Phytohormones regulate many aspects of plant life by activating transcription factors (TF) that bind sequence-specific response elements (RE) in regulatory regions of target genes. Despite their short length, REs are degenerate with a core of just 3-4bp. This degeneracy is paradoxical, as it reduces specificity and REs are extremely common in the genome. To study whether RE degeneracy might serve a biological function we developed an algorithm for the detection of regulatory sequence conservation and applied it to phytohormones REs in 45 angiosperms. Surprisingly, we found that specific RE variants are highly conserved in core hormone response genes. Experimental evidence showed that specific variants act to regulate the magnitude and spatial profile of hormonal response in Arabidopsis and tomato. Our results suggest that hormone-regulated TFs bind a spectrum of REs, each coding for a distinct transcriptional response profile. Our approach has implications for precise genome editing and for rational promoter design.

Patterning of the root xylem into protoxylem (PX) and metaxylem is regulated by auxin-cytokinin signaling and microRNA -mediated suppression of genes encoding Class III HOMEODOMAIN LEU-ZIPPER (HD-ZIPIII) proteins. We found that, in , osmotic stress via core abscisic acid (ABA) signaling in meristematic endodermal cells induces differentiation of PX in radial and longitudinal axes in association with increased expression. Similarly, in tomato, ABA enhanced PX differentiation longitudinally and radially, indicating an evolutionarily conserved mechanism. ABA increased expression of and reduced expression of the repressor ZWILLE (ZLL) (also known as ARGONAUTE10), resulting in reduced levels of all five HD-ZIPIII RNAs. ABA treatments failed to induce additional PX files in a -resistant mutant, , and in and mutants, in which expression is strongly reduced. Thus, ABA regulates xylem patterning and maturation via -regulated expression of HD-ZIPIII mRNAs and associated VND7 levels. In lateral root initials, ABA induced an increase in levels in endodermal precursors and inhibited their reduction in the future quiescent center specifically at pre-emergence stage. Hence, ABA-induced inhibition of lateral root is associated with reduced HD-ZIPIII levels.

The improvement of crop productivity under abiotic stress is one of the biggest challenges faced by the agricultural scientific community. Despite extensive research, the research-to-commercial transfer rate of abiotic stress-resistant crops remains very low. This is mainly due to the complexity of genotype × environment interactions and in particular, the ability to quantify the dynamic plant physiological response profile to a dynamic environment. Most existing phenotyping facilities collect information using robotics and automated image acquisition and analysis. However, their ability to directly measure the physiological properties of the whole plant is limited. We demonstrate a high-throughput functional phenotyping system (HFPS) that enables comparing plants' dynamic responses to different ambient conditions in dynamic environments due to its direct and simultaneous measurement of yield-related physiological traits of plants under several treatments. The system is designed as one-to-one (1:1) plant-[sensors+controller] units, i.e., each individual plant has its own personalized sensor, controller and irrigation valves that enable (i) monitoring water-relation kinetics of each plant-environment response throughout the plant's life cycle with high spatiotemporal resolution, (ii) a truly randomized experimental design due to multiple independent treatment scenarios for every plant, and (iii) reduction of artificial ambient perturbations due to the immobility of the plants or other objects. In addition, we propose two new resilience-quantifying-related traits that can also be phenotyped using the HFPS: transpiration recovery rate and night water reabsorption. We use the HFPS to screen the effects of two commercial biostimulants (a seaweed extract -ICL-SW, and a metabolite formula - ICL-NewFo1) on under different irrigation regimes. Biostimulants are considered an alternative approach to improving crop productivity. However, their complex mode of action necessitates cost-effective pre-field phenotyping. The combination of two types of treatment (biostimulants and drought) enabled us to evaluate the precision and resolution of the system in investigating the effect of biostimulants on drought tolerance. We analyze and discuss plant behavior at different stages, and assess the penalty and trade-off between productivity and resilience. In this test case, we suggest a protocol for the screening of biostimulants' physiological mechanisms of action.

The transition from pollinator-mediated outbreeding to selfing has occurred many times in angiosperms. This is generally accompanied by a reduction in traits attracting pollinators, including a reduced emission of floral scent. In Capsella, emission of benzaldehyde as a main component of floral scent has been lost in selfing C. rubella by mutation of cinnamate-CoA ligase CNL1. However, the biochemical basis and evolutionary history of this loss remain unknown, as does the reason for the absence of benzaldehyde emission in the independently derived selfer C. orientalis. We used plant transformation, in vitro enzyme assays, population genetics and quantitative genetics to address these questions. CNL1 has been inactivated twice independently by point mutations in C. rubella, causing a loss of enzymatic activity. Both inactive haplotypes are found outside Greece, the centre of origin of C. rubella, indicating that they arose before its geographical spread. By contrast, the loss of benzaldehyde emission in C. orientalis is not due to an inactivating mutation in CNL1. CNL1 represents a hotspot for mutations that eliminate benzaldehyde emission, potentially reflecting the limited pleiotropy and large effect of its inactivation. Nevertheless, even closely related species have followed different evolutionary routes in reducing floral scent. This article is protected by copyright. All rights reserved.

The periderm is a protective corky tissue that is formed through the cambial activity of phellogen cells, when the outer epidermis is damaged. Timely periderm formation is critical to prevent pathogen invasion and water loss. The outer layers of the potato periderm, the tuber skin, serves as a model to study cork development. Early in tuber development the phellogen becomes active and produces the skin. During tuber maturation it becomes inactive and the skin adheres to the tuber flesh. The characterization of potato phellogen may contribute to the management of costly agricultural problems related to incomplete skin-set and the resulting skinning injuries, and provide us with new knowledge regarding cork development in planta. A transcriptome of potato tuber phellogen isolated by laser capture microdissection indicated similarity to vascular cambium and the cork from trees. Highly expressed genes and transcription factors indicated that phellogen activation involves cytokinesis and gene reprograming for the establishment of a dedifferentiation state; whereas inactivation is characterized by activity of genes that direct organ identity in meristem and cell-wall modifications. The expression of selected genes was analyzed using qPCR in native and wound periderm at distinct developmental stages. This allowed the identification of genes involved in periderm formation and maturation.

Breeding crops for high yield and superior adaptability to new and variable climates is imperative to ensure continued food security, biomass production, and ecosystem services. Advances in genomics and phenomics are delivering insights into the complex biological mechanisms that underlie plant functions in response to environmental perturbations. However, linking genotype to phenotype remains a huge challenge and is hampering the optimal application of high-throughput genomics and phenomics to advanced breeding. Critical to success is the need to assimilate large amounts of data into biologically meaningful interpretations. Here, we present the current state of genomics and field phenomics, explore emerging approaches and challenges for multiomics big data integration by means of next-generation (Next-Gen) artificial intelligence (AI), and propose a workable path to improvement.

The earliest visual changes of leaf senescence occur in the chloroplast as chlorophyll is degraded and photosynthesis declines. Yet, a comprehensive understanding of the sequence of catabolic events occurring in chloroplasts during natural leaf senescence is still missing. Here, we combined confocal and electron microscopy together with proteomics and biochemistry to follow structural and molecular changes during Arabidopsis leaf senescence. We observed that initiation of chlorophyll catabolism precedes other breakdown processes. Chloroplast size, stacking of thylakoids, and efficiency of PSII remain stable until late stages of senescence, whereas the number and size of plastoglobules increase. Unlike catabolic enzymes, whose level increase, the level of most proteins decreases during senescence, and chloroplast proteins are overrepresented among these. However, the rate of their disappearance is variable, mostly uncoordinated and independent of their inherent stability during earlier developmental stages. Unexpectedly, degradation of chlorophyll-binding proteins lags behind chlorophyll catabolism. Autophagy and vacuole proteins are retained at relatively high levels, highlighting the role of extra-plastidic degradation processes especially in late stages of senescence. The observation that chlorophyll catabolism precedes all other catabolic events may suggest that this process enables or signals further catabolic processes in chloroplasts.

Low in macro-porosity electro-spun scaffolds are often associated with foreign body response, whilst macro-porous electro-spun scaffolds have low mechanical integrity. Herein, compressed, macro-porous and collagen (bovine Achilles tendon and human recombinant) coated electro-spun poly-ε-caprolactone scaffolds were developed and their biomechanical, in vitro and in vivo properties were assessed. Collagen coating, independently of the source, did not significantly affect the biomechanical properties of the scaffolds. Although no significant difference in cell viability was observed between the groups, collagen coated scaffolds induced significantly higher DNA concentration. In vivo, no signs of adverse tissue effect were observed in any of the groups and all groups appeared to equally integrate into the subcutaneous tissue. It is evidenced that macro-porous poly-ε-caprolactone electro-spun meshes with adequate mechanical properties and acceptable host response can be developed for biomedical applications.

Plants developed various reversible and non-reversible acclimation mechanisms to cope with the multifaceted nature of abiotic-stress combinations. We hypothesized that in order to endure these stress combinations, plants elicit distinctive acclimation strategies through specific trade-offs between reproduction and defense. To investigate acclimation strategies to combinations of salinity, drought and heat, we applied a system biology approach, integrating physiological, metabolic, and transcriptional analyses. We analyzed the trade-offs among functional and performance traits, and their effects on plant fitness. A combination of drought and heat resulted in escape strategy, while under a combination of salinity and heat, plants exhibited an avoidance strategy. On the other hand, under combinations of salinity and drought, with or without heat stress, plant fitness (i.e., germination rate of subsequent generation) was severely impaired. These results indicate that under combined stresses, plants' life-history strategies were shaped by the limits of phenotypic and metabolic plasticity and the trade-offs between traits, thereby giving raise to distinct acclimations. Our findings provide a mechanistic understanding of plant acclimations to combinations of abiotic stresses and shed light on the different life-history strategies that can contribute to grass fitness and possibly to their dispersion under changing environments.

Litchi (Litchi chinensis Sonn.) is a subtropical fruit known for its attractive red pericarp color, semi-translucent white aril and unique flavor and aroma. Rapid post-harvest pericarp browning strictly limits litchi fruit marketing. In the current research, we hypothesized that modification of litchi fruit pericarp anatomy by hormone application may reduce fruit susceptibility to post-harvest pericarp browning. In this context, we hypothesized that cytokinin treatment, known to induce cell division, may yield fruit with thicker pericarp and reduced susceptibility for fruit surface micro-crack formation, water loss and post-harvest pericarp browning. Exogenous cytokinin treatment was applied at different stages along the course of litchi fruit development and the effect on fruit pericarp anatomy, fruit maturation and postharvest pericarp browning was investigated. Interestingly, cytokinin treatment, applied 4 weeks after full female bloom (WFB), during the phase of pericarp cell division, led to mature fruit with thicker pericarp, reduced rate of post-harvest water loss and reduced susceptibility to post-harvest pericarp browning, as compared to non-treated control fruit. Histological sections ascribe the difference in pericarp anatomy to increased cell proliferation in the parenchymatic tissue and the highly-lignified brachysclereid cell layer. In contrast, exogenous cytokinin treatment applied 7 WFB, following the phase of pericarp cell division, significantly increased epidermal-cell proliferation but had no significant effect on overall fruit pericarp thickness and only minor affect on post-harvest water loss or pericarp browning. Interestingly, the late cytokinin treatment also significantly postponed fruit maturation-associated anthocyanin accumulation and chlorophyll degradation, as previously reported, but had no effect on other parameters of fruit maturation, like total soluble sugars and total titratable acids typically modified during aril maturation. In conclusion, exogenous cytokinin treatment at different stages in fruit development differentially modifies litchi fruit pericarp anatomy by induction of cell-type specific cell proliferation. Early cytokinin treatment during the phase of pericarp cell division may prolong litchi fruit storage by reducing fruit susceptibility to post-harvest water loss and pericarp browning.

KEY MESSAGE: Wild emmer allele of GNI-A1 ease competition among developing grains through the suppression of floret fertility and increase grain weight in tetraploid wheat. Grain yield is a highly polygenic trait determined by the number of grains per unit area, as well as by grain weight. In wheat, grain number and grain weight are usually negatively correlated. Yet, the genetic basis underlying trade-off between the two is mostly unknown. Here, we fine-mapped a grain weight QTL using wild emmer introgressions in a durum wheat background and showed that grain weight is associated with the GNI-A1 gene, a regulator of floret fertility. In-depth characterization of grain number and grain weight indicated that suppression of distal florets by the wild emmer GNI-A1 allele increases weight of proximal grains in basal and central spikelets due to alteration in assimilate distribution. Re-sequencing of GNI-A1 in tetraploid wheat demonstrated the rich allelic repertoire of the wild emmer gene pool, including a rare allele which was present in two gene copies and contained a nonsynonymous mutation in the C-terminus of the protein. Using an F population generated from a cross between wild emmer accessions Zavitan, which carries the rare allele, and TTD140, we demonstrated that this unique polymorphism is associated with grain weight, independent of grain number. Moreover, we showed, for the first time, that GNI-A1 proteins are transcriptional activators and that selection targeted compromised activity of the protein. Our findings expand the knowledge of the genetic basis underlying trade-off between key yield components and may contribute to breeding efforts for enhanced grain yield.

BACKGROUND: For over a century, genetic diversity of wheat worldwide was eroded by continual selection for high yields and industrial demands. Wheat landraces cultivated in Israel and Palestine demonstrate high genetic diversity and a potentially wide repertoire of adaptive alleles. While most Israeli-Palestinian wheat landraces were lost in the transition to "Green Revolution" semi-dwarf varieties, some germplasm collections made at the beginning of the 20 century survived in genebanks and private collections worldwide. However, fragmentation and poor conservation place this unique genetic resource at a high risk of genetic erosion. Herein we describe a long-term initiative to restore, conserve and characterize a collection of Israeli and Palestinian wheat landraces (IPLR). RESULTS: (i) a report on the IPLR construction (n=932).; (ii) an historical and agronomic context to this collection; (iii) characterize and assess IPLR's genetic diversity; and (iv) data comparison from two distinct sub-collections within IPLR: a collection made by N. Vavilov in 1926 (IPLR-VIR) and a later one (1979-1981) made by Y. Mattatia (IPLR-M). Though conducted in the same eco-geographic space, these two collections were subjected to considerably different conservation pathways. IPLR-M- which underwent only one propagation cycle- demonstrated marked genetic and phenotypic variability (within and between accessions) in comparison to IPLR-VIR which had been regularly regenerated over ~90 years. CONCLUSION: We postulate that long-term ex-situ conservation involving human and GxE selection may significantly reduce accession heterogeneity and allelic diversity. Results are further discussed in a broader context of pre-breeding and conservation. This article is protected by copyright. All rights reserved.

Auxin-signal transduction is mediated by the antagonistic activity of transcriptional activators and repressors. Both activators and repressors belong to gene families, but the biological importance of this complexity is not clear. Here, we addressed this question using tomato leaf development as a model by generating and analyzing mutants in multiple auxin-response components. In developing compound tomato leaves, auxin promotes leaflet formation and blade growth, and in the intercalary regions between leaflets, auxin response is inhibited by the Aux/IAA protein ENTIRE (E). e mutants form simple leaves due to ectopic blade growth in the intercalary domain. Using this unique loss-of-function phenotype and genome editing of auxin-response factor (ARF) genes, encoding auxin-response activators, we identified the contribution of specific ARFs to the e phenotype. Mutations in the related ARFs SlMP, SlARF19A, and SlARF19B, but not SlARF7, reduced the leaf blade and suppressed the e phenotype in a dosage-dependent manner that correlated with their relative expression, leading to a continuum of shapes. While single e and slmp mutants affected blade growth in an opposite manner, leaves of e slmp double mutants were similar to those of the wild type. However, the leaf shape of e slmp was more variable than that of the wild type, and it showed increased sensitivity to auxin. Our findings demonstrate that the existence of multiple auxin-response repressors and activators stabilizes the developmental output of auxin and that tuning their activity enables shape variability. The increased complexity of the auxin response therefore balances stability and flexibility in leaf patterning.

The pleiotropic and complex gibberellin (GA) response relies on targeted proteolysis of DELLA proteins mediated by a GA-activated GIBBERELLIN-INSENSITIVE DWARF1 (GID1) receptor. The tomato (Solanum lycopersicum) genome encodes for a single DELLA protein, PROCERA (PRO), and three receptors, SlGID1a (GID1a), GID1b1 and GID1b2, that may guide specific GA responses. In this work, CRISPR-Cas9-derived gid1 mutants were generated and their effect on GA responses was studied. The gid1 triple mutant was extremely dwarf and fully insensitive to GA. Under optimal growth conditions, the three receptors function redundantly and the single gid1 mutants exhibited very mild phenotypic changes. Among the three receptors, GID1a had the strongest effects on germination and growth. Yeast two-hybrid assays suggested that GID1a has the highest affinity to PRO. Analysis of lines with a single active receptor demonstrated a unique role for GID1a in protracted response to GA that was saturated only at high doses. When the gid1 mutants were grown in the field under ambient changing environments, they showed phenotypic instability, the high redundancy was lost and gid1a exhibited dwarfism that was strongly exacerbated by the loss of another GID1b receptor gene. These results suggest that multiple GA receptors contribute to phenotypic stability under environmental extremes.

Obligate root holoparasite Phelipanche aegyptiaca is an agricultural pest, which infests its hosts and feeds on the sap, subsequently damaging crop yield and quality. Its notoriously viable seed bank may serve as an ideal pest control target. The phytohormone abscisic acid (ABA) was shown to regulate P. aegyptiaca seed dormancy following strigolactones germination stimulus. Transcription analysis of signaling components revealed five ABA receptors and two co-receptors (PP2C). Transcription of lower ABA-affinity subfamily III receptors was absent in all tested stages of P. aegyptiaca development and parasitism stages. P. aegyptiaca ABA receptors interacted with the PP2Cs, and inhibited their activity in an ABA-dependent manner. Moreover, sequence analysis revealed multiple alleles in two P. aegyptiaca ABA receptors, with many non-synonymous mutations. Functional analysis of selected receptor alleles identified a variant with substantially decreased inhibitory effect of PP2Cs activity in-vitro. These results provide evidence that P. aegyptiaca is capable of biochemically perceiving ABA. In light of the possible involvement of ABA in parasitic activities, the discovery of active ABA receptors and PP2Cs could provide a new biochemical target for the agricultural management of P. aegyptiaca. Furthermore, the potential genetic loss of subfamily III receptors in this species, could position P. aegyptiaca as a valuable model in the ABA perception research field.

FTSH proteases are membrane-bound, ATP-dependent metalloproteases found in bacteria, mitochondria and chloroplasts. The product of one of the 12 genes encoding FTSH proteases in Arabidopsis, FTSH11, has been previously shown to be essential for acquired thermotolerance. However, the substrates of this protease, as well as the mechanism linking it to thermotolerance are largely unknown. To get insight into these, the FTSH11 knockout mutant was complemented with proteolytically active or inactive variants of this protease, tagged with HA-tag, under the control of the native promoter. Using these plants in thermotolerance assay demonstrated that the proteolytic activity, and not only the ATPase one, is essential for conferring thermotolerance. Immunoblot analyses of leaf extracts, isolated organelles and sub-fractionated chloroplast membranes localized FTSH11 mostly to chloroplast envelopes. Affinity purification followed by mass spectrometry analysis revealed interaction between FTSH11 and different components of the CPN60 chaperonin. In affinity enrichment assays, CPN60s as well as a number of envelope, stroma and thylakoid proteins were found associated with proteolytically inactive FTSH11. Comparative proteomic analysis of WT and knockout plants, grown at 20°C or exposed to 30°C for 6 h, revealed a plethora of upregulated chloroplast proteins in the knockout, some of them might be candidate substrates. Among these stood out TIC40, which was stabilized in the knockout line after recovery from heat stress, and three proteins that were found trapped in the affinity enrichment assay: the nucleotide antiporter PAPST2, the fatty acid binding protein FAP1 and the chaperone HSP70. The consistent behavior of these four proteins in different assays suggest that they are potential FTSH11 substrates.

The increasing demand for spelt products requires the baking industry to develop accurate and efficient tools to differentiate between spelt and bread wheat grains. We subjected a 272-sample spelt-bread wheat set to several potential diagnostic methods. DNA markers for γ-gliadin-D ( GAG56D), γ-gliadin-B ( GAG56B), and the Q-gene were used, alongside phenotypic assessment of ease-of-threshing and near-infrared spectroscopy (NIRS). The GAG56B and GAG56D markers demonstrated low diagnostic power in comparison to the Q-gene genotyping, which showed full accordance with the threshing phenotype, providing a highly accurate distinction between bread wheat and spelt kernels. A highly reliable Q classification was based on a three-waveband NIR model [Kappa (0.97), R-square (0.93)], which suggested that this gene influences grain characteristics. Our data ruled out a protein concentration bias of the NIRS-based diagnosis. These findings highlight the Q gene and NIRS as important, valuable, but simple tools for distinguishing between bread wheat and spelt.