyamine spermine. In the case of chicory this sequential addition results in a fully substituted tetracoumaroyl–spermine conjugate and points to an evolutionary advantage of these functionally enigmatic compounds.