In Arabidopsis thaliana, the genes FLOWERING LOCUS T (FT) and TERMINAL FLOWER1 (TFL1) have antagonistic roles in regulating the onset of flowering: FT activates and TFL1 represses flowering. Both encode small, closely related transcription cofactors of ;175 amino acids. Previous studies identified a potential ligand binding residue as well as a divergent external loop as critical for the differences in activity of FT and TFL1, but the mechanisms for the differential action of FT and TFL1 remain unclear. Here, we took an unbiased approach to probe the importance of residues throughout FT protein, testing the effects of hundreds of mutations in vivo. FT is surprisingly robust to a wide range of mutations, even in highly conserved residues. However, specific mutations in at least four different residues are sufficient to convert FT into a complete TFL1 mimic, even when expressed from TFL1 regulatory sequences. Modeling the effects of these mutations on the surface charge of FT protein suggests that the affected residues regulate the docking of an unknown ligand. These residues do not seem to alter the interaction with FD or 14-3-3 proteins, known FT interactors. Potential candidates for differential mediators of FT and TFL1 activities belong to the TCP (for TEOSINTE BRANCHED1, CYCLOIDEA, PCF) family of transcription factors.

In many plant species, conflicts between divergent elements of the immune system, especially nucleotide-binding oligomerization domain-like receptors (NLR), can lead to hybrid necrosis. Here, we report deleterious allele-specific interactions between an NLR and a non-NLR gene cluster, resulting in not one, but multiple hybrid necrosis cases in Arabidopsis thaliana . The NLR cluster is RESISTANCE TO PERONOSPORA PARASITICA 7 ( RPP7 ), which can confer strain-specific resistance to oomycetes. The non-NLR cluster is RESISTANCE TO POWDERY MILDEW 8 ( RPW8 ) / HOMOLOG OF RPW8 ( HR ), which can confer broad-spectrum resistance to both fungi and oomycetes. RPW8/HR proteins contain at the N-terminus a potential transmembrane domain, followed by a specific coiled-coil (CC) domain that is similar to a domain found in pore-forming toxins MLKL and HET-S from mammals and fungi. C-terminal to the CC domain is a variable number of 21- or 14-amino acid repeats, reminiscent of regulatory 21-amino acid repeats in fungal HET-S. The number of repeats in different RPW8/HR proteins along with the sequence of a short C-terminal tail predicts their ability to activate immunity in combination with specific RPP7 partners. Whether a larger or smaller number of repeats is more dangerous depends on the specific RPW8/HR autoimmune risk variant.

IntroductionFrost events lead to A$360 million of yield losses annually to the Australian wheat industry, making improvement of chilling and frost tolerance an important trait for breeding.ObjectivesThis study aimed to use metabolomics and lipidomics to explore genetic variation in acclimation potential to chilling and to identify metabolite markers for chilling tolerance in wheat.MethodsWe established a controlled environment screening assay that is able to reproduce field rankings of wheat germplasm for chilling and frost tolerance. This assay, together with targeted metabolomics and lipidomics approaches, were used to compare metabolite and lipid levels in flag leaves of two wheat varieties with contrasting chilling tolerance.ResultsThe sensitive variety Wyalkatchem showed a strong reduction in amino acids after the first cold night, followed by accumulation of osmolytes such as fructose, glucose, putrescine and shikimate over a 4-day period. Accumulation of osmolytes is indicative of acclimation to water stress in Wyalkatchem. This response was not observed for tolerant variety Young. The two varieties also displayed significant differences in lipid accumulation. Variation in two lipid clusters, resulted in a higher unsaturated to saturated lipid ratio in Young after 4 days cold treatment and the lipids PC(34:0), PC(34:1), PC(35:1), PC(38:3), and PI(36:4) were the main contributors to the unsaturated to saturated ratio change. This indicates that Young may have superior ability to maintain membrane fluidity following cold exposure, thereby avoiding membrane damage and water stress observed for Wyalkatchem.ConclusionOur study suggests that metabolomics and lipidomics markers could be used as an alternative phenotyping method to discriminate wheat varieties with differences in cold acclimation.Electronic supplementary materialThe online version of this article (10.1007/s11306-019-1606-2) contains supplementary material, which is available to authorized users.

The mechanisms underlying rootzone-localized responses to salinity during early stages of barley development remain elusive. In this study, we performed the analyses of multi-root-omes (transcriptomes, metabolomes, and lipidomes) of a domesticated barley cultivar (Clipper) and a landrace (Sahara) that maintain and restrict seedling root growth under salt stress, respectively. Novel generalized linear models were designed to determine differentially expressed genes (DEGs) and abundant metabolites (DAMs) specific to salt treatments, genotypes, or rootzones (meristematic Z1, elongation Z2, and maturation Z3). Based on pathway over-representation of the DEGs and DAMs, phenylpropanoid biosynthesis is the most statistically enriched biological pathway among all salinity responses observed. Together with histological evidence, an intense salt-induced lignin impregnation was found only at stelic cell wall of Clipper Z2, compared with a unique elevation of suberin deposition across Sahara Z2. This suggests two differential salt-induced modulations of apoplastic flow between the genotypes. Based on the global correlation network of the DEGs and DAMs, callose deposition that potentially adjusted symplastic flow in roots was almost independent of salinity in rootzones of Clipper, and was markedly decreased in Sahara. Taken together, we propose two distinctive salt tolerance mechanisms in Clipper (growth-sustaining) and Sahara (salt-shielding), providing important clues for improving crop plasticity to cope with deteriorating global soil salinization.

Arabidopsis REIL proteins are cytosolic ribosomal 60S-biogenesis factors. After shift to 10 °C, reil mutants deplete and slowly replenish non-translating eukaryotic ribosome complexes of root tissue, while controlling the balance of non-translating 40S- and 60S-subunits. Reil mutations respond by hyper-accumulation of non-translating subunits at steady-state temperature; after cold-shift, a KCl-sensitive 80S sub-fraction remains depleted. We infer that Arabidopsis may buffer fluctuating translation by pre-existing non-translating ribosomes before de novo synthesis meets temperature-induced demands. Reil1 reil2 double mutants accumulate 43S-preinitiation and pre-60S-maturation complexes and alter paralog composition of ribosomal proteins in non-translating complexes. With few exceptions, e.g. RPL3B and RPL24C, these changes are not under transcriptional control. Our study suggests requirement of de novo synthesis of eukaryotic ribosomes for long-term cold acclimation, feedback control of NUC2 and eIF3C2 transcription and links new proteins, AT1G03250, AT5G60530, to plant ribosome biogenesis. We propose that Arabidopsis requires biosynthesis of specialized ribosomes for cold acclimation.

Keywords RPW8, NLR, natural variation, autoimmunity, hybrid performance, MLKL, HET-S Barragan et al. RPW8/HR repeats and hybrid necrosis 2 SummaryHybrid offspring can look very different from their parents, including having greatly increased or decreased fitness. In many plant species, conflicts between divergent elements of the immune system can cause hybrids to express autoimmunity, a generally deleterious syndrome known as hybrid necrosis. We are investigating multiple hybrid necrosis cases in Arabidopsis thaliana that are caused by allele-specific interactions between different variants at two unlinked resistance (R) gene clusters. One is the RESISTANCE TO PERONOSPORA PARASITICA 7 (RPP7) cluster, which encodes an intracellular nucleotide binding site-leucine rich repeat (NLR) immune receptors that confer strain-specific resistance to oomycetes. The other is the RESISTANCE TO POWDERY MILDEW 8 (RPW8)/HOMOLOG OF RPW8 (HR) locus, which encodes atypical resistance proteins that can confer broad-spectrum resistance to filamentous pathogens. There is extensive structural variation in the RPW8/HR cluster, both at the level of gene copy number and at the level of C-terminal protein repeats of unknown function. We demonstrate that the number of RPW8/HR repeats correlate, albeit in a complex manner, with the severity of hybrid necrosis when these alleles are combined with specific RPP7 variants. This observation suggests that gross structural differences, rather than individual amino acid polymorphisms, guide the genetic interaction between RPW8/HR and RPP7 alleles. We discuss these findings in light of the similarity of RPW8/HR proteins with pore-forming toxins, MLKL and HET-S, from mammals and fungi.

Chilling and frost conditions impose major yield restraints to wheat crops in Australia and other temperate climate regions. Unpredictability and variability of field frost events are major impediments for cold tolerance breeding. Metabolome and lipidome profiling were used to compare the cold response in spikes of cold-tolerant Young and sensitive variety Wyalkatchem at the young microspore (YM) stage of pollen development. We aimed to identify metabolite markers that can reliably distinguish cold-tolerant and sensitive wheat varieties for future cold-tolerance phenotyping applications. We scored changes in spike metabolites and lipids for both varieties during cold acclimation after initial and prolonged exposure to combined chilling and freezing cycles (1 and 4 days, respectively) using controlled environment conditions. The two contrasting wheat varieties showed qualitative and quantitative differences in primary metabolites involved in osmoprotection, but differences in lipid accumulation most distinctively separated the cold response of the two wheat lines. These results resemble what we previously observed in flag leaves of the same two wheat varieties. The fact that this response occurs in tissue types with very different functions indicates that chilling and freezing tolerance in these wheat lines is associated with re-modelling of membrane lipid composition to maintain membrane fluidity.

29 30 WORD COUNT 31 18,932 32 2 TITLE 33 Arabidopsis REIL proteins activate ribosome biogenesis during cold acclimation 34 Running Title 35 REIL proteins activate ribosome biogenesis 36 Highlight of this study (summarizing sentence) (27 words) 37 REIL proteins affect paralog composition of eukaryotic ribosomes and suppress accumulation of 38 43S-preinitiation and pre-60S-maturation complexes, suggesting functions of ribosome 39 heterogeneity and biogenesis in plant cold acclimation. 40 Abstract (150 words) 41 Arabidopsis REIL proteins are cytosolic ribosomal 60S-biogenesis factors. After shift to 10°C, reil 42 mutants deplete and slowly replenish non-translating eukaryotic ribosome complexes of root 43 tissue, while tightly controlling the balance of non-translating 40S-and 60S-subunits. Reil 44 mutations compensate by hyper-accumulation of non-translating subunits at steady-state 45 temperature; after cold-shift, a KCl-sensitive 80S sub-fraction remains depleted. We infer that 46 Arabidopsis buffers fluctuating translation by pre-existing non-translating ribosomes before de 47 novo synthesis meets temperature-induced demands. Reil1 reil2 double mutants accumulate 43S-48 preinitiation and pre-60S-maturation complexes and have altered paralog composition of 49 ribosomal proteins in non-translating complexes. With few exceptions, e.g. RPL3B and RPL24C, 50 these changes are not under transcriptional control. Our study suggests requirement of de novo 51 synthesis of eukaryotic ribosomes for long-term cold acclimation, feedback control of NUC2 and 52 eIF3C2 transcription and links new proteins, AT1G03250, AT5G60530, to plant ribosome 53 biogenesis. We propose that Arabidopsis requires biosynthesis of specialized ribosomes for cold 54 acclimation. 55 Key words (6-10, in alphabetical order) 56 abiotic stress, Arabidopsis, cold acclimation, proteomics, REI-LIKE proteins, ribosome 57 biogenesis, roots, system analysis, transcriptomics 58 59 60 61The Arabidopsis thaliana Col-0 (Arabidopsis) REI1-LIKE (REIL) proteins, REIL1 62 (At4g31420) and REIL2 (At2g24500) are homologs of the yeast Rei1 (YBR267W) and the paralog 63 Reh1 (YLR387C) proteins. In yeast, both the Rei1 and Reh1 proteins function as ribosome 64 biogenesis factors that participate either in parallel or sequentially in the late ribosome biogenesis 65 step of cytoplasmic 60S ribosomal subunit maturation (Greber et al., 2012; Greber et al., 2016). 66 Aside from this function, these proteins are required to maintain growth at suboptimal or cold 67 temperatures. The ∆rei1 mutant is cold sensitive already at moderately suboptimal temperatures 68 of yeast. The ∆rei1 ∆reh1 double mutant is even more cold sensitive, while the yeast ∆reh1 69 mutation alone has no effect on growth in the cold (Iwase and Toh-e, 2004; Lebreton et al., 2006; 70 Parnell and Bass, 2009). Heterologous expression of Arabidopsis REIL1, but not of REIL2 partly 71 complements the cold sensitivity of the yeast ∆rei1 mutant (Schmidt et al., 2013). The Arabidopsis 72 REIL paralogs differ in st...