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.

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.

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.

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.

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.

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.

Liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) is increasingly popular for the non-targeted exploration of complex samples, where tandem mass spectrometry (MS/MS) is used to characterize the structure of unknown compounds. However, mass spectra do not always contain sufficient information to unequivocally identify the correct structure. This study investigated how much additional information can be gained using hydrogen deuterium exchange (HDX) experiments. The exchange of “easily exchangeable” hydrogen atoms (connected to heteroatoms), with predominantly [M+D]+ ions in positive mode and [M-D]− in negative mode was observed. To enable high-throughput processing, new scoring terms were incorporated into the in silico fragmenter MetFrag. These were initially developed on small datasets and then tested on 762 compounds of environmental interest. Pairs of spectra (normal and deuterated) were found for 593 of these substances (506 positive mode, 155 negative mode spectra). The new scoring terms resulted in 29 additional correct identifications (78 vs 49) for positive mode and an increase in top 10 rankings from 80 to 106 in negative mode. Compounds with dual functionality (polar head group, long apolar tail) exhibited dramatic retention time (RT) shifts of up to several minutes, compared with an average 0.04 min RT shift. For a smaller dataset of 80 metabolites, top 10 rankings improved from 13 to 24 (positive mode, 57 spectra) and from 14 to 31 (negative mode, 63 spectra) when including HDX information. The results of standard measurements were confirmed using targets and tentatively identified surfactant species in an environmental sample collected from the river Danube near Novi Sad (Serbia). The changes to MetFrag have been integrated into the command line version available at http://c-ruttkies.github.io/MetFrag and all resulting spectra and compounds are available in online resources and in the Electronic Supplementary Material (ESM).

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.

Plant U-box type E3 ubiquitin ligases (PUBs) are well known for their functions in a variety of stress responses, including immune responses and the adaptation to abiotic stresses. First linked to pollen self-incompatibility, their repertoire of roles has grown to encompass also the regulation of developmental processes. Notably, new studies provide clues to their mode of action, underline the existence of conserved PUB–kinase modules, and suggest new links to G-protein signalling, placing PUBs at the crossroads of major signalling hubs. The frequent association with membranes, by interacting and/or targeting membrane proteins, as well as through a recently reported direct interaction with phospholipids, indicates a general function in the control of vesicle transport and their cargoes. This review aims to give an overview of the most significant advances in the field, while also trying to identify common themes of PUB function.

Ceramides (CERs) are the backbone of the intercellular lipid lamellae of the stratum corneum (SC), the outer layer of the skin. Skin diseases such as atopic dermatitis, psoriasis, and aged skin are characterized by dysfunctional skin barrier and dryness which are associated with reduced levels of CERs. Replenishing the depleted epidermal CERs with exogenous CERs has been shown to have beneficial effects in improving the skin barrier and hydration. The exogenous CERs such as phyto-derived CERs (PhytoCERs) can be delivered deep into the SC using novel topical formulations. This, however, requires investigating the rate and extent of skin permeation of CERs. In this study, an LC/APCI-MS method to detect and quantify PhytoCERs in different layers of the skin has been developed and validated. The method was used to investigate the skin permeation of PhytoCERs using Franz diffusion cells after applying an amphiphilic cream containing PhytoCERs to the surface of ex vivo human skin. As plant-specific CERs are not commercially available, well-characterized CERs isolated from oat (Avena abyssinica) were used as reference standards for the development and validation of the method. The method was linear over the range of 30–1050 ng/mL and sensitive with limit of detection and quantification of 10 and 30 ng/mL, respectively. The method was also selective, accurate, and precise with minimal matrix effect (with mean matrix factor around 100%). Even if more than 85% of oat CERs in the cream remained in the cream after the incubation periods of 30, 100, and 300 min, it was possible to quantify the small quantities of oat CERs distributed across the SC, epidermis, and dermis of the skin indicating the method’s sensitivity. Therefore, the method can be used to investigate the skin permeation of oat CERs from the various pharmaceutical and cosmeceutical products without any interference from the skin constituents such as the epidermal lipids.